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Protein Phosphatase Regulatory Protein 10 Cooperates with Neurofibromin Inactivation in Myeloid Leukemogenesis

Permanent Link: http://ufdc.ufl.edu/UFE0022608/00001

Material Information

Title: Protein Phosphatase Regulatory Protein 10 Cooperates with Neurofibromin Inactivation in Myeloid Leukemogenesis
Physical Description: 1 online resource (105 p.)
Language: english
Creator: Hadjipanayis, Angela
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: aml, cml, jmml, leukemia, nf1, pediatric
Genetics (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Neurofibromatosis (NF1) is an autosomal dominant disease that affects 1 in 4000 people. Patients afflicted with NF1 present a variety of phenotypes, but the hallmark features of the disease are neurofibromas on or under the skin, cafe au lait spots, and Lisch nodules of the iris. Children with NF1 are at 200 to 500 times greater risk for developing a chronic myeloid leukemia. Often children who are diagnosed with this myeloid leukemia initially are not diagnosed with NF1 until a few years later. Therefore, there are likely more children who are affected by NF1-associated leukemia than are reported in the statistics. The focus of this proposal is to study NF1-associated myeloid leukemia, and to characterize additional genetic events in tumor progression to acute stage using human and mouse model resources. A viral based mouse model of NF1-associated leukemogenesis identified several common sites of viral integration. I investigated one site (Epi2) to see how the endogenous genes are affected and how this can contribute to myeloid leukemia. The Epi2 locus is on chromosome 6 in humans and chromosome 17 in mice. There are two genes in the Epi2 locus, PPP1R10 and MRPS18B. PPP1R10, whose protein product is PNUTS, is a serine/threonine protein phosphatase regulatory protein, and MRPS18B is a 28S mitochondrial ribosomal protein. Since both genes are located less than 1kb apart and are transcribed in opposite directions, it was possible that both genes act simultaneously in NF1 leukemogenesis, as oncogenes and/or tumor suppressors. I performed a series of experiments to test some of these hypotheses, and found evidence that PPP1R10 is a tumor suppressor gene that contributes to development of acute myeloid leukemia.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Angela Hadjipanayis.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Wallace, Margaret R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022608:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022608/00001

Material Information

Title: Protein Phosphatase Regulatory Protein 10 Cooperates with Neurofibromin Inactivation in Myeloid Leukemogenesis
Physical Description: 1 online resource (105 p.)
Language: english
Creator: Hadjipanayis, Angela
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: aml, cml, jmml, leukemia, nf1, pediatric
Genetics (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Neurofibromatosis (NF1) is an autosomal dominant disease that affects 1 in 4000 people. Patients afflicted with NF1 present a variety of phenotypes, but the hallmark features of the disease are neurofibromas on or under the skin, cafe au lait spots, and Lisch nodules of the iris. Children with NF1 are at 200 to 500 times greater risk for developing a chronic myeloid leukemia. Often children who are diagnosed with this myeloid leukemia initially are not diagnosed with NF1 until a few years later. Therefore, there are likely more children who are affected by NF1-associated leukemia than are reported in the statistics. The focus of this proposal is to study NF1-associated myeloid leukemia, and to characterize additional genetic events in tumor progression to acute stage using human and mouse model resources. A viral based mouse model of NF1-associated leukemogenesis identified several common sites of viral integration. I investigated one site (Epi2) to see how the endogenous genes are affected and how this can contribute to myeloid leukemia. The Epi2 locus is on chromosome 6 in humans and chromosome 17 in mice. There are two genes in the Epi2 locus, PPP1R10 and MRPS18B. PPP1R10, whose protein product is PNUTS, is a serine/threonine protein phosphatase regulatory protein, and MRPS18B is a 28S mitochondrial ribosomal protein. Since both genes are located less than 1kb apart and are transcribed in opposite directions, it was possible that both genes act simultaneously in NF1 leukemogenesis, as oncogenes and/or tumor suppressors. I performed a series of experiments to test some of these hypotheses, and found evidence that PPP1R10 is a tumor suppressor gene that contributes to development of acute myeloid leukemia.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Angela Hadjipanayis.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Wallace, Margaret R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022608:00001


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16be471131e8f9d0b96f6d691c65cdd4f40ed4b9







PROTEIN PHOSPHATASE REGULATORY PROTEIN 10 COOPERATES
WITH NEUROFIBROMIN INACTIVATION IN MYELOID
LEUKEMOGENESIS




















By

ANGELA HADJIPANAYIS


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008

































O 2008 Angela Hadjipanayis



































This work is dedicated to my extended family who never had the opportunity to
obtain higher education. To my parents and husband for their loving support and
belief in my abilities. To Camilynn I. Brannan whose research expertise led to the
discovery of this locus.









ACKNOWLEDGMENTS

First, I would like to thank my mentor Peggy Wallace for her indispensable support over

the years, her encouragement, and her advice on how to become a better scientist. Second, I

would like to thank my husband for taking a leap of faith in life and moving to Florida with me

when he didn't have a job at the time. Third, I would like to thank my parents for their love,

support, and advice over the years. Fourth, I would like to thank my other committee members,

Dr. Jim Resnick, Dr. Paul Oh, Dr. Stephen Hunger and Dr. Dan Driscoll for their valuable advice

on the proj ect. I also would like to thank Dr. Jennifer Embury for the tremendous effort on all the

pathology of the mice. In addition, I would like to thank Dr. Kevin Shannon and Dr. Scott Kogan

for their leukemia expertise and strategies in modeling leukemia. Lastly, I would like to thank

the lab for their help on this proj ect, their j okes, and all their personalities that made the days

seem not as long.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ........._.. _..... ._ ...............7....


LI ST OF FIGURE S .............. ...............8.....


LI ST OF AB BREVIAT IONS ............ ..... ._ .............. 11....


AB S TRAC T ............._. .......... ..............._ 13...


CHAPTER


1 INTRODUCTION ................. ...............15.......... ......


Neurofibromatosis Type 1 and JMML .............. ...............15....
M house M odels ofN fl ................... ................ .......... .......1
Molecular and Biochemical Mechanisms Underlying Myeloid Leukemogenesis. ................19
Hyperactive Ras in JMML................ ...............20.
Nfl Reconstitution Mouse Model ................. .. ...... ..... ..........2
Identification of Epi Loci in Nfl-Related Myeloid Leukemia ................ ............ .........21
Viral Insertional Mutagenesis............... ..............2
M RP S18 B .............. ...............24....
PPP 1R10 .........._.... ......._._. .... ....._... ...........2

PP 1, a Serine/Threonine Protein Phosphatase ......__....._.__._ ......._._. ..........2

2 MUTATIONAL ANALYSIS OF PPP1R10 INT LEUKEMIA ................. ............ .........28


Introducti on .................. ...............28....... ......
Materials and Methods .............. ...............29....
Patient Sam ples .............. ......... .......................2
Polymerase Chain Reaction and Sequencing of PPP1R10 Exons ................. ...............29
Restriction Fragment Length Polymorphism for Intron 15 Loss of Heterozygosity .......29
Real-Time mRNA Quantification .............. ...............30....
R e sults............ ............ ............... 1....
Discussion ................. ...............32....... ......


3 IN Y7TRO ANALYSIS OF PPP1R10 AS A POTENTIAL ONCOGENE ............................41


Introducti on ............ .......__ ...............41...
M materials and M ethods ........................ ..... .... .. ........4
Cloning of Ppp~rl0 in the MSCV Viral Vectors ....._.__._ ..... ... .__. ......._........4 1
Hematopoi eti c Cell Isol ati on and Retroviral Transducti on ................. .........._. .......42
Colony Formation Unit Granulocyte/Macrophage Colony Assay ................. ...............42












Serum Starvation of COS7 Cells ............... ...............43....
W western Blots .............. ...............43....
Re sults............. ...... ._ ...............44...
Discussion ............. ...... ._ ...............44...


4 PPP1R10 COOPERATES WITH NF1 INACTIVATION TO CAUSE ACUTE
MYELOMONOCYTIC LEUKEMIA IN A MOUSE MODEL ................. ............. .......50


Introducti on ................. ...............50.................
Materials and Methods .............. .. ...............51...

Ppp~rl0+/- Mouse Construction................ .............5
Breeding Ppp~rl0 Mice .............. ...............51...
Pathology/Sectioning/H and E Stain .............. ...............51....
Blood Smears and Manual Counts .............. ...............52....
Sudan Black Stain and MPO Stain ....._.................. ...............52. ...
Flow Cytometry ........._..... ......_. ...............53....
Nucleic Acid Extraction .............. ...............53....

Loss of Heterozygosity Analysis............... ...............53
Re sults ................ ...............54.................
Discussion ................. ...............58.................


5 DI SCUS SSION ................. ...............8.. 9......... ....


APPENDIX


A PRIMER LIST ................. ...............94................


B PFAFFL METHOD ................ ...............95........... ....


REFERENCES .............. ...............96....


BIOGRAPHICAL SKETCH ................. ...............105......... ......










LIST OF TABLES


Table page

2-1 NFl1-JMML human patient samples and Intron 15 polymorphism LOH. .......................38

2-2 JMML human samples and Intron 15 polymorphism LOH............__ .. .......__ ........39

2-3 mRNA Quantification of human leukemia RNA. ....._____ .... ... .__ ................ ....40

4-1 Ppp~rl0+/- aged mice necropsy data, M: E (2 to 8.25) ratios, and blast numbers............86

4-2 Nf+l+-Ppp~rl0+/l-aged mice necropsy data, M:E (2.3 3 to 1 3.2 5) ratios, and blast
numb ers ........._ ...... .___ ...............87....

4-3 Peripheral blood smear analysis of Nf+l+-Ppp~rl0+/- (NFl1PKO), Ppp~rl0+/-
(PKO), and Nf+l+-. ................ ...............88.___ ......

4-4 Bone marrow analysis of Nf+l+-Ppp~rl0+/- (NFl1PKO), Ppp~rl0+/- (PKO), and
Nf+l+- mice. WNL signifies within normal limits. .............. ...............88....










LIST OF FIGURES


FiMr page

2-1 Human NFl1-JMML analysis PPP1R10, Promoter Polymorphism (rsl6867845) .............33

2-2 Human NFl1-JMML analysis, PPP1R10 Exon 6 nonsense, S125X ................. ...............33

2-3 Human NFl1-JMML analysis, PPPlrl0 Exon 6 missense, S125T ................. ................33

2-4 Human NFl1-JMML analysis, PPP1R10 Exon 19 polymorphism (rs11754215). ..............33

2-5 Human NFl1-JMML PPP1R10 analysis, Intron 15 polymorphism loss of
heterozygosity. ............. ...............34.....

2-6 Rsal Digest of Intron 15 polymorphism of Control DNA' s. ................ ............ ........35

2-7 Rsal Intron 15 Polymorphism of NFl1-JMML and Control DNA. ................ ................36

2-8 Rsal Intron 15 Polymorphim Digest of JMML and an AML sample. .............. ..... .........._37

3-1 CFU-GM Colony assay of pMIG-PNUT S and pMIG in wild-type fetal liver cells..........46

3-2 Experimental Design of Colony Formation Assay. ............. ...............47.....

3-3 CFU-GM of wt PNUTS versus empty vector control. ................ .......... ...............48

3-4 Transfection of pDEST-PNUTS in COS7 cells with serum starvation and probing
signaling cascades. .............. ...............49....

4-1 Kaplan-Meier survival curve for Nf+l+-Ppp~rl0+/-, Ppp~rl0+/-, and Nf+l+- mice.........62

4-2 H and E stain sections from Nf+l+- Ppp~rl0+/- pulmonary adenocarcinoma and
mouse normal lung. ........... ..... .. ...............63..

4-3 H and E stain of sections from Nf+l+-Ppp~rl0+/- liver tumor and mouse normal
liver. ............. ...............64.....

4-4 H and E stain of sections from Nf+l+-Ppp~rl0+/- hepatocellular carcinoma and
normal liver ................. ...............65.................

4-5 H and E stain of sections from Nf+l+- Ppp~rl0+/- proliferative mesenchymal process
of the skin (top) and normal skin (bottom). ............. ...............66.....

4-6 H and E stain of sections of Ppp~rl0+/- adenocarcinoma of the salivary gland and
normal salivary gland. ........... ..... .. ...............67..

4-7 H and E stain of sections ofPpp~rl0+/- myoepithelioma, thymus and normal
thymus............... ...............68.










4-8 H and E stain of sections from Ppp~rl0+/- hemangiosarcoma and normal liver. .............69

4-9 H and E stain of sections from Ppp~rl0+/- pulmonary adenocarcinoma and normal
lung. ............. ...............70.....

4-10 H and E stain of sections from Ppp~rl 0+/- leiomyosarcoma and normal uterus............71

4-11 H and E stain of sections from Ppp~rl0+/- chloromas, showing myeloid expansion,
10X (left) and 40X (right) ................. ...............71...............

4-12 H and E stain of sections from Ppp~rl0+/- acute undifferentiated leukemia (AML-
MO) bone marrow (top, 100X) and splenomegaly ................. ...............72..............

4-13 H and E stain of sections from Nf+l+-Ppp~rl0+/- acute myeloid leukemic marrow
(AML-M4) (top) and normal bone marrow (bottom). ................... ............... 7

4-14 Peripheral Blood Smear from Ppp~rl0+/-Nf+l+- mouse, displaying abnormal
monocytes and neutrophils. ............. ...............73.....

4-15 An H and E stain of section of a granulocytic sarcoma in a Ppp~rl0+/-Nf+l+- mouse
(10X on top, 40X on bottom)............... ...............74

4-16 H and E stain of a section of a squamous cell carcinoma from a Ppp~rl0+/-Nf+l+,
4X top, 10X bottom. ............. ...............75.....

4-17 Ethidium-bromide stained polyacrylamide gel showing Nfl PCR products from
blood and bone marrow for Nf+l+-Ppp~rl0+/- mice to test for loss of heterozygosity
in the leukemic bone marrow ................. ...............76........... ...

4-18 Ethidium-Stained polyacrylamide gel showing PCR products from Ppp~rl0
genotyping in Ppp~rl0+/- mouse tissues to screen for loss of heterozygosity. ................77

4-19 Myeloperoxidase and Sudan Black stain of bone marrow sections mouse 188
(Ppp~rl0+/-), 690 (Ppp~rl0+/-Nfl+/-), and 1726 (Nf+l+-). .............. ....................7

4-20 Flow cytometry on mouse bone marrow for Ppp~rl0+/-Nf+l+- (NF lPKO), Nf+l+,
and Ppp~rl0+/- (PKO) with Grl (PE) and Macl (APC). ............. .....................7

4-21 Flow cytometry on mouse bone marrow for control bone marrow, Ppp~rl0+/-Nf+l+
(NF lPKO), Nfl+/-, and Ppp~rl0+/- (PKO) with ckit (Pacific Blue, stem cell
m arker). .............. ...............80....

4-22 Flow cytometry on mouse bone marrow from control bone marrow, Ppp~rl0+/-
Nf+l+ (NFlPKO), Nf+l+-, and Ppp~rl0+/- (PKO) with F4/80
(granulocyte/macrophage marker). .............. ...............81....

4-23 Flow cytometry on mouse bone marrow from control bone marrow, Ppp~rl0+/-
Nf+l+- (NF lPKO) and Ppp~rl0+/- (PKO) with CD19 (APC, B-cells). ................... .........82










4-24 Flow cytometry on mouse bone marrow from control bone marrow, Ppp~rl0+/-
Nf+l+- (NFl1PKO) and Ppp~rl0+/- (PKO) with TCRBeta (APC, T cell receptor). ..........83

4-25 Flow cytometry on mouse bone marrow from Ppp~rl0+/-Nf+l+- (NF lPKO), Nf+l+-
and Ppp~rl0+/- (PKO) with CD3 (Pacific Blue, T cell). ............. .....................8

4-26 An illustration of NflPKO (green), PKO (blue) and Nfl (yellow) effect the
hematopoeitic lineage. ............. ...............85.....









LIST OF ABBREVIATIONS

Acute Myeloid Leukemia

Blood

Bone Marrow

Bovine Serum Albumin

Chronic Myelomonocytic Leukemia

Chronic Myeloid Leukemia

Colony Forming Unit-Granulocyte/Macrophage

Ecotropic Proviral Integration 1

Ecotropic Proviral Integration 2

Ecotropic Proviral Integration 3

Green Fluorescent PCR

Granulocyte Macrophage Colony Stimulating Factor

Juvenile myelomonocytic leukemia

Loss of Heterozygosity

serine/threonine phosphatase regulatory protein 10

Myelodysplastic syndrome

Myeloproliferative disease

Malignant Peripheral Nerve Sheath Tumor

Murine Leukemia Virus

Neurofibromatosis type 1

NF 1 associated leukemia

Noonan Syndrome

Polymerase Chain Reaction

Ppplrl0 mouse line


AML

BL

BM

BSA

CMML

CML

CFU-GM

Epil

Epi2

Epi3

GFP

GM-C SF

JMML

LOH

PPP1R10

MD S

MPD

MPNST

MuLV

NF1

NFl1-JMML

NS

PCR

PKO









PP1 serine/threonine phosphatase 1

PPP 1R10 serine/threonine phosphatase regulatory protein 10

RT-PCR Reverse Transcription PCR

SDS Sodium Dodecyl Sulfate









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

PROTEIN PHOSPHATASE REGULATORY PROTEIN 10 COOPERATES
WITH NEUROFIBROMIN INACTIVATION IN MYELOID
LEUKEMOGENESIS

By

Angela Hadjipanayis

August 2008

Chair: Peggy Wallace
Major: Medical Sciences-Genetics

Neurofibromatosis (NF l) is an autosomal dominant disease that affects 1 in 4000 people.

Patients afflicted with NF 1 present a variety of phenotypes, but the hallmark features of the

disease are neurofibromas on or under the skin, cafe au lait spots, and Lisch nodules of the iris.

Children with NF 1 are at 200 to 500 times greater risk for developing a chronic myeloid

leukemia. Often children who are diagnosed with this myeloid leukemia initially are not

diagnosed with NF 1 until a few years later. Therefore, there are likely more children who are

affected by NFli-associated leukemia than are reported in the statistics. The focus of this

proposal is to study NFli-associated myeloid leukemia, and to characterize additional genetic

events in tumor progression to acute stage using human and mouse model resources.

A viral based mouse model of NFli-associated leukemogenesis identified several common

sites of viral integration. I investigated one site (Epi2) to see how the endogenous genes are

affected and how this can contribute to myeloid leukemia. The Epi2 locus is on chromosome 6 in

humans and chromosome 17 in mice. There are two genes in the Epi2 locus, PPP1R10 and

M~RPS18B. PPP1R10, whose protein product is PNUTS, is a serine/threonine protein

phosphatase regulatory protein, and MRPS18B is a 28S mitochondrial ribosomal protein. Since










both genes are located less than 1kb apart and are transcribed in opposite directions, it was

possible that both genes act simultaneously in NF 1 leukemogenesis, as oncogenes and/or tumor

suppressors. I performed a series of experiments to test some of these hypotheses, and found

evidence that PPP1R10 is a tumor suppressor gene that contributes to development of acute

myeloid leukemia.









CHAPTER 1
INTTRODUCTION

Neurofibromatosis Type 1 and JMML

Neurofibromatosis type 1(NF l) is an autosomal dominant disease that is characterized by a

variety of phenotypes. At the NF consensus conference of 1987, it was decided that the criteria

for clinical diagnosis required that a patient have two or more of the following symptoms

(Stumpf et al. 1988):

* Six or more cafe au lait macules on the skin over 5 mm in greatest diameter in prepubertal
individuals, and over 15 mm in greatest diameter in postpubertal individuals.

* Two or more neurofibromas of any type or one plexiform neurofibroma (benign Schwann
cell tumors).

* Freckling in the axillary (armpit) or inguinal regions (groin).

* Optic glioma. (iris hamartomas).

* A distinctive osseous lesion such as sphenoid dysplasia or thinning of long bone cortex with
or without pseudoarthrosis tibiall dysplasia)

* Two or more Lisch nodules

* A first-degree relative (parent, sib, or offspring) with NF 1 as defined by the above criteria.

NF1 patients also have an increased risk for other tumors besides those described above.

For example, in one review, 12.5 percent of the NF 1 cases had tumors in the optic pathway

(pilocytic astrocytomas) while 1.0-1.6 percent had tumors at other central nervous system sites

(www.ctf~org). For malignancies, 32.76 percent ofNF1 individuals had malignant peripheral

nerve sheath tumors (MPNSTs) while 1.76-4.2 percent had sarcomas at other sites. Another

category is leukemia; 1.7 percent had juvenile myeloid monocytic leukemia (JMML, described

further below), while 7.4 percent had myelodysplastic syndrome (MDS). There is also an

increased risk for phaeochromocytomas (adrenal tumors), gastrointestinal tumors and non-optic

brain tumors.









It is deceiving that NF 1 patients with J1VML account for a small percentage of the

malignancy types observed in NF 1 patients. This number is probably higher because J1VMVL

patients often get diagnosed with NF 1 after their initial diagnosis of leukemia. This is likely due

to the fact that J1VVL has onset very early in childhood, when most kids have not yet met

diagnostic criteria for NF 1 (Pinkel et al. 2008). Typically, in children without NF l,

lymphoblastic leukemias predominate 4: 1 over myeloid leukemias. In children with NF l, the

ratio changes to 9:20 (Bader et al. 1978). This information strongly links NF1 and J1VVL.

Another study compared leukemia patients with and without NF 1 for activating N-RAS and K-

RAS mutations in the tumor cells (Kalra et al. 1994). The results showed that leukemia patients

with NF 1 had no activating RAS mutations. However, 20% leukemias without NF 1 had

activating RAS mutations. Since loss of NF1 increases RAS activity, this suggests another link

between leukemia and NF l, via the RAS pathway. But mutations in the two genes are mutually

exclusive.

The NF1 gene, maps to chromosome 17qll.2 in humans and chromosome 11 46.06 in

the mouse (NCBI). The gene is over 98 percent conserved between the two species. In humans,

the genomic sequence spans over 280kb and its mRNA is >9 kb. There are three embedded

genes within the NF1 genomic region called EVI2B, EVI2A, and OM~GP. All three genes are

located within an intron of the NF1 gene and are transcribed in the opposite orientation to the

NF1 gene. Their roles in NF 1 and their specific protein functions are still under investigation.

The NF1 gene also undergoes alternative splicing. Specifically, there are three exons that are

alternatively spliced, exon 9a, exon 23a, and exon 48a(Geist et al. 1996; Gutmann et al. 1999;

Andersen et al. 1993; Gutmann et al. 1995). The isoforms are differentially expressed in a wide

range of tissues ranging from the cerebellum to the spleen with 9a predominantly in the brain,









48a in the muscle, and 23a ubiquitous. It has yet to be determined what roles these isoforms play

in the function of the NF1 gene and the disease, although it has been determined that inclusion of

exon 23a reduces ras-inactivating activity of the protein.

The NF 1 protein is called neurofibromin. Structurally, it is 2818 amino acids long and has

at least two conserved domains, a RAS-GAP domain and a Secl4p-like lipid domain (NCBI).

The RAS-GAP domain negatively regulates the RAS signaling pathway (NCBI). The NF 1

protein acts as a tumor suppressor protein in the sense that it functions by accelerating the

hydrolysis of RAS-GTP into RAS-GDP to prevent over-proliferation of cells. The other

conserved domain, Secl4p-like lipid binding domain, is found in secretary proteins and in lipid

regulated proteins (NCBI). The role of this and some other protein domains remains unclear in

the protein function. There is evidence that neurofibromin can be both in the cytoplasm and

nucleus (Vanderbroucke et al. 2004). The rest of this large protein is highly conserved but

additional functions have not been found.

NF 1 has proven to be somewhat complicated genetically. The mutational analysis of the

NF1 gene is difficult due to the large size of the gene and because there are many different

mutations. The NF1 consortium has categorized mutations in 246 patients (www.ctf~org). In the

consortium study, it was found chromosomal abnormalities were found in 1.6 percent of the

patients. Deletions of the entire NF1 gene were found in 7.2 percent, whereas small intragenic

deletions were found in 15.5 percent. Also, large intragenic deletions were found in 1.2 percent

of the patients, whereas small insertions accounted for 11 percent of the mutations. Missense

and nonsense mutations accounted for 11.8 percent and 17.6 percent, respectively. Finally,

putative 3'UTR mutations were found in 1.6 percent of patients, and intronic mutations affecting

RNA splicing accounted for 10.2 percent. Furthermore, it has been shown that somatic mutation









of the other allele is associated with neurofibroma, cafe au lait spots, JMML, and bone dysplasia

(Side et al. 1997; Coleman et al. 1995; Stevenson et al. 2006). Virtually all mutations can be

predicted to be disruptive or inactivating, and thus, reduction/loss of neurofibromin activity is an

initiating event in many NFli-related phenotypes.

Mouse Models of Nfl

There are no known naturally existing animal models for NF l, a hindrance to research.

Two attempts were initially made to model NF 1 in mice through knockout technology. These

two labs made mouse knockouts for the Nfl gene in different locations. One lab constructed a

mouse knockout deleting exons 30 to 32 (located after the GAP related domain) (Jacks et al

1994). 250 heterozygous mice, Nf~+lf Nfln, were followed from 7 months to 2 years of age. The

other mutation was a neo gene insertion into exon 31 (Brannan et al. 1994). The results were the

same in both labs: homozygous knockout mice were embryonic lethal and heterozygous mice

didn't develop the cardinal features of NF 1 (neurofibromas, cafe au lait, or Lisch nodules).

However, the mice did have an increased rate oftumorigenesis. It was found that 75 percent of

the heterozygous animals died as a result of the tumors over a period of 27 months compared to

15 percent of wild type animals. Jacks et al. (1994) looked into a larger series of heterozygous

animals (n=64) and found a variety of tumor types. Lymphoma presented in 14 animals,

lymphoid leukemia in 2 animals, lung carcinoma in 9 animals, hepatoma in 4 animals, and

fibrosarcoma in 3 animals. The heterozygotes. also developed a few tumors characteristic of

human NFl1. One animal had an MPNST at 21 months of age, and 12 heterozygotes. had adrenal

tumors pheochromocytomass) at 15 to 28 months of age. Seven of their heterozygotes. had

chronic myeloid leukemia at 17.7 to 27 months of age. In addition, homozygous lethality

occurred between days 12.5 to 14 dpc. (Jacks et al. 1994). Cami Brannan's exon 31 mouse model

(Nfl, Fer) was slightly different than the previous model. Dr. Brannan observed some









similarities with the Jacks mouse model, but found additional developmental defects that were

not present in the Jacks mouse model (Brannan et al. 1994). The homozygous mice, NFlFcr/Fcr

were also embryonic lethal, between 11.5 and 14.5 dpc. The NflFcrFcr embryos were studied for

developmental problems between 11.5dpc and 13.5dpc. These mice had obvious cardiac, renal,

hepatic, and skeletal muscle defects, and hyperplasia of the vertebral sympathetic ganglia

(Brannan et al. 1994).

Molecular and Biochemical Mechanisms Underlying Myeloid Leukemogenesis.

Myeloid leukemia is the overexpansion of a myeloid hematopoietic precursor (blast),

which reduces number and function of the other blood cell types. These cells can invade tissues

as well in the acute form (>30% blasts in the blood). The chronic form is <30% blasts and can be

fatal in itself, or can progress to AML. Somatic genetic mutations lead to chronic leukemia and

other genetic events cause its progression to acute stage. These are very difficult to treat for

patients. Functional analysis of genes isolated from recurring chromosomal translocation

breakpoints in leukemia cells such as AM~L1, MLL, and HOXA9 strongly implicate aberrant

transcription in leukemogenesis (Look 1997). A second group of specific genetic lesions (e.g.,

RAS and FLT3 mutations, the BCR-ABL translocation, and NF1 inactivation) undermine

hematopoietic growth control by providing hyperproliferative and survival signals (Daley GQ et

al. 1990; Largaespada et al. 1996, Kelly LM et al. 2002). Mutations in both classes of genes are

common in acute myeloid leukemia (AML). Based on these data, Gilliland and coworkers

proposed that transcription factor fusion proteins cooperate with genetic lesions that deregulate

growth-promoting signaling pathways in the progression to AML (Kelly et al. 2002). Whereas

data from mice generally support the idea that neither type of genetic lesion is sufficient to cause

AML by itself, these studies have also shown that Nfl inactivation and other genetic lesions that

result in hyperactive Ras are sufficient to induce myeloproliferative disorders (MPDs) (Le et al.










2004). However, the requirement for cooperating mutations in the pathogenesis of JMML and

other human MPDs, and the mechanisms that contribute to progression to overt AML, are poorly

characterized.

Hyperactive Ras in JMML

Somatic KRAS, NRAS, and HRAS mutations, which introduce amino acid substitutions at

codons 12, 13, and 61, are the most common dominant oncogenic mutations found in human

cancer (Bos JL 1989; Malumbres M et al. 2003). Mutant Ras proteins accumulate in the GTP-

bound conformation due to defective intrinsic GTPase activity and resistance to GAPs (Bos JL

1989; Vetter IR 2001; Donovan et al. 2002). NRAS and KRAS2 mutations are highly prevalent in

MDS, MPD, and AML (Malumbres M et al. 2003; Boguski M et al. 1993), are particularly

common in CMML (~40% of cases) (Onida F et al. 2002), and are found in ~25% of JMMLs.

(Miyauchi et al. 1994; Kalra et al. 1994) The elevated risk of JMML in children with NF 1 and

Noonan syndrome (NS) provide additional evidence that hyperactive Ras can initiate myeloid

leukemogenesis. Loss of the normal parental NF1 allele occurs in JMML cells from children

with NF l, which results in elevated levels of Ras*GTP and activation of the downstream effector

extracellular signal-regulated kinase (ERK) (Shannon KM et al. 1994; Bollag et al. 1996; Side L

et al. 1997). Approximately 50% of children with NS demonstrate germline missense mutations

in the PTPN11 gene, which encodes the SHP-2 protein tyrosine phosphatase. PTPN11 mutations

are found in nearly all NS patients with JMML, and somatic PTPN11 mutations are detected in

about 3 5% of sporadic JMML (Tartaglia M et al. 2003; Loh M et al 2004). Most of these

mutations decrease SHP-2 phosphatase activity by destabilizing an auto-inhibitory interaction

and thereby increase signaling to Ras and other downstream effectors. Overall ~90% of JMML

bone marrows have mutations in either, KRAS2, NRAS, NFl, or PTPN11, which are largely

mutually exclusive.









Nfl Reconstitution Mouse Model

Further research of the Nf 1 knockout mouse showed that when NflFcr/Fcr fetal liver cells

were transplanted into lethally irradiated recipient mice, to reconstitute their bone marrow, all of

these mice developed a chronic myeloproliferative-like syndrome similar to human JMML

(Bollag et al 1996, Largaespada et al 2004, Birnbaum et al 2000). Approximately twenty seven

percent of these mice died of complications or developed or AML. Largaespada et al. 1996, also

observed an increase in colony formation of hematopoetic cells response to

granulocyte/macrophage-colony stimulating factor (GM-CSF) (Bollag et al 1996, Largaespada et

al 2004). GM-CSF binds to receptors expressed on some myeloid lineage cells to promote their

differentiation, proliferation and cell survival (Birnbaum et al. 2000). Flow cytometry

experiments showed that these tissues had more myeloid progenitor cells as well (Largaespada et

al, 1996; Bollag et al, 1996, Birnbaum et al. 2000).

Identification of Epi Loci in Nfl-Related Myeloid Leukemia

The discovery of the Epi2 locus employed the BXH-2 mouse that is a cross between the

C57/BL6J and C3H/HeJ strains (Bedigian et al. 1984). In the parental strains, the incidence of

leukemia is low. The high incidence of AML in BXH-2 is associated with high levels of natural

expression of B-ecotropic murine leukemia virus (MuLV) that is passed from mother to

offspring in utero (Bedigian et al 1984, Blaydes et al. 2001). Ninety five percent of BXH-2 mice

die of AML by one year of age. This 8kb murine leukemia virus has a 5'and a 3' long terminal

repeat with gag, pol and eny genes (NCBI). These genes are necessary for the retrovirus's life

cycle so that it can synthesize more virus particles, package itself, and infect other cells

(Bedigian et al 1984, Blaydes et al. 2001). Also, it' s important to note that these viruses are of

non-T cell origin and replication competent. The BXH-2 mouse was used here as a tool to find

genes that cooperate with Nfl loss to cause Nfl-associated leukemia. To accomplish this task,









the BXH-2 mouse was backcrossed to the Nfl~Fer mouse that was created by Dr. Cami Brannan

(Brannan et al. 1994, Blaydes et al. 2001). It was backcrossed for 3 generations and aged until

the BXH2 Nfl+/Fer mice developed leukemia. Interestingly, fifty percent of the mice developed

AML at an average earlier time than the controls, t=5.5 months versus t=8.5 months. Tumors

were collected from all the mice and analyzed by Southern Blot analysis to test for a second

genetic hit in the Nfl locus. It was determined by Blaydes et al (2001), that 89% of the tumors

had a second hit at the Nfl locus by LOH or by viral integration into Evi2 (internal to Nfl)

integration. It was then determined that each tumor had at least 3-4 somatically acquired viral

integration (Brannan et al. 1994, Blaydes et al. 2001). This suggested that loss of Nfl on the

BXH-2 background was not sufficient to cause acute disease. In fact, it is the additional acquired

somatic mutations that are required for progression to acute disease. The actual identification of

the common sites of viral integration (presumably at the sites of cooperating genes) was done

through the use of Southern Blot analysis, a mouse mapping panel, and an ecotropic viral probe,

pAKV5 (Brannan et al. 1994, Blaydes et al. 2001). After the first isolation with probe pAKV5,

the genomic fragment was isolated and a new non-repetitive probe was made based on the

fragment isolated from the tumor. This new probe was used to test the whole collection of tumor

samples and see if the same rearrangement was also present in another tumor. Indeed, this was

the case (Brannan et al. 1994, Blaydes et al. 2001). Dr. Brannan identified three new common

sites of viral integration, termed Epil, Epi2, and Epi3. These names were abbreviated based on

the original name, ecotropic proviral integration site X where X denotes the number of the site.

In order to map the location of the viral integration on the mouse genome, a mapping mouse

panel was used in collaboration with Jenkins and Copeland's lab at the NCI (Blaydes et al.

2001). Once the three common sites of viral integration were localized, subsequent experiments









were focused on elucidating how these sites of common proviral integration cooperate with loss

of Nfl to cause AML (Brannan et al. 1994, Blaydes et al. 2001). My dissertation work focused

on the locus termed Epi2. This site was found in 2 independent tumors (33T, 419T) out of 67 in

the Brannan series, and was also seen twice in the Copeland and Jenkins' Lab. In all four cases,

the virus was inserted into M~rpsl8b in intron 1 in the same orientation (Walrath et al., in

preparation).

Viral Insertional Mutagenesis

The integration of the virus can theoretically have many effects on that region of the

genome. One mechanism is enhancer insertion, where the virus integrates either at the 3' or 5

end of a gene (McCormick et. al. 2005) and the viral enhancer causes improved efficiency of the

native promoter. Another mechanism is activation by enhancement where the virus integrates in

the 3'UTR. This is thought to increase the stability of the transitory mRNA (McCormick et. al.

2005). A third mechanism is promoter insertion, which occurs when the virus integrates

upstream or within the 5' domain of the host target gene with the same transcriptional orientation

as the target gene. This causes elevated long terminal repeat constitutive gene activation and

transcription (McCormick et al. 2005). This and the former mechanisms would cause

overexpression of the endogenous gene along the lines of oncogenes. The next mechanism is

protein truncation by transcription termination/promoter insertion where the virus integrates

within the coding region of the gene. This may result in the inactivation of the gene. The last

mechanism is also protein truncation by transcription termination at a cryptic poly A site

(McCormick et al 2005). This is thought to produce aberrant protein products exhibiting

anomalous biological function. In the Epi2 locus the virus integrated into intron 1 of Mrpsl8b

and in the same transcriptional orientation of Mrpsl8b. This could cause promoter insertion,

where one gets constitutive gene activation and transcription (McCormick et al. 2005). Another









possible theory is protein truncation by transcription termination/promoter insertion since the

integration is near the beginning of the coding region of2\~rpsl8b/Ppp~rl0.

MRPS18B

M~rpsl8b, which encodes a mitochondrial ribosomal protein, is located on chromosome 17

B3 in mouse and chromosome 6p21.3 in humans. In mice and humans, the gene has 7 exons and

a 5' as well as 3' UTR. The mouse full length mRNA is 1073 bp (NM_025878) and the human

mRNA is 1439 bp (NM_014046). The mouse protein is 254 amino acids (NP_080154), and in

humans it is 258 amino acids (NP_05765). This is one of a family of mitochondrial ribosomal

proteins encoded by nuclear genes, which aid in protein synthesis in the mitochondria.

Mitochondrial ribosomes consist of a 5, 18 and 28S subunits. M~rspl8b is one of three genes that

encode the 28S specific ribosome proteins (NCBI). MRPS18B has only one conserved domain

(encoded in exon 2), which is the ribosomal protein domain. However, it is thought that exon 1

may contain sequence that localizes the protein to the mitochondrial ribosome. IfM~RPS18B

were disrupted, then the mitochondrial ribosome, might function aberrantly, affecting energy

metablosim and apoptosis. Less than one kb upstream of2~rpsl8b, and in the opposite

transcriptional orientation is the gene Ppp~rl0. Because of its proximity, the gene was also a

candidate to be involved in NF 1 leukemogenesis.

PPP1R10

PPP1R10 consists of 20 exons in humans and 19 exons in mice. The mRNA is 4203bp in

mouse (NM_175934) and 4504bp in humans (NM_002714), although it is not clear that the

absolute transcription start has been identified. Ppp~rl0 in the mouse encodes a protein that

contains 874 amino acids (NM_175934), with the human protein being 940 amino acids long

(NP_787948). Also, the Ppplrl0 protein is 89 percent conserved between mice and humans, and

Mrpsl8b is 86 percent conserved (NCBI). It is also important to note that neither gene has yet









been implicated in any human disease. PPP1R10, whose protein product is PNUTS, has 3

conserved domains. One is in the N terminal region, a TFIIS site, which is located in exon 5, 6,

and 7. Its function in PPP1R10 activity is unknown. However, TFIIS is thought to function in

eukaryotic transcription elongation by suppressing transient pausing of the RNA polymerase.

The second conserved domain is a PPl-binding domain encoded in exons 13 and 14. This region

of the protein binds to PP 1, an important serine-threonine phosphatase (see below). The rat

PPP1R10 homolog has been shown to have an inhibitory effect on PP 1, and co-localizes with

PP1 at distinct phases during mitosis (NCBI, Allen et. al. 1998, Watanabe et. al. 2001, Kim et. al.

2003, Udho et. al. 2002). The third conserved domain is a C3H1 zinc finger encoded by exon 19,

which suggests that PPP 1R10 may affect transcription of target genes. In addition, recent

research has shown that PPP 1R10 might be involved in regulating cell death due to hypoxia or

cell stress (Lee et al. 2007).

PP1, a Serine/Threonine Protein Phosphatase

One third of all eukaryotic proteins are controlled by serine/threonine phosphatases, which

affect protein activity and localization (Ceulemans et al. 2004). Protein Phosphatase type 1(PPl),

3 5-3 8kDa, is a serine/threonine phosphatase that contributes to many important cellular functions

(Ceulemans et al. 2004, Aggen et al. 2000, Klumpp et al. 2002, Ludlow et. al. 1995): cell cycle

progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription, and

neuronal signaling. Most protein phosphorylations are reversible and there is a balance between

the phosphatases and the kinases. This phosphatase exists in many isoforms: PPlalpha,

PPlbeta/delta, as well as two from splice variants, PPlyl and PPly2. The protein isoforms are

conserved in the central three fourths of the protein, however the amino and carboxy termini are

the most divergent.









As reviewed by Ceuleman's et al (2004), PPl's catalytic subunit does not exist freely in

the cell and requires a regulatory subunit to determine specificity, sub-cellular localization, and

regulation of the phosphatase. These regulators are important in bringing PP1 into close

proximity to its substrate so that the phosphatase can be anchored in specific cellular

compartments via the targeting motifs of the regulators. The regulators, of which there are

dozens, are considered either primary or secondary based on whether the protein has or lacks a

PP1 binding site, respectively. Some primary regulators include Inhibitor-1, Inhibitor-2, NIPP-1,

PPP1R10, and SDS22. Regulatory proteins interact with PP1 via short, degenerate sequence

motifs of 4-6 residues, the PP 1 binding sites. Most of these proteins have multiple points of

interaction with PP 1 and some of them can share PP 1 interactions sites as well. It has been

theorized that PP 1 is subj ect to a combinatorial control that relies on competition of different

regulators for a combination of interaction sites. This allows the formation of large variety of

holoenzymes with distinct specific activities and substrate activities. There are also secondary

PP1 binding sites that can serve as anchor sites to promote the cooperative binding of secondary

sites with lower affinity. These secondary PP1 binding sites are called RVXF sites. Most of the

regulators contain an RVXF motif, although the binding is not associated with any

conformational changes or effects on catalytic activity.

PP1 has many cellular functions important to this project, such as in the cell cycle. First,

PP1 interacts with AURORA-B to ensure that cytokinesis works properly (Andrews et al 2000;

Sugiyam et al. 2002). Second, PP1 is involved in preventing centrosomes from splitting before

G2/M phase (Eto et al. 2002). Third, PP1 contributes to reassembly of the nuclear envelope at

the end of mitosis by dephosphorylating Laminin-B (Thompson et al. 2002). Fourth, PP1

dephosphorylates Bcl-2, an anti-apoptotic protein that is an integral membrane protein in the









endoplasmic reticulum and the mitochondria. The dephosphorylation of Bcl-2 by PP1 targets

Bcl-2 for proteosome mediated degradation (Brichese et al. 2002). Finally, PP1 has been shown

to dephosphorylate the retinoblastoma protein (pRB) in late M phase (Nelson et al. 1997).

My proj ect focused on understanding whether or how PPP 1R10 is involved in myeloid

leukemia, NF-related or otherwise. I took 3 approaches: (1) mutation analysis of PPP1R10 in

human myeloid dysplasia/leukemia samples+/-NF1 involvement, to look for oncogenic or loss-

of-function mutations (Chapter 2); (2) in vitro analysis of mouse fetal liver cells to test whether

overexpression of PPP 1R10 was tumorigenic (to test oncogene hypothesis)(Chapter 3); and (3)

characterize a Ppp~rl0 knockout mouse to test the tumor suppressor hypothesis, including the

cross of this mouse to the Nfl knockout mouse (Chapter 4)









CHAPTER 2
MUTATIONAL ANALYSIS OF PPP1R10 INT LEUKEMIA

Introduction

JMML is characterized by splenomegaly, leukocytosis, hypersensitivity to

granulocyte/macrophage stimulating factor, and absence of the Philadelphia chromosome in

tumor cells (reference diagnostic paper). There have been several genes implicated as a primary

step in JMML: NFl, the RAS gene family, and PTPN11 (a tyrosine kinase phosphatase)(Side et

al., 1997; Side et al., 1998; Tartaglia et al., 2003). Mutations in these genes are mutually

exclusive initiating genetic events, and together account for ~85% of genetically predisposed

JMML. All of these gene mutations lead to an increase in Ras signaling. The Epi2 mouse model

implicates Ppp~rl0 as a possible oncogene or tumor suppressor. Since there is so little material

from the two mouse Epi2 tumors, we chose to screen human myeloid malignancies for further

evidence that PPP1R10 might be a leukemia gene. Identification of any PPP1R10 mutations in

human myeloid leukemia samples may provide insight about whether (and how) this gene is

involved in NF 1 associated leukemia and/or JMML, myeloproliferative disorder (MPD), or

AML .

To test the hypothesis that PPP1R10 might be genetically altered, we performed

mutational analysis in NFl1-JMML, JMML, and AML tumor samples. We examined the entire

gene and found loss of heterozygosity in intron 15 in a subset of tumor samples by sequence

analysis and RFLP analysis. Additional analysis of the sequenced region revealed a putative stop

codon mutation in exon 6 of PPP1R10 in 2 out 40 leukemia samples. Real-time mRNA

quantification PCR was also performed on a subset of leukemia samples, which showed a

decrease in expression of PPP1R10 in some samples compared to a GAPDH control.











Patient Samples

DNA and RNA were extracted from tumor cells using standard methods (Thomson and

Wallace et al 2002). Most leukemia samples (bone marrow or blood cells) were obtained from

the UF tissue bank and were > 35% blasts. Tumor and germ-line samples were obtained with

IRB approval from the UF Tissue Bank (MDS (n =7), AML (n=54), CML (n=6)) and UCSF

(courtesy of Dr. Kevin Shannon, (NFl1-JMML (n=50), JMML (n=25), CMML (n=50)).

Polymerase Chain Reaction and Sequencing of PPP1R10 Exons

Tumor DNA was PCR-amplified in a reaction mixture containing 100ng for each forward

and reverse primer, 200umol dNTP's (Invitrogen), 0.5U Hotstart Taq Polymerase (Qiagen), and

lX Roche PCR buffer. See Appendix A for a list of primer sets and PCR product size for

PPP1R10, which were designed using PRIMER3 (http://fokker.wi .mit.edu/primer3/input.htm).

The following cycling conditions were used: 1 cycle of 950C for 15 minutes, 35 cycles of 950C

for 15 seconds, 600C for 30 seconds, 720C 1 minute; a final extension of 720C for 10 minutes.

All PCR products were examined on ethidium bromide stained 1.2 percent agarose gels to verify

quality and quantity of PCR product. PCR products were purified using Exosapit (Amersham

Pharmacia) and 5.8ul were used in the sequencing reaction: 2ul of 5X Sequencing Buffer, 2ul of

Big Dye 3 (ABI), and 20ng/ul of forward or reverse primers. The following cycle sequencing

conditions were used: 25 cycles of 950C for 1 minute, 500C for 30 seconds, and 600C for 4

minutes. Sequencing reactions were run on a ABI 3130XL Genetics Analyzer at the UF Center

for Epigenetics. Sequence data were analyzed using Sequencher software (Gene Codes).

Restriction Fragment Length Polymorphism for Intron 15 Loss of Heterozygosity

Sequence analysis uncovered a polymorphism in PPP1R10 intron 15 close to exon 16 and

was used for a loss of heterozygosity analysis. Loss of heterozygosity of a polymorphism can be


Materials and Methods









used to test for deletions. The heterozygosity frequency in this A/G polymorphism (rsl26443)

was reported to be .478 in Caucasians and confirmation of this was done with normal patient

DNA through RFLP. P115RsaF and P115RsaR primers (see Appendix A) were used to amplify

the intron 15 polymorphism and create an Rsal site to distinguish the alleles. With Rsal digest,

the G allele causes the 138bp to be cleaved into 26bp and 112bp fragments (two bands), where

as, the A allele does not cut with Rsal digest (one band). The digest was run on an 8% native gel

with ethidium bromide for visualization. Visualization of the RFLP on an acrylamide gel allows

for comparison of allele signal intensities. A series of germline heterozygous DNAs were used to

establish relative allele intensity, and tumor samples were compared to these DNAs. If there is a

deletion in the gene of interest in the tumor there will be a reduction in intensity of one allele

relative to the other as compared to the germline genotypes. LOH analysis was also performed at

an SNP in the shared PPP 1R10/MRPS18B promoter (primers Appendix 1), which used an Ear 1

digest.

Real-Time mRNA Quantification

Tumor cells from leukemia patients were used in the real-time quantification of PPP 1R10

expression. Tumor total RNA was serially diluted from 400ng to 25ng and subsequently used in

a RT-PCR reaction. For the reverse transcription: 200uM of dNTPs (Invitrogen), 50ng random

hexamer (Invitrogen), and 5ul of water was added to each dilution of tumor RNA. This mixture

was heated at 650C for 10min. and then placed on ice. Next, 4ul of 5X buffer, 2ul of .1M DTT,

40 units of RNase Inhibitor (Invitrogen), and 200units Superscript II (Invitrogen) was added to

each of the tubes on ice. These tubes were heated at 250C for 10 min and 500C for 40 min to

produce cDNA. For the mRNA quantification, lul of cDNA at each concentration was placed in

PCR tubes (Biorad cat. #TLSO85 1, TCS-0803), followed by the addition of 10ul of Master mix









(Biorad, contains hotstart version of a modified Tbr DNA polymerase, SYBR Green 1, optimized

PCR buffer, 5mM MgCl2, dNTP mix including (dUTP)), 100ng of forward primer, 100ng of

reverse primer, and 8ul of water. These reactions were then analyzed on a Opticon Moniter II

machine at the Center for Epigenetics: 95C for 15 min, 940C for 15sec, 550C for 30sec, Plate

Read, 720C for 1min, Go to Step 2 for 30 cycles, Melting Curve 500C to 900C, Read ever 1sec,

Hold for 10sec, 720C for 15min. The Ct number for each PPP1R10 tube was compared to the Ct

of the GAPDH gene results thorough the Pfaffl method formula (Appendix B). Primers chosen

for real-time quantification lay in different exons to avoid DNA-PCR contamination and

GAPDH was used as a housekeeping/normalizing gene (Appendix A).

Results

A set of 50 NFl1-JMML and 25 JMML (non-NFl1) myeloid leukemia samples were used in

the DNA analysis. A polymomorphism in the promoter region (rsl6867845) displayed no loss of

heterozygosity and had no mutations (Figure 2-1). A putative nonsense mutation was also

uncovered in two separate NFl1-JMML samples (Figure 2-2). This substitution in exon 6 encodes

a serine->stop at codon 125. A putative missense was also uncovered in exon 6, serine-

>threonine change at codon 125 (Figure 2-3). Another polymorphism in exon 19 (rs11754215)

also did not display loss of heterozygosity in any samples (Figure 2-4). The intron 15 SNP

(rsl1264423) did reveal potential loss of heterozygosity by sequence analysis in 6/44 NFl1-JMML

samples (Figure 2-5). To confirm this result by another method, a PCR-based forced Rsal digest

system was constructed for the polymorphism. The sequence positive NFl1-JMML samples were

re-amplified with the new primers, digested by Rsal and the products were visualized by native

polyacrylamide gel electrophoresis. The Rsal digest creates 2 fragments if the patient is

heterozygous (AG) and 1 fragment if the patient is homozygous (GG or AA). Figure 2-6, shows









the Rsal digest normal germline DNA displaying all three genotypes. Figures 2-7 and 2-8 show

digests of some tumor samples of NFl1-JMML and JMML displaying loss of heterozygosity.

More NFl1-JMML and JMML samples were tested with this assay and it was found that 6/44

NFl1-JMML and 1 1/20 JMML samples displayed loss of heterozygosity at the intron 15

polymorphism (Table 2-1 and Table 2-2).

Real time mRNA quantification on a set of leukemia samples also revealed a decrease in

PPP1R10 mRNA expression. The Ct values from the tumor RNA and from normal lymphocytes

for PPP1R10 and control GAPDH were inputed into the Pfaffl method formula (Pfaffl et al.

200 1). This formula can calculate the fold expression change of PPP1R10 in the tumor cells

compared to GAPDH using a standard curve method. Table 2-3 shows the fold change of

PPP1R10 to GAPDH is reduced in the human leukemia samples. Interestingly, there is a variable

reduction between samples, and different leukemia types (including AMLs).

Discussion

The human genetic data support the notion that PPP1R10 is a tumor suppressor gene

associated with some NFl1-JMMLs, JMMLs, and AMLs since the mutations are inactivating

(deletion, stop mutation) and the mRNA expression is reduced in tumor cells. The presence of

the PPP1R10 LOH implicates this gene in non-NFl-related leukemia suggests that this LOH is

not specific to NFli-related leukemia.

PPP1R10 is an attractive tumor candidate gene because of its regulation of PP 1, which

regulates at least 70 mammalian genes. Loss of one allele of PPP 1R10 would decrease inhibition

of PP 1 and may affect the ability of PP1 to regulate its target genes. However, it is still unknown

which target genes PP1 regulates as a result of PPP1R10 binding especially in the hematopoietic

system. Traditionally, tumor suppressors have 2 hits. Neither of the two tumors with point

changes showed LOH, however, we do not yet have evidence for the 2-hit phenomenon. Further










evidence from real-time data on one mouse Epi-2 tumor revealed a reduction in PPP1R10

mRNA expression in comparison to the other tumors without Epi-2 viral integration (Walrath,

2005). However, the mRNA reduction was not characteristic of a classic 2-hit mechanism.

Recently, several papers have reported that cooperating haploinsufficient tumor suppressors in

the mouse can be sufficient reduction in protein function to contribute to tumorigenesis

(Kamimura et al. 2007; Vives et al. 2006; Moreno-Miralles et al. 2005; Ma et al. 2005). Thus,

the data support a functional contribution by decreased PPP1R10 activity but not necessarily in a

classic tumor suppressor mechanism.







Figure 2-1. Human NFl1-JMML analysis PPP1R10, Promoter Polymorphism (rsl6867845).
C G; C T G AG CAA G TC AG G Ga AT G A G
CR~ ~ G CT G CAR G CA GT G T G; GG;





Figure 2-2. Human NFl-JMML analysis, PPP1R10 Exon 6 nonsense, S125X.


Figure 2-3. Human NFl1-JMML analysis, PPPlrl0 Exon 6 missense, S125T.


Figure 2-4. Human NFl-JMML analysis, PPP1R10 Exon 19 polymorphism (rsl 1754215).






































Figure 2-5. Human NFl-JMML PPP1R10 analysis, Intron 15 polymorphism loss of
heterozygosity.









a
J KI ~f ~ cr In r- ~ 8 ~ ~ ~ ~n N9 VI rD a re oo rr t~- N Nr- CT
8~~~~~$
00 IX100rX: DQ
LL3


Figure 2-6. Rsal Digest of Intron 15 polymorphism of Control DNA' s.










O k
Q;I
Q~cr~ In ~d BQ
~a poV1 N8"1 ~14
~S~CLLL
~ f3 t3


C~ ~a
04 In ~ ~o *e! RI
~~~96Nrr~ ~ cr
r~


Figure 2-7. Rsal Intron 15 Polymorphism of NFl1-JMML and Control DNA.












~I
8
~iD d
IN QSI


Figure 2-8. Rsal Intron 15 Polymorphim Digest of JMML and an AML sample.

















Table 2-1. NFl-JMML human patient samples and Intron 15 polymorphism LOH.










1J, 2J J AIL es ys n
2J JAILno File ITTT n pLH A





26 JMI ye e ysA


5 27J JMAIL nos Noml TT sG
61LJMAIL no Failed IT TT vesO A

35L JMAIL no Nra TT oA
33J JMAIL no FrailedITTT ye
3JJMAIL prb aldI T n LOH AG

33LJMAIL no Failed IT TT yespLH G
37JJMAIL yes Normal ITTT yes G
45J JMAIL no Failed IT TT ye

46JJMAIL no Failed IT TT vsG
7JJMAIL esFailed IT TT ves AG
9JJMAIL yes yald VTes noO
57JJMIL nos yesaLT no G
60JJMAIL no Norale IT TT ves A
35JJMAIL rb no rnaie ves G

5JJMAIL noye esA
56JJMIL yesye no AG
58JJMAIL ves Normal ITTT ves A

9LJMAIL yesn ye n A
75J JMAIL no not teten
61JJMIL noNrnal1T no A
71JJMIL n Normal ITTT e

7JJMAIL no GG
8JJMAIL no o et n


9LJMAIL no G 0

88JJMAIL no pLOH AG
6JJMAIL esGG


HM70


H189




HM78

Hh56JMAIL neg AA
Hh52 JMAIL AA
Hhl553AA
HM605 JMAIL
HM676 LOH AG
H51JMAIL po.AG
Hhl55 pLOHAG
HM660 JMAIL
Hhl48JMAIL pos.

19L pos.
20L pos.




22Lpos. AG
100 LG
1098 AL
1099 G 0
1102
1101
1238 G 0
1239
140 LG
141 AG
142 AG
143 AG
1244 LG
1245 LG
27F AG










Table 2-2. JMML human samples and Intron 15 polymorphism LOH.
Patient number Intron 15 poly. Pcr
99L pLOH AG
80L
60L GG
23L AG
102F pLOH AG
HM262 AG
98L
HM1286 pLOH AG
101F GG
HM896
HM910 pLOH AG
HM933 AG
HM1224 GG
HM920 pLOH AG
HM1236
HM773 pLOH AG
HM765 pLOH AG
HM1198
HM1158 AG
342 GG
Puente LOH AG
Schultz
Mackle
Booth
Alexande LOH AG
Jackso GG
Buck
Gillian pLOH AG
Thon LOH AG
Gonclaves









Table 2-3. mRNA Quantification of human leukemia RNA.
Tumor Sample Evi2B 3'UTR Fold Expression
polymorphism polymorphism PPPIRIO/lymphocyte
LOH? LOH?
AML 17 YES NI 1/3.57
AML 47 YES NI 1/7.29
AML 259 YES NO 1/9.43
MDS 4 YES NO 1/22.72
AML101404 YES NI 1/66.66
AML 16 YES NI 1/20
CML 20 YES NO 1/18
JMML 1 YES YES 1/6.25
AML BM NI NI 2.56/1









CHAPTER 3
IN VITRO ANALYSIS OF PPP1R10 AS A POTENTIAL ONCOGENE

Introduction

In vitro studies have been useful in elucidating the role of cancer genes and their signaling

pathways. Similarly, in vitro studies of Ppp~rl0 might elucidate its role, if any, in the signaling

pathways that contribute to cancer. One proposed mechanism for its role in AML was as an

oncogene, based on the viral Epi-2 integration upstream that could be inducing inappropriate

transcription. To test this theory, over-expression ofPpp~rl0 was studied in COS7 cells (from

African Green Monkey kidney), which were analyzed for altered expression of proteins involved

in classic signaling cascades. In addition, I studied Ppp~rl0 over-expression in wild-type mouse

fetal liver cells in a colony formation assay using (CFU-GM, specific for the

granulocyte/macrophage lineage of the hematopoietic system). These experiments are detailed

below. This work led to the conclusion that PPP1R10 is likely not an oncogene.

Materials and Methods

Cloning of 19pprl0 in the MSCV Viral Vectors

A plasmid containing the full-length mouse cDNA, pXY-Ppplrl0 (Operon), was digested

with EcoRI and NotI restriction enzymes to remove the cDNA from the pXY plasmid. The

digested product was ligated into EcoRI/NotI digested pENT4 plasmid (entry vector, Invitrogen).

The modified destination vector, pMIG, was provided by the Dr. Kevin Shannon (UCSF) and

was already modified to contain an MSCV promoter, multiple cloning site, att sites, and a GFP

transgene. Gateway BP Clonase II enzyme mix (Invitrogen) was used to transfer the Ppp~rl0

cDNA from pENT4 into the pMIG-GFP vector. The final vector was called pMIG-PNUTS and

DNA sequencing verified correct reading frame and sequence.









Hematopoietic Cell Isolation and Retroviral Transduction

Pregnant wildtype C57BL/6 females were humanely killed by CO2 inhalation at post coitus

day 14.5 (E14.5) and fetal liver cells were sterily isolated and prepared as described (Birnbaum

et al. 2000). The fetal liver cells were cultured in a stimulation medium containing Stem Span

SFEM (StemCell Technologies, Vancouver, BC, Canada), 15% FBS, 100 ng/mL stem cell factor

(SCF; Peprotech, Rocky Hill, NJ), 50 ng/mL FLT-3 ligand (Peprotech), and 100 ng/mL IL-11

(R&D Systems, Minneapolis, MN) to promote hematopoietic cell lineage growth. Bone marrow

cells were cultured in a stimulation medium containing StemSpan SFEM, 15% FBS, 100 ng/mL

SCF, 50 ng/mL IL-6, and 10 ng/mL IL-3 (both from Peprotech). Phoenix cells, used to produce

MSCV based viruses were co-transfected with empty vector (pMIG) or WT (pMIG-PNUTS)

plasmids along with plasmids encoding retroviral gag-pol and env proteins using

Lipofectamine2000 (Invitrogen, Carlsbad, CA), to package and excrete the final viral vectors

(PNUTS and empty vector). The supernatants from transfected cells, containing the viruses, were

harvested 24 hours post-infection, and used to tranduce the murine fetal liver cells for 1.5 hours.

Colony Formation Unit Granulocyte/Macrophage Colony Assay

After transduction, fetal liver cells were sorted based on GFP expression by FACSVantage

SE flow cytometer (BD Biosciences, San Jose, CA). GFP-positive fetal liver cells (PNUTS, and

vector control) were seeded on methylcellulose medium (M3231; StemCell Technologies)

containing escalating doses (0.1Ing.ml, Ing/ml and 10ng/ml) of GM-CSF (Peprotech), to test for

ability to form colonies in this media, a sign of an oncogene. Granulocyte-Macrophage colony

forming units were scored by indirect microscopy on day 7. Images were acquired using a Nikon

Coolpix 5000 camera (Torrance, CA).









Serum Starvation of COS7 Cells

To test the effect of PNUTS expression on classic oncogene signaling pathways, 10ug of

pDEST-PNUTS were transfected into two million COS7 cells (Schubbert et al 2005). Samples

were taken at specific time points after serum starvation media (DMEM, 0.01% Fetal calf serum,

lX glutamine, lX Pen-Strept) was placed on the cells. The 0 time point was taken before

addition of the starvation media. Standard protein lysates were made from all cells at all time

points (0, 24, and 48 hours) for Western blot analysis. The negative control was untransfected

cells.

Western Blots

Ten million cells from each time point were lysed in 1% NP-40 Lysis buffer (1% NP-40,

30mM NaF, 30mM b-glycerophosphate, 20mM sodium pyrophosphate, ImM sodium

orthovanadate, and a cocktail of protease inhibitors (phenylmethyl sulfonylfloride, benzamidine,

leupeptin)), and total protein was quantified (Lowry assay). Each protein sample (40ug) was

loaded onto Nupage Novex precast Midi gels (Bis-tris, SDS, MOPS denaturing polyacrylamide

gels) and electrophoresed at 175V for 1 hour in manufacturer's buffer. Proteins were electro-

transferred onto nitrocellulose membranes (Novex) for hour and 30 minutes at 300 mAmp, as

recommended by manufacturer, and blocked overnight with 5% BSA. Primary (rabbit)

antibodies [(p-mTor (1:750, Cell Signaling), p-PKCa/b (1:600, Cell Signaling), p-Stat3 (1:1000,

Cell Signaling), p-Pten (1:500, Invitrogen), p-Akt, p-Erkl/2(1:1200, Cell Signaling), pMekl/2

(1:750, Cell Signaling), b-actin (1:750, Cell Signaling), and PNUTS (1:1000, Zymed)] were

incubated at the recommended concentration with membranes in 5% BSA for 1 hour and 15

minutes at room temperature. The membranes were washed twice for 6 minutes each with TB S-

Tween (0.05%) before addition of the secondary antibody. The secondary antibody, HRP-

conjugated anti-rabbit (1:2000), was incubated with the membranes for 50 minutes and washed









3X for 6 minutes each with TBS-Tween (0.05%). Treatment of membranes with luminescent

ECL reagent (Pierce, 1:1) allowed proteins to be visualized by autoradiography (Kodak X-ray

film).

Results

Three replicates each of the fetal liver cells transduced with MSCV-pMIG and MSCV-

pMIG-PNUTS viruses were plated on methylcellulose with increasing amounts of GM-CSF, the

the ligand that stimulates the granulocyte/macrophage lineage of the hematopoietic

compartment. The resulting colonies, at day 7, were scored for size, morphology, and number in

the CFU-GM assay in order to observe alterations in granulocyte/macrophage lineage (the

pathway affected in JMML). There were no statistically significant differences between the

empty vector and PNUTS vector (Figure 3-1, 3-2, and 3-4).

In the other assay, serum-starved COS7 cells transfected with pDEST-PNUTS at 0, 6, and

12 hour time points were probed for amounts of phosphorylated versions of key signaling

molecules: p-mTor, p-PKCa/b, p-Stat3, p-Pten, p-Akt, p-Erkl/2, p-Mekl/2, b-actin (loading

control), and PNUTS (Figure 3-4). Levels of p-Stat3, p-Pten, and p-Erkl/2 were visibly

increased at the 12 hour time point, suggesting that Ppp~rl0 over-expression increases the

signaling in these pathways compared to the untransfected COS7 cells.

Discussion

If Ppp~rl0 is a classic oncogene involved in leukemia, then the CFU-GM colony assay

should detect an increase in the size, change in morphology or increase in the number of

colonies. Ppp~rl0 over-expression in Nf+l+/ fetal liver cells showed no difference in any these

measures between Ppp~rl0 and the empty vector control. This suggests that, by itself, Ppp~rl0

is not an oncogene involved in leukemia. Either other cooperating genes are needed for cytokine

independent growth, or Ppp~rl0 is a tumor suppressor gene instead (or, Ppp~rl0 is not involved









in leukemia at all). However, Le et al. 2004, showed that conditionally inactivated Nfl fetal liver

cells are sensitive to low GM-CSF, have an altered morphology, and an increased number of

colonies compared to Nf+l+/ fetal liver cells. This, confirmed Nfl as a tumor suppressor in

myeloid leukemia. A similar assay could be used to test knockdown of Ppp~rl0 (see Chapter 5).

Western analysis of phosphorylated proteins allows correlation with actively proliferating

cells. In serum starved COS7 cells, Western analysis showed a possible alteration in the MAPK

pathway in PNUTS-positive cells. Specifically, p-MEK decreased at 6 hours and then increased

at 12 hours post starvation. In addition, p-Erkl/2 was also increased 12 hours post starvation,

suggesting that Ppp~rl0 may contribute to the regulation of the Ras->Mek->Erk pathway in

COS7 cells. Specifically, increased the signaling in these pathways is positively association with

cell proliferation and cell survival. However, these results are weak compared to classic

oncogenes such as mutant H, K, or NRAS. For example, Schubbert et al. (2006) showed that

germline KRAS mutants associated with Costello Syndrome and JMML, V14I and T58I, were

defective in intrinsic GTP hydrolysis and displayed impaired responsiveness to GTPase

activating proteins. This impaired GTP hydrolysis rendered primary hematopoietic progenitors

very hypersensitive to growth factors, and deregulated signal transduction in a cell lineage-

specific manner. These mutants, along with classic others such as codon 12, induce tumorigenic

properties in the assays above.

These results indicate that Ppp~rl0 is not a classic oncogene in itself. It could however,

be a tumor suppressor gene or cooperate with other genes (as a moderate oncogene or a tumor

suppressor) to cause cancer or leukemogenesis. The serum starvation experiment showed that

Ppplrl0 could be involved in the Ras-Mek-Erk pathway, or possibly Stat3 or Pten pathways.











The CFU-GM experiment definitively showed that over-expression ofPpp~rl0 did not alter the


size, morphology or colony number as seen in other oncogenes like 1VRASGl2D.



CFU-GM Colony Assay

140

120

100-

80
-* WT Ppplrl0
60 -1 -Vector only

40

20


0 0.1 1 10
GMCSF (ng/mi)


Figure 3-1. CFU-GM Colony assay of pMIG-PNUTS and pMIG in wild-type fetal liver cells.
















MIG &MIG- a S~versions Infect Nflm Gl- ---- Sort for GFP+ cells
of wut PNUTS fetal liver


CFU-GM assay


BFU-E assay


I


Medla: Mn3231 methylcellulose
#tcelistplate: 56,250
Cytokine: GMV-CSF
Concentrations: 0, 0,01, 0.1, 1, 10 ag/mi
Count after 7 days


Medla: M3234 methylcellulose
#tcells/plate: 100,000
Cylokine: EPO
Concentrations: 0, 1, 100 agml
Count after 7 days


Figure 3-2. Experimental Design of Colony Formation Assay.




























10






Figure 3-3. CFU-GM of wt PNUTS versus empty vector control.




















_ __ I _


SII
I I
I I


I I
I I
I I
I I
I I
I I
I I


iOh 16h 1~2h


O!



I

III



-- -I





LI



~II, --" ~ I


wt PNIUTS

Olh 6h 12h


Sta~rvation (h):


I
I I
I I
I I


I
I


I ----~

I


I


p-STAT3



p-PTEN


p-ME~K


PNUTS


Figure 3-4. Transfection of pDEST-PNUTS in COS7 cells with serum starvation and probing
signaling cascades.









CHAPTER 4
PPP1R10 COOPERATES WITH NF1 INACTIVATION TO CAUSE ACUTE
MYELOMONOCYTIC LEUKEMIA IN A MOUSE MODEL

Introduction

A mouse Nfl knockout (Nfl KO +/-) created in 1994 displays no characteristic NF 1

features, but 1 1% of mice die of myelodysplasia by 17.7 to 27 months (Jacks et al., 1994).

Largaespada et al. (1996) isolated 12.5dpc fetal liver cells from another, similar knockout mouse

(Brannan et al., 1994), specifically the Nfl (embryonic lethal 13.5dpc) and transplanted

them into lethally irradiated mice. Upon bone marrow reconstitution by these donor cells, the

mice developed a JMML-like phenotype, and their bone marrow cells were hypersensitive to

granulocyte/macrophage stimulating factor, similar to that seen in JMML patients. It was then

hypothesized that additional somatic cooperating mutations were required for progression to

more acute disease. Thus, loss of the remaining NF 1 allele is required for a child with NF 1 to

develop JMML (Largaespada et al., 1996; Bollag et al., 1996), but additional somatic mutations

in other genes are necessary for progression to AML.

Since in vitro studies and previous research suggested that Ppp~rl0 was not an oncogene, I

hypothesized that Ppp~rl0 could be a tumor suppressor whose loss cooperates with Nfl

inactivation in AML. A previous student, Jessica Walrath, made a Ppp~rl0+/- mouse on the

129S2 background, utilizing the Gene Trap Consortium library of ES cells (Walrath, 2006). The

gene trap technology inserts a LacZ gene, which, through splicing, creates a null allele in the

host gene. The Consortium mapped the locations of the "trapped" genes and made these

available for a nominal fee. Jessica found that homozygosity for Ppp~rl0 knockout was a pre-

implantation lethal genotype.

I crossed Ppp~rl0+/- mice to Nf+l+- mice, and aged double heterozygotes for phenotype. I

found that these mice become ill and died between 14 and 19 months, compared to 17-27 months









seen in Nf+l+- mice. The Ppp~rl0+/-Nf+l+- mice developed acute myelomonocytic leukemia

(AML-M4) versus the Nf+l+- mice, which developed the more chronic leukemia/JMML-like

phenotype. The Ppp~rl0+/- mice were also aged for phenotype and these mice developed a

variety of single occurring solid tumors and became ill by 24 months of age. In addition to solid

tumors, the maj ority developed splenomegaly and acute undifferentiated leukemia (AML-MO).

This data support the hypothesis Ppp~rl0 cooperates with Nfl inactivation to cause AML, and

can cause a slightly different acute leukemia by itself with a high penetrance but extended

latency .

Materials and Methods

19pprl0+/- Mouse Construction

The Ppp~rl0+/- mouse was constructed by a previous student and described elsewhere

(Walrath, 2006).

Breeding 1)pprl0 Mice

129S2 Ppp~rl0+/- mice were crossed to 129S1 Nf+l+- mice and the Fl's were aged for

phenotype. There were 21 Ppp~rl0+/-, Nf+l+- (twelve females, nine males) mice aged for

phenotype as well as 10 129S2 Ppp~rl0+/- animals, and 5 129S1 Nf+l+- animals. Ppp~rl0+/-

mice were also bred for ten generations to move the knockout allele onto the 129S1 and

C57BL/6 backgrounds.

Pathology/Sectioning/H and E Stain

Mice were aged and observed for abnormal gait, hunching, lack of grooming, and or other

signs of illness. Upon observation of illness, the mice were euthanized. Full necropsies were

performed on the mice, and fixed paraffin-embedded sections were made from each tissue. These

sections were made by the UF Molecular Pathology core and subsequently stained with

Hematoxylin and Eosin.









Blood Smears and Manual Counts

At the time of observed illness, and euthanasia, five hundred microliters of blood from

each mouse were used for standard blood smear analysis. The remaining blood was used to make

DNA. Blood smear slides were stained with The Wright Geimsa stain kit (Fisher). Manual

cytology counts were performed on blood smears slides from each mouse, counting 200 cells per

field of view. In some cases, younger healthy mice had blood samples taken without euthanasia

and analyzed by the UF Animal Care Services laboratory for complete blood count. This gave us

an indication of whether the white blood cell count was increasing, well prior to death.

Sudan Black Stain and MPO Stain

Bone marrow sections were stained with Sudan Black kit for detection of myeloid cells.

Paraffin embedded were de-paraffinized, hydrated with water and then stained with the Sudan

Black Staining Kit (Sigma). Bone Marrow sections were immuno-stained using a primary mouse

monoclonal for Myeloperoxidase (ABCAM mouse monoclonal 16686-50) and a secondary

antibody, biotinylated horse anti-mouse IgG made in horse (Vector BA-2001). To de-

paraffinize/hydrate paraffin sections, sections were heated in a 60 degree Celsius oven for 15

minutes. Then, the sections were placed in xylene twice for 2 minutes each time, 100% ethanol

for 2 minutes, 95% ethanol for 2 minutes, two washes in 70% ethanol for 2 minutes, H20 for 1

minute, and PB S for 5 minutes. Sections were then incubated in 0.3% H202 for 5 minutes.

Before adding the primary antibody, the sections were incubated in 10% horse serum for 60

minutes (100ul NHS/1000ul PBS). The excess serum was blotted off and then the sections were

incubated overnight with the primary antibody, MPO mouse mAB at 1:100 at 4 degrees Celsius.

Before addition of biotinylated horse anti-mouse IgG at 1:100 (10ul of secondary AB, 10Oul

normal horse serum, 1000ul of PB S), sections were washed for 5 minutes in PB S buffer. The

biotinylated anti-mouse IgG was added for 45 minutes at RT and then sections were rinsed in 1%









horse serum for 15 minutes. Finally, the sections were incubated for 30 minutes in vectastain

reagent, then PBS for 5 minutes, DAB or Nova Red was added, and the slides were rinsed in

water, counterstained with hematoxylin, dehydrated with xylene, and slide coverslips mounted

with permanent mounting media.

Flow Cytometry

Bone marrow cells were characterized by four-color flow cytometry. Five hundred

thousand bone marrow cells were re-suspended in PBS/ .1% BSA, placed into tubes, and

incubated with the appropriate antibodies. Three-antibody combinations were used for analysis:

(1) PE-Ly~g (Abcam, ab24884), APC-CD11b (BD, 553312), Pacific blue c-kit (Biolegend,

105820); (2) PE-Ly~g, APC-CD11b, Pacific Blue F4/80 (CALTAG, MF48028); (3) Percp-CD45

(BD, 557235), APC-Terl l9 (BD, 557909), PE-CD71 (BD, 553267); (4) APC-CD19 (Biolegend,

115511), PE-CD8a (Biolegend, 100707), Pacific Blue-CD3 (Biolegend, 100214); (5) APC-

TCRBeta (Biolegend, 109212), PE-CD34 (Biolegend, 119307) (6) PE-CD13 (BD, 558745),

AexaFluor647-CD68 (AbD serotec, MCAl957A647); (7) Unstained Control. All antibodies for

flow were used according to the manufacturers recommended instructions. Flow cytometry was

performed on a BD FACS machine (BD Biosciences) with the help from UF core director Neal

Benson. Flow cytometry analysis was performed using FCS Express 3.0 software.

Nucleic Acid Extraction

DNA and RNA samples were prepared from blood, bone marrow, tissues and tumors as

previously described in Thomson and Wallace (2002). In some cases there were not enough cells

for RNA extraction, and only DNA was made.

Loss of Heterozygosity Analysis

50ng of DNA from tumors, bone marrow and blood were PCR amplified with the 3-primer

system using Pko2Ra, Pko2fb and lac2 to simultaneously genotype the mutant and wild-type









allele in Ppp~rl0 (Appendix A). Similiarly, the Nfl mutant and wildtype alleles were also

genotyped with NF31a, NF31Ib, and NeoTkp (Appendix A; Brannan et al. 1994). To PCR

amplify Ppp~rl0: 2.5ul of 10X HotMaster Buffer (Eppendorf), 200umol dNTP, 60ng Pko2ra

primer, 60 ng Pko2fb, primer, 40ng Lac2 primer, 1.25ul DMSO, 16.325ul H20, 0.125ul

Hotmaster Taq (Eppendorf). The following cycling conditions were used for Ppp~rl0: 1 cycle of

950C for 15 minutes, 35 cycles of 950C for 30 seconds, 630C for 45 seconds, 720C 45 seconds;

and a final extension of 720C for 10 minutes. To PCR amplify Nfl : 2.5ul of 10X Hotstart Buffer

(Qiagen), 200umol dNTP (Invitrogen), 65ng of NF3 1a/NF31Ib/NeoTkp, 2.5mM MgCl2, 18ul of

H20, .125ul of Hotstart Taq polymerase (Qiagen). The following cycling conditions were used

for Nfl: 1 cycle of 960C for 15 minutes, 40 cycles of 950C for 30 seconds, 550C for 30 seconds,

720C 1 minute; a final extension of 720C for 10 minutes. These PCR products were run on an 8%

native polyacrylamide gel with controls (germline DNAs), stained with ethidium bromide, to

observe whether there was loss of the wildtype allele in Nfl and Ppp~rl0. The Ppp~rl0 PCR

product sizes of wildtype/mutant alleles should be 103bp/400bp, and the Nfl wildtype/mutant

alleles should be 128bp/95bp respectively.

Results

To test if Ppp~rl0 cooperates with Nfl inactivation to cause acute leukemia in mice as a

possible tumor suppressor, Ppp~rl0+/-(129S2) mice were crossed to Nf+l+- (129S1) and aged

for phenotype. The Ppp~rl0+/-Nf+l+- mice on average died from AMML (AML-M4) at 16.5

months of age (range 14 to 19 months), versus MPD at average 19.5 months of age seen in

Nf+l+- mice (range 19 to 20 months). Two of the Ppp~rl0+/-Nf+l+- mice died of pulmonary

hemmorhage and were not included in the analysis. In addition, the Ppp~rl0+/- mice on average

died of AML (AML-MO) at 23.5 months (range 23-24 months) (Table 4-1). These are illustrated









in the Kaplan Meier survival graph (Figure 4-1). Eighty percent of Ppp~rl0+/- Nf+l+- mice

developed AMML, 15% developed MPD, and 5% developed myeloid hypoplasia (Figure 4-14,

Table 4-2). At time of death, Ppp~rl0+/- Nf+l+- mice with AMML disease had an average white

blood cell count of 15,234 compared to 9187 in Nf+l+- mice (Table 4-3, 4-4). The average

percentage of blast cells observed in the peripheral blood of these double knockout mice was

42.9% compared to 8.94% seen in the Nf+l+- mice, with 30% being the clinical cut-off for

"acute" (Table 4-3, 4-4). These mice developed splenomegaly and the leukemic cells had also

invaded other organs: kidney, eye-lid, liver, lung, gall bladder and lymph node. By peripheral

blood smear analysis, the blast cells appeared to be immature monocytes and immature

granulocytes of the granulocyte/macrophage lineage, suggesting that the mutation' s effect is at

the CFU-GM pathway (Figure 4-14, 4-26). Other than AMML, these mice also developed more

(and somewhat different) solid tumors than originally reported in Nf+l+- mice (Jacks et al. 1994).

These tumors included hemangiosarcomas of the uterus and ovary in 3 out of 19 mice, and a

proliferative mesenchymal process of the skin in 2 out of 19 mice. This latter finding appears to

consist of leukemic and immature mesenchymal cells (Figure 4-8, 4-5). Other solid tumors in the

double heterozygotes that were only observed in one each included: lung adenocarcinoma,

hepatocellular carcinoma, granulocytic sarcoma, and squamous cell carcinoma (Figure 4-2, 4-4,

4-15, and 4-16). In comparison, Nf+l+- mice also develop lung adenocarcinomas and

hepatocellular carcinomas. However, the double heterozygotes did not develop lymphomas or

adrenal tumors that are observed in the Nf+l+- mice as well. Overall, 55 percent of the mice had

solid tumors compared to the reported 25 percent of the 129S inbred background and had a

reduced latency (Jacks et al 1994; Bronson et al 1990). However, the cause of death our mice









stemmed from a dysfunctional hematopoietic system typical of acute leukemia (anemia,

bleeding).

Ppp~rl0+/- (129S2) mice lived to about 2 years of age, when they became ill and had to

be euthanized. Seven of eight of these mice had acute undifferentiated leukemia (AML-MO) and

splenomegaly, with infiltrating immature neutrophils and giant multinucleated cells (Figure 4-12,

Table 4-1). The peripheral blood smear analysis showed an increase in white blood cells, and

blast cells. On average 16, 265 white blood cells and 44% blasts (Table 4-3, 4-4). The bone

marrow histology showed irregularly shaped cells that could not be distinguished as myeloid or

lymphoid, in origin, and as well showed a 2-fold decrease in the percentage of granulocytes

compared to Nf+l+-Ppp~rl0+/-mice (Figure 4-12, Table 4-3). This result suggests that loss of

one allele ofPpp~rl0 affects the hematopoietic system at an earlier precursor than that seen in

the Nf+l+-Ppp~rl0+/- (CFU-GM). Besides leukemia, the same seven heterozygous mice

developed solid tumors, one each of the following: myoepithilioma of the salivary gland,

myoepithelioma of the thymus, hepatic hemangiosarcoma, pulmonary adenocarcinoma, pituitary

tumor, and leiomyosarcoma (Figure 4-6, 4-7, 4-8, 4-9, 4-10, 4-11). Our heterozygous Ppp~rl0

mice showed an increased rate of tumorigenesis, with over 75 percent of the mice developing

tumors compared to 25 percent of inbred 129S mice (Jacks et al. 1994).

To determine the subtype of acute leukemia, bone marrow sections were stained with

Sudan Black or mouse monoclonal myeloperoxidase antibody. Sudan Black stains acute

myelogenous leukemia (AML-M1, M2, M3, M6) strongly, weakly stains acute myelomonocytic

leukemia M4 (AMML), and positively stains auer rods. However, Sudan Black does not stain

AML-MO, AML-M7, AML-M5 and acute lymphocytic leukemia (ALL). Myeloperoxidase, on

the other hand, stains acute myelomonocytic leukemia (AML-M4) and auer rods. This antibody









does not recognize AML-M5, AML-M7, and ALL. The Ppp~rl0+/-Nf+l+- mice are thus positive

for acute myelomonocytic leukemia (AML-M4) and Ppp~rl0+/- mice have acute

undifferentiated leukemia (AML-MO) (Figure 4-19).

Tumor suppressor genes typically lose the remaining normal allele somatically in tumors,

often via large deletions, to fit Knudson' s 2-hit hypothesis (Knudson et al. 1971). These somatic

deletions can be detected in tumor DNA by loss of heterozygosity (LOH) analysis comparing the

presence of alleles in germline versus tumor at informative polymorphisms. To test for possible

LOH at both genes, we initially tried genotyping for the only known heterozygous

polymorphism, the engineered mutations that define these mice. PCR-genotyping of Nfl and

Ppp~rl0 was performed in available DNA from tumors, blood and bone marrow. The PCR

products were visualized ethidium bromide to observe relative ratio of mutant to wildtype allele,

in tumors, as compared to the germline. We were unable to get consistent results, indicating that

these 3-primer based systems are unreliable for a quantitative measure such as LOH (Figure 4-

17, 4-18).

To further confirm leukemia subtypes (AML-M4 for Ppp~rl0+/-Nf+l+-, MPD for Nf+l+-

and AML-MO for Ppp~rl0+/-), flow cytometry was performed on bone marrow cells extracted at

the time of death using hematopoietic antibodies. The Grl and Macl FACS populations usually

display increases in granulocytes and monocytes in AML-M4. The results showed an abnormal

Grl +/Macl1 population as well as a Grl (low tomed) /Macl1 population in Ppp~rl0+/-Nf+l+- bone

marrow compared to control, Nf+l+-, and Ppp~rl0+/- bone marrow (Figure 4-20). These two

populations, Grl /Macl+ and Grl (low tomed) /Macl are characteristic of granulocyte and

monocyte populations, respectively, and thus results confirmed AML-M4. In addition,

Ppp~rl0+/-Nf+l+- bone marrow displayed slight increases in c-kit and F-4/80, further confirming









that the CFU-GM point is affected in these mice (Figure 4-21, 4-22). However, although no

alteration was observed in the B-cell pathway in Ppp~rl0+/-Nf+l+- bone marrow, Ppp~rl0+/-

bone marrow displayed an increase in the B-cell marker CD19 (Figure 4-23). Alterations were

also observed in TCRbeta-positive populations for both Ppp~rl0+/-Nf+l+- and Ppp~rl0+/-,

suggesting that Ppp~rl0 affects the myeloid and lymphoid compartment and (Figure 4-24, 4-25).

This suggests that Ppplrl0 may be a possible hematopoietic stem cell gene (Figure 4-26).

Discussion

Creation of a Ppp~rl0 knockout mouse, and crossing it to the Nfl knockout mouse, has led

to the discovery of a new cancer gene involved in solid tumor formation and myeloid leukemia.

Immunostaining and flow cytometry showed that Ppp~rl0+/-Nf+l+- mice develop acute

myelomonocytic leukemia (AML-M4) with high penetrance by 20 months, and about 75 percent

of Ppp~rl0+/- mice develop acute undifferentiated leukemia (AML-MO) by age 24.

AML was classified into 7 subtypes (MO-M7), at a conference by the french, british, and

american doctors in the 1970's, called the FAB classification system (http://www.cancer.org).

AML-MO, undifferentiated acute myeloblastic leukemia, affects 5% of AML patients and has a

worse prognosis. AML-M1, acute myeloblastic leukemia with minimal differentiation, affects

15% of AML patients and has an average prognosis. AML-M2, acute myeloblastic leukemia

with maturation, affects 25% of patients and has a better prognosis. These patients often have

translocations involving chromosome 8 and 21. AML-M3, acute promyelocytic leukemia, affects

10% of AML patients and has the best prognosis of all AML subtypes. These patients often also

have translocations of chromosomes 15 and 17. AML-M4, acute myelomonocytic leukemia,

affects 20% of AML patients and has an average prognosis. These patients often have an inv (16)

genetic abnormality as well. There is another M4 subtype, M4Eos, acute myelomonocytic

leukemia with cosinophilia that affects 5% of AML subtypes and has a better prognosis. AML-










MS, acute monocytic leukemia, affects 10% of patients and has an average prognosis. These

patients also often have translocations involving chromosomes 9 and 11. AML-M6, acute

erythroid leukemia, affects 5% of AML patients and has a worse prognosis for these patients.

The last subtype, AML-M7, acute megakaryoblastic leukemia affects 5% of AML patients and

has a better prognosis. In addition, 1 out of 3 AML patients have FLT3 mutations.

Previous research has shown that a knockin allele for inv (16) predisposes mice to

impaired hematopoiesis however administering ENU mutagenesis to these mice is necessary to

cause an AMML-like disease. These mice had partial myelomonocytic differentiation in a small

percentage of cells in most mice. The maj ority of these cells are ckit+, Grl-, Macl1-, B220-, and

CD3- (Castilla et al. 1999). However, two cooperation studies with inv (16) with either loss of

ARF or a tandem duplication in FLT3 (FLT3-ITD) caused AML without ENU mutagenesis. The

inv (16); Arf-/- mice develop AML and the mutations' effect is on immature myeloid cells,

staining positive for c-kit and negative for Scal. Peripheral blood analysis of these mice shows

basophile and eosinophil granules in addition to a slight monocytic component (Moreno-Miralles

et al. 2005). In addition, inv (16) with FLT3-ITD mice develop AML with an effect on immature

myeloid cells myeloblastss and promyelocytes), which are c-kit+ and Scal- (Kim et al. 2008). In

contrast, our m odel, Ppp ~rl0+/~- Nfl +/- mi ce have an ab normal granul ocyte/m acrophage

population and an abnormal monocytic population of cells. This phenotype is a more mature

myeloid phenotype than the immature myeloid phenotype observed in the previous mouse

models.

There are no existing mouse models for AML-MO as observed in the Ppp~rl0+/- mice. It

is hypothesized that the Ppp~rl0+/- mice have other genetic alterations in genes that cooperate









with Ppp~rl0 to cause AML-MO. These could be targets of PP 1, like p53 or RB 1 or genes

unrelated to the PP 1 pathway.

All of the data suggests that Nfl has an antagonizing effect on Ppp~rl0. Nfl heterozygous

bone marrow effects mature granulocytes and macrophages and Ppp~rl0 heterozygous bone

marrow has neither lymphoid or myeloid morphology, suggesting an early hit in the

hematopoietic pathway. Therefore, Nfl/Ppp~rl0 cooperate to cause an intermediate hit in

hematopoeisis, CFU-GM. However, the effect on Nfl is synergistic since, the Ppp~rl0 mutation

allows leukemic transformation from MPD seen in Nf+l+- mice to AML-M4 in Ppp~rl0+/-

Nf+l+-.

In addition to AML-M4, fifteen percent of Ppp~rl0+/-Nf+l+- mice developed

hemangiosarcomas and ten percent developed a proliferative mesenchymal process of the skin.

Other solid tumors included squamous cell carcinoma, granulocytic sarcomas, lung

adenocarcinoma and hepatocellular carcinomas. However, the Ppp~rl0+/-Nf+l+- mice did not

develop adrenal tumors, MPNSTs or lymphomas as previously observed in Nf+l+- mice (Jacks et

al. 1994). The Ppp~rl0+/- mice, on the other hand, showed some different solid tumors:

myoepithelioma of the thymus and salivary gland, hepatic hemangiosarcoma, pulmonary

adenocarcinoma, pituitary tumor, and leiomyosarcoma. These data suggest that loss of at least

one Ppp~rl0 and Nfl allele cooperate to affect myeloid compartment development at slightly

different stages than either gene alone.

Other known tumor suppressors that are proposed to be at least indirectly related to PP 1

include p53 and RB1 (Lee et al 1992; Jacks et al. 1992; Clarke et al 1992). Mice deficient for

p53 homozygouss for knockout allele) are developmentally viable, however approximately a

fourth of mice develop hemangiosarcomas, and 80% develop lymphomas (Donehower et al.










1992). In contrast, mice deficient for RB1 die embryonically at day 16, and their hematopoietic

system myeloidd compartment) is abnormal, showing increases in immature erythrocytes.

Heterozygous RB1 mice have an increase in pituitary tumors (Lee et al 1992; Jacks et al. 1992;

Clarke et al 1992). In addition, other hemangiosarcoma mouse models have suggested a p53-

related mechanism of tumorigenesis. For example, one mouse model lacking p53, + one Ink4c or

Ink4d allele, develops hemangiosarcomas in over 50 percent of mice (Zindy et al. 2003).

Another model, pl8-/-, p53-/- displays a range of tumor types similar to that also observed in our

mice: hepatocellular carcinoma, testicular carcinoma, hemagiosarcoma, leiomyosarcoma,

fibrosarcoma, and osteosarcomas (Damo et al. 2005). In comparison, Ppp~rl0+/- Nfl+/-

phenotype includes affected hematopoeitic system, increases in hemagiosarcomas, proliferation

of leukemic cells in the skin, and a range of non-recurring tumor types. This suggests that the

tumor mechanism in the double heterozygotes involves p53 and or Rb pathways and further

investigation is necessary.

Since the 3-primer systems are unreliable for LOH analysis of tumors in our mice,

Southern blots will be performed on DNA from any available tumors or bone marrows. It is

expected that the remaining Nfl allele is lost in these mice for tumorigenesis since the previous

Nfl mouse model has shown tumors to be Nf-l-/ (Jacks et al. 1994). It is unknown whether

Ppp~rl0 will be heterozygous or homozygous in the tumors or bone marrow. However, it is

believed that Ppp~rl0 will be homozygous in the bone marrow due to tumor heterogeneity and

lack of a detectable mRNA transcript in bone marrow. Recently, several papers have reported

that cooperating haploinsufficient tumor suppressors in the mouse can be sufficient reduction in

protein function to contribute to tumorigenesis (Kamimura et al. 2007; Vives et al. 2006;

Moreno-Miralles et al. 2005; Ma et al. 2005).









This work provides evidence that Ppp~rl0 is a new cancer gene, most likely a tumor

suppressor gene, and cooperates with Nfl inactivation to cause AML-M4 and other cancers.

Future work will focus on defining the pathways that are deregulated in these cells in order to

develop targeted drug therapy for patients. This is particularly important since AML is a very

refractory cancer and few long-term effective treatments exist.


Survival Curve


Ppplrl0+/-
~NFl+/-Ppplrl0+/
~Nfl+/-


P=. 02


10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Age in months


Figure 4-1. Kaplan-Meier survival curve for Nf+l+-Ppp~rl0+/-, Ppp~rl0+/-, and Nf+l+- mice.






















LungT~~3 5 40X, 100X, and 400X


Nonnal Lung 4X, 10X, atnd 40X

Figure 4-2. H and E stain sections from Nf+l+- Ppp~rl0+/- pulmonary adenocarcinoma and
mouse normal lung.























Liver tumor 4X, 10X, and 40X


Normal Liver 4X, 10X, and 40X


Figure 4-3. H and E stain of sections from Nf+l+-Ppp~rl0+/- liver tumor and mouse normal
liver.
























HI:na cwalllla~r carcinoma, 40X, 100X, 400X


Normal Liver 4X, 10X, and 40X


Figure 4-4. H and E stain of sections from Nf+l+-Ppp~rl0+/- hepatocellular carcinoma and
normal liver.




































Figure 4-5. H and E stain of sections from Nf+l+- Ppp~rl0+/- proliferative mesenchymal
process of the skin (top) and normal skin (bottom).






















Agagggregggyj of salivary gland 2X, 10X, and 40X


Figure 4-6. H and E stain of sections of Ppp~rl0+/- adenocarcinoma of the salivary
gland and normal salivary gland.






















Myoepithelioma 4X, 10X, and 40X


Normal Thymus 4X, 10X, and 40X


Figure 4-7. H and E stain of sections ofPpp~rl0+/- myoepithelioma, thymus and normal
thymus.






































Nonnal Liver 4X, 10X, 40X

Figure 4-8. H and E stain of sections from Ppp~rl0+/- hemangiosarcoma and normal
liver.


Hepatic ~ J~~~~ 4X, 40X























Pulllnunar) Adam;A1D3ga 4X, 10X, and 40X


Nornal Lung 4X, 10X and 40X


Figure 4-9. H and E stain of sections from Ppp~rl0+/- pulmonary adenocarcinoma and normal
lung.






















Leiomyosarcoma 4X, 10X, and 40X


Normal Uterus 4X, 10X, and 40X


Figure 4-10. H and E stain of sections from Ppp~rl0+/- leiomyosarcoma and normal uterus.


Figure 4-11. H and E stain of sections from Ppp~rl0+/- chloromas, showing myeloid expansion,
10X (left) and 40X (right).













































Figure 4-12. H and E stain of sections from Ppp~rl0+/- acute undifferentiated leukemia (AML-
MO) bone marrow (top, 100X) and splenomegaly. Bottom picture shows spleens from
a Ppp~rl0+/- mouse with AML MO (left) and a normal mouse (right). Scale is in cm.























Acute leukemia, 4X.


Figure 4-13. H and E stain of sections from Nfl+/-Ppp~rl0+/- acute myeloid leukemic marrow
(AML-M4) (top) and normal bone marrow (bottom).
















~ CFU-Gmyeloblast >promyelocyte myelocytee >metamyelocyt
CFU-GM CUG e>band> segmenters granulocytess)
(granulocyte/macrophage) CUM myeloblast >monoblast >promonocyte >monocyte


Figure 4-14. Peripheral Blood Smear from Ppp~rl0+/-Nf+l+- mouse, displaying
abnormal monocytes and neutrophils.










































Figure 4-15. An H and E stain of section of a granulocytic sarcoma in a Ppp~rl0+/-Nf+l+-
mouse (10X on top, 40X on bottom).













































Figure 4-16. H and E stain of a section of a squamous cell carcinoma from a Ppp~rl0+/-Nf+l+,
4X top, 10X bottom.










4------------Mouse Tails-----------------9


Figure 4-17. Ethidium-bromide stained polyacrylamide gel showing Nfl PCR products from
blood and bone marrow for Nf+l+-Ppp~rl0+/- mice to test for loss of heterozygosity
in the leukemic bone marrow.












5: E E cl EEclE m
rl CP m P3 m
-~~"gg o\Q151~8 S;
~-rOl(~hOQ


vi te L.L
~8++


Figure 4-18. Ethidium-Stained polyacrylamide gel showing PCR products from Ppp~rl0
genotyping in Ppp~rl0+/- mouse tissues to screen for loss of heterozygosity.

















'188 MPO 690 MPO


188 Sudan Black 692 Sudan Black '1726 Sudan Black

Figure 4-19. Myeloperoxidase and Sudan Black stain of bone marrow sections mouse 188
(Ppp~rl0+/-), 690 (Ppp~rl0+/-Nfl+/-), and 1726 (Nf+l+-).












In rloe 0011csl


I A T1Ti~001,fda~l


rn'

Id
4;
LU 102
a


100 10 10 r
APC-A


103 10"


APC-A


Control BM


NF1PKO


NF1


50 ::


1j lo 10" 1
APC A


PKO



Figure 4-20. Flow cytometry on mouse bone marrow for Ppp~rl0+/-Nf+l+- (NF lPKO), Nf+l+/
and Ppp~rl0+/- (PKO) with Grl (PE) and Macl (APC). Blue box indicates
granulocyte population and red box indicates monocyte population.




























toO t0' loZ so' 10
APC-A


IA Tutle TX1 4cs I


o0.07%~ 4.42"(





'o 10 ACA o' s


0.88%


0.65%


,3


B


44sk


Q to
cu

o to
F
U
m
n


I


lo' lo'
PE-A

NF1PKO


NF1


Control BM


tloo lo' tor 10a 1
PE-A


PKO


Figure 4-21. Flow cytometry on mouse bone marrow for control bone marrow, Ppp~rl0+/-Nf+l+

(NF lPKO), Nfl+/-, and Ppp~rl0+/- (PKO) with ckit (Pacific Blue, stem cell marker).












TC T~ake 42le
0.29% 0.17%


14
10


iis
o

o_


A Tube 002.!cs ]
10^ 12.74%b 3.55%







iNFI


Control BM


Figure 4-22. Flow cytometry on mouse bone marrow from control bone marrow, Ppp~rl0+/-
Nf+l+ (NFlPKO), Nf+l+-, and Ppp~rl0+/- (PKO) with F4/80
(granulocyte/macrophage marker).


A Tube 002.focs
10 1.07%6 9.89%







C 11 10 ACA[1



NFlPKO


'I100 lo' 1o' 1o" 1o<
PE-A


o 238% 3.18%







100 1
PE-A

PKO














Ic tube OU4.les (


B Tus r.r.a


I ~ Tu~e irUcL Ics]


10d



S10


too


103
Q
g lo2
Q
10'


10" 10
PerCP-A


NFl1PKO


S104


100~
I00 1' roL 1(o 10*
PerCP-A


1o' to'
PerCP-A


Control BM


PKO


Figure 4-23. Flow cytometry on mouse bone marrow from control bone marrow, Ppp~rl0+/-
Nf+l+- (NFl1PKO) and Ppp~rl0+/- (PKO) with CD19 (APC, B-cells).











Cu Ok.cTune 006.Fes |
10 12.72%, i 0.06%


l 10




o 0.03%4

r r PerCP-A I 0


A'a Tub 05 an


10




0" i

lo


104

103


1U 11


10 10 101
PerCP-A


11 12 13
10PorCP A 0

NFlPKO

LBE~ube__005.fcs


Control BM


NFI


101 102 103
APC-A
PKO


Figure 4-24. Flow cytometry on mouse bone marrow from control bone marrow, Ppp~rl0+/-
Nf+l+- (NFl1PKO) and Ppp~rl0+/- (PKO) with TCRBeta (APC, T cell receptor).


SA Tu~be 005.fes












B Tube 005 Ice |


IA Tulbe 005 cs 1


tu
$ lo



100


04
10


(i 10 -

10100


l P -A
NF1


1o' to'


10` P0 -A l 10

NFlPKO


10"


,25 0.4204

S100


100 10' 102 103 104
PE-A

PKO

Figure 4-25. Flow cytometry on mouse bone marrow from Ppp~rl0+/-Nf+l+- (NF lPKO),
Nf+l+- and Ppp~rl0+/- (PKO) with CD3 (Pacific Blue, T cell).
















~3--~3- ~3T-Lymphocyte

CLP BIr B -~- -Lympocyl



MEP ~rC~--r O ~ IErythroCyte
MematoosettBFU-E OFU-E
dem cel(S)CFU-s C M P-- errucl
Meg-CF IPlatelets
S no I EosinophilW

GMP Eo CFC


Nutcrophile




Mast-CFC


Figure 4-26. An illustration of NflPKO (green), PKO (blue) and Nfl (yellow) effect the
hematopoeitic lineage.






















Table 4-1. Ppolrl0+/- aged mice necropsy data, M: E (2 to 8.25) ratios, and bl ist numbers.
PK(OHets IDOS ISex IAge~ Ncropsy findings T otal no. ofsolid tumors IBone mlarrow diagnosisl Peripheral blood cytology Finaldinoi
19)5 1905/1 IP F 2yr IMyeepitlimala (Ddx ACA), salivary gla~nd. presamptive 2 AUL AML AUL'
Moptliuametastaticathymas
LkmIapresumptiv), splacn
LumIa presumptive), pcrirenal LN

I194 LEWUS:IllF 12yr Hetpatiche~mmalan~isoa (grosand miers) 3 AUL A.ML AUL
Pulmonaryd urcinem (micro}

LumIa presumptlve) spleen (gmros ad murm)

191 I(WO5:IF 12yr pimuilar* runrIpIII ss e1iLII I AUIL AUL AUL

Lkmia puremptrve), lung
Lumia presumptrve), small ntestine

192 L1~0o1il IF 2Z yr INo stgmfcant lessons 0 M E 2:1 (WNL) M1DS? Normal

188 1916/1iP 2 yr Rounad cEll tumrsR TNTC, visceral lad theracic ravity M:E 4.2 I AUL'

204 Llk20: Ihl IM yr T~hrmbosis wsth fabrinoid neeurmus, heant Mv:E: 3 68:1 (AUL) AUIL AUL
Mylodleukmia pearly diI~reai~atd, spleen 1
MylId ukemia poorly di~terentiated. kiidncy

199 LEW20/llIM 2yr1 Mhyelead icukmla prerumptive) 1ung 0 M:E51: 1 AUL AUL
Mye~ cleadlpeukma pum ptive), sphen

19)7 L(W2Del IM~ 12yr IMyelead leukemrnan Heart lung. liver. LN, 5
pteof SE M:E 8 25 I (AUL AUL AUL
Mylid ukemia poorly differnentiatd. splee
sailr o 1. have poo


















Table 4-2. Nfl+/-Ppplrl0+/-aged mice necropsy data, M:E (2.33 to 13.25) ratios, and blast

numbers.
PKUl NFI dueble hral DOS bea age Neupn f~aing $u. tSutid Tuester Sar Marren resull6 Finaldign
El llhi I hlt1a~aitnuae-t urkr 1b L ramia.pbiticd hfur~l lninfarous

; 1:15 l('hi LI I' gavlnrulladheriurner I SFl lETnlnll hhnalarme w~ilalunrirr


M .us Ik.; rrirr 1:1,e 4.* :Asf
Itpr e lu~lato r lie a.trr


B Yill FuII I I* In eruprinpi pke is~ \ff0 Vi


$ 5-1i I I) Ibubmu, Ing w shl hart.rrsae 7.1 U.=\4 L.L
Matrrianiukre.in rr plhen phasul harwrrurrun

.iv9 1l F I* pnditeralihtmemdy press I ~l f SM 4L
pkrmlrnal:. pkweinrdlr
halr il.JdrleIslrhened
Leakernet sthnrt lert sps..u


I ~ ~ ~ L F"2 1 Liakerromn i Inr pbgina. urtyirry Mffo %|


rXI4>1 41 I I T usemu InLu~net~.phersrulaum U U.L?1 .LL


171 U!Z I F Faimlr? hrartwelurin fil ur4 Bldnr L keR blmurwrwo.:s3)d rri

r"I 44 n 2 I F (^ fltrwarn twncirtre b' ii4 .LL

ui.I. u.. .. i. .. ar 1 911l

III442 ) Ir MI 4I M:.uOvci~c.p~aabaun bY~ ".f 0L


ilelti.1 1:i )- ( Lecraubr u Ia g rins I ML)5~ .CLLI
lIsmop~r~uartemsnsur


Illiefi i1 I: F I-) lIhrsndlwrarturelnua I M


Rxpcrhal leA2:ml

I1vi'1l 11 I i. I It $quman Ri eipapillnetal' I *rrhidbrinpp*i



II if.41L 3 I: I) My~I~tlddismu luIslarM. diblraiderplann Wre

CrL Jdu~e 434: r of ul mu~leb
IlpmkrhrnslW~i

11!I1:a :91.' F blitr irutem eia. ory, L. lqdee
rr CI*TL71 asknerL trplasU

Ilhli~h 1291 ( II prelfratubrmrwe ylmralrs I II"J4 411t
M ~k~culmleaknsirvetashe


Il~lie '-) : F Hi 1bhle rs .t:s I :;1.I kL
Iraseryth srrama oI hear marro
n.i karws 'rr 1 min irielesnraph~l
Hewinitinure ulrnis pilarinaltanppin

lllbeha : :2 : hlo iaem.rsa I kiL.)R nLL
ItalkshandkyPphabhra ckstachmbree
raia dt.ar:.ruta seranuluickr


































87
















Table 4-3. Peripheral blood smear analysis of Nfl+/-Ppplrl0+/- (NFl1PKO), Ppplrl0+/- (PKO),

and Nfl+/-. The yellow color signifies the average count for all mice for that

compartment.

YI~bb




















Boar Marrow o~!




















AI llIAmal. ra rtl riad iBlr ~ r~l- 11. Im sir mgrkrast* .lna garsblw pri l-mnmla+ ir.r~lidasisp strnri r! i.4I re.




u .II




~~~1 '1 1Il I





574 DEC II
NF oa als r tri cad ME imauemgkrvre auin rn lss eca oalmced prcn ~t eretgasfia igoi
171 338 I127 45 10 8 2I30a 7" M
171 I2 20 21 0629 229*, 5 2 odrae

Noma centrol


~nlr952 I 10 60 15 t2.5l 3lu 144I. 6s~ I' .s 41 11 96"* W









CHAPTER 5
DISCUS SION

Previous work in Dr. Brannan's lab has elucidated three common loci that cooperate with

Nfl inactivation to cause acute leukemia. The focus of this thesis has been one such locus, Epi2.

The candidate gene I investigated at this locus was PPP1R10 located on human chromosome

6q21.3. Experiments were designed to help answer the question that if PPP1R10 is a cancer

gene, is it an oncogene or a tumor suppressor gene.

The mouse retrovirus integrated in intron 1 of2~rpsl8b in the same transcriptional

orientation. However, since Ppp~rl0 is less than one kilobase away, in the opposite orientation

of the virus, it was hypothesized that this gene may also be affected, particularly by the viral

LTR. Based on the location of the virus, the most likely mechanisms were protein truncation of

M~rpsl8b or down-regulation of Ppp~rl0 through the viral LTR integrating in a transcription

factor-binding site. PPP1R10 seemed to be a more attractive candidate because of its ability to

regulate PP1, a serine threonine phosphatase. PPP1R10, whose protein product is PNUTS, is a

serine/threonine phosphatase regulator that binds PP1, regulates PPlI's enzymatic activity and

targets PP 1 to the nucleus. PP 1, a maj or phosphatase, regulates over 70 mammalian genes in the

cell, however the downstream effectors of PNUTS are still unknown or PNUTS/PP 1 complex.

The goal of the first specific aim was to sequence PPP1R10 and M~RPS1B and look for

any genetic abnormalities in human leukemia samples. In PPP1R10, two possible point

mutations were found, a serine to stop (nonsense) and a serine to threonine (missense) change at

codon 125 in exon 6. However, we have not been able to determine if these are somatic or not.

Other polymorphisms previously validated were also found, one in the promoter (rsl6867845)

and one in exon 19 (rs 11754215). Both of these displayed no loss of heterozygosity in human

leukemia samples. One polymorphism appeared to display loss of heterozygosity in an intron 15










SNP by sequence analysis. This finding was further confirmed by constructing an induced Rsal

restriction fragment length polymorphism at the site. This would indicate PPP1R10, may have a

role in leukemia through a tumor suppressor mechanism. Results, suggested LOH in other

samples, although validation with both methods needs to be done and other SNPs need to be

examined as well.

The in vitro experiments were aimed at testing Ppp~rl0 as a possible oncogene. The first

experiment was to over-express PNUTS in COS7 cells, starve the cells and probe for increases or

decreases in different effectors in cancer-related signaling pathways. Western blots showed

subtle effects in p-Stat3, p-Pten, p-Erkl/2, and p-Mekl/2 but these were not considered to be

representative of oncogenic activation. Further work on downstream effectors of Ppp lrl0 could

focus on probing these pathways by down-regulating PNUTS through siRNA or a conditional

gene targeted Ppp~rl0 mouse, to study its role in different tissues. This is important since

PNUTS has not been studied in hematopoietic cells.

To further confirm that Ppp~rl0 is not an oncogene, a CFU-GM assay was performed by

over-expressing Ppp~rl0 in murine wild-type fetal liver cells and then plating the cells with

increasing amounts of GM-CSF. The expected results for an oncogene were increased sensitivity

to GM-CSF at low concentrations, with increasing number of colonies as well as an altered cell

morphology. The results displayed no altered morphology of the colonies and no cytokine

independent growth via GM-CSF. These experiments provide more evidence that PPP1R10 is

not an oncogene.

To further study the hypothesis of tumor suppressor, a Ppp~rl0 gene targeted Gene Trap

mouse knockout was created by Jessica Walrath, a previous graduate student. Dr. Walrath

showed that homozygosity for Ppp~rl0 is a pre-implantation lethal mutation (Walrath 2006).










Many tumor suppressor genes when knocked out are embryonic lethal (e.g Nfl, Nf2), consistent

with Ppp~rl0 being a tumor suppressor gene (Jacks et al. 1994). Jessica also had evidence of

decreased Ppp~rl0 RNA levels in an Epi2 tumor (and somewhat less decreased in a few non

Epi2 tumors) but the RNA was not absent (Walrath, 2006). This was similar to my human tumor

data and is consistent with tumor suppressor but possibly having an effect through

heterozygosity (haploinsufficiency) Further evidence fitting a hypothesi s of tumor suppressor

was found when 21 Ppp~rl0+/-Nf+l+- mice I generated developed cancer at a very high

frequency. Results showed a phenotype different than observed originally in Nf+l+- mice, which

develop a minor percentage of MPD or pheochromocytomas between 17 and 27 months of age

(Jacks et al. 1994). Eighty two percent of Ppp~rl0+/-Nf+l+- mice developed AML-M4 and

between 14 and 21 months. In addition, fifteen percent of these mice developed

hemangiosarcomas of the ovary and uterus, and ten percent had a proliferation mesenchymal

process of the skin. In Ppp~rl0+/- mice, clinical symptoms were not observed until 24 months of

age. These mice then became ill and rapidly declined due to AML-MO. Seventy-five percent of

these mice also developed one or more non-recurring solid tumors, suggesting an overall

increase in tumorigenesis in other tissues as well. This supports the hypothesis that Ppplrl0 is a

tumor suppressor whose loss has high penetrance in certain tissues such as bone marrow. Further

these mice are novel models for AML-MO and AML-M4.

Based on this work and the literature, we hypothesize that loss of Ppp~rl0 causes

abnormal regulation of PP 1, and increased phospho-p53. Recent literature has shown that PP 1

dephosphorylates p53 at serine 15 and serine 37 (Li et al. 2006). The dephosphorylation of these

sites changes transcriptional activity and apoptotic activity of p53 to the active form (Li et al.

2006). Phosphorylated p53 (inactive) has been associated with tumorigenesis (Feng et al. 2008).









In our model, loss of Ppp~rl0 decreases PP1 binding with p53, which interferes with

dephosphorylation of p53, raising the amount of phosphorylated p53. This would cause effects in

DNA repair, apoptosis, and the other processes regulates by p53, which go awry when it is

inactivated as in many cancers. Alternative explanation is that the pRB pathway is affected, since

PP1 is known to dephosphorylate (inactivate) this important tumor suppressor in other tissues

(Krucher et al. 2006). However, mouse model phenotypes fit with p53 involvement better (Zindy

et al. 2003; Damo et al. 2005).

Future work on this proj ect will be geared towards making a conditional knockout for

Ppp~rl0. The PP 1 binding site that encompasses exons 13 and 14 will be flexed in order to

ensure that all isoforms of Ppp~rl0 lack of ability to regulate PP l. This mouse will be crossed to

an Mxl-Cre mouse, whose Cre expression is inducible and affects the hematopoietic system,

spleen, and liver (Kuhn et al. 1995). Expression of Cre will be induced with plpC inj section, or

interferon, and then mice will be followed for development of AML. Other tissues could be

studied by crossing the conditional Ppplrl0 mouse to other Cre genes driven by tissue-specific

promoters. For example, hemangiosarcomas are endothelial in origin. Thus, crossing the

conditional Ppp~rl0 knockout to a Cre transgenic mouse driven by an endothelial-promoter such

as ICAM2 or Tie2 (the latter of which already exists) (Cowan et al.1998; Kisanuki et al. 2001).

This could provide an excellent model for hemagiosarcoma, which could be used to understand

pathways involved or test treatments. We could also test effects of background strain on

penetrance, latency, and or tumor repertoire (i.e. identify modifier genes).

Another area of research will focus on the pathways that are deregulated in the mouse bone

marrow by loss or reduction of Ppp~rl0 with or without Nfl We could use siRNA for Ppp Irl0O

+/- Nfl in hematopoeitic precursor cell lines (e.g DC13) or primary cells, or possibly establish









cell lines from the tumors or from non-transformed bone marrow cells, for in vitro studies. These

experiments may be carried out with Western blots and or phosphoFlow cytometry developed by

the Nolan lab at Stanford (Krutzik et al. 2005). Some candidate pathways include: p53, pRB,

PKA, and the Ras pathway. Once the deregulated signaling pathway(s) is discovered,

therapeutics can be identified to help treat patients with these leukemias. This could be

specifically targeted to the PP 1 level, or utilize therapies already being developed for the

pathways implicated.



























Primer Name Primer Seunc enomic or cDNA Reion of gee Mse or Hs
PNUT romF 5-CAGGACAGGAATIGACGGAAA-3'geocprmtrH
PNUT romR 5-CTAGCACCTCCCTHICCTCTG-3'gnocprmtrH
PNUTl ro2F 5'TACCAATCCTGGGTGAGAAATG-3' eocprmtrH
PNUTl ro2R 5-CCGCAAAATHITACCCACTAGA-3' eocprmtrH
P115RsAF 5-CCCTAAACTCATCCCCCTAGT-3 geoiFP intron 15 H
P115RsaR 5-CTGAAAGAAGAACAAAAAAAATCAGTA-3 geoi FP intron 15 H
lac2 5-CAAGGCGATTAAGTTGGGTAACG-3' geoi xn2Me
Pko2fb 5-CGAAGGACCGTCACCACATAAC-3 geoi xn2Me
Pko2fa 5-CGAAGGACCGTCACCACATAAC-3 geoi xn2Me
NF31a 5'GTATTGAATTGAAGCACCHITGTHG-I enoicMs
NF31b 5-CTGCCCAAGGCTCCCCCG-3' eocMe
Neo~k 5-GCGTGHICGAATTCGCCAATG-3' eocMe
PPPex2a 5'TTGTGHICCTTIATCCCAGGT-3' gnmceo 2 H
PPPex2b 5-CTTCGGCCACAGATHICAAG-3' geoixn2 H
PPPex2c 5-CTGCTTGGGACTTGAAATCTG-3' geoixn2 H
PPPex2d 5'-THGCGACGTCAGCACCT-3' gnmceo 2 H
PPPex3F 5'TCCTCTTACCATAGAAACCACCA-3' geoixn3 H
PPPex3R 5-CAAAAAGGGGTAAGACTCACC-3' geoixn3 H
PPPex4F 5-GGCTGCTTCATITCTAACCCTA-3' geoi xn4H
PPPex4R 5-GCTTACACCHICCCATICCAA-3' geoi xn4H
PPPex5F 5-CTGCCTGCCTGCAGATITAT-3' gnmceo 5 H
PPPex5R 5'AAAGGTACCTGCTTGAGATGG-3' geoixn5 H
PPPex6F 5-GCCTCCTGGCATHIACCHIT-3' geoi xn6H
PPPex6R 5'AAAGCCCATGAGAGAGCAGA-3' geoi xn6H
PPPex7F 5-GCATITCCCCTHICTGTCAC-3' gnmceo 7 H
PPPex7R 5'AGGACTAAGGAGCHIACCAGCA-3' geoixn7 H
PPPex8F 5'TCCCAGTCTGCTTCACCTCT-3' geoixn8 H
PPPex8R 5-GAACGGAACHIGGCATGACT-3' geoixn8 H
PPPex9a 5-GTCTGGGGTTTGAGHICAGC-3' geoixn9 H


genomc exo 11 H
genomi exon12 H
3' geoixn12 H
genomi exon13 H
genomi exon13 H
enomic exon4 H
enomic exon4 H
genomi exon15 H


genomi exon20 H
genomi exon21 H
3' gpenomic lexon 20 H
i-3' gpenomic lexon 20 H


PPPex9b
PPPex10a


PPPex12F
PPPex12R
PPPex13a
PPPex13b
PPPex14F
PPPex14R
PPPex15F
PPPex15R
PPPex16F
PPPex16R
PPPex17F


PPPex17R
PPPex18F
PPPex18R
PPPex19a



PPPex19b
PPPex19e
PPPex19f
PPPex20F
PPPex20R
PPPmtron4F


5'-CATTCAGAGCCTGAGAATATGG-3'


!eol
!en l
!eol
ren l


exon 9
exon 10
exol


5'-CTGTGTHIGCAGGTGTAGGATT
5'-CTTCTGGGAGCCCATACCTT-3'
5'-CACAATAGCCAAGCCCCTTT-3'
5'-ACCCAAGATCCCTCCHITCAG-i
5'-HICTGCTTCCAGCCTCHITG-3'


5'-TCTCACCTGTGTT


5'-CAATGACCATCACAACHICCA-3'
5'-TGAATICGGTGCATCTTTCA-3'
5'-ACAGTGAGGAGAGCGAGCTT-3'
5'-CHITCGCCATGGTTGTTACC-3'
5'-CCCAAGCATCTGCTTGATCTT-3'
5'-GCTCATGCTGTGHIACTCTTGTTT
5'-AAGCAGATAAGCCCATICCA-3'
5'-GGCAGTTCCTGGTAGCTGAT-3'


:enomic


exon 15
exon 16
exon 16
exon 17
exon 17
exon 18
exon 18
exon 19


exon 19


!enomic


-3'


5'-GACCCCCAAATGGACGAG-3'
5'-CCACCCATGCTACCACCA-3'
5'-GCATTGCCCTCAGCTATTTC-:
5'-GAAAATGGGCCTCACAGAAG
5'-CCGCCAACGTCAATAAATCT-


:enomic


[ntron 4SNP


Ppplrl~cDNAIR
Ppplrl~cDNA2F
Ppplrl~cDNA2R
Ppplrl~cDNA3F
Ppplrl~cDNA3R
Ppplrl~cDNA4F
Ppplrl~cDNA4R
Ppplrl~cDNA5F
Ppplrl~cDNA5R
Ppplrl~cDNA6F
Ppplrl~cDNA6R
Ppplrl~cDNA7F
Ppplrl~cDNA7R
Ppplrl~cDNA8F
Ppplrl~cDNA8R
GAPDH-5'


5'-CCTCTCCCTAAATGGAGHIGGG-3'
5'-CTCATCCAGCATHICCGH-3' '
5'-TAGACCCCAAAGAACTGCTA-3 '
5'-GTGCTGACGTCGCAAAAA-3'
5'-ATTGGCATCAGTCCTTGTCAG-3 '
5'-GCAAGTCAAGTGAGGATGAAG-3'
5'-CTGTCCCAGGCATCAAAAHI-3'
5'-CTGGGTTTTCTGGATGCTCTC-3'
5'-CAAGATCAAAGACTTCGGGG-3'


exon 2
exon 2
exon 4
exon 2
exon 8
exon 7
exon 10
exon 10
exon 15
exon 14
exon 17
exon 17
exon 20



exons 203
exons 213


:DNA
:DNA
:DNA
:DNA
:DNA
:DNA
:DNA


5'-TCATCATCTCTGCCCCCTCTG-3'


Hs/Mse
Hse/Mse


PPPex2-3R


APPENDIX A

PRIMER LIST









APPENDIX B
PFAFFL METHOD



Ratio= (E,,)DCPtargetYcontrol-sample2)

(Erer)DCPref(control-samples)









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BIOGRAPHICAL SKETCH

Angela Hadjipanayis was born in Winnipeg, Manitoba, Canada, in 1975. She is the

daughter of George and Paraskevi Hadjipanayis. Her father is from Cyprus and is a Professor of

physics at the University of Delaware in Newark, Delaware. Her mother is from Patras, Greece

and is the head librarian at St. Marks High School in Wilmington, Delaware. She has one sibling,

Costas Hadjipanayis who is a neurosurgeon in the Department of Neurosurgery at Emory

University in Atlanta, Georgia. Angela is also married to Joseph Phillips, who has a Ph.D. in

physics and holds a postdoctoral position at the University of Florida with Dr. Weihong Tan.





PAGE 1

1 PROTEIN PHOSPHATASE REGULATO RY PROTEIN 10 COOPERATES WITH NEUROFIBROMIN INACTIVATION IN MYELOID LEUKEMOGENESIS By ANGELA HADJIPANAYIS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

PAGE 2

2 2008 Angela Hadjipanayis

PAGE 3

3 This work is dedicated to my extended family who ne ver had the opportunity to obtain higher education. To my parents and husband for their loving support and belief in my abilities. To Camilynn I. Bra nnan whose research e xpertise led to the discovery of this locus.

PAGE 4

4 ACKNOWLEDGMENTS First, I would like to tha nk m y mentor Peggy Wallace for her indispensa ble support over the years, her encouragement, and her advice on how to become a bette r scientist. Second, I would like to thank my husband for taking a leap of faith in life and moving to Florida with me when he didnt have a job at the time. Third, I would like to thank my parents for their love, support, and advice over the years. Fourth, I would like to thank my other committee members, Dr. Jim Resnick, Dr. Paul Oh, Dr. Stephen Hunger a nd Dr. Dan Driscoll for their valuable advice on the project. I also would like to thank Dr. Je nnifer Embury for the trem endous effort on all the pathology of the mice. In addition, I would like to thank Dr. Kevin Shannon and Dr. Scott Kogan for their leukemia expertise and strategies in modeling leukemia. Lastl y, I would like to thank the lab for their help on this project, their jokes, and all their personalities that made the days seem not as long.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................7LIST OF FIGURES .........................................................................................................................8LIST OF ABBREVIATIONS ........................................................................................................ 11ABSTRACT ...................................................................................................................... .............13 CHAP TER 1 INTRODUCTION .................................................................................................................. 15Neurofibromatosis Type 1 and JMML ................................................................................... 15Mouse Models of Nf1 .............................................................................................................18Molecular and Biochemical Mechanisms Underlying Myeloid Leukemogenesis. ................ 19Hyperactive Ras in JMML ......................................................................................................20Nf1 Reconstitution Mouse Model ..........................................................................................21Identification of Epi Loci in Nf1-Related Myeloid Leukemia ...............................................21Viral Insertional Mutagenesis .................................................................................................23MRPS18B ...............................................................................................................................24PPP1R10 ....................................................................................................................... ..........24PP1, a Serine/Threonine Protein Phosphatase ........................................................................252 MUTATIONAL ANALYSIS OF PPP1R10 IN LEUKEMI A ............................................... 28Introduction .................................................................................................................. ...........28Materials and Methods ...........................................................................................................29Patient Samples ...............................................................................................................29Polymerase Chain Reaction and Sequencing of PPP1R10 Exons .................................. 29Restriction Fragment Length Polymorphism for Intron 15 Loss of Heterozygosity .......29Real-Time mRNA Quantification ...................................................................................30Results .....................................................................................................................................31Discussion .................................................................................................................... ...........323 IN VITRO ANALYSIS OF PPP1R1 0 AS A POTENTIAL ONCOGENE ............................ 41Introduction .................................................................................................................. ...........41Materials and Methods ...........................................................................................................41Cloning of Ppp1r10 in the MSCV Viral Vectors ............................................................41Hematopoietic Cell Isolation and Retroviral Transduction .............................................42Colony Formation Unit Granulocyte/Macrophage Colony Assay .................................. 42

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6 Serum Starvation of COS7 Cells .....................................................................................43Western Blots ..................................................................................................................43Results .....................................................................................................................................44Discussion .................................................................................................................... ...........444 PPP1R10 COOPERATES WITH NF1 INACTIVATION TO CAUSE ACUTE MYELOMONOCYTIC LEUKEMIA IN A MOUSE MODEL ............................................. 50Introduction .................................................................................................................. ...........50Materials and Methods ...........................................................................................................51Ppp1r10+/Mouse Construction ..................................................................................... 51Breeding Ppp1r10 Mice ..................................................................................................51Pathology/Sectioning/ H and E Stain ............................................................................... 51Blood Smears and Manual Counts .................................................................................. 52Sudan Black Stain and MPO Stain .................................................................................. 52Flow Cytometry ...............................................................................................................53Nucleic Acid Extraction ..................................................................................................53Loss of Heterozygosity Analysis ..................................................................................... 53Results .....................................................................................................................................54Discussion .................................................................................................................... ...........585 DISCUSSION .................................................................................................................... .....89 APPENDIX A PRIMER LIST ........................................................................................................................94B PFAFFL METHOD ................................................................................................................95REFERENCES .................................................................................................................... ..........96BIOGRAPHICAL SKETCH .......................................................................................................105

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7 LIST OF TABLES Table page 2-1 NF1-JMML human patient samples and Intron 1 5 polymorphism LOH. ......................... 382-2 JMML human samples and Intron 15 polymorphism LOH. .............................................. 392-3 mRNA Quantification of human leukemia RNA. .............................................................. 404-1 Ppp1r10+/aged mice necropsy data, M: E (2 to 8.25) ratios, and blast numbers............ 864-2 Nf1+/-Ppp1r10+/-aged mice necropsy data, M:E ( 2.33 to 13.25) ratios, and blast numbers. ...................................................................................................................... .......874-3 Peripheral blood smear analysis of Nf1 +/-Ppp1r10 +/(NF1PKO), Ppp1r10+/(PKO), and Nf1+/-. .............................................................................................................884-4 Bone marrow analysis of Nf1+/-Ppp1r10+/(NF1PKO), Ppp1r10+/(PKO), and Nf1+/mice. WNL signifies within normal limits. ............................................................ 88

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8 LIST OF FIGURES Figure page 2-1 Human NF1-JMML analysis PPP1R10, Promoter Polymorphism (rs16867845). ............332-2 Human NF1-JMML analysis, PPP1R10 Exon 6 nonsense, S125X. .................................. 332-3 Human NF1-JMML analysis, PPP1r10 Exon 6 missense, S125T. .................................... 332-4 Human NF1-JMML analysis, PPP1R10 Exon 19 polymorphism (rs11754215). ..............332-5 Human NF1-JMML PPP1R10 analysis, Intron 15 polymorphism loss of heterozygosity. ............................................................................................................... ....342-6 RsaI Digest of Intron 15 pol ymorphism of Control DNAs. .............................................352-7 RsaI Intron 15 Polymorphism of NF1-JMML and Control DNA. ....................................362-8 RsaI Intron 15 Polymorphim Dige st of JMML and an AML sample. ...............................373-1 CFU-GM Colony assay of pMIG-PNUTS and pMIG in wild-type fetal liver cells. .........463-2 Experimental Design of Colony Formation Assay. ........................................................... 473-3 CFU-GM of wt PNUTS vers us empty vector control. ...................................................... 483-4 Transfection of pDEST-PNUTS in COS7 cells with serum starvation and probing signaling cascades. .............................................................................................................494-1 Kaplan-Meier survival curve for Nf1+/-Ppp1r10 +/-, Ppp1r10 +/-, and Nf1 +/mice. ........ 624-2 H and E stain sections from Nf1+/Ppp1r10+/pulmonary adenocarcinoma and mouse normal lung. ............................................................................................................634-3 H and E stain of sections from Nf1+/-Ppp1r10+/liver tumor and mouse normal liver. ........................................................................................................................ ...........644-4 H and E stain of sections from Nf1+/-Ppp1r10+/hepatocellular carcinoma and normal liver. ................................................................................................................. ......654-5 H and E stain of sections from Nf1+/Ppp1r10+/proliferative mesenchymal process of the skin (top) and normal skin (bottom). ....................................................................... 664-6 H and E stain of sections of Ppp1r10 +/adenocarcinoma of the salivary gland and normal salivary gland. ........................................................................................................ 674-7 H and E stain of sections of Ppp1r10 +/myoepithelioma, thymus and normal thymus. ....................................................................................................................... ........68

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9 4-8 H and E stain of sections from Ppp1r 10+/hemangiosarcoma and normal liver. ............. 694-9 H and E stain of sections from Ppp1r10+/pulmonary adenocarcinoma and normal lung. ......................................................................................................................... ..........704-10 H and E stain of sections from Ppp1r10+/leiomyosarcoma and normal uterus. ............. 714-11 H and E stain of sections from Ppp1r10+/chloromas, showin g myeloid expansion, 10X (left) and 40X (right). .................................................................................................714-12 H and E stain of sections from Ppp1r10+/acute undifferentiated leukemia (AMLM0) bone marrow (top, 100X) and splenomegaly ............................................................. 724-13 H and E stain of sections from Nf1+/-Ppp1r10+/acute myeloid leukemic marrow (AML-M4) (top) and normal bone marrow (bottom). ....................................................... 734-14 Peripheral Blood Smear from Ppp1r10+/-Nf1+/mouse, displaying abnormal monocytes and neutrophils. ...............................................................................................734-15 An H and E stain of section of a granulocytic sarcoma in a Ppp1r10+/-Nf1 +/mouse (10X on top, 40X on bottom). ............................................................................................ 744-16 H and E stain of a section of a squamous cell carcinoma from a Ppp1r10+/-Nf1+/, 4X top, 10X bottom. ..........................................................................................................754-17 Ethidium-bromide stained polyacrylamide gel showing Nf1 PCR products from blood and bone marrow for Nf1+/-Ppp1r10+/mice to test for loss of heterozygosity in the leukemic bone marrow. ............................................................................................ 764-18 Ethidium-Stained polyacrylamid e gel showing PCR products from Ppp1r10 genotyping in Ppp1r10+/mouse tissues to screen for loss of heterozygosity. ................ 774-19 Myeloperoxidase and Sudan Black st ain of bone marrow sections mouse 188 ( Ppp1r10+/-), 690 ( Ppp1r10 +/-Nf1+/-), and 1726 ( Nf1+/-). ............................................. 784-20 Flow cytometry on mouse bone marrow for Ppp1r10+/-Nf1+/(NF1PKO), Nf1+/, and Ppp1r10+/(PKO) with Gr1 (PE) and Mac1 (APC). ................................................. 794-21 Flow cytometry on mouse bone marrow for control bone marrow, Ppp1r10+/-Nf1+/ (NF1PKO), Nf1+/-, and Ppp1r10+/(PKO) with ckit (Pacific Blue, stem cell marker). ...................................................................................................................... ........804-22 Flow cytometry on mouse bone marrow from control bone marrow, Ppp1r10+/Nf1+/ (NF1PKO), Nf1+/-, and Ppp1r10 +/(PKO) with F4/80 (granulocyte/macrophage marker). .................................................................................... 814-23 Flow cytometry on mouse bone marrow from control bone marrow, Ppp1r10+/Nf1+/(NF1PKO) and Ppp1r10+/(PKO) with CD19 (APC, B-cells). ............................ 82

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10 4-24 Flow cytometry on mouse bone marrow from control bone marrow, Ppp1r10+/Nf1+/(NF1 PKO) and Ppp1r10+/(PKO) with TCRBeta (A PC, T cell receptor). ..........834-25 Flow cytometry on mouse bone marrow from Ppp1r10+/-Nf1 +/(NF1PKO), Nf1+/and Ppp1r10+/(PKO) with CD3 (P acific Blue, T cell). .................................................. 844-26 An illustration of Nf1PKO (green), PKO (blue) and Nf1 (yellow) effect the hematopoeitic lineage. .......................................................................................................85

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11 LIST OF ABBREVIATIONS AML Acute Myeloid Leukemia BL Blood BM Bone Marrow BSA Bovine Serum Albumin CMML Chronic Myelomonocytic Leukemia CML Chronic Myeloid Leukemia CFU-GM Colony Forming Unit-Granulocyte/Macrophage Epi1 Ecotropic Proviral Integration 1 Epi2 Ecotropic Proviral Integration 2 Epi3 Ecotropic Proviral Integration 3 GFP Green Fluorescent PCR GM-CSF Granulocyte Macrophage Colony Stimulating Factor JMML Juvenile myelomonocytic leukemia LOH Loss of Heterozygosity PPP1R10 serine/threonine phosph atase regulatory protein 10 MDS Myelodysplastic syndrome MPD Myeloproliferative disease MPNST Malignant Peripheral Nerve Sheath Tumor MuLV Murine Leukemia Virus NF1 Neurofibromatosis type 1 NF1-JMML NF1 associated leukemia NS Noonan Syndrome PCR Polymerase Chain Reaction PKO Ppp1r10 mouse line

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12 PP1 serine/threonine phosphatase 1 PPP1R10 serine/threonine phosph atase regulatory protein 10 RT-PCR Reverse Transcription PCR SDS Sodium Dodecyl Sulfate

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13 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PROTEIN PHOSPHATASE REGULATO RY PROTEIN 10 COOPERATES WITH NEUROFIBROMIN INACTIVATION IN MYELOID LEUKEMOGENESIS By Angela Hadjipanayis August 2008 Chair: Peggy Wallace Major: Medical Sciences-Genetics Neurofibromatosis (NF1) is an autosomal dominant disease th at affects 1 in 4000 people. Patients afflicted with NF1 present a variety of phenotypes, but the hallmark features of the disease are neurofibromas on or unde r the skin, caf au lait spots, and Lisch nodules of the iris. Children with NF1 are at 200 to 500 times great er risk for developing a chronic myeloid leukemia. Often children who are diagnosed w ith this myeloid leukemia initially are not diagnosed with NF1 until a few years later. Ther efore, there are likely more children who are affected by NF1-associated leukemia than are re ported in the statistics. The focus of this proposal is to study NF1-associated myeloid leukemia, and to characterize additional genetic events in tumor progression to acute stag e using human and mouse model resources. A viral based mouse model of NF1-associat ed leukemogenesis iden tified several common sites of viral integration. I investigated one site (Epi2) to see how the endogenous genes are affected and how this can contribute to myeloid leukemia. The Epi2 locus is on chromosome 6 in humans and chromosome 17 in mice. There are two genes in the Epi2 locus, PPP1R10 and MRPS18B PPP1R10, whose protein product is PNUT S, is a serine/threonine protein phosphatase regulatory protein, a nd MRPS18B is a 28S mitochondr ial ribosomal protein. Since

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14 both genes are located less than 1kb apart and are transcribed in opposite directions, it was possible that both genes act simultaneously in NF 1 leukemogenesis, as oncogenes and/or tumor suppressors. I performed a series of experime nts to test some of these hypotheses, and found evidence that PPP1R10 is a tumor suppressor gene that c ontributes to development of acute myeloid leukemia.

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15 CHAPTER 1 INTRODUCTION Neurofibromatosis Type 1 and JMML Neurofibrom atosis type 1(NF1) is an autosoma l dominant disease that is characterized by a variety of phenotypes. At the NF consensus conference of 1987, it was decided that the criteria for clinical diagnosis required that a patient have two or more of the following symptoms (Stumpf et al. 1988): Six or more caf au lait macules on the skin over 5 mm in greatest diameter in prepubertal individuals, and over 15 mm in greatest diam eter in postpubert al individuals. Two or more neurofibromas of any type or one plexiform neurofibroma (benign Schwann cell tum ors). Freckling in the axillary (armpit) or inguinal regions (groin). Optic glioma. (iris hamartomas). A distinctive osseous lesion such as sphenoid dysplasia or thinning of long bone cortex with or without pseudoarthrosis (tibial dysplasia) Two or more Lisch nodules A first-degree relative (parent, sib, or offspring) with NF1 as defined by the above criteria. NF1 patients also have an increased risk for other tum ors besides those described above. For example, in one review, 12.5 percent of th e NF1 cases had tumors in the optic pathway (pilocytic astrocytomas) while 1.0-1.6 percent had tumors at other central nervous system sites (www.ctf.org). For malignancies, 32.76 percent of NF1 individuals had malignant peripheral nerve sheath tumors (MPNSTs) while 1.76-4.2 percent had sarcomas at other sites. Another category is leukemia; 1.7 percent had juvenile myeloid monocytic leukemia (JMML, described further below), while 7.4 per cent had myelodysplastic syndrome (MDS). There is also an increased risk for phaeochromocytomas (adrenal tumors), gastrointestin al tumors and non-optic brain tumors.

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16 It is deceiving that NF1 patients with JMML account for a small percentage of the malignancy types observed in NF1 patients. This number is probably higher because JMML patients often get diagnosed with NF1 after their initial diagnosis of leukemia. This is likely due to the fact that JMML has onset very early in childhood, when most kids have not yet met diagnostic criteria for NF1 (Pinkel et al. 2008). Typically, in children without NF1, lymphoblastic leukemias predominate 4:1 over my eloid leukemias. In children with NF1, the ratio changes to 9:20 (Bader et al. 1978). This information strongly links NF1 and JMML. Another study compared leukemia patients with and without NF1 for activating N-RAS and KRAS mutations in the tumor cells (Kalra et al. 19 94). The results showed that leukemia patients with NF1 had no activating RAS mutations. However, 20% leukemias without NF1 had activating RAS mutations. Since loss of NF1 increases RAS activity, th is suggests another link between leukemia and NF1, via the RAS pathway. But mutations in the two genes are mutually exclusive. The NF1 gene, maps to chromosome 17q11.2 in humans and chromosome 11 46.06 in the mouse (NCBI). The gene is over 98 percent conserved between the two species. In humans, the genomic sequence spans over 280kb and its mRNA is >9 kb. There are three embedded genes within the NF1 genomic region called EVI2B EVI2A and OMGP All three genes are located within an intron of the NF1 gene and are transcribed in the opposite orientation to the NF1 gene. Their roles in NF1 and their specific protein functions are still under investigation. The NF1 gene also undergoes alterna tive splicing. Specifically, th ere are three exons that are alternatively spliced, exon 9a, exon 23a, and e xon 48a(Geist et al. 1996; Gutmann et al. 1999; Andersen et al. 1993; Gutmann et al. 1995). The isoforms are differentially expressed in a wide range of tissues ranging from the cerebellum to th e spleen with 9a predominantly in the brain,

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17 48a in the muscle, and 23a ubiquitous. It has yet to be determined what roles these isoforms play in the function of the NF1 gene and the disease, although it has been determined that inclusion of exon 23a reduces ras-inactivati ng activity of the protein. The NF1 protein is called neur ofibromin. Structurally, it is 2818 amino acids long and has at least two conserved domains, a RAS-GAP domain and a Sec14p-like lipid domain (NCBI). The RAS-GAP domain negatively regulates the RAS signaling pathway (NCBI). The NF1 protein acts as a tumor suppressor protein in th e sense that it functions by accelerating the hydrolysis of RAS-GTP into RA S-GDP to prevent over-proliferation of cells. The other conserved domain, Sec14p-like lipid binding domain, is found in s ecretory proteins and in lipid regulated proteins (NCBI). The ro le of this and some other prot ein domains remains unclear in the protein function. There is evidence that neurofibromin can be both in the cytoplasm and nucleus (Vanderbroucke et al. 2004). The rest of this large pr otein is highly conserved but additional functions have not been found. NF1 has proven to be somewhat complicated genetically. The mutational analysis of the NF1 gene is difficult due to the la rge size of the gene and because there are many different mutations. The NF1 consortium has categorized mu tations in 246 patients ( www.ctf.org). In the consortium study, it was found chromosomal a bnormalities were found in 1.6 percent of the patients. Deletions of the entire NF1 gene were found in 7.2 percent, whereas small intragenic deletions were found in 15.5 percent. Also, large intragenic deletions were found in 1.2 percent of the patients, whereas small insertions account ed for 11 percent of the mutations. Missense and nonsense mutations accounted for 11.8 percent and 17.6 percent, respectively. Finally, putative 3UTR mutations were found in 1.6 percent of patients, and intronic mutations affecting RNA splicing accounted for 10.2 percent. Furtherm ore, it has been shown that somatic mutation

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18 of the other allele is associated with neurofib roma, caf au lait spots, JMML, and bone dysplasia (Side et al. 1997; Coleman et al. 1995; Stevenso n et al. 2006). Virtually all mutations can be predicted to be disruptive or inactivating, and thus, reduction/loss of neurofibromin activity is an initiating event in many NF1-related phenotypes. Mouse Models of Nf1 There are no known naturally ex isting anim al models for NF 1, a hindrance to research. Two attempts were initially made to mode l NF1 in mice through knockout technology. These two labs made mouse knockouts for the Nf1 gene in different locations. One lab constructed a mouse knockout deleting exons 30 to 32 (located after the GAP related domain) (Jacks et al 1994). 250 heterozygous mice, Nf1+/Nf1n31, were followed from 7 months to 2 years of age. The other mutation was a neo gene insertion into exon 31 (Brannan et al. 1994) The results were the same in both labs: homozygous knockout mice were embryonic lethal a nd heterozygous mice didnt develop the cardinal featur es of NF1 (neurofibromas, caf au lait, or Lisch nodules). However, the mice did have an increased rate of tumorigenesis. It was found that 75 percent of the heterozygous animals died as a result of the tumors over a period of 27 months compared to 15 percent of wild type animals. Jacks et al. ( 1994) looked into a larger series of heterozygous animals (n=64) and found a variety of tumor t ypes. Lymphoma presented in 14 animals, lymphoid leukemia in 2 animals, lung carcinoma in 9 animals, hepatoma in 4 animals, and fibrosarcoma in 3 animals. The heterozygotes al so developed a few tumo rs characteristic of human NF1. One animal had an MPNST at 21 mont hs of age, and 12 heterozygotes had adrenal tumors (pheochromocytomas) at 15 to 28 months of age. Seven of their heterozygotes had chronic myeloid leukemia at 17.7 to 27 months of age. In addition, homozygous lethality occurred between days 12.5 to 14 dpc. (Jacks et al. 1994). Cami Brannans exon 31 mouse model (Nf1, Fcr) was slightly different than the previous model. Dr. Brannan observed some

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19 similarities with the Jacks mouse model, but found additional developmental defects that were not present in the Jacks mouse model (B rannan et al. 1994). The homozygous mice, NF1Fcr/Fcr, were also embryonic lethal, between 11.5 and 14.5 dpc. The Nf1Fcr/Fcr embryos were studied for developmental problems between 11.5dpc and 13.5dpc. These mice had obvious cardiac, renal, hepatic, and skeletal muscle defects, and hype rplasia of the vertebral sympathetic ganglia (Brannan et al. 1994). Molecular and Biochemical Mechanisms Underlying My eloid Leukemogenesis. Myeloid leukemia is the overexpansion of a myeloid hematopoietic precursor (blast), which reduces number and function of the other bl ood cell types. These cells can invade tissues as well in the acute form (>30% blasts in the blo od). The chronic form is <30% blasts and can be fatal in itself, or can progress to AML. Somatic genetic mutations lead to chronic leukemia and other genetic events cause its progression to acut e stage. These are very difficult to treat for patients. Functional analysis of genes isolat ed from recurring chromosomal translocation breakpoints in leukemia cells such as AML1, MLL, and HOXA9 strongly implicate aberrant transcription in leukemogenesis (Look 1997). A s econd group of specific genetic lesions (e.g., RAS and FLT3 mutations, the BCR-ABL translocation, and NF1 inactivation) undermine hematopoietic growth control by providing hyperpro liferative and survival signals (Daley GQ et al. 1990; Largaespada et al. 1996, Kelly LM et al. 2002). Mutations in both classes of genes are common in acute myeloid leukemia (AML). Ba sed on these data, Gilliland and coworkers proposed that transcription factor fusion proteins cooperate with genetic lesions that deregulate growth-promoting signaling pathwa ys in the progression to AML (Kelly et al. 2002). Whereas data from mice generally support the idea that neither type of genetic lesion is sufficient to cause AML by itself, these studies have also shown that Nf1 inactivation and other genetic lesions that result in hyperactive Ras are sufficient to induce myeloproliferative disorders (MPDs) (Le et al.

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20 2004). However, the requirement for cooperating mu tations in the pathogenesis of JMML and other human MPDs, and the mechanisms that cont ribute to progression to overt AML, are poorly characterized. Hyperactive Ras in JMML Som atic KRAS, NRAS, and HRAS mutations, which introduce amino acid substitutions at codons 12, 13, and 61, are the most common domin ant oncogenic mutations found in human cancer (Bos JL 1989; Malumbres M et al. 2003). Mutant Ras proteins accumulate in the GTPbound conformation due to defectiv e intrinsic GTPase activity and resistance to GAPs (Bos JL 1989; Vetter IR 2001; Donovan et al. 2002). NRAS and KRAS2 mutations are highly prevalent in MDS, MPD, and AML (Malumbres M et al. 2003; Boguski M et al. 199 3), are particularly common in CMML (~40% of cases) (Onida F et al. 2002), and are found in ~25% of JMMLs. (Miyauchi et al. 1994; Kalra et al. 1994) The el evated risk of JMML in children with NF1 and Noonan syndrome (NS) provide additional eviden ce that hyperactive Ras can initiate myeloid leukemogenesis. Loss of the normal parental NF1 allele occurs in JMML cells from children with NF1, which results in elevated levels of RasGTP and activation of the downstream effector extracellular signal-regulated ki nase (ERK) (Shannon KM et al. 1994; Bollag et al. 1996; Side L et al. 1997). Approximately 50% of children w ith NS demonstrate germline missense mutations in the PTPN11 gene, which encodes the SHP-2 protein tyrosine phosphatase. PTPN11 mutations are found in nearly all NS pa tients with JMML, and somatic PTPN11 mutations are detected in about 35% of sporadic JMML (Tartaglia M et al. 2003; Loh M et al 2004). Most of these mutations decrease SHP-2 phosphatase activity by destabilizing an auto-i nhibitory interaction and thereby increase signaling to Ras and other downstream effectors. Overall ~90% of JMML bone marrows have mutations in either, KRAS2, NRAS, NF1 or PTPN11 which are largely mutually exclusive.

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21 Nf1 Reconstitution Mouse Model Further research of the Nf1 knoc kout m ouse showed that when Nf1Fcr/Fcr fetal liver cells were transplanted into lethally irradiated recipient mice, to reconstitute their bone marrow, all of these mice developed a chronic myeloprolifera tive-like syndrome similar to human JMML (Bollag et al 1996, Largaespada et al 2004, Birnba um et al 2000). Approximately twenty seven percent of these mice died of complications or developed or AML. Larg aespada et al. 1996, also observed an increase in colony forma tion of hematopoetic cells response to granulocyte/macrophage-colony stimulating factor (GM-CSF) (Bollag et al 1996, Largaespada et al 2004). GM-CSF binds to receptors expressed on some myeloid lineage cells to promote their differentiation, proliferation a nd cell survival (Birnbaum et al. 2000). Flow cytometry experiments showed that these tissues had more myeloid progenitor cells as well (Largaespada et al, 1996; Bollag et al, 1996, Birnbaum et al. 2000). Identification of Epi Loci in Nf1-Related Myeloid Leukemia The discovery of the Epi2 locus em ployed the BXH-2 mouse that is a cross between the C57/BL6J and C3H/HeJ strains (Bedigian et al. 1984). In the parental strains, the incidence of leukemia is low. The high incidence of AML in B XH-2 is associated with high levels of natural expression of B-ecotropic murine leukemia viru s (MuLV) that is passed from mother to offspring in utero (Bedigian et al 1984, Blaydes et al. 2001). Nine ty five percent of BXH-2 mice die of AML by one year of age. This 8kb murine leukemia virus has a 5and a 3 long terminal repeat with gag, pol and env genes (NCBI). Thes e genes are necessary for the retroviruss life cycle so that it can synthesize more virus particles, package itself, and infect other cells (Bedigian et al 1984, Blaydes et al. 2001). Also, its important to note that these viruses are of non-T cell origin and replication competent. The BXH-2 mouse was used here as a tool to find genes that cooperate with Nf1 loss to cause Nf1-associated leukemia. To accomplish this task,

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22 the BXH-2 mouse was backcrossed to the Nf1+/Fcr mouse that was created by Dr. Cami Brannan (Brannan et al. 1994, Blaydes et al 2001). It was backcrossed fo r 3 generations and aged until the BXH2 Nf1+/Fcr mice developed leukemia. Interestingl y, fifty percent of the mice developed AML at an average earlier time than the cont rols, t=5.5 months versus t=8.5 months. Tumors were collected from all the mice and analyzed by Southern Blot analysis to test for a second genetic hit in the Nf1 locus. It was determined by Blaydes et al (2001), that 89% of the tumors had a second hit at the Nf1 locus by LOH or by viral integration into Evi2 (internal to Nf1) integration. It was then determined that each tu mor had at least 3-4 somatically acquired viral integrations (Brannan et al. 1994, Blaydes et al. 2001). Th is suggested that loss of Nf1 on the BXH-2 background was not sufficient to cause acute di sease. In fact, it is the additional acquired somatic mutations that are required for progressi on to acute disease. The actual identification of the common sites of viral inte gration (presumably at the site s of cooperating genes) was done through the use of Southern Blot analysis, a mouse mapping panel, and an ecotropic viral probe, pAKV5 (Brannan et al. 1994, Blaydes et al. 2001). After the firs t isolation with probe pAKV5, the genomic fragment was isolated and a ne w non-repetitive probe was made based on the fragment isolated from the tumor. This new prob e was used to test the whole collection of tumor samples and see if the same rearrangement was also present in another tumo r. Indeed, this was the case (Brannan et al. 1994, Blaydes et al. 2001) Dr. Brannan identified three new common sites of viral integration, termed Epi1 Epi2 and Epi3 These names were abbreviated based on the original name, ecotropic provir al integration site X where X denotes the number of the site. In order to map the location of the viral in tegration on the mouse genome, a mapping mouse panel was used in collaboration with Jenkins and Copelands lab at th e NCI (Blaydes et al. 2001). Once the three common sites of viral integr ation were localized, subsequent experiments

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23 were focused on elucidating how these sites of common proviral integra tion cooperate with loss of Nf1 to cause AML (Brannan et al. 1994, Blaydes et al. 2001). My dissertation work focused on the locus termed Epi2 This site was found in 2 independent tumors (33T, 419T) out of 67 in the Brannan series, and was also seen twice in the Copeland and Jenkins La b. In all four cases, the virus was inserted into Mrps18b in intron 1 in the same orie ntation (Walrath et al., in preparation). Viral Insertional Mutagenesis The integ ration of the virus can theoretical ly have many effects on that region of the genome. One mechanism is enhancer insertion, wher e the virus integrates either at the 3 or 5 end of a gene (McCormick et. al. 2005) and the viral enhancer causes improved efficiency of the native promoter. Another mechanism is activati on by enhancement where the virus integrates in the 3UTR. This is thought to increase the st ability of the transitory mRNA (McCormick et. al. 2005). A third mechanism is promoter inserti on, which occurs when the virus integrates upstream or within the 5 domain of the host target gene with the same tr anscriptional orientation as the target gene. This causes elevated l ong terminal repeat constitu tive gene activation and transcription (McCormick et al. 2005). This and the former mechanisms would cause overexpression of the endogenous gene along the lin es of oncogenes. The next mechanism is protein truncation by transcription termination/promoter insertion where th e virus integrates within the coding region of the gene. This may result in the inactivation of the gene. The last mechanism is also protein truncation by transcri ption termination at a cryptic poly A site (McCormick et al 2005). This is thought to produce aberrant protein products exhibiting anomalous biological function. In the Epi2 lo cus the virus intergra ted into intron 1 of Mrps18b and in the same transcriptional orientation of Mrps18b. This could cause promoter insertion, where one gets constitutive gene activation and transcription (McCormick et al. 2005). Another

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24 possible theory is protein trunc ation by transcription terminati on/promoter insertion since the integration is near the begi nning of the coding region of Mrps18b/Ppp1r10 MRPS18B Mrps18b, which encodes a m itochondrial ribosomal protein, is located on chromosome 17 B3 in mouse and chromosome 6p21.3 in humans. In mice and humans, the gene has 7 exons and a 5 as well as 3 UTR. The mouse full length mRNA is 1073 bp (NM_025878) and the human mRNA is 1439 bp (NM_014046). The mouse protein is 254 amino acids (NP_080154), and in humans it is 258 amino acids (NP_05765). This is one of a family of mitochondrial ribosomal proteins encoded by nuclear ge nes, which aid in protein s ynthesis in the mitochondria. Mitochondrial ribosomes consist of a 5, 18 and 28S subunits. Mrsp18b is one of three genes that encode the 28S specific ribosome proteins (N CBI). MRPS18B has only one conserved domain (encoded in exon 2), which is the ribosomal pr otein domain. However, it is thought that exon 1 may contain sequence that localizes the pr otein to the mitochondrial ribosome. If MRPS18B were disrupted, then the mitochondrial ribosome, might function aberrantly, affecting energy metablosim and apoptosis. Less than one kb upstream of Mrps18b, and in the opposite transcriptional orientation is the gene Ppp1r10. Because of its proximity, the gene was also a candidate to be involved in NF1 leukemogenesis. PPP1R10 PPP1R10 consists of 20 exons in hum ans and 19 exons in mice. The mRNA is 4203bp in mouse (NM_175934) and 4504bp in humans (NM_002714), although it is not clear that the absolute transcription start has been identified. Ppp1r10 in the mouse encodes a protein that contains 874 amino acids (NM_175934), with the human protein being 940 amino acids long (NP_787948). Also, the Ppp1r10 protein is 89 percent conserved between mice and humans, and Mrps18b is 86 percent conserved (NCB I). It is also important to note that neither gene has yet

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25 been implicated in any human disease. PPP1 R10, whose protein product is PNUTS, has 3 conserved domains. One is in the N terminal region, a TFIIS site, which is located in exon 5, 6, and 7. Its function in PPP1R10 ac tivity is unknown. However, TF IIS is thought to function in eukaryotic transcription elongati on by suppressing transient pausing of the RNA polymerase. The second conserved domain is a PP1-binding do main encoded in exons 13 and 14. This region of the protein binds to PP1, an important seri ne-threonine phosphatase (see below). The rat PPP1R10 homolog has been shown to have an inhibitory effect on PP1, and co-localizes with PP1 at distinct phases during m itosis (NCBI, Allen et. al. 1998, Wa tanabe et. al. 2001, Kim et. al. 2003, Udho et. al. 2002). The third conserved domain is a C3H1 zinc finger encoded by exon 19, which suggests that PPP1R10 may affect transcrip tion of target genes. In addition, recent research has shown that PPP1R10 might be involved in regulati ng cell death due to hypoxia or cell stress (Lee et al. 2007). PP1, a Serine/Threonine Protein Phosphatase One third of all eukaryotic proteins are cont rolled by serine/threonine phosphatases, which affect protein activ ity and localization (Ceulemans et al. 2004). Protein P hosphatase type 1(PP1), 35-38kDa, is a serine/threonine phosphatase that contributes to many important cellular functions (Ceulemans et al. 2004, Aggen et al. 2000, Klumpp et al. 2002, Ludlow et. al. 1995): cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription, and neuronal signaling. Most protein phosphorylations are reversible and there is a balance between the phosphatases and the kinases. This phospha tase exists in many isoforms: PP1alpha, PP1beta/delta, as well as tw o from splice variants, PP1y1 and PP1y2. The protein isoforms are conserved in the central three fourths of the protein, however the amino and carboxy termini are the most divergent.

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26 As reviewed by Ceulemans et al (2004), PP1s catal ytic subunit does no t exist freely in the cell and requires a regulatory subunit to determine specificit y, sub-cellular localization, and regulation of the phosphatase. These regulators are important in bringing PP1 into close proximity to its substrate so that the phospha tase can be anchored in specific cellular compartments via the targeting motifs of the re gulators. The regulators, of which there are dozens, are considered either primary or seconda ry based on whether the protein has or lacks a PP1 binding site, respectively. So me primary regulators include Inhibitor-1, Inhibitor-2, NIPP-1, PPP1R10, and SDS22. Regulatory proteins interact with PP1 via short, degenerate sequence motifs of 4-6 residues, the PP1 binding sites. Mo st of these proteins have multiple points of interaction with PP1 and some of them can share PP1 interactions sites as well. It has been theorized that PP1 is subject to a combinatoria l control that relies on competition of different regulators for a combination of inte raction sites. This allows th e formation of large variety of holoenzymes with distinct specif ic activities and substrate activities. There are also secondary PP1 binding sites that can serve as anchor site s to promote the coopera tive binding of secondary sites with lower affinity. These secondary PP1 bi nding sites are called RVXF sites. Most of the regulators contain an RVXF motif, although the binding is not associated with any conformational changes or eff ects on catalytic activity. PP1 has many cellular functions important to this project, such as in the cell cycle. First, PP1 interacts with AURORA-B to ensure that cytokinesis works properl y (Andrews et al 2000; Sugiyam et al. 2002). Second, PP1 is involved in preventing centrosomes from splitting before G2/M phase (Eto et al. 2002). Third, PP1 contri butes to reassembly of the nuclear envelope at the end of mitosis by dephosphorylating Lamini n-B (Thompson et al. 2002). Fourth, PP1 dephosphorylates Bcl-2, an anti-apo ptotic protein that is an inte gral membrane protein in the

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27 endoplasmic reticulum and the mitochondria. The dephosphorylation of Bcl-2 by PP1 targets Bcl-2 for proteosome mediated degradation (Brichese et al. 2002) Finally, PP1 has been shown to dephosphorylate the retinoblast oma protein (pRB) in late M phase (Nelson et al. 1997). My project focused on understanding whether or how PPP1R10 is involved in myeloid leukemia, NF-related or otherwise. I took 3 approaches: (1) mutation analysis of PPP1R10 in human myeloid dysplasia/leukemia samples+/-NF 1 involvement, to look for oncogenic or lossof-function mutations (Chapter 2); (2) in vitro anal ysis of mouse fetal live r cells to test whether overexpression of PPP1R10 was tumorigenic (to te st oncogene hypothesis) (Chapter 3); and (3) characterize a Ppp1r10 knockout mouse to test the tumor suppressor hypothesis, including the cross of this mouse to the Nf 1 knockout mouse (Chapter 4)

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28 CHAPTER 2 MUTATIONAL ANALYSIS OF PPP1R10 IN LEUKEMI A Introduction JMML is ch aracterized by splenomegaly, leukocytosis, hypersensitivity to granulocyte/macrophage stimulating factor, and absence of the Philadelphia chromosome in tumor cells (reference diagnostic paper). There ha ve been several genes implicated as a primary step in JMML: NF1 the RAS gene family, and PTPN11 (a tyrosine kinase phosphatase)(Side et al., 1997; Side et al., 1998; Tartaglia et al., 2003). Mutations in these genes are mutually exclusive initiating gene tic events, and together account fo r ~85% of genetically predisposed JMML. All of these gene mutations lead to an increase in Ras signaling. The Epi2 mouse model implicates Ppp1r10 as a possible oncogene or tumor suppressor. Since ther e is so little material from the two mouse Epi2 tumors, we chose to screen human myeloid malignancies for further evidence that PPP1R10 might be a leukemia gene. Identification of any PPP1R10 mutations in human myeloid leukemia samples may provide insight about whet her (and how) this gene is involved in NF1 associated leuk emia and/or JMML, myeloprolif erative disorder (MPD), or AML. To test the hypothesis that PPP1R10 might be genetically altered, we performed mutational analysis in NF1-JMML, JMML, and AML tumor samples. We examined the entire gene and found loss of heterozygosity in intron 15 in a subset of tumor samples by sequence analysis and RFLP analysis. Additional analysis of the sequenced region revealed a putative stop codon mutation in exon 6 of PPP1R10 in 2 out 40 leukemia samples. Real-time mRNA quantification PCR was also performed on a subset of leukemia samples, which showed a decrease in expression of PPP1R10 in some samples compared to a GAPDH control.

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29 Materials and Methods Patient Samples DNA and RNA were extracted from tumor ce lls using standard methods (Thomson and Wallace et al 2002). Most leukemia samples (bone marrow or blood cells) were obtained from the UF tissue bank and were > 35% blasts. Tumo r and germ-line samples were obtained with IRB approval from the UF Tissue Bank (MDS (n =7), AML (n=54), CML (n=6)) and UCSF (courtesy of Dr. Kevin Shannon, (NF1-JMML (n=50), JMML (n=25), CMML (n=50)). Polymerase Chain Reaction and Sequencing of PPP1R10 Exons Tum or DNA was PCR-amplified in a reaction mixture containing 100ng for each forward and reverse primer, 200umol dNTP s (Invitrogen), 0.5U Hotstart Taq Polymerase (Qiagen), and 1X Roche PCR buffer. See Appendix A for a lis t of primer sets and PCR product size for PPP1R10 which were designed using PRIMER3 (h ttp://fokker.wi.mit.edu/primer3/input.htm). The following cycling conditions were used: 1 cycle of 95 C for 15 minutes, 35 cycles of 95 C for 15 seconds, 60 C for 30 seconds, 72 C 1 minute; a final extension of 72C for 10 minutes. All PCR products were examined on ethidium brom ide stained 1.2 percent agarose gels to verify quality and quantity of PCR product. PCR produc ts were purified using Exosapit (Amersham Pharmacia) and 5.8ul were used in the sequencing reaction: 2ul of 5X Se quencing Buffer, 2ul of Big Dye 3 (ABI), and 20ng/ul of forward or re verse primers. The following cycle sequencing conditions were used: 25 cycles of 95 C for 1 minute, 50C for 30 seconds, and 60 C for 4 minutes. Sequencing reactions were run on a AB I 3130XL Genetics Analyzer at the UF Center for Epigenetics. Sequence data were analy zed using Sequencher software (Gene Codes). Restriction Fragment Length Polymorphism for Intron 15 Loss of Heteroz ygosity Sequence analysis uncove red a polymorphism in PPP1R10 intron 15 close to exon 16 and was used for a loss of heterozygosity analysis. Loss of heterozygosity of a polymorphism can be

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30 used to test for deletions. The heterozygosity frequency in this A/G polymorphism (rs126443) was reported to be .478 in Caucasians and conf irmation of this was done with normal patient DNA through RFLP. P115RsaF and P115RsaR primers (see Appendix A) were used to amplify the intron 15 polymorphism and create an RsaI site to distinguish the allele s. With RsaI digest, the G allele causes the 138bp to be cleaved into 26bp and 112bp fragments (two bands), where as, the A allele does not cut with Rsa1 digest (o ne band). The digest was run on an 8% native gel with ethidium bromide for visua lization. Visualization of the RFLP on an acrylamide gel allows for comparison of allele signal in tensities. A series of germline he terozygous DNAs were used to establish relative allele intensity, and tumor samp les were compared to th ese DNAs. If there is a deletion in the gene of interest in the tumor th ere will be a reduction in intensity of one allele relative to the other as compared to the germlin e genotypes. LOH analysis was also performed at an SNP in the shared PPP1R10/MRPS18B promoter (primers Appendix 1), which used an Ear 1 digest. Real-Time mRNA Quantification Tu mor cells from leukemia patients were used in the real-time qua ntification of PPP1R10 expression. Tumor total RNA was se rially diluted from 400ng to 25ng and subsequently used in a RT-PCR reaction. For the reverse transcript ion: 200uM of dNTPs (Invitrogen), 50ng random hexamer (Invitrogen), and 5ul of water was added to each dilution of tumor RNA. This mixture was heated at 65 C for 10min. and then placed on ice. Next, 4ul of 5X buffer, 2ul of .1M DTT, 40 units of RNase Inhibitor (Inv itrogen), and 200units Superscrip t II (Invitrogen) was added to each of the tubes on ice. Thes e tubes were heated at 25 C for 10 min and 50 C for 40 min to produce cDNA. For the mRNA quantification, 1ul of cDNA at each concentration was placed in PCR tubes (Biorad cat. #TLS0851, TCS-0803), follo wed by the addition of 10ul of Master mix

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31 (Biorad, contains hotstart version of a modi fied Tbr DNA polymerase, SYBR Green 1, optimized PCR buffer, 5mM MgCl2, dNTP mix including (dUTP)), 100ng of forward primer, 100ng of reverse primer, and 8ul of water. These reactio ns were then analyzed on a Opticon Moniter II machine at the Center for Ep igenetics: 95C for 15 min, 94 C for 15sec, 55C for 30sec, Plate Read, 72 C for 1min, Go to Step 2 for 30 cycles, Melting Curve 50 C to 90 C, Read ever 1sec, Hold for 10sec, 72 C for 15min. The Ct number for each PPP1R10 tube was compared to the Ct of the GAPDH gene results thorough the Pfaffl met hod formula (Appendix B). Primers chosen for real-time quantification lay in different exons to avoid DNA-PCR contamination and GAPDH was used as a housekeeping/normalizing gene (Appendix A). Results A set of 50 NF1-JMML and 25 JMML (non-NF1) m yeloid leukemia samples were used in the DNA analysis. A polymomorphism in the promoter region (rs16867845) displayed no loss of heterozygosity and had no mutations (Figure 2-1). A putative nonsense mutation was also uncovered in two separate NF1-JMML samples (Fi gure 2-2). This substitution in exon 6 encodes a serine->stop at codon 125. A putative missense was also uncovered in exon 6, serine>threonine change at codon 125 (Figure 2-3) Another polymorphism in exon 19 (rs11754215) also did not display loss of heterozygosity in any samples (Figure 2-4). The intron 15 SNP (rs1264423) did reveal potential lo ss of heterozygosity by sequence analysis in 6/44 NF1-JMML samples (Figure 2-5). To confirm this result by another method, a PCR-base d forced RsaI digest system was constructed for the polymorphism. The sequence positive NF1-JMML samples were re-amplified with the new primers, digested by RsaI and the products were visualized by native polyacrylamide gel electrophoresis The RsaI digest creates 2 fragments if the patient is heterozygous (AG) and 1 fragment if the patie nt is homozygous (GG or AA). Figure 2-6, shows

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32 the RsaI digest normal germline DNA displaying all three genotypes. Figures 2-7 and 2-8 show digests of some tumor samples of NF1-JMML and JMML displaying loss of heterozygosity. More NF1-JMML and JMML samples were tested with this assay and it was found that 6/44 NF1-JMML and 11/20 JMML samples displayed loss of heterozygosity at the intron 15 polymorphism (Table 2-1 and Table 2-2). Real time mRNA quantification on a set of leuke mia samples also revealed a decrease in PPP1R10 mRNA expression. The Ct values from th e tumor RNA and from normal lymphocytes for PPP1R10 and control GAPDH were inputed into the Pfaffl method formula (Pfaffl et al. 2001). This formula can calculate the fold expression change of PPP1R10 in the tumor cells compared to GAPDH using a standard curve method. Table 2-3 shows the fold change of PPP1R10 to GAPDH is reduced in the human leukemia sample s. Interestingly, th ere is a variable reduction between samples, and different leukemia types (including AMLs). Discussion The hum an genetic data support the notion that PPP1R10 is a tumor suppressor gene associated with some NF1-JMMLs, JMMLs, a nd AMLs since the mutations are inactivating (deletion, stop mutation) and the mRNA expression is reduced in tumor cells. The presence of the PPP1R10 LOH implicates this gene in non-NF1-re lated leukemia suggests that this LOH is not specific to NF1-related leukemia. PPP1R10 is an attractive tumor candidate gene because of its regulation of PP1, which regulates at least 70 mammalian genes. Loss of one allele of PPP1R10 would decrease inhibition of PP1 and may affect the ability of PP1 to regul ate its target genes. However, it is still unknown which target genes PP1 regulates as a result of PPP1R10 binding especially in the hematopoietic system. Traditionally, tumor suppressors have 2 hits. Neither of the two tumors with point changes showed LOH, however, we do not yet have evidence for the 2-hit phenomenon. Further

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33 evidence from real-time data on one mouse Epi-2 tumor revealed a reduction in PPP1R10 mRNA expression in comparison to the other tumors without Epi2 viral integration (Walrath, 2005). However, the mRNA reduction was not charac teristic of a classic 2-hit mechanism. Recently, several papers have reported that coop erating haploinsufficient tumor suppressors in the mouse can be sufficient reduction in prot ein function to contribu te to tumorigenesis (Kamimura et al. 2007; Vives et al. 2006; Moreno-Miralles et al 2005; Ma et al. 2005). Thus, the data support a functional contribution by decr eased PPP1R10 activity but not necessarily in a classic tumor suppressor mechanism. Figure 2-1. Human NF1-JMML analysis PPP1R10, Promoter Polymorphism (rs16867845). Figure 2-2. Human NF1-JMML analysis, PPP1R10 Exon 6 nonsense, S125X. Figure 2-3. Human NF1-JMML analys is, PPP1r10 Exon 6 missense, S125T. Figure 2-4. Human NF1-JMML analysis PPP1R10 Exon 19 polymorphism (rs11754215).

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34 Figure 2-5. Human NF1-JMML PPP1R10 anal ysis, Intron 15 polymorphism loss of heterozygosity.

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35 Figure 2-6. RsaI Digest of Intr on 15 polymorphism of Control DNAs.

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36 Figure 2-7. RsaI Intron 15 Polymor phism of NF1-JMML and Control DNA.

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37 Figure 2-8. RsaI Intron 15 Polymorphim Digest of JMML and an AML sample.

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38 Table 2-1. NF1-JMML human patient samp les and Intron 15 polymorphism LOH. Sample Name Diagnosis Mo. 7 LOH/NF1 Mutation PTPN11done? Intron15 poly LOH1J, 32J JMML yes yes no 2J JMML no Failed IVTTno pLOH AG 18J JMML yes yes no 10L JMML no Normal IVTTyes 29J JMML yes yes yes pLOH AG 29J EBV 26J JMML yes yes yes AG 28J JMML no Failed IVTTyes 27J JMML no Normal IVTTyes GG 61L JMML no Failed IVTTyes 35J 35L JMML no Normal IVTTno AG 33J JMML no Failed IVTTyes 34J JMML prob. Failed IVTTno pLOH AG 33L JMML no Failed IVTTyes pLOH AG 37J JMML yes Normal IVTTyes 45J JMML no Failed IVTTyes 46J JMML no Failed IVTTyes GG 47J JMML yes Failed IVTTyes AG 49J JMML yes yes no 57J JMML no yes no GG 60J JMML no Normal IVTTyes AG 35J JMML prob. no rna yes 51J JMML no yes yes AG 56J JMML yes yes no AG 58J JMML yes Normal IVTTyes 58J EBV 89L JMML yes yes no AA 75J JMML no not tested no 61J JMML no Normal IVTTno 71J JMML no Normal IVTTyes 74J JMML yes Normal IVTTyes 77J JMML no GG 83J JMML no 91L JMML no GG 88J JMML no pLOH AG 63J JMML yes GG HM695 HM725 HM170 HM756 HM853 HM189 HM520 HM549 HM654 HM678 HM609 HM546 JMML neg AA HM552 JMML AA HM553 AA HM605 JMML HM676 pLOH AG HM571 JMML pos. AG HM558 pLOH AG HM660 JMML HM548 JMML pos. HM263 pos. HM264 pos. 19L pos. 20L pos. 17L pos. AG 18L pos. 22L pos. AG 1100 AG 1098 AA 1099 GG 1102 1101 1238 GG 1239 1240 AG 1241 AG 1242 AG 1243 AG 1244 AG 1245 AG 27F AG 28F

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39 Table 2-2. JMML human samples and Intron 15 polymorphism LOH. Patient numberIntron 15 poly. Pcr 99LpLOH AG 80L 60LGG 23LAG 102FpLOH AG HM262AG 98L HM1286pLOH AG 101FGG HM896 HM910pLOH AG HM933AG HM1224GG HM920pLOH AG HM1236 HM773pLOH AG HM765pLOH AG HM1198 HM1158AG 342GG PuentepLOH AG Schultz Mackley Booth AlexanderpLOH AG JacksonGG Buck GillianpLOH AG ThongpLOH AG Gonclaves

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40 Table 2-3. mRNA Quantificati on of human leukemia RNA.

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41 CHAPTER 3 IN VITRO ANALYSIS OF PPP1R1 0 AS A POTENTIAL ONCOGENE Introduction In vitro studies have been useful in elucidating the role of cancer ge nes and their signaling pathways. Sim ilarly, in vitro studies of Ppp1r10 might elucidate its role, if any, in the signaling pathways that contribute to cancer. One proposed mechanism for its role in AML was as an oncogene, based on the viral Epi-2 integration upstream that c ould be inducing inappropriate transcription. To test this theory, over-expression of Ppp1r10 was studied in COS7 cells (from African Green Monkey kidney), which were analyzed for altered expression of proteins involved in classic signaling cascad es. In addition, I studied Ppp1r10 over-expression in wild-type mouse fetal liver cells in a colony formation assay using (CFU-GM, specific for the granulocyte/macrophage lineage of the hematopoiet ic system). These experiments are detailed below. This work led to the conclusion that PPP1R10 is likely not an oncogene. Materials and Methods Cloning of Ppp1r10 in the MSCV Viral Vecto rs A plasmid containing the fulllength mouse cDNA, pXY-Ppp1r 10 (Operon), was digested with EcoRI and NotI restric tion enzymes to remove the cDNA from the pXY plasmid. The digested product was ligated into EcoRI/NotI di gested pENT4 plasmid (ent ry vector, Invitrogen). The modified destination vector, pMIG, was provided by the Dr. Kevin Shannon (UCSF) and was already modified to contain an MSCV promoter, multiple cloning site, att sites, and a GFP transgene. Gateway BP Clonase II enzyme mi x (Invitrogen) was used to transfer the Ppp1r10 cDNA from pENT4 into the pMIG-GFP vector. The final vector was called pMIG-PNUTS and DNA sequencing verified correct reading frame and sequence.

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42 Hematopoietic Cell Isolation and Retroviral Transduction Pregnant wildtype C57BL/6 females were humanely killed by CO2 inhalation at post coitus day 14.5 (E14.5) and fetal liver cell s were sterily isolat ed and prepared as described (Birnbaum et al. 2000). The fetal liver cells were cultured in a stimul ation medium containing StemSpan SFEM (StemCell Technologies, Vancouver, BC, Canada), 15% FBS, 100 ng/mL stem cell factor (SCF; Peprotech, Rocky Hill, NJ), 50 ng/mL FLT-3 ligand (Pepro tech), and 100 ng/mL IL-11 (R&D Systems, Minneapolis, MN) to promote hema topoietic cell lineage growth. Bone marrow cells were cultured in a stimulation medium containing StemSpan SFEM, 15% FBS, 100 ng/mL SCF, 50 ng/mL IL-6, and 10 ng/mL IL-3 (both from Peprotech). Phoenix cells, used to produce MSCV based viruses were co-transfected with empty vector (pMIG) or WT (pMIG-PNUTS) plasmids along with plas mids encoding retroviral gag-pol and env proteins using Lipofectamine2000 (Invitrogen, Carlsbad, CA), to package and excrete the final viral vectors (PNUTS and empty vector). The supernatants from transfected cells, containing the viruses, were harvested 24 hours post-infection, and used to tra nduce the murine fetal liver cells for 1.5 hours. Colony Formation Unit Granulocyte/Macrophage Colony Assay After transduction, fetal liver cells w ere sorted based on GFP expression by FACSVantage SE flow cytometer (BD Biosciences, San Jose, CA). GFP-positive fetal liver cells (PNUTS, and vector control) were seeded on me thylcellulose medium (M3231; StemCell Technologies) containing escalating doses (0.1ng.ml, 1ng/ml and 10ng/ ml) of GM-CSF (Pepro tech), to test for ability to form colonies in this media, a sign of an oncogene. Gra nulocyte-Macrophage colony forming units were scored by indirect micros copy on day 7. Images were acquired using a Nikon Coolpix 5000 camera (Torrance, CA).

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43 Serum Starvation of COS7 Cells To test the effect of PNUTS expression on classic oncogene signaling pathways, 10ug of pDEST-PNUTS were transfected in to two million COS7 cells (Schubbert et al 2005). Samples were taken at specific time points after serum starvation media (DMEM, 0.01% Fetal calf serum, 1X glutamine, 1X Pen-Strept) was placed on the cells. The 0 time point was taken before addition of the starvation media. Standard protein lysates were made from all cells at all time points (0, 24, and 48 hours) for Western blot an alysis. The negative cont rol was untransfected cells. Western Blots Ten m illion cells from each time point were lysed in 1% NP-40 Lysis buffer (1% NP-40, 30mM NaF, 30mM b-glycerophosphate, 20mM sodium pyrophosphate, 1mM sodium orthovanadate, and a cocktail of protease inhibitors (phenylmethylsulfonylfloride, benzamidine, leupeptin)), and total protein was quantified (Lowry assay). Each protein sample (40ug) was loaded onto Nupage Novex precast Midi gels (Bis-tris, SDS, MOPS denaturing polyacrylamide gels) and electrophoresed at 175V for 1 hour in ma nufacturers buffer. Proteins were electrotransferred onto nitrocellulose membranes (N ovex) for 1hour and 30 minutes at 300 mAmp, as recommended by manufacturer, and blocked ove rnight with 5% BSA. Primary (rabbit) antibodies [(p-mTor (1:750, Cell Signaling), p-PK Ca/b (1:600, Cell Signaling), p-Stat3 (1:1000, Cell Signaling), p-Pten (1:500, Invitrogen), p-Akt, p-Erk1/2( 1:1200, Cell Signaling), pMek1/2 (1:750, Cell Signaling), b-actin (1:750, Cell Signaling), and PNUTS (1:1000, Zymed)] were incubated at the recommended concentration with membranes in 5% BSA for 1 hour and 15 minutes at room temperature. The membranes we re washed twice for 6 minutes each with TBSTween (0.05%) before addition of the s econdary antibody. The secondary antibody, HRPconjugated anti-rabbit (1:2000), was incubated wi th the membranes for 50 minutes and washed

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44 3X for 6 minutes each with TBS-Tween (0.05%). Treatment of membranes with luminescent ECL reagent (Pierce, 1:1) allowed proteins to be visualized by au toradiography (Kodak X-ray film). Results Three rep licates each of the fetal liver cells transduced with MSCV-pMIG and MSCVpMIG-PNUTS viruses were plat ed on methylcellulose with in creasing amounts of GM-CSF, the the ligand that stimulates the granulocyt e/macrophage lineage of the hematopoietic compartment. The resulting colonies, at day 7, were scored for size, morphology, and number in the CFU-GM assay in order to observe alteratio ns in granulocyte/macrophage lineage (the pathway affected in JMML). There were no st atistically significant differences between the empty vector and PNUTS vector (Figure 3-1, 3-2, and 3-4). In the other assay, serum-starved COS7 cells transfected with pD EST-PNUTS at 0, 6, and 12 hour time points were probed for amounts of phosphorylated versions of key signaling molecules: p-mTor, p-PKCa/b, p-Stat3, p-Pten, p-Akt, p-Erk1 /2, p-Mek1/2, b-actin (loading control), and PNUTS (Figure 3-4). Levels of p-Stat3, p-Pten, and p-Erk1/2 were visibly increased at the 12 hour ti me point, suggesting that Ppp1r10 over-expression increases the signaling in these pathways compared to the untransfected COS7 cells. Discussion If Ppp1r10 is a classic oncogene involved in leukem ia, then the CFU-GM colony assay should detect an increase in the size, change in morphology or increase in the number of colonies. Ppp1r10 over-expression in Nf1+/+ fetal liver cells showed no difference in any these measures between Ppp1r10 and the empty vector control. This suggests that, by itself, Ppp1r10 is not an oncogene involved in leukemia. Either other cooperating genes are needed for cytokine independent growth, or Ppp1r10 is a tumor suppressor gene instead (or, Ppp1r10 is not involved

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45 in leukemia at all). However, Le et al. 2004, showed that conditionally inactivated Nf1 fetal liver cells are sensitive to low GM-CSF, have an al tered morphology, and an increased number of colonies compared to Nf1+/+ fetal liver cells. This, confirmed Nf1 as a tumor suppressor in myeloid leukemia. A similar assay could be used to test knockdown of Ppp1r10 (see Chapter 5). Western analysis of phosphorylat ed proteins allows correlati on with actively proliferating cells. In serum starved COS7 cells, Western anal ysis showed a possible alteration in the MAPK pathway in PNUTS-positive cells. Specifically, p-ME K decreased at 6 hours and then increased at 12 hours post starvation. In addition, p-Erk1/2 was also increased 12 hours post starvation, suggesting that Ppp1r10 may contribute to the regulation of the Ras->Mek->Erk pathway in COS7 cells. Specifically, increased the signaling in these pathways is positively association with cell proliferation and cell surviv al. However, these results are weak compared to classic oncogenes such as mutant H, K, or NRAS. For example, Schubbert et al. (2006) showed that germline KRAS mutants associat ed with Costello Syndrome a nd JMML, V14I and T58I, were defective in intrinsic GTP hydr olysis and displayed impaired responsiveness to GTPase activating proteins. This impaired GTP hydrolys is rendered primary hematopoietic progenitors very hypersensitive to growth factors, and deregulated signal transduction in a cell lineage specific manner. These mutants, along with clas sic others such as codon 12, induce tumorigenic properties in the assays above. These results indicate that Ppp1r10 is not a classic oncogene in itself. It could however, be a tumor suppressor gene or cooperate with other genes (as a moderate oncogene or a tumor suppressor) to cause cancer or le ukemogenesis. The serum starva tion experiment showed that Ppp1r10 could be involved in the Ras-Mek-Erk pathway, or possibly Stat3 or Pten pathways.

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46 The CFU-GM experiment definitively showed that over-expression of Ppp1r10 did not alter the size, morphology or colony number as seen in other oncogenes like NRASG12D. CFU-GM Colony Assay0 20 40 60 80 100 120 140 00.1110 GMCSF (ng/ml) WT Ppp1r10 Vector only Figure 3-1. CFU-GM Colony assa y of pMIG-PNUTS and pMIG in wild-type fetal liver cells.

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47 Figure 3-2. Experimental Desi gn of Colony Formation Assay.

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48 Figure 3-3. CFU-GM of wt PNUTS versus empty vector control.

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49 Figure 3-4. Transfection of pD EST-PNUTS in COS7 cells with serum starvation and probing signaling cascades.

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50 CHAPTER 4 PPP1R10 COOPERATES WITH NF1 INACTIVATION TO CAUSE ACUTE MYELOMONOCYTIC LEUKEMIA IN A MOUSE MODEL Introduction A m ouse Nf1 knockout ( Nf1 KO +/-) created in 1994 disp lays no characteristic NF1 features, but 11% of mice die of myelodysplasia by 17.7 to 27 m onths (Jacks et al., 1994). Largaespada et al. (1996) isolated 12.5dpc fetal liver cells from another, similar knockout mouse (Brannan et al., 1994), specifically the Nf1 fcr /fcr (embryonic lethal 13.5dpc) and transplanted them into lethally irradiated mice. Upon bone marrow reconstitution by these donor cells, the mice developed a JMML-like phenotype, and thei r bone marrow cells were hypersensitive to granulocyte/macrophage stimulating factor, similar to that seen in JMML patients. It was then hypothesized that additional somatic cooperating mutations were required for progression to more acute disease. Thus, loss of the remaining NF1 allele is required for a child with NF1 to develop JMML (Largaespada et al., 1996; Bollag et al., 1996), but additional somatic mutations in other genes are necessary for progression to AML. Since in vitro studies and previous research suggested that Ppp1r10 was not an oncogene, I hypothesized that Ppp1r10 could be a tumor suppressor whose loss cooperates with Nf1 inactivation in AML. A previous student, Jessica Walrath, made a Ppp1r10+/mouse on the 129S2 background, utilizing the Gene Trap Consortium library of ES cells (Walrath, 2006). The gene trap technology inserts a LacZ gene, which, through splicing, creates a null allele in the host gene. The Consortium mapped the locations of the trapped genes and made these available for a nominal fee. Jessica found that homozygosity for Ppp1r10 knockout was a preimplantation lethal genotype. I crossed Ppp1r10+/mice to Nf1+/mice, and aged double heterozygotes for phenotype. I found that these mice become ill and died betwee n 14 and 19 months, compared to 17-27 months

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51 seen in Nf1+/mice. The Ppp1r10+/Nf1+/mice developed acute myelomonocytic leukemia (AML-M4) versus the Nf1+/mice, which developed the more chronic leukemia/JMML-like phenotype. The Ppp1r10+/mice were also aged for phenotype and these mice developed a variety of single occurring solid tumors and became ill by 24 months of age. In addition to solid tumors, the majority developed splenomegaly and acute undifferentia ted leukemia (AML-M0). This data support the hypothesis Ppp1r10 cooperates with Nf1 inactivation to cause AML, and can cause a slightly different acute leukemi a by itself with a high penetrance but extended latency. Materials and Methods Ppp1r10+/Mouse Construction The Ppp1r10+/m ouse was constructed by a previ ous student and described elsewhere (Walrath, 2006). Breeding Ppp1r10 Mice 129S2 Ppp1r10+/m ice were crossed to 129S1 Nf1+/mice and the F1s were aged for phenotype. There were 21 Ppp1r10+/-, Nf1+/(twelve females, nine males) mice aged for phenotype as well as 10 129S2 Ppp1r10+/animals, and 5 129S1 Nf1+/animals. Ppp1r10+/mice were also bred for ten generations to move the knockout allele onto the 129S1 and C57BL/6 backgrounds. Pathology/Sectioning/H and E Stain Mice were aged and observed for abnorm al ga it, hunching, lack of gr ooming, and or other signs of illness. Upon observation of illness, the mice were euthanized. Full necropsies were performed on the mice, and fixed paraffin-embedded sections were made from each tissue. These sections were made by the UF Molecular Pathology core and subsequently stained with Hematoxylin and Eosin.

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52 Blood Smears and Manual Counts At the tim e of observed illness, and euthan asia, five hundred microliters of blood from each mouse were used for standard blood smear analysis. The remaining blood was used to make DNA. Blood smear slides were stained with Th e Wright Geimsa stain kit (Fisher). Manual cytology counts were performed on blood smears slid es from each mouse, counting 200 cells per field of view. In some cases, younger healthy mi ce had blood samples taken without euthanasia and analyzed by the UF Animal Care Services la boratory for complete blood count. This gave us an indication of whether the white blood cell count was increasing, well prior to death. Sudan Black Stain and MPO Stain Bone m arrow sections were stained with Suda n Black kit for detection of myeloid cells. Paraffin embedded were de-paraffi nized, hydrated with water and then stained with the Sudan Black Staining Kit (Sigma). B one Marrow sections were immunostained using a primary mouse monoclonal for Myeloperoxidase (ABCAM mo use monoclonal 16686-50) and a secondary antibody, biotinylated horse anti-mouse IgG made in horse (Vector BA-2001). To deparaffinize/hydrate paraffin sections, sections were heated in a 60 degree Celsius oven for 15 minutes. Then, the sections were placed in xyl ene twice for 2 minutes each time, 100% ethanol for 2 minutes, 95% ethanol for 2 minutes, tw o washes in 70% ethanol for 2 minutes, H20 for 1 minute, and PBS for 5 minutes. Secti ons were then incubated in 0.3% H2O2 for 5 minutes. Before adding the primary antibody, the sections were incubated in 10% horse serum for 60 minutes (100ul NHS/1000ul PBS). The excess serum wa s blotted off and then the sections were incubated overnight with the primary antibody, MP O mouse mAB at 1:100 at 4 degrees Celsius. Before addition of biotinylat ed horse anti-mouse IgG at 1:100 (10ul of secondary AB, 10ul normal horse serum, 1000ul of PBS), sections were washed for 5 minutes in PBS buffer. The biotinylated anti-mouse IgG was added for 45 minutes at RT and then sections were rinsed in 1%

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53 horse serum for 15 minutes. Finall y, the sections were incubated for 30 minutes in vectastain reagent, then PBS for 5 minutes, DAB or Nova Red was added, and the slides were rinsed in water, counterstained with hematoxylin, dehydra ted with xylene, and slide coverslips mounted with permanent mounting media. Flow Cytometry Bone m arrow cells were characterized by four-color flow cytometry. Five hundred thousand bone marrow cells were re-suspended in PBS/ .1% BSA, placed into tubes, and incubated with the appropriate antibodies. Thre e-antibody combinations we re used for analysis: (1) PE-Ly6g (Abcam, ab24884), APC-CD11b (BD, 553312), Pacific blue c-kit (Biolegend, 105820); (2) PE-Ly6g, APC-CD11b, Pacific Blue F4 /80 (CALTAG, MF48028); (3) Percp-CD45 (BD, 557235), APC-Ter119 (BD, 557909), PE-CD 71 (BD, 553267); (4) APC-CD19 (Biolegend, 115511), PE-CD8a (Biolegend, 100707), Pacific Blue-CD3 (Biolegend, 100214); (5) APCTCRBeta (Biolegend, 109212), PE-CD34 (B iolegend, 119307) (6) PE-CD13 (BD, 558745), AexaFluor647-CD68 (AbD serotec, MCA1957A647); (7) Unstained Control. All antibodies for flow were used according to the manufacturers recommended instructions. Flow cytometry was performed on a BD FACS machine (BD Biosciences) with the help from UF core director Neal Benson. Flow cytometry analysis was performed using FCS Express 3.0 software. Nucleic Acid Extraction DNA and RNA sa mples were prepared from blood, bone marrow, tissues and tumors as previously described in Thomson and Wallace (2 002). In some cases there were not enough cells for RNA extraction, and only DNA was made. Loss of Heterozygosity Analysis 50ng of DNA from tumors, bone marrow and blood were PCR amplified with the 3-primer system using Pko2Ra, Pko2fb and lac2 to simu ltaneously genotype the mutant and wild-type

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54 allele in Ppp1r10 (Appendix A). Similiarly, the Nf1 mutant and wildtype alleles were also genotyped with NF31a, NF31b, and NeoTkp (Appe ndix A; Brannan et al. 1994). To PCR amplify Ppp1r10: 2.5ul of 10X HotMaster Buffer (E ppendorf), 200umol dNTP, 60ng Pko2ra primer, 60 ng Pko2fb primer, 40ng Lac2 primer, 1.25ul DMSO, 16.325ul H20, 0.125ul Hotmaster Taq (Eppendorf). The followi ng cycling conditions were used for Ppp1r10: 1 cycle of 95C for 15 minutes, 35 cycles of 95 C for 30 seconds, 63 C for 45 seconds, 72 C 45 seconds; and a final extension of 72 C for 10 minutes. To PCR amplify Nf1: 2.5ul of 10X Hotstart Buffer (Qiagen), 200umol dNTP (Invitrogen), 65ng of NF31a/NF31b/NeoTkp, 2.5mM MgCl2, 18ul of H20, .125ul of Hotstart Taq polymerase (Qiagen). The following cycling conditions were used for Nf1: 1 cycle of 96 C for 15 minutes, 40 cycles of 95 C for 30 seconds, 55 C for 30 seconds, 72C 1 minute; a final extension of 72C for 10 minutes. These PCR products were run on an 8% native polyacrylamide gel with controls (germ line DNAs), stained with ethidium bromide, to observe whether there was loss of the wildtype allele in Nf1 and Ppp1r10. The Ppp1r10 PCR product sizes of wildtype/mutant al leles should be 103bp/400bp, and the Nf1 wildtype/mutant alleles should be 128bp/95bp respectively. Results To test if Ppp1r10 cooperates with Nf1 inactivation to cause acute leukemia in mice as a possible tumor suppressor, Ppp1r10+/-(129S2) mice were crossed to Nf1+/(129S1) and aged for phenotype. The Ppp1r10+/-Nf1+/mice on average died from AMML (AML-M4) at 16.5 months of age (range 14 to 19 months), versus MPD at average 19.5 months of age seen in Nf1+/mice (range 19 to 20 months). Two of the Ppp1r10+/Nf1+/mice died of pulmonary hemmorhage and were not included in the analysis. In addition, the Ppp1r10+/mice on average died of AML (AML-M0) at 23.5 months (range 2324 months) (Table 4-1). These are illustrated

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55 in the Kaplan Meier survival gra ph (Figure 4-1). Eighty percent of Ppp1r10+/Nf1+/mice developed AMML, 15% developed MPD, and 5% developed myeloid hypoplasia (Figure 4-14, Table 4-2). At time of death, Ppp1r10+/Nf1+/mice with AMML disease had an average white blood cell count of 15,234 compared to 9187 in Nf1+/mice (Table 4-3, 4-4). The average percentage of blast cells observed in the peripheral blood of these double knockout mice was 42.9% compared to 8.94% seen in the Nf1+/mice, with 30% being the clinical cut-off for acute (Table 4-3, 4-4). These mice developed splenomegaly and the leukemic cells had also invaded other organs: kidney, eye-lid, liver, lung, gall bladder and lymph node. By peripheral blood smear analysis, the blast cells appeared to be immature monocytes and immature granulocytes of the granulocyte/ macrophage lineage, suggesting that the mutations effect is at the CFU-GM pathway (Figure 4-14, 4-26). Other than AMML, these mice also developed more (and somewhat different) solid tumo rs than originally reported in Nf1+/mice (Jacks et al. 1994). These tumors included hemangiosarcomas of the uterus and ovary in 3 out of 19 mice, and a proliferative mesenchymal process of the skin in 2 out of 19 mice. This latter finding appears to consist of leukemic and immature mesenchymal cells (Figure 4-8, 4-5). Other solid tumors in the double heterozygotes that were only observed in one each in cluded: lung adenocarcinoma, hepatocellular carcinoma, granulocytic sarcoma, and squamous cell carcinoma (Figure 4-2, 4-4, 4-15, and 4-16). In comparison, Nf1 +/mice also develop lung adenocarcinomas and hepatocellular carcinomas. However, the double heterozygotes did not develop lymphomas or adrenal tumors that are observed in the Nf1+/mice as well. Overall, 55 percent of the mice had solid tumors compared to the reported 25 pe rcent of the 129S inbr ed background and had a reduced latency (Jacks et al 1994 ; Bronson et al 1990). However, the cause of death our mice

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56 stemmed from a dysfunctional he matopoietic system typical of acute leukemia (anemia, bleeding). Ppp1r10+/(129S2) mice lived to about 2 years of age, when they became ill and had to be euthanized. Seven of eight of these mice ha d acute undifferentiated leukemia (AML-M0) and splenomegaly, with infiltrating immature neutroph ils and giant multinucleated cells (Figure 4-12, Table 4-1). The peripheral blood smear analysis showed an in crease in white blood cells, and blast cells. On average 16, 265 wh ite blood cells and 44% blasts (Table 4-3, 4-4). The bone marrow histology showed irregularly shaped cells th at could not be distinguished as myeloid or lymphoid, in origin, and as well showed a 2-fold decrease in the percentage of granulocytes compared to Nf1+/-Ppp1r10+/-mice (Figure 4-12, Table 4-3). This result suggests that loss of one allele of Ppp1r10 affects the hematopoietic system at an earlier precursor than that seen in the Nf1+/Ppp1r10+/(CFU-GM). Besides leukemia, the same seven heterozygous mice developed solid tumors, one each of the following: myoepithilioma of the salivary gland, myoepithelioma of the thymus, hepatic hemangi osarcoma, pulmonary adenocarcinoma, pituitary tumor, and leiomyosarcoma (Figure 4-6, 4-7, 4-8, 4-9, 4-10, 4-11). Our heterozygous Ppp1r10 mice showed an increased rate of tumori genesis, with over 75 percent of the mice developing tumors compared to 25 percent of inbred 129S mice (Jacks et al. 1994). To determine the subtype of acute leukemia, bone marrow sections were stained with Sudan Black or mouse monoclonal myeloperoxi dase antibody. Sudan Black stains acute myelogenous leukemia (AML-M1, M2, M3, M6) str ongly, weakly stains acute myelomonocytic leukemia M4 (AMML), and positively stains auer rods. However, Suda n Black does not stain AML-M0, AML-M7, AML-M5 and acute lymphoc ytic leukemia (ALL). Myeloperoxidase, on the other hand, stains acute my elomonocytic leukemia (AML-M4) and auer rods. This antibody

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57 does not recognize AML-M5, AML-M7, and ALL. The Ppp1r10+/-Nf1 +/mice are thus positive for acute myelomonocytic leukemia (AML-M4) and Ppp1r10+/mice have acute undifferentiated leukemia (AML-M0) (Figure 4-19). Tumor suppressor genes typically lose the remaining normal a llele somatically in tumors, often via large deletions, to fit Knudsons 2-hi t hypothesis (Knudson et al. 1971). These somatic deletions can be detected in tumor DNA by loss of heterozygosity (LOH) analysis comparing the presence of alleles in germline versus tumor at informative polymorphisms. To test for possible LOH at both genes, we initially trie d genotyping for the only known heterozygous polymorphism, the engineered mutations th at define these mice. PCR-genotyping of Nf1 and Ppp1r10 was performed in available DNA from tumors, blood and bone marrow. The PCR products were visualized ethidium bromide to obse rve relative ratio of muta nt to wildtype allele, in tumors, as compared to the germline. We were unable to get consistent results, indicating that these 3-primer based systems are unreliable for a quantitative measure such as LOH (Figure 417, 4-18). To further confirm leukemia subtypes (AML-M4 for Ppp1r10+/-Nf1+/-, MPD for Nf1 +/and AML-M0 for Ppp1r10+/-), flow cytometry was performed on bone marrow cells extracted at the time of death using hematopoietic antibodie s. The Gr1 and Mac1 FACS populations usually display increases in granulocytes and monocytes in AML-M4. The results showed an abnormal Gr1+/Mac1+ population as well as a Gr1 (low to med) /Mac1+ population in Ppp1r10+/-Nf1+/bone marrow compared to control, Nf1 +/-, and Ppp1r10+/bone marrow (Figure 4-20). These two populations, Gr1+/Mac1+ and Gr1 (low to med) /Mac1+, are characteristic of granulocyte and monocyte populations, respectively, and thus results confirmed AML-M4. In addition, Ppp1r10+/Nf1+/bone marrow displayed slight increase s in c-kit and F-4/80, further confirming

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58 that the CFU-GM point is affected in thes e mice (Figure 4-21, 4-22). However, although no alteration was observed in the B-cell pathway in Ppp1r10+/Nf1+/bone marrow, Ppp1r10+/bone marrow displayed an increase in the B-cell marker CD19 (Figure 4-23). Alterations were also observed in TCRbeta-positive populations for both Ppp1r10 +/-Nf1+/and Ppp1r10 +/-, suggesting that Ppp1r10 affects the myeloid and lymphoid compartment and (Figure 4-24, 4-25). This suggests that Ppp1r10 may be a possible hematopoietic stem cell gene (Figure 4-26). Discussion Creation of a Ppp1r10 knockout m ouse, and crossing it to the Nf1 knockout mouse, has led to the discovery of a new cancer gene involved in solid tumor formation and myeloid leukemia. Immunostaining and flow cytometry showed that Ppp1r10+/Nf1+/mice develop acute myelomonocytic leukemia (AML-M4) with high penetrance by 20 months, and about 75 percent of Ppp1r10 +/mice develop acute undifferentia ted leukemia (AML-M0) by age 24. AML was classified into 7 subtypes (M0-M7), at a conferen ce by the french, british, and american doctors in the 1970s, called the FAB classification system (http://www.cancer.org). AML-M0, undifferentiated acute myeloblastic leuke mia, affects 5% of AML patients and has a worse prognosis. AML-M1, acute myeloblastic leukemia with minimal differentiation, affects 15% of AML patients and has an average progn osis. AML-M2, acute myeloblastic leukemia with maturation, affects 25% of patients and has a better prognosis. These patients often have translocations involving chromosome 8 and 21. AML-M3, acute promyelocytic leukemia, affects 10% of AML patients and has the best prognosis of all AML subtypes. These patients often also have translocations of chromosomes 15 and 17. AML-M4, acute myelomonocytic leukemia, affects 20% of AML patients and ha s an average prognosis. These pati ents often have an inv (16) genetic abnormality as well. There is another M4 subtype, M4Eos, acute myelomonocytic leukemia with eosinophilia that affects 5% of AML subtypes and has a better prognosis. AML-

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59 M5, acute monocytic leukemia, affects 10% of patients and has an average prognosis. These patients also often have translocations i nvolving chromosomes 9 and 11. AML-M6, acute erythroid leukemia, affects 5% of AML patient s and has a worse prognosis for these patients. The last subtype, AML-M7, acute megakaryoblas tic leukemia affects 5% of AML patients and has a better prognosis. In additi on, 1 out of 3 AML patients have FLT3 mutations. Previous research has shown that a knockin allele for inv (16) predisposes mice to impaired hematopoiesis however administering E NU mutagenesis to these mice is necessary to cause an AMML-like disease. These mice had pa rtial myelomonocytic differentiation in a small percentage of cells in most mice. The majority of these cells are ckit+, Gr1-, Mac1-, B220-, and CD3(Castilla et al. 1999). However, two coopera tion studies with inv (16) with either loss of ARF or a tandem duplication in FLT3 (FLT3-ITD) caused AML without ENU mutagenesis. The inv (16); Arf -/mice develop AML and the mutations effect is on immature myeloid cells, staining positive for c-kit and negative for Sca1. Peripheral blood analysis of these mice shows basophile and eosinophil granules in addition to a slight monocy tic component (Moreno-Miralles et al. 2005). In addi tion, inv (16) with FLT3 -ITD mice develop AML with an effect on immature myeloid cells (myeloblasts and promyelocytes), which are c-kit+ and Sca1(Kim et al. 2008). In contrast, our model, Ppp1r10+/Nf1 +/mice have an abnormal granulocyte/macrophage population and an abnormal monocytic population of cells. This phenotype is a more mature myeloid phenotype than the immature myeloid phenotype observed in the previous mouse models. There are no existing mouse models for AML-M0 as observed in the Ppp1r10 +/mice. It is hypothesized that the Ppp1r10+/mice have other genetic altera tions in genes that cooperate

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60 with Ppp1r10 to cause AML-M0. These could be targets of PP1, like p53 or RB1 or genes unrelated to the PP1 pathway. All of the data suggests that Nf1 has an antagonizing effect on Ppp1r10. Nf1 heterozygous bone marrow effects mature gra nulocytes and macrophages and Ppp1r10 heterozygous bone marrow has neither lymphoid or myeloid mo rphology, suggesting an early hit in the hematopoietic pathway. Therefore, Nf1/Ppp1r10 cooperate to cause an intermediate hit in hematopoeisis, CFU-GM. However, the effect on Nf1 is synergistic since, the Ppp1r10 mutation allows leukemic transformation from MPD seen in Nf1+/mice to AML-M4 in Ppp1r10+/Nf1+/-. In addition to AML-M4, fifteen percent of Ppp1r10+/-Nf1+/mice developed hemangiosarcomas and ten percent developed a pr oliferative mesenchymal process of the skin. Other solid tumors included squamous cel l carcinoma, granulocytic sarcomas, lung adenocarcinoma and hepatocellular carcinomas. However, the Ppp1r10+/Nf1+/mice did not develop adrenal tumors, MPNSTs or lymphomas as previously observed in Nf1+/mice (Jacks et al. 1994). The Ppp1r10+/mice, on the other hand, showed some different solid tumors: myoepithelioma of the thymus and salivar y gland, hepatic hemangiosarcoma, pulmonary adenocarcinoma, pituitary tumor, and leiomyosarco ma. These data suggest that loss of at least one Ppp1r10 and Nf1 allele cooperate to affect myeloid compartment development at slightly different stages than either gene alone. Other known tumor suppressors that are proposed to be at least indirectly related to PP1 include p53 and RB1 (Lee et al 1992; Jacks et al. 1992; Clar ke et al 1992). Mice deficient for p53 (homozygous for knockout allele) are developm entally viable, however approximately a fourth of mice develop hemangiosarcomas, a nd 80% develop lymphomas (Donehower et al.

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61 1992). In contrast, mice deficient for RB1 die embryonically at day 16, and their hematopoietic system (myeloid compartment) is abnormal, showing increases in immature erythrocytes. Heterozygous RB1 mice have an increase in pituitary tumors (Lee et al 1992; Jacks et al. 1992; Clarke et al 1992). In addition, other hemangi osarcoma mouse models have suggested a p53related mechanism of tumorigenesis. For example, one mouse model lacking p53, + one Ink4c or Ink4d allele, develops hemangiosarcomas in over 50 percent of mice (Zindy et al. 2003). Another model, p18-/-, p53-/displays a range of tumor types si milar to that also observed in our mice: hepatocellular carcinoma, testicular carcinoma, hemagiosarcoma, leiomyosarcoma, fibrosarcoma, and osteosarcomas (Damo et al. 2005). In comparison, Ppp1r10+/Nf1+/phenotype includes affected hematopoeitic system, increases in hemagiosarcomas, proliferation of leukemic cells in the skin, and a range of non -recurring tumor types. This suggests that the tumor mechanism in the double heterozygotes involves p53 and or Rb pathways and further investigation is necessary. Since the 3-primer systems are unreliable for LOH analysis of tumors in our mice, Southern blots will be performed on DNA from any available tumors or bone marrows. It is expected that the remaining Nf1 allele is lost in these mice for tumorigenesis since the previous Nf1 mouse model has shown tumors to be Nf1-/(Jacks et al. 1994). It is unknown whether Ppp1r10 will be heterozygous or homozygous in the tumors or bone marrow. However, it is believed that Ppp1r10 will be homozygous in the bone marrow due to tumor heterogeneity and lack of a detectable mRNA transcript in bone marrow. Recently, several papers have reported that cooperating haploinsufficient tumor suppresso rs in the mouse can be sufficient reduction in protein function to contribute to tumorigenesis (Kamimura et al. 2007; Vives et al. 2006; Moreno-Miralles et al. 2005; Ma et al. 2005).

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62 This work provides evidence that Ppp1r10 is a new cancer gene, most likely a tumor suppressor gene, and cooperates with Nf1 inactivation to cause AM L-M4 and other cancers. Future work will focus on defining the pathways th at are deregulated in these cells in order to develop targeted drug therapy for patients. This is particularly important since AML is a very refractory cancer and few long-term effective treatments exist. Survival Curv e 0 20 40 60 80 100 120 10111213141516171819202122232425 A g e in month s Ppp1r10+/NF1+/-Ppp1r10+/ Nf1+/P=.02 Figure 4-1. Kaplan-Meier survival curve for Nf1+/-Ppp1r10 +/-, Ppp1r10 +/-, and Nf1 +/mice.

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63 Figure 4-2. H and E stain sections from Nf1+/Ppp1r10+/pulmonary adenocarcinoma and mouse normal lung.

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64 Figure 4-3. H and E stain of sections from Nf1+/-Ppp1r10+/liver tumor and mouse normal liver.

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65 Figure 4-4. H and E stain of sections from Nf1+/-Ppp1r10+/hepatocellular carcinoma and normal liver.

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66 4X 4X 10X 10X 40X 40X Figure 4-5. H and E stain of sections from Nf1+/Ppp1r10+/proliferative mesenchymal process of the skin (top) and normal skin (bottom).

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67 Figure 4-6. H and E stain of sections of Ppp1r10 +/adenocarcinoma of the salivary gland and normal salivary gland.

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68 Normal Thymus 4X, 10X, and 40X Myoepithelioma 4X, 10X, and 40X Figure 4-7. H and E stain of sections of Ppp1r10 +/myoepithelioma, thymus and normal thymus.

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69 Figure 4-8. H and E stain of sections from Ppp1r10+/hemangiosarcoma and normal liver.

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70 Figure 4-9. H and E stain of sections from Ppp1r10+/pulmonary adenocarcinoma and normal lung.

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71Normal Uterus 4X, 10X, and 40X Leiomyosarcoma 4X, 10X, and 40X Figure 4-10. H and E stain of sections from Ppp1r10+/leiomyosarcoma and normal uterus. Figure 4-11. H and E stain of sections from Ppp1r10+/chloromas, showing myeloid expansion, 10X (left) and 40X (right).

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72 Splenomegaly 100X Figure 4-12. H and E stain of sections from Ppp1r10+/acute undifferentiated leukemia (AMLM0) bone marrow (top, 100X) and splenomegaly. Bottom picture shows spleens from a Ppp1r10+/mouse with AML M0 (left) and a nor mal mouse (right). Scale is in cm.

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73 Figure 4-13. H and E stain of sections from Nf1 +/-Ppp1r10 +/acute myeloid leukemic marrow (AML-M4) (top) and normal bone marrow (bottom). Normal neutrophil Abnormal neutrophilCFU-GM CFU-G CFU-M(granulocyte/macrophage)myeloblast>promyelocyte>myelocyte>metamyelocyt e>band> segmenters(granulocytes) myeloblast>monoblast>promonocyte>monocyte Figure 4-14. Peripheral Blood Smear from Ppp1r10 +/-Nf1+/mouse, displaying abnormal monocytes and neutrophils.

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74 10X 40X Figure 4-15. An H and E stain of sec tion of a granulocytic sarcoma in a Ppp1r10+/Nf1+/mouse (10X on top, 40X on bottom).

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75 Figure 4-16. H and E stain of a section of a squamous cell carcinoma from a Ppp1r10+/-Nf1+/, 4X top, 10X bottom. 4X 10X

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76 Figure 4-17. Ethidium-bromide stained polyacrylamide gel showing Nf1 PCR products from blood and bone marrow for Nf1+/-Ppp1r10+/mice to test for loss of heterozygosity in the leukemic bone marrow.

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77 Figure 4-18. Ethidium-Stained polyacry lamide gel showing PCR products from Ppp1r10 genotyping in Ppp1r10+/mouse tissues to screen for loss of heterozygosity.

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78 Figure 4-19. Myeloperoxidase and Sudan Bl ack stain of bone marrow sections mouse 188 ( Ppp1r10+/-), 690 ( Ppp1r10 +/-Nf1+/-), and 1726 ( Nf1+/-).

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79 Figure 4-20. Flow cytometry on mouse bone marrow for Ppp1r10+/-Nf1 +/(NF1PKO), Nf1+/, and Ppp1r10+/(PKO) with Gr1 (PE) and Ma c1 (APC). Blue box indicates granulocyte population and red box indicates monocyte population.

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80 Figure 4-21. Flow cytometry on mouse bone marrow for control bone marrow, Ppp1r10+/-Nf1+/ (NF1PKO), Nf1+/-, and Ppp1r10+/(PKO) with ckit (Pacific Blue, stem cell marker).

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81 Figure 4-22. Flow cytometry on mouse bone marrow from control bone marrow, Ppp1r10+/Nf1+/ (NF1PKO), Nf1+/-, and Ppp1r10 +/(PKO) with F4/80 (granulocyte/macrophage marker).

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82 Figure 4-23. Flow cytometry on mouse bone marrow from control bone marrow, Ppp1r10+/Nf1+/(NF1PKO) and Ppp1r10+/(PKO) with CD19 (APC, B-cells).

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83 Figure 4-24. Flow cytometry on mouse bone marrow from control bone marrow, Ppp1r10+/Nf1+/(NF1PKO) and Ppp1r10+/(PKO) with TCRBeta (APC, T cell receptor).

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84 Figure 4-25. Flow cytometry on mouse bone marrow from Ppp1r10+/-Nf1 +/(NF1PKO), Nf1+/and Ppp1r10+/(PKO) with CD3 (Pacific Blue, T cell).

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85 Figure 4-26. An illustration of Nf1PKO (green), PKO (blue) and Nf1 (yellow) effect the hematopoeitic lineage.

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86 Table 4-1. Ppp1r10+/aged mice necropsy data, M: E (2 to 8.25) ratios, and blast numbers.

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87 Table 4-2. Nf1+/-Ppp1r10+/-aged mice necropsy data, M:E ( 2.33 to 13.25) ratios, and blast numbers.

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88 Table 4-3. Peripheral blood smear analysis of Nf1 +/-Ppp1r10 +/(NF1PKO), Ppp1r10+/(PKO), and Nf1+/-. The yellow color signifies the average count for all mice for that compartment. Table 4-4. Bone marrow analysis of Nf1+/-Ppp1r10+/(NF1PKO), Ppp1r10 +/(PKO), and Nf1+/mice. WNL signifies within normal limits.

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89 CHAPTER 5 DISCUSSION Previous work in Dr. Brannans lab has elucid ated three common loci that cooperate with Nf1 inactiva tion to cause acute le ukemia. The focus of this thesis has been one such locus, Epi2 The candidate gene I investigated at this locus was PPP1R10 located on human chromosome 6q21.3. Experiments were designed to help answer the question that if PPP1R10 is a cancer gene, is it an oncogene or a tumor suppressor gene. The mouse retrovirus inte grated in intron 1 of Mrps18b in the same transcriptional orientation. However, since Ppp1r10 is less than one kilobase away, in the opposite orientation of the virus, it was hypothesized that this gene may also be affected, pa rticularly by the viral LTR. Based on the location of the virus, the most likely mechanisms were protein truncation of Mrps18b or down-regulation of Ppp1r10 through the viral LTR integr ating in a transcription factor-binding site. PPP1R10 seemed to be a more attractive ca ndidate because of its ability to regulate PP1, a serine threonine phosphatase. PPP1R10, whose protein product is PNUTS, is a serine/threonine phosphatas e regulator that binds PP1, regulat es PP1s enzymatic activity and targets PP1 to the nucleus. PP1, a major phospha tase, regulates over 70 mammalian genes in the cell, however the downstream effectors of P NUTS are still unknown or PNUTS/PP1 complex. The goal of the first specific aim was to sequence PPP1R10 and MRPS18B and look for any genetic abnormalities in human leukemia samples. In PPP1R10 two possible point mutations were found, a serine to stop (nonsense) and a serine to threonine (missense) change at codon 125 in exon 6. However, we have not been able to determine if these are somatic or not. Other polymorphisms previously validated were also found, one in the promoter (rs16867845) and one in exon 19 (rs11754215). Both of these di splayed no loss of heterozygosity in human leukemia samples. One polymorphism appeared to display loss of heterozygosity in an intron 15

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90 SNP by sequence analysis. This finding was furt her confirmed by constructing an induced RsaI restriction fragment length polymorphism at the site. This would indicate PPP1R10 may have a role in leukemia through a tumor suppressor m echanism. Results, suggested LOH in other samples, although validation with both methods needs to be done and other SNPs need to be examined as well. The in vitro experiments were aimed at testing Ppp1r10 as a possible oncogene. The first experiment was to over-express PNUTS in COS7 cel ls, starve the cells and probe for increases or decreases in different effectors in cancer-rel ated signaling pathways. Western blots showed subtle effects in p-Stat3, p-Pt en, p-Erk1/2, and p-Mek1/2 but thes e were not considered to be representative of oncogenic ac tivation. Further work on downstr eam effectors of Ppp1r10 could focus on probing these pathways by down-regulating PNUTS through siRNA or a conditional gene targeted Ppp1r10 mouse, to study its role in differe nt tissues. This is important since PNUTS has not been studie d in hematopoietic cells. To further confirm that Ppp1r10 is not an oncogene, a CF U-GM assay was performed by over-expressing Ppp1r10 in murine wild-type fetal liver cells and then plating the cells with increasing amounts of GM-CSF. The expected results for an oncogene were increased sensitivity to GM-CSF at low concentrations, with increasing number of colonies as well as an altered cell morphology. The results displayed no altered morphology of the colonies and no cytokine independent growth via GM-CSF. These e xperiments provide more evidence that PPP1R10 is not an oncogene. To further study the hypothesis of tumor suppressor, a Ppp1r10 gene targeted Gene Trap mouse knockout was created by Jessica Walrath, a previous graduate student. Dr. Walrath showed that homozygosity for Ppp1r10 is a pre-implantation lethal mutation (Walrath 2006).

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91 Many tumor suppressor genes when knocked out are embryonic lethal (e.g Nf1, Nf2), consistent with Ppp1r10 being a tumor suppressor gene (Jacks et al. 1994). Jessica al so had evidence of decreased Ppp1r10 RNA levels in an Epi2 tumor (and somewhat less decreased in a few non Epi2 tumors) but the RNA was not absent (Walra th, 2006). This was similar to my human tumor data and is consistent with tumor supp ressor but possibly having an effect through heterozygosity (haploinsufficiency). Further ev idence fitting a hypothesis of tumor suppressor was found when 21 Ppp1r10+/-Nf1+/mice I generated developed cancer at a very high frequency. Results showed a phenotype di fferent than observed originally in Nf1 +/mice, which develop a minor percentage of MPD or pheochr omocytomas between 17 and 27 months of age (Jacks et al. 1994). Eighty two percent of Ppp1r10+/-Nf1+/mice developed AML-M4 and between 14 and 21 months. In addition, fi fteen percent of these mice developed hemangiosarcomas of the ovary and uterus, and ten percent had a prol iferation mesenchymal process of the skin. In Ppp1r10+/mice, clinical symptoms were not observed until 24 months of age. These mice then became ill and rapidly declin ed due to AML-M0. Seventy-five percent of these mice also developed one or more non-r ecurring solid tumors, suggesting an overall increase in tumorigenesis in ot her tissues as well. This supports the hypothesis that Ppp1r10 is a tumor suppressor whose loss has high penetrance in certain tissues such as bone marrow. Further these mice are novel models for AML-M0 and AML-M4. Based on this work and the litera ture, we hypothesize that loss of Ppp1r10 causes abnormal regulation of PP1, and increased phos pho-p53. Recent literature has shown that PP1 dephosphorylates p53 at serine 15 an d serine 37 (Li et al. 2006). The dephosphorylation of these sites changes transcriptional activ ity and apoptotic activity of p53 to the active form (Li et al. 2006). Phosphorylated p53 (inactive) has been as sociated with tumorigenesis (Feng et al. 2008).

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92 In our model, loss of Ppp1r10 decreases PP1 binding with p53 which interferes with dephosphorylation of p53, raising the amount of phosphorylated p53. This would cause effects in DNA repair, apoptosis, and the other processes regulates by p53, which go awry when it is inactivated as in many cancers. Alternative explana tion is that the pRB pathway is affected, since PP1 is known to dephosphorylate (inactivate) this important tumor suppressor in other tissues (Krucher et al. 2006). However, mouse model phenotypes fit with p53 involvement better (Zindy et al. 2003; Damo et al. 2005). Future work on this project will be g eared towards making a conditional knockout for Ppp1r10. The PP1 binding site that encompasses exons 13 and 14 will be floxed in order to ensure that all isoforms of Ppp1r10 lack of ability to regulate PP1. This mouse will be crossed to an Mx1-Cre mouse, whose Cre expression is i nducible and affects the hematopoietic system, spleen, and liver (Kuhn et al. 1995 ). Expression of Cre will be i nduced with pIpC injection, or interferon, and then mice will be followed for development of AML. Other tissues could be studied by crossing the conditional Ppp1r10 mouse to other Cre genes driven by tissue-specific promoters. For example, hemangiosarcomas ar e endothelial in orig in. Thus, crossing the conditional Ppp1r10 knockout to a Cre transgenic mouse dr iven by an endothelial-promoter such as ICAM2 or Tie2 (the latter of which alrea dy exists) (Cowan et al .1998; Kisanuki et al. 2001). This could provide an excellent model for hemagiosarcoma, which could be used to understand pathways involved or test treatments. We c ould also test effects of background strain on penetrance, latency, and or tumor repert oire (i.e. identify modifier genes). Another area of research will focus on the pa thways that are deregulated in the mouse bone marrow by loss or reduction of Ppp1r10 with or without Nf1. We could use siRNA for Ppp1r10 +/Nf1 in hematopoeitic precursor cell lines (e.g DC13) or primary cells, or possibly establish

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93 cell lines from the tumors or from non-transformed bone marrow cells, for in vitro studies. These experiments may be carried out with Western blots and or ph osphoFlow cytometry developed by the Nolan lab at Stanford (Krutzik et al. 2005) Some candidate pathways include: p53, pRB, PKA, and the Ras pathway. Once the deregul ated signaling pathwa y(s) is discovered, therapeutics can be identified to help treat patients with these leukemias. This could be specifically targeted to the PP1 level, or ut ilize therapies already being developed for the pathways implicated.

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94 APPENDIX A PRIMER LIST Primer Name Primer Sequence Genomic or cDNARegion of geneMse or HsPNUTSprom1F5'-CAGGACAGGAATTGACGGAAA-3' genomic promoter Hs PNUTSprom1R5'-CTAGCACCTCCCTTTCCTCTG-3' genomic promoter Hs PNUTSprom2F5'-TACCAATCCTGGGTGAGAAATG-3' genomic promoter Hs PNUTSprom2R5'-CCGCAAAATTTTACCCACTAGA-3' genomic promoter Hs P115RsAF 5'-CCCTAAACTCATCCCCCTAGT-3' genomic RFLP intron 15 Hs P115RsaR 5'-CTGAAAGAAGAACAAAAAAAATCAGTA-3 genomic RFLP intron 15 Hs lac2 5'-CAAGGCGATTAAGTTGGGTAACG-3' genomic exon 2 Mse Pko2fb 5'-CGAAGGACCGTCACCACATAAC-3' genomic exon 2 Mse Pko2fa 5'-CGAAGGACCGTCACCACATAAC-3' genomic exon 2 Mse NF31a 5-'GTATTGAATTGAAGCACCTTTGTTTG-3' genomic Mse NF31b 5'-CTGCCCAAGGCTCCCCCG-3' genomic Mse NeoTkp 5'-GCGTGTTCGAATTCGCCAATG-3' genomic Mse PPPex2a 5'-TTGTGTTCCTTTTATCCCAGGT-3' genomic exon 2 Hs PPPex2b 5'-CTTCGGCCACAGATTTCAAG-3' genomic exon 2 Hs PPPex2c 5'-CTGCTTGGGACTTGAAATCTG-3' genomic exon 2 Hs PPPex2d 5'-TTTGCGACGTCAGCACCT-3' genomic exon 2 Hs PPPex3F 5'-TCCTCTTACCATAGAAACCACCA-3' genomic exon 3 Hs PPPex3R 5'-CAAAAAGGGGTAAGACTCACC-3' genomic exon 3 Hs PPPex4F 5'-GGCTGCTTCATTTCTAACCCTA-3' genomic exon 4 Hs PPPex4R 5'-GCTTACACCTTCCCATTCCAA-3' genomic exon 4 Hs PPPex5F 5'-CTGCCTGCCTGCAGATTTAT-3' genomic exon 5 Hs PPPex5R 5'-AAAGGTACCTGCTTGAGATGG-3' genomic exon 5 Hs PPPex6F 5'-GCCTCCTGGCATTTACCTTT-3' genomic exon 6 Hs PPPex6R 5'-AAAGCCCATGAGAGAGCAGA-3' genomic exon 6 Hs PPPex7F 5'-GCATTTCCCCTTTCTGTCAC-3' genomic exon 7 Hs PPPex7R 5'-AGGACTAAGGAGCTTACCAGCA-3' genomic exon 7 Hs PPPex8F 5'-TCCCAGTCTGCTTCACCTCT-3' genomic exon 8 Hs PPPex8R 5'-GAACGGAACTTGGCATGACT-3' genomic exon 8 Hs PPPex9a 5'-GTCTGGGGTTTGAGTTCAGC-3' genomic exon 9 Hs PPPex9b 5'-CATTCAGAGCCTGAGAATATGG-3' genomic exon 9 Hs PPPex10a 5'-GGGGTGAGTCGGAGATGTT-3' genomic exon 10 Hs PPPex10b 5'TCAAAGTACATCTTCCCCACTTT-3' genomic exon 10 Hs PPPex11a 5'-CTGTGTTTGCAGGTGTAGGATT-3' genomic exon 11 Hs PPPex11b 5'-CTTCTGGGAGCCCATACCTT-3' genomic exon 11 Hs PPPex12F 5'-CACAATAGCCAAGCCCCTTT-3' genomic exon 12 Hs PPPex12R 5'-ACCCAAGATCCCTCCTTTCAG-3' genomic exon 12 Hs PPPex13a 5'-TTCTGCTTCCAGCCTCTTTG-3' genomic exon 13 Hs PPPex13b 5'-CCTCGTTCAGTTTCATCCAA-3' genomic exon 13 Hs PPPex14F 5'-CCTGCCGACAGTAAATGTGA-3' genomic exon 14 Hs PPPex14R 5'-CAGGAGTGTCCAAGCACTCA-3' genomic exon 14 Hs PPPex15F 5'-TCTCACCTGTGTTGTTCCTGA-3' genomic exon 15 Hs PPPex15R 5'-CAATGACCATCACAACTTCCA-3' genomic exon 15 Hs PPPex16F 5'-TGAATTCGGTGCATCTTTCA-3' genomic exon 16 Hs PPPex16R 5'-ACAGTGAGGAGAGCGAGCTT-3' genomic exon 16 Hs PPPex17F 5'-CTTTCGCCATGGTTGTTACC-3' genomic exon 17 Hs PPPex17R 5'-CCCAAGCATCTGCTTGATCTT-3' genomic exon 17 Hs PPPex18F 5'-GCTCATGCTGTGTTACTCTTGTTT-3' genomic exon 18 Hs PPPex18R 5'-AAGCAGATAAGCCCATTCCA-3' genomic exon 18 Hs PPPex19a 5'-GGCAGTTCCTGGTAGCTGAT-3' genomic exon 19 Hs PPPex19b 5'-GGACGATGTCCACTGCTGTT-3' genomic exon 19 Hs PPPex19c 5'-ATGAAGGCCCTGGTGGTAG-3' genomic exon 19 Hs PPPex19b 5'-GAGACGGGTACCTCACGTTC-3' genomic exon 19 Hs PPPex19e 5'-GACCCCCAAATGGACGAG-3' genomic exon 20 Hs PPPex19f 5'-CCACCCATGCTACCACCA-3' genomic exon 21 Hs PPPex20F 5'-GCATTGCCCTCAGCTATTTC-3' genomic exon 20 Hs PPPex20R 5'-GAAAATGGGCCTCACAGAAG-3' genomic exon 20 Hs PPPintron4F5'-CCGCCAACGTCAATAAATCT-3' genomic intron 4 SNP Hs PPPintron4R5'-GAGAGGCGCTTTTGCTAAGA-3' genomic intron 4 SNP Hs Ppp1r10cDNA1F5'-AACAAACAAGCCTCAGCAACA-3' cDNA exon 2 Mse Ppp1r10cDNA1R5'-CCTCTCCCTAAATGGAGTTGGG-3' cDNA exon 2 Mse Ppp1r10cDNA2F5'-CTCATCCAGCATTTCCGTT-3' cDNA exon 2 Mse Ppp1r10cDNA2R5'-TAGACCCCAAAGAACTGCTA-3' cDNA exon 4 Mse Ppp1r10cDNA3F5'-GTGCTGACGTCGCAAAAA-3' cDNA exon 2 Mse Ppp1r10cDNA3R5'-ATTGGCATCAGTCCTTGTCAG-3' cDNA exon 8 Mse Ppp1r10cDNA4F5'-GCAAGTCAAGTGAGGATGAAG-3' cDNA exon 7 Mse Ppp1r10cDNA4R5'-CTGTCCCAGGCATCAAAATT-3' cDNA exon 10 Mse Ppp1r10cDNA5F5'-CTGGGTTTTCTGGATGCTCTC-3' cDNA exon 10 Mse Ppp1r10cDNA5R5'-CAAGATCAAAGACTTCGGGG-3' cDNA exon 15 Mse Ppp1r10cDNA6F5'-GGAGGGCAAGCTGAGAG-3' cDNA exon 14 Mse Ppp1r10cDNA6R5'-TTATGGGAAGCATGGGAG-3' cDNA exon 17 Mse Ppp1r10cDNA7F5'-CTCCAAGCTGCCTCCAGTT-3' cDNA exon 17 Mse Ppp1r10cDNA7R5'-GTCGAGGAGGAAAYGAGCCA-3' cDNA exon 20 Mse Ppp1r10cDNA8F5'-CTCCCGGACCATACCACAGA-3' cDNA exon 20 Mse Ppp1r10cDNA8R5'-CTACCACCCAGGGGTCAATGG-3' cDNA exon 21 Mse GAPDH-5' 5'-TCATCATCTCTGCCCCCTCTG-3' cDNA Hs/Mse GAPDH-3' 5'-GCCTGCTTCACCACCTTCTTG-3' cDNA Hse/Mse PPPex2-3F 5'-TTGAGTTTTGGGTCCTGGTT-3' cDNA exons 2-3 Hs/Mse PPPex2-3R5'-TGGAAATCCCATCCACACTT-3' cDNA exons 2-3 Hse/Mse

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95 APPENDIX B PFAFFL METHOD Ratio= (E target )DCPtarget(control-sample) (Eref)DCPref(control-samples)

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105 BIOGRAPHICAL SKETCH Angela Hadjipanayis was born in Winnipeg, Manitoba, Canada, in 1975. She is the daughter of George and Paraskevi Hadjipanayis. Her father is from Cyprus and is a Professor of physics at the University of Delaware in Newark Delaware. Her mother is from Patras, Greece and is the head librarian at St. Marks High School in Wilmingt on, Delaware. She has one sibling, Costas Hadjipanayis who is a neurosurgeon in the Department of Neurosurgery at Emory University in Atlanta, Georgia. Angela is also married to Joseph Phillips, who has a Ph.D. in physics and holds a postdoctoral position at the Un iversity of Florida with Dr. Weihong Tan.