The Role of PALB2 in BRCA1/2-Mediated DNA Repair and Tumor Suppression DISSERTATION Presented in Partial Fulfillment of the Requ

The role of PALB2 in BRCA1/2-mediated DNA repair and tumor suppression

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Dongju Park

Graduate Program in Molecular , Ce llular and Deve lopmenta l B iology

The Ohio State University

2017

Dissertation Committee:

Dr Thomas Ludwig, Advisor

Dr Jeffrey Parvin

Dr Kay Huebner

Dr Mark Parthun

Dongju Park

2017

Abstract

Germline mutations in the cancer susceptibility genes BRCA1 or BRCA2 confer an increased risk of developing breast, ovarian and pancreatic cancer. BRCA1 and BRCA2 are key molecules in DNA damage repair, specifically repair by homologous recombination (HR). Accumulating DNA damage, due to dysfunction of the BRCA genes, leads to genome instability and increased cancer risk. In 2006, a novel protein,

PALB2 (partner and localizer of BRCA2) was identified as a DNA repair pathway component (1). PALB2 co-localizes with BRCA1 and BRCA2 at DNA damage sites and is thought to act as an adaptor protein that mediates the BRCA1-BRCA2 interaction (2).

Indeed, prior studies showed that BRCA1 and PALB2 direct ly interact through their coile d-coil doma in. The C-terminal WD40 domain of PALB2 binds to BRCA2 and the

BRC repeats of BRCA2 recruit a central protein for HR, the Rad51 recombinase.

Recently, mutations in the PALB2 gene were reported in the tumors of breast and pancreatic cancer patients. Interestingly, some of these mutations could inhibit its direct binding of PALB2 to BRCA1 or BRCA2 thus disrupting DNA repair. Furthermore, missense mutations in the coiled-coil domain of BRCA1 (M1400V, L1407P and

M1411T) that mediates the interaction with PALB2 have been reported among familial

breast cancer patients, which also could increase the risk of pancreatic cancer. Therefore,

we hypothesized that physical and functional interaction between BRCA1 and BRCA2

through the linker protein PALB2 is required for BRCA1/2-mediated HR and tumor

suppression. To test this hypothesis, we first determined whether PALB2 is a bonafide

tumor-suppressor by deleting Palb2 in the pancreas specifically in genetically engineered

KrasL SL -G12D/+; p53L SL -R270H/+; Pdx1-Cre mouse model (now referred to as KPC mice) and

observing these animals for pancreatic tumors in comparison with Brca1flex/flex-KPC or

Brca2 flex/flex-KPC pancreatic tumor mouse models. Homozygous deletion of Palb2 in the pancreas of KPC mice accelerated pancreatic tumor development compared to KPC mice, similarly to Brca1flex/flex-KPC and Brca2 flex/flex-KPC anima ls. However, the

histopathology of tumors was different between the groups (KPC vs Palb2 flex/flex-KPC vs

Brca1flex/flex-KPC vs Brca2flex/flex-KPC). Pancreatic tumor cell lines established from Palb2

flex/flex-KPC mice showed hypersensitivity to DNA damaging agents such as interstrand cross-linkers, Mitomycin C (MMC) and Cisplatin and poly ADP ribose polymerase inhibitor (PARP inhibitor) to an extent similar to that of cells from Brca1flex/flex-KPC or

Brca2flex/flex-KPC tumors, compared to cells generated from KPC tumors. In addition,

Palb2 flex/flex-KPC mice in vivo treated with MMC showed clearly prolonged survival.

These results suggest that PALB2 plays a key role in BRCAs mediated HR and tumor

suppression.

Next, we determined if the BRCA1-PALB2 interaction per se is critical for BRCA1- mediated tumor suppression by generating Brca1 L1363P point mutant knock-in mice

iii

(L1407P in human sequence) that abolishes the binding of BRCA1 to PALB2. Mouse

embryonic fibroblasts (MEFs) from homozygous Brca1 L1363P mutant animals exhibit hypersensitivity to DNA damaging agents (MMC, PARP-inhibitor and ionizing radiation

(IR)), and following IR mutant cells fail to recruit the Rad51 recombinase to sites of

DNA damage, implying a defect of DSBs repair by HR. Accumulation of unresolved

DNA damage induces hyperactivation of p53 and its downstream target p21 in

Brca1L1363P/L1363P primary MEFs resulting in impaired proliferation and premature

senescence. While Brca1-/- null mice are early embryonic lethal, homozygous

Brca1L1363P/L1363P mice are viable. However, mutant mice exhibit growth retardation to

various extents. Some mutant animals were extremely small, developed aplastic anemia

and died within a month. All other Brca1L1363P/L1363P animals developed T-cell acute

lymphoblastic leukemia (T-ALL) with an average latency of 3 months. Interestingly, the major it y of T-ALLs (53%) from Brca1L1363P/L1363P animals acquired activating Notch1

mutations such as discovered in patients with T-ALLs. The phenotypes observed in

Brca1L1363P/L1363P cells and mice recapitulate clinical phenotypes seen in Fanconi Anemia

(FA) patients. Therefore, this mouse model can provide insights for developing new

therapies for FA. Furthermore, t his results demonstrate the importance of the BRCA1-

PALB2 interact ion in vivo and suggests that the interaction is essential for BRCA1- mediated tumor suppression.

Dedication

Dedicated to my parents, husband and friends

Vita

2003-2007...... Bachelor of Science in Biotechnology,

Catholic University of Korea

2007-2010...... Research Assistant, Department of

Molecular Medic ine , Colle ge of Medic ine ,

Ewha Womans University

2010-2011...... Visiting Scholar, Department of Molecular

Neurobiology, The Ohio State University

2011-Present ...... Graduate Research Associate, Molecular

Cellular and Developmental Biology

Graduate Program, Department of Cancer

Biology & Genetics. The Ohio State

Univers ity

Publications

1. Park, J., Kim, W., Kim, J., Park, M., Nam, J., Yun, C., Kwon, Y., and Jo, I. (2011) Chk1 and

Hsp90 cooperatively regulate phosphorylation of endothelial nitric oxide synthase at serine 1179.

Free Radical Biology and Medicine 51 (12), 2217-2226

2. Park, J., Park, M., Byun, C., and Jo, I. (2012) c-Jun N-terminal kinase 2 phosphorylates endothelial nitric oxide synthase. Biochemical and biophysical research communications, 417 (1),

340-345

3. Yoon,S., Park, D., Ryu, J., Ozer, G., Tep, C., Shin, Y., Lim, T., Kunwar, A., Walton, J.,

Nelson, R., Nagahara, A., Tuszynski, M., and Huang, K. (2012) JNK3 perpetuates metabolic stress induced by Abeta Peptides. Neuron 75 (5), 824-837

Fields of Study

Major Fie ld: Molecular, Cellular and Developmental Biology

vii

Table of Conte nts

Abstract ...... ii

Dedication ...... v

Vita ...... vi

Publications ...... viii

Fields of Study ...... vii

Table of Contents ...... viii

List of Tables ...... ix

List of Figures ...... x

CHAPTER 1 Introduction...... 1

CHAPTER 2 Palb2 pancreatic tumor model in compar ison with Brca1/2 tumor models 15

CHAPTER 3 Ablation of BRCA1-PALB2 interaction phenocopies Fanconi Anemia.... 44

CHAPTER 4 Conclusions and future directions...... 100

BIBLIOGRAPHY ...... 107

viii

List of Tables

Table 1 Notch1 mutations detected by exome sequencing from Brca1L1363P/ L 1363P; p53+/+ thymic tumor...... 97

Table 2 Notch1 mutations detected by exome sequencing from Brca1L1363P/ L 1363P; p53+/- thymic tumor...... 98

Table 3 Notch1 mutations detected by exome sequencing from Brca1L1363P/ L 1363P; p53-/- thymic tumor...... 99

List of Figures

Figure 1.1 Model of PALB2 and BRCA2 working synergistically to stimulate homologous recombination...... 10

Figure 1.2 Patient-derived BRCA1 mutations abolished the BRCA1-PALB2

associat ion ...... 11

Figure 1.3 HR protein interactions and domains...... 12

Figure 1.4 Pancreatic cancer initiation and progression ...... 13

Figure 1.5 Fanconi anemia/BRCA pathway...... 14

Figure 2.1 Pancreas-specific deletion of Palb2, Brca1 or Brca2 early in development

results in smaller pancreata ...... 31

Figure 2.2 Concomitant expression of mutant KrasG12D and p53R270H cooperate with

Palb2, Brca1 or Brca2 loss in the pancreatic ductal cells to promote PDAC

tumorigenesis...... 33

Figure 2.3 Pancreatic cystic lesions resembling MCNs are unique to Palb2 and Brca1-

mutant anima ls...... 36

Figure 2.4 Primary tumor cells from Palb2-KPC, Brca1-KPC and Brca2-KPC solid

tumors exhibit hypersensitivity to DNA damaging agents...... 38

Figure 2.5 Interstrand crosslinking agents inhibit Palb2-KPC and Brca2-KPC tumor growth in vivo ...... 41

Figure 3.1 Generation of the Brca1L1363P knock-in mice ...... 70

Figure 3.2 Brca1L1363P/L1363P MEFs have defective HDR and hypersensitivity to

interstrand cross linker...... 73

Figure 3.3 Brca1L1363P mutation induces premature senescent phenotype in pMEFs ...... 78

Figure 3.4 Brca1L1363P/ L1363P mice show a variety of phenotypic abnormalities ...... 80

Figure 3.5 ~13% of Brca1L1363P/ L1363P animals developed bone marrow failure ...... 85

Figure 3.6 p53 deletion partially rescues hematopoietic defects in Brca1L1363P/L1363P

anima ls ...... 89

Figure 3.7 Brca1L1363P/L1363P animals developed T-lymphoblastic lymphoma/ leukemia

between 2 and 4 months ...... 91

Figure 3.8 Notch1 mutations were present in 21 of 26 tumors analyzed by sequencing . 95

CHAPTER 1

Introduction

1.1 Overview

Maintenance of genome integrity is indispensable for all living organisms. DNA damage, abnormal DNA replication and improper cell division lead to an unstable genome, which eventually can cause cancer. Pancreatic cancer is the fourth most common cause of cancer death in United States due to absence of reliable detection methods and effective treatments. Approximately, 5-10% of pancreatic cancer is familial; BRCA1, PALB2 and

BRCA2 are among established pancreatic susceptibility genes (3). Recent studies found that BRCA1, PALB2 and BRCA2 proteins cooperate in DNA damage repair by formation of a complex, and their role is critical to maintain genome integrity (2, 4, 5).

Since genome instability is a hallmark of cancer, formation of the BRCA1-PALB2-

BRCA2 complex could be inextricably connected with their tumor suppressive functions.

Double-strand DNA breaks (DSBs) are deleterious DNA lesions. DSB repair can take place through two major pathways: Homologous recombination (HR) and non- homologous end joining (NHEJ). HR is an error-free pathway preferentially occurring

1 during late S phase and G2 phase in which the sister chromatid is used as the repair template. In contrast, NHEJ is an error-prone pathway which can occur throughout the cell cycle. During NHEJ, the ends of a DSB might be slightly modified and then direct ly joined without a homologous template causing sma ll insertions or deletions (6). BRCA1,

BRCA2 and PALB2 are essentia l proteins for HR. At the DSB, the 5’ strands are resected by the MRN (MRE11-RAD50-NBS1) complex and CtBP-interacting protein (CtIP) to generate 3' single-stranded DNA (ssDNA). Replication protein A (RPA), a ssDNA- binding protein, f irst coats the ssDNA. BRCA1 recruits PALB2 and BRCA2 to DSBs forming a BRCA1-PALB2-BRCA2 complex, which facilitate assembly of the RAD51 recombinase filament on 3' DNA overhangs. PALB2-BRCA2 stabilizes and activates

RAD51-ssDNA filament to promote DNA strand invasion, stimulat ing HR (6, 7) [Figure

1.1.].

As mentioned above, BRCAs are crit ica l for HR. Indeed, BRCAs-def ic ient ce lls have highly unstable genomes, resulting in extensive aneuploidy and chromosomal rearrangements (8). Since the BRCA1 mutations implicated in familial breast and pancreatic cancer are usually frameshift or nonsense mutations, many tumor-associated alleles encode truncated proteins that have lost the coiled-coil domain. Furthermore, in some breast cancer families, tumor susceptibility can be ascribed to missense mutations that cause a single amino acid substitution in the coiled-coil domain of BRCA1

(Met1400Val, Leu1407Pro and Met 1411Thr). Interestingly, these sequences in BRCA1 were shown to interact with the coiled-coil domain of PALB2 (4, 6) [Figure 1.2.].

PALB2 was first identified as a binding partner of BRCA2 and shown to be required for localization of BRCA2 to sites of DNA damage, and thus crucial for BRCA2 function in

HR (1). The coile d-coil domain at the N-terminus of PALB2 interacts with the coiled-coil domain of BRCA1 and the C-terminal WD repeats of PALB2 bind to the N-terminus of

BRCA2 (6) [Fig. 1.3.]. Down regulation of PALB2 by siRNA suppresses HR in a manner similar to BRCA1 and BRCA2 depletion (2). Furthermore, like BRCA1 and BRCA2,

monoallelic mutations in PALB2 confer familial susceptibility to pancreatic cancer (9, 10),

while bia lle lic PALB2 lesions can cause Fanconi Anemia (FA), a genome instability

syndrome that predisposes patients to cancer (11). The mounting evidence that PALB2 is

required for HR and functions as a pancreatic susceptibility gene in some patients

suggests that it may also be important for BRCA1/2-mediated tumor suppression.

/Understanding the functions of PALB2 that suppress tumor development is therefore

critical to develop novel and improved treatments for pancreatic tumors. In Chapter 2, I

describe the experiments showing that the PALB2 gene is a bonafide pancreatic tumor

suppressor by delet ing Palb2 in the mouse pancreas and comparing the resulting tumor

histopathology with Brca1 and Brca2 pancreatic tumor mode ls. Using primary pancreatic

cancer cells generated from four different pancreatic tumor mouse models (Brca1flex/flex-

KPC, Palb2 flex/flex-KPC, Brca2 flex/flex-KPC and KPC), I also describe a comparative analysis of drug sensitivity. In Chapter 3, I provide evidence for the critical role of

BRCA1-PALB2 interaction in BRCA1 mediated HR and FA disease. In this chapter, I introduce general knowledge of pancreatic cancer, FA disease and the FA/BRCA pathway.

1.2. Pancreatic ductal adenocarcinoma

1.2.1. Epidemiology

Pancreatic cancer, the fourth most common cause of cancer death in the United States, is often called the "silent killer". Location of the pancreas in the deep abdomen and an absence of symptoms until it reaches an advanced stage make t his disease diff icult to diagnose at a curable stage. Thus, most patients are diagnosed at advanced stages of tumorigenesis, showing only 8% survival rate at 5 years (12). Therefore, a strategy for prevention is an important goal. However, the prevention of pancreatic cancer may not be possible through avoidance of risk factors because direct causes of this cancer have not been identified except for cases of hereditary pancreatic cancer. Although some studies have indicated that tobacco smoking, obesity and diabetes could be risk factors for pancreatic cancer, it cannot fully explain variations in the incidence of pancreatic cancer among different countries (13-17). Approximately 5~10% of pancreatic cancer is hereditary with 80% penetrance. The common genetic alterations in hereditary pancreatic cancer are activating inactivation of the tumor suppressor genes such as breast cancer susceptibility gene 1 and 2 (BRCA1 and BRCA2), partner of localizer of BRCA2 (PALB2)

[8], ataxia telangiectasia mutated (ATM), tumor protein 53 (TP53) (18), cyclin-dependent kinase inhibitor 2A (CDKN2A/p16) (19), mothers against decapentaplegic homology 4

(MADH4/SMAD4) (20) and serine/threonine kinase 11 (STK1/LKB1)1 (21).

In 1995, Schutte et al. found by representational difference analysis, a homozygous deletion of the BRCA2 locus on chromosome 13q12.3 in a pancreatic carcinoma (22).

This first discovery makes BRCA2, a candidate tumor suppressor gene, not only for breast cancer but also for pancreatic cancer. Since then, many researchers have examined

an association between BRCAs mutations and pancreatic cancer. The Breast Cancer

Linkage Consortium and Thompson et al reported statistically significantly increased risk for pancreatic cancer in BRCA1 mutation carriers (Relative risk=2.26, 95% CI=1.26-4.06,

P=0.004) and in BRCA2 mutation carriers (Relative risk=3.51, 95% CI=1.87-6.58,

P=0.0012) based on their cross-sectional study (23, 24). In 2006, after discovery of

PALB2 as a linker protein between BRCA1 and BRCA2, Jones et al. ident if ie d PALB2 as

another pancreatic cancer susceptibility gene. They reported that PALB2 is the second

most mutated gene for hereditary pancreatic cancer other than BRCA2 (9).

1.2.2 Progression of pancreatic ductal adenocarcinoma

There are two different types of pancreatic cancer, exocrine and endocrine tumors, based

on where the tumor begins. More than 95% of malignant pancreatic neoplasms arise in

the exocrine pancreas. Among different types of exocrine pancreatic cancer,

approximately 85% of cases are pancreatic ductal adenocarcinoma (PDAC) which is a

malignant neoplasm and originates from the pancreatic ductal epithelium. There are three

distinct precursor lesions of PDAC: pancreatic intraepithelial neoplasia (PanIN),

intraductal papillary mucinous neoplasm (IPMN) and mucinous cystic neoplasm (MCN)

(25).

PanIN is the most common precursor and can be histologically classified into four

different stages based on cellular atypia: PanIN-1A, PanIN-1B, PanIN-2, and PanIN-3. In

PanIN-1A, the lowest grade lesions, ductal epithelium is elongated and produce abundant mucin. These epithelial lesions form papillary or micropapillary architecture in PanIN-1B.

The papillary mucinous epithelial lesions show some nuclear abnormalities (i.e. some loss of polarity and crowding) in PanIN-2. These cells bud off into lumen with cribr if or ming luminal necrosis and atypica l mit os is in PanIN-3. Although some PDAC are associated with hereditary mutations, most cases are associated with somatic mutations. Elevation of mutation frequency and variety correlate with the progression of

PanIN. Molecular analysis has shown that PanIN-1 lesions frequently possess KRAS and

INK4A/p16 mutations. PanIN3 lesions are more likely to express mutations of KRAS, p53 and SMAD4 (26, 27) [Figure 1.4.].

Other precursor lesions of PDAC are IPMN and MCN which are 2 distinct entities of pancreatic mucin-producing cystic neoplasms. IPMN arises from the main pancreatic duct or its branches and is characterized by papillary neoplastic neoplasia, cyst formation and mucin secretion. Some of IPMNs can transform from benign to malignant tumor.

Based on the degree of invasiveness, IPMN can be classified into two groups, invasive and noninvasive IPMN. This separation is critical since it affects surgical outcome. MCN is histologically characterized by the presence of ovarian-like stroma which is positive for both estrogen receptor (ER) and progesterone receptor (PR). It is composed of columnar, muc in-producing epithelium, supported by ovarian-like stroma and can develop into mucinous cystadenocarcinomas (25, 28).

1.3 Fanconi Anemia

1.3.1 Fanconi anemia disease

Fanconi anemia (FA) is a rare genetic disorder associated with bone marrow failure

(BMF), acute myelogenous leukemia and cancer development. It was originally described by the Swiss pediatrician Guido Fanconi in 1927 and he reported a family with three affected siblings who developed anemia and congenital anomalies (29, 30).

Patients with FA show extremely heterogeneous clinical phenotypes including hematopoietic issues (i.e. aplastic anemia and progressive BMF) that are the hallmark of the disease and developmental abnormalities including short stature, abnormal thumbs, microcephaly, growth retardation, skin pigmentation, hypoplasia of the sexual organs.

This disease is caused by bia lle lic mutat ions in one of FA genes. Until now, 21 FA genes have been identified. Proteins encoded by FA genes are involved in interstrand DNA crosslink (ICL) repair, called the FA/BRCA pathway. Thus, there are two major cellular characteristics of the FA pathology: the chromosome fragility and the hypersensitivity to

ICL-inducing chemicals such as mitomycin C (MMC), diepoxybutane and cis-platinum, which are used to diagnose FA (31).

Recently, it became more important to classify FA genes into two different groups based

on presence of patients with BMF and chromosome fragility: bonaf ide FA genes and FA-

like genes. Out of 21 FA genes, only 16 are classif ied as bonafide FA genes (FANCA, B,

C, D1, D2, E, F, G, I, J, L, N, O, P, Q, T and V). FANCM, O, R, S are FA-like genes

because the small number of patients have been reported without BMF (31, 32).

1.3.2. FA/BRCA pathway

FA proteins encoded by FA genes cooperate in the FA/BRCA1 pathway, which resolves

ICLs and DNA lesions that block DNA replication and transcription. The pathway coordinates various steps for ICL repair: lesion recognition, DNA incision, lesion bypass and lesion repair (33-35) [Figure 1.5.].

Lesion recognition

When ICL lesions encounter replication forks FANCM-FAAP24 heterodimer recognize

the damage and initiate the FA/BRCA pathway. The heterodimer functions as a landing

pad for FA core complex that consist of 14 proteins (FANCA, B, C, E, F, G, L, M, T,

FAAP100, MHF1, MHF2, FAAP20 and FAAP24). The core complex mono-

ubiquitinates and activates two other FA proteins, FANCD2 and FANCI, which is central

for the FA/BRCA pathway (33-35).

DNA incision

The mono-ubiquitinated FANCD2-FANCI complex is relocalized to a converged fork

near the ICL to control nucleolytic incision. The FANCD2-Ub recruits the nuclease

scaffold protein SLX4/FANCP, which activates structure specific nucleases, ERCC1-

XPF (also known as ERCC4 or FANCQ), MUS81-EME1 and SLX1. The ERCC1-XPF nicks the lagging strand DNA adjacent to the stalled fork resulting in a DSB on the lagging strand and the crosslinked nucleotide tethered leading strand (33-35).

Lesion bypass

Translesion DNA synthesis (TLS) polymerases such as REV1 or Polζ bypass the unhooked cross-linked oligonucleotides and restore a nascent strand, which functions as a template for HR repair (33-35).

Lesion repair by HR and adduct excision

To complete ICL repair, the DSB caused by the nucleolytic incision step must be repaired by HR. As mentioned above, a DSB end-resection factor, CtIP initiates HR with the

MRN complex generating 3’ overhang single strand DNA (ssDNA). The unstable ssDNA is protected by RPA and the BRCA1 (FANCS) - PALB2 (FANCN) - BRCA2 (FANCD1) complex is recruited to sites of DNA damage, which promotes RAD51 loading. The

RAD51 coated ssDNA nucleofilaments invade into a homologous DNA template. After

DSB repair by HR, the remaining adduct is removed by nucleotide excision repair (NER) and the deubiquitinating enzyme, USP1-UAF1 complex lastly removes monoubiquitin from FANCD2-FANCI (33-35).

Figure 1.1. Model of PALB2 and BRCA2 working synergistically to stimulate homologous recombination (7)

Figure 1.2. Patient-derived BRCA1 mutations abolished the BRCA1-PALB 2 association (5)

Figure 1.3. HDR protein interactions and domains (6)

Figure 1.4. Pancreatic cancer initiation and progression (27)

Figure 1.5. Fanconi anemia/BRCA pathway (35)

CHAPTER 2

Palb2 pancreatic tumor model in comparison with Brca1/2 tumor mode ls

2.1 Introduction

Pancreatic cancer is often called a “silent killer” due to the lack of specific symptoms

during early stages of the disease. Pancreatic cancer is often diagnosed at a late stage causing limitation of treatment and extremely low survival rates. Previous studies have shown that some people who carry germline mutations of BRCA1 or BRCA2 developed pancreatic cancer, although BRCA1/2 are best known as breast and ovarian cancer susceptibility genes (24, 36). Hence, we cannot rule out the risk of pancreatic cancers in people with BRCA1/2 mutations. Recently, PALB2 was also identified as a breast and pancreatic cancer susceptibility gene. Exome sequencing analysis revealed truncating mutations of PALB2 in patients with familial pancreatic cancer (9, 10). PALB2 is known to play a role in DNA repair, especially in homologous recombination (HR) by cooperating with BRCA1 and BRCA2. PALB2 co-localizes with BRCA1 and BRCA2 at

DNA damage sites and functions as a linker protein that mediates BRCA1-BRCA2

15 interaction (2, 4, 5, 7, 37). Disruption of its binding to BRCA1 or BRCA2 cause defective

DNA repair, which could result in genome instability and tumor susceptibility. Indeed, peripheral blood karyotyping data showed that PALB2 mutant carriers have significantly increased chromosomal aberrations (38). In addition, cells with PALB2 silenced by siRNA showed a defect in repair of DNA double strand breaks as well as sensitivity to the inter-strand cross-linker, Mitomycin C (MMC) (2). These observations imp ly that

PALB2 is crit ical in maint ain ing genome integrit y through its interaction w ith BRCA1 and

BRCA2 during the DNA damage response. This notion is supported by the identification of

PALB2 mutations in patients cancers that inhibit its direct interaction with BRCA1 or

BRCA2, thus disrupting DNA repair (39). Furthermore, missense mutations within the

coiled-coil (CC) domain of BRCA1 that ablate interaction with the CC domain of PALB2

have been reported among familial breast cancer patients, and could also increase the risk of

pancreatic cancer (4). Taken together, this suggests that the role of the adaptor protein -

PALB2, may be crit ical for BRCA1/2-mediated tumor suppression.

Since germ-line mutations of BRCA1, BRCA2 and PALB2 have been found among familial

pancreatic cancer cases (9, 10, 24, 36, 40), we hypothesized that PALB2 plays a key role in

BRCA-mediated tumor suppression by connecting BRCA1 to BRCA2. Understanding the functions of PALB2 that suppress tumor development is therefore critical to develop novel and improved treatments against pancreatic tumors. To test this hypothesis, we inactivated

Palb2 specifically in the pancreatic duct epithelium and monitored whether these mice would develop pancreatic cancer.

Palb2 deletion with concomitant expression of mutant KrasG12D and p53R270H in pancreatic ducta l epithe lia l cells promotes the development of pancreatic ductal adenocarcinoma 16

(PDAC), the most common malignancy of the pancreas. Here, we describe a new Palb2 mouse model of pancreatic cancer and compare it to Brca1- and Brca2- defic ient pancreat ic tumor models. We also explored and examined how inactivation of the three different genes

(Palb2, Brca1 and Brca2) affects anticancer drug activity.

2.2. Material and Methods

2.2.1. Histological analysis

Dissected tissues were fixed in 10% neutral-buffered formalin solution for 24-48 hrs and transferred to 70% ethanol. Tissues were processed, embedded in paraffin, sectioned at

4µm onto positively charged slides, deparaffinized, rehydrated, and stained with hematoxylin and eosin.

2.2.2. Immunohistochemistry

For ER and PR staining, formalin fixed paraffin-embedded (FFPE) tumor tissue sections were deparaffinized in xylene and rehydrated in a graded alcohol series followed by quenching with 0.3% H2O2 for 30 minutes. Next, washed slides were heated in a pressure

cooker containing 1L Tr is-EDTA buffer (10mM Tris base [pH9.0], and 1mM EDTA

[pH8.0]) to unmask antigen for 15 minutes and cooled down for 30 minutes in an ice bath.

Slides were blocked in blocking solution (150µl goat serum in PBS) for an hour and incubated with diluted primary antibodies (ER (1:500, Santa Cruz, SC-542) and PR

(1:200, DAKO, A0098)) in blocking buffer overnight at 4°C. Slides were washed and incubated in diluted biotinylated secondary antibody solution for 30 minutes at room

17 temperature. Immunolabeling were detected using the VECTASTAIN ABC reagent kit

(Vector Labs) following standard protocols. Counterstaining with Harris hematoxylin

solution were performed after detection.

For amylase and CK19 immunohistochemistry, sections were stained using a Bond Rx

autostainer (Leica). Briefly, slides were baked at 65⁰C for 15 minutes and the automated

system performed dewaxing, rehydration, antigen retrieval, blocking, primary antibody

incubation, post primary antibody incubation, detection (DAB or RED), and

counterstaining using Bond reagents (Leica). Slides were then removed from the

machine, dehydrated through a graded alcohol series and xylene, mounted and

coverslipped. Antibodies for the following markers were diluted in antibody diluent

(Leica): amylase (1:400, CST 3796) and rat antibody- cytokeratin 19 (TROMA-III)

(1:150, Developmental Studies Hybridoma Bank, University of Iowa).

2.2.3. Establishment of primary pancreatic tumor cells

Mice showing overt pathological signs were euthanized and underwent autopsy. All

major organs were processed for histopathology. In addition, pieces of tumor tissue were

collected, cut into smaller pieces with a pair of scalpels and trypsinized. After

neutralization with complete medium (DMEM supplemented with 10% FBS, 100units

penicillin/100µg/mL streptomycin, 2mM L-Glutamine, and 0.25µg/ ml Plas moc in), the

chopped tumors were dispersed by passing through a syringe several times and placed

onto a gelatinized 10cm dish. Tumor cells were cultured at 37⁰C in 5% CO2/95% humidity until they were established.

2.2.4. Karyotype Analysis

Cells were incubated in medium with or without DNA damaging agents (Mitomycin C

40ng/ml or Olaparib 1µM) for 16hrs and treated with 0.05µg/ml KaryoMax colcemid

(GIBCO) for 2hrs. Cells were harvested, incubated in pre-warmed 0.56% KCL solution for 30 minutes at 37⁰C and fixed in Carnoy’s solution (75% methanol and 25% acetic acid). Metaphase spreads were prepared and stained in 0.5% Giemsa solution and analyzed on a Zeiss micr oscope w ith a 100X objective under oil.

2.2.5. Allograft assay

For subcutaneous injection of tumor cells into nude mice, cultured tumor cells (50~60% confluent) were harvested by trypsinization. After cell counting, cells were resuspended in 1% FBS in PBS solution. 0.3X106 cells/100µl were injected subcutaneously in the

dorsal side of the upper hind limb of nude mice. After 10 to 14 days, when the tumor size

was approximately 100mm3 in volume vehicles, MMC (5mg/kg) or Cisplatin (6mg/kg)

were injected into mice intraperitoneally. Tumor size was measured using calipers every alternate day after drug injection.

2.3. Results

2.3.1. Pancreas-specific deletion of Palb2, Brca1 or Brca2 early in development results in smaller pancreata

Deletion of the mouse Palb2 gene results in early embryonic lethality, as does Brca1 and

Brca2 knock-out, indicating that all three tumor suppressor gene products, Palb2, Brca1

19 and Brca2 are essentia l for embryonic via bilit y (41). To circumvent the embryonic lethality and to study the role of these tumor suppressors in pancreatic development and malignant transformation, we specifica lly deleted Palb2, Brca1 or Brca2 in the pancreas using Cre-LoxP recombination technique. For this purpose, we used conditional knockout alleles of Palb2 (Palb2fx2-3, obtained from Dr. Bing Xia, Cancer Institute of New

Jersey) (42), Brca1 (Brca1fx2) (43) and Brca2 (Brca2fx3-4, previously generated in our

laboratory), in combination with the well characterized Pdx1-Cre transgene that has been extensively used for modeling of PDACs in mice. The Pdx1-Cre transgene is expressed in the epithelial lineages of the embryonic pancreas (which includes both exocrine and endocrine lineages) and continues to be expressed throughout adulthood (44). We intercrossed heterozygous Palb2flex2-3, Brca1flex2 or Brca2flex3-4 anima ls to obta in

homozygous animals of respective genotypes. Homozygous Palb2fx/fx, Brca1fx/fx, or

Brca2fx/fx anima ls w ith Pdx1-Cre transgene were born at expected frequency (data not

shown). These animals developed into healthy adults with overall body size and weight

gain similar to the control littermates (data not shown). In the meantime, we dissected

pancreata from some of these homozygotes to analyze the histo-architecture of the

pancreata in detail. Surpr is ingly, pancreata from Palb2fx/fx; Pdx1 Cre, Brca1fx/fx; Pdx1 Cre and Brca2fx/fx; Pdx1 Cre animals were reduced in size and weight compared to the control

littermates [Figure 2.1.A. and 2.1.B.]. However, the overall histopathology of the

pancreata was normal and not significantly different from those of control pancreata.

Subsequently, we performed Southern blot analysis of genomic DNA from these

pancreata to determine the status of recombination of the conditional Brca1fx/fx and

Brca2fx/fx alleles. Southern blot analysis confirmed that the “floxed” condit iona l a lle les of

Brca1 and Brca2 were fully recombined in pancreata of heterozygous Brca1fx/+ and

Brca2fx/+ mice. However, no recombination product was detected in pancreata of

Brca1fx/fx and Brca2fx/fx anima ls [Figure 2.1.C.]. To verify that the lack of recombination

of the Brca- alleles is not due to the lack of Pdx1 Cre transgene expression in these

pancreata, we also bred the conditional Rosa26rlacz reporter allele into these animals. In contrast to the conditional Brca1 or Brca2 knock-out alle les , the conditional Rosa26rlacz

allele was fully recombined in Brca1fx/fx and Brca2fx/fx anima ls as demonstrated by staining of the pancreata with X-gal [Figure 2.1.C.]. In summary, deleting Palb2, Brca1 or Brca2 early during embryonic pancreatic development results in smaller pancreata

likely because pancreatic progenitor cells deficient in Palb2, Brca1 or Brca2 functions

are non-viable and eliminated.

2.3.2. Inactivation of Palb2, Brca1 or Brca2 in pancreatic ductal cells promotes the

development of PDAC

PALB2, BRCA1 and BRCA2 proteins are involved in DNA damage repair, primarily

through their roles in homologous recombination (HR). PALB2 is a linker pr ote in

physically and functionally connecting BRCA1 and BRCA2 during HR (2). In the

context of an intact p53-induced DNA damage checkpoint, the accumulation of

chromosomal abnormalities as a result of loss of PALB2, BRCA1 and BRCA2

culminates in cell death. Hence, for cells that have lost PALB2, BRCA1 or BRCA2 to

undergo neoplastic transformation, first, they would have to overcome the DNA damage

21 induced checkpoint by inactivation of p53. As mentioned above, pancreatic progenitor cells that lack Palb2, Brca1 or Brca2 functions are eliminated during embryonic development, presumably via p53-induced apoptosis. Most p53 mutations identified from

tumors are missense and typically affect the DNA binding domain. The R273H mutation

(R270H in the mouse) is one of the hot-spot mutations in the human p53 gene (45).

Previous reports showed that the mutant p53 R270H protein has a dominant-negative

inhibition effect on wild type p53. More specifically, heterozygous mutant allele of

p53R270H delayed transcriptional activation of its downstream target genes and inhibited

p53 dependent apoptosis (46). Therefore, we chose the “conditional knock-in” LSL-

p53R270H mutant allele, LSL- p53R270H, for our pancreatic tumor mouse models.

Activating mutations in the Kras gene (e.g., KrasG12D) are the most frequent mutations

found in human PDACs with some studies reporting a prevalence rate as high as 90% (25,

27). Also, in agreement with the hypothesis that KrasG12D mutations are likely to be

involved in PDAC initiation, these mutations are frequently found in early precursor

les ions of PDAC, such as pancreatic intraepithelial lesions (PanINs) (25). Hence, we

decided to delete Palb2, Brca1 or Brca2 concomitant with mutant KrasG12D expression - using a “conditional knock-in” mutant allele, LSL-KrasG12D, and henceforth simply

referred as KrasG12D (47).

We generated cohorts of animals that are Palb2fx/fx; KrasG12D; Pdx1 Cre (n=7), Brca1fx/fx;

KrasG12D; Pdx1 Cre (n=6) or Brca2fx/fx; KrasG12D; Pdx1 Cre (n=23). The heterogeneity,

distribution and progression of PanIN lesions into PDACs seen among these cohorts are

similar to those previously reported for KrasG12D; Pdx1 Cre anima ls. Therefore, deleting

22 only Palb2, Brca1 or Brca2 from the pancreata expressing mutant KrasG12D is not

sufficient to increase the incidence or shorten the latency of PanIN appearance; moreover,

among these animals, the PanINs do not seem to progress into PDAC any faster than in

animals expressing KrasG12D alone (data not shown).

Next, we deleted Palb2, Brca1 or Brca2 in pancreatic ductal cells in animals concomitantly expressing mutant KrasG12D and p53R270H. We hypothesized that

preventing apoptosis induced by p53 activation upon loss of Palb2, Brca1 or Brca2

functions may allow resulting genomic instability to accumulate in KrasG12D mutant pancreata. This, in turn, could help to promote and accelerate PDAC progression in these triple-mutant animals. Previously, Hingorani et al. reported a mouse model of PDAC in which p53R172H/+; KrasG12D; Pdx1-Cre anima ls developed PDAC with a median survival

R270H/+ G12D (T50) of 20 weeks (47). Similar ly, we generated our own p53 ; Kras ; Pdx1-Cre control cohort (now referred to as KPC mice) and in our hands they developed PDAC with a median survival of 24.5 weeks. In contrast, triple mutant animals that were

Palb2fx/fx; p53R270H/+; KrasG12D; Pdx1-Cre, Brca1fx/fx; p53R270H/+; KrasG12D; Pdx1-Cre or

Brca2fx/fx; p53R270H/+; KrasG12D; Pdx1-Cre (now referred to as Palb2-KPC, Brca1-KPC or

Brca2-KPC respectively) developed PDAC with a much shorter median survival of 10

weeks, 12 weeks and 13.5 weeks, respectively [Figure 2.2.A. and 2.2.B.]. Palb2-KPC

mice became moribund slightly sooner than Brca1-KPC or Brca2-KPC anima ls. One

possible explanation for the shorter latency could be because most of the Palb2-KPC

mice have tumor developing in the head of the pancreas where they are more likely to

grow into the bile-duct and cause jaundice [Figure 2.2.B]. 21 of 23 Palb2-KPC anima ls

23 had a solid tumor in the head of the pancreas and in 14 cases, the tumor caused blockage of the bile-duct. In summary, based on the tumor-free survival data, we can conclude that concomitant loss of p53 and Palb2, Brca1 or Brca2 tumor-suppressor functions cooperate

to dramatically augment tumorigenic potential of oncogenic mutations, such as KrasG12D.

PanINs are well-known precursor of PDAC. Classical PanIN precursor lesions within the

pancreatic ductules displayed characteristic mucinous epithelia with varying degree of

atypia. As the PanINs progressed into more advanced stages (PanIN-2 and 3), the degree

of atypia in these epithelia increased dramatically and they eventually transformed into

invasive PDAC. With the progression of the disease, the PanINs advanced into full-

blow n PDAC that eventually engulfed the entire pancreas and began to invade the nearby

organs within the peritoneal cavity, such as the duodenum and the spleen (25). We

compared the histology of the pancreatic tumors that developed in the triple-mutant

anima ls (Palb2-KPC, Brca1-KPC or Brca2-KPC) to that of the double-muta nt anima ls

(KPC). Histopathology of moribund mice identified solid tumor in pancreas with some

PanIN lesions. Acinar ductal metaplasia (ADM) is a precursor of PanIN lesions, and cells

undergoing ADM express both acinar (amylase) and ductal (cytokeratin 19) markers (25,

48). Immuno-hist ochemica l (IHC) ana lys is of amylase and cytokeratin 19 (CK19)

demonstrated ADM structures in PanIN lesions indicating that PDAC has progressed

from ADM induced PanIN lesions [Figure 2.2.D.]. The PDAC progression among Palb2-

KPC, Brca1-KPC or Brca2-KPC anima ls was indist inguis hable from those that

developed in KPC animals except that the tumors developed and progressed much faster.

2.3.3. Pancreatic cystic lesions resembling MCNs are unique to Palb2 and Brca1- mutant animals

As described earlier, Brca1-KPC animals developed PDAC via the classical PanIN route.

In addit ion, these anima ls invaria bly presented with cysts which were grossly visible

upon dissection. These cystic lesions were frequently numerous and multilocular, some

as large as two to three cm in size and yielded as much as a few milliliters of serous fluid

with hemorrhagic components and cellular debris [Figure 2.3.A.]. In a few instances, the

animals became moribund simply from the cysts which had enlarged to the extent that

they began to compress nearby organs causing great discomfort to the animals. As tumors

progressed, the cysts often became engulfed by the tumors and frequently several large

tumors merged to give rise to gigantic confluent tumors. Some of these cystic lesions

display the hallmarks of mucinous cystic neoplasm (MCN). These cysts were found in

the tail and body of pancreas and they were circumscribed by ovarian-like stroma with

wavy nuclei and often expressed steroid hormone receptors, namely estrogen receptor

(ER) and progesterone receptor (PR) (49, 50) [Figure 2.3.B.].

Unlike Brca1-KPC animals, KPC and Brca2-KPC did not show the cystic lesions.

However, of 23 cases, 6 of Palb2-KPC animals developed the cystic lesions which were surrounded by ER and PR positive ovarian-like stroma [Figure 2.3.B]. Based on these observations, in contrast to KPC and Brca2-KPC animals, Brca1-KPC and some of

Palb2-KPC animals develop MCNs which are cystic precursor lesions of PDAC, in addition to classical PanINs. Furthermore, while several Palb2-KPC tumors with cysts resemble Brca1-KPC more closely, most Palb2-KPC tumors without cysts are similar to

Brca2-KPC tumors. The mixture of Brca1-KPC and Brca2-KPC tumor types could be

because Palb2 functions as a linker protein between Brca1 and Brca2.

2.3.4. Primary tumor cells from Palb2-KPC, Brca1-KPC and Brca2-KPC solid

tumors exhibit hypersensitivity to DNA damaging agents

As stated earlier, Palb2, Brca1 and Brca2 play important roles in DNA repair, mainly in

the HR pathway (6). Therefore, deletions or hypomorphic mutations in these genes cause

genome instability resulting in ce lls w ith hypersensitivity to DNA damaging drugs such

as interstrand cross linking agents (ICLs agents) (e.g., Mitomycin C and Cisplatin) and

poly ADP ribose polymerase inhibitors (PARP inhibitors) (e.g., Olaparib). Based on

previous reports, we expected chromosomal instability and hypersensitivity of primary

tumor cells isolated from Palb2-KPC, Brca1-KPC, or Brca2-KPC solid tumors to DNA

damaging agents. To verify this, we generated multiple cell lines from each mouse model

(KPC, Palb2-KPC, Brca1-KPC, and Brca2-KPC) and performed karyotype analysis. As

expected, metaphases from Palb2-, Brca1- or Brca2- deleted cells showed increased

number of chromosomal aberration even without DNA damaging drug treatment

compared to KPC control cells. After Mitomycin C (MMC) or Olaparib treatment, the

number of chromosomal abnormalities of Palb2-, Brca1- or Brca2- deleted cells were

dramatically increased. Metaphases from those cells had various types of chromosomal

aberration including breaks, gaps and exchanges [Figure 2.4.A and 2.4.B]. Although

Palb2, Brca1 and Brca2 play roles together in the HR pathway by forming a complex, it

remains unclear how the deletion of each individual gene affects their sensitivity to

26 different DNA damaging agents. To test this, we compared drug sens it ivit y of tumor cells from each group (KPC, Palb2-KPC, Brca1-KPC, and Brca2-KPC). Palb2-, Brca1- or

Brca2- deleted cells showed hypersensitivity to MMC, Cisplatin and Olaparib to a similar

extent but KPC cells were resistant to those drugs. However, we did not observe differences of sensitivity to other chemotherapy drugs such as Paclitaxel and Fluorouracil

(5-FU) among 4 different groups of tumor cells (KPC, Palb2-KPC, Brca1-KPC, and

Brca2-KPC) [Figure 2.4.C].

2.3.5. Inte rstrand crosslinking agents inhibit Palb2-KPC, Brca1-KPC and Brca2-

KPC tumor growth in vivo

As described above, Palb2-, Brca1- or Brca2- deleted tumor cells exhibit dramatically elevated sensitivity to DNA damaging drugs. Based on this observation, we hypothesized that the DNA damaging drugs could inhibit growth of Palb2-, Brca1- or Brca2- deleted tumor cells specifically. To test this hypothesis, we performed allograft assays. KPC,

Palb2-KPC, Brca2-KPC pancreatic tumor cells were injected subcutaneously in the dorsal side of the upper hind limb of nude mice. When the subcutaneous tumor sizes reached 100mm3, MMC or Cisplatin were injected intraperitoneally. Regardless of genotype, there was no significant difference in tumor growth in vehic le treated group.

Consistent with the in vitro cell studies, MMC or Cisplatin treated Palb2-, Brca1- or

Brca2- deleted tumors grew much slower than KPC tumors [Figure 3.5.A and 3.5.B]. We also confirmed MMC drug efficacy using the Palb2-KPC endogenous model. MMC treated Palb2-KPC anima ls showed clear prolonging of survival [Figure 3.5.C.].

2.4. Discussion

Germline mutations in BRCA1 or BRCA2 genes increase pancreatic cancer risks as well as breast and ovarian cancer (24, 36, 51, 52). As PALB2 mutations have been observed in families with breast and pancreatic cancer, the PALB2 gene is a lso a cancer susceptibility gene (9, 10). Proteins encoded by these three different pancreatic tumor susceptibility genes (PALB2, BRCA1 and BRCA2) cooperate during HR by forming a complex. In the complex, PALB2 physically intereacts with and connects BRCA1 and BRCA2 (2). Thus,

we hypothesized that the role of the PALB2 linker protein, may be critical for BRCA1/2-

mediated tumor suppression. To test this hypothesis, we generated a Palb2-defic ient

pancreatic tumor mouse model. Here, we show that loss of Palb2 cooperates with mutant

p53R270H and KrasG12D in the development of PDACs, as do the Brca1- and Brca2- KPC pancreatic tumor mouse models previously generated in our lab.

As for Brca1 and Brca2, constitutive knock out of Palb2 in mice leads to embryonic lethality, indicating that the full length gene products are essential for cellular viability and proliferation in the early embryo. In this study, we observed that pancreas specific deletion of Palb2, Brca1 or Brca2 results in decreased organ size. Consistent with this observation, recombination of the floxed alleles was not detectable in homozygous conditional animals suggesting that loss of Palb2, Brca1 or Brca2 in early pancreatic progenitor cells is incompatible with cell viability. Eventually, the organ will repopulate from stem cells in which recombination of the conditional alleles has not taken place. The essentia l role of Palb2, Brca1 and Brca2 genes in pancreas explains why mutations in these genes are associated with an increased risk of hereditary pancreatic cancer.

Palb2-KPC mice developed PDACs and became moribund with shorter latency than

Brca1-KPC or Brca2-KPC, possibly because >90% of Palb2-KPC tumors originated in

the head of the pancreas obstructing the bile duct. We have seen a similar phenotype with

Brca2Δex3-KPC mice (data not shown), where at least 70% of animals presented with

jaundice because of bile-duct blockage from tumors located in the head of the pancreas. It

has been reported that patient derived missense mutations located in Exon3 of BRCA2

abolished or dramatically reduced the PALB2-BRCA2 interaction (1). Therefore, the

specif ic tumor or igin in the head of the pancreas in Palb2-KPC and Brca2Δex3-KPC mice might be due to ablation of PALB2-BRCA2 interaction.

Brca1-KPC and Brca2-KPC tumors were quite different in terms of formation of cystic les ions. While a lmost a ll Brca1-KPC animals developed large cysts with PanIN derived

PDAC, no cystic lesions were observed in Brca2-KPC and KPC mice. Interestingly,

Palb2-KPC tumors showed a mixture of Brca1-KPC and Brca2-KPC tumor phenotypes regarding the presence of cysts. 6 of 23 Palb2-KPC tumors had large cysts with PDAC, which closely resemble the Brca1-KPC tumors. However, the remainder of the tumors cyst-free, like Brca2-KPC or KPC tumors. This implies the role of PALB2, as a linker protein between BRCA1 and BRCA2, in BRCA1/2 mediated tumor suppression.

Loss of heterozygosity (LOH) is often detected in tumors developing in BRCAs mutant carriers indicating loss of the w ild type alle le is a cr it ica l step t o init iate carcinogenesis

(53). However, we did not observe LOH in heterozygous Brca1- or Brca2-KPC mouse models. Among pancreatic tumor patients w it h PALB2 and BRCA mutations, LOH has also been reported (54). Therefore, using our Palb2-KPC mouse model, we tested

29 whether LOH is required for pancreatic tumor development in Palb2fx/+; KPC animals.

Like Brca1fx/+; KPC or Brca2fx/+; KPC mice, tumor latency of Palb2fx/+; KPC anima ls

was similar with KPC animals and the tumors maintained the intact wild type allele,

which supports funct iona l HR [Figure 2.4.].

To evaluate the inhibition of proliferation of tumor cells, drug sensitivity was determined

by MTT assay. Although we expected generally elevated sensitivity to DNA damaging

drugs from Palb2-, Brca1- or Brca2- deficient tumor cells, any possible differences in sensitivity to specific drugs among Palb2-, Brca1- or Brca2- deficient tumor cells should be determined. In this study, using 10 different primary tumor cells (2X KPC, 2X

Brca1fx/fx; KPC, 2X Brca2 fx/fx; KPC, 2X Palb2 fx/fx; KPC and 2X Palb2 fx/+; KPC), we measured the degree of sensitivity to different chemotherapy drugs. Palb2-, Brca1- or

Brca2- defic ient tumor cells in comparison to KPC or Palb2 fx/+; KPC tumor cells are exquisitely sensitive to DNA damage inducing agents including olaparib, MMC and

Cis plat in. However, these cells did not show differential sens it ivity to non-DNA damage-

inducing drugs (5-Fluorouracil and Paclitaxel) that are routinely used in PDAC therapy

regimens of PDAC. Based on the in-vitro experiments, we also confirmed efficacy of

MMC and cisplatin treatment in vivo. In summary, these preclinical results show DNA-

damaging agents are effective and may be particularly useful in the treatment of PALB2-,

BRCA1- or BRCA2- deficient pancreatic tumors.

Figure 2.1. Pancreas-specific deletion of Palb2, Brca1 or Brca2 early in development

results in smalle r pancre ata (A) Macroscopic views of pancreata of Palb2c/+; Pdx1 Cre and Palb2c/c; Pdx1 Cre. (B) Morphometry analysis on Brca1c/c; Pdx1 Cre and Brca2c/c;

Pdx1 Cre showed that Brca1- or Brca2- deleted pancreata volume were smaller than age

matched controls. (C) Detection of Cre-mediated recombination activity in pancreas

using Pdx1 Cre; Rosa26-LacZ mice. (D) Southern blot analysis detected recombined

Brca1 conditional allele in pancreas of Brca1c/+; Pdx1 Cre anima ls. Condit iona l alle le

from pancreas from Brca1c/-; Pdx1 Cre animals remained unrecombined (T: tail genomic

DNA and P: pancreatic genomic DNA).

CONTINUED

Figure 2.1. CONTINUED Pancreas-specific deletion of Palb2, Brca1 or Brca2 early in development results in smaller pancreata

e 1.5 m u l o v

s 1.0 a e r

c n a p 0.5 e

v i t a l e

R 0.0 l d d o e e tr t t n le le o e e C d d 1 2 a a rc rc C B B

CONTINUED

Figure 2.2. Concomitant expression of mutant KrasG12D and p53R270H cooperate with

Palb2, Brca1 or Brca2 loss in the pancreatic ductal cells to promote PDAC tumorigenesis (A) Kaplan-Me ier tumor-free survival curve of KPC, Brca1-KPC, Palb2-

KPC and Brca2-KPC. (B) Gross appearance of pancreatic tumor of Palb2-KPC anima l.

Large solid tumor in the head of pancreas growing into bile duct. (C) H&E (Hematoxylin and eosin) analysis of the histopathology of PDAC of KPC, Brca1-KPC, Palb2-KPC and

Brca2-KPC. (D) Immunohistochemistry double staining of amylase and cytokeratin 19 detected acinar ductal metaplasia (ADM) lesions.

CONTINUED

Figure 2.2. CONTINUED Concomitant expression of mutant KrasG12D and p53R270H cooperate with Palb2, Brca1 or Brca2 loss in the pancreatic ductal cells to promote

PDAC tumorigenesis A

CONTINUED

Figure 2.2. CONTINUED Concomitant expression of mutant KrasG12D and p53R270H cooperate with Palb2, Brca1 or Brca2 loss in the pancreatic ductal cells to promote

PDAC tumorigenesis C

Figure 2.3. Pancreatic cystic lesions resembling MCNs are unique to Palb2 and

Brca1-mutant animals (A) Gross morphology pictures of a primary tumor in pancreas of

Brca1-KPC and Palb2-KPC. Yellow arrow indicates the presence of a large cyst in

pancreas. (B) H&E staining of cystic lesions in Brca1-KPC and Palb2-KPC pancreatic

tumor. Immunohistochemistry for ER and PR showed epithelial cells associated with

ovarian-like stroma.

CONTINUED

Figure 2.3. CONTINUED Pancreatic cystic lesions resembling MCNs are unique to

Palb2 and Brca1-mutant animals

CONTINUED 37

Figure 2.4. Primary tumor cells from Palb2-KPC, Brca1-KPC and Brca2-KPC solid tumors exhibit hypersensitivity to DNA damaging agents (A, B) Karyotype analysis.

Palb2-KPC, Brca1-KPC and Brca2-KPC pancreatic tumor cells showed increased

sensitivity compared to KPC tumor cells to MMC and Olaparib. (C) Drugs toxicity was

measured by MTT assay. Each drug was treated for 72hrs before MTT was carried out.

For Olaparib treatment, due to short half-life, fresh drug media was changed every 24hrs.

CONTINUED

Figure 2.4. CONTINUED Primary tumor cells from Palb2-KPC, Brca1-KPC and

Brca2-KPC solid tumors exhibit hypersensitivity to DNA damaging agents A

CONTINUED

Figure 2.4. CONTINUED Primary tumor cells from Palb2-KPC, Brca1-KPC and

Brca2-KPC solid tumors exhibit hypersensitivity to DNA damaging agents

Figure 2.5. Interstrand crosslinking agents inhibit Palb2-KPC and Brca2-KPC tumor

growth in vivo (A, B) Growth curves of allograft tumors in nude mouse models. MMC

and Cisplatin treatment inhibited growth of Palb2-KPC, Brca1-KPC and Brca2-KPC

tumors. (C) Kaplan-Meier tumor-free survival curve of Palb2-KPC with or without

MMC. MMC treatment prolonged survival of Palb2-KPC animals (5mg/kg, every 3

weeks injection)

CONTINUED

Figure 2.5. CONTINUED Interstrand crosslinking agents inhibit Palb2-KPC and

Brca2-KPC tumor growth in vivo A

CONTINUED

Figure 2.5. CONTINUED Interstrand crosslinking agents inhibit Palb2-KPC and

Brca2-KPC tumor growth in vivo C

CHAPTER 3

Ablation of BRCA1-PALB2 interaction phenocopies Fanconi Anemia

3.1. Abstract

Germline mutations of BRCA1 or BRCA2 genes confer an increased risk of developing

breast, ovarian and pancreatic cancer. These genes are the best characterized breast

cancer susceptibility genes and are known to be crucial components in DNA damage

repair, specifically homologous recombination (HR). PALB2 links BRCA1 and BRCA2

in HR of DNA double strand breaks (DSBs). PALB2 co-localizes with BRCA1 and

BRCA2 at sites of DNA damage and is thought to act as an adaptor protein that mediates

the BRCA1-BRCA2 interaction. Indeed, prior studies showed that BRCA2 interacts with

PALB2 through its C-terminal WD40 domains. BRCA2 contains BRC repeats which can recruit a central protein for HR, the Rad51 recombinase. Recently, mutations in the

PALB2 gene were reported among breast and pancreatic cancer patients. Furthermore, missense mutations in the coiled-coil domain of BRCA1 (M1400V, L1407P and

M1411T) that mediate the interaction with PALB2 have been reported among familial

breast cancer patients. Therefore, we hypothesized that the interaction between BRCA1

44 and PALB2 is critical for BRCA1-mediated HR and tumor suppression. To test this hypothesis, we generated Brca1 L1363P mutant mice (equivalent to the human BRCA1

L1407P variant). Mouse embryonic fibroblasts (MEFs) from homozygous Brca1 L1363P mutant animals exhibit hypersensitivity to DNA damaging agents (MMC, PARP- inhibitor and ionizing radiation (IR)), and following IR mutant cells fail to recruit the

Rad51 recombinase to sites of DNA damage, implying a defect of DSBs repair by HR.

Accumulation of unresolved DNA damage induces hyperactivation of p53 and its downstream target p21 in Brca1L1363P/L1363P primary MEFs resulting in impa ired proliferation and premature senescence. While Brca1 null mice are early embryonic lethal, homozygous Brca1L1363P/L1363P mice are viable. However, mutant mice exhibit

growth retardation to various extents. Some mutant animals were extremely small,

developed aplastic anemia and died within a month. All other Brca1L1363P/L1363P anima ls developed T-acute lymphoblastic leukemia (T-ALL) with an average latency of 3 months.

Interestingly, the majority of T-ALL from Brca1L1363P/L1363P animals acquired activating

Notch1 mutations. The phenotypes observed in mutant cells and mice recapitulate clinical phenotypes seen in FA.

3.2. Introduction

Fanconi Anemia (FA) is a rare autosomal recessive or X-linked genetic disease caused by biallelic mutations in one of the FA genes. Until now, 21 FA genes have been ident if ied and proteins encoded by these genes are involved in DNA repair and replication.

Chromosomal fragility caused by a defective DNA repair pathway in FA cells lead to

45 heterogeneous clinical phenotypes. One of the key features of FA phenotypes is early- onset bone marrow failure (BMF). Accumulation of unrepaired DNA lesions activates a pro-apoptotic pathway, which induces hematopoietic stem cell (HSC) depletion. Thus, most FA patients develop BMF to various extents. Another hallmark of FA is hypersensitivity to interstrand cross linking agents (ICL) such as Mitomycin C (MMC),

Diepoxybutane (DEB) and Cisplatin suggesting that FA genes are essential for ICL repair.

Accordingly, sensitivity of fibroblasts to ICL agents is used for FA disease diagnosis. In addition, FA patients also develop congenital abnormalities including short stature, abnormal thumbs, microcephaly, hyper-/hypo-pigmentation (café au lait spots) and exhibit increased risk of cancers (30, 34, 55).

ICLs are very toxic to the cell as they are difficult to repair because the two DNA strands are covalently linked, preventing DNA replication, transcription and cell division. To resolve deleterious ICLs, cells evolved the FA/BRCA pathway in which three classic

DNA repair pathways are cooperatively combined; nucleotide excision repair (NER), translesion synthesis (TLS), and homologous recombination (HR). Once cells sense ICL mediated stalled replication fork, the FA core complex that is composed of 8 FA proteins

(FANCA/B/C/E/F/G/L/M) is recruited to the ICL lesion and monoubiquitinates FANCD2 and its binding partner, FANCI. The monoubiquitination of FANCD2-FANCI complex is a central step in the FA pathway, which promotes downstream nucleolytic incisions and

TLS. Double-strand breaks (DSBs) created by nucleolyt ic inc is ion are then repaired by

HR. Among FA genes, BRCA1/FANCS, BRCA2/FANCD1, PALB2/FANCN and

RAD51C/FANCO are critical for HR (31, 34, 35). BRCA1 is localized to DSBs by

46 interaction with RAP80-Abraxas through its C-terminal BRCT domain, recruiting

PALB2 and BRCA2 sequentially (56). At the site of the DSB, they form a complex

functioning as a scaffold to recruit the RAD51 recombinase, a central protein for HR. In

the BRCA1-PALB2-BRCA2 complex, PALB2 acts as the adaptor protein between

BRCA1 and BRCA2. BRCA1 interacts with PALB2 through its coiled-coil domain and

PALB2 binds to BRCA2 through its C-terminal WD40-domain. BRCA2 has several BRC

repeats that can interact with RAD51 (4-6).

It is well-known that monoallelic mutat ions in the BRCA1 gene predispose women to

breast and ovarian cancer (52). However, biallelic BRCA1 mutations were recently

reported in two patients with FA characteristics establishing BRCA1 as a new Fanconi

Anemia (FA) gene (FANCS) (51, 57). Both patients carried a deleterious BRCA1

mutation on one allele and a missense mutation within the C-terminal BRCT domain on

the other allele. Although BMF is a major hallmark of FA, neither of the two patients

showed spontaneous BMF, but had ovarian and breast cancer (51, 57). Of note, our lab

reported previously on the phenotypes of two Brca1 knock-in mouse models; Brca1

S1598F within the first BRCT repeat and a truncating mutation in Brca1 exon11 (58, 59).

While both of these animals showed increased spontaneous tumor development including

mammary tumors, they did not exhibit increased mortality due to BMF.

It has previously been reported that tumor associated missense mutations in the coiled-

coil domain of BRCA1 ( i.e. M1400V, L1407P and M1411T) ablate the BRCA1-PALB2

interaction (4). Based on these observations, we considered that whether PALB2

regulates and/or mediates the BRCA1 tumor suppressor activity. To test this hypothesis

47 and to examine the role of the BRCA1-PALB2 interaction in normal and malignant development, we have used a knock-in strategy and generated cells and mice that are defective for the BRCA1-PALB2 interaction (The human BRCA1 L1407P mutation corresponds to Brca1 L1363P in mice). Surprisingly, the phenotypes observed in mutant cells and mice closely resemble clinical features of FA patients. Until now, to study FA, many researchers have generated cell lines and mouse models by bia lle lic de let ion of FA genes. However, although cells from FA patients or from FA mouse model showed sensitivity to ICL agents, FA mouse models do not recapitulate clinical phenotypes of FA disease such as spontaneous BMF. Here, we report the first mouse model of FA that fully recapitulates FA. Therefore, this mouse model can provide insights for developing new therapeutic targets to treat FA-patients.

3.3. Material and Me thods .

3.3.1. Targeted mutagenesis and mouse generation

The homology arms of the Brca1 L1363P targeting constructs were derived from subcloned fragments of murine 129/Sv genomic DNA. The leucine at pos it ion 1363 residue in exon 13 was changed to proline by site directed mutagenesis. In the final constructs, the homology fragment was interrupted in intron 12 by insertion of a loxP- flanked neomycin selection marker cassette that contains a SpeI restrict ion s ite. In addition, a thymidine kinase cassette was included in the construct as a negative selection marker. The targeting construct was linearized with NotI and electroporated into 129/Sv embryonic stem (ES) cells. After drug selection, genomic DNA from drug-resistant ES

48 clones was digested with SpeI and correctly targeted heterozygous ES subclones

(Brca1L1363P-neo/+) were ident if ied by Southern blotting. To produce chimeric mice, the

targeted ES cells were injected into C57BL/6 blastocysts. Chimeric males were crossed

to wildtype females to obtain heterozygous animals (Brca1L1363P-neo/+). The loxP-flanked neomycin cassette was excised from the targeted allele by mating Brca1L1363P-neo/+

animals with ROSA-Cre transgenic mice to obtain Brca1L1363P/+ mice.

3.3.2. Establishment of mouse embryonic fibroblasts

Heterozygous Brca1L1363P/+ animals were intercrossed and pregnant females were

euthanized at E.13.5 to dissect the embryos. After removal of the head and evisceration,

the remainder of the embryo was finely minced, trypsinized and neutralized. The cells

were dispersed by passing through a syringe several times and placed into a 15ml tube.

Cells in the top part of the tube were collected, leaving behind the large chunks at the

bottom, and plated onto gelatinized 10cm dishes (passage #0). pMEFs were cultured in

DMEM supplemented with 10% FBS, 100 units penicillin/100µg/mL streptomycin, 2mM

L-Glutamine, and 0.25µg/ml Plasmocin at 37 °C in 5% CO2/95% humidity. To establish immortalized MEFs (immMEFs), early passage pMEFs (passage #2 or #3 were

transfected with SV40 large T antigen plasmid using Lipofectamine 2000 (Invitrogen).

3.3.3. Immunofluorescence staining

MEFs were grown on poly-L-lysine coated glass coverslips and subjected to 10Gy of

ionizing radiation (IR). Cells were fixed in 4% ice cold paraformaldehyde/PBS (PFA) for

15minutes at 1hr after irradiation (10Gy), permeabilized in 0.2% TritonX-100/PBS for 10 minutes and blocked in 5% BSA/PBS for 30 minutes. For pericentrin and α-tubulin double immunostaining, ice-cold 100% methanol was used for fixation. Cells were then stained with diluted primary antibodies, ABRAXAS 1:3000, BRCA1 1:500, RAD51

(Novus biological, NBP1-90983, 1:300) and γH2AX (Millipore, Cat# 05-636, 1:5000),

Pericentrin (Abcam, Cat#ab4448, 1:250) and α-tubulin (Sigma, Cat# T6199, 1:1000) for

an hour at room temperature. The stained cells were washed three times in 1XPBS-T

(0.1% tween 20), incubated with Alexa Fluor 594 goat anti-rabbit or Alexa Fluor 488

goat anti-mouse (Invitrogen, 1:400), stained with Hoechst 33342 (Life technologies, REF

H3570) and then mounted onto a glass slide with Aqua-Poly/Mount medium

(Polysciences Inc.).

3.3.4. Co-immunoprecipitaton

pOZ-mouse PALB2 FLAG-HA or empty vector was transfected into immMEFs using

Jet-pei transfection reagents (Polyplus). After 48hrs incubation, cells were lysed in low

salt NP40 lysis buffer (10mM Hepes, pH7.6, 0.25M NaCl, 0.1% NP40, 5mM EDTA,

10% Glycerol) on ice for 20 minutes, followed by centrifugation at 13,000 rpm for

10minutes at 4°C. 500µg of lysates were incubated with Brca1 antibody or HA-magnetic

beads overnight and the precipitates were resolved on 6% SDS-poly acrylamide gels.

Transferred blots were probed with HA (Roche, Cat# 11-867-423-001, 1:1000) or

BRCA1 (1:2000) antibody.

3.3.5. Cytogenetic Analysis

Cells were incubated in medium with or without DNA damaging agents (Mitomycin C

40ng/ml or Olaparib 1µM) for 16hrs and treated with 0.05µg/ml KaryoMax colcemid

(GIBCO) for 2hrs. Cells were harvested, incubated in pre-warmed 0.56% KCL solution for 30minutes at 37°C and fixed in Carnoy’s solution (75% methanol and 25% acetic acid). Metaphase spreads were prepared and stained in 0.5% Giemsa solution and analyzed on a Zeiss microscope w ith a 100X objective under oil.

3.3.6. DR-GFP assay pMEFs carrying the DR-GFP reporter in the Pim1 locus were generated. To induce a clean double-strand break, the I-SceI expression vector or empty vector was electroporated at 230V, 930µF by using a Bio-Rad genepulsarII. After 48 hours of transfection, cells were harvested and GFP positive cells were counted by flow cytometry.

3.3.7. Histological analysis

Tissue samples were collected from euthanized animals, fixed in 10% formalin for

24~48hrs and embedded in paraffin. Tissues were cut into 4µm sections and stained with hematoxylin and eosin.

3.3.8. Annexin V analysis

Mice (1month of age) were irradiated (5Gy) and thymi were collected 4 hours after irradiat ion. thymi were placed on 70µm cell strainer atop a 50ml tube and crushed using a

51 plunger of a small syringe. The flow through cells were washed and resuspended in PBS.

1X106 cells were stained with Annexin V-FITC (Trevigen) and Annexin-V pos it ive cells were analysed by flow cytometry, us ing a BD LSR II.

3.3.9. Colony Forming Assay

Bone marrow cells were flushed from the hind limb of one month old mice and 1X105

cells were seeded in 30mm dishes in complete Methocult medium (Stem Cell

Technologies, M3434). Cells were cultured at 37°C in 5% CO2 for 7–10 days and the

number of colonies were counted.

3.3.10. Immunohistochemistry

Paraffin embedded tissue sections were generated and stained using a Bone Rx

autostainer (Leica). Brief ly, slides were baked at 65°C for 15minutes and software

automatically performed dewaxing, rehydration, antigen retrieval, blocking, primary

antibody incubation, post primary antibody incubation, detection (DAB), and

counterstaining using Bond reagents (Leica). Samples were then manually dehydrated through a graded alcohol series and xylene and mounted. CD3 (Abcam, cat#ab166669,

1:150) and GP100 (Abcam, cat#ab137078, 1:300) antibodies were diluted in antibody

diluents (Leica)

3.3.11. Quantitative Real-Time PCR

Total RNA was isolated from bone marrow of one month old mice and cDNA was

synthesized via SuperScript III Reverse Transcriptase (Invitrogen). TaqMan® gene

expression assay was used for qRT-PCR (p21 Mm00432448_ m1 and Gapdh.

M99999915_g1).

3.3.12. Flow cytometry

Thymi and femurs were collected in Eppendorf tubes containing RPMI media. Thymi

were mechanically dissociated through a 70 µm cell strainer to obtain single cell

suspensions. Bone marrow cells were physically separated from the femur using PBS

wash and were passed through a 70 µm cell strainer to obtain single cell suspensions. Red

blood cell (RBC) lysing buffer was used to lyse RBCs. After washing with PBS, cells

were counted using hematocytometer. Cells (2x106) were pipetted for a flow panel.

Conjugated antibodies for CD3-FITC (Cat# 553062), CD4-FITC (Cat# 553047),

TER119-FITC (Cat# 557915), Gr1-FITC (Cat# 553127), CD117-PE-Cy7 (Cat# 558163),

CD4-APC (Cat# 553051), CD4-APC-Cy7 (Cat# 552051), and CD44-PE (Cat# 553134) were purchased from BD BioSciences. Conjugated antibodies for CD11b-PerCP/Cy5.5

(Cat# 101228), Sca1-BV421 (Cat# 108128), CD69-FITC (Cat # 104506), Gr1-AF700

(Cat# 108422), CD25-APC (Cat# 102012), CD8-BV421 (Cat# 100738), TCRβ-PE-Cy7

(Cat# 109222), and NK1.1-PerCP/Cy5.5 (Cat# 108728) were purchased from BioLegend.

Conjugated antibodies for CD5-FITC (REF# 11-0051-83) and CD34-AF700 (REF# 56-

0341-82) were purchased from e Bioscience. All flow cytometry was performed using a

BD LSR II. The results were analyzed using Flowjo.

3.3.13. Genome Sequencing

Genomic sequencing was performed on DNA isolated from formalin-fixed paraffin-

embedded (FFPE) sections of mouse thymic lymphomas and on blood samples. An

amplicon-based panel was custom-designed that targeted 29 genes previously known to

be mutated in human T-lymphoblastic lymphoma/acute lymphoblastic leukemia or in

mouse models of T-ALL, including the entire coding regions of Notch1, Phf6, Pten, the

Ras genes and Trp53 as well as the BRCT and PALB2-binding domains of Brca1 and the

corresponding interacting domain on Palb2. Sequencing libraries were constructed using

the TruSeq Custom Amplicon Low Input kit (Illumina, San Diego, CA) and sequencing

performed on the Miseq platform using Reagent kit v2/500 cycles (Illumina). A mean

read depths of 600X was achieved with a demonstrated sensitivity of 3% variant allele

fraction (VAF) established by comparison with non-tumor samples and by bioinformatics

criteria. Analysis was performed using Miseq reporter (Illumina) and NextGEne software

(SoftGenetics, State College, PA).

3.4 Results

3.4.1. Brca1 L1363P mouse generation

To examine whether the BRCA1-PALB2 interaction is required for BRCA1 mediated

HDR and tumor suppression, we generated a mouse model expressing a breast cancer 54 associated BRCA1 mutation L1407P (L1363P in mice) that was shown to ablate the binding of BRCA1 to PALB2 (4). The Brca1 L1363P-neo construct contained the

L1363P mutation in exon 13 and a loxP-flanked selection marker cassette in intron 12

(Figure 3.1.A.). The targeting construct was electroporated into wild type 129/SV mouse

embryonic stem (ES) cells and neomycin resistant colonies which had undergone

homologous integration were identified by Southern blot analysis (Figure 3.1.B.). The

targeted ES cells (Brca1L1363P-neo/+) were injected into C57BL/6 blastocysts to obtain

chimeric mice and these mice were bred to produce heterozygous animals. The loxP-

flanked neomycin cassette was removed from the targeted allele through crossing it to

Rosa Cre mice. Genotyping for Brca1 L1363P was performed by PCR and the s ingle

point mutation was confirmed by direct sequencing (Figure 3.1.C. and 3.1.D.).

Heterozygous animals were intercrossed and homozygous Brca1L1363P/L1363P were born with the expected Mendelian frequency. Although homozygous mutant mice are viable, they are considerably smaller in size compared to littermate controls (Figure 2A and 2B).

In addition, Brca1L1363P/L1363P embryos and placentas at E.13.5 were evidently smaller than the Brca1+/+ or Brca1L1363P/+ littermates indicating that the BRCA1 L1363P mutation adversely affects embryonic development (data not shown).

3.4.2. Brca1 L1363P/L1363P MEFs are defective in HDR

The steady-state levels of the mutant Brca1 L1363P protein were similar to wild type

Brca1 protein. Both wild type and mutant BRCA1 proteins become hyper-phosphorylated

after exposure to ionizing irradiation (IR), suggesting that damage-induced

55 phosphorylation of BRCA1 which is an early event in the DNA damage response is

independent of the BRCA1-PALB2 interaction (Figure 3.2.A.). The BRCA1 and BARD1

proteins form a heterodimer through their N-terminal RING domain, which stabilizes

both proteins (60-62). BARD1 proteins were stable and comparably phosphorylated upon

IR in Brca1+/+, Brca1L1363P/ +, Brca1L1363P/L1363P immortalized mouse embryonic

fibroblasts (immMEFs) (Figure 3.2.A.). To confirm whether the BRCA1 L1363P

mutation ablates its interaction with PALB2, we performed reciprocal

immunoprecipitation (IP). Flag-HA tagged mouse wild type f ull lengt h Palb2 cDNA

plasmid was transfected into Brca1+/+ and Brca1L1363P/ L1363P immMEFs and whole cells lysates from Brca1+/+ or Brca1L1363P/ L1363P were immunoprecipitated (IPed) with a Brca1 antibody or HA magnetic beads and then immunoblotted with HA or Brca1 antibodies respectively. As shown in Figure 3.2.B., wild type Brca1 efficiently interacted with HA-

PALB2, whereas mutant Brca1 L1363P failed to do so, indicat ing that the mutant

BRCA1 L1363P does not bind to PALB2.

BRCA1 recruitment to sites of DNA damage is mediated by the RAP80-Abraxas

comple x through binding of phosphorylated Abraxas to the tandem repeats of BRCT

repeats of BRCA1 (56, 63-65). Therefore, mutant BRCA1-BRCT repeat cells

(Brca1S1598F/S1598F) failed to assemble BRCA1 foci after irradiation (IR) (58). We

hypothesized that mutant Brca1 L1363P can still form irradiation induced foci (IRIF) since mutant Brca1 L1363P has intact BRCT repeats. Indeed, no significant differences were observed in Abraxas and Brca1 IRIF between Brca1+/+ and Brca1L1363P/L1363P

immMEFs (Figure 3.2.D. and 3.2.E.). Efficient Brca1 foci formation suggests that

56 ablation of the Brca1-Palb2 interaction does not influence binding of Abraxas to the

BRCT repeats of Brca1. To further test if the BRCT domain of mutant Brca1 L1363P

protein can associate with its binding partners, we immunoprecipitated Brip1 from

Brca1+/+ and Brca1L1363P/L1363P immME Fs, and immunoblotted for Brca1. Both wild type and mutant Brca1 L1363P proteins were co-IPed by Brip1 (data not shown) indicating

phosphor-ligand binding to the BRCT repeats occurs independent of the Brca1/Palb2

interaction.

It has been reported that BRCA1 associates with BRCA2 through PALB2, and the

BRCA1-PALB2 interaction is required for BRCA2 mediated RAD51 localization, which

is an essential step in HDR (2, 4, 66). Thus, we tested whether ablation of the BRCA1-

PALB2 interaction would affect recruitment of RAD51 to the sites of DNA damage. As expected, Rad51 foci formation was markedly impaired in Brca1L1363P/L1363P immMEFs ,

although there was no difference in recruitment of wild type and mutant Brca1 proteins to

sites of DNA damage (Figure 3.2.F and 3.2.G.). The impaired IRIF formation for Rad51

in Brca1L1363P/L1363P immMEFs suggests a HDR defect. Therefore, we measured HDR by

us ing a DR-GFP reporter assay. The DR-GFP reporter gene has 2 nonfunctional GFP

genes: SceGFP, which is disrupted by insertion of the I-SceI endonuclease recognition

sequence, and iGFP, which contains a 5’ and 3’ end truncated GFP. Expression of I-SceI

induces a DSB in the SceGFP, which will undergo HDR using iGFP as a template

resulting in GFP-positive cells that can be quantified by flow cytometry (67, 68).

Previous ly, our lab reported HDR defects in Brca1S1598F/S1598F cells by DR-GFP assay and

RAD51 IRIF formation (58). Thus, Brca1S1598F/S1598F cells were used as a negative

57 control. Brca1+/+; DR-GFP or Brca1L1363P/+; DR-GFP, Brca1L1363P/L1363P; DR-GFP,

Brca1S1598F/S1598F; DR-GFP primary MEFs were transfected with I-SceI vectors by

electroporation and GFP-positive cells were quantitated after 48 hours. In control

Brca1+/+ or Brca1L1363P/+; DR-GFP pMEFs, approximately 1.1% of cells were GFP positive following I-SceI expression. However, in both Brca1L1363P/L1363P; DR-GFP and

Brca1S1598F/S1598F; DR-GFP cells , GFP positive cells were dramatically reduced indicating significantly impaired HDR efficiency (Figure 3.2.H.). However, Brca1S1598F/S1598F; DR-

GFP cells had consistently more GFP positive cells than Brca1L1363P/L1363P; DR-GFP cells. In agreement with this observation, the impairment of RAD51 IRIF formation was more severe in Brca1L1363P/L1363P than Brca1 S1598F/S1598F cells (data not shown). Next, we treated immMEFs with a PARP inhibitor, olaparib, that induces DSBs and karyotype analysis was conducted. Both Brca1L1363P/L1363P and Brca1S1598F/S1598F immMEFs exhibited hypersensitivity to olaparib compared to Brca1+/+ or Brca1L1363P/+ immMEFs, however, we did not detect differential sensitivity to olaparib between the two Brca1 mutants

(Figure 3.2.I. and 3.2.J.). Together, these data shows that the BRCA1-PALB2 interaction is essential for HDR and Brca1L1363P/L1363P cells have less residual HDR than

Brca1S1598F/S1598F. In addition, mutant Brca1L1363P/L1363P pMEFs showed impaired

proliferation, premature senescence and centrosome amplification compared with

Brca1+/+ or Brca1L1363P/+ pMEFs, which are mediated by hyper-activation of p53 (Figure

3.2.C, 3.3.A., 3.3.B., and 3.3.C.).

3.4.3. Brca1 L1363P/L1363P MEFs exhibit hypersensitivity to interstrand cross linking

agents

One of the characteristics of FA is that cells derived from FA patients show pronounced

sensitivity to interstrand crosslinking (ICL) agents such as diepoxibutane (DEB),

mitomycin C (MMC), and cisplatin. It is well known that BRCA1-, BRCA2- or PALB2-

deficent cells exhibit hypersensitivity to ICL-inducing agents (11, 51, 57). However, it is

still unclear whether the BRCA1-PALB2 interaction is required for effic ient ICL repair.

Cytogenetic analysis showed that Brca1L1363P/L1363P and Brca1S1598F/S1598F MEFs have an elevated basal level of chromosomal aberrations and that both mutants are extremely

sensitive to MMC, indicating that both the BRCA1-PALB2 interaction and functional

BRCT repeats of BRCA1 is critical for ICL repair (Figure 3.2.I. and 3.2.J.). If primary

cells in the animal require BRCA1 for repair of ICLs, then BRCA1-deficient mice should

be sensitive to these agents. To test for sensitivity, adult mice were injected with MMC

(5mg/kg bodyweight) into the peritoneal cavity. No effect was observed in either

Brca1+/+ or Brca1L1363P/+ anima ls (n=6) , while a ll Brca1L1363P/ L1363P anima ls (n=6) died within a few days (T50=6 days). (Figure 3.4.G) Histology upon death showed complete

depletion of bone marrow cells in Brca1L1363P/L1363P (data not shown). Similar ly,

Brca1L1363P/L1363P animals were found to be hypersensitive to irradiation, as all mutant

animals (n=12) died within 16 days (T50=9 days) following whole body irradiation with

6.5 Gy.

3.4.4. Brca1 L1363P/L1363P mice display phenotypes of mice with hyper-activation of p53

Cells from FA patients that are defective in the FA/BRCA pathway show constitutive activation of p53 due to accumulation of unresolved DNA damage and endogenous stress

(69). There are several studies describing phenotypes of mice with increased p53 activity.

The phenotypes include smaller body size, dark footpads and tail skin, an elevated level of thymocyte apoptosis, and cerebellar hypoplasia (70, 71). As aforementioned, all

Brca1L1363P/L1363P animals were smaller than littermates (Brca1+/+ or Brca1L1363P/+) and the vast majority had kinky tails (Figure 3.4.A. and 3.4.B.). Deletion of p53 partially ameliorated the decreased weight phenotype of mutant Brca1L1363P/L1363P but

Brca1L1363P/L1363P; p53+/– or Brca1L1363P/L1363P; p53–/–mice were still cons istent ly sma ller

than control mice (Brca1+/+; p53+/–, Brca1+/+; p53–/–, Brca1L1363P/+; p53–/– or

Brca1L1363P/+; p53–/–) (data not shown).

Brca1L1363P/L1363P mice exhibited dark pigmentation in the footpads and tails and the pigment accumulation was in e pidermis of mutant animals (Figure 3.4.C. and 3.4.D.).

McGowan et al. reported stabilization of p53 stimulates the proliferation of melanocytes in the epidermis (70). Thus, to test whether pigmentation of mutant mice is caused by an increased number of melanocytes, we performed immunohistochemistry staining for

GP100, a melanocyte marker. We observed an elevated number of melanocytes in the tail epidermis of mutant mice but not in dermis (Figure 3.4.D.). Next, we tested if the observed increase of melanocytes in the epidermis of Brca1L1363P/L1363P mice is caused by

stabilization and hyperactivation of p53 melanocytes by generating Brca1L1363P/L1363P;

p53+/– and Brca1L1363P/L1363P; p53–/–mice. Remarkably, the pigmentary phenotype was

60 reduced in Brca1L1363P/L1363P; p53+/– animals and fully reversed in Brca1L1363P/L1363P;p53–/–

anima ls. (Figure 3.4.C.)

At four weeks of age, Brca1L1363P/L1363P anima ls exhibit a much sma ller thymus compared

to age matched controls (data not shown). To determine the level of apoptosis in the

thymus, Annexin V staining was performed. In mutant Brca1L1363P/L1363P mice, the number of apoptotic thymocytes was two-fold higher than controls at the basal level.

Although the numbers of apoptotic cells were dramatically elevated in both control and

mutant group after irradiation, only mutant mice showed increased number of Annexin V

pos it ive cells. Deletion of one allele of Trp53 gene attenuated the irradiation induced

apoptosis in Brca1L1363P/L1363P; p53+/– anima ls , indicating that IR induced cell death was mediated by p53 (Figure 3.4.F.).

Finally, mutant Brca1L1363P/ L1363P animals had prominent midbrain areas due to hypoplasia of the cerebellum and the cortex, which could also be reversed by a deletion of one copy of the p53 gene in Brca1L1363P/L1363P; p53+/– anima ls (Figure 3.4.E.).

Taken together, these phenotypes demonstrate hyper-activation of p53 possibly caused by

DNA repair defects in Brca1L1363P/L1363P mutant animals, which is observed in FA cells

(72).

3.4.5. Brca1 L1363P/L1363P mice develop bone marrow failure – indicative of FA-like

phenotype

13% of Brca1L1363P/L1363P mutant animals that were extremely small from birth developed spontaneous bone marrow failure and died within a month. Their bone tissue sections

61 presented aplastic anemia and variable hypocellular marrow with depletion of all cell

types (Figure 3.5.A. and 3.5.B.). In prenatal mice, hematopoietic stem cells (HSCs) are

actively cycling in the fetal liver to generate blood cells for oxygen transport and to the

development of the immune system (73). Rapidly growing cells inc luding HSCs can be more severely affected by DNA damage repair defects than other cell types. Unrepaired

DNA damages causes activation of the p53/p21 pathway arresting cells at the G0/G1 phase of the cell cycle, which ultimately impairs HSCs pool expansion. Recently, a report showed higher p21 level in FA fetal liver and patient bone marrow samples which could explain why FA patients undergo bone marrow failure (BMF) early in life (69). Five

Brca1L1363P/L1363P mice which died perinatally showed decreased extramedullary

hematopoiesis compared to controls (Figure 3.5.C.). Based on this observation, we expected a severe reduction of HSCs in older mutant animals, which could be triggered by hyperactivation of p53. We analyzed HSCs population with LSK marker (Lin- Sca-

1+c-kit+) using animals at 1 month of age. As expected, Brca1L1363P/L1363P mice showed a marked decrease in HSCs populations compared to control mice. We also examined

HSCs in other Brca1 mutant animals, Brca1tr/tr (59) and Brca1S1598F/S1598F (58, 59), which

have HDR defects but did not develop bone marrow failure. Interestingly, Brca1tr/tr

animals showed a 40 % reduction in HSCs w hile Brca1S1598F/S1598F had normal levels of

HSCs. (Figure 3.5.D.) The Brca1tr protein is truncated after the first 924 amino ac ids due to the insertion of a stop codon within exon 11 and therefore should not contain c oiled- coil and BRCT tandem repeats. We previously reported that Brca1tr protein is very

unstable and not detectable (59). Therefore, we questioned how Brca1tr/tr animals could

62 have more HSCs than Brca1L1363P/L1363P. Since wild type BRCA1 cells express an

alternative splice variant which lacks exon 11 (BRCA1∆ex11), we checked whether

Brca1tr/tr cells can express BRCA1∆ex11 protein that would contain a funct ional c oiled-coil

domain (74). Western blot showed that BRCA1+/+ and Brca1tr/tr immMEFs both express

the BRCA1∆ex11 protein isoform, whic h may partially rescue defective HSCs in Brca1tr/tr

anima ls (data not shown). Of note, the two FA pat ie nts wit h bia lle lic BRCA1 mutations did not show any signs of BMF and BRCA1 alleles of both patients had a deleterious

variation and a missense mutation in the BRCT repeats in trans (51, 57). Consistent with a previous report (69), when compared to control mice, decreased frequency of the HSC populations inversely correlated with levels of p21 mRNA (Figure 3.5.E.). We also measured progenitor activity by the colony forming unit assay (CFU). Plating of 1X105

total bone marrow cells from Brca1L1363P/L1363P rarely yielded colonies in culture and the

few colonies obtained were very small and contained only a few cells (Figure 3.5.F. and

3.5.G.). Even plat ing 1X106 total bone marrow cells from Brca1L1363P/L1363P anima ls

showed no improvement of colony formation (data not shown). Exhaustion of HSCs and scanty CFU formation in Brca1L1363P/L1363P mice were partially rescued by deletion of p53

gene suggesting that reduced HSCs in Brca1L1363P/L1363P bone marrow are caused by

hyperactivation of p53 (Figure 3.6.A., 3.6.B., and 3.6.C.). Consistent with reduced

numbers of HSCs in BRCA1tr/tr animals (Figure 3.5.D.), BRCAtr/tr bone marrow cells

showed a 50% reduct ion in CFU (Figure 3.5.F. and 3.5.G.). Altogether, these data indicate that the BRCA1-PALB2 interaction is essential for proliferation and the

expansion of the HSC population. Although both Brca1L1363P/L1363P and Brca1S1598F/S1598F

63 immMEFs showed a HDR defect, for HSC proliferation and expansion, functional BRCT repeats are dispensable, which implies a role of BRCA1 in HSCs beyond HDR. This can also expla in why the two BRCA1-FA (FANC-S) patients identified so far did not develop

bone marrow failure.

3.4.6. Brca1 L1363P/L1363P mice develop T-lymphoblastic lymphoma/leukemia

Most homozygous mutant mice (47 of 54) developed progressive wasting and upon

necropsy displayed massive thymic expansion with infiltration of lymphoblastic leukemia within 2 to 4 months of age (Figure 3.7.A. and 3.7.D.). Leukemic cells invaded into the lung and pleura and the invading cells were CD3 positive T cells (Figure 3.7.E). Flow cytometry performed in three Brca1L1363P/ L1363P cases showed that the leuke mic blast cells

were immature T cells, double positive for CD4 and CD8 and negative for CD 34,

consistent with lymphoblastic lymphoma/leukemia (Figure 3.7.F. and 3.7.G.). Peripheral

blood from Brca1L1363P/L1363P animals with thymic tumor showed high white blood cell

(WBC) count composed mostly of lymphoblasts (CD4+ CD8+). The lymphoblast cells

were also observed in the bone marrow (Figure 3.7.B. and 3.7.G.). Since the majority of

FA patients have defective HSCs, they are at high-r isk of hematologic malignancies, mostly acute myeloid leukemia (AML). Although lymphoid leukemia is rare in people with FA, there is a case report of T-cell acute lymphoblastic leukemia in a BRCA2- mutant FA patient (75).

At 4 weeks of age, no circulating lymphoblasts were detected in mutant animals.

However, complete blood counts from mutant mice displayed persistent macrocytosis,

64 elevated mean corpuscular volume (MCV) and decreased hemoglobin levels over time, indicating that Brca1L1363P/L1363P mice develop macrocytic anemia (Figure 3.7.C.).

3.4.7. Thymic tumor in Brca1L1363P/L1363P mice acquired NOTCH1 mutation

The most common recurrently mutated gene in the thymic neoplasms from

Brca1L1363P/L1363P mice was in Notch1 (25 of 29 cases, 86.2%), with tumors in 18 mice showing multiple presumed pathogenic Notch1 variants. Lymphomas from 9 of the mice had tumor-associated mutations in the NOD/heterodimerization domain (HD) at locations that matched those most commonly seen in human T-LBL/ALL. These included the most common change in human neoplasms (L1668P in 5) as well as L1574Q, V1578M,

I1670S, C1682R, A1686D, and A1691P in 1 tumor each (Figure. 3.8., Table 1, Table 2 and Table 3). Truncating or frameshift mutations in the C-terminal TAD and PEST domains of Notch1 were also common, similar to the findings seen in most human T-

ALL (76). Based on a comparison of variant allele frequency (VAF) and tumor percentage, presumed subclonal/oligoclonal or multiclonal patterns of Notch1 mutations were seen in 14 lymphoma samples. There was a higher frequency of pathogenic HD mutations in Brca1L1363P/L1363P tumors on a p53 wild-type background (5/7) as compared to p53 heterozygous (2/14) and p53-/- null backgrounds (2/7, p=.01).

3.5. Dis cuss ion

In this chapter, we analysed a knock-in mouse model expressing the patient derived

Brca1 mutation L1363P (mouse equivalent of the human BRCA1 L1407P mutation),

65 which developed FA-like hematopoietic defects and animals succumb to T-ALL with

100% penetrance. Recently, FA genes were divided into two different groups: bona fide

FA genes and FA-like genes (32). The BRCA1 gene, which was recently identified as the

FANC-S gene, was excluded from bonafide FA genes because two individuals with

bialle lic BRCA1 mutations did not dis play the c linica l characteristics of FA,

hematopoietic defects and bone marrow failure (51, 57). However, the role of BRCA1 in

hematopoiesis remains unclear. There are two studies reported last year that showed

development of BMF or hematopoietic malignancy in Brca1 deleted or mutated mouse

models, which strengthen our study with the evidence that BRCA1 have critical functions

in hematopoietic function (77, 78)

Previous ly, our lab reported on several different Brca1 mutant mouse models (i.e. exon11

truncation, I26A – non-functional E3 ubiquitin ligase activity, S1598F – inability to bind

with its interacting partners (Abraxas, Brip1 and CtIP)) (58, 59, 79). From those anima l

models, we did not observe any signs of hematopoietic defect. While Brca1I26A/I26A mice,

lacking E3 ubiquitin ligase activity, were still proficient in tumor suppression, other

mutant Brca1 mice (Brca1tr/tr and Brca1S1598F/S1598F) exhibite d increased spontaneous

tumor development including mammary tumors (58, 59). Interestingly, the two BRCA1

FA patients who did not dis play hematopoietic defects or spontaneous bone marrow

failure were diagnosed with breast and ovarian cancer. Both patients carried a deleterious

as well as a missense BRCA1 mutation in trans. The locations of the missense mutations

were in the C-terminal BRCT domain, similar to our Brca1S1598F/S1598F mouse model that developed mammary tumor (51, 57). However, we also showed that the levels of HSCs in

Brca1S1598F/S1598F mice were comparable with wild type age-matched controls. These results can explain why BRCA1 FA patients did not develop BMF and suggest that

although functional BRCA1 BRCT repeats are essential for tumor suppression, they are dispensable in the hematopoietic system. However, Mgbemena et al. recently generated a

knock-in mouse model expressing the BRCA1 5382insC alle le , which is the common

Ashkenazi Jewish founder mutation. This mutation leads to frameshift resulting in

expression of a truncated BRCA1 protein lacking the C-terminal BRCT repeats (78). Due

to the lethality of homozygous mutant animals, they generated Brca1flox/5382insC anima ls

and deleted the floxed allele in HSCs using the induc ible Mx1-Cre (78). In contrast to our

Brca1S1598F/S1598F mouse that had no HSC defects, Brca1flox/5382insC anima ls showed a dramatic reduction in HSCs. This suggests that the full length BRCT repeats per se have functions in hematopoiesis besides ability to interact with its binding partners.

Interestingly, the Brca1tr/tr mice had an intermediate phenotype in the level of HSCs and it was puzzling that their HSCs leve l could be higher than in Brca1L1363P/L1363P animals.

Therefore, we hypothesized that expression of an alternative splice variant, the

BRCA1∆ex11 protein, with contains a functional coiled-coil domain might partially rescue the severe hematopoietic defect in Brca1tr/tr anima ls. To test this, we checked expression of the BRCA1∆ex11 protein in Brca1L1363P/L1363P and Brca1ex11tr/ex11tr immMEFs. Consistent

with our previous report, the unstable truncated BRCA1tr protein was not detectable.

However, we observed stable expression of the BRCA1∆ex11 protein from Brca1+/+,

Brca1tr/tr and Brca1L1363P/L1363P cells (data not shown).

Although various functions of BRCA1 have been reported until now including checkpoint enforcement, DNA repair, chromatin modification and transcriptional regulation, BRCA1 is generally considered an HR protein, first and foremost. Therefore, as previously reported and shown here, most Brca1 mutant mice and cells have HR defects although to different extents (58, 59). All three different Brca1 mouse models

(Brca1L1363P/L1363P, Brca1tr/tr and Brca1S1598F/S1598F) had non-functional HR and exhibited

sensitivity to DNA damaging agents, causing activation and stabilization of p53 in cells.

Nevertheless, there are differences in phenotypes of each mouse model. During early

spermatogenesis, both Brca1L1363P/L1363P and Brca1tr/tr faile d to se lf-renew male germ stem cells, spermatogonia. In contrast, Brca1S1598F/S1598F males did not exhibit a

spermatogonial stem cell phenotype and developed normally through completion of the

meiotic divisions. Instead, haploid round spermatids from Brca1S1598F/S1598F testes were

unable to differentiate into mature sperms. Collectively, full-length BRCA1 protein as well as the BRCA1-PALB2 interaction appears to be essential for self-renewal of spermatogonia, while functional BRCT repeats are dispensable. Interestingly, while oogenesis was normal in Brca1tr/tr and Brca1S1598F/S1598F females resulting in normal fertility, Brca1L1363P/L1363P females were sterile. We observed formation of very few follic les in young mice (four weeks of age), but no follicles were found in the ovaries of adult Brca1L1363P/L1363P animals (data not shown). Thus, the BRCA1-PALB2 interaction

has a critical role in formation and maintenance of germ cells in both sexes. We also

observed decreased number of mammary stem cells in Brca1L1363P/L1363P female. This indicates that at least three different stem cell populations (HSCs, germ stem cells and

68 mammary stem cells) are affected in Brca1L1363P/L1363P animals implying the importance of a functional coiled-coil domain of BRCA1 and its interaction with PALB2 in different

types of stem cell formation and maintenance beyond.

Finally, we generated and characterized a novel mouse model of FA by introducing a

point mutation into the coiled-coil domain of BRCA1 ablating its interaction with PALB2.

Homozygous Brca1L1363P/L1363P display both spontaneous BMF and the rapid development of thymic lymphoma/leukemia. Although two patients with cancer and some FA-like features have been reported with biallelic mutations in BRCA1, these patients were not reported to show BMF (51, 57). Therefore, this new model can provide new insights into FA BMF, as distinct from the other features of FA. Specifically, this mouse model replicates the progressive decline in the numbers of HSCs characteristic of

FA. It, thus, provides an ideal model system to investigate the changes in HSCs that occur with progressive loss of hematopoiesis.

Figure 3.1. Generation of the Brca1 L1363P knock-in mice (A) Schematic diagram of the

mouse Brca1 locus and the procedure used to generate the knock-in mutant mice. Wild

type Brca1 allele encompassing exon 7~15 is shown at the top. The targeting construct

contains neomycin cassette flanked by loxP sites (closed triangles) and inserted into

intron 12, a single point mutation (asterisk) in exon 13 and a thymidine kinase cassette as

a negative selection marker. The wavy line in targeting constructs represents vector

sequence. After homologous recombination, the targeted Brca1L1363P-Neo alle le is diagrammed in the middle. Insertion of the neomycin cassette introduced an additional

SpeI restriction enzyme site to the targeted mutant allele, which used for southern blot.

Cre mediated neomycin cassette recombination is shown below. Arrows are genotyping pr imers. (B) Southern blot result of the representative ES recombinant clones after digests with SpeI. The additional introduction of a SpeI in neomycin cassette results in reduction of ~20kb SpeI germ-line fragment to ~6kb knock in fragment. (C) PCR analys is from tail DNA is shown for the respective genotypes. The positions of the PCR primers are shown as arrows in A. (D) Sanger sequencing confirmed the Brca1L1363P

mutation.

CONTINUED

Figure 3.1. CONTINUED Generation of the Brca1 L1363P knock-in mice

CONTINUED

Figure 3.1. CONTINUED Generation of the Brca1 L1363P knock-in mice

Figure 3.2. Brca1L1363P/L1363P M EFs have defective HDR and hypersensitivity to

interstrand cross linker (A) Western blot analysis of Brca1 and Bard1 proteins in

Brca1L1363P/L1363P immMEFs. Cells were treated with 10Gy IR and harvested after 2hrs recovery. (B) Mutant BRCA1 LP protein fails to interact with PALB2. pOZ-mouse

PALB2 FLAG-HA was transfected into Brca1+/+ or into Brca1L1363P/L1363P immMEFs for

48hrs. Whole cell lysates were subjected to immunoprecipitation (IP) with HA or

BRCA1 antibodies followed by immunoblot analyses with indicated antibodies. Empty vector transfected lysates were used as a negative control. (C) Brca1L1363P/L1363P pMEFs

show impaired proliferation. Brca1+/+ and Brca1L1363P/ L 1363P MEFs (passage 1) were

seeded onto 96 well plate (1000 cells per well). The proliferation rate was measured by

MTT assay for four consecutive days. (D, E, F, G) Recruitment of Abraxas, Brca1 and

Rad51 to sites of DNA damage in Brca1L1363P/L1363P immMEFs. 100 cells were counted

from three independent immMEFs lines per genotype. (H) Brca1L1363P/L1363P MEFs are

defective in HDR. Brca1+/+, Brca1L1363P/L1363P, Brca1S1598F/S1598F pMEFs (passage 1)

containing DR-GFP reporter gene were electroporated with either empty vector or IsceI

expression vector. GFP positive cells were quantified by flow cytometry. (I, J)

Brca1L1363P/L1363P immMEFs exhibit hypersensitivity to Poly (ADP-Ribose) Polymerase

(PARP) Inhibitor, olaparib and interstrand cross linker, Mitomycin C (MMC) compared

to Brca1+/+ or Brca1L1363P/+ immMEFs. 25 metaphases were analyzed from two different

MEFs lines per genotype. Error bars represent S.E.M and p-values were calculated by

unpaired t-test (**** p<0.0001).

CONTINUED

Figure 3.2. CONTINUED Brca1L1363P/L1363P M EFs have defective HDR and hypersensitivity to interstrand cross linker

CONTINUED

Figure 3.2. CONTINUED Brca1L1363P/L1363P M EFs have defective HDR and hypersensitivity to interstrand cross linker

D E

F G

CONTINUED

Figure 3.2. CONTINUED Brca1L1363P/L1363P M EFs have defective HDR and hypersensitivity to interstrand cross linker H

CONTINUED 76

Figure 3.2. CONTINUED Brca1L1363P/L1363P M EFs have defective HDR and hypersensitivity to interstrand cross linker J

Figure 3.3. Brca1L1363P mutation induces premature senescent phenotype in pMEFs

(A) β-galactosidase staining on pMEFs (passage 3). 0.3X106 cells were seeded and

incubated in β-gal solution overnight. Quantification of 3 different clones was pooled

from each genotype. (B) Pericentrin and α-tubulin immunofluorescent staining on pMEFs

(passage 2 & 3). 200 cells were counted from each cell line. 6 individual cells were

examined. (C) Stabilization of p53 protein in Brca1L1363P/L1363P pMEFs (passage 3). Cells were treated with 10Gy IR. After 1hr recovery, cells were lysed and western blotting was performed. Error bars represent S.E.M and p-values were calculated by unpaired t-test

(**** p<0.0001).

CONTINUED

Figure 3.3. CONTINUED Brca1 L1363P mutation induces premature senescent

phenotype in pMEFs

Figure 3.4. Brca1L1363P/ L1363P mice show a variety of phenotypic abnormalities (A, B)

Brca1L1363P/L1363P mice have smaller body size, darker skin and kinky tail. Twenty of 4-5 weeks old mice were weighed in each genotype. (C) Brca1L1363P/L1363P mice have darker pigmentation on footpads and this phenotype was rescued by deletion of p53 gene. (D)

Hematoxylin and eosin (H&E) and IHC for GP100 stained tail skins show increased the number of melanocytes producing more mela nin in Brca1L1363P/L1363P epidermis. (E)

Brca1L1363P/L1363P mice have prominent colliculus area (dotted yellow line), which rescued

by deletion of p53. (F) Annexin V assay showed significant increase apoptotic

thymocytes in Brca1LP/LP mice. 5 Gy X-ray was treated to 4 weeks old animals and

thymocytes were collected after 4hrs recovery. (G) Kaplan-Meier survival curve of

Brca1LP/LP mice treated with MMC or IR. Homozygous Brca1L1363P/L1363P mice were extremely sensitive to MMC and IR. Error bars represent S.E.M and p-values were calculated by unpaired t-test (**** p<0.0001).

CONTINUED

Figure 3.4. CONTINUED Brca1L1363P/ L1363P mice show a variety of phenotypic abnormalities A

CONTINUED

Figure 3.4. CONTINUED Brca1L1363P/ L1363P mice show a variety of phenotypic abnormalities

CONTINUED

Figure 3.4. CONTINUED Brca1L1363P/ L1363P mice show a variety of phenotypic abnormalities E

CONTINUED

Figure 3.4. CONTINUED Brca1L1363P/ L1363P mice show a variety of phenotypic abnormalities F

Figure 3.5. ~13% of Brca1L1363P/ L1363P animals developed bone marrow failure (A)

Kaplan-Meier survival curve of Brca1L1363P/L1363P mice that died of bone marrow failure in a month. (B) H&E stained bone tissue showed depletion of marrow cells in

Brca1L1363P/L1363P mice. (C) Liver H&E staining at postnatal day one exhibits decreased

extramedullary hematopoiesis in Brca1L1363P/L1363P mice died perinatally. (D) Flow

cytometry analysis for LSK cells count at 4 weeks of age showed reduced HSCs in

Brca1L1363P/L1363P mice. (E) Quantitative real-time PCR analysis showed inverse correlation of p21 mRNA level with LSK numbers. RNA was isolated from total bone marrow cell pellets (F, G) Colony-forming unit (CFU) capacity of mouse bone marrow at

4 weeks of age is correlated to the LSK numbers from each animal. 1X105 total bone marrow cells were seeded in methocult medium and cultured for 7 to 14 days. Error bars represent S.E.M and p-values were calculated by unpaired t-test (**** p<0.0001).

CONTINUED

Figure 3.5. CONTINUED ~13% of Brca1L1363P/ L1363P animals de ve lope d bone marro w failure A

B C

CONTINUED

Figure 3.5. CONTINUED ~13% of Brca1L1363P/ L1363P animals de ve lope d bone marro w failure

CONTINUED

Figure 3.5. CONTINUED ~13% of Brca1L1363P/ L1363P animals de ve lope d bone marro w failure F

Figure 3.6. p53 deletion partially rescues hematopoietic defects in Brca1L1363P/L1363P

animals. (A) Flow cytometry analysis for LSK cell counts from 4 weeks old mice. p53 deletion increased the number of LSK cells in Brca1L1363P/L1363P animals. (B) Real time

PCR analysis of p21 from total bone marrow at 4 week of age. p53 deletion leads to

decrease p21 le vel. (C) CFU counting from 1X105 total bone marrow cells. p53 deletion partially rescue colony forming capacity of homozygous mutant cells.

CONTINUED

Figure 3.6. CONTINUED p53 deletion partially rescues hematopoietic defects in

Brca1L1363P/ L1363P animals

Figure 3.7. Brca1L1363P/L1363P animals developed T-lymphoblastic lymphoma/

leukemia between 2 and 4 months. (A) Kaplan-Meier survival curve of

Brca1L1363P/L1363P mice that developed massive thymic expansion. (B) Write Giemsa staining of blood film from Brca1+/+ control and moribund Brca1L1363P/L1363P mouse. Less

number of erythrocytes and large number of lymphoblast cells were observed in

peripheral blood from moribund Brca1L1363P/L1363P mice. (C) Brca1L1363P/ L 1363P mice

develop macrocytic anemia. Macrocytosis was detected in mutant animals from 1 week

and maintained for their entire life. No differences in hemoglobin (Hb) levels were

detected from 1 week to 4 weeks of age between control and mutant group. However, at

12weeks, mutant animals showed very low Hb counts compared to control. (D) H&E

staining on normal thymus and thymic tumor. Normal thymus fr om contr ol a nima ls

contains distinct cortex and medulla (C: cortex, M: medullar). Cells from thymic mass

was completely replaced with the large nucleolated lymphoblasts. (E) H&E stained lung

sections showed invasion of thymic tumor into lung. The invaded blast cells are CD3

positive T cells. (F, G) Flow cytometry analysis from total bone marrow revealed that the

tumor cells are CD34- , CD4+ and CD8+.

CONTINUED

Figure 3.7. CONTINUED Brca1L1363P/L1363P animals developed T-lymphoblastic lymphoma/ leukemia between 2 and 4 months A

CONTINUED 92

Figure 3.7. CONTINUED Brca1L1363P/L1363P animals developed T-lymphoblastic lymphoma/ leukemia between 2 and 4 months D

CONTINUED

Figure 3.7. CONTINUED Brca1L1363P/L1363P animals developed T-lymphoblastic lymphoma/ leukemia between 2 and 4 months

Figure 3.8. Notch1 mutations were present in 26 of 30 tumors analyzed by

sequencing. Mutations patterns were similar to those seen in human lymphoblastic

lymphoma/leukemia, including 11 mutations in the HD domain (6 L1660P mutations, an

ortholog of the human hotspot L1678P mutation) and 6 frameshift or nonsense mutations

in the C-terminal TAD/PEST domains. A distinct finding, compared to most human

cases, was the frequent finding of N-termina l missense mutations in the EGF-like

domains, associated with other more typical oncogenic mutations in the same neoplasm.

CONTINUED

Figure 3.8. CONTINUED Notch1 mutations were present in 26 of 30 tumors analyzed by sequencing

thymic tumor. thymic

+/+ p53

;

P 1363

P/ L 1363 L

Brca1

sequencing from

exome by detected tations mu

Notch1

. Table 1

thymic tumor.

- +/ p53

; P 1363 P/ L 1363 L Brca1 sequencing from tations detected by exome exome by detected tations mu

Notch1

Table 2

thymic tumor.

/ - p53

; P

1363 P/ L

1363 L

Brca1

sequencing from

exome

tations detected by mu

Notch1

Table 3

CHAPTER 4

Conclusions and Future Directions

It was reported that germline mutations of either of the two breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, also increase the risk of familial pancreatic cancer (23, 36, 80, 81). In addition, the PALB2 (a partner and localizer of BRCA2) gene was identified as a pancreatic tumor susceptibility gene (9). Recent studies found that

BRCA1, BRCA2 and PALB2 proteins cooperate in DNA damage repair by forming a complex (BRCA1-PALB2-BRCA2), and their role is critical to maintain genome integrity (1, 2, 5). Since genome instability is a hallmark of cancer, formation of this complex could be inextricably connected with the BRCA tumor suppression function.

BRCAs have been implicated in multiple aspects of the DNA damage response, including homologous recombination (HR) of double-strand DNA breaks and several distinct cell cycle checkpoints (82). Indeed, the genomes of BRCAs-deficient cells are highly unstable, resulting in extensive aneuploidy and chromosomal rearrangements (8). Since the BRCA1 lesions implicated in familial breast and pancreatic cancer are mainly frameshift or nonsense mutations, many tumor-associated alleles encode truncated proteins that have lost the coiled-coil domain. Furthermore, in some breast cancer families, tumor susceptibility can be ascribed to missense mutations by a single amino

100 acid substitution in the coiled-coil domain of BRCA1 (Met1400Val, Leu1407Pro and

Met1411Thr). Interestingly, these sequences in BRCA1 were shown to interact with the coile d-coil domain of PALB2 (5).

PALB2 was first identified as a binding partner of BRCA2 and shown to be required for the localization of BRCA2 to sites of DNA damage, and thus crucial for BRCA2 function in HR (1). PALB2 harbors a series of C-terminal WD repeats that bind the N-terminus of

BRCA2. In addition, the coiled-coil region at the N-terminus of PALB2 interacts with the coile d-coil domain of BRCA1. Down regulation of PALB2 by siRNA suppresses HR in a similar manner to BRCA1 and BRCA2 depletion (2). Like BRCA1 and BRCA2, monoallelic mutations in PALB2 confer familial susceptibility to breast, ovarian and pancreatic cancer (9, 10), while bia lle lic PALB2 lesions cause FA subtype N (FANC-N)

(11). The evidence that PALB2 is cr it ica l for HR and functions as a breast and pancreatic susceptibility gene suggest that it may also be important for BRCAs-mediated tumor suppression. We hypothesized that PALB2 is essential for BRCAs mediated tumor suppression by physically linking BRCA1 and BRCA2. Therefore, we predicted that tiss ue-specif ic de let ion of Palb2 or inhibit ing its interact ion wit h BRCA1 will result in tumorigenesis. In this study, we tested these hypothesis and inactivated Palb2 specifically in the pancreatic ductal epithelium to generate an animal model for Palb2-def ic ient pancreatic cancer. We also generated a point mutant knock-in mouse (BRCA1 L1363P) to ablate the BRCA1-PALB2 interaction, and determined whether these animals develop tumors.

101

In Chapter 2, we described the Palb2-defic ient pancreatic tumor mouse model. Using

KrasLNL-G12D/+; Pdx1-Cre animals generated by Hingorani and colleagues for human

ductal adenocarcinoma (47), we generated a cohort of Palb2fx/fx; KrasLNLG12D/+; Pdx1-

Cre. These animals developed pancreatic tumor but tumor latency was similar with

KrasLNLG12D/+; Pdx1-Cre anima ls, indicat ing that the deletion of Palb2 gene in the ductal epithelium of the pancreas is not sufficient to increase the incidence or shorten the tumor latency. Likewise, neither Brca1fx/fx; KrasLNLG12D/+; Pdx1-Cre nor Brca2fx/fx;

KrasLNLG12D/+; Pdx1-Cre anima ls exhibited accelerated tumor development compared to

KrasLNLG12D/+; Pdx1-Cre mice. BRCA1 tumors frequently harbor p53 gene mutations and p53 deficiency accelerates tumor formation in mice bearing Brca1 mutations (6, 83-85) since the loss of p53 function allows the cells to regain a proliferative phase. Therefore, we concomitantly activated mutant p53R270H and KrasG12D and inactivated Palb2.

Palb2fx/fx; p53LNL-R270H/+; KrasLNL-G12D/+; Pdx1-Cre animals developed tumors more

LNLR270H/+ LNLG12D/+ rapidly (T50=71days) than p53 ; Kras ; Pdx1-Cre anima ls

fx/fx (T50=172days). These animals also became moribund slightly faster than Brca1 ;

LNLR270H/+ LNLG12D/+ fx/fx LNLR270H/+ p53 ; Kras ; Pdx1-Cre (T50=83days) or Brca2 ; p53 ;

LNLG12D/+ Kras ; Pdx1-Cre (T50=96days) due to development of tumors in the head of pancreas leading faster to obstruction of the biliary duct. Interestingly, Palb2- deficient pancreatic tumors exhibited Brca1- deficient tumor like- or Brca2- defic ient tumor like- features in terms of the presence of different PDAC precursor lesions. While almost all

Brca1 deficie nt pancreatic tumors developed two different precursor lesions (mucinous cystic neoplasm (MCN) and PanINs), MCN were never seen in Brca2 deficient

102

pancreatic tumors. Interestingly, approximately 26% of Palb2fx/fx; p53 LNLR270H/+;

KrasLNLG12D/+; Pdx1-Cre mice developed large cysts surrounded by ovarian-like stroma in pancreata, which resemble closely the Brca1 deficie nt tumors. However, the remaining

other animals developed PDACs without any cysts. The underlying reason for the observed dichotomy in precursor lesions between the Brca1-deficient and Brca2- deficient tumors and the mixture of both in Palb2 deficient tumors will require further

investigation. A possible explanation could be differential sensitivity of different ductal

epithelial cells to the loss of BRCA1 and BRCA2.

By generating pancreatic tumor mouse models, we aimed to develop novel and improved

treatments against pancreatic cancer. Using primary tumor cells established from our

mouse models, we investigated sensitivity to mult iple dr ugs. Palb2-, Brca1- or Brca2-

defic ient KPC tumor cells exhibited hypersensitivity to DNA damaging agents (MMC,

Cis platin and Olaparib) in comparison with KPC tumor cells, but not to other

chemotherapeutic drugs (5-FU and Paclitaxel) that are commonly used to treat pancreatic

cancer. The DNA damaging drugs als o inhibited tumor growth in vivo result ing in

decreased tumor burden and prolonged survival of treated animals. These preclinical

results indicate that DNA damaging agents are effective and may be particularly useful in

the treatment of PALB2-, BRCA1- or BRCA2- deficient pancreatic tumors.

Although the DNA damaging agents have shown clinically efficacy in PALB2-, BRCA1-

or BRCA2- cancers, tumor cells frequently will develop resistance to these drugs and the

tumor will recur and progress. Therefore, it will be critical to study the mechanisms of

acquired resistance. Improved preclinical models like our Palb2-, Brca1- or Brca2-

103

deficient pancreatic tumor mouse models combined with powerful high-throughput

screening techniques will help to understand and overcome mechanisms of drug

resistance. By understanding the mechanisms, we could identify novel therapeutic targets

in recurrent tumors.

As described above, PALB2 is a bona-fide tumor suppressor gene. To determine whether

the tumor suppression function of PALB2 is ascribed to its binding to BRCA1 and

BRCA2, we generated point mutant knock-in mice (BRCA1 L1363P) to ablate the

BRCA1-PALB2 interaction. Unlike other Brca1 mutant animals that develop a variety of

tumors, homozygous Brca1L1363P/L1363P animals showed congenital abnormalities (smaller

body size, pigmentation in skin and hypoplasia in sexual organs) and developed aplastic

anemia causing spontaneous bone marrow failure (BMF). The mice also developed T-

acute-lymphoblastic leukemia/lymphoma (T-ALL) wit h 100% penetrance. These

phenotypes recapitulate Fanconi Anemia (FA) disease which is caused by biallelic

mutations in FA genes. BRCA1/FANCS, PALB2/FANCN and BRCA2/FANCD1 were also

identified to be FA genes. Although researchers tried to generate FA disease mouse

models by mutating different FA genes, none of the mode ls tr uly phenocopies the HSC

defects and spontaneous BMF observed in FA. Therefore, our Brca1L1363P/L1363P mouse appears to be the first FA disease mouse model which can be used to develop novel or improved therapies.

Due to the loss of all mutant Brca1L1363P/L1363P anima ls to T-ALL or BMF, we were unable to determine whether the BRCA1-PALB2 interaction is crit ica l for tumor

suppression. To circumvent this issue, conditional Brca1 animals can be utilized. By

104

deleting a conditional Brca1 alle le in an organ specific manner, we will express only

mutant Brca1L1363P in t he epithe lia l ce lls of t he pancreas- or ma mmary gland. We are

currently generating an experimental cohort of Brca1L1363P/co; Wap cre females for mammary tumor development. If conditional Brca1L1363P/co females develop mammary tumors with the same latency, penetrance, and phenotype as conditional Brca1- null females, then we will conclude that the coiled-coil domain and the BRCA1-PALB2

interaction is essential for tumor suppression of mammary tumorigenesis by BRCA1. On the other hand, if tumor formation is not observed in the conditional Brca1L1363P/co cohort

(similar to the Brca1WT cohort), we will conclude that the coiled-coil domain and the

BRCA1-PALB2 interaction is largely dispensable for BRCA1-mediated tumor

suppression. If, however, the coiled-coil domain and the BRCA1-PALB2 interaction is

required for some but not all of the tumor suppression functions of BRCA1, then we may

observe tumor formation in Brca1L1363P/co females that is significantly reduced (longer latency and/or lower frequency) with respect to Brca1-null mice and significantly increased with respect to the control Brca1WT mice.

In chapter 3, we als o compared hematopoietic stem cells (HSCs) defects among three

different BRCA1 mutant animals (Brca1L1363P/L1363P, Brca1S1598F/S1598F and Brca1tr/tr).

Interestingly, depending on the location of the mutation, these animals showed different hematopoietic phenotypes. While the number of HSCs in Brca1L1363P/L1363P animals was dramatically reduced, Brca1S1598F/S1598F mice had normal le vels of HSCs and Brca1tr/tr

mice showed an intermediate phenotype. The two reported BRCA1 FA patients did not

display hematopoietic defects or BMF which are characteristic of FA. Thus, BRCA1 is

105

currently not considered to be a bona-fide FA gene. However, the women were diagnosed

with breast and ovarian cancer. It is noteworthy, that in both cases, full length BRCA1

proteins with missense mutation in the BRCT repeats would be expressed, similar to our

tumor-prone BRCA1-S1598F model. In summary, BRCA1 is a multifaceted protein and the phenotypic analysis of specific point mutations will help to further understand its role in normal and malignant development.

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