Srilatha Simhadri

Requirement of Heterogeneous Nuclear Ribonucleoprotein C for BRCA Gene Expression and Homologous Recombination

Background Heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) is a core component of 40S ribonucleoprotein particles that bind pre-mRNAs and influence their processing, stability and export. Breast cancer tumor suppressors BRCA1, BRCA2 and PALB2 form a complex and play key roles in homologous recombination (HR), DNA double strand break (DSB) repair and cell cycle regulation following DNA damage. Methods PALB2 nucleoprotein complexes were isolated using tandem affinity purification from nuclease-solubilized nuclear fraction. Immunofluorescence was used for localization studies of proteins. siRNA-mediated gene silencing and flow cytometry were used for studying DNA repair efficiency and cell cycle distribution/checkpoints. The effect of hnRNP C on mRNA abundance was assayed using quantitative reverse transcriptase PCR. Results and Significance We identified hnRNP C as a component of a nucleoprotein complex containing breast cancer suppressor proteins PALB2, BRCA2 and BRCA1. Notably, other components of the 40S ribonucleoprotein particle were not present in the complex. hnRNP C was found to undergo significant changes of sub-nuclear localization after ionizing radiation (IR) and to partially localize to DNA damage sites. Depletion of hnRNP C substantially altered the normal balance of repair mechanisms following DSB induction, reducing HR usage in particular, and impaired S phase progression after IR. Moreover, loss of hnRNP C strongly reduced the abundance of key HR proteins BRCA1, BRCA2, RAD51 and BRIP1, which can be attributed, at least in part, to the downregulation of their mRNAs due to aberrant splicing. Our results establish hnRNP C as a key regulator of BRCA gene expression and HR-based DNA repair. They also suggest the existence of an RNA regulatory program at sites of DNA damage, which involves a unique function of hnRNP C that is independent of the 40S ribonucleoprotein particles and most other hnRNP proteins.

Benzo[a]pyrene diol epoxide–deoxyguanosine adducts are accurately bypassed by yeast DNA polymerase ζ in vitro

The possible role of bypass DNA polymerase zeta in mutagenic translesion synthesis past benzo[a]pyrene (BP) 7,8-diol-9,10-epoxide (DE) N(2)-deoxyguanosine (dG) adducts has been examined. We prepared 59-mer DNA templates containing dG adducts derived from trans opening of enantiomers of BP DE-2, in which the 7-hydroxyl group and epoxide oxygen are trans. The 10S-BP DE-dG and 10R-BP DE-dG adducts derive from the (+)- and (-)-DE-2 enantiomers, respectively. The adducted dG is located at a site identified as a G-->T mutational hotspot in random mutagenesis studies of (+)-BP DE-2 in Chinese hamster V-79 cells. Yeast pol zeta (complex of Gst-Rev3p and Rev7p) formed extension products (total of all lengths) of 71, 74 and 88% of a primer annealed to the 10S-BP DE-dG, 10R-BP DE-dG and non-adducted 59-mer templates, respectively. However, only 18 and 19% of the primer was extended to the full-length product on 10S-BP DE-dG and 10R-BP DE-dG adducted templates compared to 55% of the primer on the non-adducted template. A major 34-mer product corresponding to primer elongation up to and including the base before the adduct indicated that nucleotide incorporation opposite both adducts was strongly blocked. Full-length products were isolated from gels and subjected to PCR amplification and cloning. Sequence analysis of more than 300 clones of these full-length products on each template showed that only the correct dCMP was incorporated opposite both the adducted and non-adducted G-hotspot in the template. This corresponds to a probability of mutation lower than 0.3%, the limit of detection, and demonstrates the remarkable fidelity of yeast pol zeta in translesion synthesis past these BP DB-dG lesions in vitro.

Male Fertility Defect Associated with Disrupted BRCA1-PALB2 Interaction in Mice

Background: BRCA1 and PALB2 interact with each other to promote homologous recombination and DNA double strand break repair. Results: Mice with abrogated PALB2-BRCA1 interaction show male fertility defect. Conclusion: PALB2 and BRCA1 function together to ensure normal male meiosis. Significance: This work demonstrates the importance of the PALB2-BRCA1 interaction in vivo and reveals a novel role of PALB2 in sex chromosome synapsis. PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis.

Compromised BRCA1|[ndash]|PALB2 interaction is associated with breast cancer risk

The major breast cancer suppressor proteins BRCA1 and BRCA2 play essential roles in homologous recombination (HR)-mediated DNA repair, which is thought to be critical for tumor suppression. The two BRCA proteins are linked by a third tumor suppressor, PALB2, in the HR pathway. While truncating mutations in these genes are generally pathogenic, interpretation of missense variants remains a challenge. To date, patient-derived missense variants that disrupt PALB2 binding have been identified in BRCA1 and BRCA2; however, there has not been sufficient evidence to prove their pathogenicity in humans, and no variants in PALB2 that disrupt either its BRCA1 or BRCA2 binding have been reported. Here we report on the identification of a novel PALB2 variant, c.104T>C (p.L35P), that segregates in a family with a strong history of breast cancer. Functional analyses showed that L35P abrogates the PALB2–BRCA1 interaction and completely disables its abilities to promote HR and confer resistance to platinum salts and PARP inhibitors. Whole-exome sequencing of a breast cancer from a c.104T>C carrier revealed a second, somatic, truncating mutation affecting PALB2, and the tumor displays hallmark genomic features of tumors with BRCA mutations and HR defects, cementing the pathogenicity of L35P. Parallel analyses of other germline variants in the PALB2 N-terminal BRCA1-binding domain identified multiple variants that affect HR function to varying degrees, suggesting their possible contribution to cancer development. Our findings establish L35P as the first pathogenic missense mutation in PALB2 and directly demonstrate the requirement of the PALB2-BRCA1 interaction for breast cancer suppression.

BRAF Fusion as a Novel Mechanism of Acquired Resistance to Vemurafenib in BRAFV600E Mutant Melanoma

Purpose: Many patients with BRAFV600E mutant melanoma treated with BRAF inhibitors experience a rapid response, but ultimately develop resistance. Insight into the mechanism of resistance is critical for development of more effective treatment strategies. Experimental Design: Comprehensive genomic profiling of serial biopsies was performed in a patient with a BRAFV600E mutant metastatic melanoma who developed resistance to vemurafenib. An AGAP3–BRAF fusion gene, identified in the vemurafenib-resistant tumor, was expressed in BRAFV600E melanoma cell lines, and its effect on drug sensitivity was evaluated. Results: Clinical resistance to vemurafenib in a melanoma harboring a BRAFV600E mutation was associated with acquisition of an AGAP3–BRAF fusion gene. Expression of the AGAP3–BRAF fusion in BRAFV600E mutant melanoma cells induced vemurafenib resistance; however, these cells remained relatively sensitive to MEK inhibitors. The patient experienced clinical benefit following treatment with the combination of a BRAF and a MEK inhibitor. Rebiopsy of the tumor at a later time point, after BRAF and MEK inhibitors had been discontinued, showed loss of the AGAP3–BRAF fusion gene. Mixing experiments suggest that cells harboring both BRAFV600E and AGAP3–BRAF only have a fitness advantage over parental BRAFV600E cells during active treatment with a BRAF inhibitor. Conclusions: We report acquisition of a BRAF fusion as a novel mechanism of acquired resistance to vemurafenib in a patient with melanoma harboring a BRAFV600E mutation. The acquisition and regression of clones harboring this fusion during the presence and absence of a BRAF inhibitor are consistent with rapidly evolving clonal dynamics in melanoma. Clin Cancer Res; 23(18); 5631–8. ©2017 AACR.

Protein-lysine methyltransferases G9a and GLP1 promote responses to DNA damage

Upon induction of DNA breaks, ATM activation leads to a cascade of local chromatin modifications that promote efficient recruitment of DNA repair proteins. Errors in this DNA repair pathway lead to genomic instability and cancer predisposition. Here, we show that the protein lysine methyltransferase G9a (also known as EHMT2) and GLP1 (also known as EHMT1) are critical components of the DNA repair pathway. G9a and GLP1 rapidly localizes to DNA breaks, with GLP1 localization being dependent on G9a. ATM phosphorylation of G9a on serine 569 is required for its recruitment to DNA breaks. G9a catalytic activity is required for the early recruitment of DNA repair factors including 53BP and BRCA1 to DNA breaks. Inhibition of G9a catalytic activity disrupts DNA repair pathways and increases sensitivity to ionizing radiation. Thus, G9a is a potential therapeutic target in the DNA repair pathway.

PALB2 connects BRCA1 and BRCA2 in the G2/M checkpoint response

The G2/M checkpoint inhibits mitotic entry upon DNA damage, thereby preventing segregation of broken chromosomes and preserving genome stability. The tumor suppressor proteins BRCA1, PALB2 and BRCA2 constitute a BRCA1–PALB2–BRCA2 axis that is essential for homologous recombination (HR)-based DNA doublestrand break repair. Besides HR, BRCA1 has been implicated in both the initial activation and the maintenance of the G2/M checkpoint, while BRCA2 and PALB2 have been shown to be critical for its maintenance. Here we show that all three proteins can play a significant role in both checkpoint activation and checkpoint maintenance, depending on cell type and context, and that PALB2 links BRCA1 and BRCA2 in the checkpoint response. The BRCA1–PALB2 interaction can be important for checkpoint activation, whereas the PALB2–BRCA2 complex formation appears to be more critical for checkpoint maintenance. Interestingly, the function of PALB2 in checkpoint response appears to be independent of CHK1 and CHK2 phosphorylation. Following ionizing radiation, cells with disengaged BRCA1–PALB2 interaction show greatly increased chromosomal abnormalities due apparently to combined defects in HR and checkpoint control. These findings provide new insights into DNA damage checkpoint control and further underscore the critical importance of the proper cooperation of the BRCA and PALB2 proteins in genome maintenance.

Evidence of Intertissue Differences in the DNA Damage Response and the Pro-oncogenic Role of NF-κB in Mice with Disengaged BRCA1–PALB2 Interaction

The BRCA1-PALB2-BRCA2 axis plays an essential role in DNA homologous recombination repair, defect in which drives genome instability and cancer development. How cells with defects in this pathway respond to DNA damage in vivo and how tumors develop from these cells remain poorly defined. Here, we analyzed several aspects of the DNA damage response in multiple tissues of Palb2-mutant mice in which the interaction between PALB2 and BRCA1 is disengaged. Without any challenge, the mutant mice showed increased endogenous DNA damage. Following ionizing radiation, the mutant mice displayed higher levels of DNA breaks and stronger induction of p53 and p21, but continued DNA synthesis, reduced apoptosis, and accelerated tumor development. The differences in p21 induction, DNA synthesis, and apoptosis between wild-type and mutant mice were substantially more pronounced in the mammary gland than in the intestine, suggesting a potential contributing factor to the increased risk and the tissue specificity of BRCA/PALB2-associated tumor development. Moreover, the mutant mice showed higher levels of reactive oxygen species and constitutive activation of NF-κB, an antiapoptotic transcription factor inducible by both DNA damage and oxidative stress. Treatment of the mutant mice with an inhibitor of NF-κB reactivated apoptosis and delayed tumor development following radiation. Thus, our results also suggest a prosurvival and pro-oncogenic role of NF-κB in PALB2-mutant cells.Significance: This study explores novel tumor suppression mechanisms of the BRCA1-PALB2 DNA damage response pathway and implicates NF-κB activation as a protumorogenic event and possible therapeutic target. Cancer Res; 78(14); 3969-81. ©2018 AACR.

Abstract 2470: Compromised BRCA1-PALB2 interaction is associated with breast cancer risk

The major breast cancer suppressor proteins BRCA1 and BRCA2 play essential roles in homologous recombination (HR)-mediated DNA repair, which is thought to be critical for tumor suppression. The two BRCA proteins are physically and functionally linked by a third tumor suppressor, PALB2 (partner and localizer of BRCA2), in the HR pathway. Heterozygous PALB2 mutation carriers have increased risk of breast, ovarian and pancreatic cancer. While truncating mutations in BRCA genes are generally pathogenic, interpretations of missense variants remains a challenge. To date, patient-derived missense variants that disrupt PALB2 binding have been identified in BRCA1 and BRCA2; however, there has not been sufficient evidence to establish their pathogenicity in humans. Variants in PALB2 that disrupt either its BRCA1 or BRCA2 binding have not been reported. Here, we report on the identification of a novel PALB2 variant, c.104T>C [p.L35P], that segregated in a family with a strong history of breast cancer. Functional analyses showed that L35P abrogates the PALB2-BRCA1 interaction, resulting in impaired HR and sensitivity to platinum salts and PARP inhibitors. Whole-exome sequencing of breast tumor from a c.104T>C carrier revealed a somatic, truncating mutation in the second allele of PALB2, with the tumor displays hallmark genomic features of tumors with BRCA mutations and HR defects. Using a combination of traditional clinical genetics, tumor whole-exome sequencing and in-depth functional analyses, we have provided direct evidence to cement the pathogenicity of L35P. Parallel analyses of other germline variants in the PALB2 N-terminal BRCA1-binding domain also identified multiple variants that affect HR function to varying degrees, suggesting their possible contribution to cancer development. Our findings establish p.L35P as the first pathogenic missense mutation in PALB2 identified to date and directly demonstrate the requirement of the PALB2-BRCA1 interaction for breast cancer suppression. Citation Format: Tzeh Keong Foo, Marc Tischkowitz, Srilatha Simhadri, Talia Boshari, Kathleen A. Burke, Samuel H. Berman, Nadia Zayed, Yuan Chun Ding, Susan L. Neuhausen, Britta Weigelt, Jorge S. Reis-Filho, William D. Foulkes, Bing Xia. Compromised BRCA1-PALB2 interaction is associated with breast cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2470. doi:10.1158/1538-7445.AM2017-2470

Abstract 2487: Functional characterization of the BRCA2 variant, K3326X

Individuals with germline mutations in the breast cancer susceptibility gene, BRCA2, have an approximate 70% risk of developing breast cancer, a 30% risk of developing ovarian cancer, a 20-fold increased risk of developing prostate cancer, and a 10-fold risk of developing pancreatic cancer during their lifetime. Loss of function germline mutations in BRCA2 affect its role in the homologous recombination (HR) DNA repair pathway leading to significant genomic instability. In addition to deleterious truncating mutations, several sequence variants, collectively called Variants of Unknown Significance (VUS), have been identified and are distributed along its length. One such variant is rs11571833, a nonsense mutation in the last exon (c.9976A>T, K3326X), resulting in the loss of the C-terminal 93 amino acid residues in BRCA2. This truncated variant has been previously described as a polymorphism that does not increase susceptibility to breast and ovarian cancers, and as a neutral unclassified variant non-deleterious to its function. However, recent studies identified K3326X to be enriched in breast cancer cases and to increase the risk for lung, pancreatic, ovarian, and upper aero-digestive tract cancers. Preliminary data, obtained from the Rutgers Cancer Institute of New Jersey breast cancer case-control study, identified K3326X enrichment in 1.25% of cases compared to 0.7% of controls. Several of the carriers had second primaries and displayed a trend toward increased number of family members diagnosed with colon cancer. Notably, K3326X was also identified in 1.38% (11 of 796) of our histologically-diverse cohort of genomically-profiled tumors that included cancers of the breast, ovarian/fallopian tube, lung, vulvar, cancer of unknown primary, and one breast cancer case having prolonged response to platinum-based therapy. Thus, K3326X may represent a functional loss of wild type BRCA2 function, as we observe concomitant loss of heterozygosity at this locus. In a preliminary study, we evaluated the K3326X variant, in vitro, in a functional DR-GFP-based reporter assay measuring HR. Our data reveal the BRCA2 K3326X variant to be impaired in the HR pathway indicating a loss of wild-type protein function. We will also evaluate cell viability of the K3326X variant in the presence of DNA damaging drugs like cisplatin, poly-ADP ribose polymerase inhibitors and mitomycin C. Future studies will also incorporate a retrospective evaluation of tumor specimens that have undergone comprehensive genomic profiling. These data would indicate that BRCA2 K3326X represents a functional hypomorphic variant that may have implications in therapeutic approaches and cancer risk evaluations across a spectrum of tumor types. Citation Format: Srilatha R. Simhadri, Sonia C. Dolfi, Atul Kulkarni, Bing Xia, Shridar Ganesan, Kim M. Hirshfield. Functional characterization of the BRCA2 variant, K3326X [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2487. doi:10.1158/1538-7445.AM2017-2487