Research progress of relationship between DNA homologous recombination repair and breast cancer

Background: Germline mutations in the BRCA1 and BRCA2 genes lead to hereditary breast cancers that are defective in homologous recombination (HR) repair and sensitive to DNA damaging agents. HR deficiency (HRD) also occurs in sporadic breast cancers, but its incidence and etiology are unclear. Genomic signatures of HRD recently were employed as biomarkers in clinical trials with modest success. We posited that sporadic breast cancers displaying functional HR deficiency would harbor genetic alterations affecting HR DNA repair genes. To test this hypothesis, we applied a functional assay to define lack of competent HR DNA repair and sequenced the exomes of consecutive sporadic breast cancers. Methods: We developed an assay to assess the ability of cancer cells to localize RAD51 into sub nuclear foci in response to ex-vivo irradiation (IR) in fresh sporadic breast cancer tissue specimens from 60 patients. RAD51 focus formation was compared between mock and IR conditions to determine relative fold induction. Twenty-nine tumors with sufficient DNA underwent whole-exome sequencing. Structural genomic signatures of HRD (i.e. large state transitions (LST), telomeric imbalance (NtAI), loss-of-heterozygositiy (LOH)) and a previously reported mutational signature related to BRCA1/2 hereditary breast cancers were assessed. HR deficient tumors were defined as those with both RAD51 foci defects and a genomic signature of HR deficiency (LST>15 or presence of a BRCA mutational signature). Somatic, germ-line, and copy number changes in HR genes were investigated. BRCA1 methylation status was determined. Results: Seventeen of 60 (28%) tumors displayed defective RAD51 recruitment following ex-vivo IR (RAD51-DEF). RAD51-DEF was seen in all breast cancer subtypes , including 7 of 33 (21%) ER+/HER2-, 4 of 14 (29%) HER2+, and 6 of 13 (48%) triple-negative cases. Of the 29 sequenced tumors, 13 (45%) were RAD51-DEF and 16 (55%) were competent for inducing RAD51 foci. LST was elevated in 10 tumors (LST >15) and associated with RAD51-DEF (p=0.02), whereas NtAI (p=0.10) and LOH (p=0.052) did not show a significant association with RAD51-DEF. The BRCA1/2 mutational signature was evident in 4 tumors, all were RAD51-DEF (p=0.03) and 2 were BRCA2 mutated. Nine of 29 (31%) sequenced tumors were determined to have HRD by the RAD51 assay and presence of a genomic scar. Eight of these 9 (88%) cases with HRD had a genetic alteration of both alleles of a bona fide HR gene due to a pathogenic mutation (somatic or germline) coupled with loss of heterozygosity or a homozygous deletion compared to 1 (5%) tumor without HRD (p<0.001). Both alleles of a gene were affected for BRCA2 (n=4), FAAP100 (n=2), CHEK2 (n=1), TP53BP1 (n=1) and BRCA1 (n=1). BRCA1 gene promoter methylation was found not to be significantly associated with HRD. Conclusion: Combined functional and genomic analyses of breast tumors demonstrated that genetic loss of an HR gene may underpin HRD in sporadic breast cancers. Our findings warrant further comprehensive genetic assessment (somatic, germline, and copy number) of HR genes as potential biomarker for HR-directed therapies. Citation Format: Powell SN, Riaz N, Mutter RW, Ng CKY, Delsite R, Piscuoglio S, King TA, Martelotto L, Sakr R, Brogi E, Edelweiss M, Lim R, Higginson D, Weigelt B, Lee W, Reis-Filho JS. A functional assay for homologous recombination (HR) DNA repair and whole exome sequencing reveal that HR-defective sporadic breast cancers are enriched for genetic alterations in DNA repair genes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-03.

Background: BRCA1 and BRCA2 are involved in homologous recombination (HR) DNA repair and are germ-line cancer pre-disposition genes that result in the hereditary breast and ovarian cancer (HBOC) syndrome. Whether germ-line or somatic alterations in these genes or other members of the HR pathway and if mono- or bi-allelic alterations of HR-related genes have a phenotypic impact in breast and other cancers remains to be fully elucidated. Here we took a combined genomic and functional approach to identify the role of mutations in HR-related genes and their impact on HR DNA repair. Methods: Whole-exome sequencing and Affymetrix SNP6 array data from 8,178 tumors, comprising 24 different cancer types including breast cancer, were retrieved from The Cancer Genome Atlas (TCGA). We identified the prevalence of missense and pathogenic (frame-shift, nonsense, start/stop codon and splice site variants) somatic and germline mutations in 102 HR-related genes curated from the literature. For each mutation, we determined if the alterations were bi-allelic. We evaluated genomic signatures of HR-deficiency in each tumor using large-scale state transitions (LSTs) and a mutational signature of HR-deficiency (signature 3). An independent set of 24 fresh sporadic breast cancer tissue specimens from our institution was subjected to i) an ex-vivo assay that assesses the ability of cancer cells to form RAD51 foci in response to ex-vivo irradiation (IR), and ii) whole exome-sequencing to define whether RAD51 deficient tumors would display LSTs, signature 3 and bi-allelic inactivation of HR-related genes. Results: 13% and 5% of all TCGA cases displayed pathogenic mono- and bi-allelic alterations of HR-related genes, respectively. Of the biallelic alterations, only 45% occurred in traditional BRCA1/2 associated hereditary cancers (HBOCs, namely breast, ovarian and prostate cancer). Bi-allelic, but not mono-allelic, pathogenic genetic alterations in HR-related genes were significantly associated with genomic evidence of HR deficiency across cancer types, in HBOCs and within breast cancer. In HBOCs, bi-allelic alterations in HR-related genes were mutually exclusive (p=0.02). In breast cancer, bi-allelic inactivation of HR DNA repair-related genes was observed in 9.8%, of which 7.8% involved a germline pathogenic mutation and 2.0% were solely somatic. In breast cancer, in addition to BRCA1 and BRCA2, bi-allelic inactivation of PALB2 (0.2%), ATM (1.1%) and POLQ (0.3%) were found to be associated with genomic features of HR deficiency. In the 24 additional breast cancers, 9 were classified by the functional ex-vivo RAD51 assay as HR-deficient, 8 of which displayed bi-allelic inactivation of one HR-related gene, whereas only 1 of the 15 HR-proficient breast cancers harbored bi-allelic inactivation of HR-related genes (p<0.001). Conclusion: Bi-allelic germline and somatic alterations of HR-related genes in addition to BRCA1 and BRCA2 are present in breast and other cancer types. Irrespective of the gene, these bi-allelic alterations are associated with HR deficiency as defined by genomic methods and functional assays, expanding the potential opportunities for therapies targeting HR DNA repair defects. Citation Format: Riaz N, Blecua P, Lim RS, Shen R, Higginson DS, Weinhold N, Norton L, Weigelt B, Powell SN, Reis-Filho JS. Bi-allelic alterations in homologous recombination (HR) DNA repair-related genes as the basis for HR defects in human cancers: A pan-cancer genomics and functional analysis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD8-09.

Individuals with breast cancer susceptibility genes(BRCA) germline mutations have a significantly increased lifetime risk for breast cancer, BRCA-mutant cancer cells have abnormal homologous recombination repair of DNA.Inhibition of Poly (ADP-ribose) polymerase (PARP) has shown marked benefit for breast cancer with homologous recombination deficiency, whether driven by defects in BRCA1, BRCA2, or other pathway components. Currently, under U. S.Food and Drug Administration approval of PARP inhibitor on ovarian cancer, clinical trials for PARP inhibitor are being conducted to assess whether PARP inhibitor as monotherapy or in combination with other drugs could benefit more breast cancer patients. Besides the studies of PARP inhibitor as monotherapy in breast cancer patients, researches in investigating platinum-PARP inhibitor combination in BRCA mutated breast cancer or triple negative breast cancer were conducted. In addition, many studies of immunotherapy combined with PARP inhibitor in breast cancer are ongoing. However, resistance to PARP inhibitors and the toxicity of the drugs remain challenges. This review will discuss the utility and unsolved problems of PARP inhibitor in breast cancer as well as the potential future directions. Key words: Breast neoplasms; Poly(ADP-ribose) polymerases; Enzyme Inhibitors; Research progress

Background: Identification of germline mutations in hereditary breast cancer (HBC) genes can have profound benefits in the treatment of breast cancer (BC) and manging risk for patient and the family. In addition, sequencing of the tumours can reveal clinically relevant somatic features, such as homologous recombination deficiency (HRD) and mutational signatures, that can guide treatment which are not discernible by germline sequencing alone. We hypothesised that current standard of care where women diagnosed with BC are not routinely offered tumour sequencing is suboptimal as fails to exploit all therapeutic vulnerabilities which can be exploited in the treatment of BC. The MAGIC study is the first prospective trial in Australia of unselected invasive BCs in general oncology practice combining both germline and tumour sequencing. Methods: The MAGIC study included a total of 651 consecutive consented patients presenting with non-metastatic BC between June 2020 to March 2023. It included invasive BC, high-grade DCIS and pleomorphic LCIS. The sequencing was conducted in two phases; for phase one, 157 cases underwent whole genome sequencing (WGS) on both germline and matched BC DNA while for phase two, 494 cases underwent only germline whole exome sequencing. Germline variants were interrogated for pathogenic variants in BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, BRIP1, RAD51B, RAD51C, RAD51D, MLH1, MSH2, MSH6, PMS2, CDH1, PTEN, STK11, TP53 and NTHL1. Tumour homologous recombination (HR) repair deficiency was calculated using HRDetect (Nat Med 2017;23:517). For BCs showing a high HRDetect score ( >0.75) all HBC gene promoter CpG islands were assessed for hypermethylation using the Twist NGS Methylation system. Mutational signatures were calculated from somatic mutations identified from whole genome sequenced BCs using the DeconstructSig package in R (Genome Biol. 2016;17:31) and referenced against the COSMIC v2 signature catalogue. Results: Actionable germline variants in any HBC gene were identified in 7.7% of cases (7.8% of invasive and 6.1% of in situ BC) with 5.2% having an actionable mutation in an HR pathway gene (BRCA1, BRCA2, PALB2 and RAD51C). Most cases with pathogenic variants showed bi-allelic inactivation in the tumour. All BRCA1, BRCA2 and PALB2 tumours showed high HRDetect and/or HRD scores consistent with loss of HR repair function, indicating them to be the underlying hereditary driver of the BC. One BC with a pathogenic PMS2 variant showed retention of the wild-type allele with no evidence of a mismatch repair mutational signature suggesting PMS2 was not the driver of this BC. Of the 157 BC in phase one that underwent WGS and did not have a germline pathogenic HBC gene variant, 16% (18/117 invasive and 3/13 DCIS) had an HRDetect score of >0.7 indicative of a homologous recombination repair defect and therefore biologically a “BRCA-like” tumour. Of these 21 BRCA-like cases, only 3 invasive BCs had clear somatic bi-allelic inactivation of an HBC gene that could explain the high HRDetect score (2 BRCA1 and 1 BRCA2). One of the 21 BRCA-like cases had a germline variant of unknown significance (VUS) in an HBC gene (a BRCA2 missense variant in an ER+ tumour. The tumour showed loss of the wild-type allele). Of the BRCA-like cases, 71% were ER positive which included 3 invasive lobular cancers. Conclusion: Tumour sequencing identified three times as many cases that might be eligible for HR repair defect targeted therapy than germline testing alone (16% versus 5.2%). Unlike a previous study (Ann Oncol., 2019 30:1071-1079), the majority of the BRCA-like BCs were ER+ with no evidence of a VUS in a known HBC gene suggesting these are driven by novel germline or somatic mutations in genes involved in DNA HR repair. Citation Format: Ian Campbell, Lisa Devereux, Kirsten Hogg, Luxi Lal, Michelle Sinclair, Lesley Stafford, Magnus Zethoven, Anita Skandarajah, Paul James, Geoffrey Lindeman, Bruce Mann, Dilanka De Silva. The MAGIC study: Universal whole genome tumour and germline sequencing of newly diagnosed breast cancer identifies a high proportion of ER+ BRCA-like tumours [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-08-11.

Objectives: Approximately, 5-10% of breast cancer (BC) are related to inherited germline mutations. Homologous recombination repair (HRR) of DNA is the most important theory in hereditary cancer, in which BRCA1/2 are the most recognized and tested genes. Genetic testing for hereditary BC has changed significantly. Increasing evidence suggests parallel multigene testing of HRR gene. Methods: NGS-based germline multigene-panel testing of 105 HRR gene was performed on 159 high risk BC patients with at least one of the following risk factors: triple negative BC, early onset (≤45y), family history of BC and bilateral BC. Capture-base targeted sequencing was performed on white blood cells using a panel consisting of 105 genes related to homologous recombination repair of DNA. Results: Among the 159 patients of median age 40, All the patients had early stage infiltrating ductal carcinoma. 12 patients (7.55%) had pathogenic BRCA1/2 mutation. We investigated the prevalence of 21 pathogenic germline pathogenic mutations beyond BRCA1/2 from 21 patients spanning 13 HRR genes, resulting in a prevalence rate of 13.21% for pathogenic germline mutations in high risk germline BRCA1/2 WT BC patients. PALB2 is the most common gene with pathogenic mutation (n=5). In these 21 patients, 18 (85.71%) were diagnosed with BC before the age of 45, within 7 patients (33.33%) even younger than 35 years old. 7 of them (33.33%) had bilateral BC; one had triple negative BC (4.76%) and 5 patient (23.81%) had a family history of BC. Nearly half of the patients (n=9) presented at least 2 of the risk factors. Conclusions: Our study confirms the clinical significance of testing non-BRCA genes, and suggests multigene panel testing for patients at risk for hereditary BC as a routine in patients with those risk factors, especially young and bilateral BC patients even without family history of BC. Citation Format: Xiaying Kuang, Nan Shao, Ying Lin. Clinical significance of pathogenic variants in germline BRCA1/2 wild type patients at risk for hereditary breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 813.

Introduction: Germline pathogenic variants in genes in the homologous recombination (HR) pathway such as BRCA1, BRCA2, ATM, PALB2, RAD51C and RAD51D confer an increased risk of breast and ovarian cancers. Screening for germline variants in these cancer susceptibility genes is critical as prophylactic measures and monitoring can be performed in these individuals to minimize their risk of developing breast and/or ovarian cancers. More recently, it has been recognized that cancers arising in carriers of germline pathogenic (P)/ likely pathogenic (LP) variants in HR genes often show a homologous recombination (HR) deficient (HRD) phenotype and can be targeted with platinum agents or PARP inhibitors. RAD51B is a RAD51 paralog that binds to RAD51C and functions as a heterodimer in the HR pathway. Whilst RAD51B germline variants have been reported in isolated cases of breast and ovarian cancers, it is unclear as to whether RAD51B P/LP germline variants would confer predisposition to these cancer types. Materials and Methods: We screened 9,287 consecutive unselected cancer patients who consented for both tumor and germline testing using the MSK-IMPACT platform for the presence of RAD51B germline truncating variants. Selected RAD51B germline mutant breast cancers identified by MSK-IMPACT were subjected to whole-exome sequencing (WES) analysis to determine the dominant mutational signatures using DeconstructSigs. Functional assays were performed to ascertain the impact of RAD51B loss on HR DNA repair and PARP inhibitor sensitivity using genome editing methods in non-malignant breast epithelial cell lines. Results: Out of the 9,287 cancer patients, we detected likely pathogenic loss of function RAD51B germline variants in 11 patients (0.12%), which was similar to the frequency detected in the gnomAD database (0.09%). All female carriers of RAD51B loss of function variants (n=8) had breast or ovarian cancers (8/1,619 breast or ovarian cancers, 0.5%). The observed carrier frequencies of germline truncating variants in RAD51B was statistically enriched in breast and ovarian cancer patients compared to individuals in the gnomAD database (0.5% vs 0.09% P=0.0003; odds ratio = 5.06 (95% CI: 2.1-10.3)). Although segregation studies were not available, 9/11 of the RAD51B germline loss of function variant carriers had a personal or family history of breast or ovarian cancers. All five breast and ovarian cancers from RAD51B mutation carriers investigated by WES were found to harbor RAD51B bi-allelic inactivation through loss of heterozygosity of the RAD51B wild-type allele. In addition, these five cases were found to display genomic features of HRD including high large-scale state transition scores and a dominant mutational signature 3. CRISPR/Cas9 genome editing of RAD51B in a non-malignant breast epithelial cell model revealed that RAD51B deficient cells display PARP inhibitor sensitivity similar to that reported in BRCA1 and BRCA2 deficient cell lines. Conclusion: RAD51B loss-of-function germline variants confer susceptibility to breast and ovarian cancer development. Breast and ovarian cancers occurring in the context of RAD51B germline mutations harbor bi-allelic inactivation of RAD51B through loss-of-heterozygosity of the wild-type allele, genomic features of HRD and are likely sensitive to PARP inhibition. Albeit rarely germline mutated, RAD51B should be considered as an addition to clinical germline testing panels for hereditary breast and ovarian cancer syndrome patients. <!–EndFragment→ Citation Format: Diana SReis-FilhoReis-Filho Mandelker, Semanti Mukherjee, Jeremy Setton, Pier Selenica, Yelena Kemel, Ozge Ceyhan-Birsoy, Margaret Sheehan, Kaitlyn Tkachuk, David N Brown, Simon Powell, Britta Weigelt, Mark E Robson, Nadeem Riaz, Kenneth Offit, Jorge S Reis-Filho. RAD51B loss-of-function variants confer susceptibility to hereditary breast and ovarian cancers and result in tumors with genomic features of homologous recombination repair defects [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-09-01.

Epithelial cancers arise through acquisition of mutation in multiple genes causing either gain or loss of functions that give a phenotypic advantage to the affected cell and acquisition of key features of malignancy commonly described as the “hallmarks of cancer”. This acquisition of sufficient mutational burden to acquire these “hallmarks” may be accelerated by deficiency in one or more of number of partially redundant DNA repair mechanisms that have evolved to maintain genome stability. Many forms of chemotherapy and radiation therapy have long exploited tumor's relative deficiencies in the DNA damage response and in DNA repair specifically. Recently more specific inhibitors of DNA repair and DNA damage response effectors have been developed and tested in preclinical models and early phase clinical trials. These have shown proof of concept for the principle of tumor specific “synthetic lethality” where the synergistic combination of a therapeutically induced deficiency in one form of genome stability mechanism with a tumor specific constitutive defect in a mutually dependant from DNA repair leads to high specific tumor cell killing. This principle has been found to apply to the combination of small molecule induced inhibition of PARP1 and PARP2 in combination with tumor cell intrinsic defects in the homologous recombination (HR) DNA repair mechanism. A number of genes involved in HR, including BRCA1, BRCA2, ATM and PALB2 lead to elevated risk of breast cancer when mutated in the germline. The tumors that develop in these contexts have, as a result, functional defects in HR. Recent evidence has also suggested that a significant proportion of breast cancers show distinctive patterns of genome instability and changes in DNA damage induced repair protein expression phenotypes that indicate loss of function of HR DNA repair. Recent correlative biology studies in breast cancer and ovarian cancer trials show correlation between these emerging biomarkers with efficacy of platinum based chemotherapy, also known to target deficiency in HR. The further investigation of PARP inhibitors in breast cancer has been slowed by an initial focus of interest in the larger sporadic breast cancer population and the difficulties in identifying a likely maximum effect population in this wider population without robust assays for defects in the DNA damage response. Progress has also been hampered by the difficulty of finding tolerable combinations of these agents with chemotherapy. Differences have been uncovered between PARP inhibitor agents both in terms of potency and mechanisms of action related to presence or absence of trapping of PARP in complex with DNA that may differentially effect efficacy in targeting HR DNA repair defects or potentiation of DNA damaging chemotherapeutics. The small molecule Iniparib has been found to have no significant PARP inhibitory effect and in consequence the results with this agent in breast cancer should not be regarded as having tested any role for PARP inhibition in this context. Focus in late phase clinical trials of PARP inhibitors in breast cancer is currently returning to potent PARP inhibitors and the original proof of concept germline mutation carrier population. Here crucial questions may still remain around prevalence of mechanisms of acquired resistance demonstrated to occur in preclinical models through somatic reversion of mutations in BRCA1 and BRCA2, selection for loss of function of genes such as 53BP1 that stimulate alternative use of Non-homologous end-joining (NHEJ) repair and overexpression of the P-glycoprotein drug efflux transporter. In this lecture I will review the preclinical and emerging biomarker data described above and their relevance to current and imminent clinical trials exploring PARP inhibition in breast cancer. Citation Format: Andrew Tutt. Exploiting DNA repair deficiencies in breast cancer using PARP inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA19.

Myeloproliferative neoplasms (MPNs) present with excessive production of terminally differentiated blood cells derived from myeloid progenitor cells. 5-10% of Philadelphia (Ph)-negative MPNs, which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), may eventually progress to acute myeloid leukemia (AML) with poor outcome. The lack of curative results with conventional therapy demonstrates an urgent need to identify pathways perturbed in MPNs which can be targeted. Previously, complete responses were observed in MPN patients in a trial combining poly (ADP-ribose) polymerase (PARP) inhibitor ABT-888 with carboplatin and topotecan which was associated with in vitro PARP inhibitor sensitivity (McDevitt et al., ASH 2011). To identify patients who will be sensitive to PARP inhibition, we investigated mechanisms underlying this favorable response. PARP inhibitors block repair of DNA single stand breaks, allowing these lesions to persist. The ensuing increase in DNA double stranded breaks (DSBs) cannot be repaired in cells defective in DNA homologous recombination (HR) repair and results in an exquisite death response of the malignant clone. Previously, we have observed using SNP-A arrays that MPNs have frequent deletions including genes involved in repairing DNA DSBs and cross-links: 13q (BRCA2), 9q (FANCC) and 11p (FANCF) (McDevitt et al., ASH 2011). In this study, we examined the prevalence of inactivation in HR repair genes (BRCA1, BRCA2, Fanconi Anemia genes, ATR, ATM, BLM) via epigenetic silencing in MPN and AML patients using methylation-specific PCR (MSP). BRCA1 quantitative MSP (qMSP) revealed promoter hypermethylation in 9% of 59 MPN patients which associated with decreased BRCA1 transcript. BRCA1 methylation was also observed in AML, and additional samples will be examined to clarify the role of epigenetic silencing in disease progression. Hypermethylation of FANCC (3%) and FANCL (3%) was observed in 30 AML samples at low frequencies, and has not been observed in the MPN samples examined thus far. Ongoing studies include SNP arrays to determine if epigenetic silencing complements allelic loss to generate HR repair defects, DNA damage foci formation assays with PARP inhibition to investigate functional defects in HR repair in primary cells isolated from patients in the ABT-888 trial. In addition, karyotypic profiles will be investigated to determine if complex cytogenetics are associated with repressed HR repair. In addition to studying how defects in HR repair, either via promoter methylation or allelic loss, contribute to disease pathogenesis, our studies will also determine if epigenetic silencing of HR genes can be used to identify a subpopulation of MPN patients that will respond favorably to PARP inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4025. doi:1538-7445.AM2012-4025

Objective To analyze the impact of Helicobacter pylori standard strain (Hp P12) and its virulence factor vacuolating cytotoxin A (VacA) on DNA damage and homologous recombination (HR) repair in a human gastric epithelial cell line (GES-1). Methods Strains of Hp P12 and vacA gene knockout Hp P12 (Hp P12 ΔvacA) were respectively used to infect GES-1 cells at a multiplicity of infection of 100. GES-1 cells treated with etoposide (50 μmol/L) or mitomycin (0.5 μg/ml) for 2 h were used as positive control. Western blot and immunofluorescence were performed to detect the expression of DNA damage marker protein γH2AX and key HR repair proteins (Rad51, pMRE11, CtIP and pCtIP) and the recruitment of them at DNA damage sites. Human embryonic kidney HEK-293 (DR-GFP) cells were infected with Hp P12 and Hp P12 ΔvacA strains to verify the impact of VacA on HR repair efficiency. Results The expression and recruitment of γH2AX and key HR repair proteins (Rad51, pMRE11, CtIP and pCtIP) were increased in Hp P12-infected cells as compared with that in uninfected and Hp P12 ΔvacA-infected cells (all P<0.05). To evaluate the HR repair efficiency, I-SceⅠ plasmid-transfected HEK-293 (DR-GFP) cells were infected with Hp P12 and Hp P12 ΔvacA and the results showed that green fluorescent protein (GFP)-positive cells were decreased after infection, especially in Hp P12 ΔvacA-infected cells (both P<0.05). Conclusions Hp P12 infection could cause DNA damage and promote HR repair in GES-1 cells, in which the virulence factor VacA played an important role. Key words: Helicobacter pylori; Homologous recombination repair; GES-1 cell; Vacuolating cytotoxin A (VacA)

The BRCA2 breast cancer tumor suppressor is involved in the repair of double strand breaks and broken replication forks by homologous recombination through its interaction with DNA repair protein Rad51. Cells defective in BRCA2.FANCD1 are extremely sensitive to mitomycin C (MMC) similarly to cells deficient in any of the Fanconi anemia (FA) complementation group proteins (FANC). These observations suggest that the FA pathway and the BRCA2 and Rad51 repair pathway may be linked, although a functional connection between these pathways in DNA damage signaling remains to be determined. Here, we systematically investigated the interaction between these pathways. We show that in response to DNA damage, BRCA2-dependent Rad51 nuclear focus formation was normal in the absence of FANCD2 and that FANCD2 nuclear focus formation and mono-ubiquitination appeared normal in BRCA2-deficient cells. We report that the absence of BRCA2 substantially reduced homologous recombination repair of DNA breaks, whereas the absence of FANCD2 had little effect. Furthermore, we established that depletion of BRCA2 or Rad51 had a greater effect on cell survival in response to MMC than depletion of FANCD2 and that depletion of BRCA2 in FANCD2 mutant cells further sensitized these cells to MMC. Our results suggest that FANCD2 mediates double strand DNA break repair independently of Rad51-associated homologous recombination.

Background: Homologous recombinant repair (HRR) deficit and the associated sensitivity to poly (ADP-ribose) polymerase inhibitors (PARPi) has been well studied in breast, ovarian, prostate, and pancreatic cancers, but very little is known about it in other cancer types. Objectives: We sought to understand the spectrum of HRR mutations in various cancer types, with the goal of identifying therapeutic targets in lesser-explored cancers. Materials and Methods: In this retrospective study conducted between January 2021 and December 2022, we analyzed a cohort of 659 patients with various cancer types with mutations in 15 HRR genes using next generation sequencing, at 4baseCare Onco Solutions Pvt. Ltd., Bengaluru, Karnataka, India. Results: We identified a total of 825 gene variants, including 366 likely pathogenic/pathogenic mutations (44.4%), with BRCA1 (196 variants; 22.8%), BRCA2 (183 variants; 21.3%), and ATM (157 variants; 18.3%) being prevalent. Germline and somatic mutations were prevalent in BRCA1 (114 variants; 60.3%) and BRCA2 (46 variants; 24%), respectively. Recurrent mutations were identified in 8 genes, including BRCA1, BRCA2, BRIP1, BARD1, CDK12, CHEK2, PALB2 and RAD54L. BRCA1 and BRCA2 mutations were observed in breast, gynecologic, and musculoskeletal cancers; ATM and BRCA2 in gastrointestinal and biliary tract cancers, respiratory, and head-and-neck cancers; BRCA2 and CDK12 in genitourinary cancers. Additionally, co-occurring mutations (in genes such as BRCA1-BRIP1, ATM-BRCA2, ATM-BRIP1) and known therapeutically significant mutations were identified. Conclusions: The presence of therapeutically significant HRR mutations across a broad spectrum of cancer types in our study suggests that these mutations can possibly be targeted, especially in cancers where there is a paucity of therapeutic targets. Further, non-BRCA HRR genes, such as ATM and CDK12, could play a more prominent role than previously recognized.

The tumor suppression function of p53 is mostly conferred by its transactivation activity, which is inactivated by p53 mutations in approximately 50% of human cancers. In cancers harboring wild type p53, the p53 transactivation activity may be compromised by other mechanisms. Identifying the mechanisms by which wild type p53 transactivation activity can be abrogated may provide insights into the molecular etiology of cancers harboring wild type p53. In this report, we show that BCCIP, a BRCA2 and CDKN1A-interacting protein, is required for the transactivation activity of wild type p53. In p53 wild type cells, BCCIP knock down by RNA interference diminishes the transactivation activity of p53 without reducing the p53 protein level, inhibits the binding of p53 to the promoters of p53 target genes p21 and HDM2, and reduces the tetrameric formation of p53. These data demonstrate a critical role of BCCIP in maintaining the transactivation activity of wild type p53 and further suggest down-regulation of BCCIP as a novel mechanism to impair the p53 function in cells harboring wild type p53.

Src homology 2 domain-containing phosphatase (SHP2), which is encoded by proto-oncogene PTPN11, is one of the transmembrane protein-tyrosine phosphatases. SHP2 has an important function in signal transduction pathways and activities of cells through the regulation of tyrosine phosphorylation level of intracellular proteins. The status of SHP2 activation is closely connected with the regulation of hormone levels, state of invasion and metastasis of tumor, development and progression of tumor stem cells of breast cancer, as well as signal pathways including Ras/ERK and PI3K/Akt/mTOR. Gene knockout or gene silencing expression helps inhibit tumor growth, irreversibly hindering the ability of the tumor to regain stem cells and disturb the signal pathways of the invasion and metastasis of breast cancer. Recent studies have shown that SHP2 may help in bringing anticancer drugs to a higher level. This article concentrates on the research progress in relationship of SHP2 with invasion and metastasis of breast cancer.

Background PARP inhibitors have proven efficacy in breast cancer patients with germline BRCA1 or BRCA2 mutations (gBRCAmt) but also have potential to be effective in cancers with defects in homologous recombination (HR) DNA repair. Moreover, synergy between PARP inhibition and immune checkpoint blockade is expected based on increased mutation burden, neoantigen expression, and immunogenic cell death. Triple negative breast cancer (TNBC) or low estrogen receptor (ER)-positive breast cancers may have diverse defects in HR repair. Methods This is a window of opportunity, serial biopsy trial of olaparib and durvalumab before standard neoadjuvant chemotherapy for TNBC or low ER+ breast cancer (NCT03594396). Patients had clinical stage II/III TNBC or low ER+ HER2- breast cancers where ER was expressed in 10% or lower in tumor cells. Olaparib 300mg bid was given for 4 weeks without rest. One dose of durvalumab 1500mg was given on day 15. Study tumor biopsy and blood sample were taken before the study treatment, after 2 weeks of olaparib before durvalumab, and 2 weeks after durvalumab at the completion of 4 weeks of olaparib. FDG-PET/MRI was taken at baseline, after 2 weeks, after 4 weeks of study treatment, and computed tomography (CT) scan at baseline and after 4 weeks. After the study treatment, standard neoadjuvant chemotherapy with 4 cycles of doxorubicin+cyclophosphamide followed by 4 cycles of docetaxel was given. Primary endpoint was the changes of tumor biology detected by serial tumor biopsy. Secondary endpoints were pathological complete response (pCR) rate, response rate, prediction of early metabolic response by functional HR status, and safety. Functional HR deficiency was assessed by counting RAD51 foci as a marker for HR repair in the baseline tumor tissues before and after 30Gy irradiation to induce DNA damage. Results Fifty-four female patients were enrolled (median age 40 years, range 24-68). ER was negative (TNBC) in 43 patients and low-positive in 11 patients. Clinical stage was II in 25 patients and III in 29 patients, and axillary lymph node involvement was in 47 patients. gBRCAmt was assessed in 53 patients and 16 (30.1%) harbored pathogenic mutations. Functional HR status as measured by RAD51 foci formation was deficient in 27 (50%) patients and proficient in 27 (50%) patients. Functional HR deficiency was related with early metabolic response by FDG-PET after 2 weeks of olaparib (response in 17/27 HR-deficient tumors [63.0%] vs. 7/27 HR-proficient tumors [25.9%]; p=.006). After 4 weeks of olaparib and durvalumab, HR-deficiency was still related to metabolic response (23/27 vs. 17/27, respectively; p=.062) but HR-proficient tumors also showed metabolic decline. Moreover, HR deficiency was also related with RECIST response measured by CT after 4 weeks (17/27 vs. 9/27; p=.029). As of June 2021, 40 patients completed the neoadjuvant treatment and surgery; among those, 30 achieved pCR (pCR rate 75%). Among 13 patients with gBRCAmt who underwent surgery, 11 achieved pCR (84.6%). Updated results on pCR will be presented. Conclusions Olaparib and durvalumab followed by standard neoadjuvant chemotherapy shows very high pCR rate in TNBC or low ER+ stage II/III breast cancer. Functional HR status as measured by RAD51 foci formation was predictive of early metabolic response to olaparib. Genomic and transcriptomic analyses are underway in the samples before, during, and after olaparib and durvalumab. Citation Format: Seock-Ah Im, Kyung-Hun Lee, Ahrum Min, Daewon Lee, Tae Yong Kim, Han Suk Ryu, Jiwon Koh, Gi-Jeong Cheon, Yoon-Jung Shin, Yujin Kim, Hyeong-Gon Moon, Wonshik Han, Han-Byoel Lee, Morgan Diolaiti, David Quigley, Alan Ashworth, Nariya Cho. Window of opportunity trial of neoadjuvant olaparib and durvalumab for triple negative or low ER-positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD15-08.

Purpose: The standard treatment for organ-confined prostate cancer (PC) is surgery or radiation, and locally advanced PC is typically treated with radiotherapy alone or in combination with androgen-deprivation therapy. Here, we investigated whether Stat5a/b participates in regulation of double-strand DNA break repair in PC, and if Stat5 inhibition may provide a novel strategy to sensitize PC to radiation therapy. Experimental Design: Stat5a/b regulation of DNA repair in PC was evaluated by comet assay and clonogenic survival assay, followed by assays specific to homologous recombination (HR) DNA repair and nonhomologous end-joining (NHEJ) DNA repair. For HR DNA repair, Stat5a/b regulation of Rad51 and the mechanisms underlying the regulation were investigated in PC cells, xenograft tumors, and patient-derived PCs ex vivo in 3D explant cultures. Stat5a/b induction of Rad51 and HR DNA repair and responsiveness to radiation were evaluated in vivo in mice bearing PC xenograft tumors. Results: Stat5a/b is critical for Rad51 expression in PC via Jak2-dependent mechanisms by inducing Rad51 mRNA levels. Consistent with this, genetic knockdown of Stat5a/b suppressed HR DNA repair while not affecting NHEJ DNA repair. Pharmacologic Stat5a/b inhibition potently sensitized PC cell lines and PC tumors to radiation, while not affecting radiation sensitivity of the neighboring tissues. Conclusion: This work introduces a novel concept of a pivotal role of Jak2-Stat5a/b signaling for Rad51 expression and HR DNA repair in PC. Inhibition of Jak2-Stat5a/b signaling sensitizes PC to radiation and, therefore, may provide an adjuvant therapy for radiation to reduce radiation-induced damage to the neighboring tissues. Citation Format: Cristina Maranto, Vindhya Udhane, Ayush Dagvadorj, David T. Hoang, Lei Gu, Vitali Alexeev, Kareem Malas, Karmel Cardenas, Mateusz Koptyra, Jonathan R. Brody, Ulrich Rodeck, Carmen Bergom, Ken A. Iczkowski, Ken Jacobsohn, William See, Sara M. Schmitt, Marja T. Nevalainen. Stat5a/b blockade sensitizes prostate cancer to radiation through inhibition of Rad51 and DNA repair [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B073.