PARP Inhibitor Sensitivity Research Articles

Multi-scale characterisation of homologous recombination deficiency in breast cancer

BackgroundHomologous recombination is a robust, broadly error-free mechanism of double-strand break repair, and deficiencies lead to PARP inhibitor sensitivity. Patients displaying homologous recombination deficiency can be identified using ‘mutational signatures’. However, these patterns are difficult to reliably infer from exome sequencing. Additionally, as mutational signatures are a historical record of mutagenic processes, this limits their utility in describing the current status of a tumour.MethodsWe apply two methods for characterising homologous recombination deficiency in breast cancer to explore the features and heterogeneity associated with this phenotype. We develop a likelihood-based method which leverages small insertions and deletions for high-confidence classification of homologous recombination deficiency for exome-sequenced breast cancers. We then use multinomial elastic net regression modelling to develop a transcriptional signature of heterogeneous homologous recombination deficiency. This signature is then applied to single-cell RNA-sequenced breast cancer cohorts enabling analysis of homologous recombination deficiency heterogeneity and differential patterns of tumour microenvironment interactivity.ResultsWe demonstrate that the inclusion of indel events, even at low levels, improves homologous recombination deficiency classification. Whilst BRCA-positive homologous recombination deficient samples display strong similarities to those harbouring BRCA1/2 defects, they appear to deviate in microenvironmental features such as hypoxic signalling. We then present a 228-gene transcriptional signature which simultaneously characterises homologous recombination deficiency and BRCA1/2-defect status, and is associated with PARP inhibitor response. Finally, we show that this signature is applicable to single-cell transcriptomics data and predict that these cells present a distinct milieu of interactions with their microenvironment compared to their homologous recombination proficient counterparts, typified by a decreased cancer cell response to TNFα signalling.ConclusionsWe apply multi-scale approaches to characterise homologous recombination deficiency in breast cancer through the development of mutational and transcriptional signatures. We demonstrate how indels can improve homologous recombination deficiency classification in exome-sequenced breast cancers. Additionally, we demonstrate the heterogeneity of homologous recombination deficiency, especially in relation to BRCA1/2-defect status, and show that indications of this feature can be captured at a single-cell level, enabling further investigations into interactions between DNA repair deficient cells and their tumour microenvironment.

An activity-based functional test for identifying homologous recombination deficiencies across cancer types in real time

Homologous recombination (HR)-mediated DNA repair is a prerequisite for maintaining genome stability. Cancer cells displaying HR deficiency (HRD) are selectively eliminated by poly(ADP-ribose) polymerase inhibitors (PARPis). To date, sequencing of HR-associated genes and analyzing genome instability have been used as clinical predictions for PARPi therapy. However, these genetic tests cannot reflect dynamic changes in the HR status. Here, we have developed a virus- and activity-based functional assay to quantify real-time HR activity directly. Instead of focusing on a few HR-associated genes, our functional assay detects endpoint HR activity and establishes an activity threshold for identifying HRD across cancer types, validated by PARPi sensitivity and BRCA status. Notably, this fluorescence-based assay can be applied to primary ovarian cancer cells from patients to reflect their level of HRD, which is associated with survival benefits. Thus, our work provides a functional test to predict the response of primary cancer cells to PARPis.

Differential DNA damage repair and PARP inhibitor vulnerability of the mammary epithelial lineages

It is widely assumed that all normal somatic cells can equally perform homologous recombination (HR) and non-homologous end joining in the DNA damage response (DDR). Here, we show that the DDR in normal mammary gland inherently depends on the epithelial cell lineage identity. Bioinformatics, post-irradiation DNA damage repair kinetics, and clonogenic assays demonstrated luminal lineage exhibiting a more pronounced DDR and HR repair compared to the basal lineage. Consequently, basal progenitors were far more sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis) in both mouse and human mammary epithelium. Furthermore, PARPi sensitivity of murine and human breast cancer cell lines as well as patient-derived xenografts correlated with their molecular resemblance to the mammary progenitor lineages. Thus, mammary epithelial cells are intrinsically divergent in their DNA damage repair capacity and PARPi vulnerability, potentially influencing the clinical utility of this targeted therapy.

B-390 Next-generation Sequencing Panel to Detect Homologous Recombination Deficiency (HRD) Biomarkers in Brazilian Ovarian Cancer Samples

Abstract Background Homologous recombination deficiency (HRD) has been used as a biomarker of PARP inhibitors (PARPi) sensitivity in ovarian cancer (OC), especially in high-grade serous (HGS) types. Initially, only likely pathogenic (LPV) and pathogenic variants (PV) in BRCA1/2 were screened to define PARPi eligibility. However, LPV and PV are shown in less than 20% of OC, which restricted PARPi therapies to a small group. Advances in OC biology knowledge identified other markers that also contribute to HRD. Nowadays, is estimated that almost 50% of OC have HRD. When HRD is present, DNA damage is not properly repaired and the accumulated genomic changes over time are called Genomic Scars (GS). In this sense, Next Generation Sequencing (NGS) is important in GS detection due to searching different loci simultaneously, allowing an estimate of the GS score related to the HRD. So, this work aims to describe the profile of samples requesting an NGS test to detect HRD biomarkers. Methods Were evaluated anonymized data of 76 formalin-fixed, paraffin-embedded (FFPE) samples with OC diagnosis. The samples’ quality was evaluated with TapeStation 4200 and Qubit Fluorometer. Libraries were prepared with AmoyDx® HRD Focus Kit and sequenced with NextSeq 500. Data analysis was performed with ANDAS software. Results The HRD-positive percentage was 59,2% (45/76), being mostly BRCA wild type. Almost 15% of all samples presented PV or LPV in BRCA1 or 2, which was more frequent in BRCA1 (∼82%). The allele frequencies (AF) detected ranged from 5% to 83%. Two of the detected variants (classified as LPV) are not yet registered in databases. The HRD-negative group represented 34,2% (26/76), and 6,6% of performed tests had inconclusive results. Some requests (data not included) were not performed due to insufficient material, low quality, or FFPE block age greater than two years, which are limiting factors of this kind of test. Conclusion The availability of validated HRD testing options is essential to extend the reach to patients who may benefit from PARPi. In this analysis, most of the BRCA1/2 PV or LPV had AF above 50%, which can help oncologists to suggest germline PV investigation for these patients and also their families. Besides, our data showed a slightly higher HRD-positive rate than reported in the literature, although other works have already described rates above 60%. It reinforces the importance to monitor the HRD status of Brazilian samples to obtain a more assertive profile to the clinical practice.

ZEB1 promotes non-homologous end joining double-strand break repair.

Repair of DSB induced by IR is primarily carried out by Non-Homologous End Joining (NHEJ), a pathway in which 53BP1 plays a key role. We have discovered that the EMT-inducing transcriptional repressor ZEB1 (i) interacts with 53BP1 and that this interaction occurs rapidly and is significantly amplified following exposure of cells to IR; (ii) is required for the localization of 53BP1 to a subset of double-stranded breaks, and for physiological DSB repair; (iii) co-localizes with 53BP1 at IR-induced foci (IRIF); (iv) promotes NHEJ and inhibits Homologous Recombination (HR); (v) depletion increases resection at DSBs and (vi) confers PARP inhibitor (PARPi) sensitivity on BRCA1-deficient cells. Lastly, ZEB1's effects on repair pathway choice, resection, and PARPi sensitivity all rely on its homeodomain. In contrast to the well-characterized therapeutic resistance of high ZEB1-expressing cancer cells, the novel ZEB1-53BP1-shieldin resection axis described here exposes a therapeutic vulnerability: ZEB1 levels in BRCA1-deficient tumors may serve as a predictive biomarker of response to PARPis.

Histone methyltransferase KMT5C drives liver cancer progression and directs therapeutic response to PARP inhibitors.

Epigenetic plasticity is a major challenge in cancer-targeted therapy. However, the molecular basis governing this process has not yet been clearly defined. Despite the considerable success of poly(ADP-ribose) polymerase inhibitors (PARPi) in cancer therapy, the limited response to PARPi, especially in HCC, has been a bottleneck in its clinical implications. Herein, we investigated the molecular basis of the histone methyltransferase KMT5C (lysine methyltransferase 5C) that governs PARPi sensitivity and explored a potential therapeutic strategy for enhancing PARPi efficacy. We identified KMT5C, a trimethyltransferase of H4K20, as a targetable epigenetic factor that promoted liver tumor growth in mouse de novo MYC/Trp53-/- and xenograft liver tumor models. Notably, induction of KMT5C by environmental stress was crucial for DNA repair and HCC cell survival. Mechanistically, KMT5C interacted with the pivotal component of homologous recombination repair, RAD51, and promoted RAD51/RAD54 complex formation, which was essential for the activation of dsDNA breaks repair. This effect depended on the methyltransferase activity of KMT5C. We further demonstrated that the function of KMT5C in promoting HCC progression was dependent on RAD51. Importantly, either a pharmacological inhibitor (A196) or genetic inhibition of KMT5C sensitized liver cancer cells to PARPi. KMT5C played a vital role in promoting liver cancer progression by activating the DNA repair response. Our results revealed a novel therapeutic approach using the KMT5C inhibitor A196, concurrent with olaparib, as a potential HCC therapy.

Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity

DNA single‐strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair‐defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5‐chloro‐2′‐deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA‐defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.

Heterochromatin-dependent Replication Stress: A Lesson from IDH1/2 Mutants.

Oncogenic point mutants of isocitrate dehydrogenases 1 and 2 (IDH2) generate 2-hydroxyglutarate (2HG), which inhibits lysine demethylases and increases heterochromatin. Tumor cells expressing IDH mutants are sensitive to poly(ADP) ribose polymerase (PARP) inhibitors, offering an opportunity to eliminate IDH-driven tumor cells in therapy. Expression of an oncogenic IDH1 mutant in cells leads to aberrant heterochromatin formation at DNA breaks and impairs DNA repair through homologous recombination (HR), providing a possible explanation for the PARP inhibitor sensitivity of IDH mutant cells. However, a recent study published in Molecular Cell shows that IDH mutant tumors do not display the genomic alterations associated with HR defects. Instead, IDH mutants induce heterochromatin-dependent DNA replication stress. Furthermore, PARP is activated by the replication stress induced by IDH mutants and required for suppressing the ensuing DNA damage, providing an alternative model to explain the susceptibility of IDH mutant cells to PARP inhibitors. This study presents a new example of oncogene-induced and heterochromatin-dependent replication stress, and a role of PARP in the response to the stress, extending the molecular basis for PARP-targeted therapy.

An AlphaFold2 map of the 53BP1 pathway identifies a direct SHLD3-RIF1 interaction critical for shieldin activity.

53BP1 is a chromatin-binding protein that promotes DNA double-strand break repair through the recruitment of downstream effectors including RIF1, shieldin, and CST. The structural basis of the protein-protein interactions within the 53BP1-RIF1-shieldin-CST pathway that are essential for its DNA repair activity is largely unknown. Here, we used AlphaFold2-Multimer (AF2) to predict all possible pairwise combinations of proteins within this pathway and provide structural models of seven previously characterized interactions. This analysis also predicted an entirely novel binding interface between the HEAT-repeat domain of RIF1 and the eIF4E-like domain of SHLD3. Extensive interrogation of this interface through both in vitro pulldown analysis and cellular assays supports the AF2-predicted model and demonstrates that RIF1-SHLD3 binding is essential for shieldin recruitment to sites of DNA damage, and for its role in antibody class switch recombination and PARP inhibitor sensitivity. Direct physical interaction between RIF1 and SHLD3 is therefore essential for 53BP1-RIF1-shieldin-CST pathway activity.

Abstract B076: IGFBP3 promotes resistance to olaparib via modulating EGFR signaling in advanced prostate cancer

Abstract Background: Castration-resistant prostate cancer (CRPC) is an incurable disease and a leading cause of cancer death in men worldwide. Olaparib (Lynparza) was among the first PARP inhibitors (PARPi) approved for the treatment of CRPC tumors harboring DNA repair defects. However, clinical resistance to PARPi’s has been documented. The mechanisms underlying resistance to PARPi’s remain elusive. To study acquired resistance, we developed olaparib-resistant LN-OlapR and 2B-OlapR cell lines generated through chronic olaparib treatment of the olaparib-sensitive cell lines LNCaP and C4-2B, respectively. RNA-seq revealed IGFBP3 is overexpressed in both OlapR cell lines. IGFBP3 overexpression is correlated with poor clinical outcome and is thought to participate in DNA repair pathways. IGFBP3 plays a key role in nonhomologous end joining (NHEJ) repair through a ternary complex with EGFR and DNA-PKcs. The IGFBP3/EGFR signaling axis is thought to modulate NHEJ repair and could have implications for PARPi sensitivity. We hypothesize that increased IGFBP3 expression promotes PARPi resistance by enhancing DNA repair capacity. Methods: RNA-sequencing and gene set enrichment analysis were used to determine the expression profile changes in resistant cells compared to parental cells. Real time PCR and western blots confirmed the expression of DNA damage repair genes such as γH2AX, EGFR, and DNA-PKcs. ELISA was used to determine IGFBP3 secretion. RNAi was used to inhibit IGFBP3 and EGFR expression. Gefitinib was used to inhibit EGFR activity. Cell viability assays were used to assess cell growth. Results: Transcriptomic profiling revealed that IGFBP3 is highly expressed in resistant models. We verified increased levels of IGFBP3 RNA and protein in both OlapR models. We found that RNAi inhibition of IGFBP3 increases γH2AX and cleaved-PARP protein levels in the resistant models, which suggests accumulation of DNA double strand breaks (DSBs) leading to genomic instability and cell death. We discovered increased phosphorylation of EGFR and DNA-PKcs in the resistant cells. Furthermore, silencing/inhibiting IGFBP3 and EGFR reduces OlapR cell viability and resensitizes resistant cells to treatment. Conclusions: Our findings demonstrated that inhibiting IGFBP3 and EGFR aids in PARPi sensitivity in the resistant setting. Future work will utilize OlapR models to study how the IGFBP3/EGFR/DNA-PKcs protein complex promotes the development of resistance. Understanding the role of IGFBP3 in PARPi resistance will enhance our ability to re-sensitize resistant CRPC to PARPi therapeutics. Citation Format: Amy R. Leslie, Allen C. Gao, Alan P. Lombard, Shu Ning. IGFBP3 promotes resistance to olaparib via modulating EGFR signaling in advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B076.

Abstract PR014: Genetic determinants of PARP inhibitor sensitivity and resistance in prostate cancer

Abstract Prostate cancer (PCa) patients harboring BRCA1/2 mutations are often sensitive to PARP inhibition. However, genomic alterations in other DNA damage response (DDR) genes have not been consistently predictive of clinical response to PARP inhibition. In this study, we preformed genome-wide CRISPR/Cas9 loss-of-function screens in four BRCA-proficient PCa cell lines with the goal of expanding the use of PARP inhibitors (PARPis) beyond BRCA1/2 mutations and finding new synthetically lethal interactions. We provided a systematic view of the genetic determinants and mechanisms underlying PARPi sensitivity and resistance in PCa beyond BRCA1/2 alterations. We identified previously unknown genes whose loss has a profound impact on PARPi response, serving as new predictive biomarkers for PARP inhibition if mutated or deleted in PCa or therapeutic targets through pharmacologic inhibition in combination with PARPis. Specifically, we discovered two genes RNASEH2B and MMS22L, located on frequently deleted genomic regions on chromosomes 13q (up to 17%) and 6q (up to 14%) respectively in PCa, loss of which confers BRCA-like synthetic lethality to PARP inhibition. On the other hand, we found that inactivation of TP53 and RB1, two commonly mutated tumor suppressor genes in PCa, may compromise PARP inhibitor response. Mechanistically, loss of RNASEH2B confers PCa cells sensitivity to PARP inhibition due to impaired ribonucleotide excision repair and PARP trapping. When co-deleted with RB1 on the same genomic region, however, cells lose their sensitivity, in part, through E2F1-induced BRCA2 expression, thereby enhancing homologous recombination repair (HRR) capacity. Loss of MMS22L renders PCa cells hypersensitive to PARP inhibition by disrupting RAD51 loading required for HRR. However, this response is TP53-dependent. Inactivation of TP53 renders cells resistant to PARPis through increased BRCA2 expression, a target of TP53-E2F7 mediated transcriptional repression. Our findings may inform the use of PARPi beyond BRCA1/2-deficient tumors, but also indicate that current biomarkers guided by mutations of a single gene overlook concurrent genomic alterations.These results may explain some of the disparate clinical results from PARP inhibition due to interaction between multiple genomic alterations and support a comprehensive genomic testing to determine who may benefit from PARP inhibition. In addition, we found that loss of CHEK2 confers resistance rather than sensitivity to PARP inhibition through increased expression of BRCA2, which enhances HRR capacity sufficient for the repair of PARPi-induced double-strand DNA breaks and cell survival. Combined PARP and ATR inhibition overcomes PARPi resistance caused by CHEK2 loss. Our findings support reevaluation of current biomarkers for PARP inhibition in PCa treatment. Citation Format: Takuya Tsujino, Tomoaki Takai, Chenkui Miao, Fu Gui, Adam S. Kibel, Li Jia. Genetic determinants of PARP inhibitor sensitivity and resistance in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR014.

Tandem duplicator phenotype: A novel targetable subgroup in pancreatic cancer?

4161 Background: Validated biomarkers are urgently needed to provide a more precise approach in pancreatic cancer (PDAC). Homologous recombination deficiency (HRD) is a heterogenous cohort in PDAC and predicts platinum and PARP inhibitor sensitivity. The BRCA-1 genotype associates with distinct tandem duplications (TDs), however non-BRCA 1 TD phenotypes (TDP) have been observed across tumour types and may be associated with perturbations in CDK12, FBXW17, and CCNE1. Here, we characterize the prevalence and outcomes of TDP in a large series of prospectively sequenced PDAC. Methods: Whole-genome sequencing was performed in 191 early stage resected and 275 advanced PDAC cases in the PanCuRx initiative and the COMPASS trial at the Ontario Institute for Cancer Research and Princess Margaret Cancer Centre. Tumors underwent laser capture enrichment prior to sequencing. TD scores were calculated as previously described by Menghi et al, and HRDetect scores assigned. Samples were classified as classical or basal-like by Moffitt. Outcomes of patients with TDP tumors were evaluated. Tumors that are nonHRD and nonTDP are referred to as typicals. Results: Of 466 cases, 46 were identified as TDP (9.9%; 17 resected, 29 advanced). Subgroups of TDP by etiology included BRCA1 (n=8; 6 germline, 2 somatic), somatic CCNE1 (n=1), and unknown (n=37; no identified alterations in TDP-related genes). Pathogenic germline variants were not found in unique genes other than BRCA1. HRD TDP was associated with a significantly smaller TD size compared to nonHRD TDP (median 12 kb vs. 125 kb, p<0.0001) and median TD load of 114. NonHRD TDP genomes exhibited elevated TD load (median 70 vs. 13, p<0.0001) and HRDetect scores (p<0.0001) compared with typicals. NonHRD TDP was not prognostic in resected PDAC disease (p=0.6361) compared with typicals. We observed a trend towards improved response to 1st-line platinum therapy in nonHRD TDP vs. typicals (p=0.066) for patients with advanced disease. When stratified for only ‘classical’ RNA subtype cases as per Moffitt classification (HRD TDP:2 basal-like, 8 classical, 1 unknown; nonHRD TDP: 9 basal-like, 25 classical, 1 unknown), platinum therapy was correlated with better response in non-HRD TDP vs. typicals (n= 10, 70% ORR, p=0.0108) highlighting the chemoresistance of the basal-like subgroup. Superior survival was not observed in this small cohort. Conclusions: In PDAC, the etiology of nonHRD TDP is unclear but may represent a potential marker for platinum and DNA damage response agents. Further investigation is warranted with a larger sample size.

A phase II study of olaparib and durvalumab in patients with IDH-mutated cholangiocarcinoma.

4099 Background: IDH1/2 mutations (m) occur in approximately 25% of cholangiocarcinomas (CCAs) (20% intrahepatic, < 5% extrahepatic) and result in the accumulation of R-2-hydroxyglutarate (R2HG). R2HG, suppresses homologous recombination repair and as a result IDH1/2m are hypothesized to predict PARP inhibitor sensitivity. PARP inhibition upregulates PD-L1 and the combination is thought to be synergistic. We conducted a phase II trial of olaparib with durvalumab for patients with IDH-mutated cholangiocarcinoma. Methods: Patients with advanced IDHm CCA were enrolled in this phase II open-label study (NCT03991832). Patients received olaparib 300 mg twice daily continuously and durvalumab 1500 mg IV every 4 weeks. Simon’s optimal two-stage design was used, with an interim analysis planned for efficacy. 10 patients were accrued in the first stage, with a plan for expansion if ≥2 responses were observed. The primary objective was to determine the overall response rate (RECIST 1.1) and disease control rate (DCR). Secondary objectives included progression-free survival, overall survival, and safety of the olaparib and durvalumab combination. Results: 10 patients with IDH1m were enrolled in this study between January 2020-August 2021. Median age was 63.5 years (range 49-78), and 50% were female. All patients had a baseline ECOG PS of 0-1. 40% of patients had prior surgical resections and subsequently developed recurrent disease. 20% of patients had ³2 lines of prior chemotherapy, with 90% receiving prior platinum. Median duration of treatment was 1.95 months (range 1.8-13.5). There were no treatment-related grade 3-4 toxicities. Any grade toxicities included fatigue (100%), nausea (60%), anemia (20%), hypothyroidism (20%), elevated liver enzymes (20%). We did not observe any complete or partial responses. The DCR was 30% with 3 patients demonstrating stable disease. One patient remained on treatment for 13.5 months, and was eventually taken off study due to clinical progression. The median PFS was 1.97 months (95% CI 1.73-3.93). Given that no responses were seen in the initial 10 patients, the study did not proceed to Stage 2. Conclusions: While the combination of olaparib and durvalumab was well-tolerated, this study was discontinued after Stage I due to lack of efficacy. IDH1/2m may not induce a BRCAness phenotype and further correlative studies are planned to evaluate this. Clinical trial information: NCT03991832 .

ABRAXAS1 orchestrates BRCA1 activities to counter genome destabilizing repair pathways—lessons from breast cancer patients

It has been well-established that mutations in BRCA1 and BRCA2, compromising functions in DNA double-strand break repair (DSBR), confer hereditary breast and ovarian cancer risk. Importantly, mutations in these genes explain only a minor fraction of the hereditary risk and of the subset of DSBR deficient tumors. Our screening efforts identified two truncating germline mutations in the gene encoding the BRCA1 complex partner ABRAXAS1 in German early-onset breast cancer patients. To unravel the molecular mechanisms triggering carcinogenesis in these carriers of heterozygous mutations, we examined DSBR functions in patient-derived lymphoblastoid cells (LCLs) and in genetically manipulated mammary epithelial cells. By use of these strategies we were able to demonstrate that these truncating ABRAXAS1 mutations exerted dominant effects on BRCA1 functions. Interestingly, we did not observe haploinsufficiency regarding homologous recombination (HR) proficiency (reporter assay, RAD51-foci, PARP-inhibitor sensitivity) in mutation carriers. However, the balance was shifted to use of mutagenic DSBR-pathways. The dominant effect of truncated ABRAXAS1 devoid of the C-terminal BRCA1 binding site can be explained by retention of the N-terminal interaction sites for other BRCA1-A complex partners like RAP80. In this case BRCA1 was channeled from the BRCA1-A to the BRCA1-C complex, which induced single-strand annealing (SSA). Further truncation, additionally deleting the coiled-coil region of ABRAXAS1, unleashed excessive DNA damage responses (DDRs) de-repressing multiple DSBR-pathways including SSA and non-homologous end-joining (NHEJ). Our data reveal de-repression of low-fidelity repair activities as a common feature of cells from patients with heterozygous mutations in genes encoding BRCA1 and its complex partners.

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells

The PSMC3IP-MND1 heterodimer promotes meiotic D loop formation before DNA strand exchange. In genome-scale CRISPR-Cas9 mutagenesis and interference screens in mitotic cells, depletion of PSMC3IP or MND1 causes sensitivity to poly (ADP-Ribose) polymerase inhibitors (PARPi) used in cancer treatment. PSMC3IP or MND1 depletion also causes ionizing radiation sensitivity. These effects are independent of PSMC3IP/MND1's role in mitotic alternative lengthening of telomeres. PSMC3IP- or MND1-depleted cells accumulate toxic RAD51 foci in response to DNA damage, show impaired homology-directed DNA repair, and become PARPi sensitive, even in cells lacking both BRCA1 and TP53BP1. Epistasis between PSMC3IP-MND1 and BRCA1/BRCA2 defects suggest that abrogated D loop formation is the cause of PARPi sensitivity. Wild-type PSMC3IP reverses PARPi sensitivity, whereas a PSMC3IP p.Glu201del mutant associated with D loop defects and ovarian dysgenesis does not. These observations suggest that meiotic proteins such as MND1 and PSMC3IP have a greater role in mitotic DNA repair.

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells