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'Supplementary Table S2- Somatic mutations identified in the tumors from individuals III.6; III.7 and III.8 in family 1 and individual III.1 in family 2 among 193 DNA repair genes.'

This file includes supplementary Figure S1, Supplementary Figure S2 and Supplementary Figure S3. It also includes Supplementary Table S1 - RAD51D c.620C

'Supplementary Table3 -Presence of allelic imbalanced alleles in RAD51D and TP53 somatic mutations in RAD51D c.620C>T;p.S207L HGSC carriers.'

RAD51D is a key player in DNA repair by homologous recombination (HR), and RAD51D truncating variant carriers have an increased risk for ovarian cancer. However, the contribution of nontruncating RAD51D variants to cancer predisposition remains uncertain. Using deep sequencing and case–control genotyping studies, we show that in French Canadians, the missense RAD51D variant c.620C>T;p.S207L is highly prevalent and is associated with a significantly increased risk for ovarian high-grade serous carcinoma (HGSC; 3.8% cases vs. 0.2% controls). The frequency of the p.S207L variant did not significantly differ from that of controls in breast, endometrial, pancreas, or colorectal adenocarcinomas. Functionally, we show that this mutation impairs HR by disrupting the RAD51D–XRCC2 interaction and confers PARP inhibitor sensitivity. These results highlight the importance of a functional RAD51D–XRCC2 interaction to promote HR and prevent the development of HGSC. This study identifies c.620C>T;p.S207L as the first bona fide pathogenic RAD51D missense cancer susceptibility allele and supports the use of targeted PARP-inhibitor therapies in ovarian cancer patients carrying deleterious missense RAD51D variants. Cancer Res; 77(16); 4517–29. ©2017 AACR.

Multiple myeloma (MM) is a hematological malignancy that emerges from antibody-producing plasma B cells. Proteasome inhibitors, including the FDA-approved bortezomib (BTZ) and carfilzomib (CFZ), are frequently used for the treatment of MM patients. Still, a significant proportion of MM patients are refractory or develop resistance to this class of inhibitors, which represents a significant challenge in the clinic. Thus, identifying factors that determine the potency of proteasome inhibitors in MM is of paramount importance to bolster their efficacy in the clinic. Using genome-wide CRISPR-based screening, we identified a subunit of the mitochondrial pyruvate carrier (MPC) complex, MPC1, as a common modulator of BTZ response in two distinct human MM cell lines in vitro. We noticed that CRISPR-mediated deletion or pharmacological inhibition of the MPC complex enhanced BTZ/CFZ-induced MM cell death with minimal impact on cell cycle progression. In fact, targeting the MPC complex compromised the bioenergetic capacity of MM cells, which is accompanied by a reduced proteasomal activity, thereby exacerbating BTZ-induced cytotoxicity in vitro. Importantly, we observed that the RNA expression levels of several players in pyruvate metabolism were altered in advanced stages of MM wherein they correlated with poor prognosis for MM patients. Collectively, this study highlights the importance of the MPC complex for the survival of MM cells and their responses to proteasome inhibitors. These findings establish mitochondrial pyruvate metabolism as a potential target for the treatment of MM, and a hitherto unappreciated strategy to increase the efficacy of proteasome inhibitors in the clinic.

Interstrand DNA crosslinks (ICLs) represent complex lesions that block essential biological processes, including DNA replication, recombination, and transcription. Several pathways have been involved in ICL repair, in particular nucleotide excision repair (NER), translesion DNA synthesis (TLS), Fanconi anemia (FA), and homologous recombination (HR). Still, the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genome-wide screening, we identified the poorly characterized C1orf112 (also known as Apolo1) as a novel sensitizer to the clinically relevant ICL-inducing agent mafosfamide. Consistently, we noted that low expression of C1orf112 correlates with increased sensitivity to a series of ICL agents and PARP inhibitors in a panel of cell lines. We showed that lack of C1orf112 does not impact the initial recruitment and ubiquitylation of FANCD2 at the ICL site but rather impairs the resolution of RAD51 from ICL-induced DSBs, thereby compromising homology-directed DNA repair pathways. Our proximal mapping of C1orf112 protein neighbours coupled to structure-function analysis revealed that C1orf112, through its WCF motif, forms a complex with the N-terminal domain of the AAA+ ATPase FIGNL1 and regulates the interaction of FIGNL1 with RAD51. Our work establishes the C1orf112-FIGNL1 complex as an integral part of the HR-mediated response to ICLs by regulating the unloading of RAD51 during ICL repair.

Background Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we developed a strategy to curtail these drawbacks through inhibition of an mTORC1 target, the eukaryotic initiation factor 4A (eIF4A). Methods We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival and apoptosis, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP). Results eIF4i exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. eIF4Ai reduces mitochondrial membrane potential (MMP), the rate of ATP synthesis from mitochondrial respiration and glycolysis, and the expression of the antiapoptotic factors BCL2 and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity. Conclusions We discovered that AML cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. Our work indicates that eIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis. Furthermore, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.

Genome-wide CRISPR screens have been extremely useful in identifying therapeutic targets in diverse cancers by defining genes that are essential for malignant growth. However, most CRISPR screens were performed in vitro and thus cannot identify genes that are essential for interactions with the microenvironment in vivo. Here, we report genome-wide CRISPR screens in 2 in vivo murine models of acute myeloid leukemia (AML) driven by the KMT2A/MLLT3 fusion or by the constitutive coexpression of Hoxa9 and Meis1. Secondary validation using a focused library identified 72 genes specifically essential for leukemic growth in vivo, including components of the major histocompatibility complex class I complex, Cd47, complement receptor Cr1l, and the β-4-galactosylation pathway. Importantly, several of these in vivo–specific hits have a prognostic effect or are inferred to be master regulators of protein activity in human AML cases. For instance, we identified Fermt3, a master regulator of integrin signaling, as having in vivo–specific dependency with high prognostic relevance. Overall, we show an experimental and computational pipeline for genome-wide functional screens in vivo in AML and provide a genome-wide resource of essential drivers of leukemic growth in vivo.

Defects in DNA repair genes have been extensively associated with cancer susceptibility. Germline pathogenic variants (GPV) in genes involved in homologous recombination repair pathways predispose to cancers arising mainly in the breast and ovary, but also other tissues. The RAD51 paralogs RAD51C and RAD51D were included in this group 10 years ago when germline variants were associated with non-BRCA1/2 familial ovarian cancer. Here, we have reviewed the landscape of RAD51C and RAD51D germline variants in cancer reported in the literature during the last decade, integrating this list with variants identified by in-house patient screening. A comprehensive catalog of 341 variants that have been classified applying ACMG/AMP criteria has been generated pinpointing the existence of recurrent variants in both genes. Recurrent variants have been extensively discussed compiling data on population frequencies and functional characterization if available, highlighting variants that have not been fully characterized yet to properly establish their pathogenicity. Finally, we have complemented this data with relevant information regarding the conservation of mutated residues among RAD51 paralogs and modeling of putative hotspot areas, which contributes to generating an exhaustive update on these two cancer predisposition genes.

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability but little is known about how HP1 is controlled. Here, we show that the zinc finger protein POGZ promotes the presence of HP1 at DNA double‐strand breaks (DSBs) in human cells. POGZ depletion delays the resolution of DSBs and sensitizes cells to different DNA‐damaging agents, including cisplatin and talazoparib. Mechanistically, POGZ promotes homology‐directed DNA repair by retaining the BRCA1/BARD1 complex at DSBs in an HP1‐dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal. Pogz haploinsufficiency (Pogz(+)/delta) results in developmental delay, impaired intellectual abilities, hyperactive behaviour and a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Pogz(+)/delta mice are further radiosensitive and accumulate DSBs in diverse tissues, including the spleen and brain. Altogether, our findings identify POGZ as an important player in homology‐directed DNA repair both in vitro and in vivo.

It was hypothesized that variants in underexplored homologous recombination repair (HR) genes could explain unsolved multiple-case breast cancer (BC) families. We investigated HR deficiency (HRD)-associated mutational signatures and second hits in tumor DNA from familial BC cases. No candidates genes were associated with HRD in 38 probands previously tested negative with gene panels. We conclude it is unlikely that unknown HRD-associated genes explain a large fraction of unsolved familial BC.

The homologous recombination repair (HRR) pathway repairs DNA double-strand breaks in an error-free manner. Mutations in HRR genes can result in increased mutation rate and genomic rearrangements, and are associated with numerous genetic disorders and cancer. Despite intensive research, the HRR pathway is not yet fully mapped. Phylogenetic profiling analysis, which detects functional linkage between genes using coevolution, is a powerful approach to identify factors in many pathways. Nevertheless, phylogenetic profiling has limited predictive power when analyzing pathways with complex evolutionary dynamics such as the HRR. To map novel HRR genes systematically, we developed clade phylogenetic profiling (CladePP). CladePP detects local coevolution across hundreds of genomes and points to the evolutionary scale (e.g., mammals, vertebrates, animals, plants) at which coevolution occurred. We found that multiscale coevolution analysis is significantly more biologically relevant and sensitive to detect gene function. By using CladePP, we identified dozens of unrecognized genes that coevolved with the HRR pathway, either globally across all eukaryotes or locally in different clades. We validated eight genes in functional biological assays to have a role in DNA repair at both the cellular and organismal levels. These genes are expected to play a role in the HRR pathway and might lead to a better understanding of missing heredity in HRR-associated cancers (e.g., heredity breast and ovarian cancer). Our platform presents an innovative approach to predict gene function, identify novel factors related to different diseases and pathways, and characterize gene evolution.

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability, in particular by promoting homologous recombination (HR)-mediated DNA repair. However, little is still known about how HP1 is controlled during this process. Here, we describe a novel function of the POGO transposable element derived with ZNF domain protein (POGZ) in the regulation of HP1 during the DNA damage response in vitro. POGZ depletion delays the resolution of DNA double-strand breaks (DSBs) and correlates with an increased sensitivity to different DNA damaging agents, including the clinically-relevant Cisplatin and Talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair pathways by retaining the BRCA1/BARD1 complex at DSBs, in a HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal and Pogz haplo-insufficiency (Pogz+/Δ) results in a developmental delay, impaired intellectual abilities, a hyperactive behaviour as well as a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Importantly, Pogz+/Δ mice are radiosensitive and accumulate DSBs in diverse tissues, including the spleen and the brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo, with clinical implications for the WHSUS.

Promoting the macroautophagy/autophagy-mediated degradation of specific proteins and organelles can potentially be utilized to induce apoptosis in cancer cells or sensitize tumor cells to therapy. To examine this concept, we enriched for autophagosomes from histone deacetylase inhibitor (HDACi)-sensitive U937 lymphoma cells and isogenic HDACi-resistant cells. Mass spectrometry on autophagosome-enriched fractions revealed that HDACi-resistant cells undergo elevated pexophagy, or autophagy of the peroxisome, an organelle that supports tumor growth. To disturb peroxisome homeostasis, we enhanced pexophagy in HDACi-resistant cells via genetic silencing of peroxisome exportomer complex components (PEX1, PEX6, or PEX26). This consequently sensitized resistant cells to HDACi-mediated apoptosis, which was rescued by inhibiting ATM/ataxia-telangiectasia mutated (ATM serine/threonine kinase), a mediator of pexophagy. We subsequently engineered melanoma cells to stably repress PEX26 using CRISPR interference (CRISPRi). Melanoma cells with repressed PEX26 expression showed evidence of both increased pexophagy and peroxisomal matrix protein import defects versus single guide scrambled (sgSCR) controls. In vivo studies showed that sgPEX26 melanoma xenografts recurred less compared to sgSCR xenografts, following the development of resistance to mitogen-activated protein kinase (MAPK)-targeted therapy. Finally, prognostic analysis of publicly available datasets showed that low expression levels of PEX26, PEX6 and MTOR, were significantly associated with prolonged patient survival in lymphoma, lung cancer and melanoma cohorts. Our work highlighted that drugs designed to disrupt peroxisome homeostasis may serve as unconventional therapies to combat therapy resistance in cancer. Abbreviations: ABCD3/PMP70: ATP binding cassette subfamily D member 3; ACOX1: acyl-CoA oxidase 1; AP: autophagosome; COX: cytochrome c oxidase; CQ: chloroquine; CRISPRi: clustered regularly interspaced short palindromic repeats interference; DLBCL: diffuse large B-cell lymphoma; GO: gene ontology; dCas9: Cas9 endonuclease dead, or dead Cas9; HDACi: histone deacetylase inhibitors; IHC: Immunohistochemistry; LAMP2: lysosomal associated membrane protein 2; LCFAs: long-chain fatty acids; LFQ-MS: label-free quantitation mass spectrometry; LPC: lysophoshatidylcholine; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PBD: peroxisome biogenesis disorders; PTS1: peroxisomal targeting signal 1; ROS: reactive oxygen species; sgRNA: single guide RNA; VLCFAs: very-long chain fatty acids; Vor: vorinostat; WO: wash-off.

Hematological malignancies are broadly divided into myeloid and lymphoid neoplasms, reflecting the two major cellular lineages of the hematopoietic system. It is generally rare for hematological malignancies to spontaneously progress with a switch from myeloid to lymphoid lineage. We describe the exceptional case of a patient who sequentially developed myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), and B-cell acute lymphoblastic leukemia (B-ALL), as well as our investigation into the underlying pathogenesis. Using whole-exome sequencing (WES) performed on sorted CMML and B-ALL cell fractions, we identified both common and unique potential driver mutations, suggesting a branching clonal evolution giving rise to both diseases. Interestingly, we also identified a germline variant in the cancer susceptibility gene CHEK2. We validated that this variant (c.475T > C; p.Y159H), located in the forkhead-associated (FHA) domain, impairs its capacity to bind BRCA1 in cellulo. This unique case provides novel insight into the genetics of complex hematological diseases and highlights the possibility that such patients may carry inherited predispositions.

Hematological malignancies are broadly divided into myeloid and lymphoid neoplasms, reflecting the two major cellular lineages of the hematopoietic system. It is generally rare for hematological malignancies to spontaneously progress with a switch from myeloid to lymphoid lineage. We describe the exceptional case of a patient who sequentially developed myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), and B-cell acute lymphoblastic leukemia (B-ALL), as well as our investigation into the underlying pathogenesis. Using whole-exome sequencing (WES) performed on sorted CMML and B-ALL cell fractions, we identified both common and unique potential driver mutations, suggesting a branching clonal evolution giving rise to both diseases. Interestingly, we also identified a germline variant in the cancer susceptibility gene CHEK2. We validated that this variant (c.475T > C; p.Y159H), located in the forkhead-associated (FHA) domain, impairs its capacity to bind BRCA1 in cellulo. This unique case provides novel insight into the genetics of complex hematological diseases and highlights the possibility that such patients may carry inherited predispositions.

Four-and-a-half LIM domain protein 2 (FHL2) is an adaptor molecule regulating various cellular processes, including signal transduction, transcription, and cell survival. Although involved in inflammation and immune responses, its role in the germinal center reaction and B cell maturation remains unknown. We found that FHL2−/− mouse spleens displayed enlarged follicles with more B cells. When a T cell–dependent immune response was elicited using SRBC, FHL2−/− germinal center area was enhanced 2-fold compared with wild type (WT), concomitant with expanded dark zones. Nevertheless, the SRBC-induced rise in spleen IgG1 expression, and plasma IgG1 levels observed in WT were absent in FHL2−/− mice, and circulating plasma cells were also reduced in FHL2−/−. This could be explained by deficient upregulation of spleen activation-induced cytidine deaminase mRNA. Interestingly, FHL2−/− B cells successfully underwent class-switch recombination in vitro, and both activation-induced cytidine deaminase induction and IgG1 response to SRBC were equivalent in B cell–deficient μMT mice transplanted with WT or FHL2−/− bone marrow, suggesting that the defects observed in FHL2−/− mice were not B cell intrinsic. However, spleen lysates from FHL2−/− mice revealed a disturbed spleen microenvironment, with reduced CXCL12 and CXCL13 levels compared with WT. Our data suggest that spleen FHL2 expression is essential for a normal germinal center reaction and proper induction of class-switch recombination in response to a T cell–dependent Ag, leading to the emergence of Ab producing plasma cells. This could be due to the regulation of spleen cytokine production by FHL2.

Several immune regulatory cell types participate in the protection against autoimmune diseases such as autoimmune diabetes. Of these immunoregulatory cells, we and others have shown that peripheral CD4-CD8- double negative (DN) T cells can induce antigen-specific immune tolerance. Particularly, we have described that diabetes-prone mice exhibit a lower number of peripheral DN T cells compared to diabetes-resistant mice. Identifying the molecular pathways that influence the size of the DN T cell pool in peripheral lymphoid organs may thus be of interest for maintaining antigen-specific immune tolerance. Hence, through immunogenetic approaches, we found that two genetic loci linked to autoimmune diabetes susceptibility, namely Idd2 and Idd13, independently contribute to the partial restoration of DN T cell proportion in secondary lymphoid organs. We now extend these findings to show an interaction between the Idd2 and Idd13 loci in determining the number of DN T cells in secondary lymphoid organs. Using bioinformatics tools, we link potential biological pathways arising from interactions of genes encoded within the two loci. By focusing on cell cycle, we validate that both the Idd2 and Idd13 loci influence RAD51 expression as well as DN T cell progression through the cell cycle. Altogether, we find that genetic interactions between Idd2 and Idd13 loci modulate cell cycle progression, which contributes, at least in part, to defining the proportion of DN T cells in secondary lymphoid organs.

RAD51D is a key player in DNA repair by homologous recombination (HR), and RAD51D truncating variant carriers have an increased risk for ovarian cancer. However, the contribution of nontruncating RAD51D variants to cancer predisposition remains uncertain. Using deep sequencing and case–control genotyping studies, we show that in French Canadians, the missense RAD51D variant c.620C>T;p.S207L is highly prevalent and is associated with a significantly increased risk for ovarian high-grade serous carcinoma (HGSC; 3.8% cases vs. 0.2% controls). The frequency of the p.S207L variant did not significantly differ from that of controls in breast, endometrial, pancreas, or colorectal adenocarcinomas. Functionally, we show that this mutation impairs HR by disrupting the RAD51D–XRCC2 interaction and confers PARP inhibitor sensitivity. These results highlight the importance of a functional RAD51D–XRCC2 interaction to promote HR and prevent the development of HGSC. This study identifies c.620C>T;p.S207L as the first bona fide pathogenic RAD51D missense cancer susceptibility allele and supports the use of targeted PARP-inhibitor therapies in ovarian cancer patients carrying deleterious missense RAD51D variants. Cancer Res; 77(16); 4517–29. ©2017 AACR.

Maintaining genomic integrity is essential to preserve normal cellular physiology and to prevent the emergence of several human pathologies including cancer. The breast cancer susceptibility gene 2 (BRCA2, also known as the Fanconi anemia (FA) complementation group D1 (FANCD1)) is a potent tumor suppressor that has been extensively studied in DNA double-stranded break (DSB) repair by homologous recombination (HR). However, BRCA2 participates in numerous other processes central to maintaining genome stability, including DNA replication, telomere homeostasis and cell cycle progression. Consequently, inherited mutations in BRCA2 are associated with an increased risk of breast, ovarian and pancreatic cancers. Furthermore, bi-allelic mutations in BRCA2 are linked to FA, a rare chromosome instability syndrome characterized by aplastic anemia in children as well as susceptibility to leukemia and cancer. Here, we discuss the recent developments underlying the functions of BRCA2 in the maintenance of genomic integrity. The current model places BRCA2 as a central regulator of genome stability by repairing DSBs and limiting replication stress. These findings have direct implications for the development of novel anticancer therapeutic approaches.

DNA double‐strand breaks (DSBs) can be repaired by two major pathways: non‐homologous end‐joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)‐based approach, we identify 11 high‐confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ‐mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1‐, RIF1‐, and REV7‐dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR. Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision‐making process during DSB repair.

SUMMARYDNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of a previously undescribed factor, FAM35A/SHDL2, as a novel effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B-cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1- and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and another previously uncharacterized protein, C20orf196/SHDL1, which promotes NHEJ and limits HR. Together, these results establish FAM35A as a novel effector of REV7 in controlling the decision-making process during DSB repair.

Introduction Targeted genome editing using the CRISPR/Cas9 technology is becoming a major area of research due to its high potential for the treatment of genetic diseases. Our understanding of this approach has expanded in recent years yet several new challenges have presented themselves as we explore the boundaries of this exciting new technology. Chief among these is improving the efficiency but also the preciseness of genome editing. The efficacy of CRISPR/Cas9 technology relies in part on the use of one of the major DNA repair pathways, Homologous recombination (HR), which is primarily active in S and G2 phases of the cell cycle. Problematically, the HR potential is highly variable from cell type to cell type and most of the cells of interest to be targeted in vivo for precise genome editing are in a quiescent state. Conclusion In this review, we discuss the recent advancements in improving targeted CRISPR/Cas9 based genome editing and the promising ways of delivering this technology in vivo to the cells of interest.

Activation induced deaminase (AID) initiates somatic hypermutation and class switch recombination of the Ig genes in antigen-activated B cells, underpinning antibody affinity maturation and isotype switching. AID can also be pathogenic by contributing to autoimmune diseases and oncogenic mutations. Moreover, AID can exert noncanonical functions when aberrantly expressed in epithelial cells. The lack of specific inhibitors prevents therapeutic applications to modulate AID functions. Here, we have exploited our previous finding that the HSP90 molecular chaperoning pathway stabilizes AID in B cells, to test whether HSP90 inhibitors could target AID in vivo. We demonstrate that chronic administration of HSP90 inhibitors decreases AID protein levels and isotype switching in immunized mice. HSP90 inhibitors also reduce disease severity in a mouse model of acute B-cell lymphoblastic leukemia in which AID accelerates disease progression. We further show that human AID protein levels are sensitive to HSP90 inhibition in normal and leukemic B cells, and that HSP90 inhibition prevents AID-dependent epithelial to mesenchymal transition in a human breast cancer cell line in vitro. Thus, we provide proof-of-concept that HSP90 inhibitors indirectly target AID in vivo and that endogenous human AID is widely sensitive to them, which could have therapeutic applications.

Appropriate repair of DNA lesions and the inhibition of DNA repair activities at telomeres are critical to prevent genomic instability. By fuelling the generation of genetic alterations and by compromising cell viability, genomic instability is a driving force in cancer and aging1, 2. Here we identify MAD2L2 (also known as MAD2B or REV7) through functional genetic screening as a novel factor controlling DNA repair activities at mammalian telomeres. We show that MAD2L2 accumulates at uncapped telomeres and promotes non-homologous end-joining (NHEJ)-mediated fusion of deprotected chromosome ends and genomic instability. MAD2L2 depletion causes elongated 3′ telomeric overhangs, implying that MAD2L2 inhibits 5′ end-resection. End-resection blocks NHEJ while committing to homology-directed repair (HDR) and is under control of 53BP1, RIF1 and PTIP3. Consistent with MAD2L2 promoting NHEJ-mediated telomere fusion by inhibiting 5′ end-resection, knockdown of the nucleases CTIP or EXO1 partially restores telomere-driven genomic instability in MAD2L2-depleted cells. Control of DNA repair by MAD2L2 is not limited to telomeres. MAD2L2 also accumulates and inhibits end-resection at irradiation (IR)-induced DNA double-strand breaks (DSBs) and promotes end-joining of DSBs in multiple settings, including during immunoglobulin class switch recombination (CSR). These activities of MAD2L2 depend on ATM kinase activity, RNF8, RNF168, 53BP1 and RIF1, but not on PTIP, REV1 and REV3, the latter two acting with MAD2L2 in translesion synthesis (TLS)4. Together our data establish MAD2L2 as a critical contributor to the control of DNA repair activity by 53BP1 that promotes NHEJ by inhibiting 5′ end-resection downstream of RIF1.

GFI1 is a transcriptional regulator expressed in lymphoid cells, and an “oncorequisite” factor required for development and maintenance of T-lymphoid leukemia. GFI1 deletion causes hypersensitivity to ionizing radiation, for which the molecular mechanism remains unknown. Here, we demonstrate that GFI1 is required in T cells for the regulation of key DNA damage signaling and repair proteins. Specifically, GFI1 interacts with the arginine methyltransferase PRMT1 and its substrates MRE11 and 53BP1. We demonstrate that GFI1 enables PRMT1 to bind and methylate MRE11 and 53BP1, which is necessary for their function in the DNA damage response. Thus, our results provide evidence that GFI1 can adopt non-transcriptional roles, mediating the post-translational modification of proteins involved in DNA repair. These findings have direct implications for treatment responses in tumors overexpressing GFI1 and suggest that GFI1’s activity may be a therapeutic target in these malignancies., The transcription factor GFI1 mediates the DNA damage response (DDR) of T cells through a yet unknown mechanism. Here the authors show that GFI1 can adopt non-transcriptional roles during DDR, enabling PRMT1 to bind and methylate the DNA repair proteins MRE11 and 53BP1.

Background: Four-and-a-half LIM domain protein 2 (FHL2) is an adaptor molecule that regulates signalling cascades and gene transcription. We have uncovered vasculoprotective and atheroprotective effects of FHL2 knockout in mice. Since B cells could regulate these processes, we investigated the potential role of FHL2 in B cell function and activity. Methods and Results: Under basal conditions, FHL2-/- mice presented a mild splenomegaly (84.0±9.0 vs 68.0±5.0mg), associated with bigger spleen follicles (1.6 fold) and higher proportions of spleen B cells (56.0±2.0 vs 40.0±9.0%) vs WT mice. Flow cytometry confirmed significantly higher (p Conclusion: These results suggest that FHL2 plays a critical role in spleen antibody production in response to a T-dependent antigen, possibly by affecting activation or antigen presentation by Fo T helper cells.

RAD51D is a key player in DNA repair by homologous recombination (HR), and

Biallelic inactivation of BRCA1 or BRCA2 is associated with a pattern of genome-wide mutations known as signature 3. By analyzing [sim]1, 000 breast cancer samples, we confirmed this association and established that germline nonsense and frameshift variants in PALB2, but not in ATM or CHEK2, can also give rise to the same signature. We were able to accurately classify missense BRCA1 or BRCA2 variants known to impair homologous recombination (HR) on the basis of this signature. Finally, we show that epigenetic silencing of RAD51C and BRCA1 by promoter methylation is strongly associated with signature 3 and, in our data set, was highly enriched in basal-like breast cancers in young individuals of African descent.

Shutting Down Repair to Protect Cells repair DNA double-strand breaks (DSBs) by halting the cell cycle and activating the machinery involved in mending the breaks. However, during mitosis neither the DNA damage checkpoint nor DSB repair occur, apparently leaving the cell extremely vulnerable to DSBs. Orthwein et al. (p. 189 , published online 20 March) found that the DSB response was blocked by the phosphorylation of two crucial repair factors, RNF8 and PB531, preventing their recruitment to the site of damage. Restoring DSB repair during mitosis caused end-to-end chromosome fusions, which are catastrophic for chromosome segregation and normal cell division, explaining why the repair machinery is shut down during cell division.

53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone ‘code’ produced by DSB signalling. This study shows that 53BP1 recruitment to sites of DNA damage involves dual recognition of H4K20me2 and H2AK15 histone ubiquitination; the ubiquitin mark and the surrounding epitope on H2A are read by a region of 53BP1 designated the ubiquitination-dependent recruitment motif. The key DNA damage response protein 53BP1 acts by binding to chromatin at the site of a double-strand break. Previous studies suggested that 53BP1 acts after a ubiquitination event promoted by RNF168, although its recruitment to breaks was thought to depend only on histone H4K20 methylation. Daniel Durocher and colleagues now show that 53BP1 recruitment involves the recognition of both H4K20me2 and histone H2AK15 ubiquitination. The ubiquitin mark, and the surrounding context on histone H2A, are read by a region of 53BP1 that the authors designate the ubiquitination-dependent recruitment motif.

DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.

The expanding repertoire of programmable nucleases such as Cas9 brings new opportunities in genetic medicine1–3. In many cases, these nucleases are engineered to induce a DNA double-strand break (DSB) to stimulate precise genome editing by homologous recombination (HR). However, HR efficiency is nearly always hindered by competing DSB repair pathways such as non-homologous end-joining (NHEJ). HR is also profoundly suppressed in non-replicating cells, thus precluding the use of homology-based genome engineering in a wide variety4 of cell types. Here, we report the development of a genetically encoded inhibitor of 53BP1 (known as TP53BP1), a regulator of DSB repair pathway choice5. 53BP1 promotes NHEJ over HR by suppressing end resection, the formation of 3-prime single-stranded DNA tails, which is the rate-limiting step in HR initiation. 53BP1 also blocks the recruitment of the HR factor BRCA1 to DSB sites in G1 cells4,6. The inhibitor of 53BP1 (or i53) was identified through the screening of a massive combinatorial library of engineered ubiquitin variants by phage display7. i53 binds and occludes the ligand binding site of the 53BP1 Tudor domain with high affinity and selectivity, blocking its ability to accumulate at sites of DNA damage. i53 is a potent selective inhibitor of 53BP1 and enhances gene targeting and chromosomal gene conversion, two HR-dependent reactions. Finally, i53 can also activate HR in G1 cells when combined with the activation of end-resection and KEAP1 inhibition. We conclude that 53BP1 inhibition is a robust tool to enhance precise genome editing by canonical HR pathways.

DNA double-strand break repair by homologous recombination is initiated by the formation of 3' single-stranded DNA (ssDNA) overhangs by a process termed end resection. Although much focus has been given to the decision to initiate resection, little is known of the mechanisms that regulate the ongoing formation of ssDNA tails. Here we report that DNA helicase B (HELB) underpins a feedback inhibition mechanism that curtails resection. HELB is recruited to ssDNA by interacting with RPA and uses its 5'-3' ssDNA translocase activity to inhibit EXO1 and BLM-DNA2, the nucleases catalyzing resection. HELB acts independently of 53BP1 and is exported from the nucleus as cells approach S phase, concomitant with the upregulation of resection. Consistent with its role as a resection antagonist, loss of HELB results in PARP inhibitor resistance in BRCA1-deficient tumor cells. We conclude that mammalian DNA end resection triggers its own inhibition via the recruitment of HELB.

Activation induced deaminase (AID) initiates somatic hypermutation and class switch recombination of the Ig genes in antigen-activated B cells, underpinning antibody affinity maturation and isotype switching. AID can also be pathogenic by contributing to autoimmune diseases and oncogenic mutations. Moreover, AID can exert noncanonical functions when aberrantly expressed in epithelial cells. The lack of specific inhibitors prevents therapeutic applications to modulate AID functions. Here, we have exploited our previous finding that the HSP90 molecular chaperoning pathway stabilizes AID in B cells, to test whether HSP90 inhibitors could target AID in vivo. We demonstrate that chronic administration of HSP90 inhibitors decreases AID protein levels and isotype switching in immunized mice. HSP90 inhibitors also reduce disease severity in a mouse model of acute B-cell lymphoblastic leukemia in which AID accelerates disease progression. We further show that human AID protein levels are sensitive to HSP90 inhibition in normal and leukemic B cells, and that HSP90 inhibition prevents AID-dependent epithelial to mesenchymal transition in a human breast cancer cell line in vitro. Thus, we provide proof-of-concept that HSP90 inhibitors indirectly target AID in vivo and that endogenous human AID is widely sensitive to them, which could have therapeutic applications.

Background and goal: RAD51D is a key player in DNA repair by homologous recombination (HR) and carriers of truncating RAD51D mutations have an increased risk for ovarian cancer (OC). However, the contribution of non-truncating RAD51D variants to cancer predisposition remains uncertain. We sought to fully characterize the previously described missense RAD51D variant c.620C>T;p.S207L in order to elucidate its role in OC. Methods: A clinical panel screening was used to identify the RAD51D variant c.620C>T;p.S207L in two French Canadian (FC) kindred affected with familial High Grade Serous Cancer (HGSC) of the ovary or endometrium. High resolution melting, TaqMan genotyping and Sanger sequencing were used to genotype the p.S207L variant in a series of unselected cases of HGSC of the ovary and endometrium, breast, pancreas and colorectal cancer and healthy controls, all of a FC origin. Whole exome sequencing (WES) was performed to study the genetic signature characterizing RAD51D associated tumors. RAD51 foci formation and CRISPR-Cas9-stimulated and HR-mediated gene targeting assays were used to assess HR activity of RAD51D-S207L mutated CHO cells. HR activity in RAD51D-S207L mutated human cells was tested by a DR-GFP assay. The effect of RAD51D p.S207L on RAD51D-XRCC2 interactions was analyzed by co-immunoprecipitation and quantified in-vivo in a single cell colocalization assay. Sensitivity to PARP inhibitors (PARPi) was evaluated in a cell survival assay. Results: Using deep sequencing and case-control genotyping studies, we showed that the missense RAD51D variant c.620C>T;p.S207L is over-represented in the French Canadian population affected by HGSC of the ovary (3.8% cases vs 0.002% controls; p < 0.0001).The frequency of the p.S207L variant did not differ from that of controls in breast, endometrial, pancreas and colorectal adenocarcinomas. A common haplotype shared by all the carriers suggested a founder origin for c.620C>T;p.S207L mutation. WES analysis of RAD51D tumor profiles revealed the presence of signature 3 which is known to be associated with HR defects. Functionally, we show that this mutation impairs HR by disrupting the RAD51D-XRCC2 interaction and confers PARP-inhibitor sensitivity to CHO cells. Conclusions: This work identifies RAD51D p.S207L as the first bona fide pathogenic missense susceptibility allele for HGSC of the ovary and supports the use of targeted PARPi therapies in OC patients carrying missense RAD51D mutations. Citation Format: Barbara Rivera, Massimo R. Di Iorio, Jessica Frankum, Javad Nadaf, Somayyeh Fahiminiya, Suzanna L. Arcand, David Burk, Damien Grapton, Eva Tomiak, Valerie Hastings, Nancy Hamel, Rabea Wagener, Olga Aleynikova, Sylvie Giroux, Fadi F. Hamdan, Alexandre Orthwein, George Zogopoulos, Francois Rousseau, Albert Berghuis, Diane M. Provencher, Guy A. Rouleau, Jacques L. Michaud, Anne-Marie Mes-Masson, Jacek Majewski, Susanne Bens, Reiner Siebert, Steven Narod, Mohammad Akbari, Chris J. Lord, Patricia N. Tonin, Alexandre Dionne-Laporte, William D. Foulkes. A functionally null RAD51D missense mutation is strongly associated with ovarian carcinoma [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 2479. doi:10.1158/1538-7445.AM2017-2479

Activation induced deaminase (AID) plays a central role in adaptive immunity by initiating the processes of somatic hypermutation (SHM) and class switch recombination (CSR). On the other hand, AID also predisposes to lymphoma and plays a role in some autoimmune diseases, for which reasons AID expression and activity are regulated at various levels. Post-translational mechanisms regulating the amount and subcellular localization of AID are prominent in balancing AID physiological and pathological functions in B cells. Mechanisms regulating AID protein levels include stabilizing chaperones in the cytoplasm and proteins efficiently targeting AID to the proteasome within the nucleus. Nuclear export and cytoplasmic retention contribute to limit the amount of AID accessing the genome. Additionally, a number of factors have been implicated in AID active nuclear import. We review these intertwined mechanisms proposing two scenarios in which they could interact as a network or as a cycle for defining the optimal amount of AID protein. We also comparatively review the expression levels of AID necessary for its function during the immune response, present in different cancers as well as in those tissues in which AID has been implicated in epigenetic remodeling of the genome by demethylating DNA.

The enzyme activation-induced deaminase (AID) deaminates deoxycytidine at the immunoglobulin genes, thereby initiating antibody affinity maturation and isotype class switching during immune responses. In contrast, off-target DNA damage caused by AID is oncogenic. Central to balancing immunity and cancer is AID regulation, including the mechanisms determining AID protein levels. We describe a specific functional interaction between AID and the Hsp40 DnaJa1, which provides insight into the function of both proteins. Although both major cytoplasmic type I Hsp40s, DnaJa1 and DnaJa2, are induced upon B-cell activation and interact with AID in vitro, only DnaJa1 overexpression increases AID levels and biological activity in cell lines. Conversely, DnaJa1, but not DnaJa2, depletion reduces AID levels, stability and isotype switching. In vivo, DnaJa1-deficient mice display compromised response to immunization, AID protein and isotype switching levels being reduced by half. Moreover, DnaJa1 farnesylation is required to maintain, and farnesyltransferase inhibition reduces, AID protein levels in B cells. Thus, DnaJa1 is a limiting factor that plays a non-redundant role in the functional stabilization of AID.

Hsp90 stabilizes and prevents degradation of cytoplasmic activation-induced deaminase., Activation-induced deaminase (AID) is the mutator enzyme that initiates somatic hypermutation and isotype switching of the antibody genes in B lymphocytes. Undesired byproducts of AID function are oncogenic mutations. AID expression levels seem to correlate with the extent of its physiological and pathological functions. In this study, we identify AID as a novel Hsp90 (heat shock protein 90 kD) client. We find that cytoplasmic AID is in a dynamic equilibrium regulated by Hsp90. Hsp90 stabilizes cytoplasmic AID, as specific Hsp90 inhibition leads to cytoplasmic polyubiquitination and proteasomal degradation of AID. Consequently, Hsp90 inhibition results in a proportional reduction in antibody gene diversification and off-target mutation. This evolutionarily conserved regulatory mechanism determines the functional steady-state levels of AID in normal B cells and B cell lymphoma lines. Thus, Hsp90 assists AID-mediated antibody diversification by stabilizing AID. Hsp90 inhibition provides the first pharmacological means to down-regulate AID expression and activity, which could be relevant for therapy of some lymphomas and leukemias.

International audience; The enzyme activation-induced deaminase (AID) triggers antibody diversification in B cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, and its nuclear entry requires active import mediated by a conformational nuclear localization signal. We also identify in its C terminus a determinant for AID cytoplasmic retention, which hampers diffusion to the nucleus, competes with nuclear import and is crucial for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.

Gag proteins are necessary and sufficient to direct human immunodeficiency virus type 1 (HIV-1) particle assembly and budding. Recent evidence suggests that Gag targeting to late endosomal/multivesicular body (LE/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE/MVB, where it accumulated, thus accounting for the majority of LE/MVB-associated Gag. Importantly, this accumulation of Gag in LE/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-β-cyclodextrin. These results point towards the PM as being the primary site of productive HIV-1 assembly in cells that also support Gag accumulation in intracellular compartments.

The chromatin-based response to DNA double-strand breaks (DSBs) is characterized by the accumulation of DNA repair factors and DNA damage signaling molecules within a large domain that surrounds the lesion. This response is largely dependent on ATM-dependent phosphorylation but also on chromatin ubiquitylation. Indeed, the RNF8 and RNF168 E3 ubiquitin ligases are necessary for the accumulation of proteins such as 53BP1, BRCA1 and RAD18 at sites of DNA damage. The critical importance of this pathway is manifested by the observation that biallelic mutations in the RNF168 gene results in an ataxia-telangiectasia-like syndrome referred to as RIDDLE. We are interested in understanding how this pathway is regulated and how ubiquitin-dependent protein interactions orchestrate the hierarchy of protein recruitment at sites of DNA lesions. I will present our current understanding on the regulation and organization of this signaling cascade and in particular, I will focus my presentation on addressing how the pathway underpinned by RNF168 is regulated during the course of the cell cycle. Citation Format: Alexandre Orthwein, Daniel Durocher. Regulation of the RNF168-dependent response to DNA double-strand breaks. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA12. doi:10.1158/1538-7445.CANSUSC14-IA12