Abstract Ataxia telangiectasia and Rad3-related kinase (ATR) is essential to the faithful replication of DNA in rapidly dividing cells such as cancer cell lines. ATR is activated in response to single-strand DNA breaks that result from increased replication stress caused by defects in DNA repair mechanisms and/or damaged DNA. ATR stabilizes and restarts the stressed replication forks, suppresses origin firing, activates cell cycle checkpoints, and facilitates DNA repair. In fact, ATR activity contributes to the tolerance of chronic replication stress in cells transformed by oncogenes rendering cancer cells more dependent on ATR activity for survival. Systemic exposure to ATR inhibitors (ATRis) in clinical studies have demonstrated dose-limiting toxicities that reduce their clinical utility. Therefore, precision antibody-drug conjugates (ADCs) for targeted delivery of ATR payloads could bypass the systemic toxicities associated with oral administration. There is significant overlap between cancers with ATRi sensitizing defects in DNA repair mechanisms with those expressing high levels of PD-L1 surface antigens including stomach, breast, lung and colon cancers. Our personalized antibody-drug conjugate approach couples surface targeted therapeutics to tumors with increased sensitivity to the delivered payload to improve the therapeutic index. We have discovered multiple derivatizable proprietary ATRis with desirable physicochemical properties that demonstrate low nM potency in cancer cell lines with ATRi sensitizing mutations. Furthermore, the ATRi payloads maintained in vitro cytotoxic potency in cells harbouring clinically relevant acquired resistance mutations in Topoisomerase 1, whereas exatecan did not. Once conjugated to the anti-PD-L1 monoclonal antibody, the resulting ADCs demonstrated selective picomolar cellular growth inhibition and excellent metabolic stability in mouse plasma. Preclinical in vivo studies in mice demonstrate robust efficacy and selectivity with PD-L1-ATRi as single agent relative to IgG-ATRi. The in vitro and in vivo data presented herein demonstrate the efficacy of an ADC with an ATRi mono-payload and illustrate the potential for combinations with standard of care chemotherapeutic agents such as gemcitabine or PARP inhibitors. Citation Format: Anne Roulston, Martin Duplessis, Francois Denis, Hugo Poirier, Shou Yun Yin, Sara Fournier, Roselyn Kryczka, Jessica Desjardins, Nancy Laterreur, Abira Rajah, Alexanne Bonneau-Fortin, Bingcan Liu, Alejandro Alvarez-Quilon, Philipe Mochirian, Ismael Samudio. Novel ATR inhibitor payloads for antibody-drug conjugates targeting PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5602.

Abstract It was recently shown that inhibition of polo-like kinase 4 (PLK4) induces synthetic lethality in cancers with chromosome 17q-encoded TRIM37 copy number gain due to cooperative regulation of centriole duplication and mitotic spindle nucleation. We show here that chromosome 17q/TRIM37 gain is a defining feature of high-risk neuroblastoma and renders patient-derived cell lines hypersensitive to the novel PLK4 inhibitor RP-1664. We demonstrate that centriole amplification at low doses of RP-1664 contributes to this sensitivity in a TRIM37 -independent fashion. CRISPR screens and live cell imaging reveal that upon centriole amplification, neuroblastoma cells succumb to multipolar mitoses due to an inability to cluster or inactivate supernumerary centrosomes. RP-1664 monotherapy showed robust anti-tumor activity in 14/15 human neuroblastoma-derived xenograft models, and significantly extended survival in a transgenic MYCN -driven murine model of neuroblastoma. RP-1664 combined with GD2-directed chemoimmunotherapy resulted in maintained complete responses in 6/9 mice with established MYCN -driven murine neuroblastomas. These data support clinical development of PLK4 inhibitors for high-risk neuroblastoma and other cancers with somatically acquired TRIM37 overexpression.

Leveraging WRN helicase dependency in microsatellite instability (MSI) cancers offers a synthetic lethal (SL) therapeutic opportunity, with several WRN inhibitors in development. However, the hypermutator nature of MSI tumors creates strong evolutionary pressure for rapid resistance. Here, we apply a multimodal functional genomics framework integrating base editing screens and deep mutational scanning to map on-target resistance to two clinical WRN inhibitors, HRO761 and VVD-214. We identify discrete resistance hotspots within WRN and demonstrate that single-allele (heterozygous) mutations at the drug-binding site are sufficient to abrogate WRN inhibitor-induced cytotoxicity. Resistance profiles diverged between HRO761 and VVD-214, revealing mutations that impair one but preserve sensitivity to the other. Genome-wide CRISPR screens further identified non-homologous end joining (NHEJ) factors and the checkpoint phosphatase WIP1 as tractable synthetic vulnerabilities that potentiate WRN inhibition. Together, these findings establish a framework for resistance-aware deployment of WRN inhibitors through rational drug selection, therapeutic switching, and combination strategies. ### Competing Interest Statement MEO, NL, JB, CF, JTFY and were employees of Repare Therapeutics at the time part of the data was collected and analyzed. NL, CF and AA-Q, are currently employees of DCx Biotherapeutics. JB is currently an employee of Servier Pharmaceuticals. JTFY is currently an employee of AstraZeneca. Other authors declare no competing interest. Canadian Institutes of Health Research, https://ror.org/01gavpb45, FRN:DV2-197674, CRP-196961 Cancer Research Society, #1275778 Fonds de Recherche du Québec - Santé

Synthetic lethality (SL) provides a treatment paradigm for targeting cancer with alterations in driver genes that are not conventionally druggable, including loss-of-function (LoF) mutations in tumor suppressor genes and gain-of-function (GoF) alterations in oncogenes. We undertook a series of genome-wide CRISPR screens using functionally validated isogenic cell lines and also conducted a large-scale SL analysis using data from the cancer dependency map (DepMap). We charted SL interactions across 15 genetic alterations characteristic of diseases with high incidence and unmet clinical need: FBXW7, CCNE1, CDK12, ARID1A, KMT2D, DNMT3A, TET2, KEAP1, STK11, IDH1, SF3B1, SRSF2, U2AF1, chromosome 18q loss, and chromosome 13q loss. We show validation of several SL interactions between tractable targets with cancer drivers, including ARID1A and the hexosamine biosynthetic pathway aminotransferase GFPT1, STK11 with CAMK protein kinase family members including MARK2, FBXW7 and the CDK1 regulatory kinase PKMYT1, and CCNE1 amplification and the anaphase promoting complex or cyclosome (APC/C). In summary, this study offers a rich resource of genetic interactions across cancer drivers enabling the discovery of new biological insights and drug targets for future therapeutic development. ### Competing Interest Statement All authors were employees of Repare Therapeutics when this data was collected and analyzed. J.D., N.L., A.R., J.L., A.R., C.F., and A.A.Q. are currently employees of DCx Biotherapeutics. J.B. is currently an employee of Servier Pharmaceutics. C.B. is currently an employee of Epitopea. S.J. and L.L. are currently employees of Leapfrog Bio. T.G.R., M. Zimmermann, and J.T.F.Y. are currently employees of AstraZeneca. A.L. is currently an employee of DropGenie. J.H.L. is currently an employee of Zymeworks Inc. M.C.M. is currently an employee of Frontier Discovery Inc. A.V. is currently an employee of Bayer.

A novel therapeutic strategy was recently proposed for high-risk neuroblastoma carrying copy number gain of the TRIM37 gene: centriole loss upon inhibition of polo-like kinase 4 (PLK4), while tolerated by normal cells, induces aberrant mitotic spindle formation and p53-dependent cell death in TRIM37-overexpressing cells. Interestingly, while full PLK4 inhibition causes centriole loss, partial inhibition is known to elevate centriole numbers. Here we show using a novel selective PLK4 inhibitor RP-1664 that both centriole loss and amplification contribute to hypersensitivity of neuroblastoma cells. Whereas inactivation of TRIM37 and TP53 rescues neuroblastoma cell death at higher concentrations of RP-1664, at lower doses cell death is TRIM37/TP53-independent. With CRISPR screens and live cell imaging we demonstrate that upon centriole amplification, neuroblastoma cells succumb to multipolar mitoses due to inability to cluster or inactivate supernumerary centrosomes. In vivo, RP-1664 shows robust efficacy in neuroblastoma xenografts at doses consistent with centriole amplification. ### Competing Interest Statement This study was funded by Repare Therapeutics. All authors are current or former employees of Repare Therapeutics and receive(d) salary and/or equity compensation.

Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

Activation-induced cytidine deaminase (AID) catalyzes the deamination of deoxycytidines within Immunoglobulin (Ig) genes to induce somatic hypermutation (SHM) and class switch recombination (CSR) 1,2. AID-induced deoxyuracils within Ig loci are recognized and processed by subverted base excision and mismatch repair pathways that ensure a mutagenic outcome in B lymphocytes 3–8. However, it is unclear why DNA repair pathways that remove deoxyuracil from DNA are not efficient at faithfully repairing AID-induced lesions. Here, we identified through a genome-wide CRISPR screen that FAM72A, a protein with no ascribed function, is a major determinant for the error-prone processing of deoxyuracil. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a−/− mice have reduced CSR and SHM frequencies. The SHM spectrum in B cells from Fam72a−/− mice is opposite to that observed in Ung2−/− mice 9, suggesting that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2 resulting in reduced UNG2 activity, and significantly reduced protein levels in the G1 phase, coinciding with peak AID activity. This effect leads to increased genome-wide deoxyuracils in B cells. By antagonizing UNG2, FAM72A therefore increases U•G mispairs that engage mutagenic mismatch repair promoting error-prone processing of AID-induced deoxyuracils. This work shows that FAM72A bridges base-excision repair and mismatch repair to modulate antibody maturation.

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers.

BRCA1/2 -mutated cancer cells must adapt to the genome instability caused by their deficiency in homologous recombination. Identifying and targeting these adaptive mechanisms may provide new therapeutic strategies. Here we present the results of genome-scale CRISPR/Cas9-based synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells that identified the gene encoding CIP2A as essential in a wide range of BRCA1 - and BRCA2 -mutated cells. Unlike PARP inhibition, CIP2A-deficiency does not cause accumulation of replication-associated DNA lesions that require homologous recombination for their repair. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient cells and tumors, indicating that targeting this mitotic chromosome stability process represents an attractive synthetic-lethal therapeutic strategy for BRCA1 - and BRCA2 -mutated cancers.

AbstractAmplification of the gene encoding cyclin E (CCNE1) is an oncogenic driver in several malignancies and is associated with chemoresistance and poor prognosis. To uncover therapeutic targets forCCNE1-amplified tumors, we undertook genome-scale CRISPR/Cas9-based synthetic lethality screens in cellular models ofCCNE1amplification. Here, we report that increasingCCNE1dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumor regressions when combined with gemcitabine in models ofCCNE1-amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively inCCNE1overexpressing-cells, promoting early mitosis in cells undergoing DNA synthesis.CCNE1overexpression disrupts CDK1 homeostasis at least in part through an early activation of the FOXM1/MYBL2/MuvB-dependent mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy forCCNE1-amplified cancers.

The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 30 DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 30 -blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 30 blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

The ATM kinase is a master regulator of the DNA damage response to double-strand breaks (DSBs) and a well-established tumour suppressor whose loss is the cause of the neurodegenerative and cancer-prone syndrome Ataxia-Telangiectasia (A-T). A-T patients and Atm −/− mouse models are particularly predisposed to develop lymphoid cancers derived from deficient repair of RAG-induced DSBs during V(D)J recombination. Here, we unexpectedly find that specifically disturbing the repair of DSBs produced by DNA topoisomerase II (TOP2) by genetically removing the highly specialised repair enzyme TDP2 increases the incidence of thymic tumours in Atm −/− mice. Furthermore, we find that TOP2 strongly colocalizes with RAG, both genome-wide and at V(D)J recombination sites, resulting in an increased endogenous chromosomal fragility of these regions. Thus, our findings demonstrate a strong causal relationship between endogenous TOP2-induced DSBs and cancer development, confirming these lesions as major drivers of ATM-deficient lymphoid malignancies, and potentially other conditions and cancer types.

The response to DNA replication stress in eukaryotes is under the control of the ataxia-telangiectasia and Rad3-related (ATR) kinase. ATR responds to single-stranded (ss) DNA to stabilize distressed DNA replication forks, modulate DNA replication firing and prevent cells with damaged DNA or incomplete DNA replication from entering into mitosis. Furthermore, inhibitors of ATR are currently in clinical development either as monotherapies or in combination with agents that perturb DNA replication. To gain a genetic view of the cellular pathways requiring ATR kinase function, we mapped genes whose mutation causes hypersensitivity to ATR inhibitors with genome-scale CRISPR/Cas9 screens. We delineate a consensus set of 117 genes enriched in DNA replication, DNA repair and cell cycle regulators that promote survival when ATR kinase activity is suppressed. We validate 14 genes from this set and report genes not previously described to modulate response to ATR inhibitors. In particular we found that the loss of the POLE3/POLE4 proteins, which are DNA polymerase e accessory subunits, results in marked hypersensitivity to ATR inhibition. We anticipate that this 117-gene set will be useful for the identification of genes involved in the regulation of genome integrity, the characterization of new biological processes involving ATR, and may reveal biomarkers of ATR inhibitor response in the clinic.

DNA repair by homologous recombination (HR) is essential for genomic integrity, tumor suppression, and the formation of gametes. HR uses DNA synthesis to repair lesions such as DNA double-strand breaks and stalled DNA replication forks, but despite having a good understanding of the steps leading to homology search and strand invasion, we know much less of the mechanisms that establish recombination-associated DNA polymerization. Here, we report that C17orf53/HROB is an OB-fold-containing factor involved in HR that acts by recruiting the MCM8-MCM9 helicase to sites of DNA damage to promote DNA synthesis. Mice with targeted mutations in Hrob are infertile due to depletion of germ cells and display phenotypes consistent with a prophase I meiotic arrest. The HROB-MCM8-MCM9 pathway acts redundantly with the HELQ helicase, and cells lacking both HROB and HELQ have severely impaired HR, suggesting that they underpin two major routes for the completion of HR downstream from RAD51. The function of HROB in HR is reminiscent of that of gp59, which acts as the replicative helicase loader during bacteriophage T4 recombination-dependent DNA replication. We therefore propose that the loading of MCM8-MCM9 by HROB may similarly be a key step in the establishment of mammalian recombination-associated DNA synthesis.

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini 1 , 2 . This function of 53BP1 requires interactions with PTIP 3 and RIF1 4 – 9 , the latter of which recruits REV7 (also known as MAD2L2) to break sites 10 , 11 . How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases 12 , 13 , and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

DNA double-strand breaks (DSBs) elicit the so-called DNA damage response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI) combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within “repair foci.” Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.

Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders.

Poster presentado en EMBO Conference: The DNA damage response in cell physiology and disease, celebrada en Cape Sounio (Grecia), del 7 al 11 de octubre de 2013