Synthetic lethality (SL) underlies the success of PARP1 inhibitors (PARPi) in treating homologous recombination (HR)-deficient cancers, yet their broader applicability beyond HR deficiency remains poorly defined. Here, we performed an in vivo CRISPR screen that identifies FANCA deficiency as a driver of tumor progression and PARPi SL, validated across diverse human cancer models. Notably, FANCA loss does not impair HR but instead disrupts FEN1 recruitment to replication forks, leading to defective Okazaki fragment maturation, lagging-strand single-strand DNA gap accumulation, and RPA exhaustion upon PARPi treatment. Additionally, FANCA loss in oncogene-expressing cells promotes transcription-replication conflict (TRC) accumulation selectively on the lagging strand and sensitizes HR-proficient cells to PARPi, a phenotype reversible by RNA polymerase II inhibition or RNase H overexpression. Together, these findings identify FANCA deficiency as a context-specific PARPi vulnerability and establish FANCA as a key suppressor of TRCs required for genomic stability under oncogenic replication stress.

The essential role of polymerase theta (Polθ)-mediated end joining (TMEJ), an alternative double strand break repair pathway, has been primarily studied in homologous recombination (HR)-deficient contexts( 1 , 2 ). Here, we uncover an indispensable role for TMEJ in HR-proficient mammalian cells during the repair of interstrand crosslinks (ICLs). We show that Polθ is recruited downstream of canonical ICL repair steps-including ICL unhooking, RAD51 loading, and RAD51 ubiquitylation-and localizes to sites of unresolved HR through interactions with ubiquitylated RAD51 filaments. Using genomic scar profiling and targeted ICL repair assays, we find that TMEJ resolves a minor subset of lesions that are not amenable to HR repair, such as clustered ICLs that can induce two-ended replication fork collapse. These findings reveal a RAD51 ubiquitylation-dependent mechanism for Polθ recruitment and establish TMEJ as a hierarchically deployed DNA repair pathway that safeguards genome stability when HR is insufficient to resolve replication-associated DNA damage. Short Summary:Polθ is recruited via RAD51 ubiquitylation to resolve clustered ICLs that generate HR-refractory replication fork collapse.

Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS–STING-mediated signalling and tumour suppression 1 – 3 . However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA) 4 – 10 . Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11–RAD50–NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1–RIPK3–MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation of ZBP1 in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis.

Abstract Background: Bladder cancer (BC) remains a common and deadly disease, with 83,190 cases and 32,350 deaths projected in the U.S. in 2024. We previously showed bromodomain and extra-terminal protein inhibitors (BETi) are potent in multiple preclinical models of BC. Our bulk RNA-seq and RT-qPCR data showed BETi significantly reduces expression of homologous recombination (HR) genes, such as RAD51 and RBBP8, which could activate alternative DNA repair pathways including non-homologous end joining (NHEJ) and theta-mediated end joining (TMEJ). Specifically, DNA breaks are repaired through TMEJ by polymerase theta (Polθ) utilizing microhomology templates. Recently published data revealed Polθ inhibition is synthetically lethal when other DDR genes are concomitantly lost or inhibited. Thus, in this study, we sought to elucidate whether BETi-induced HR gene repression impacts TMEJ and POLQ expression, and to identify combinations with BETi that best leverage Polθ-mediated synthetic lethality. Methods: 5637 and J82 BC cells were pretreated with birabresib for 24 h and then transfected via electroporation with a Cas9 ribonucleoprotein, a gRNA targeting LBR2 and a 996 bp HR donor. Transfected cells were treated with birabresib for 24H, and DDR pathway function was assessed by digital droplet PCR. BC cells were synchronized at the G1/S transition with 2 mM thymidine incubated with birabresib alone. After 48 h, cells were harvested, and changes to target gene expression (i.e., POLQ) was assessed by RT-qPCR. Next, the same cells were treated with 8 ascending doses of the BETi birabresib (0.1–100 μM) alone or in combination with either the PARP inhibitor olaparib, CtIP inhibitor triapene, or RAD51 inhibitor RI-1 (10 nM–200 μM). After 72-96 h, CellTiter-Glo™ measured cell viability, and Compusyn v1 calculated combination index (CI) scores where <1.0 indicated synergism and >1.0 indicated antagonism. RT-qPCR was conducted to evaluate expression changes to POLQ after combination treatment. Results: After 48 h, birabresib significantly reduced TMEJ in both 5637 and J82 BC cells (49% reduction, P=0.008 and 41% reduction, P=0.004 both n=3), but did not significantly impact HR or NHEJ. POLQ mRNA expression was most reduced in synchronized 5637 and J82 BC cells after treatment with birabresib alone (46% reduction, P=0.006, n=3, and 38% reduction, P=0.001, n=3). Birabresib combined with olaparib was the most synergistic combination in both 5637 and J82 BC cells (CI = 0.83 and 0.29), but birabresib combined with triapene was also synergistic (CI = 0.91 and 0.90). In J82 cells, birabresib significantly reduced POLQ expression (27% reduction, P=0.008, n=3), but olaparib did not. In both 5637 and J82 cells combined birabresib and olaparib increased POLQ expression (41% increase and 5% increase, ns, n=3). Combined birabresib and triapene displayed a similar trend (46% increase and 17% increase, ns, n=3). Conclusions: These preliminary data support further exploration of POLQ expression increases caused by combined BETi+DDRi treatment in preclinical models of BC. Citation Format: Ryan M. Kemper, Bhavika C. Chirumamilla, Dennis A. Simpson, Manfred Meng, Gaorav P. Gupta, Daniel J. Crona. BET inhibition sensitizes preclinical models of bladder cancer to DDR inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B012.

Abstract Oncogene-induced replication stress leads to endogenous DNA damage, activating the cGAS/STING signaling pathway, a critical player in tumor suppression. The exact mechanism underlying cGAS activation, however, remains unclear due to the continual inhibition of cGAS by its high-affinity interaction with the histone acidic patch (AP), which sterically prevents its activation by double-stranded DNA (dsDNA). In this investigation, we elucidate the significant role of the DNA double-strand break sensor, Mre11, in regulating cGAS activation and thereby inhibiting mammary tumorigenesis. Our data reveal that the binding of the Mre11-Rad50-Nbn (MRN) complex to nucleosome fragments is crucial to liberate cGAS from AP-mediated sequestration, facilitating its subsequent activation by dsDNA. Consequently, Mre11 emerges as a vital component in the cGAS activation process, responding to oncogenic stress, cytosolic dsDNA, and ionizing radiation. Moreover, we highlight the ramifications of Mre11-dependent cGAS activation which fosters ZBP1/RIPK3/MLKL-mediated necroptosis, a vital process in curtailing oncogenic proliferation and breast tumorigenesis. Significantly, our study identifies a strong correlation between the downregulation of ZBP1 in human triple-negative breast cancer and increased genomic instability, suppressed immune response, and adverse patient prognosis. Our findings firmly establish Mre11 as a pivotal mediator connecting DNA damage to cGAS activation, thereby facilitating tumor suppression through ZBP1-dependent necroptosis, offering a promising avenue for targeted breast cancer therapies. Citation Format: Minguk Jo, Rashmi J. Kumar, Chien-Chu Lin, Joshua A. Boyer, Jamshaid A. Shahir, Katerina Fagan-Solis, Dennis A. Simpson, Cheng Fan, Christine E. Foster, Anna M. Goddard, Lynn M. Lerner, Simon W. Ellington, Qinhong Wang, Ying Wang, Alice Y. Ho, Pengda Liu, Charles M. Perou, Qi Zhang, Robert K. McGinty, Jeremy E. Purvis, Gaorav P. Gupta. Mre11 mediates cGAS activation and tumor suppression through ZBP1-dependent necroptosis in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A012.

Abstract DNA polymerase theta (Pol θ), functions in theta-mediated end joining (TMEJ), an error-prone DNA double strand break repair pathway. Pol θ is synthetic lethal with homologous recombination (HR) factors, and this finding has led to the development of Pol θ inhibitors and clinical trials to determine their effectiveness in HR-deficient tumors. Despite this, the biological functions of Pol q that promote genome integrity in normal cells are poorly understood. Here, we elucidate an essential role for Pol θ during mammalian interstrand crosslink (ICL) repair. We show that Pol θ is recruited downstream of ICL nucleolytic incision, BRCA2-dependent Rad51 loading, and FBXO5-mediated Rad51 ubiquitylation at sites of unsuccessful HR. Genomic scar analyses reveal that TMEJ is specifically required for repair of clustered ICLs, which are not amenable to HR-mediated repair. Thus, Pol q may be the predominant repair pathway for clustered lesions that generate two-ended double strand breaks during DNA replication. These findings suggest that Pol θ has essential roles in DNA repair in HR proficient cells, suggesting that Pol θ inhibitors may also be beneficial in the treatment of HR proficient tumors. Citation Format: Chelsea M. Smith, Dennis Simpson, Wanjuan Feng, Gaorav Gupta. Polymerase theta (Pol θ) is essential to repair a subset of DNA breaks in HR proficient cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B004.

Supplementary Table S1 from Ataxia Telangiectasia-Mutated–Dependent DNA Damage Checkpoint Functions Regulate Gene Expression in Human Fibroblasts

Homologous recombination (HR)-deficiency induces a dependency on DNA polymerase theta (Polθ/ Polq )-mediated end joining, and Polθ inhibitors (Polθi) are in development for cancer therapy. BRCA1 and BRCA2 deficient cells are thought to be synthetic lethal with Polθ, but whether distinct HR gene mutations give rise to equivalent Polθ-dependence, and the events that drive lethality, are unclear. In this study, we utilized mouse models with separate Brca1 functional defects to mechanistically define Brca1-Polθ synthetic lethality. Surprisingly, homozygous Brca1 mutant, Polq −/− cells were viable, but grew slowly and had chromosomal instability. Brca1 mutant cells proficient in DNA end resection were significantly more dependent on Polθ for viability; here, treatment with Polθi elevated RPA foci, which persisted through mitosis. In an isogenic system, BRCA1 null cells were defective, but PALB2 and BRCA2 mutant cells exhibited active resection, and consequently stronger sensitivity to Polθi. Thus, DNA end resection is a critical determinant of Polθi sensitivity in HR-deficient cells, and should be considered when selecting patients for clinical studies.

Supplementary Table S2 from Ataxia Telangiectasia-Mutated–Dependent DNA Damage Checkpoint Functions Regulate Gene Expression in Human Fibroblasts

Supplementary Table S4 from Ataxia Telangiectasia-Mutated–Dependent DNA Damage Checkpoint Functions Regulate Gene Expression in Human Fibroblasts

Supplementary Table S7 from Ataxia Telangiectasia-Mutated–Dependent DNA Damage Checkpoint Functions Regulate Gene Expression in Human Fibroblasts

Supplementary Table S6 from Ataxia Telangiectasia-Mutated–Dependent DNA Damage Checkpoint Functions Regulate Gene Expression in Human Fibroblasts

Supplementary Table S3 from Ataxia Telangiectasia-Mutated–Dependent DNA Damage Checkpoint Functions Regulate Gene Expression in Human Fibroblasts

Supplementary Table S5 from Ataxia Telangiectasia-Mutated–Dependent DNA Damage Checkpoint Functions Regulate Gene Expression in Human Fibroblasts

Many cancer therapies, including radiotherapy, induce DSBs as the major driving mechanism for inducing cancer cell death. Thus, modulating DSB repair has immense potential for radiosensitization, although such interventions must be carefully designed to be tumor selective to ensure that normal tissue toxicities are not also increased. Here, we review mechanisms of error-prone DSB repair through a highly efficient process called end joining. There are two major pathways of end-joining repair: non-homologous end joining (NHEJ) and alternative end joining (a-EJ), both of which can be selectively upregulated in cancer and thus represent attractive therapeutic targets for radiosensitization. These EJ pathways each have therapeutically targetable pioneer factors - DNA-dependent protein kinase catalytic subunit (DNA-PKcs) for NHEJ and DNA Polymerase Theta (Pol θ) for a-EJ. We summarize the current status of therapeutic targeting of NHEJ and a-EJ to enhance the effects of radiotherapy - focusing on challenges that must be overcome and opportunities that require further exploration. By leveraging preclinical insights into mechanisms of altered DSB repair programs in cancer, selective radiosensitization through NHEJ and/or a-EJ targeting remains a highly attractive avenue for ongoing and future clinical investigation.

Purpose/Objective(s) Alterations in DNA damage response (DDR) genes are prevalent in human cancers. How these genetic changes modify responses to DNA-directed therapeutics (DDTs) is poorly understood. Here we describe an in vivo CRISPR screening platform to systemically identify DDR gene mutations that confer sensitivity or resistance to different classes of DDTs in a transgenic breast cancer model. Materials/Methods Murine breast tumors were induced by mammary intraductal injection of Rosa26LSL-Myc;LSL-Cas9;Trp53flox/flox 6-10 week old female mice with 5 × 105 transduction units of lentivirus expressing Cre recombinase and a library of small guide RNAs targeting 310 DDR genes ("DDR-CRISPR" library). Mammary tumors were analyzed for sgRNA representation by amplicon-based next generation sequencing. A pool of 39 DDR-CRISPR mammary tumor lines was orthotopically injected into a cohort of female NOD/RAG1−/- mice. When tumors reached 8-10mm in maximal dimension, cohorts of six tumors each were treated with the following DDTs: doxorubicin/hydroxydaunorubicin, Paclitaxel, Capecitabine, Olaparib, AZD6738, 2Gyx4, and 8Gyx1. Tumors were harvested when they reached 15mm in maximal dimension. Statistically significant changes in sgRNA abundance in drug treated tumors versus mock treated controls were identified using two-tailed t-tests. FANCA-knockout clones of MDA-MB-231 cells were generated by dual Cas9-sgRNA targeting. Sensitivity to Olaparib and AZD6738 were assessed by colony forming assay and in vivo by tumor growth analyses in female NOD/RAG1−/− mice. Results Mock-treated mammary tumor pools contained approximately 500 sgRNAs targeting 230 distinct DDR genes. Each type of DDT resulted in distinct yet reproducible fluctuations in sgRNA representation, likely reflecting the variable biological effects of DDR gene mutations on therapeutic sensitivity. Olaparib and AZD6738 treatments resulted in depletion of sgRNAs targeting genes in the canonical homologous recombination (HR) repair pathway (e.g., Brca2, Mre11, Nbn, and Rad54b). However, non-HR genes such as FANCA were also significantly depleted by PARP and ATR inhibitor treatments. Using an isogenic MDA-MB-231 cell line model, we show that FANCA deficiency does not impair HR but results in significant accumulation of single-stranded gaps upon PARP and ATR inhibitor treatment. Single and dual treatment with PARP and/or ATR inhibitors resulted in substantial effects on tumor growth delay and mouse survival. Conclusion Our findings illustrate that genetic mixtures of DDR-deficient breast cancers generated through an in vivo CRISPR screen can be used to efficiently quantify the impact of DDR gene mutations on therapeutic sensitivity. Using this platform, we discovered FANCA deficiency as a determinant of sensitivity to PARP and ATR inhibitors due to an accumulation of single-stranded DNA gaps, despite being proficient in HR repair.

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays ('PathSig-dPCR') for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.