Extrachromosomal circular DNA (ecDNA) is frequently generated within the nucleus, contributing to genome dynamics and heterogeneity, thereby promoting cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here, using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes as key drivers of ecDNA production. Following DNA segmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively mediates ecDNA production by joining the free ends of the DNA fragments. Furthermore, ecDNA from patient tumors harbors junction sites with a LIG4 signature. Notably, disruption of either LIG4 or the BRCA1-A complex in cancer cells impairs ecDNA-mediated adaptation, hindering the development of resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.

Abstract Targeted therapies are designed to eliminate cancer cells by directly inhibiting oncogenic driver proteins. In addition to their primary inhibitory effects on oncogenic signaling, these agents frequently impose collateral cellular stresses, such as DNA damage. KRAS-targeted therapies, particularly KRAS G12C inhibitors (G12Ci), represent a major therapeutic advance but remain limited in efficacy. Previous reports of targeted therapy-induced DNA damage, including studies of TKIs and MAPK inhibitors, have primarily been based on cytotoxic dosing conditions. Far less is known about whether DNA damage can also be induced by targeted therapies in less sensitive cancer models, particularly under sublethal doses that better mimic clinical responses. Failure to repair DNA damage can lead to chromosomal instability (CIN) and chromosomal aberrations. CIN is widely recognized to promote tumor evolution by enhancing cellular plasticity and adaptability, thereby contributing to therapeutic resistance and metastatic progression. However, it remains unknown how KRAS G12C inhibition influences CIN and whether G12Ci-induced CIN might generate unique, exploitable vulnerabilities.In this study, we profiled 15 KRAS G12C-mutant NSCLC cell lines representing diverse mutational backgrounds. We treated these models with the KRAS G12Ci LY3499446 and comprehensively assessed their DNA damage responses, CIN phenotypes, and sensitivity screening to combination therapies with agents that perturb chromosomal stability. We observed heterogeneous induction of DNA damage and CIN across these cell lines. Notably, we identified the strongest correlation between G12Ci-induced CIN and synergistic interaction with the Aurora kinase A inhibitor (AURKAi) LSN3321213. Machine learning-based single-cell image tracking and DNA barcoding analyses revealed that AURKA inhibition alone causes mitotic arrest followed by mitotic slippage, allowing cells to evade death, whereas combined G12Ci and AURKAi treatment triggers catastrophic mitotic cell death. Mechanistically, we found that G12Ci stabilizes Cyclin B1 through mitotic activation of ATR/ATM DNA repair signaling, thereby prolonging mitotic arrest. Under conditions of combined inhibition of KRAS G12C and AURKA, in which Cyclin B1 degradation is impaired, cells fail to exit mitosis and undergo catastrophic cell death. Together, our findings identify CIN as a predictive marker of response to combined KRAS G12C and AURKA inhibition, providing mechanistic rationale to enhance the therapeutic window of AURKA inhibitors when used with targeted therapies. Citation Format: Chendi Li, Varuna Nangia, Melissa Vieira, Anahita Nimbalkar, Christopher Graser, Jeremy Chang, Mohammad Syed, Yi Shen, Radhika Koranne, Lee Zou, Franziska Michor, Sabrina L. Spencer, Aaron N. Hata. Targeted therapy-induced chromosomal instability dictates mitotic dependency on Aurora kinase A [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 6773.

Abstract Chordoma is a rare bone cancer with a high rate of recurrence and no approved systemic therapies. Target discovery efforts have uncovered complex genomic rearrangements, alterations in DNA damage response (DDR) and chromatin-remodeling genes, and a reliance on DDR pathways that stabilize stalled replication forks. Additionally, a subset of chordomas is highly infiltrated by multicellular immune aggregates, but the underlying mechanisms driving an antitumor immune response remain unclear. We hypothesized that replication stress presents an actionable therapeutic vulnerability in chordoma and that targeting this pathway could induce lethal DNA damage and immunogenic cell death. To assess the effects of exacerbating replication stress in chordoma, a panel of 14 cell lines was treated with the DNA synthesis inhibitor gemcitabine or the ATR inhibitor elimusertib (BAY 1895344). Most lines were highly sensitive, with 9 of 14 showing EC50 values below 10 nM (gemcitabine) or 100 nM (elimusertib). Biochemical analysis showed that gemcitabine-induced DNA damage leads to activation of Chk1 in an ATR-dependent manner, with DNA fiber assays revealing decreased replication fork speed, symmetry, and stability following ATR inhibition (ATRi). Sensitivity to ATRi or gemcitabine was mirrored in chordoma PDX models, with ATRi promoting 85–90% tumor growth inhibition in sensitive models and gemcitabine driving tumor regressions. Exceptional synergy was observed in vitro when these therapies were combined, with significant reductions in cell viability, enhanced DNA double-strand breaks, and near-complete S-phase collapse. Moreover, treatment with either drug resulted in the accumulation of double-stranded DNA in the cytoplasm, along with upregulation of type I interferon, immunomodulatory chemokines CXCL10 and CCL5, and cell surface PD-L1. These findings are consistent with a model where ATRi or gemcitabine treatment promotes lethal DNA damage and immunogenic cell death in chordoma, which may be further augmented by PD-1 checkpoint blockade. Citation Format: Nindo Punturi, Arijit Ghosh, Caitlin King, Wendy Leung, Joan B. Levy, Lee Zou, Gregory M. Cote, Dan Freed. Targeting replication stress promotes immunogenic cell death in chordoma [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 252.

The enhancer lysine acetyltransferases CBP/p300 are compelling targets for multiple myeloma therapy. Chemical inhibition of these multidomain factors, either through the bromodomain or the catalytic acetyltransferase domain, show promising activity in pre-clinical models. Chemical degradation is the only modality that can completely disrupt all functional domains. Our previous attempts to induce CBP/p300 targeted degradation led to a potent tool compound, dCBP-1. Here we comprehensively demonstrate across a large panel of cell lines how CBP/p300 degradation compares to inhibition, with pronounced selective antiproliferative activity toward multiple myeloma. We use chemical linker optimization strategies to create a compound with better pharmacokinetic properties. Through these we define an advanced analog of dCBP-1, dCBP-30, that has improved potency and improved in vivo properties including oral bioavailability. dCBP-30 led to potent and sustained loss of CBP and p300, potent inhibition of several myeloma-specific dependency programs, and elicits tumor reduction in xenograft models.

Cyclic GMP-AMP synthase (cGAS), a DNA sensor that activates type-I interferon responses, is restrained in the nucleus through chromatin binding, but its impact on DNA metabolism remains unknown. Here we show that chromatin-bound cGAS impedes DNA replication forks unless countered by ATM. Upon ATM loss, chromatin-bound cGAS slows replication forks, increases nascent DNA fragmentation and activates cytosolic cGAS. Remarkably, all these effects are alleviated upon the loss of cGAS chromatin binding, suggesting that ATM enables tolerance to chromatin-bound cGAS. Mechanistically, ATM, backed by ATR, releases cGAS from chromatin by phosphorylating MRE11. ATR inhibition in ATM-deficient cells exacerbates replication stress, causing synthetic lethality and stimulated interferon response. In ATM-deficient cancer cells, cGAS dictates replication stress and ATR inhibitor sensitivity, highlighting its potential as a biomarker for ATR-targeted therapy. Together, our findings uncover a regulatory circuit in which ATM and chromatin-bound cGAS jointly maintain the homeostasis of replication and cGAS signalling in cycling cells.

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine cancer that is typically metastatic upon diagnosis and has poor overall survival. Here we report that the inactivation of ABL tyrosine kinases impairs the outgrowth of metastatic SCLC tumors, resulting in prolonged animal survival. ABL inactivation increases the accumulation of transcription-replication conflicts (TRCs), compromises replication fork progression, and impairs the function of proteins implicated in transcription-coupled homologous recombination, including RAD51 and RAD52. Mechanistically, ABL-mediated tyrosine phosphorylation of RAD52 and RAD51 prevents the accumulation of TRCs and promotes replication fork progression, respectively. Because ABL inactivation increased DNA damage, we evaluated whether blocking the activity of DNA damage-repair pathways in the presence of ABL inhibitors might synergize to promote SCLC cell death. Concurrent inactivation of ABL and ATR, the primary responder to replication stress, synergistically inhibits SCLC cell growth in vitro and impairs metastatic outgrowth over single-agent-treated mice. Thus, co-inactivation of ABL and DNA damage-repair pathways might be exploited to inhibit outgrowth of SCLC metastases.

Abstract Chemoresistance is the leading cause of poor prognosis in triple-negative breast cancer (TNBC), yet the underlying mechanisms remain unknown. To reveal metabolic drivers of de novo chemoresistance in TNBC, we analyzed pretreatment primary tumor biopsies, employing quantitative proteomics and metabolomics. Chemoresistant TNBCs exhibit hallmarks of oxidative phosphorylation (OXPHOS) and altered nucleotide metabolism linked to overexpression of the mitochondrial sirtuin, SIRT5. Through gain- and loss-of-function studies and stable isotope tracing, we demonstrate that SIRT5 induces a coordinated metabolic switch that redirects glycolysis to the pentose phosphate pathway, thereby augmenting nucleotide pools, while enhancing glutaminolysis to support OXPHOS. Mechanistically, SIRT5 enhances conversion of 6-phospho-D-gluconate to ribulose-5-phosphate through demalonylation of 6-phosphogluconate dehydrogenase (6-PGD), and coordinately activates oncogenic c-MYC to promote glutamine utilization and dependence. Concurrently, SIRT5-induced nucleotide deregulation induces replication stress and hypersensitivity to ATR checkpoint activation, and ATR inhibition synergistically reverses chemoresistance in TNBC. Thus, elevated SIRT5 orchestrates a coordinated metabolic switch to expand nucleotide pools and drive chemoresistance, while producing ATR checkpoint dependence that represents a metabolic vulnerability of SIRT5-overexpressing TNBC. Graphical Abstract

TRDMT1 is an RNA methyltransferase that catalyzes 5-methylcytosine (m5C) formation in R-loops to promote transcription-coupled homologous recombination (TC-HR). Although TRDMT1 inhibition selectively kills BRCA1-deficient cancer cells, broader cancer dependencies on TRDMT1 remain unclear. Here, a TRDMT1 inhibitor (TRDMT1i) sensitivity screen across a large panel of cancer cell lines identifies loss of MLH1 or PMS2, two components of the MutLα mismatch repair (MMR) complex frequently inactivated in tumors, as key determinants of TRDMT1 dependency. In contrast, MutLβ and MutSα/β are dispensable for TRDMT1i resistance, revealing a unique MMR-independent function of MutLα. Mechanistically, TRDMT1 and MutLα independently recognize DNA–RNA hybrids and cooperatively suppress co-transcriptional R-loops genome-wide in undamaged cells, with m5C directing pathway choice. Furthermore, MutLα suppresses R-loops through its ATPase and endonuclease activities and through recruitment of EXO1. Combined loss of TRDMT1 and MLH1 causes extensive R-loop accumulation and transcription replication conflicts (TRCs), impairing replication fork progression, inducing DNA damage, and driving apoptosis-mediated synthetic lethality. Importantly, TRDMT1i suppresses growth of MLH1-deficient tumors by inducing TRCs in vivo, suggesting a potential therapeutic strategy for targeting MutLα-deficient tumors. These studies not only expand our understanding of cancer dependency on TRDMT1, but also identify a promising strategy to exploit TRCs in cancer therapy. DNA strand break repair can be assisted by RNA dependent mechanism. Here the authors show that the RNA methyltransferase TRDMT1 and the mismatch repair complex MutLα independently recognize DNA-RNA hybrids and cooperatively suppress co-transcriptional R-loops, with m5C directing pathway choice.

Transitions of cancer cells between distinct cell states, which are typically driven by transcription reprogramming, fuel tumor plasticity, metastasis, and therapeutic resistance. Whether the transitions between cell states can be therapeutically targeted remains unknown. Here, using the epithelial-to-mesenchymal transition (EMT) as a model, we show that the transcription reprogramming during a cell-state transition induces genomic instability through R-loops and transcription-replication conflicts and that the cell-state transition cannot occur without the ATR kinase, a key regulator of the replication stress response. ATR inhibition during EMT not only increased transcription- and replication-dependent genomic instability, but also disrupted transcription reprogramming. Unexpectedly, ATR inhibition elevated R-loop-associated DNA damage at the SNAI1 gene, a key driver of the transcription reprogramming during EMT, triggering ATM- and Polycomb-mediated transcription repression of SNAI1. Beyond SNAI1, ATR also suppressed R-loops and antagonized repressive chromatin at a subset of EMT genes. Importantly, inhibition of ATR in tumors undergoing EMT reduced tumor growth and metastasis, suggesting that ATR inhibition eliminates cancer cells in transition. Thus, during EMT, ATR not only protects genome integrity but also enables transcription reprogramming, revealing that ATR is a safeguard of cell-state transitions and a target to suppress tumor plasticity.

Targeted therapies eliminate cancer cells by inhibiting oncogenic signaling; however, tumor cells often evade cytotoxicity through proteomic and epigenetic reprogramming that enables survival. These adaptive responses may create collateral cellular stresses, such as DNA damage, that can be therapeutically exploited. When unresolved, DNA damage leads to chromosomal instability (CIN), a potential source of vulnerability. Whether KRAS inhibition induces DNA damage or CIN in KRAS -mutant non-small cell lung cancer (NSCLC) has not been established. Here, we show that the KRAS G12C inhibitor LY3499446 induces CIN in KRAS -mutant NSCLC cell lines. A targeted compound screen revealed that the extent of CIN induction by KRAS G12C inhibition strongly correlates with therapeutic synergy with the selective Aurora kinase A inhibitor LSN3321213. Mechanistically, KRAS G12C inhibition stabilizes cyclin B1 during mitosis through activation of mitotic ATR/ATM signaling. In the presence of Aurora Kinase A inhibition, cyclin B1 stabilization delays mitotic exit and diverts cell fate from mitotic slippage or division toward mitotic catastrophe. Together, our findings identify CIN as a predictive marker of response to combined KRAS G12C and Aurora Kinase A inhibition, providing mechanistic rationale to enhance the therapeutic window of AURKA inhibitors when used with targeted therapies.

BACKGROUND:Approximately 10% of cancers achieve replicative immortality through a telomerase-independent mechanism of telomere maintenance, termed Alternative Lengthening of Telomeres (ALT). ALT is particularly prevalent in certain subtypes of malignant gliomas, such as IDH-mutant astrocytoma and pediatric glioblastoma, and frequently co-occurs with ATRX (ATRX chromatin remodeler) inactivating mutations. Although ALT is an adaptive mechanism through which cancer cells achieve proliferative immortality, the elevated levels of replication stress observed in ALT tumors constitute a potential therapeutic vulnerability. METHODS:Leveraging CRISPR/Cas9 screening data from the Cancer Dependency Mapping Project, coupled with patient-derived cell lines and xenografts, we identified SMARCAL1 as a novel synthetic lethal vulnerability in ATRX-deficient glioma models that engage ALT. Using complementary molecular assays for DNA damage, telomere maintenance, and telomeric replication stress, we define the mechanisms underlying cytotoxicity induced by SMARCAL1 depletion in ALT-positive glioma cells. RESULTS:Our data demonstrate the annealing helicase SMARCAL1 is a highly specific synthetical lethal vulnerability in cancers that use ALT. SMARCAL1 localizes to ALT-associated PML (Promyelocytic leukemia protein) bodies in ALT-positive glioma cell lines, including IDH-mutant astrocytomas. SMARCAL1 depletion, via doxycycline-induced RNAi, led to a hyperactivation of the ALT phenotype, high levels of DNA double-strand breaks in G2 phase, and cell death via mitotic catastrophe. In mice bearing intracranial xenografts derived from high-grade IDH-mutant astrocytoma, inducible SMARCAL1 depletion prolonged animal survival. CONCLUSIONS:Our findings demonstrate that the molecular processes orchestrating ALT-mediated telomere maintenance constitute a targetable synthetic lethal vulnerability that can be exploited by SMARCAL1 inhibition, thus supporting the future development of small molecule inhibitors of SMARCAL1 as anti-cancer therapeutics.

Figure S3: (A) Kaplan-Meier plot for PFS vs time on study where patients were stratified based on p53 status of the tumor. The p53 status was available for 15 patients in the study. Patients from T4 was not included in this analysis.

Figure S1: (A) Study schema. Patients were recruited into one of four cohorts based on NGS or IHC. Patients received twice-per week infusions of berzosertib at the recommended phase 2 dose. Paired biopsies were collected for cohorts 1 to 3 for translational studies. Six patients were allowed per cohort; however, patients could be replaced if paired biopsies were unsuccessful. *For cohort T3, gene alterations included: germline BRCA1/2 mutations, other homologous repair (HR) alterations (e.g., somatic BRCA1/2, BARD1, BRIP1, CDK12, CHEK2, FANCA, FANCC, FANCE, FANCF, FANCM, MRE11A, NBN, PALB2, RAD51B, RAD51C and RAD51D), MYC amplification, FBXW7 truncating or missense mutations, CCNE1 amplification, ARID1A mutations. (B) Kaplan-Meier plot showing Progression free survival of patients in each cohort

Cancer cells deficient in BRCA1/2 have impaired DNA repair, making them sensitive to PARP inhibitors (PARPis). In this issue of Genes & Development, Seppa and colleagues (doi:10.1101/gad.352421.124) investigated how BRCA1 protects single-stranded DNA gaps from nucleolytic processing. They showed that PARPi-induced gaps are rapidly resected by several exonucleases bidirectionally and filled by translesion synthesis. In BRCA1-deficient cells, gaps become larger and persistent due to excessive resection. These gaps do not convert to DNA double-stranded breaks (DSBs) via endonuclease activity but cause DSBs through replication fork collisions in a cell cycle-dependent manner. This research clarifies how BRCA1 loss contributes to PARPi sensitivity in BRCA mutant tumors.

Figure S4. H-score of pre- and on-treatment biopsy stained with anti-SLFN11 antibody for each patient is shown. Patients from cohort 4 was not included in the analysis. Patients from T4 was not included in this analysis.

R-loops are dynamic three-stranded nucleic acid structures that form naturally during transcription. These structures typically arise when the newly synthesized RNA hybridizes with the DNA template strand, displacing the non-template DNA strand. R-loops are not only found at protein-coding genes but also in regions producing non-coding RNAs, such as telomeres, centromeres, ribosomal DNA genes, and transfer RNA genes. While R-loops are regulated by both the process of transcription and chromatin structures, they also play a critical role in modulating transcription and influencing the chromatin landscape. Moreover, the interactions between R-loops, transcription, and chromatin are essential for maintaining genome stability and are often disrupted in various human diseases. In this review, we will explore recent insights into the intricate relationship between R-loops and transcription, as well as their crosstalk with chromatin.

DNA double-strand breaks (DSB) are among the most deleterious forms of DNA damage and, if unresolved, result in DNA mutations and chromosomal aberrations that can cause disease, including cancer. Repair of DSBs by homologous recombination requires extensive nucleolytic digestion of DNA ends in a process known as DNA-end resection. In recent years, progress has been made in understanding how this process is initiated, but the later stages of this process-long-range DNA-end resection-are not well understood. Many questions remain in terms of how the DNA helicases and endonucleases that catalyse this process are regulated, a key step to avoiding spurious activity in the absence of breaks. The importance of DNA-end resection in human disease is highlighted by several human genetic syndromes that are caused by mutations or deficiencies in key proteins involved in this process. Here, using high-throughput microscopy coupled with a cDNA 'chromORFeome' library, we identified ZNF280A as an uncharacterized chromatin factor that is recruited to breaks and essential for DNA DSB repair. Lack of ZNF280A drives genomic instability and substantial sensitivity to DNA-damaging agents. Mechanistically, we demonstrate that ZNF280A promotes long-range DNA-end resection by facilitating the recruitment of the BLM-DNA2 helicase-nuclease complex to DNA DSB sites, enhancing efficiency of the enzymatic activity of this complex at DNA damage sites. ZNF280A is therefore essential for DNA-end resection and DNA repair by homologous recombination. Importantly, ZNF280A is hemizygously deleted in a human genetic condition, 22q11.2 distal deletion syndrome. Features of this condition include congenital heart disease, microcephaly, immune deficiency, developmental delay and cognitive deficits-features that are associated with other human syndromes caused by defects in genes involved in DNA repair. Remarkably, cells from individuals with a 22q11.2 distal deletion have defects in DNA-end resection and homologous recombination, resulting in increased incidence of genomic instability. These phenotypes are rescued by reintroduction of ZNF280A, providing evidence of defective DNA repair as a potential mechanistic explanation for several clinical features associated with this human condition.

AbstractPurpose: Preclinical studies have identified molecular correlates of sensitivity to ATR inhibition. This translational study was designed to test the ATR inhibitor berzosertib in patients with advanced solid tumors carrying alterations in ATRX, ataxia-telangiectasia–mutated (ATM), genes conferring replication stress (RS), or SDH. Patients and Methods: Patients were recruited to four cohorts: T1: ATRX-mutant leiomyosarcoma; T2: ATM-mutant solid tumors; T3: solid tumors with mutations in RS-associated genes; and T4: SDH-deficient gastrointestinal stromal tumors (GIST). Patients were treated with berzosertib 240 mg/m2 intravenously twice per week. Pretreatment and on-treatment biopsies were obtained in cohorts T1 to T3. Results: Patients with SDH-mutant GIST had the longest median progression-free survival (PFS; 229 days) with stable disease as the best response. Patients in the other cohorts experienced progressive disease within 4 months. There was no significant difference in PFS comparing outcomes in patients with/without mutations in ATM or RS genes. Decreased pS345-CHK1 levels in on-treatment biopsies indicated target engagement by berzosertib and were accompanied by substantial increases in levels of DNA damage (γ-H2AX) and RS (pKAP1) markers in a subset of patients. However, these biomarker changes did not translate to clinical benefit. In contrast, in cohorts T1 to T3, increased expression of SLFN11 on treatment correlated with clinical benefit (HR = 0.045; 95% confidence interval, 0.005–0.400). Conclusions: Across cohorts, only patients with SDH-mutant GIST experienced prolonged disease control. Despite evidence of target engagement, patients enrolled to all other cohorts had short PFS, suggesting rapid adaptation to ATR inhibitor monotherapy. Among these patients, those with tumors expressing SLFN11 during berzosertib exposure derived the most clinical benefit.