Ataxia Telangiectasia Mutated (ATM) kinase deficiency results in cancer susceptibility and drug hypersensitivity. Deficiency in either the BRCA1 interacting A complex or XRCC4/Ligase 4 confers resistance to Topoisomerase I or PARP1 inhibitors in ATM-deficient cells. This suggests that BRCA1-A directs toxicity to fork-damaging agents in ATM mutated cells via illegitimate end-joining. Here, we show that ATM inhibition triggers combined SUMO and ubiquitin mediated BRCA1-A damaged fork recognition to restrict end-resection and cause Topoisomerase I inhibitor hypersensitivity. BRCA1-A deficient cells display elevated chromatin accessibility and nuclease activity at damaged forks, coupled with restored resection and drug resistance. Electron microscopy evidence demonstrates that ATM inhibition prevents replication fork reversal, which is restored by BRCA1-A loss to generate substrates for end resection. These findings reveal that BRCA1-A enforces a restrictive chromatin state to suppress the genesis of resection substrates, implicating fork reversal as a key determinant of chemotherapy response in ATM deficient cells.

CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy, specifically aromatase inhibitors or selective estrogen receptor degraders or modulators, have significantly improved outcomes for advanced estrogen receptor-positive (ER) breast cancer; however, therapeutic resistance remains a primary cause of mortality. Overcoming CDK4/6i treatment resistance is an urgent problem. Here, we demonstrate in ER PyMT-BO1 and triple-negative 4T1 murine models that ribociclib (LEE011) monotherapy reduces primary mammary fat pad (MFP) tumor burden through a mechanism partially dependent on CD8 T cell function. However, LEE011 monotherapy fails to decrease tumor burden in bone colonization models, indicating site-specific resistance within the bone microenvironment. We identified doxorubicin as a synergistic partner that, when combined with LEE011, inhibits tumor cell proliferation and suppresses myeloid-specific arginase 1 (ARG1) expression. While doxorubicin monotherapy reduces tumor burden, its efficacy in bone is often compromised by off-target bone loss and pro-metastatic TGF-β signaling. Notably, the combination of LEE011 and doxorubicin successfully reduces bone metastatic burden, reverses treatment-induced osteoclast activity, and decreases the infiltration of ARG1 immunosuppressive myeloid cells. Furthermore, this combination therapy remodels the bone niche to significantly enhance the efficacy of adoptive T cell immunotherapy. Collectively, these results suggest that the synergy between CDK4/6i and doxorubicin represents a promising therapeutic strategy to overcome the protective signals of the bone microenvironment in metastatic breast cancer.

Targeting replication-associated DNA repair mechanisms, including the control of ADP-ribosylation by PARP1/2 and PARG, is a powerful therapeutic approach for cancer. However, the mechanisms by which PARG inhibition impacts DNA replication remain unclear. Here, we combine isolation of proteins on nascent DNA (iPOND) with quantitative proteomics and functional assays to investigate replication fork dynamics upon acute PARG inhibition. We find that FET family proteins (FUS, EWS, and TAF15) are recruited to replication forks in a PAR-dependent manner, forming condensates that slow fork progression and promote fork reversal. FET proteins control fork dynamics in response to some, but not all, replication stresses. FUS inactivation leads to unrestrained fork progression via RECQ1 and PRIMPOL, increased single-stranded DNA gaps, genome instability, and synthetic lethality with BRCA1 deficiency. These findings reveal that FET protein assemblies modulate replication stress responses, influencing genome stability and the cellular response to cancer therapeutics targeting PARylation pathways.

Resolving complex topological structures at replication forks is essential for faithful DNA replication, yet the underlying mechanisms remain poorly understood. Evidence from diverse eukaryotes suggests that condensin – best known for driving chromosome condensation in mitosis – may also operate during S phase to alleviate torsional stress in cooperation with topoisomerases. Here, we show in budding yeast and human cells that condensin binds stressed replication forks, where it cooperates with topoisomerases I and II to promote nascent DNA resection and restart replication. Our data indicate that condensin acts together with topoisomerase I at reversed forks to convert positive supercoils into topological DNA structures that are relaxed by topoisomerase II, enabling fork restart. These findings reveal an evolutionarily conserved role for condensin in resolving topological constraints at arrested forks, reminiscent of its function in chromosome segregation, and suggest that this activity helps prevent the formation of toxic chromosome structures during fork arrest and reversal.