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.

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.

TP53 mutations are found in 10% to 15% of myeloid neoplasms and are associated with a dismal prognosis. Although hypomethylating agents (HMAs), such as decitabine, are active in TP53-mutated myeloid neoplasms (TP53-MN), mutation clearance is rarely complete and nearly all patients relapse. Molecular determinants of response to HMAs in TP53-MN are poorly understood. Here, we show that decitabine induces replicative stress with decreased replication fork progression, induction of single-strand DNA breaks, and activation of the ataxia telangiectasia mutated-Rad3-related (ATR) pathway. Resolution of decitabine-induced replication stress is impaired in TP53-mutated acute myeloid leukemia (AML) cells, representing a potential therapeutic vulnerability. Indeed, the combination of decitabine and ATR inhibition (ATRi) induces synthetic lethality that is selective for TP53-AML and due, in part, to induction of mitotic catastrophe. Interestingly, this synergistic lethality was not observed with azacitidine or treatment with GSK3685032, a potent DNA methyltransferase 1 inhibitor, both of which produce a comparable level of global hypomethylation to decitabine. Treatment with decitabine and an ATR inhibitor reduces leukemia burden and prolongs survival in in vivo mouse models of TP53-mutated AML. Collectively, these findings show that TP53 loss generates a selective vulnerability to decitabine-induced replication stress, with the combination of ATRi and decitabine showing promise as a new therapeutic approach for TP53-MN.