175 | CYCLIN D1 OVEREXPRESSION INDUCES REPLICATION STRESS AND MICROHOMOLOGY‐MEDIATED END‐JOINING DEPENDENCE FOR DNA DAMAGE REPAIR IN MANTLE CELL LYMPHOMA

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Oncogene expression can cause replication stress (RS), leading to DNA double-strand breaks (DSB) that require repair through pathways such as homologous recombination (HR), non-homologous end joining (NHEJ), and microhomology-mediated end-joining (MMEJ, the only DSB repair pathway active in mitosis). Cyclin D1 (CCND1) is a known oncoprotein overexpressed in cancer; however, its role in RS is unknown. Using mantle cell lymphoma (MCL) as a naturally occurring model of cyclin D1 overexpression, we examined its impact on RS and DSB-repair mechanisms. First, the expression level of Cyclin D1 positively correlated with heightened RS (indicated by elevated p-RPA-S33 levels) and increased DNA damage (assessed by γ-H2AX), especially in the mitotic phase, across diverse MCL cell lines. Reporter assays demonstrated that the MMEJ pathway was specifically upregulated upon Cyclin D1 overexpression, whereas no significant changes were observed in the HR and NHEJ pathways. Consistent with this, increased MMEJ activity, assessed via foci formation and immunoblotting of DNA polymerase theta expression (POLΘ, encoded by POLQ gene), the primary polymerase for the MMEJ pathway, correlated with CCND1 overexpression. CRISPRi-mediated CCND1 knockdown reduced POLQ mRNA expression and the dependency of MCL cells on the MMEJ pathway for survival. Mechanistically, Cyclin D1 binds to the POLQ gene promoter and activates transcription, driving POLΘ-mediated MMEJ that is essential to withstand cyclin D1-induced RS. We then assessed the dependency of MCL cells on MMEJ-mediated DSB repair for cell survival. As expected, genetic deletion or pharmacological inhibition of POLΘ in MCL cells led to increased RS and increased unrepaired DNA damage during mitosis, ultimately leading to chromosomal instability, mitotic catastrophe, and apoptosis. Additionally, the deficiency of POLΘ in MCL cells resulted in increased levels of single-strand DNA, another critical biomarker for RS. Second, we found that ATM deficiency, the second most common genetic alteration in MCL, further increased the cellular dependence on MMEJ. Interestingly, the accumulation of unrepaired DNA damage during mitosis was notably increased in ATM-deficient Cyclin D1-overexpressed cells. When POLΘ was inhibited, a significant antitumor effect was evident in MCL cells, including ibrutinib-resistant MCL, both in vitro and in vivo, and this effect was enhanced by ATM deficiency. Further, simultaneous inhibition of both ATM and POLΘ was synthetic lethal in MCL cell lines in vitro and in vivo and primary tumor cells from patients with MCL. In summary, Cyclin D1 overexpression leads to a substantial increase in RS, intensifying the reliance on MMEJ-mediated mitotic DSB repair. POLΘ is crucial in mitigating the adversities of CCND1-driven RS. Targeting the MMEJ pathway via POLΘ inhibition, particularly in the presence of Cyclin D1 overexpression and ATM deficiency, emerges as a promising therapeutic approach in MCL. Research funding declaration: Predolin Biobank Keywords: non-Hodgkin; other basic and translational science; molecular targeted therapies No potential sources of conflict of interest.

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