Cyclin D1 Overexpression in Mantle Cell Lymphoma Causes Cellular Dependence on Microhomology-Mediated End-Joining

Oncoprotein-driven human cancers exhibit replication stress (RS), leading to an increase in DNA double-strand breaks (DSB). These DSBs are repaired by one of the three main pathways - homologous recombination repair (HR), non-homologous end-joining (NHEJ), and microhomology-mediated end joining (MMEJ). Cyclin D1 (CCND1) is one of the most overexpressed oncoproteins and is universally overexpressed in mantle cell lymphoma (MCL). While CCND1 overexpression drives cells into the S-phase, its specific effects on RS and DSB repair pathways are poorly understood. Here, we investigated the impact of CCND1 overexpression on RS and DSB repair mechanisms in MCL.

First, the expression level of CCND1 positively correlated with heightened RS, as indicated by elevated p-RPA-S33 and p-RPA S8 levels, and increased DNA damage, assessed by g-H2AX, especially in the mitotic phase, across diverse cell models, including MCL. Since CCND1-overexpressed cells proliferate rapidly, we hypothesized that some DNA repair pathways might be upregulated to meet the demand for repairing the excess RS-driven DSBs. Reporter assays demonstrated that the MMEJ pathway was specifically upregulated upon CCND1 overexpression, whereas no significant changes were observed in the HR and NHEJ pathways. Furthermore, CRISPR interference (CRISPRi)-mediated knockdown of CCND1 in MCL cells led to a significant reduction in MMEJ activity. This finding suggests the specificity of CCND1 in mediating the upregulation of the MMEJ pathway. Consistent with this, increased MMEJ activity, assessed via foci formation and immunoblotting of DNA polymerase theta expression (POLQ, encoded by POLQ gene), the primary polymerase for the MMEJ pathway, correlated with CCND1 overexpression, suggesting the crucial role of MMEJ in DSB repair processes under CCND1-overexpressed conditions.

Sincethe MMEJ pathwayplays a pivotal role in repairing DSB during mitosis, we assessed the dependency of MCL cells on MMEJ-mediated DSB repair for cell survival. As expected, genetic deletion or pharmacological inhibition (ART558 and novobiocin) of POLQ 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 POLQ 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 after CCND1 overexpression in MCL, further increased the cellular dependence on MMEJ. Interestingly, the accumulation of unrepaired DNA damage during mitosis was notably increased in ATM-deficient CCND1-overexpressed cells. When POLQ 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 (AZD0156) and POLQ was synthetic lethal in MCL cell lines and primary tumor cells from patients with MCL.

Mechanistically, we observed the direct binding of CCND1 to the POLQ promoter and a corresponding upregulation of POLQ mRNA in CCND1-overexpressing cells. Consistently, a significant overexpression of POLQ mRNA was observed in primary MCL cells, compared to other non-Hodgkin lymphoma cells without CCND1 overexpression. Moreover, the positive correlation between POLQ and CCND1 expression was further validated using syngeneic B-lymphoma cells with and without CCND1 overexpression. Importantly, CRISPRi-mediated CCND1 knockdown reduced POLQ mRNA expression and the dependency of MCL cells on the MMEJ pathway for survival. In addition, ATM deficiency showed a further increase of endogenous POLQ expression and foci formation in cells with CCND1 overexpression, suggesting ATM negatively regulates the MMEJ pathway, especially in the CCND1 overexpressed background.

In summary, CCND1 overexpression leads to a substantial increase in RS, intensifying the reliance on MMEJ-mediated mitotic DSB repair. Within MCL, POLQ is crucial in mitigating the adversities of CCND1-driven RS. Our data suggest that targeting the MMEJ pathway via POLQ inhibition, particularly in the presence of CCND1 overexpression and ATM deficiency, emerges as a promising therapeutic approach in oncoprotein-driven hematologic malignancies such as MCL.

D'Andrea:PrimeFour Therapeutics: Consultancy; Impact Therapeutics: Consultancy; Deerfield Management Company L.P.: Consultancy; AbbVie: Consultancy; Schrödinger Inc.: Consultancy; Servier Bio-Innovation LLC: Consultancy; Covant Therapeutics: Membership on an entity's Board of Directors or advisory committees; Impact Therapeutics: Membership on an entity's Board of Directors or advisory committees; Impact Therapeutics: Current holder of stock options in a privately-held company; PrimeFour Therapeutics: Current holder of stock options in a privately-held company; Bristol Myers Squibb: Research Funding; EMD Serono: Research Funding; Moderna: Research Funding; Tango Therapeutics: Research Funding.