Mukkavalli, Sirisha

Integration of the high-risk human papillomavirus 16 (HPV16) genome into the host chromosome, frequently driven by microhomology-mediated end joining (MMEJ), is a critical step in the carcinogenesis of HPV-associated tumors. However, the mechanisms by which viral oncoproteins manipulate the error-prone MMEJ pathway remain poorly defined. Here, we demonstrate that the HPV16 E6 oncoprotein upregulates MMEJ to facilitate viral genome integration. This heightened MMEJ activity is driven by a marked increase in the protein levels of DNA polymerase theta (PolΘ), a central enzyme of the MMEJ pathway. Mechanistically, we show that the elevation of PolΘ levels in response to HPV16 E6 expression is dependent on the host E3 ubiquitin ligase UBE3A/E6AP but is independent of p53 degradation. E6 redirects UBE3A to enhance the ubiquitination and degradation of RAD23A, a shuttle protein required for delivering polyubiquitinated PolΘ to the proteasome. Consequently, the loss of functional RAD23A phenocopies the effect of HPV16 E6, leading to PolΘ protein stabilization and increased MMEJ activity. By elucidating the E6-UBE3A-RAD23A-PolΘ axis, our findings reveal a mechanism through which HPV manipulates the host DNA repair machinery to promote its integration and oncogenic potential.

Oncogene expression can cause replication stress (RS), leading to DNA double-strand breaks (DSBs) that require repair through pathways such as homologous recombination, nonhomologous end-joining, and microhomology-mediated end-joining (MMEJ). Cyclin D1 (encoded by CCND1 ) is a well-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 the impact of cyclin D1 on RS and DSB repair mechanisms. Cyclin D1 overexpression elevated RS, increased DNA damage, especially during mitosis, and caused specific upregulation of MMEJ. Furthermore, cyclin D1 activated polymerase theta ( POLQ ) transcription by binding its promoter loci, driving POLΘ-mediated MMEJ that is essential to withstand cyclin D1–induced RS. Moreover, concurrent ATM deficiency further intensified RS, enhanced POLQ expression, and heightened reliance on MMEJ-mediated DNA damage repair. Consequently, inhibition of POLΘ in cyclin D1–overexpressed settings further exacerbated RS, causing single-strand DNA gap accumulations and chromosomal instability, ultimately leading to apoptosis, an effect amplified in ATM-deficient cells. Targeting MMEJ via POLΘ inhibition is therefore an effective strategy in the context of cyclin D1 overexpression and ATM deficiency and may provide a unique therapeutic approach for treating MCL and other malignancies characterized by similar alterations.

SUMMARY Ewing sarcoma (EwS) is a group of bone and soft tissue cancers in children and young adults. Since EwS cells have pronounced sensitivity to radiation and chemotherapy-induced DNA damage, the role of the oncoprotein, EWS-FLI1, in DNA repair is likely. Here, we demonstrate that EWS-FLI1 causes a defect in microhomology-mediated end-joining (MMEJ) repair. EWSR1 is a splicing factor that promotes the faithful splicing of the POLQ pre-mRNA, required for the expression of POLΘ, a critical protein in the MMEJ pathway. Expression of EWS-FLI1, or loss of EWSR1, causes exon 25 skipping of the POLQ transcript, decreased POLΘ expression, impaired MMEJ, and cellular sensitivity to inhibitors of the Fanconi Anemia (FA), NHEJ, or HR pathways, through the mechanism of synthetic lethality. Knockdown of EWS-FLI1 expression restores POL0 mitotic foci and increases MMEJ activity. Inhibitors of the FA, NHEJ, or HR therefore may provide a targeted therapy for patients with EwS. Highlights - Ewing sarcoma tumors have a deficiency in POLθ expression and a corresponding loss of MMEJ activity - EWSR1 is a splicing factor that interacts with other splicing factors such as FUBP1 and KHSRP/FUBP2 to accurately splice the POLQ mRNA. - The EWS-FLI1 fusion oncoprotein, or loss of EWSR1, causes a splicing defect, leading to exon25 skipping of the POLQ pre-mRNA and loss of POLθ expression - The MMEJ deficiency of EwS cells results in cellular sensitivity to inhibitors of Fanconi Anemia, Homologous Recombination or Non-Homologous End-Joining - Exon 25 skipping of POLQ mRNA is a predictive biomarker for HR inhibitors in human cancers

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.