Polymerase Theta Inhibition Impairs Tumor Growth and Enhances Melphalan-Induced DNA Damage in Multiple Myeloma

Backgroud: Multiple myeloma (MM) is an aggressive hematologic malignancy characterized by frequent genomic instability. Although the acquisition of genomic alterations drives disease progression, the accumulation of DNA lesions fosters an increased reliance of MM cells on superfluous DNA damage response (DDR) mechanisms. Microhomology-mediated end-joining (MMEJ) is an error-prone backup DNA repair pathway. Recent studies have identified DNA polymerase theta (POLQ), the principal component of MMEJ, as a synthetic lethal target in homologous recombination-deficient tumor models. However, it remains unclear whether POLQ plays a role in neoplasms like MM that possess intact major DDR pathways. Melphalan is still a fundamental DNA-damaging agent in MM treatment. Melphalan-induced DNA damage can eliminate a large proportion of myeloma cells. However, it can also elicit compensatory activation of the DDR in surviving cells, which may result in an inadequate treatment response and chemotherapy resistance. In this study, we aimed to investigate the dependency of MM cells on the POLQ-mediated MMEJ pathway and to determine whether POLQ inhibition could enhance the cytotoxicity of melphalan in MM treatment.

Methods: To understand the addiction of MM cells to the POLQ-mediated MMEJ pathway, we explored the expression level of the POLQ protein in MM and assessed its prognostic relevance. We demonstrated the impacts of POLQ inhibition on genomic stability and proliferation of MM cells through the shRNA interference plasmid and the pharmacological inhibitor. To elucidate the role of POLQ-mediated MMEJ in repairing melphalan-induced DNA damage, we employed western blotting and the EJ2-GFP report assay to evaluate the activation of the MMEJ pathway following melphalan exposure. We analyzed the synergistic cytotoxicity of POLQ inhibition and melphalan in MM, both in vitro and in vivo. The impacts of combination therapy on DNA damage and mitotic catastrophe were examined by comet assay, western blotting, and immunofluorescence.

Results: We found a significant upregulation of POLQ expression in MM cell lines and primary myeloma cells from MM patients as compared to normal plasma cells. The results from public MM datasets showed that a higher level of POLQ was associated with advanced ISS stage and a poor prognosis. POLQ inhibition resulted in an elevated level of γH2AX, the hallmark of DNA lesions, and reduced cell viability in MM cell lines, suggesting that MM cells required the superfluous POLQ-mediated MMEJ pathway to survive in the turmoil of genomic instability. We observed a further increase in POLQ expression and MMEJ repair efficiency following melphalan treatment, which indicated that MMEJ was involved in the repair of melphalan-induced DNA damage. We subsequently showed that knockdown of POLQ by shRNA could significantly enhance the cytotoxicity of melphalan in vitro. The Bliss drug synergy scoring assay revealed a synergistic effect between POLQ inhibitor novobiocin and melphalan. Remarkably, the synergistic therapeutic efficacy was maintained in the subcutaneous MM xenograft mouse model. Mechanistically, the concurrent treatment with novobiocin and melphalan induced extensive DNA damage, as observed in the comet assay. The increased levels of phosphorylated histone H3 in the combination treatment group suggested that a significant number of MM cells with severe genomic damage were driven into mitosis. The presence of micronuclei, grape-shaped nuclei, and multipolar spindle confirmed that the combination therapy triggered mitotic catastrophe in MM cells.

Conclusion: Our study showed that the POLQ-mediated MMEJ pathway was not only essential for coping with spontaneous genomic instability but also for the repair of melphalan-induced damage in MM. The inhibition of POLQ enhanced the cytotoxicity of melphalan by increasing unrepaired DNA lesions and inducing lethal mitotic catastrophe. Therapeutic strategies combining melphalan with POLQ inhibitors may have the potential to improve treatment outcomes for patients with MM.