The role of HUWE1 in DNA replication and genome stability in multiple myeloma

Multiple Myeloma (MM) is a haematological malignancy of terminally differentiated B cells. Despite significant advances in treatment options, development of resistance and toxic side effects are still a major clinical problem, and the disease remains incurable. This highlights the need for a deeper understanding of the pathogenesis of MM and the identification of novel therapeutic targets. Dysregulated expression of the E3 ligase HUWE1 has been reported in MM and HUWE1 has subsequently been identified as a potential therapeutic target. Recent genomic studies also uncovered HUWE1 as a novel mutational driver gene in MM, further implicating the E3 ligase in MM pathogenesis. HUWE1 is known to regulate a myriad of proteins involved in key cellular processes including DNA repair, replication and apoptosis. The aim of this study was to investigate the role of HUWE1 in replication and DNA damage response (DDR) pathways in MM. Expression of HUWE1 was demonstrated to increase in a stepwise manner across plasma cell dyscrasias, and high expression in MM patients was found to confer a shorter overall survival. Knockdown of HUWE1 using shRNA technology demonstrated significantly reduced cellular proliferation compared to the control which was underpinned by a S phase arrest. Replication proteins were identified as a novel substrate of HUWE1, which was found to catalyse the polyubiquitination of the replication protein. Further investigation found that this HUWE1-mediated ubiquitination was essential for loading of these proteins to sites of DNA damage induced by replicative stress and ultimately resulted in a DNA damage repair defect. Another area of this study focused on delineating the functional consequences of clinically relevant HUWE1 mutations which are observed in approximately 5% of MM patients. Patients witha HUWE1 mutation were observed to have significantly higher number of co-occurrent non-synonymous and somatic mutations. Analysis of a panel of MM cell lines with wild-type or mutant HUWE1, identified a similar phenotype in which HUWE1 mutant cell lines exhibited higher mutation rates as determined by the PIG-A gene mutation assay and increased incidence of micronuclei formation, two characteristics indicative of genomic instability. This loss of genome stability was determined to be underpinned by a dampened DNA repair response. This work outlines a clear role for HUWE1 in genome stability and highlights that exploitation of the replication stress response may represent a therapeutic vulnerability for MM patients with a HUWE1 mutation.