Targeting HUWE1 in Multiple Myeloma Leads to Impaired Replicative Stress Response and Enhanced Sensitivity to Bortezomib

Abstract

Introduction Despite continuous advances in therapies over the last few decades, Multiple Myeloma (MM) remains an incurable malignancy with almost all patients developing relapsed/refractory disease. This emphasizes the need for novel approaches that can improve the efficacy of current standard of care treatments. Previous work from our lab and others has identified dysregulation of the E3 ligase HUWE1 in MM and highlighted it's potential as a therapeutic target. Through its interaction with a diverse set of substrates, HUWE1 is implicated in many key cellular processes including stress responses and DNA replication and repair pathways and is being increasingly recognised as a major regulator of MYC activity. In this study, we uncover a novel role for HUWE1 in DNA replication and repair and assess the combination of HUWE1 and proteasome inhibition as a potential therapeutic strategy in MM. Methods MM cell lines were transfected with SMARTvector Inducible Human HUWE1 shRNA or a non-targeting control (NTC) shRNA (Dharmacon, USA). Replicative stress was induced by treatment with 2 mM Hydroxyurea (HU) and assessed using immunofluorescence staining and iPOND (isolation of proteins on nascent DNA) analysis. Viability was assessed using CellTiter® Glo and combination indices (CI) calculated using CompuSyn software. Commercially available HUWE1 inhibitor BI8622 (MedChemExpress) and novel HUWE1 inhibitors synthesised in house were used throughout the study. Results HUWE1 knockdown and/or inhibition in MM cell lines led to an accumulation of cells in S phase, consistent with previous studies indicating that HUWE1 is required for effective DNA replication. Using proteomic profiling and co-immunoprecipitation, we identified and validated Replication Protein A, 70 kDa (RPA70) as a novel substrate of HUWE1. RPA70 is a subunit of the heterotrimeric RPA complex which plays a critical role in the recruitment of DNA repair proteins. RPA70 is the first subunit to translocate to sites of DNA damage where it binds to DNA and recruits the other subunits (RPA32 and RPA14) forming the complex, thus allowing for initiation of DNA repair. We observed significantly less K63-linked ubiquitination of RPA70 (p=0.00213) in the absence of HUWE1, with an associated decrease in both phosphorylation (p=0.0064) and localisation of RPA subunit RPA32 to DNA following treatment with hydroxyurea (HU) to induce replicative stress. Subsequent iPOND analysis determined that this is associated with reduced recruitment of repair proteins, including RAD51, to DNA. Moreover, induced replicative stress in HUWE1 depleted/inhibited cells resulted in significantly higher levels of DNA damage at 6hrs (p=0.00421) and 24hrs (p=0.0219) post HU treatment. A recent study demonstrated that HUWE1 ubiquitination of MYC promotes the formation of MYC multimers that protect stalled replication forks to limit the formation of double strand breaks (DSB) (Nature 2022;612:148-155). In line with this we also observe decreased ubiquitination of MYC in the HUWE1 knockdown cell line compared to the NTC following HU treatment. Finally, we assessed the efficacy of combining HUWE1 inhibitors with bortezomib which is known to impair DSB repair. Dual inhibition of HUWE1 and proteasome activity resulted in synergistic effects (CI values < 1) and a corresponding increase in DNA damage compared to HUWE1 inhibitor or bortezomib alone as measured by levels of ƴH2AX. Conclusion We have identified RPA as a novel substrate for HUWE1 and demonstrate that targeting HUWE1 results in increased replication stress and a dampened DNA repair capacity, underpinned by reduced recruitment of repair proteins. This work outlines a clear role for HUWE1 in genome stability in MM cells and highlights that HUWE1 inhibitors represent a novel anti-myeloma strategy that acts in synergy with bortezomib to exacerbate DNA damage.