Proteasome Shuttle Factor and DNA Repair Protein Rad23A Is a Potential Therapeutic Target in Multiple Myeloma

Background and Rationale

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by clonal expansion in the bone marrow. MM cells exhibit extensive DNA damage, necessitating constant repair for survival and proliferation. Additionally, they rely on an overactive proteasome pathway to degrade the large amount of immunoglobulins they produce. Consequently, DNA-damaging chemotherapeutic drugs and proteasome inhibitors are standard treatments for MM. However, acquired resistance to these drugs is common. RAD23 Homolog A (Rad23A) is a multifunctional protein recognized for its involvement in the nucleotide excision repair (NER) and proteasome pathways. Here, we show that inhibition of Rad23A triggers potent anti-MM activity using both in vitro and in vivo models of MM.

Results

We first analyzed MM patient gene expression databases and found that Rad23A was highly expressed in MM patient compared to normal controls. The high expression of Rad23A was inversely correlated with patient overall survival (n=1614; p<1E−16). qPCR and immunoblotting confirmed that Rad23A levels were higher in MM patient cells and MM cell lines versus normal cells. CRISPR/Cas9-mediated knockout (KO) of Rad23A in MM.1R, MM.1S, and AMO1 cells, as well as shRNA knockdown (KD) in H929 and ANBL6-WT cells, resulted in decreased cell growth. Notably, decreased cell growth was also observed in cells resistant to the proteasome inhibitor bortezomib (ANBL6-BR). Western blot analysis revealed that Rad23A KO and KD cells had elevated levels of K48-linked polyubiquitylated proteins, indicating an inhibited proteasome system. Depletion of Rad23A activated the DNA damage pathway, evidenced by elevated gamma-H2AX signals in immunofluorescence staining and increased Rad51 foci formation observed under confocal microscopy. This led to elevated endoplasmic reticulum stress response signaling (BiP, p-eIF2α) and apoptosis (Cleaved-Caspase3, PARP, p21). Increased Caspase 3/7 activity was observed in Rad23A KO and KD cells using live-cell imaging system (IncuCyte), indicating caspase-related apoptosis activation. Flow cytometry showed that Rad23A depletion triggered cell cycle arrest, with cells accumulating in the G2/M phase. Proteomic analysis showed that Rad23A-KO cells downregulated 225 proteins and upregulated 642 proteins compared to WT cells. Upregulated proteins were involved in interferon-alpha response, apoptosis, and the p53 pathway, while downregulated proteins were associated with DNA repair, TNF-α signaling via NF-κB, and cell cycle checkpoints. Finaly, mice xenografted with Rad23A inducible-knockout MM1R cells exhibited reduced tumor growth (p<0.0001) and prolonged survival (p<0.0001) compared to those engrafted with WT-MM1R cells.

Conclusion

Our data demonstrate the therapeutic potential of the proteasome shuttle factor and DNA repair protein Rad23A, providing a preclinical basis for developing Rad23A inhibitors to treat MM.