Inhibition of Endoplasmic Reticulum Associated Degradation Is Cytotoxic to Relapsed Refractory Multiple Myeloma through Altered Proapoptotic Signaling

Multiple myeloma (MM), a plasma cell neoplasm, is the second most common hematologic malignancy with a median overall survival of 6.5-10 years. In MM, malignant plasma cells secrete excess immunoglobulins, which are associated with renal failure, low blood counts and bone lesions. Due to their high level of protein synthesis and trafficking, MM cells are highly reliant on endoplasmic reticulum (ER) stress response pathways, including ER associated degradation pathway (ERAD). ERAD is a multiprotein complex that degrades misfolded proteins from the ER as well as regulates secreted and cell membrane associated proteins. Inhibiting ERAD in MM leads to activation of the unfolded protein response (UPR) and induction of apoptosis. Proteasome inhibitors (PIs) disrupt ERAD activity by inhibiting ubiquitin-proteasome degradation of ERAD substrates and are part of the current standard of care for frontline therapy for MM. However, almost all patients eventually develop resistance to PIs resulting in relapsed/refractory disease. While prior studies have shown that targeting alternative proteins in the ERAD pathway is an effective therapeutic strategy to overcome PI resistance, the identification of small molecule inhibitors of ERAD has been limited with traditional screening approaches or chemical modulation that is not specific to the ERAD pathway. To address these limitations, we developed a cell-based screen using an inducible ERAD substrate, null Hong Kong alpha-1 antitrypsin (NHK), to identify inhibitors of ERAD substrate degradation.

We screened over 2200 compounds from the FDA repurposing library and identified one inhibitor, omaveloxolone (RTA408), that prevents ERAD-mediated degradation of luminal and membrane substrates. RTA408 was originally described as a E3 ligase inhibitor, which is approved to treat Friedreich’s Ataxia. When applied in eleven multiple myeloma cell lines, we observe cytotoxicity with an average half maximal inhibition (IC₅₀) of approximately 270 nanomolar. MM cytotoxicity is additive with lenalidomide or dexamethasone. RTA408 is cytotoxic at nanomolar concentrations in primary malignant plasma cells derived from patients with de novo or PI refractory disease, whereas non-malignant CD3 (T cells) or CD11b (myeloid cells) are spared. We have applied our inhibitors in a xenograft transplant model with MM.1s cells, which have been shown to mimic relapsed refractory disease.

Treatment with RTA408 prevents degradation of the endogenous ERAD substrate lambda light chain immunoglobulin and activates the UPR within 30 minutes in MM cells, a faster onset than PIs. In addition, while PI treatment leads to stabilization of c-MYC in MM cells, the activation of the UPR by RTA408 is associated with the degradation of c-MYC, suggesting differential regulation of cytosolic proteins. RTA408 cytotoxicity is mediated by altered cell death receptor signaling leading to caspase 8 activation in the extrinsic apoptotic pathway. Inhibition of cell death receptor complex assembly rescues the cytotoxicity mediated by RTA408, showing that alternative targeting of ERAD can modulate MM cell surface receptor trafficking and activity.

These studies identify a novel inhibitor for ERAD substrate degradation, which may represent an alternative therapeutic approach in relapsed refractory MM. RTA408 is efficacious in primary malignant plasma cells from patients with PI resistance and represents an alternative mechanism to target ER protein degradation with unique effects on proapoptotic signaling and degradation of cytosolic proteins. Further studies are planned for ERAD target protein identification, evaluate ERAD substrate regulation of proapoptotic signaling, and test the application of this inhibitor with combinatorial MM therapeutic approaches.