ONC201 Overcomes Chemotherapy Resistance By Upregulation of Bim in Multiple Myeloma

In multiple myeloma, disease relapse and drug resistance occurs in the majority of myeloma patients after standard treatment despite recent improvements offered by new therapies. Therefore, there is an urgent need for new drugs that can overcome drug resistance and prolong patient survival after failure of standard therapies. ONC201, the founding member of a novel class of anti-tumor agents called impridones, has selective preclinical efficacy against a variety of tumor types. It is currently in phase I and phase II clinical trials for patients with advanced solid tumors and hematological malignancies. Given the pronounced sensitivity of B-cell lymphomas to ONC201, we assessed the efficacy of ONC201 in preclinical models of multiple myeloma. We treated human myeloma cell lines and primary myeloma cells isolated from bone marrow aspirates of myeloma patients with ONC201 for 72 hours. CellTiter-Glo Luminescent and annexin-V binding assays for assessing myeloma cell viability and apoptosis were performed, along with immunoblotting for cleavage of caspases, phosphorylation of signaling kinases, and expression of pro- or anti-apoptotic proteins. ONC201 treatment decreased myeloma cell viability, with IC50 values that were 1 μM to 1.5 μM, even in high risk myeloma cell line RPMI8226. The status of TP53 did not appear to affect the efficacy of ONC201, as MM.1S or NCI-H929 cells with wild-type TP53 and OPM-2 or RPMI8226 with mutated TP53 had a similar sensitivity towards ONC201. These results agree with prior reports in other tumor types that have demonstrated that the efficacy of ONC201 is independent of TP53. Western blot analysis showed increased apoptosis, cleavage of caspase-9, caspase-3, and PARP.

We also found that ONC201 induced expression of the pro-apoptotic protein Bim in myeloma cells, which can occur downstream of ERK inactivation. Knockdown of Bim expression in myeloma cells by shRNAs abrogated ONC201-induced apoptosis. Phosphorylation of Bim at Ser69 by Erk1/2 has been shown to promote proteasomal degradation of Bim. In accordance with this mechanism, we observed that ONC201 treatment reduced levels of phosphorylated Erk1/2, an indicator of Erk1/2 kinase activity, and Bim pSer69. In addition, ONC201 induced apoptosis in dexamethasome-, bortezomib-, and carfilzomib-resistant myeloma cell lines with the same efficacy as in wild-type cells.

As a rational strategy to increase the efficacy of ONC201 by enhancing its inhibition of proteasome-mediated Bim degradation, we tested combinations of ONC201 with proteasome inhibitors bortezomib or carfilzomib. These combinations were synergistic in reducing cell viability and enhancing Bim expression and PARP cleavage in myeloma cells. Overall, these findings demonstrate that ONC201 inhibits the Erk1/2 signaling pathway and induces Bim expression to induce apoptosis in multiple myeloma regardless of resistance to standard-of-care therapies. Our studies suggest that ONC201 should be evaluated clinically in relapsed/refractory multiple myeloma.