P-201: Induction of pyroptosis for the treatment of multiple myeloma

Introduction: Pyroptosis is a novel type of cell death and induction of pyroptosis by small molecule chemicals has been proven to be a promising strategy for the treatment of hematologic malignancies, including acute myeloid leukemia (Johnson DC et al., Nat Med, 2018). However, little is known about myeloma cell pyroptosis. We recently found that proteasome inhibitors, the most important class of the mainstay anti-myeloma agents, trigger pyroptosis in a BAX and GSDME-dependent manner (Liang J et al., Acta Pharmacol Sin, 2023). Gasdermins are the executor of pyroptosis but the detailed regulatory mechanism is not well known. Novel agents to induce MM cell pyroptosis remained discovered.

Methods: We applied phase contrast microscopy and ELISA to evaluate myeloma cell pyroptosis; RNA dequencing was performed to find out associated genes involved in clioquinol (CLQ)-induced MM cell pyroptosis. Western blotting was performed to examine GSDME activation and associated protein changes. Immunoprecipitation/immunoblotting was performed to examine the interaction between BAX, GSDME and IFIT1/T3. MM-cell line-derived myeloma xenografts were established by injection s.c MM cel lines into nude mice.

Results: In the present study, we found that Clioquinol (CLQ), an anti-parasitic drug that has been proven to leukemia and myeloma cell apoptosis and autophagy, could also induce myeloma cell pyroptosis, featured with appearance of balloon-like morphology, leakage of lactate dehydrogenase (LDH) and cytochrome C, in both MM cell lines and primary cells. Moreover, CLQ induces MM cell pyroptosis through Caspase-3-mediated cleavage of GSDME. To find out the underlying mechanism, we performed an RNA sequencing on CLQ-induced pyroptotic cells. The results showed that interferon-inducible genes including IFIT1 and IFIT3 were strikingly upregulated by CLQ. IFIT1/T3 was downregulated in both primary and MM cell lines and they could be induced by CLQ. However, neither IFIT1 nor IFIT3 could induce MM cell pyroptosis. In contrast, both IFIT1 and IFIT3 could significantly enhance GSDME activation induced by CLQ. We further found that IFIT1/T3 bound to GSDME and increased the mitochondrial distribution of N-GSDME. Furthermore, we found that BAX bound to N-GSDME that could be markedly increased by IFIT1/T3 and IFIT1/T3 facilitated the translocalization of N-GSDME to mitochondria in the presence of BAX. Lastly, we found that CLQ displayed synergistic effects in myeloma cell pyroptosis with venetoclax, a specific inhibitor of Bcl-2 and an activator of BAX. The combination of CLQ and venetoclax displayed potent anti-MM activity in vivo in association with the induction of IFIT1/T3 and MM cell pyroptosis.

Conclusions: This study therefore not only illustrates IFIT1/T3 is a important factor to enhance CLQ-induced myeloma cell pyroptosis. We also show the potential application of CLQ/VEN in myeloma therapy.