PIKfyve inhibition in myeloma disrupts autophagy and the lysosome, increasing MHC expression and cholesterol metabolism Open Access

• PIK001 is a novel small-molecule inhibitor of PIKfyve with potent in vitro and ex vivo anti-MM activity.

• Autophagy disruption via PIKfyve inhibition upregulates MHC Class I in MM, with potential implications in tumor immunity.

We previously reported a chemo-genomics screen that unexpectedly identified Phosphatidylinositol-3-phosphate 5 kinase (PIKfyve) as a vulnerable target in multiple myeloma (MM). PIKfyve is an essential regulator of lysosomal function and autophagy. Given the high basal necessity of autophagy in MM for sustainable immunoglobulin synthesis, targeting autophagy holds clinical potential as a novel therapeutic avenue. Here, we report the development and characterization of PIK001 and analogues, potent and selective novel small-molecule inhibitors of PIKfyve. PIK001 demonstrated potent anti-MM activity in vitro, as well as synergistic activity with established anti-MM agents (including venetoclax and selinexor), while retaining efficacy in lenalidomide-resistant models. Multi-omic characterization of isogenic cell lines sensitive / resistant to PIK001 identified a catalytic domain mutation (PIKFYVEN1939K) and heterogenous alterations in autophagy capabilities. Importantly, we noted that PIK001 exposure also resulted in significantly increased cholesterol metabolism and upregulation of MHC Class I expression, with potential implications in tumor immunity. Beyond MM, PIKfyve inhibition also shows selective cytotoxicity in acute myeloid leukemia, melanoma, and renal cancer, highlighting broader therapeutic potential. These findings establish PIKfyve inhibition as a valid target for MM and other hematologic malignancies, provide insights into mechanisms of sensitivity and resistance, and a compelling foundation for further pre-clinical (particularly with respect to the role of cholesterol metabolism and tumor immunity) and clinical development.