P-217: ULK3-mediated autophagy is a tractable therapeutic target for the treatment of multiple myeloma.

Introduction: Multiple myeloma (MM) remains incurable despite advances in therapy. Our group is the first to demonstrate that ULK3-mediated autophagy in MM is a key program that sustains cell survival upon treatment. MM is characterized by high basal levels of autophagy and, currently, specific ULK3 inhibitors are lacking. Here we have generated novel multiple kinase inhibitors that can block ULK3 activity and demonstrate their efficacy in vitro, in vivo and in ex vivo human specimens.

Methods: We performed RNASeq analysis of CD138+ MM patient cells collected at the Moffitt Cancer Center (n=815) and demonstrated ULK3 is highly expressed across the disease spectrum. We also show it is crucial for MM progression using CRISPR and in vivo preclinical models (5TGM1Luc, U266Luc).
We characterized novel small molecule inhibitors SG3014/MA9060 that target multiple kinases including ULK3 with nanomolar potency and demonstrate their impact on MM cell viability. We also use a novel ex vivo platform developed at Moffitt to demonstrate the efficacy of these reagents and their effect on ULK3 mediated autophagy.

Results: ULK3 protein levels correlate with MM patients’ progression stages. In fact, refractory patients have increased autophagy activity and significantly higher expression of ULK3. Genetic ablation of ULK3 by CRISPR guides in U266/ 8226/ MM1.S cell lines results in rapid cessation of the downstream autophagy mediators (ULK1, ATG13, pATG13). Importantly, using a vesicle labelling tool (CytoID), we demonstrated shutdown of autophagy in ULK3 knockout cells. We also show that MA9060/SG3014 decrease ULK3 levels and autophagy as measured by immunoblot and CytoID. Using an in vivo model of MM progression in the skeleton (U266) we observed that MA9060 reduced tumor burden, protected against myeloma induced bone disease and significantly extended overall survival (CTRL untreated n=65 days vs MA9060 n=110 days). Importantly, we noted no overt toxicity and protected effect against myeloma-induced bone disease. Ex vivo, we demonstrated the efficacy of MA9060 for the treatment of CD138+ MM derived from newly diagnosed and refractory patients both as a single agent and in combination with standard of care therapies such as the proteasome inhibitor, carfilzomib.

Conclusions: ULK3 is a key regulator of autophagy in multiple myeloma and its genetic or pharmacological inhibition significantly limits MM viability making it an attractive therapeutic target for the treatment of the disease.