Validation of a small molecule inhibitor of PDE6D-RAS interaction with potent anti-leukemic effects

RAS mutations prevalent in high-risk leukemia have been linked to relapse and chemotherapy resistance. Efforts to directly target RAS proteins have been largely unsuccessful. However, since RAS-mediated transformation is dependent on signaling through the RAS-related C3 botulinum toxin substrate (RAC) small GTPase, we hypothesized that targeting RAC may be an effective therapeutic approach in RAS mutated tumors. Here we describe multiple small molecules capable of inhibiting RAC activation in acute lymphoblastic leukemia cell lines. One of these, DW0254, also demonstrates promising anti-leukemic activity in RAS-mutated cells. Using chemical proteomics and biophysical methods, we identified the hydrophobic pocket of phosphodiester 6 subunit delta (PDE6D), a known RAS chaperone, as a target for this compound. Inhibition of RAS localization to the plasma membrane upon DW0254 treatment is associated with RAC inhibition through a phosphatidylinositol-3-kinase/AKT-dependent mechanism. Our findings provide new insights on the importance of PDE6D-mediated transport for RAS-dependent RAC activation and leukemic cell survival. ### Competing Interest Statement Dr. Williams has been funded by the NIH. He is or was recently a member on a Board of Directors or advisory committees for: Bluebird bio, Orchard Therapeutics, Novartis, Beam Therapeutics, Emerging Therapy Solutions, Geneception, and BioMarin. Additionally, he is the Co-founder of Alerion Biosciences and Orchard Therapeutics. Dr. Anighoro, Dr. Autelitano, Dr. Beaumont, Dr. Klingbeil and Dr. Ermann declare present or past employment by Evotec while engaged in the research project. Dr. De Vita declares present employment by Novartis Institute for Biomedical Research. The remaining authors declare no conflict of interest.