Mechanism of Action of a Novel Golgi-Targeted Anti-Cancer Agent PROJECT SUMMARY To cure cancer we need new, actionable targets for therapeutics that are orthogonal to current treatment strategies to enable new, combinatorial treatment regimens. The Golgi GOLPH3 pathway has emerged as an attractive target. Genes encoding GOLPH3 pathway proteins (GOLPH3, MYO18A, PITPNC1, and PI4KB) have all been identified as frequently amplified and over-expressed in common human cancers, acting to drive cancer (including lung, breast, colorectal, and prostate cancers). Furthermore, the function of the GOLPH3 pathway is unique among known cancer promoting genes in that the GOLPH3 pathway functions in vesicle exit from the Golgi for trafficking to the plasma membrane. Genetic interference with the GOLPH3 pathway (including depletion of PI4P, GOLPH3, MYO18A, or PI4KIII?) kills cancerous cells in culture and in vivo, while relatively sparing normal cells. Therefore, the GOLPH3 pathway is an attractive target for novel therapeutics. We have identified a small molecule inhibitor of the GOLPH3 pathway using a high-content, image-based, phenotypic, high-throughput screen. This compound acts on MYO18A to impair Golgi GOLPH3 pathway function and to preferentially kill cancerous cells. Here, we propose experiments to determine the mechanism of action of this small molecule by identifying the direct, mechanistic target. Thus, we will provide new insight into mechanisms of oncogenic transformation by the GOLPH3 pathway, regulation of the GOLPH3 pathway, and enable further medicinal chemistry for hit-to-lead optimization with the goal of developing a first in class therapeutic agent.