PROJECT SUMMARY The project addresses the problem of drug resistance in cancer which is arguably the most important problem facing patients with advanced cancer. While advances have been made in targeted therapy and immunotherapy, over 600,000 Americans will die in 2018 from cancer. Over the last two decades, we discovered TRAIL receptor DR5 and resistance mechanisms in cancer, identified drug synergies, and discovered small molecule ONC201 as a first-in-class TRAIL pathway inducer. Based on the novelty of ONC201, its emerging mechanism of action, the specific impact my lab can have on the field and on patients, this proposal will focus in depth on ONC201 preclinical mechanistic directions. ONC201 has progressed as a monotherapy into multiple clinical trials with various tumor types. Our studies are providing important basic information regarding the mechanism of action of ONC201 involving TRAIL induction after dual blockade of ERK and Akt converging on Foxo3a to activate TRAIL, and an integrated stress response that involves eIF2-alpha dependent ATF4/CHOP-mediated induction of TRAIL death receptor 5. ONC201 depletes colorectal cancer stem cells and with dose intensification in mice we observed anti-metastasis effects, inhibition of cell migration, and infiltration by NK and T cells into treated tumors (recently published by Wagner et al., J. Clin. Invest., 2018). Our data has led to a change in clinical dosing in all open clinical trials including at Fox Chase Cancer Center (NCT02609230). Our specific aims include: Aim #1: Investigate ONC201 effects on the tumor microenvironment through NK and T cells leading to anti-tumor and anti-metastasis effects. Aim #2: Investigate the role of the immediate binding target for ONC201, the sub-family of dopamine receptors DRD2/DRD3, in mediating its anti- tumor effects. We will explore novel connections between antagonism of the putative specific drug binding target dopamine receptor D2 and D3, the TRAIL and integrated stress pathway mechanism triggered by ONC201, their status in normal vs tumor cells, and sensitive vs resistant cells or tumors from patients exposed to ONC201. Our studies include in depth mechanism analysis of the immune stimulatory effects of ONC201, including analysis of immune infiltration by different immune cell subsets, various cytokines involved in attracting immune cells to tumors or those that may be potentially immune- suppressive, and use of TRAIL and DR5 knockout as well as NCR1-GFP mice with GFP(+) NK cells to analyze host tumor interactions of ONC201 (or ONC201 analogue) treated tumors. We explore ONC201 resistance mechanisms through molecular profiling of tumor specimens from ONC201 trials and critically assess their role in preclinical models.