ONC201 was initially identified as an inducer of cell death through the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. The compound is being tested in patients with a variety of tumor types, including those of the breast. Both triple negative breast cancer (TNBC) cells and non-TNBC cells are sensitive to ONC201. In a subset of TRAIL-sensitive TNBC cells, ONC201 treatment leads to the induction of apoptosis in a TRAIL-dependent manner. In the majority of TRAIL-resistant non-TNBC cells, ONC201’s effects are anti-proliferative rather than apoptotic. In vivo , the apoptotic response to ONC201 leads to efficacy of the compound while the anti-proliferative response does not (Ralff et al., Mol Cancer Ther ., 2017). Here we worked to identify strategies to convert the response of cancer cells to ONC201 from anti-proliferative to apoptotic. ONC201 treatment primed TRAIL-resistant non-TNBC cells to undergo TRAIL-dependent cell death. Increases in the mRNA and surface protein levels of death receptor 5 (DR5) as well as decreases in the expression of multiple anti-apoptotic proteins occured in ONC201-treated cells. We also observed small but significant increases in TRAIL mRNA and surface protein in non-TNBC cells following treatment with ONC201. Despite apoptotic priming and the induction of TRAIL, the effect of the compound in the non-TNBC cells remains anti-proliferative but not apoptotic. We hypothesized that the level of TRAIL induced by ONC201 in these cells, known to be TRAIL-resistant, was insufficient. Remarkably, the addition of exogenous recombinant human TRAIL (rhTRAIL) converted the response of TRAIL-resistant non-TNBC cells to ONC201 from anti-proliferative to apoptotic. Cleaved PARP, caspase-3, caspase-9, and caspase-8 were observed in cells treated with ONC201 followed by rhTRAIL, but not in cells treated with ONC201 or rhTRAIL alone. Propidium iodide staining and quantification of the subG 1 population via flow cytometry further confirmed these findings. Similar observations about the ability of rhTRAIL to convert the cellular response to ONC201 from anti-proliferative to apoptotic were made in cancer cells from other tumor types. Importantly, rhTRAIL did not convert the response of normal fibroblasts to ONC201 from anti-proliferative to apoptotic. Addition of a DR5 agonistic antibody to ONC201 treated non-TNBC cells also converted the response of the non-TNBC cells to ONC201 from anti-proliferative to apoptotic. Ongoing experiments are currently testing the combination of ONC201 and rhTRAIL in vivo . These findings describe a strategy for potently converting the response of a cancer cell to ONC201 from anti-proliferative to apoptotic. Clinical application of this combination therapy may result in tumor regressions in patients with decreased sensitivity to ONC201 as a single agent. Citation Format: Marie D. Ralff, Jocelyn E. Ray, Avital Lev, Lanlan Zhou, David T. Dicker, Wafik S. El-Deiry. Recombinant human TRAIL or a DR5 agonistic antibody convert the response of non-triple negative breast cancer cells to ONC201 from anti-proliferative to apoptotic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 258.