Genetic and Pharmacological Screens Converge in Identifying FLIP, BCL2, and IAP Proteins as Key Regulators of Sensitivity to the TRAIL-Inducing Anticancer Agent ONC201/TIC10

Abstract

ONC201/TIC10 is a small-molecule inducer of the TRAIL gene under current investigation as a novel anticancer agent. In this study, we identify critical molecular determinants of ONC201 sensitivity offering potential utility as pharmacodynamic or predictive response markers. By screening a library of kinase siRNAs in combination with a subcytotoxic dose of ONC201, we identified several kinases that ablated tumor cell sensitivity, including the MAPK pathway-inducer KSR1. Unexpectedly, KSR1 silencing did not affect MAPK signaling in the presence or absence of ONC201, but instead reduced expression of the antiapoptotic proteins FLIP, Mcl-1, Bcl-2, cIAP1, cIAP2, and survivin. In parallel to this work, we also conducted a synergy screen in which ONC201 was combined with approved small-molecule anticancer drugs. In multiple cancer cell populations, ONC201 synergized with diverse drug classes, including the multikinase inhibitor sorafenib. Notably, combining ONC201 and sorafenib led to synergistic induction of TRAIL and its receptor DR5 along with a potent induction of cell death. In a mouse xenograft model of hepatocellular carcinoma, we demonstrated that ONC201 and sorafenib cooperatively and safely triggered tumor regressions. Overall, our results established a set of determinants for ONC201 sensitivity that may predict therapeutic response, particularly in settings of sorafenib cotreatment to enhance anticancer responses.

Figure 1. siRNA screen identifies kinase regulators of ONC201 sensitivity

A) Decrease in cell viability in HCT116 cells associated with the siRNA alone (y-axis) or the difference in observed and predicted activity in cell viability associated with the combination of ONC201 treatment (1 µM) and knockdown by siRNA (x-axis). (B) Cell viability in HCT116 cells following ONC201 treatment (1 µM) and/or siRNA knockdown at 24 or 48 hours post-treatment (n=3). Quantification (top panel) and raw data (bottom panels) are shown. *P < 0.05 compared to 48 hours post-ONC201 treatment and control siRNA by Student’s two-tailed t test. (C) Network analysis of ONC201 kinase regulators (blue) and putative mechanism of action (green). (D) Western blot analysis of HCT116 cells treated with DMSO or ONC201 (5 µM) with or without siRNA-mediated knockdown of KSR1 (60 hours). (E) Sub-G1 DNA content analysis following treatment with ONC201 (5 µM) or sorafenib at indicated concentrations (72 hours, n=3). * P< 0.05 by student’s two-tailed t test.

Figure 2. Identification of synergistic combinations of FDA-approved small molecule anti-cancer drugs with ONC201

A) Reduction in cell viability in response to ONC201 (1 µM), approved agents, or the combination (72 hours, n=3). The difference between the observed activity and the sum of the monoagent activities are shown in blue. Only combinations yielding >20% observed activity over the predicted activity are shown. (B) Prioritization of combinatorial data by efficacy and synergistic activity (n=2). The prioritization criterion was defined as the lower right shaded quadrant. Data for the prioritized monoagent and combinations are shown in (C). Each set of 4 bars represents a distinct synergistic data point under a given combination of drug concentrations, along with its associated monoagent and vehicle controls. The data sets are sorted with increasing combinatorial efficacy from left to right.

Figure 3. ONC201 synergizes with sorafenib in HepG2 human HCC cells

A) Cell viability of hepatocellular and renal cell carcinoma cell lines (72 hours, n=3). (B) Sub-G1 DNA content analysis in HepG2 cells following treatment with ONC201 (5 µM) or sorafenib at indicated concentrations (72 hours, n=3). * P< 0.05 by student’s two-tailed t test. (C) DAPI staining of HepG2 cells treated with ONC201 (5 µM), sorafenib (40 µM), or the combination (72 hours). Apoptotic nuclei are indicated by yellow triangles. (D) Waterfall plot showing tumor volume in HepG2 xenografts at 14 days following treatment initiation relative to tumor size prior to treatment initiation. Treatments were ONC201 (25 mg/kg PO on day 1 and 7) and sorafenib (40 mg/kg PO on days 1–5, 8–12) (n≥8). (E) Number of mice with complete tumor regressions (n=10).

Figure 4. Sorafenib and KSR1 regulate FLIP, IAP, and Bcl-2 proteins

A) Surface TRAIL and surface DR5 by flow cytometry following treatment with ONC201 (72 hours, 10 µM, n =3). *P< 0.05 by student’s two-tailed t test compared to all other conditions. (B) Western blot analysis of HepG2 cells treated with ONC201 (5 µM), sorafenib (40 µM), or the combination (72 hours). (C) Western blot analysis of HepG2 or HCT116 cell treated with sorafenib (40 µM) for 12 hours. (D) Western blot analysis of HCT116 cells with or with siRNA-mediated knockdown of KSR1. (E) Effect of FLIP, Mcl-1, or KSR1 siRNA-mediated knockdown on sensitivity to ONC201 in HCT116 and cells (72 hours, n=3). *P < 0.05 compared to control siRNA under the same treatment conditions by Student’s two-tailed t test. Confirmation of knockdown by Western blot is shown in the left panel.