ONC201 synergistically induces cell death in triple negative breast cancer with CDK 4/6 and MEK inhibition

Background: Triple negative breast cancer (TNBC) has a relative paucity of effective targeted therapies compared to other molecular subtypes, and with poor prognosis. Thus, new targeted therapeutics are needed. ONC201 is a first-in-class small molecule that induces caspase-mediated apoptosis in cancers. It recently showed efficacy in pediatric glioma, harboring the potential to be translated in advanced breast cancer. Here we evaluated the efficacy of ONC201 in TNBC cell lines, investigated proteomic/genomic markers that predict efficacy, and performed a synthetic lethal RNAi screening to identify rational combinational targets. Method: The CellTiter-Blue (CTB) cell viability or sulforhodamine B assay was used to measure the range of half-maximal inhibitory concentrations (IC50) of ONC201 in TNBC cell lines. 2D/3D high-throughput RNAi kinome screening was performed using ONC201-sensitive CAL51, resistant HCC70 cells. The top genes noted to augment ONC201 sensitivity by siRNA were analyzed using both protein-protein interactome analysis (STRING V.11) and Ingenuity Pathway Analysis (IPA) to identify contributing canonical pathways. Combinational anti-proliferative activity of ONC201 and target inhibitors were evaluated by CTB cell viability assay and quantified by CalcuSyn software. Identified potential partners were subsequently tested using 3D ex vivo spheroid assay, tumor xenograft animal model to measure tumor growth inhibition (TGI). Five NOD/SCID mice were treated per each group. Mice were treated with 50mg/kg twice daily dosing of ONC201, and/or 25mg /kg of palbociclib after tumor grew to 100mm3. Reverse phase protein array (RPPA) of both ONC201 treated and non-treated TNBC cell lysates was performed to identify pathways affecting the sensitivity to ONC201 by comparing the effect of protein level changes (individual and set) using 3-way ANOVA, R program. Results: Twenty TNBC cell lines exhibited varying IC50 values (2 μM to 40 μM). No correlation between Vanderbilt subtypes and IC50 was observed. RNAi kinome screening identified 65 overlapping target kinases. Pathway analyses revealed PI3K/Akt, MEK/ERK, CDK 2/4/6 as potential combination therapeutic partners. In the in vitro study, the MEK inhibitor trametinib and CDK 4/6 inhibitor palbociclib consistently showed synergistic TGI with ONC201 (combination index values [CI] = 0.1 - 0.7). AKT inhibitor MK-2206 showed moderate combinational TGI (CI = 0.5 - 0.9). PI3K inhibitors PF04691052, buparlisib, and dactolisib showed cell type-specific combinational TGI. RPPA analysis did not reveal direct apoptosis-regulators mediators of sensitivity to ONC201, yet identified 7 potential predictive markers (fibronectin, pHER2, PAR, PLK1, pRb, SOD2, and EMA) of resistance/sensitivity. It also provided the rationale to study CDK 4/6 inhibition as a combination partner. Ex vivo study of ONC201/palbociclib (CDK 4/6 inhibitor) treatment inhibited of the colony formation in CAL51 cells. Interestingly, ONC201/palbociclib did not induce synergy in HCC70 cells, while the ONC-201/trametinib pair was synergistic in both cell lines. CAL51 tumor xenograft in vivo study confirmed the synergy of ONC201/palbociclib. Conclusion: MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib demonstrated synergy with ONC201 yet palbociclib only in ONC201 sensitive CAL51. Detailed mechanisms of synergy are being investigated that can further guide clinical translation. Citation Format: Bora Lim, Christine Peterson, Elin Cho, Alexander Davis, Troy Pearson, Huey Liu, Minha Hwang, Debu Tripathy, Naoto T Ueno, Jangsoon Lee. ONC201 synergistically induces cell death in triple negative breast cancer with CDK 4/6 and MEK inhibition [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-14.