Imipridone (Onc201) And Radiation Therapy Combination Shows Promise In Breast Cancer Treatment

Imipridone (IMIP) is an orally active DRD2 small molecule antagonist which has shown efficacy in multiple preclinical and early phase clinical studies in advanced cancers. IMIPs work by inducing caspase-dependent apoptosis that involves activation of the integrated stress response (ATF4/CHOP) pathway, up-regulation of TRAIL, and activation of death receptors 4 and 5 (DR4/DR5). Radiation (RT) requires the DR5 pathway to induce apoptosis, leading to the hypothesis that IMIP may produce a synergistic effect when combined with RT in breast cancer. In vitro experiments were done utilizing 7 breast cancer cell lines. We treated human cancer cells (2-10 x 103 per well) in 6-8 replicates with radiation alone, IMIP alone, or the combination. RT (0-8 Gy) was delivered via a Gamma-cell 40 exactor and IMIP was given at varying doses 0-10 μM. Alamar Blue cell viability assay was performed after treatment. Cell lines found to respond to either therapy were used in clonogenic assays to test for synergy. We assessed apoptotic pathways using Western blot analysis of levels of cleaved PARP (cPARP) and cleaved caspase 3 (cC3). In vivo studies utilizing the 4T1 breast cancer mouse models compared outcomes of RT alone, IMIP alone, and the combination. Mice were sacrificed at a pre-specified endpoint and lung metastases were counted in this metastatic model. Finally the role of specific immune cells was assessed by using depletion antibodies prior to administration of combination therapy. Multiple cell lines showed decreased cell viability after combination therapy. When IMIP (1 uM) was given 12 hours prior to radiation (8 Gy), there was a significant decrease in MB468 cell viability compared to no treatment (36.9%, P<0.0001), RT alone (30.5%, p<0.0001) and IMIP alone (26.9%, p<0.0001). No significant decrease was achieved with either treatment alone compared to no treatment. Clonogenic assay using MB468 revealed a reduction in the surviving fraction at every RT dose (2, 4, 6, and 8 Gy) in the presence of IMIP. Western blot analysis showed increasing cPARP with increasing IMIP at 2 Gy and increasing cC3 with increasing doses of IMIP at 4 and 8 Gy. In vivo studies utilizing 4T1 in Balb/c mice demonstrated that combination RT (6 Gy) and IMIP (48 mg/kg given 3x weekly) decreased tumor growth and prolonged survival compared to either treatment alone. Combination therapy also led to decreased lung metastases. In vivo immune cell depletion revealed that CD8 cells were necessary for the improved effects of combination therapy. Adding IMIPs to radiation exhibited a significant synergistic apoptotic effect on breast cancer cells in vitro, which was translated into slower tumor growth, decreased metastases, and improved survival in the in vivo mouse breast cancer model. This synergistic effect is CD8 T cell-mediated. These data support the use of this combination therapy in early phase clinical trials.