Abstract 2664: Differential activation of the integrated stress response correlates with anti-tumor activity of imipridones ONC201 and ONC206 in pediatric sarcomas

Cure rates for pediatric sarcomas have minimally improved with intensification of genotoxic chemotherapy. We have previously shown that activation of the Integrated Stress Response (ISR), a cellular stress response that can lead to apoptosis, is a novel, effective means of selectively inducing the death of rhabdomyosarcoma (RMS) cells. However, context-specific wiring of homeostatic components such as protein chaperones dictates unique barriers to ISR activation. We hypothesized that just as HSP70 inhibition can preferentially activate the ISR in RMS, but not Ewing sarcoma, alternative small molecules might highlight dependencies unique to other sarcoma subtypes. ONC201 is a selective dopamine receptor (DRD2/3) antagonist that has previously shown antitumor activity through ISR activation in solid tumor models. ONC206 is a more potent analogue of ONC201 that was shown to be effective in Ewing sarcoma cell lines with sub-micromolar IC50. We used these molecules to test the hypothesis that DRD2/3 antagonism can activate the ISR in pediatric sarcomas, identifying a new therapeutic strategy. We measured the growth inhibitory effects of ONC201 and ONC206 across a panel of four rhabdomyosarcoma (RMS) and three Ewing sarcoma (ES) patient-derived cell lines. Across all sarcoma cell lines tested, ONC201 and ONC206 inhibited growth with low micromolar and sub-micromolar IC50 values respectively. ES cell lines were more sensitive than RMS lines, with mean ONC201 IC50 of 7.7 micromolar vs 3.2 micromolar (p = 0.0016). To explore this difference, we measured ISR activation by immunoblot. The ISR initiates with phosphorylation of eIF2α, leading to a halt in protein translation. RMS cells showed a strong increase in phosphorylated eIF2α one hour after treatment with either drug that was sustained through 24 hours, but attenuated by 48 hours of treatment. ES cells, however, did not show increased eIF2α phosphorylation until 48 hours of treatment, at which point levels of phosphorylation were much higher than in RMS cells. Correspondingly, PARP cleavage was relatively modest in RMS cells, but robust in ES cells at 48 hours of treatment. We conclude that two phenotypes of ISR activation – early and attenuated, versus late and sustained – correlate with the therapeutic efficacy of these agents. Our data suggest that regulation of the ISR downstream of DRD2/3 antagonism underlies differential response to two clinically relevant agents. The development of biomarkers of the stre