MTORC1/2 inhibition enhances STX-478, a PIK3CA H1047R mutant inhibitor, in PIK3CA H1047R mutant colorectal cancer

Background: STX-478, a PIK3CA mutant selective inhibitor, that we have previously shown to have promise in treating PIK3CA H1047R mutant colorectal cancer (CRC) models. A high throughput drug screen was performed and identified AZD2014, an MTORC1/2 inhibitor, to have enhanced response with STX-478. Here, we examine the combination of PI3K inhibition with MTORC1/2 inhibition in PIK3CA H1047R mutant colorectal cancer (CRC). Methods: CRC cell lines SW48 and SW48PK (Horizon Discovery; SW48 with a PIK3CA H1047R mutation) were treated with varying concentrations of STX-478 (250-750 nM) and AZD2014 (20-250 nM) and viability measured by WST was performed. PIK3CA H1047R mutant PDCOs were treated, imaged, and response was determined by measuring the change in longest diameter of the organoids over time. Glass's Delta was used as a statistical test to measure the effect size of the different treatment groups. Mouse derived cancer organoids (MDCOs) were derived from Fc 1 Apc fl/+ Pik3ca H1047R murine colon tumors. MDCOs/PDCOs and CRC cell lines were treated with 750 nM or 500 nM STX-478 and 100 nM AZD2014, respectively, and collected for immunoblotting for apoptosis and PI3K proteins. Results: Both SW48 and SW48PK had a significant reduction in cell viability with treatment of single agent AZD2014 at 100 nM (SW48 p<0.001, SW48PK p=0.005). When treated with combination of STX-478 and AZD2014, both cell lines had a significant reduction in cell viability (500 nM STX-478 with 100 nM AZD2014; SW48 p=0.002, SW48PK p<0.001). For SW48PK, a significant reduction of phosphorylation of ribosomal protein S6 (RPS6; p<0.001), AKT (p<0.001), and 4EBP1 (p=0.002) were observed after treatment with the combination, while no significant difference was seen in SW48. MDCOs had a significant decrease in RPS6 compared to control and single agent STX-478 (p<0.001). In PIK3CA H1047R mutant PDCOs, a significant reduction in relative change in diameter was observed in the combination treatment compared to control (relative change in diameter control versus combination: 30.2% vs -0.96%; Glass's Delta = 1.67). PIK3CA H1047R mutant PDCOs also had a significant decrease in phosphorylation of 4EBP1 (p<0.001) and RPS6 (p<0.001). Conclusions: The combination of STX-478 and AZD2014 enhanced response in both 2D and 3D PIK3CA H1047R mutant CRC models and shows promise for combining MTORC1/2 with PIK3CA inhibition for PIK3CA mutant CRC. Future work should investigate STX-478 and MTORC1/2 inhibition in PIK3CA mutant CRC in vivo .