Abstract 371: Co-existent <i>PIK3CA</i> and <i>ARID1A</i> mutations lead to enhanced sensitivity to PI3K inhibition through PUMA induction by FOXO3a and p53/p21

Abstract Background: PIK3CA (PK) mutations occur in ∼18% of colorectal cancer (CRC) cases. However, an effective means to target these cancers in clinic remains a challenge. Co-alterations in PIK3CA and ARID1A (AD) occur in ∼1% of all cancer cases. The Z1F arm of the NCI MATCH clinical trial demonstrated an improved response rate for the pan-PI3K inhibitor, copanlisib (cop) in subjects with PK and AD mutant cancers. Recent studies have shown that cop treatment leads to p53 independent induction of PUMA by FOXO3a in CRC cell lines. Furthermore, ARID1A is suspected to regulate the levels of p53/p21 and c-MYC, which are important for the induction of apoptosis. Here we further evaluate this potential mechanism of action. Methods: AD was knocked out from human 2D CRC cells SW48 and SW48PIK3CA-H1047R (SW48PK) using CRISPR as previously described (SW48AD; SW48PKAD). Flow cytometry (FC) was performed for apoptosis (Annexin V and PI) after 48 hours of cop treatment at 10nM and 50nM (PI3K and MTOR IC50 respectively). Western blot (WB) was performed after cop treatment for 6 hours and 24 hours to determine the protein levels of the anti-apoptotic protein MCL-1, pro-apoptotic protein PUMA, and proteins that activate PUMA (FOXO3a, p53, p21 and c-MYC). Results: An increase in apoptosis was observed in SW48PKAD cells compared to SW48PK with 50nM cop treatments for 48 hours (p=0.02). MCL-1 protein expression at baseline was lower in SW48PKAD compared to SW48PK cells. This also decreased significantly by 6 hours with cop (p&amp;lt;0.05). PUMA expression was significantly increased in SW48PKAD after 6 hours cop 10nM (p&amp;lt;0.05) and 50nM (p&amp;lt;0.005) compared to SW48PK, where there was a delayed increase in PUMA levels by 24 hours (50nM; p&amp;lt;0.005). FOXO3a was significantly increased in SW48PKAD at 6 hours (10 and 50nM; p&amp;lt;0.005), indicating the potential for the induction of PUMA to be secondary to FOXO3a. Among FOXO3a targets, c-MYC showed no difference in its expression but there was higher expression of p53 in SW48PKAD compared to SW48PK without cop which significantly decreased after 24 hours of cop at 50nM (p&amp;lt;0.005). p21, which is present downstream of p53 and helps induce apoptosis in both p53-dependent and independent manners, was also highly expressed without cop and decreased after 24 hours in SW48PKAD compared to SW48PK (p&amp;lt;0.005). Conclusions: Co-alterations in PIK3CA and ARID1A lead to enhanced sensitivity to PI3K inhibition through PUMA induction by FOXO3a due to aberrant expression of p53/p21 caused by ARID1A loss. Further validation studies are underway utilizing PK and AD altered patient-derived cancer organoids. Citation Format: Shirsa Udgata, Jordan N. Stoecker, Xingqi Shen, Cheri A. Pasch, Dustin A. Deming. Co-existent PIK3CA and ARID1A mutations lead to enhanced sensitivity to PI3K inhibition through PUMA induction by FOXO3a and p53/p21 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 371.