Abstract A014: Oncogenic <i>PIK3CA</i> on therapeutic resistance and cellular heterogeneity in malignant vascular cancer
Authors: Donghee Lee, Emma Kozurek, Md Abdullah, Rong Li, Joanne Kim, Erin B. Dickerson, Jong Hyuk Kim
Journal: Cancer Research (2024)

Abstract Angiosarcoma is an extremely rare and aggressive cancer that originates from endothelial cells lining blood or lymphatic vessels. The PI3K-AKT pathway plays a crucial role in angiogenesis and pathogenesis of this vascular cancer. Multi-omics studies have identified oncogenic mutations in the PIK3CA, PIK3R1, and PTEN genes involved in the PI3K-AKT pathway in human and canine angiosarcomas. Recently, the FDA approved alpelisib, a PI3K-alpha specific inhibitor, for breast cancer patients with PIK3CA mutations. However, less than 30% of patients treated with alpelisib showed an overall response. Thus, understanding how PIK3CA mutant cells acquire therapeutic resistance is of high importance, yet it remains largely unexplored. In this study, we aimed to determine the molecular effects of PIK3CA mutations on therapeutic resistance against PI3K inhibitors in angiosarcomas. Specifically, PIK3CA H1047R mutations were induced in DHSA-1426 and COSB canine hemangiosarcoma cell lines using CRISPR/Cas9-mediated gene editing, and mutant clones were clonally selected. RNA-seq analysis from the PIK3CA mutant cells and 74 canine hemangiosarcoma tissue samples demonstrated that the activation of the oncogenic PI3K pathway increased cytokine and chemokine secretion, possibly contributing to an immunogenic and inflammatory tumor phenotype. Alpelisib completely abrogated AKT phosphorylation and decreased cytokine and chemokine expression in PIK3CA mutant cells. However, alpelisib showed only moderate potency in inhibiting cell proliferation of the mutant cells, demonstrating partial resistance. Subsequently, our single-cell RNA-seq analysis identified drug-resistant cell clusters within the total cell population. Marker gene analysis revealed that the resistant cell clusters upregulated genes associated with the G protein pathway (GNAI1, VAV3, and GNG11), adenylyl cyclase pathway (PDE4D and PDE7B), calcium signaling pathway (RYR3 and CAMK2D), TGF-beta pathway (TGFB2 and TGFBR2), and the anti-apoptotic gene BCL2. These pathways are all associated with RAS-MAPK signaling, and we confirmed enhanced phosphorylation of ERK upon alpelisib treatment through western blotting. Further, pseudo-time analysis using Monocle 3 indicated that the resistant cell clusters shared a similar differentiation time point with the sensitive cell clusters within the PIK3CA mutant population. Our single-cell trajectory data suggest that therapeutic resistance to alpelisib could be independent of differentiation level. Instead, it may involve an evolutionary program controlled by specific selective pressures between resistant and sensitive cells, potentially associated with immune regulatory mechanisms. Our ongoing work is evaluating whether a cellular autonomous program that regulates cytokine secretion induced by PIK3CA mutation contributes to therapeutic resistance in the tumor microenvironment through in vivo experiment. This study will provide insights into potential mechanisms governing the cellular evolution process of PIK3CA mutant cells, which promote drug resistance. Citation Format: Donghee Lee, Emma Kozurek, Md Abdullah, Rong Li, Joanne Kim, Erin B. Dickerson, Jong Hyuk Kim. Oncogenic PIK3CA on therapeutic resistance and cellular heterogeneity in malignant vascular cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A014.