Abstract 2450: Transforming intestinal stem cells into metastatic cancer Stem cells by aberrantly activated cytokine-STAT5A signaling

Abstract Background: Lgr5 intestinal stem cells (ISC) are augmented by colorectal (CRC), but it is not clear if the aberrant Lgr5 ISCs result in the metastatic phenotypes of CRC, suggesting that epistasis-involved mutations in CRC suppressor genes or oncogene control Lgr5 ISC transformation into cancer stem cells (CSC). Somatic mutations in STAT5A or B are highly associated with CRC. Cytokine-STAT5 action is essential for Lgr5 ISC stemness, and enhanced STAT5 phosphorylation can induce Lgr5 ISC differentiation into secretory niche cells. Using human and mouse tumoroids, and organoid xerograph models, we aim to determine the effects of STAT5 oncogenic activation on Lgr5 ISC malignant and metastatic phenotypes. Methods: Colorectal surgical specimen from CRC and hepatic metastatic CRC patients were collected to perform RNAseq or single cell RNAseq (scRNAseq) analysis. Compound-mutant mice with STAT5 loss or gain of function in Apcmin+/- mice, were generated by crossing Apcmin+/- mice with Lgr5CreER and Rs26CreER, and Stat5f/f (loss of function of Stat5) or icS5f/f (gain of function of STAT5A) mice. CRISPR/Cas9 gene editing was applied to engineer APC mutations in human epithelial cell lines or Apc mutations in Lgr5 ISCs. Apc-mutant Lgr5 ISCs or tumoroids from Apcmin+/- mice combined with STAT5 loss or gain of function were differentiated, then were under intra-abdominal xenotransplantation. APC-mutant intestinal epithelial cell lines were used to determined CSC migration in the presence of STAT5 activation. STAT5 inhibitors were used to treat Apcmin+/- mice or tumoroids. Results: Human subject studies revealed that hepatic metastatic CRC patients exhibited the robustly increased STAT5A and/or Lgr5 colonic cancer cells. Murine studies showed that constitutively STAT5A activation (Ca-pYSTAT5) in colonic Apcmin+/- CSCs promoted CSC migration, invasion and metastasis. In vivo migration assay showed that transient STAT5A activation in the APC-mutant intestinal epithelia accelerated the migration of APC-mutant epithelia. Ex Vivo tumoroid xenotransplantation showed that Ca-pYSTAT5 resulted in tumoroid invasion into liver. Lineage tracing analysis showed that Ca-pYSTAT5 expanded Lgr5 CSCs in tumoroids and drove Lgr5 CSCs to hepatic metastasis. Importantly, Stat5 deletion in Lgr5 ISCs abolishes colon cancer orgnoids invasion. Pharmacological inhibition of STAT5 dimerization leads to Lgr5 cancer organoid degeneration and partially annihilated intestinal adenoma in Apcmin+/-. CHIP-seq analysis showed that Ca-pYSTAT5 increased an aberrant high level of Bcl6 expression by inducing super-enhancer activation in the Bcl-6 locus. Conclusion: Aberrantly activated Cytokine-STAT5A signaling is required for ISC malignancy and CSC hepatic metastasis. STAT5 blockade could be important to impair CRC metastasis. Citation Format: Haifeng Li, Wen Gao, Ruixue Liu, Xiaonan Han. Transforming intestinal stem cells into metastatic cancer Stem cells by aberrantly activated cytokine-STAT5A signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2450.