Abstract LB172: A cytosolic complex between exportin-7(XPO7) and ste20-like kinase (SLK) regulates PI3K-AKT-mTOR signaling in cholangiocarcinoma

Abstract

Abstract Cholangiocarcinoma (CCA) is a deadly malignancy with an evolving therapeutic landscape. Although nucleic acid-based evaluations have unveiled some druggable targets, limited options are available for the majority of patients with this disease. We therefore evaluated biliary exosomes by mass spectrometry from 16 patients with CCA, which revealed a 17-fold enhancement of the nuclear export protein XPO7 in CCA-derived biliary exosomes. IHC analysis of XPO7 expression in CCA tumors from 170 patients unexpectedly demonstrated intense cytoplasmic staining in 30% of the samples, which correlated with abbreviated survival. Within the cytosol, we demonstrate that XPO7 exists in a molecular complex with the serine/threonine kinase SLK. shRNA-mediated knockdown of either XPO7 or SLK abrogated tumor organoid formation in vitro, and reduced orthotopic tumor formation and substantially altered tumor morphology using athymic mice. A kinome screen of FDA-approved drugs revealed tivozanib inhibits SLK (IC50= 81.8 nM), which we confirmed using crystallography. Tivozanib treatment reduced tumor organoid formation in vitro and induced tumor regression in vivo using murine xenografts with resulting morphology similar to XPO7 and SLK knockdown tumors. Importantly, site-directed mutagenesis of SLK was used to confirm the observed phenotypic alterations secondary to SLK inhibition. Signal transduction studies with SLK knockdown in CCA cell lines demonstrated downregulation of PI3K-AKT-mTOR signaling with further effects on DNA damage resulting in G2 cell cycle arrest. Moreover, tivozanib demonstrated anti-tumor activity and increased apoptosis in established CCA patient-derived xenografts with cytoplasmic XPO7 expression. Using ex vivo tumor slice cultures, tivozanib significantly increased tumor cell apoptosis in XPO7/SLK-expressing tumors (N=3) but not in tumors without this expression pattern (N=6). Further evaluation of these tivozanib-treated tumor slice cultures demonstrated a reduction in the expression of phosphorylated mTOR (S2448), AKT (T308), S6 (S235/236) in the high XPO7/SLK-expressing tumors but not in tumors without this expression pattern. Lastly, tivozanib monotherapy demonstrated in vivo efficacy with arrest of tumor progression (RECIST stable disease) in an ongoing clinical trial (NCT04645160). Citation Format: Priyanka P. Desai, Tahsin M. Khan, Reed I. Ayabe, Emily Smith, Surajit Sinha, Ashley Rainey, Yuri Lin, Tracey Pu, Kenneth Lubrice, Leila Sarvestani, Sarfaraz Akmal, Shahyan Rehman, Kirsten Remmert, Michael Yaffe, Craig Thomas, John Brognard, Jonathan M. Hernandez. A cytosolic complex between exportin-7(XPO7) and ste20-like kinase (SLK) regulates PI3K-AKT-mTOR signaling in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB172.