Abstract 263: KITlow cells mediate Imatinib resistance and disease persistence in gastrointestinal stromal tumor

Abstract

Abstract INTRODUCTION: Gastrointestinal stromal tumor (GIST) is commonly driven by oncogenic KIT mutations that are effectively targeted by Imatinib (IM). However, IM does not cure GIST; adjuvant therapy only delays recurrence in high-risk tumors. Therefore, we hypothesized that GIST contains cells with primary IM resistance, representing a critical target for overcoming disease persistence. METHODS: Gene Set Enrichment Analysis (GSEA) was performed with publicly available data (GSE1596636). Human GIST cell lines (GIST-T1 and 882) and primary KIT-mutant GIST (IRB #090401) were analyzed by fluorescence-activated cell sorting (FACS) to sort KITloCD34+ (KITlo) vs KIThiCD34+ (KIThi) subpopulations using anti-human KIT and CD34 antibodies. RNA was extracted from cell lysates for analysis by quantitative RT-PCR. Cell viability was assessed by CellTiter-Glo or MTT assays following drug treatments. RESULTS: We performed GSEA on 27 matched GISTs comparing pre- and post-neoadjuvant IM treatment (RTOG S0132). Post-IM samples had 50% lower KIT expression (P=0.002) and were enriched in “cancer stem cell” and Axl/Gas6/NF-κB signaling gene signatures. Similarly, in vitro IM treatment of GIST cell lines resulted in 2-fold reduction of KIT expression and 1.9 to 4.4-fold increased expression of stem-associated transcription factors (SATFs: OCT4, SOX2, KLF4, NANOG). Parallel FACS analysis of IM-treated GIST cell lines (125 nM, 72-h) demonstrated the presence of a KITlo subpopulation (GIST-T1: 0.75%; GIST882: 4%) while untreated cells had 3- to 11-fold fewer KITlo cells. Primary human GISTs also had KITlo cells by FACS (4-8%; N=4), suggesting that the population is not an artifact of in vitro culture. KITlo GIST882 cells were IM-resistant (IC50: KITlo 3641 nM vs KIThi 180 nM) and significantly overexpressed all SATFs by qRT-PCR (2.0 to 4.1-fold; P<0.001) consistent with RTOG S0132 analysis. Moreover, RNAseq confirmed that KITlo cells (relative to KIThi) are enriched in factors present in the “cancer stem cell” gene signature identified by GSEA (KLF4, CCL5, ATF3, JUN, IFIT1, PMP22). Lastly, we tested candidate drugs against targets overexpressed in IM-treated tumors. Unsorted cells were pre-treated with IM (185 nM, 48-h) to enrich for KITlo cells. Subsequent treatment with R428 (AXL inhibitor) or bardoxolone (NF-κB inhibitor) resulted in 70% (R428, 5 μM) and 80% (bardoxolone, 5 μM) cell death. Finally, FACS-sorted KITlo cells were sensitive to both drugs (50% and 88% killing, respectively). CONCLUSIONS: KITlo cells are a distinct subpopulation in human GIST with intrinsic IM-resistance and may represent a novel mechanism of GIST persistence. These cells overexpress stem cell transcriptional programs, including the Axl/Gas6/NF-κB pathway, which represent novel therapeutic targets in vitro. Further studies are needed to explore the in vivo efficacy of combination or sequential targeting of KITlo cells in GIST. Citation Format: Sudeep Banerjee, Chih-Min Tang, Mayra Yebra, Kwat Medetgul, Adam M. Burgoyne, Pablo Tamayo, Robert Wechsler-Reya, Jason K. Sicklick. KITlow cells mediate Imatinib resistance and disease persistence in gastrointestinal stromal tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 263.