Unraveling the Mechanisms of Sensitivity to Anti-FGF Therapies in Imatinib-Resistant Gastrointestinal Stromal Tumors (GIST) Lacking Secondary KIT Mutations

SIMPLE SUMMARY: Acquired resistance of gastrointestinal stromal tumors (GISTs) to imatinib mesylate (IM) is one of the most critical challenges in GIST therapy. Here we show that a long-term culture of GIST T-1 cells with IM induces clonal heterogeneity resulting in the appearance of cancer cells exhibiting activation of the FGFR-signaling pathway which was associated with KIT loss. The first one was due to the overexpression of FGFR1/2 and increased production of FGF-2 ligand. These events maintained GIST resistance to IM and rendered these GIST cells highly sensitive to all types of pan-FGFR-inhibitors used in the current study. Knockout of FGFR2 in this GIST subclone significantly attenuated pro-apoptotic and anti-proliferative activities of infigratinib (BGJ 398) both in vitro and in vivo, thereby suggesting the activation of FGFR-signaling pathway via FGFR2-mediated axis as the predominant molecular mechanism in these GIST cells. Collectively, the extended inhibition of KIT-signaling in GISTs induces clonal heterogeneity of cancer cells and might change the tumor’s sensitivity to FGFR-inhibitors due to selection of cancer cells with an FGFR-overactivated pathway. ABSTRACT: We showed previously that inhibition of KIT signaling in GISTs activates FGFR-signaling pathway rendering cancer cells resistant to receptor tyrosine kinase inhibitor (RTKi) imatinib mesylate (IM) (Gleevec) despite of absence of secondary KIT mutations and thereby illustrating a rationale for the combined (e.g., KIT- and FGFR-targeted) therapies. We show here that long-term culture of IM-resistant GISTs (GIST-R1) with IM substantially down-regulates KIT expression and induces activation of the FGFR-signaling cascade, evidenced by increased expression of total and phosphorylated forms of FGFR1 and 2, FGF-2, and FRS-2, the well-known adaptor protein of the FGF-signaling cascade. This resulted in activation of both AKT- and MAPK-signaling pathways shown on mRNA and protein levels, and rendered cancer cells highly sensitive to pan-FGFR-inhibitors (BGJ 398, AZD 4547, and TAS-120). Indeed, we observed a significant decrease of IC50 values for BGJ 398 in the GIST subclone (GIST-R2) derived from GIST-R1 cells continuously treated with IM for up to 12 months. An increased sensitivity of GIST-R2 cells to FGFR inhibition was also revealed on the xenograft models, illustrating a substantial (>70%) decrease in tumor size in BGJ 398-treated animals when treated with this pan-FGFR inhibitor. Similarly, an increased intra-tumoral apoptosis as detected by immunohistochemical (IHC)-staining for cleaved caspase-3 on day 5 of the treatment was found. As expected, both BGJ 398 and IM used alone lacked the pro-apoptotic and growth-inhibitory activities on GIST-R1 xenografts, thereby revealing their resistance to these TKis when used alone. Important, the knockdown of FGFR2, and, in much less content, FGF-2, abrogated BGJ 398′s activity against GIST-R2 cells both in vitro and in vivo, thereby illustrating the FGF-2/FGFR2-signaling axis in IM-resistant GISTs as a primary molecular target for this RTKi. Collectively, our data illustrates that continuous inhibition of KIT signaling in IM-resistant GISTs lacking secondary KIT mutations induced clonal heterogeneity of GISTs and resulted in accumulation of cancer cells with overexpressed FGF-2 and FGFR1/2, thereby leading to activation of FGFR-signaling. This in turn rendered these cells extremely sensitive to the pan-FGFR inhibitors used in combination with IM, or even alone, and suggests a rationale to re-evaluate the effectiveness of FGFR-inhibitors in order to improve the second-line therapeutic strategies for selected subgroups of GIST patients (e.g., IM-resistant GISTs lacking secondary KIT mutations and exhibiting the activation of the FGFR-signaling pathway).