220 Discovery of selective inhibitors of fibroblast growth factor receptor (FGFR): Antitumor activity in cellular and xenograft tumor models with FGFR activation

Abstract

Background: Deregulated FGF signaling promotes oncogenesis in several tumor types including gastric, bladder, endometrial, breast and multiple myeloma. Tumor genomic analyses of these cancers has identified amplifications, translocations, and activating mutations in FGFR1, FGFR2, and FGFR3. Inhibitors selective for the FGFR family provide an opportunity to target diverse cancer subtypes driven by FGFR activation while avoiding potential complications from VEGFR/PDGFR inhibition and anti-angiogenic therapy. Methods: Small molecule inhibitors of FGFR family kinases were identified by high-throughput screening and lead optimization using in vitro enzymatic assays. FGFR2 activation was measured in cultured cells and in xenograft tumors using phosphorylation of FGFR and downstream signaling proteins FRS2, ERK, and AKT. Cell viability was assessed by measuring cellular ATP levels. Xenograft tumors were grown in nude mice and compounds dosed by oral gavage. Results: FGFR inhibitors were identified with potent biochemical activity against FGFR1, FGFR2, and FGFR3 (IC 50 10-100 nM). X-ray crystallographic studies with FGFR2 demonstrated that the compounds bind in the ATP binding pocket. Cell viability assays were used to identify FGFR-dependent tumor cell lines. Most of these lines have genetic alterations in FGFR family members such as the colon adenocarcinoma line NCI-H716 which contains an amplification of FGFR2. Treatment of NCI-H716 cells with FGFR inhibitors blocks phosphorylation of FGFR2 and the downstream proteins FRS2, ERK, and AKT (IC 50 10-100 nM) as well as a broader phosphotyrosine signaling network that includes the HER family kinases. In vivo pharmacodynamic studies with orally bioavailable compounds demonstrated target inhibition in NCI-H716 xenograft tumors as assessed by a reduction in pFGFR2. Efficacy studies in the NCI-H716 xenograft model showed up to 70% tumor regression at well tolerated doses. Distinct chemical subseries were identified that selectively inhibit FGFR2 vs. other family members or that inhibit FGFR2 with mutations at the gatekeeper residue V584, a common source of resistance to kinase inhibitor therapy. Conclusions: In FGFR-driven tumor models, FGFR selective inhibitors block receptor activation and downstream signaling, reduce cell viability in vitro, and inhibit the growth of xenograft tumors. These results support advancement of FGFR selective inhibitors for the treatment of select cancer subtypes identified by tumor genomics.