The N550K/H Mutations in FGFR2 Confer Differential Resistance to PD173074, Dovitinib, and Ponatinib ATP-Competitive Inhibitors

Abstract

We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2(mutant) endometrial cancers (ECs). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2(mutant) cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2(N550K) in JHUEM-2 cells (FGFR2(C383R)) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2(N550K) can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2(N550K). We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors

“…FGFR4 has been reported to play a very important role in metastasis, drug resistance, and poor prognosis (23,(68)(69)(70); therefore FIIN-2 and FIIN-3, with good FGFR4 potency, show promising potential for application in many FGFR-dependent cancer types such as breast cancer (63,71) and hepatocellular carcinoma (72,73). In addition, they are capable of overcoming the valine-to-methionine gatekeeper mutation in H2077 and H1581 cell lines, although similar mutations found in patients' specimens have been demonstrated experimentally to confer resistance to the leading clinical FGFR inhibitors (19,48,50).…”

“…Activation of FGFR-dependent signaling pathways can stimulate tumor initiation, progression, and resistance to therapy. Translocation events implicating the FGFR1 gene and various fusions of FGFR1 are found in myeloproliferative syndromes (12); chromosomal translocations of FGFR1 or FGFR3 and the transforming acidic coiled-coil genes (TACC1 or TACC3) are oncogenic in glioblastoma multiforme, bladder cancer, head and neck cancer, and lung cancer (13)(14)(15)(16); oncogenic mutations of FGFR2 and FGFR3 are observed in lung squamous cell carcinoma; FGFR2 N549K is observed in 25% of endometrial cancers; FGFR3 t(4;14) alterations are reported in 15-20% of multiple myeloma (17)(18)(19); FGFR4 Y367C mutation in the transmembrane domain drives constitutive activation and enhanced tumorigenic phenotypes in a breast carcinoma cell line (20)(21)(22); and K535 and E550 mutants are reported to activate FGFR4 in rhabdomyosarcoma (23). FGFR amplification is reported in various cancers (24,25): FGFR1 is amplified in colorectal, lung, and renal cell cancers (26,27); FGFR2 is amplified in gastric cancer and colorectal cancer (28,29); FGFR3 is commonly amplified in bladder cancer and also is reported for cervical, oral, and hematological cancers (30)(31)(32); and FGFR4 is amplified in hepatocellular carcinoma, gastric cancer, pancreatic cancer, and ovarian cancer (33)(34)(35)(36)(37).…”

“…The FGFR2 V564I gatekeeper mutant was isolated as a resistant clone in a FGFR2 Ba/F3 screen of dovitinib and also was reported to confer resistance to the multitargeted drug ponatinib (19). In humans the FGFR4 V550L gatekeeper mutation was detected in 9% (4/43) of embryonal rhabdomyosarcoma tumors (49), and the FGFR4 V550M mutation was detected in 13% (2/15) of neuroendocrine breast carcinomas (50).…”

FGFR a promising druggable target in cancer: Molecular biology and new drugs

“…In MM cell lines, a gatekeeper mutation in FGFR3 (FGFR3 V555M) is involved in resistance to AZD4547 and PD173074 (both Pan-FGFR Inhibitors) (Chell et al, 2013). In endometrial cell lines, several FGFR2 point mutations (especially V564I) have been reported to confer different degrees of resistance to multi-target inhibitors (Dovitinib, Ponatinib and PD173074) (Byron et al, 2013). Moreover, a gatekeeper mutation in FGFR1 (V561M) has also been described in squamous cell lung cancer and breast cancer, suggesting that it could be involved in resistance to multi-target inhibitors like Lucitanib because but the same cell line maintained the susceptibility to AZD4547 (which target the same mutation), that is due to a different construct-flexibility between the drugs, AZD4547 affinity is preserved by V561M FGFR1 due to a flexible linker that allows multiple inhibitor binding modes (Sohl et al, 2015).…”

ERK Signal Suppression and Sensitivity to CH5183284/Debio 1347, a Selective FGFR Inhibitor

“…Acquired mutations in target genes or downstream components are major mechanisms of resistance (5)(6)(7)(8)(9)(10)(11)(12)(13). Although acquired FGFR mutations have not yet been identified in patients, several FGFR mutations that confer resistance to FGFR inhibitors have been reported (7,14). Because cancer cells continue to utilize the pathway to which they are originally addicted, they acquire some genetic alterations to reactivate the pathway.…”