Efficacy of futibatinib, an irreversible fibroblast growth factor receptor inhibitor, in FGFR-altered breast cancer

Abstract

Several alterations in fibroblast growth factor receptor (FGFR) genes have been found in breast cancer; however, they have not been well characterized as therapeutic targets. Futibatinib (TAS-120; Taiho) is a novel, selective, pan-FGFR inhibitor that inhibits FGFR1-4 at nanomolar concentrations. We sought to determine futibatinib's efficacy in breast cancer models. Nine breast cancer patient-derived xenografts (PDXs) with various FGFR1-4 alterations and expression levels were treated with futibatinib. Antitumor efficacy was evaluated by change in tumor volume and time to tumor doubling. Alterations indicating sensitization to futibatinib in vivo were further characterized in vitro. FGFR gene expression between patient tumors and matching PDXs was significantly correlated; however, overall PDXs had higher FGFR3-4 expression. Futibatinib inhibited tumor growth in 3 of 9 PDXs, with tumor stabilization in an FGFR2-amplified model and prolonged regression (> 110 days) in an FGFR2 Y375C mutant/amplified model. FGFR2 overexpression and, to a greater extent, FGFR2 Y375C expression in MCF10A cells enhanced cell growth and sensitivity to futibatinib. Per institutional and public databases, FGFR2 mutations and amplifications had a population frequency of 1.1%-2.6% and 1.5%-2.5%, respectively, in breast cancer patients. FGFR2 alterations in breast cancer may represent infrequent but highly promising targets for futibatinib.

Figure 1

Fibroblast growth factor receptor (FGFR) alterations in breast cancer patient-derived xenografts (PDXs). (a) FGFR expression and genomic alterations from 22 PDXs generated from 21 patients. (b) Relative protein expression of breast cancer PDXs determined by reverse phase protein array (RPPA). (c) Relative FGFR1-4 mRNA expression in patient samples and matching PDX models. RNA expression is presented in log2-normalized reads per kilobase million (RPKM). A 2-sided paired Student t test was used to calculate P-values. (d) The correlation of expression between FGFR genes in patient samples and those in matching PDX models is shown. RNA expression is presented in log2-normalized RPKM. The Pearson correlation coefficient (r) was used to measure the statistical association between 2 variables.

Figure 2

Response of patient-derived xenografts (PDXs) to the fibroblast growth factor receptor (FGFR) inhibitor futibatinib. (a–i) Female mice bearing breast cancer PDXs (n = 5 per group) were treated orally with the vehicle control or futibatinib (15 mg/kg/day). The treatment was stopped at 28 days or when the tumor diameter reached 1.5 cm³, except for mice bearing the PDX.007 model, in which the treatment was stopped on day 110. Growth (left) and event-free survival duration (right, defined as days until tumor doubling) curves are shown for each model. Growth curve graphs show the mean change in TV (mm³) ± the standard errors of the means. (j) Tumor volume of mice in control and futibatinib groups were measured at the last day of vehicle treatment. Bars, error bars, and dots show mean tumor volume (mm³), SEM, and individual tumor volume of each mouse, respectively. C, control; F, futibatinib. (k) Relative tumor growth tumor volume treatment/control ratios (TV T/C ratio) were calculated on day 21. End point tumor volumes (TV) were calculated at the last day of vehicle treatment. Event free survival (EFS) was calculated at the last day of futibatinib treatment. *This model does not have RNAseq data.

Figure 3

Effects of FGFR2 alterations on cell sensitivity to fibroblast growth factor receptor (FGFR) inhibitors and FGFR signaling. Normal mammary epithelial MCF10A cells were transduced with FGFR2-BICC1 fusion, FGFR2 Y375C mutation, FGFR2 wild type (WT), and the vector control. (a) Cell viability was measured by a sulforhodamine B (SRB) assay. Bars show the mean OD570 ± the standard errors of the means. (b) Cells were cultured for 3 weeks. Cell colonies were stained, and the total colony area was quantitated. The comparisons of the total colony areas of the FGFR2 WT, FGFR2-BICC1, and FGFR2 Y375C cell lines to the vector control cell line. Bars show the mean total colony areas ± the standard errors of the means. Below, representative stained plates are shown. (c) Cells were and treated with a serial dilution of futibatinib for 4 days. Cell viability was assessed with an SRB assay, and the half-maximal inhibitory concentration (IC₅₀) values were calculated. Bars show the mean IC₅₀ values ± the standard errors of the means. (d) While being starved, cells were treated with fibroblast growth factor (FGF1) for 24 h and various doses of futibatinib for 4 days. Cell viability was assessed with an SRB assay, and IC₅₀ values were calculated. Bars show mean IC₅₀ values ± the standard errors of the means. (e) Cells were starved for 24 h and treated with futibatinib at 0.2 µM for 3 h 45 min, followed by FGF1 for 15 min. Immunoblotting was performed using antibodies against FGFR2, p-Akt (S473), Akt, p-ERK1/2 (T202/Y204), ERK1/2, and β-actin. (f) PDX.007CL cells were plated in spheroid plates and treated a panel of FGFR inhibitors for five days. A luminescence assay was used to determine cell viability and IC₅₀ values were calculated. (g) MCF10A vector control and FGFR2 Y375C cells were treated with a panel of FGFR inhibitors for four days. Sulforhodamine B assay was used to determine cell viability and IC₅₀ values were calculated.

Figure 4

FGFR mutations in patients with breast cancer. Breast cancer data in MD Anderson Cancer Center (MDACC), MSK-IMPACT, Metastatic Breast Cancer (MBC) Project, and The Cancer Genomics Project (TCGA) databases were downloaded and compiled. (a) The bars show the percentages of somatic FGFR mutations in patients with breast cancer in each of these databases. (b) The 4 databases were analyzed together, and all mutations on the individual genes were illustrated using the cBioPortal MutationMapper tool. (c) Percentages of samples with FGFR2 amplification (AMP) in each database. (d) Distribution of FGFR2 Y375C mutations in different tissues in the Catalogue of Somatic Mutations in Cancer (COSMIC) database.