RLY-4008, the First Highly Selective FGFR2 Inhibitor with Activity across FGFR2 Alterations and Resistance Mutations

Abstract

Oncogenic activation of fibroblast growth factor receptor 2 (FGFR2) drives multiple cancers and represents a broad therapeutic opportunity, yet selective targeting of FGFR2 has not been achieved. Although the clinical efficacy of pan-FGFR inhibitors (pan-FGFRi) validates FGFR2 driver status in FGFR2 fusion-positive intrahepatic cholangiocarcinoma, their benefit is limited by incomplete target coverage due to FGFR1- and FGFR4-mediated toxicities (hyperphosphatemia and diarrhea, respectively) and the emergence of FGFR2 resistance mutations. RLY-4008 is a highly selective, irreversible FGFR2 inhibitor designed to overcome these limitations. In vitro, RLY-4008 demonstrates >250- and >5,000-fold selectivity over FGFR1 and FGFR4, respectively, and targets primary alterations and resistance mutations. In vivo, RLY-4008 induces regression in multiple xenograft models-including models with FGFR2 resistance mutations that drive clinical progression on current pan-FGFRi-while sparing FGFR1 and FGFR4. In early clinical testing, RLY-4008 induced responses without clinically significant off-isoform FGFR toxicities, confirming the broad therapeutic potential of selective FGFR2 targeting.

Significance: Patients with FGFR2-driven cancers derive limited benefit from pan-FGFRi due to multiple FGFR1-4-mediated toxicities and acquired FGFR2 resistance mutations. RLY-4008 is a highly selective FGFR2 inhibitor that targets primary alterations and resistance mutations and induces tumor regression while sparing other FGFRs, suggesting it may have broad therapeutic potential. See related commentary by Tripathi et al., p. 1964. This article is featured in Selected Articles from This Issue, p. 1949.

Figure 1.

RLY-4008 is a potent and selective irreversible inhibitor of FGFR2. A, Sequence alignment of the kinase domains of FGFR1–4 indicates a high degree of similarity among paralogs. RLY-4008 binding site residues are boxed; residues shown in pink identify amino acid differences between FGFR2 and paralogs within this region. Numbering refers to the FGFR2 IIIc isoform. B, Chemical structure of RLY-4008, N-(4-(4-amino-5-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)methacrylamide. C, Crystal structure of RLY-4008 in complex with FGFR2 (PDB: 8STG). Protein is shown in green, and inhibitor carbons are shown in magenta. The Cys491 sulfur is shown in gold and as a covalent adduct with RLY-4008. D and E, Rate of covalent labeling of FGFR2 (red) and FGFR1 (blue) by RLY-4008 (D) and futibatinib (E) as measured by intact mass over time. Triplicate biological replicates are reported. F, RLY-4008 concentration-dependent modification rate against FGFR2 (red) and FGFR1 (blue). RLY-4008 against FGFR2: k_inact = 6.45 × 10⁻² per second; K_I = 1.87 μmol/L; k_inact/K_I = 3.45 × 10⁻² per second/(μmol/L). RLY-4008 against FGFR1: k_inact = 2.33 × 10⁻³ per second; K_I = 6.14 μmol/L; k_inact/K_I = 3.79 × 10⁻⁴ per second/(μmol/L). G, Fold change in biochemical IC₅₀ values of the indicated inhibitors between FGFR2 and FGFR1, FGFR3, and FGFR4. Average fold change of three independent experiments each containing two biological replicates is reported. Error bars indicate SD. H, TREEspot depicting selectivity of RLY-4008 screened against 468 kinases via KINOMEscan (DiscoverX, Eurofins). At the test concentration of 500 nmol/L, three kinases showed greater than 75% inhibition: FGFR2 (94.1%), MEK5 (92.4%), and MKNK2 (89%). Image generated using TREEspot Software Tool and reprinted with permission from KINOMEscan, a division of DiscoveRx Corporation, ^©DiscoverX Corporation 2010.

Figure 2.

RLY-4008 inhibits FGFR2-mediated signaling and proliferation in cells. A, Inhibition of FGFR2-mediated signaling in SNU-16 cells. Cells were incubated with RLY-4008 for 2 hours prior to lysis and analysis via pFGFR2 (Y653/654) and pERK (T202/Y204) HTRF (PerkinElmer). B, Inhibition of FGFR2-mediated signaling and induction of apoptosis in SNU-16 cells. Immunoblots of cell lysates generated from cells treated with DMSO (Control), IC₅₀, or IC₉₀ concentrations of RLY-4008 (as determined by pFGFR2 HTRF) for the indicated times. Samples were analyzed via traditional Western or via WES (Protein Simple). Loading controls were actin (traditional) and actin and vinculin (WES). cl, cleaved; p, phospho; t, total. C, Viability IC₅₀ values for RLY-4008 in FGFR2-, FGFR1-, FGFR3-, and FGFR4-dependent cancer cell lines (Supplementary Table S3). Cells were treated for 96 hours and cellular viability was assayed using CellTiter-Glo (Promega). Average IC₅₀ of two independent experiments each containing two biological replicates is reported. Error bars indicate SD. * Indicates that IC₅₀ was not reached in this cell line (maximum RLY-4008 concentration = 10 μmol/L).

Figure 3.

Treatment with RLY-4008 leads to dose-dependent inhibition of FGFR2 and tumor regression in multiple FGFR2-altered tumor models and spares FGFR1 in vivo. A–F, Refer to boxed legend. A, C, E, and G, Dotted line indicates tumor volume prior to initiation of treatment. B, D, and F, Dotted line indicates 10% pFGFR2/tFGFR2 (90% inhibition of pFGFR2). A, Antitumor activity of RLY-4008 compared with pemigatinib and futibatinib in an FGFR2–TTC28 iCCA patient-derived xenograft (PDX) model (n = 6/group). Data are mean ± SEM. B, Dose-dependent inhibition of FGFR2 in FGFR2–TTC28 tumors. Animals were sacrificed, and tumors were harvested at the indicated time points after the final dose on the third day of dosing. Tumor lysates were analyzed via pFGFR2 (Y653/654) ELISA and tFGFR2 HTRF; pFGFR2 normalized to tFGFR2 is reported (n = 3/group). Free plasma concentration of RLY-4008 is reported. Data are mean ± SEM. Fut, futibatinib; Pem, pemigatinib. C, Antitumor activity of RLY-4008 compared with futibatinib in the SNU-16 gastric cancer xenograft model (n = 7/group). Data are mean ± SEM. D, Dose-dependent inhibition of FGFR2 in SNU-16 tumors. Animals were sacrificed and tumors were harvested at the indicated time points after the final dose on the fourth day of dosing. Tumor lysates were analyzed via pFGFR2 (Y653/654) and tFGFR2 HTRF; pFGFR2 normalized to tFGFR2 is reported (n = 3/group). Free plasma concentration of RLY-4008 is reported. Data are mean ± SEM. E, Antitumor activity of RLY-4008 in the AN3CA endometrial cancer xenograft model (n = 8/group). Data are mean ± SEM. F, Dose-dependent inhibition of FGFR2 in AN3CA tumors. Animals were sacrificed and tumors were harvested at the indicated time points after the final dose on the third day of dosing. Tumor lysates and plasma were analyzed and reported as in B (n = 3/group). G, Antitumor activity of RLY-4008 and pan-FGFRi futibatinib, pemigatinib, erdafitinib, and infigratinib in an FGFR2–OPTN iCCA cell line–derived xenograft model, ICC13–7 (n = 8/group). Data are mean ± SEM. H, RLY-4008 spares FGFR1 in vivo. Two hours after the final dose of the study shown in G, blood was collected from all animals for serum phosphate analysis (n = 8/group). Data are mean ± SEM. ***, P < 0.0001, one-way ANOVA. b.i.d., twice daily; q.d., once daily; t.i.d., three times daily.

Figure 4.

RLY-4008 is active on mutations associated with acquired resistance to pan-FGFRi. A, Acquired resistance mutations in the FGFR2 kinase domain are commonly found in patients with FGFR2 fusion– or rearrangement–positive iCCA treated with pan-FGFRi. The graph indicates the number of times the indicated mutant allele was detected in tissue or ctDNA in 23 patients (out of 46) who developed FGFR2 kinase domain mutations at progression on pan-FGFRi. Figure art adapted from Varghese et al. and patient data are from Goyal et al. (23, 34). B, Heat map displaying the fold change in potency (IC₅₀) for the indicated inhibitors against the indicated FGFR2 mutant as compared with FGFR2 WT. Numbering of mutant residues refers to the FGFR2 IIIc isoform to remain consistent with the usage of this nomenclature. Following 2 hours of incubation with the compound, FGFR2 inhibition was determined via pFGFR2 (Y653/654) HTRF assay, and IC₅₀ values against FGFR2 WT and FGFR2 mutants were calculated. The average fold change of three independent experiments each containing two biological replicates was used to derive a heat map in GraphPad Prism. Fold change of one indicates equivalent potency on FGFR2 WT and the indicated FGFR2 mutant. C–E, Dotted line indicates tumor volume prior to initiation of treatment. C, Antitumor activity of RLY-4008 compared with futibatinib, pemigatinib, erdafitinib, and infigratinib in the ICC13-7–FGFR2^V564F xenograft model (n = 8/group). Only RLY-4008 leads to tumor regression. Data are mean ± SEM. D, Following 28 days of treatment on the indicated inhibitors, animals on pan-FGFRi in the study shown in C were changed to treatment with RLY-4008 10 mg/kg once daily. Tumor regression was observed in all animals receiving RLY-4008. Data are mean ± SEM. E, Antitumor activity of RLY-4008 compared with futibatinib, pemigatinib, erdafitinib, and infigratinib in the AN3CA (FGFR2^K310R;N549K) endometrial cancer xenograft model (n = 7/group). Only RLY-4008 and futibatinib treatment lead to tumor regression. Data are mean ± SEM. F, RLY-4008 overcomes acquired resistance to pemigatinib in vivo. Antitumor activity of pemigatinib followed by RLY-4008 in an FGFR2–TTC28 iCCA patient-derived xenograft model. Animals were dosed with pemigatinib for 40 days, followed by treatment with RLY-4008 from days 42–98. Each line represents one animal. b.i.d., twice daily; q.d., once daily; t.i.d., three times daily.

Figure 5.

Clinical response in an FGFRi-naive iCCA patient with liver and lymph node metastases. The patient was treated with RLY-4008 at 70 mg once daily, the RP2D. A, Summary of key patient and disease characteristics. B, CT scans of liver and lymph node metastases at baseline (top) and after 163 days of RLY-4008 treatment (bottom) show profound tumor regression. C, Serum phosphate over the course of treatment with RLY-4008. The shaded area represents the normal range for serum phosphate (0.8–1.5 mmol/L). q.d., once daily; RP2D, recommended phase II dose.

Figure 6.

Clinical response in a patient with pan-FGFRi–resistant iCCA with liver, bone, and lung metastases. The patient was treated with RLY-4008 starting at 30 mg once daily. A, Summary of key patient and disease characteristics. B, ctDNA analysis demonstrated complete clearance of FGFR2^N549K, FGFR2^N549D, and FGFR2^{V564 L} clones by day 30. C, Left: CT scans of right lobe liver metastasis (arrow) and multifocal liver lesions (circled) at baseline (top) and on day 57 of RLY-4008 treatment (bottom) show a rapid, marked reduction in tumor volume. Right: CT scans of manubrium bone lesion at baseline (top) and on day 57 of RLY-4008 treatment (bottom) show complete regression of lesion. D, Serum phosphate during treatment with RLY-4008. Shaded area represents the normal range for serum phosphate (0.8–1.5 mmol/L). ND, not detectable; q.d., once daily.

Figure 7.

Clinical response in a patient with metastatic salivary gland carcinoma with squamous features and metastases in the lung, liver, and spine. The patient was treated with RLY-4008 at 70 mg once daily, the RP2D. A, Summary of key patient and disease characteristics. B, Liver lesions, left: CT scans of liver metastases on the dome at baseline (top; arrows) and day 64 of RLY-4008 treatment (bottom; arrow indicates remaining lesion with marked reduction in volume) show dramatic regression of lesions. Liver lesions, right: CT scans of diffuse metastatic lesions within the liver parenchyma at baseline and on day 64 of RLY-4008 treatment show a near complete regression of such lesions (circles). Lung lesions: CT scan of lung metastases at baseline and on day 64 of RLY-4008 treatment show overall decrease in lung metastases and near resolution of a lesion (arrow). C, Serum phosphate over the course of treatment with RLY-4008. The shaded area represents the normal range for serum phosphate (0.8–1.5 mmol/L). q.d., once daily; RP2D, recommended phase II dose.