FGFR1 Kinase Domain Research Articles

Understanding and Overcoming Resistance to Selective FGFR Inhibitors across FGFR2-Driven Malignancies.

Understanding resistance to selective FGFR inhibitors is crucial to improve the clinical outcomes of patients with FGFR2-driven malignancies. We analyzed sequential ctDNA, ± whole-exome sequencing, or targeted next-generation sequencing on tissue biopsies from patients with tumors harboring activating FGFR2 alterations progressing on pan-FGFR-selective inhibitors, collected in the prospective UNLOCK program. FGFR2::BICC1 Ba/F3 and patient-derived xenograft models were used for functional studies. Thirty-six patients were included. In cholangiocarcinoma, at resistance to both reversible inhibitors (e.g., pemigatinib and erdafitinib) and the irreversible inhibitor futibatinib, polyclonal FGFR2 kinase domain mutations were frequent (14/27 patients). Tumors other than cholangiocarcinoma shared the same mutated FGFR2 residues, but polyclonality was rare (1/9 patients). At resistance to reversible inhibitors, 14 residues in the FGFR2 kinase domain were mutated-after futibatinib, only the molecular brake N550 and the gatekeeper V565. Off-target alterations in PI3K/mTOR and MAPK pathways were found in 11 patients, often together with on-target mutations. At progression to a first FGFR inhibitor, 12 patients received futibatinib or lirafugratinib (irreversible inhibitors), with variable clinical outcomes depending on previous resistance mechanisms. Two patients with TSC1 or PIK3CA mutations benefited from everolimus. In cell viability assays on Ba/F3 and in pharmacologic studies on patient-derived xenografts, irreversible inhibitors retained better activity against FGFR2 kinase domain mutations, with lirafugratinib active against the recalcitrant V565L/F/Y. At progression to FGFR inhibitors, FGFR2-driven malignancies are characterized by high intra- and interpatient molecular heterogeneity, particularly in cholangiocarcinoma. Resistance to FGFR inhibitors can be overcome by sequential, molecularly oriented treatment strategies across FGFR2-driven tumors.

The Irreversible FGFR Inhibitor KIN-3248 Overcomes FGFR2 Kinase Domain Mutations.

FGFR2 and FGFR3 show oncogenic activation in many cancer types, often through chromosomal fusion or extracellular domain mutation. FGFR2 and FGFR3 alterations are most prevalent in intrahepatic cholangiocarcinoma (ICC) and bladder cancers, respectively, and multiple selective reversible and covalent pan-FGFR tyrosine kinase inhibitors (TKI) have been approved in these contexts. However, resistance, often due to acquired secondary mutations in the FGFR2/3 kinase domain, limits efficacy. Resistance is typically polyclonal, involving a spectrum of different mutations that most frequently affect the molecular brake and gatekeeper residues (N550 and V565 in FGFR2). Here, we characterize the activity of the next-generation covalent FGFR inhibitor, KIN-3248, in preclinical models of FGFR2 fusion+ ICC harboring a series of secondary kinase domain mutations, in vitro and in vivo. We also test select FGFR3 alleles in bladder cancer models. KIN-3248 exhibits potent selectivity for FGFR1-3 and retains activity against various FGFR2 kinase domain mutations, in addition to being effective against FGFR3 V555M and N540K mutations. Notably, KIN-3248 activity extends to the FGFR2 V565F gatekeeper mutation, which causes profound resistance to currently approved FGFR inhibitors. Combination treatment with EGFR or MEK inhibitors potentiates KIN-3248 efficacy in vivo, including in models harboring FGFR2 kinase domain mutations. Thus, KIN-3248 is a novel FGFR1-4 inhibitor whose distinct activity profile against FGFR kinase domain mutations highlights its potential for the treatment of ICC and other FGFR-driven cancers.

Hyperglycemia activates FGFR1 via TLR4/c-Src pathway to induce inflammatory cardiomyopathy in diabetes

Protein tyrosine kinases (RTKs) modulate a wide range of pathophysiological events in several non-malignant disorders, including diabetic complications. To find new targets driving the development of diabetic cardiomyopathy (DCM), we profiled an RTKs phosphorylation array in diabetic mouse hearts and identified increased phosphorylated fibroblast growth factor receptor 1 (p-FGFR1) levels in cardiomyocytes, indicating that FGFR1 may contribute to the pathogenesis of DCM. Using primary cardiomyocytes and H9C2 cell lines, we discovered that high-concentration glucose (HG) transactivates FGFR1 kinase domain through toll-like receptor 4 (TLR4) and c-Src, independent of FGF ligands. Knocking down the levels of either TLR4 or c-Src prevents HG-activated FGFR1 in cardiomyocytes. RNA-sequencing analysis indicates that the elevated FGFR1 activity induces pro-inflammatory responses via MAPKs–NFκB signaling pathway in HG-challenged cardiomyocytes, which further results in fibrosis and hypertrophy. We then generated cardiomyocyte-specific FGFR1 knockout mice and showed that a lack of FGFR1 in cardiomyocytes prevents diabetes-induced cardiac inflammation and preserves cardiac function in mice. Pharmacological inhibition of FGFR1 by a selective inhibitor, AZD4547, also prevents cardiac inflammation, fibrosis, and dysfunction in both type 1 and type 2 diabetic mice. These studies have identified FGFR1 as a new player in driving DCM and support further testing of FGFR1 inhibitors for possible cardioprotective benefits.

First-308: Phase III study of tinengotinib versus physician's choice in patients with FGFR-altered, chemotherapy- and FGFR inhibitor–refractory/relapsed cholangiocarcinoma.

TPS575 Background: Fibroblast growth factor (FGFR) alterations occur in 10-15% of adult patients with advanced intrahepatic cholangiocarcinoma (CCA) and 1-2% of adult patients with advanced extrahepatic cholangiocarcinoma. The first generation FGFR inhibitors (FGFRi) pemigatinib and futibatinib, have been approved for the treatment of advanced CCA with FGFR2 fusions or rearrangements after systemic chemotherapy. However, disease progression occurs within 6-9 months. Secondary polyclonal mutations in the FGFR kinase domain represent a prominent acquired resistance mechanism. Tinengotinib, a novel multi-kinase inhibitor with high potency against a variety of FGFR2 kinase domain mutations, has shown promising clinical benefit in subjects with FGFR-altered metastatic CCA who were heavily pretreated with chemotherapy and FGFRi(s) in phase I/II clinical trials. (NCT03654547, NCT04742959, NCT04919642). Methods: FIRST-308 is a phase III, randomized, global multicenter study to evaluate the efficacy and safety of oral tinengotinib versus Physician’s Choice in subjects with FGFR-altered, chemotherapy- and FGFRi-refractory/relapsed CCA. Approximately 200 subjects will be enrolled in US, Europe, and Asia. Key eligibility criteria include age ≥ 18 years, ECOG performance status 0 or 1, documentation of FGFR2 fusion/rearrangement gene status, prior treatment with at least one line of chemotherapy and exactly one prior FDA-approved FGFRi for unresectable or metastatic disease. The study includes Part A and Part B. The Part A is to select a dose for Part B. Eligible subjects will be randomized in a 2:2:1 ratio to receive tinengotinib 8 mg QD, tinengotinib 10 mg QD or Physician's Choice (FOLFOX or FOLFIRI) in Part A or 2:1 in Part B to receive the recommended Part B dose or Physician's Choice. The stratification factors at randomization include geographic region, prior lines of chemotherapy (1 or ≥ 2) and Physician’s choice. Tinengotinib will be administered orally QD in 28-day cycles. Subjects will continue to receive study treatment until confirmed disease progression, unacceptable toxicity, withdrawal of consent, or termination of study by Sponsor, whichever occurs first. For Part A, the primary endpoint is safety/tolerability; secondary endpoints include objective response rate (ORR), duration of response (DOR) and PK analysis. For Part B, the primary endpoint is progression-free survival (PFS); secondary endpoints include overall survival (OS), ORR, DOR, safety, quality of life and population PK. Exploratory endpoints will assess the correlations between baseline FGFR2 alterations by cfDNA and efficacy, and exposure-response in terms of efficacy and safety. Study is open for enrollment. Clinical trial information: NCT05948475 .

Association of FGFR2 structural alterations in intrahepatic cholangiocarcinomas with female gender and younger age.

537 Background: FGFR2 fusions and other rearrangements are targetable alterations due to the advent of FGFR2 kinase domain inhibitors, and are considerably more common in intrahepatic cholangiocarcinomas (iCCA) than other cancer types. Understanding clinical factors associated with FGFR2 fusions and other rearrangements may guide precision oncology efforts. Prior studies indicate that FGFR2 alterations are more common in younger patients, but it remains unknown whether other demographics are specifically associated with FGFR2 structural alterations. Here, we reviewed the frequency of FGFR2 structural alterations in 8,898 iCCA patients from the AACR Genie and the Foundation Medicine databases. Methods: We reviewed the Foundation Medicine (n= 7,934) and AACR GENIE database (n=964) for intrahepatic cholangiocarcinomas. We identified iCCAs with FGFR2 structural alterations and mutations in other common driver genes and analyzed their distribution by age and sex. Results: FGFR2 structural alterations were more frequent in female (10.4%) compared to male (6.4%) patients across both cohorts. FGFR2 structural alterations had an association with an odds ratio of 2.5 (95% CI 1.6-3.7) and 1.7 (95% CI-2.0) with the female gender in the AACR GENIE and Foundation Medicine databases, respectively. The frequency of FGFR2 fusions/rearrangement was highest during reproductive years (up to age 40) and subsequently declined with age. This decline was greater in females than males. Mutations in other genes associated with iCCA including non-fusion FGFR2 alterations, and mutations in ARID1A, KRAS, and TP53 were not associated with gender or age. Conclusions: Our results show that FGFR2 structural alterations are significantly enriched in younger female patients with iCCA. Clinical assessment of FGFR2 alterations should be prioritized in this patient population, and future clinical studies in this molecular subtype should ensure inclusive enrollment practices. Further research is warranted to determine if the pathogenesis of FGFR2 structural alterations are driven by female biology including the involvement of sex hormones and the potential impact on the development of acquired resistance to FGFR2 kinase inhibitors.

Overcoming drug resistance of cancer cells by targeting the FGF1/FGFR1 axis with honokiol or FGF ligand trap.

Chemoresistance of cancer cells, resulting from various mechanisms, is a significant obstacle to the effectiveness of modern cancer therapies. Targeting fibroblast growth factors (FGFs) and their receptors (FGFRs) is becoming crucial, as their high activity significantly contributes to cancer development and progression by driving cell proliferation and activating signaling pathways that enhance drug resistance. We investigated the potential of honokiol and FGF ligand trap in blocking the FGF1/FGFR1 axis to counteract drug resistance. Using PEAQ-ITC, we verified direct interaction of honokiol with the FGFR1 kinase domain. We then demonstrated the effect of FGF1/FGFR1 inhibition on taltobulin resistance in cells expressing FGFR1. Finally, we generated drug-resistant clones by prolonged exposure of cells with negligible FGFR levels to taltobulin alone, taltobulin and honokiol, or taltobulin and FGF ligand trap. We demonstrated for the first time a direct interaction of honokiol with the FGFR1 kinase domain, resulting in inhibition of downstream signaling pathways. We revealed that both honokiol and FGF ligand trap prevent FGF1-dependent protection against taltobulin in cancer cells expressing FGFR1. In addition, we showed that cells obtained by long-term exposure to taltobulin are resistant to both taltobulin and other microtubule-targeting drugs, and exhibit elevated levels of FGFR1 and cyclin D. We also found that the presence of FGF-ligand trap prevents the development of long-term resistance to taltobulin. Our results shed light on how blocking the FGF1/FGFR1 axis by honokiol and FGF ligand trap could help develop more effective cancer therapies, potentially preventing the emergence of drug-resistant relapses.

EX VIVO DRUG SENSITIVITY EVALUATION OF A RARE ZMYM2::FGFR1+ LEUKEMIA PATIENT

Introduction: 8p11 myeloproliferative syndrome (EMS) is a hematologic malignancy caused by a translocation of FGFR1, most commonly joined with zinc- finger domain ZNF198 (ZMYM2). Pemigatinib is approved for FGFR fusion positive myeloid/lymphoid neoplasms (MLNs). Material and Methods: We analyzed samples and clinical data from a patient with a ZMYM2::FGFR1 translocation. Ex vivo drug sensitivity assays examined patient mononuclear cells isolated from blood and bone marrow. ZMYM2::FGFR1 transformed BaF3 cells were evaluated with single agent and drug combination panels. Sanger sequencing of the FGFR portion of the fusion sought to identify potential Ponatanib resistance mutations. Results: This case involves a 36-year-old man with 8p11 myeloproliferative syndrome and a ZMYM2::FGFR1 fusion along with a RUNX1R201Q mutation treated with HyperCVAD with Ponatinib, then HyperCVAD with Ponatinib and Bortezomib followed by Pemigatinib. Hematologic response suggested efficacy of Pemigatinib alone. Over time, the patient developed resistance to Ponatinib while maintaining sensitivity to Pemigatinib. Sequencing identified an amino acid substitution (FGFR1F686L; ZMYM2::FGFR1F1171L) within the kinase domain that could contribute to Ponatinib resistance. Bortezomib and Axitinib exhibited high efficacy on the patient’s sample. Ex vivo drug assays performed on ZMYM2::FGFR1 transformed BaF3 cells confirmed the high effectiveness of Pemigatinib and Ponatinib. Other FGFR inhibitors, including Olverematinib, AZD4547, Axitinib, Cediranib, Dovitinib, and Lenvatinib, showed exquisite sensitivity. Conclusions: Ex vivo drug sensitivity assays demonstrated the high efficacy of FGFR inhibitors in ZMYM2::FGFR1 fusion-positive leukemia cells and a fusion- expressing cell line. Mutations in the FGFR1 kinase domain (ZMYM2::FGFR1F1171L) may contribute to Ponatinib insensitivity. Further investigations are needed to determine if Pemigatinib alone is sufficient for treating ZMYM2::FGFR1 fusion-positive patients.

Landscape of Clinical Resistance Mechanisms to FGFR Inhibitors in FGFR2-Altered Cholangiocarcinoma.

FGFR inhibitors are effective in FGFR2-altered cholangiocarcinoma, leading to approval of reversible FGFR inhibitors, pemigatinib and infigratinib, and an irreversible inhibitor, futibatinib. However, acquired resistance develops, limiting clinical benefit. Some mechanisms of resistance have been reported, including secondary FGFR2 kinase domain mutations. Here, we sought to establish the landscape of acquired resistance to FGFR inhibition and to validate findings in model systems. We examined the spectrum of acquired resistance mechanisms detected in circulating tumor DNA or tumor tissue upon disease progression following FGFR inhibitor therapy in 82 FGFR2-altered cholangiocarcinoma patients from 12 published reports. Functional studies of candidate resistance alterations were performed. Overall, 49 of 82 patients (60%) had one or more detectable secondary FGFR2 kinase domain mutations upon acquired resistance. N550 molecular brake and V565 gatekeeper mutations were most common, representing 63% and 47% of all FGFR2 kinase domain mutations, respectively. Functional studies showed different inhibitors displayed unique activity profiles against FGFR2 mutations. Interestingly, disruption of the cysteine residue covalently bound by futibatinib (FGFR2 C492) was rare, observed in 1 of 42 patients treated with this drug. FGFR2 C492 mutations were insensitive to inhibition by futibatinib but showed reduced signaling activity, potentially explaining their low frequency. These data support secondary FGFR2 kinase domain mutations as the primary mode of acquired resistance to FGFR inhibitors, most commonly N550 and V565 mutations. Thus, development of combination strategies and next-generation FGFR inhibitors targeting the full spectrum of FGFR2 resistance mutations will be critical.

First in human (FIH) phase 1/1b study evaluating KIN-3248, a next-generation, irreversible pan-FGFR inhibitor (FGFRi), in patients (pts) with advanced urothelial carcinoma (UC) and other solid tumors harboring FGFR2 and/or FGFR3 gene alterations.

TPS593 Background: FGFR1-4 gene alterations are infrequent across solid tumors though preclinical and clinical evidence indicate activating alterations drive oncogenesis and tumor growth. They can be found in up to 20% of patients with UC and up to 35% of upper tract UC. Reversible FGFRi are approved for the treatment of pts with locally advanced or metastatic urothelial carcinoma (UC) with susceptible FGFR2 or FGFR3 genetic alterations (erdafitinib) or metastatic CCA harboring FGFR2 gene fusions or rearrangements (pemigatinib and infigratinib). A critical limitation of current clinical-stage FGFRi is the emergence of secondary, on-target resistance mutations (mutn) that reduce duration of response, and indeed, about 70% of CCA patients pre-treated with FGFRi exhibit secondary FGFR2 kinase domain resistance mutn at the time of relapse. KIN-3248 is a next-generation, selective, irreversible, small molecule pan-FGFRi, structurally designed to inhibit primary FGFR oncogenic alterations as well as secondary kinase domain mutn associated with disease progression. Preclinically, KIN-3248 has favorable pharmaceutical properties, is well-tolerated with continuous, daily oral administration in GLP toxicology studies and is efficacious against primary FGFR2 and FGFR3 oncogenic driver alterations as well as secondary FGFR2 resistance mutn (e.g., gatekeeper and molecular brake) in human cancer cell and PDX models. Methods: This is a FIH, multicenter, non-randomized Ph1 study of KIN-3248 in adult pts with advanced and metastatic solid tumors (AMST) harboring FGFR2 and/or FGFR3 gene alterations. KIN-3248 is given PO QD continuously in 28-day cycles until drug intolerance or disease progression. Part A is a dose-escalation assessing single agent KIN-3248 via a BOIN design to determine the MTD/RP2D. Part B will evaluate a selected dose of KIN-3248 in 3 cohorts of pts (UC, CCA, or other AMST), each driven by specified FGFR alterations—FGFRi-naïve and -pretreated pts are eligible in both parts. Enrollment criteria include ECOG PS 0-1, intact organ function, prior receipt of standard treatment or medical judgment that such is not appropriate. Pts may have measurable or evaluable disease. Key exclusion criteria include known active brain metastases and active/uncontrolled HBV/HCV. Planned sample size is ~120 pts. Primary endpoints are safety/tolerability (Part A), and preliminary antitumor activity: objective response rate, disease control rate, duration of response, & duration of stable disease (Part B). Secondary objectives include pharmacokinetic and pharmacodynamic assessments including measures of FGFR pathway modulation. The study is actively enrolling patients in the US and globally. Clinical trial information: NCT05242822 .

Use of Apatinib as a Bait to Fish its Unexpected Kinase Targets from the Hepatocellular Carcinoma Druggable Kinome

Apatinib is a tyrosine kinase inhibitor that cognately blocks the kinase activity of vascular endothelial growth factor receptor (VEGFR) signaling for the treatment of advanced gastric cancer (GC). However, the drug is also clinically found to reposition a significant suppressing potency on hepatocellular carcinoma (HCC). In this study, we reported the successful use of Apatinib as a bait to fish its potential kinase targets from the HCC druggable kinome pool. In the procedure, cell viability assays observed that the Apatinib has a potent cytotoxicity on human HCC cell lines. Dynamics simulations and affinity scoring systematically created an intermolecular interaction profile of Apatinib with ontologically enriched kinases in the HCC druggable kinome, from which the top-hit kinases were considered as potential candidates. It is revealed that the inhibitor has a weak potency on the well-established HCC target of ErbB pathway, but exhibits potent activity against some known targets or regulators of HCC. In particular, kinase assays substantiated that Apatinib can effectively inhibit four FGFR family members with moderate or high activity. In addition, the clinical FGFR1 gatekeeper mutation V561M was also observed to considerably impair the inhibitory activity, thus causing a drug resistance. Molecular modeling suggested that the Apatinib adopts two distinct binding modes to separately interact with wild-type and gatekeeper-mutant FGFR1 kinase domain.

First in human (FIH) phase 1/1b study evaluating KIN-3248, a next-generation, irreversible pan-FGFR inhibitor (FGFRi), in patients (pts) with advanced cholangiocarcinoma (CCA) and other solid tumors harboring FGFR2 and/or FGFR3 gene alterations.

TPS637 Background: FGFR1-4 gene alterations are infrequent across solid tumors though preclinical and clinical evidence indicate activating alterations drive oncogenesis and tumor growth. Pharmacological inhibition of FGFR1-4 leads to tumor shrinkage and disease control. Reversible FGFRi are approved for the treatment of pts with locally advanced or metastatic CCA harboring FGFR2 gene fusions or rearrangements (pemigatinib and infigratinib) or metastatic urothelial carcinoma (UC) with susceptible FGFR2 or FGFR3 genetic alterations (erdafitinib). A critical limitation of current clinical-stage FGFRi is the emergence of secondary, on-target resistance mutations (mutn) that reduce duration of response, and indeed, about 70% of CCA patients treated with either reversible or irreversible FGFRi exhibit secondary FGFR2 kinase domain resistance mutn at the time of relapse. KIN-3248 is a next-generation, selective, irreversible, small molecule pan-FGFRi, structurally designed to inhibit primary FGFR oncogenic alterations as well as secondary kinase domain mutn associated with disease progression. Preclinically, KIN-3248 has favorable pharmaceutical properties, is well-tolerated with continuous, daily oral administration in GLP toxicology studies and is efficacious against primary FGFR2 and FGFR3 oncogenic driver alterations as well as secondary FGFR2 resistance mutn (e.g., gatekeeper and molecular brake) in human cancer cell and PDX models. Methods: This is a FIH, multicenter, non-randomized Ph1 study of KIN-3248 in adult pts with advanced and metastatic solid tumors (AMST) harboring FGFR2 and/or FGFR3 gene alterations. KIN-3248 is given PO QD continuously in 28-day cycles until drug intolerance or disease progression. Part A is a dose-escalation assessing single agent KIN-3248 via a BOIN design to determine the MTD/RP2D; Part B will evaluate a selected dose of KIN-3248 in 3 cohorts of pts (CCA, UC, or other AMST), each driven by specified FGFR alterations—FGFRi-naïve and -pretreated pts are eligible in both parts. Enrollment criteria include ECOG PS 0-1, intact organ function, prior receipt of standard treatment or medical judgment that such is not appropriate. Pts may have measurable or evaluable disease. Key exclusion criteria include known active brain metastases and active/uncontrolled HBV/HCV. Planned sample size is ~120 pts. Primary endpoints are safety/tolerability (Part A), and preliminary antitumor activity: objective response rate, disease control rate, duration of response, and duration of stable disease (Part B). Secondary objectives include pharmacokinetic and pharmacodynamic assessments including measures of FGFR pathway modulation. The study is actively enrolling patients in the US and globally. Clinical trial information: NCT05242822 .

Design and rationale of a first-in-human (FIH) phase 1/1b study evaluating KIN-3248, a next-generation, irreversible (irrev), pan-FGFR inhibitor (FGFRi), in adult patients with solid tumors harboring FGFR2 and/or FGFR3 gene alterations (NCT05242822).

TPS9601 Background: FGFR1-4 gene alterations are observed in approximately 7% of all human cancers. There are currently 3 FDA-approved, reversible FGFRi for treatment of patients w/previously treated, locally advanced or metastatic (met) cholangiocarcinoma (CCA) harboring FGFR2 gene fusions/rearrangements (pemigatinib and infigratinib) or met urothelial carcinoma (UC) w/susceptible FGFR2 or FGFR3 genetic alterations (erdafitinib). A major limitation of approved and clinical-stage FGFRi is emergence of secondary, on-target resistance mutations (mutn) that reduce duration of response. Up to 67% of CCA patients treated with either reversible or irrev FGFRi exhibit secondary FGFR2 kinase domain resistance mutn at the time of relapse. KIN-3248 is a next-generation, selective, irrev, small molecule pan-FGFRi, structurally designed to inhibit primary FGFR oncogenic alterations as well as secondary kinase domain mutn associated w/disease progression. Preclinically, KIN-3248 has favorable pharmaceutical properties, is well-tolerated with continuous, daily oral administration in 28d GLP toxicology studies in rats and beagle dogs and is efficacious against primary FGFR2 and FGFR3 oncogenic driver alterations as well as secondary FGFR2 resistance mutn (e.g., gatekeeper and molecular brake) in human cancer cell and PDX models in vitro and in vivo. Methods: This is a FIH, multicenter, non-randomized Ph1 study of KIN-3248 in adult pts with advanced & metastatic solid tumors (AMST) harboring FGFR2 and/or FGFR3 gene alterations. KIN-3248 is given po qd continuously in 28-day cycles until drug intolerance or disease progression. Planned sample size is approx. 120 pts: Part A is a dose-escalation to MTD for pts w/AMST having either FGFR2 and/or FGFR3 alterations. Part A assesses single agent KIN-3248; Part B will evaluate a selected dose of KIN-3248 in 3 cohorts of pts (ICC, UC, or other AMST), each driven by specified FGFR alterations. Standard Ph1 enrollment criteria are required (ECOG PS 0-1, normal organ function, prior receipt of standard treatment or medical judgment that such is not appropriate). Pts may have measurable or evaluable disease. Key exclusion criteria include known active brain metastases and active/uncontrolled HBV/HCV. FGFRi-naïve & -pretreated patients are both eligible. Primary endpoints are safety/tolerability (Part A), and preliminary antitumor activity: objective response rate, disease control rate, duration of response, & duration of stable disease (Part B). Secondary objectives include pharmacokinetic and pharmacodynamic assessments including measures of FGFR pathway modulation. Enrollment is expected to commence in April 2022. Clinical trial information: NCT05242822.

Activity of KIN-3248, a next-generation pan-FGFR inhibitor, against acquired FGFR-gatekeeper and molecular-brake drug resistance mutations.

461 Background: Oncogenic FGFR (FGFR1, FGFR2, FGFR3, and FGFR4) gene alterations are observed in approximately 7% of all human cancers and present as point mutations, small intragenic deletions, genomic amplifications, or chromosomal rearrangements/ gene fusions. FGFR2 gene fusions and FGFR3 activating alterations are predicted oncogenic drivers in 10-20% of cholangiocarcinoma and 20-35% of urothelial cancers, respectively. While currently approved FGFR inhibitors (e.g., erdafitinib, pemigatinib, infigratinib) and those in development (e.g., futibatinib) provide clinical benefit in these cancer indications, disease progression typically occurs within 6-8 months of starting treatment and is often associated with the emergence of on-target resistance mutations within the FGFR kinase domain. KIN-3248 is a next-generation, irreversible, small molecule, pan-FGFR inhibitor designed to target clinically relevant primary FGFR driver alterations and secondary resistance mutations, including FGFR2 and FGFR3 gatekeeper, molecular brake, and activation loop mutations. Methods: KIN-3248 was evaluated across wild-type FGFR family members and clinically relevant fusions and kinase domain resistance mutations in vitro. In addition, KIN-3248 activity was assessed in FGFR-driven and FGFR inhibitor-resistant human gastrointestinal xenograft tumor models. Results: KIN-3248 exhibited low nanomolar biochemical potency against wild-type FGFR family members as well as mutants associated with resistance to FGFR inhibitors (IC50 3.9 – 24.1 nM). Consistently, KIN-3248 was active in human FGFR2-PHGDH fusion-positive CCLP-1 and FGFR2-OPTN fusion-positive ICC13-7 cholangiocarcinoma cell lines engineered to express wild-type or clinically relevant gatekeeper, molecular brake, and activation loop mutant alleles (EC50 2.4 – 9.9 nM). Lastly, KIN-3248 led to dose-dependent tumor growth inhibition and regressions in FGFR inhibitor-resistant, patient-derived gastric cancer and cholangiocarcinoma models harboring secondary FGFR2 kinase domain mutations. This efficacy was accompanied by both pharmacodynamic biomarker modulation, downstream pathway inhibition, and apoptotic cell death across models in vitro and in vivo. Conclusions: KIN-3248 is a next-generation, irreversible, orally available, small molecule pan-FGFR inhibitor that overcomes clinically observed secondary mutations in FGFR2 and FGFR3 known to drive resistance to both reversible and irreversible FGFR inhibitors and associated with disease progression. Its highly-selective, potent and broad-spectrum activity against mutations in both the FGFR2 and FGFR3 kinase domains – including gatekeeper, molecular brake, and activation loop alterations – is unique among FGFR inhibitors and has the potential to extend the clinical response of cancer patients with FGFR-altered tumors.

Abstract P02-02: First results of RLY-4008, a potent and highly selective FGFR2 inhibitor in a first-in-human study in patients with FGFR2-altered cholangiocarcinoma and multiple solid tumors

Abstract INTRODUCTION: Oncogenic FGFR2 alterations (fusions/rearrangements, amplifications, mutations) are key drivers in cholangiocarcinoma (CCA) and multiple solid tumors. Current pan-FGFR inhibitor (FGFRi) therapy is limited by off-isoform toxicity and acquired FGFR2 kinase domain resistance mutations. RLY-4008 is a highly selective and potent oral inhibitor designed to target both FGFR2 driver and resistance mutations. We initiated a first-in-human study in advanced solid tumors patients (pts) to define the safety, pharmacokinetics (PK) and efficacy of RLY-4008 (NCT04526106). METHODS: Adult pts received RLY-4008 QD or BID on a 4-week cycle following a BOIN escalation design. Adverse events (AEs), PK, ctDNA and anti-tumor activity (RECIST 1.1) were assessed. RESULTS: As of 16AUG21, 45 pts (35 CCA; 10 other) have been treated with RLY-4008 at total daily doses of 30-200 mg (18 pts BID; 27 pts QD). 44 pts had oncogenic FGFR2 alterations (26 fusions/13 mutations/5 amplifications). The median number of prior anti-neoplastic therapies was 3 (range 1-15). 94% (33/35) of CCA pts had prior chemotherapy and 69% (24/35) had prior FGFRi. 56% (9/16) CCA pts with prior FGFRi and evaluable ctDNA had ≥1 FGFR2 resistance mutation at baseline, most commonly at positions 549 (8/9), 617 (3/9), or 564 (2/9). RLY-4008 had rapid absorption (Tmax 1-7h), half-life to support QD dosing (18-34 h), dose-dependent exposure (AUC; Cmax) and predicted FGFR2 occupancy >85% across dose levels. The MTD has not been defined, and QD dose exploration continues to select the optimal biologically efficacious dose. AEs occurring in >20% of pts include stomatitis (49%), palmar-plantar erythrodysesthesia (PPE, 38%), dry mouth (29%), and nail toxicities (22%), majority of which were ≤Gr 2. 6 pts had Gr 1-2 retinopathy, which resolved in all cases. 5 AEs were considered dose limiting toxicities: 4 in BID (rash/PPE/mucositis/hyperbilirubinemia) and 1 in QD (retinopathy). No Gr 4/5 drug-related AEs were seen. 25 pts remain on treatment (range 1-37 weeks). RLY-4008 showed broad anti-tumor activity across dose levels and FGFR2 alterations with radiographic tumor reductions of ≥10% in 59% pts (19/32; -11% to -83%). Activity was seen in FGFRi-naïve, FGFR2-fusion+ CCA with PRs in 50% of pts (3/6, 2 confirmed and 1 pending confirmation; -56% to -83%). Activity was also seen in FGFRi pre-treated FGFR2-fusion+ CCA pts (N=16) with 16 SD, including 9 pts with tumor reduction ≥10% (from -12% to -35%). Of the FGFRi pre-treated FGFR2-fusion+ CCA patients with detectable FGFR2 resistance mutations in ctDNA at baseline, 78% (7/9) were undetectable at C2D1. CONCLUSION: RLY-4008 demonstrates promising safety, tolerability, and clinical activity in FGFR2-altered solid tumor pts, including those who progressed on prior FGFRi therapy. Consistent with the FGFR2-selective mechanism, minimal off-isoform toxicity (FGFR1-hyperphosphatemia; FGFR4-diarrhea) was seen. These encouraging data validate selective targeting of FGFR2 and suggest that RLY-4008 has potential to overcome resistance to FGFRi. Citation Format: Lipika Goyal, Mitesh Borad, Vivek Subbiah, Amit Mahipal, Suneel Kamath, Kabir Mody, Robin Katie Kelley, Richard Kim, Vaibhav Sahai, Anthony El-Khoueiry, Efrat Dotan, Oleg Schmidt-Kittler, Jinshan Shen, Kai Yu Jen, Alicia Deary, Wei Guo, Mahesh Padval, Cori Ann J. Sherwin, Charles Ferte, Beni Wolf, Alison M. Schram. First results of RLY-4008, a potent and highly selective FGFR2 inhibitor in a first-in-human study in patients with FGFR2-altered cholangiocarcinoma and multiple solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P02-02.

FGFR2 Extracellular Domain In-Frame Deletions Are Therapeutically Targetable Genomic Alterations That Function as Oncogenic Drivers in Cholangiocarcinoma.

We conducted next-generation DNA sequencing on 335 biliary tract cancers and characterized the genomic landscape by anatomic site within the biliary tree. In addition to frequent FGFR2 fusions among patients with intrahepatic cholangiocarcinoma (IHCC), we identified FGFR2 extracellular domain in-frame deletions (EID) in 5 of 178 (2.8%) patients with IHCC, including two patients with FGFR2 p.H167_N173del. Expression of this FGFR2 EID in NIH3T3 cells resulted in constitutive FGFR2 activation, oncogenic transformation, and sensitivity to FGFR inhibitors. Three patients with FGFR2 EIDs were treated with Debio 1347, an oral FGFR1/2/3 inhibitor, and all showed partial responses. One patient developed an acquired L618F FGFR2 kinase domain mutation at disease progression and experienced a further partial response for 17 months to an irreversible FGFR2 inhibitor, futibatinib. Together, these findings reveal FGFR2 EIDs as an alternative mechanism of FGFR2 activation in IHCC that predicts sensitivity to FGFR inhibitors in the clinic. SIGNIFICANCE: FGFR2 EIDs are transforming genomic alterations that occur predominantly in patients with IHCC. These FGFR2 EIDs are sensitive to FGFR inhibition in vitro, and patients with these alterations benefited from treatment with FGFR inhibitors in the clinic.This article is highlighted in the In This Issue feature, p. 2355.

PATH-22. COMPREHENSIVE ANALYSIS OF DIVERSE LOW-GRADE NEUROEPITHELIAL TUMORS WITH FGFR1 ALTERATIONS REVEALS A DISTINCT MOLECULAR SIGNATURE OF ROSETTE-FORMING GLIONEURONAL TUMOR

Abstract The FGFR1 gene encoding fibroblast growth factor receptor 1 has emerged as a frequently altered oncogene in the pathogenesis of multiple low-grade neuroepithelial tumor (LGNET) subtypes including pilocytic astrocytoma (PA), dysembryoplastic neuroepithelial tumor (DNT), rosette-forming glioneuronal tumor (RGNT), and extraventricular neurocytoma (EVN). These activating FGFR1 alterations in LGNET can include tandem duplication of the exons encoding the intracellular tyrosine kinase domain, in-frame gene fusions most often with TACC1 as the partner, or hotspot missense mutations within the tyrosine kinase domain (either p.N546 or p.K656). However, the specificity of these different FGFR1 events for the various LGNET subtypes and accompanying genetic alterations are not well defined, nor are the histopathologic features of pilocytic astrocytomas with FGFR1 alterations versus those harboring the more common BRAF mutations or fusions. Here we performed comprehensive genomic and epigenomic characterization on a diverse cohort of 30 LGNET with FGFR1 alterations. We identified that RGNT harbors a distinct epigenetic signature compared to other LGNET with FGFR1 alterations, and is uniquely characterized by FGFR1 kinase domain hotspot missense mutations in combination with either PIK3CA or PIK3R1 mutation, often with accompanying NF1 or PTPN11 mutation. In contrast, EVN harbors its own distinct epigenetic signature and is characterized by FGFR1-TACC1 fusion as the solitary pathogenic alteration. Additionally, DNT and PA are characterized by either kinase domain tandem duplication or hotspot missense mutations, occasionally with accompanying NF1 or PTPN11 mutation, but lacking the accompanying PIK3CA or PIK3R1 mutation that characterizes RGNT. The glial component of LGNET with FGFR1 alterations typically has a predominantly oligodendroglial morphology, and many of the pilocytic astrocytomas with FGFR1 alterations lack the biphasic pattern, piloid processes, and Rosenthal fibers that characterize pilocytic astrocytomas with BRAF mutation or fusion. Together, this analysis refines the classification and histopathologic spectrum of LGNET with FGFR1 alterations.

Comprehensive analysis of diverse low-grade neuroepithelial tumors with FGFR1 alterations reveals a distinct molecular signature of rosette-forming glioneuronal tumor

The FGFR1 gene encoding fibroblast growth factor receptor 1 has emerged as a frequently altered oncogene in the pathogenesis of multiple low-grade neuroepithelial tumor (LGNET) subtypes including pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor (DNT), rosette-forming glioneuronal tumor (RGNT), and extraventricular neurocytoma (EVN). These activating FGFR1 alterations in LGNET can include tandem duplication of the exons encoding the intracellular tyrosine kinase domain, in-frame gene fusions most often with TACC1 as the partner, or hotspot missense mutations within the tyrosine kinase domain (either at p.N546 or p.K656). However, the specificity of these different FGFR1 events for the various LGNET subtypes and accompanying genetic alterations are not well defined. Here we performed comprehensive genomic and epigenomic characterization on a diverse cohort of 30 LGNET with FGFR1 alterations. We identified that RGNT harbors a distinct epigenetic signature compared to other LGNET with FGFR1 alterations, and is uniquely characterized by FGFR1 kinase domain hotspot missense mutations in combination with either PIK3CA or PIK3R1 mutation, often with accompanying NF1 or PTPN11 mutation. In contrast, EVN harbors its own distinct epigenetic signature and is characterized by FGFR1-TACC1 fusion as the solitary pathogenic alteration. Additionally, DNT and pilocytic astrocytoma are characterized by either kinase domain tandem duplication or hotspot missense mutations, occasionally with accompanying NF1 or PTPN11 mutation, but lacking the accompanying PIK3CA or PIK3R1 mutation that characterizes RGNT. The glial component of LGNET with FGFR1 alterations typically has a predominantly oligodendroglial morphology, and many of the pilocytic astrocytomas with FGFR1 alterations lack the biphasic pattern, piloid processes, and Rosenthal fibers that characterize pilocytic astrocytomas with BRAF mutation or fusion. Together, this analysis improves the classification and histopathologic stratification of LGNET with FGFR1 alterations.

Abstract 1097: FGFR2 in-frame indels: A novel targetable alteration in intrahepatic cholangiocarcinoma

Abstract Biliary cancers include intrahepatic cholangiocarcinoma (IHCC), extrahepatic cholangiocarcinoma (EHCC), and gallbladder cancer (GBC). These cancers are clinically, pathologically, and biologically heterogeneous, but are all aggressive with poor prognosis and few available therapies. Potentially actionable genomic alterations described to date include IDH1 mutations, FGFR2 fusions, BRAF V600E mutations, HER2 amplification/mutation, and BRCA1/2 mutations. FGFR inhibitors have demonstrated encouraging efficacy in patients with FGFR2 oncogenic fusions; however, the efficacy of these inhibitors in patients with other types of FGFR alterations is less clear. We analyzed somatic alterations in 272 patients with biliary cancers (135 IHCCs, 34 EHCCs, and 59 GBCs) that underwent targeted OncoPanel sequencing at Dana-Farber Cancer Institute. The identified oncogenic alterations were similar to those previously reported in cholangiocarcinoma genomic landscapes. Mutational signature analyses found genomic features of microsatellite instability in 5 cases, APOBEC in 4 cases, and homologous recombination repair deficiency in 7 cases. Clinicopathological and outcome data for these patients are being collected to evaluate for associations with genomic findings. 16 FGFR2 translocations involving intron 17 were identified in the IHCC cohort (14% of evaluable IHCCs). Surprisingly, 5 IHCCs (4% of evaluable cases) harbored extracellular domain FGFR2 indels, of which p.W290_I291delinsC was previously reported to show oncogenic activity in vitro. Two cases harbored an identical FGFR2 p.H167_N173del. In vitro expression of this deletion resulted in oncogenic transformation, and that this growth could be inhibited by FGFR inhibitors. The two patients with FGFR2 p.H167_N173del were treated with an FGFR-1/2/3 inhibitor (Debio-1347), and both achieved durable partial response (PR) of over 11 months. We followed the first patient with tumor sequencing at five timepoints during her care and found that at resistance to Debio-1347, she developed an FGFR2 kinase domain mutation (p.L617F) that was subsequently demonstrated to confer resistance to Debio-1347 in vitro. This patient was then treated with a second FGFR inhibitor and experienced a PR lasting 17 months. At resistance to this second FGFR inhibitor, the tumor was biopsied and found to harbor a previously undetected BRAF p.L597Q mutation. The second patient remains on active treatment. Our data show that extracellular domain FGFR2 in-frame indels are rare but targetable novel oncogenic alterations in IHCC. An expanded search of AACR Project GENIE data found 18 extracellular FGFR2 in-frame indels in diverse tumor types, supporting further functional evaluation and clinical targeting of these indels across tumor types. Citation Format: Yvonne Y. Li, James M. Cleary, Srivatsan Raghavan, Liam F. Spurr, Qibiao Wu, Lei Shi, Lauren K. Brais, Maureen Loftus, Lipika Goyal, Anuj K. Patel, Atul B. Shinagare, Thomas E. Clancy, Geoffrey Shapiro, Ethan Cerami, William R. Sellers, William C. Hahn, Matthew Meyerson, Nabeel Bardeesy, Andrew D. Cherniack, Brian M. Wolpin. FGFR2 in-frame indels: A novel targetable alteration in intrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1097.

Abstract 5933: Biochemical characterization of FGFR2-PPHLN1 and BCR-FGFR1, drivers of epithelial and hematological malignancies

Abstract Fibroblast growth factor receptors (FGFRs) are part of the receptor tyrosine kinase (RTK) family and are essential in the activation of various downstream signaling pathways, which are necessary for cell differentiation and proliferation. However, mutation and translocation of FGFRs leads to aberrant activation of signaling, which often results in cancer. This work focuses on the FGFR2-PPHLN1 fusion protein, a driver of intrahepatic cholangiocarcinoma (ICC), and the BCR-FGFR1 fusion protein, a driver of stem cell leukemia/lymphoma (SCLL). Both FGFR2-PPHLN1 and BCR-FGFR1 are poorly characterized, resulting in few therapies and clinical advancements for patients positive for these oncogene-driven neoplasms. Here, we aim to characterize these two FGFR fusion proteins, and analyze therapeutic options to treat these malignancies. Both FGFR2-PPHLN1 and BCR-FGFR1 contain a coiled-coil dimerization domain fused to a constitutively activated kinase provided by either FGFR2 or FGFR1, respectively. This work shows the reliance of these FGFR fusion proteins on the tyrosine kinase activity of the respective FGFR, as kinase dead mutants were not biologically active. Additionally both FGFR2-PPHLN1 and BCR-FGFR1 expressing cells exhibited an over activation of the RAS/MAPK and JAK/STAT pathways, indicating the importance of these pathways in FGFR1-PPHLN1 driven ICC, and BCR-FGFR1 driven SCLL, respectively. Transformed cells expressing FGFR2-PPHLN1 are sensitive to FGFR inhibitor BGJ398. However, this sensitivity is abolished when a kinase activating mutation is introduced with FGFR2(N549K)-PPHLN1. Residue N549 in the FGFR2 kinase domain functions as part of a molecular brake; mutation of this residue disrupts the auto-inhibitory network of hydrogen bonds, leading to increased kinase activation. The loss in sensitivity to BJG398 treatment for cells expressing FGFR2(N549K)-PPHLN1 indicates that BGJ398 treatment alone may be ineffective once secondary mutations are established in the kinase domain. Preliminary data suggests that transformed cells expressing either FGFR2-PPHLN1, or kinase activated FGFR2(N549K)-PPHLN1, may be sensitive to treatment with FGFR inhibitor TAS-120, suggesting that TAS-120 treatment could be beneficial for patients with FGFR2-PPHLN1 driven ICC. Furthermore, we establish BCR-FGFR1 as a client of the Hsp90 chaperone complex. Transformed cells expressing BCR-FGFR1 are sensitive to the Hsp90 inhibitor, Ganetespib, and also respond to combined treatment with Ganetespib and FGFR inhibitor BGJ398, suggesting novel clinical treatment options for patients positive for this fusion. Collectively, we show that the tyrosine kinase activity provided by FGFR2 or FGFR1, respectively is required for the biological activity of these fusion proteins, and we establish novel treatment options for patients with FGFR2-PPHLN1 or BCR-FGFR1 driven malignancies. Citation Format: Malalage Nicole Peiris, Fangda Li, April N. Meyer, Dalida Warda, Daniel J. Donoghue. Biochemical characterization of FGFR2-PPHLN1 and BCR-FGFR1, drivers of epithelial and hematological malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5933.

Acquired On-Target Clinical Resistance Validates FGFR4 as a Driver of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide with no clinically confirmed oncogenic driver. Although preclinical studies implicate the FGF19 receptor FGFR4 in hepatocarcinogenesis, the dependence of human cancer on FGFR4 has not been demonstrated. Fisogatinib (BLU-554) is a potent and selective inhibitor of FGFR4 and demonstrates clinical benefit and tumor regression in patients with HCC with aberrant FGF19 expression. Mutations were identified in the gatekeeper and hinge-1 residues in the kinase domain of FGFR4 upon disease progression in 2 patients treated with fisogatinib, which were confirmed to mediate resistance in vitro and in vivo. A gatekeeper-agnostic, pan-FGFR inhibitor decreased HCC xenograft growth in the presence of these mutations, demonstrating continued FGF19-FGFR4 pathway dependence. These results validate FGFR4 as an oncogenic driver and warrant further therapeutic targeting of this kinase in the clinic. SIGNIFICANCE: Our study is the first to demonstrate on-target FGFR4 kinase domain mutations as a mechanism of acquired clinical resistance to targeted therapy. This further establishes FGF19-FGFR4 pathway activation as an oncogenic driver. These findings support further investigation of fisogatinib in HCC and inform the profile of potential next-generation inhibitors.See related commentary by Subbiah and Pal, p. 1646.This article is highlighted in the In This Issue feature, p. 1631.