FGFR Inhibitor Research Articles

Fibroblast growth factor receptor inhibitors mitigate the neuropathogenicity of Borrelia burgdorferi or its remnants ex vivo.

In previous studies, we showed that fibroblast growth factor receptors (FGFRs) contribute to inflammatory mediator output from primary rhesus microglia in response to live Borrelia burgdorferi. We also demonstrated that non-viable B. burgdorferi can be as pathogenic as live bacteria, if not more so, in both CNS and PNS tissues. In this study we assessed the effect of live and non-viable B. burgdorferi in inducing FGFR expression from rhesus frontal cortex (FC) and dorsal root ganglion (DRG) tissue explants as well as their neuronal/astrocyte localization. Specific FGFR inhibitors were also tested for their ability to attenuate inflammatory output and apoptosis in response to either live or non-viable organisms. Results show that in the FC, FGFR2 was the most abundantly expressed receptor followed by FGFR3 and FGFR1. Non-viable B. burgdorferi significantly upregulated FGFR3 more often than live bacteria, while the latter had a similar effect on FGFR1, although both treatments did affect the expressions of both receptors. FGFR2 was the least modulated in the FC tissues by the two treatments. FGFR1 expression was more prevalent in astrocytes while FGFR2 and FGFR3 showed higher expression in neurons. In the DRG, all three receptor expressions were also seen, but could not be distinguished from medium controls by immunofluorescence. Inhibition of FGFR1 by PD166866 downregulated both inflammation and apoptosis in both FC and DRG in response to either treatment in all the tissues tested. Inhibition of FGFR1-3 by AZD4547 similarly downregulated both inflammation and apoptosis in both FC and DRG in response to live bacteria, while with sonicated remnants, this effect was seen in one of the two FC tissues and 2 of 3 DRG tissues tested. CCL2 and IL-6 were the most downregulated mediators in the FC, while in the DRG it was CXCL8 and IL-6 in response to FGFR inhibition. Downregulation of at least two of these three mediators was observed to downregulate apoptosis levels in general. We show here that FGFR inhibition can be an effective anti-inflammatory treatment in antibiotic refractive neurological Lyme. Alternatively, two biologics may be needed to effectively curb neuroinflammation and pathology in the CNS and PNS.

439 Extracellular-in-frame deletions and kinase domain duplications are novel, gain-of-function mutations in fibroblast growth factor receptor genes in cancer

OBJECTIVES/GOALS: There are gain-of-function genomic alterations in FGFR genes that guide personalized treatment in some patients with cholangiocarcinoma (10%) and bladder cancer (30%) who can benefit from targeted therapies. We sought to evaluate other genomic alterations in cancer involving FGFRs and assess whether they are gain-of-function. METHODS/STUDY POPULATION: We collaborated with Foundation Medicine Inc (FMI), for the assessment of 300,000 sequenced tumors and a retrospective analysis of recent publications, to identify novel candidate FGFR alterations. We propose to transiently transfect HEK293T cells with an empty vector (EV), FGFR1-4 wild-type (WT), and these variants and use a luminescent-proximity based high-throughput assay, AlphaLISA, and Western blot to assess FGFR and phosphorylated downstream signaling proteins, FRS2, AKT and ERK, and their sensitivity to FGFR inhibitors: pemigatinib, erdafitinib, futibatinib, RLY-4008, and TYRA-200. RESULTS/ANTICIPATED RESULTS: Through our collaboration we identified >100 novel candidate FGFR1-4 variants of unknown significance (VUS) including extracellular-in-frame deletions (EIDs), kinase domain duplications (KDDs), insertions/deletions (INDELs), short number variants (SNVs), and truncations. Immunoblot analysis confirmed the presence of desired EV, FGFR WT, and VUS’ in HEK293T cells. We anticipate the FGFR EIDs and KDDs to display an increased presence of in the respective pFGFR, pFRS2, pERK, and pAKT as compared to the EV and FGFR WT by both immunoblot and AlpahLISA analysis. Additionally, we anticipate the VUS’ to be sensitive to FGFR inhibitors: pemigatinib, erdafitinib, futibatinib, RLY-4008, and TYRA-200 using the AlphaLISA assay. DISCUSSION/SIGNIFICANCE: These findings suggest that the novel FGFR VUS’ are capable of constitutive activation of FGFR kinase activity, and they preliminary demonstrate that these newly identified FGFR alterations are therapeutically targetable. Thus, providing rationale for further clinical evaluation to identify new cohorts of FGFR inhibitor responders.

Abstract 1930: Combination of KRASG12C and FGFR1 inhibitors as a resistance-overcoming therapeutic strategy for maximizing therapeutic impact of KRASG12C inhibitors

Abstract INTRODUCTION: Lung cancer is the leading cause of cancer-related deaths worldwide. KRAS is the most frequent mutated driver gen in lung adenocarcinoma (LUAD). KRASG12C inhibitors (G12Ci) have revealed promising results in the clinic, having being approved two G12Ci for the treatment of LUAD patients, which marks the first approved targeted therapy for KRAS-mutant tumors. Nevertheless, these therapies face the same limitation as other targeted therapies, the therapeutic potential of these inhibitors can be impaired by resistance mechanisms. Deciphering resistance mechanisms to G12Ci is of prime relevance to predict which patients may benefit from these therapies and to propose resistance-overcoming therapeutic strategies for maximizing therapeutic impact of these inhibitors. MATERIAL AND METHODS: We generated 10 acquired resistant LUAD cell lines to G12Ci (Sotorasib and Adagrasib), exposing sensitive cells to increasing concentrations of drugs. All the acquired resistant models were characterized at the proteomic (phospho-Array and Western Blot) and transcriptomic (WTS) levels to find signatures that define the resistance.The efficacy of G12Ci was tested in vitro in a panel of 8 cell lines and 7 PDX-derived organoids (PDXDO) models and in vivo in 7 patient-derived xenografts (PDX) models to classify them as resistant, sensitive or partially sensitive. The efficacy of the combination of FGFR1 and G12Ci was tested in vitro in the panel of 10 acquired resistant cell lines but also in the parental cell lines and PDXDOs (sensitive and intrinsic resistant) and in vivo in the intrinsic resistant PDXs. RESULTS AND DISCUSSION: The characterization of 10 acquired resistant cell lines at the proteomic and transcriptomic levels showed differences in the expression and/or activation of Fibroblast Growth Factor Receptors (FGFRs) in more than 50% of them. The combination of KRASG12C and FGFR1 inhibitors in all acquired resistant cell lines showed efficacy, regardless of whether acquired resistance was mediated or not by FGFR1 overexpression/overactivation.In addition, the combination of KRASG12C and FGFR1 inhibitors showed more efficacy than the monotherapies in most intrinsically resistant cell lines, PDXDOs and PDXs models. Finally, we tested the combination with FGFR1i in sensitive or partially responsive models, observing an improvement in efficacy compared to monotherapy with G12Ci. CONCLUSIONS: The activation or overexpression of FGFR1 acts as a mechanism of acquired resistance to KRASG12C inhibitors.-The combination of FGFR and KRASG12C inhibitors is effective as an acquired/intrinsic resistance-overcoming therapeutic strategy in cell lines, PDXDOs and PDXs models.-The combination of FGFR and KRASG12C inhibitors is also synergistic for sensitive models, which could maximize therapeutic impact of KRASG12C inhibitors. Citation Format: Alba Santos, Patricia Plaza, Marta Jimenez, David Gómez-Sánchez, Luis Paz-Ares, Irene Ferrer. Combination of KRASG12C and FGFR1 inhibitors as a resistance-overcoming therapeutic strategy for maximizing therapeutic impact of KRASG12C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1930.

Abstract 1228: Selective FGFR4 inhibitor Irpagratinib (ABSK011) exhibits broad synergistic and combinatory anti-tumor effects with other therapeutic agents in preclinical HCC models

Abstract Background: Hepatocellular carcinoma (HCC) is the most common primary liver cancer, ranking as the sixth most prevalent cancer and the third leading cause of death worldwide. Approved therapeutic drugs, including chemotherapy, anti-angiogenesis agents and immunotherapy-based combination regimen (atezolizumab plus bevacizumab), have shown suboptimal clinical benefits for patients with HCC. There are critical unmet medical needs for additional therapeutic options to address this challenge. Deregulation of FGF19-FGFR4 signaling accounts for roughly 30% of HCC and plays a pivotal role in driving HCC tumorigenesis. To date, several FGFR4 inhibitors have advanced to the clinical stage for the treatment of HCC patients. Among them, a recently discovered novel and highly selective inhibitor, Irpagratinib (ABSK011), has demonstrated the potential to become a first-in-class or best-in-class FGFR4 inhibitor. It showed promising anti-tumor activity in an ongoing phase Ib clinical study, with an ORR of 40.7% observed in FGF19 overexpressed late line HCC patients in cohorts treated with BID regimens. To further expand the therapeutic potential of irpagratinib, we carried out an array of combination treatments and revealed its broad synergistic effects with various immune and targeted agents in treating HCC. Methods: We evaluated the combination effects of Irpagratinib with a variety of therapeutic agents, covering both targeted and immune-oncology agents, in preclinical HCC in vivo models. The tested agents, including lenvatinib, atezolizumab plus bevacizumab, anti-PD-(L)1, SHP2 inhibitors and EGFR antagonists, were assessed across a range of preclinical models, such as cell-derived or patient-derived xenografts, engineered syngeneic models and humanized models. Results: Synergistic anti-tumor effects of irpagratinib were observed in combination with the indicated agents, demonstrating more profound effects than either single agent alone in the selected preclinical in vivo models. This resulted in significant effects on inducing tumor regression or inhibiting tumor progression. Conclusions: In summary, these findings collectively illustrate very broad synergistic anti-tumor effects of irpagratinib when combined with various other therapeutic agents. These results may pave the road for potential novel combinatory therapeutic strategies that could expand the utility of irpagratinib and provide innovative and more effective therapies to HCC patients. Citation Format: Nannan Zhang, Bin Shen, Zhidong He, Cheng Dai, Jie Wang, Jie Zhang, Manqi Liu, Yongxian Zhang, Zhui Chen. Selective FGFR4 inhibitor Irpagratinib (ABSK011) exhibits broad synergistic and combinatory anti-tumor effects with other therapeutic agents in preclinical HCC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1228.

Abstract 1965: 3HP-2827-high selective inhibitor of FGFR2, a best-in-class therapy for FGFR2-driven solid tumors

Abstract FDA-approved pan-FGFR inhibitors bring encouraging results in solid tumors. However, resistance mechanisms and side effect profiles may limit their clinical utility. We designed 3HP-2827, a highly selective and potent small molecule inhibitor of FGFR2, to treat solid tumors harboring FGFR2 alterations, including amplifications, mutations, and fusions to overcome these limitations. 3HP-2827 inhibits FGFR2 enzyme activity with more than 1623-fold selectivity over FGFR1 and FGFR3. 3HP-2827 inhibits cell proliferation with IC50 <10 nM and 30 nM in FGFR2 amplified and FGFR2-mutant cancer cell lines, respectively. 3HP-2827 demonstrates high kinome selectivity for FGFR2 against a panel of 485 human kinases, resulting in 90.1% inhibition of FGFR2, and no other kinases showed greater than 75% inhibition. 3HP-2827 leads only modest activity at 10 µM against PDE4D2 and LCK in 44 distinct common adverse drug reaction targets, supporting its high degree of safety. 3HP-2827 also demonstrates a dose-dependent inhibition of phosphorylation of FGFR2 and FGFR2 signaling pathway nodes, including ERK and AKT, in the FGFR2-dependent cancer cell line. In vivo, 3HP-2827 demonstrates potent antitumor activity in cell- and patient-derived xenograft mouse models, including FGFR2-amplified gastric cancer, FGFR2-mutant (N549K) endometrial cancer, FGFR2 fusion ICC and gastric cancers. Strikingly, 3HP-2827 demonstrates superior activity in patient-derived ICC and gastric cancer, cell-derived endometrial cancer (CDX) in tumor and pFGFR2 inhibitions. In the ICC PDX model, 3HP-2827 also induces the regression of pemigatinib-resistant tumors. 3HP-2827 is well tolerated in the toxicology studies, and no hyperphosphatemia or tissue mineralization is observed at 150 mg/kg/kg/day in rats and 120 mg/kg/day in dogs. The data support that 3HP-2827, a potential to be a best-in-class selective FGFR2 inhibitor, will be studying clinically at the beginning of 2024. Citation Format: Susan S. Liu-Chen, Jufang Lin, Jingyu Hu, Yanchao Liu, Weibo Zhang, Zhixiong Zhang, Yan Ding, Jinqiu Liu, Kevin Chen, Shaojing Hu. 3HP-2827-high selective inhibitor of FGFR2, a best-in-class therapy for FGFR2-driven solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1965.

Abstract 1968: Discovery of a highly potent and selective FGFR2 inhibitor for FGFR2-driven cancers

Abstract Fibroblast growth factor receptor 2 (FGFR2) is a clinically validated target and frequently altered in many solid tumors. The resulting oncogenic driver alterations often drive multiple solid tumors. Therefore, targeting FGFR2 has broad therapeutic potential. Currently, only pan-FGFR inhibitors are approved for FGFR2-driven intrahepatic cholangiocarcinoma, but not for other FGFR2-driven cancers. Due to the severe on-target toxicity associated with inhibition of FGFR1 and FGFR4, the use of the pan-FGFR inhibitors has been very limited. Thus, developing a selective FGFR2 inhibitor addresses the unmet medical need as the treatment options for FGFR2-altered cancers remain limited. Here we report the discovery and characterization of a highly selective FGFR2 inhibitor, ACE-16229210. This molecule potently inhibits FGFR2 (IC50 = 6.2 nM) and exhibits excellent selectivity (>133-fold) over other FGFR1 and FGFR4 in biochemical assays. It also potently (IC50 <1 nM) and selectively (≥500-fold) inhibits FGFR2-induced ERK phosphorylation in multiple cancer cell lines harboring FGFR2 fusions, amplification, and mutations, but not those harboring FGFR1, FGFR3, or FGFR4 genetic alterations (IC50 > 460 nM). Furthermore, this molecule, when evaluated in a panel of 24 cell lines, demonstrates potent anti-proliferative activity against the cell lines harboring FGFR2, but not FGFR3, and especially FGFR1, or FGFR4 genetic alterations. Thus, ACE-16229210 is a potent and selective FGFR2 inhibitor. Interestingly, it exhibits the most potent activity against gatekeeper and molecular brake mutations followed by fusions, amplification, and activation loop mutations. This molecule shows a robust broad spectrum of antitumor activity with significant tumor regression at low doses (1-10 mg/kg) in several tumor xenograft and PDX models representing the major FGFR2 relevant tumor histologies including gastric, breast, ovarian, and endometrial cancers harboring clinically important FGFR2 driver alterations with a well-defined pharmacokinetic/pharmacodynamic relationship. This molecule also demonstrates strong synergistic effects when combined with irinotecan and fulvestrant in a gastric tumor model and a breast cancer PDX model harboring FGFR2 amplification, respectively. Finally, ACE-16229210 did not significantly affect serum phosphorus levels in animals at the exposure levels that are >100-fold higher than the efficacious AUC, further confirming its excellent selectivity over FGFR1. Taken together, these in vitro and in vivo studies show that ACE-16229210 is a potent and selective FGFR2 inhibitor with the potential to be developed into a more effective treatment option for multiple cancers harboring FGFR2 oncogenic driver alterations. Citation Format: Wenqian Li, Bin Liu, Junmei Wang, Tinggui Yin, Kuo-Long Yu, Sanjeev Kumar, Zhiming Wen, Genshi Zhao, Weitao Pan. Discovery of a highly potent and selective FGFR2 inhibitor for FGFR2-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1968.

Abstract 4575: FGFR3 altered bladder cancer exhibits a lineage-dependent vulnerability

Abstract Fibroblast Growth Factor 3 (FGFR3) genomic alterations are oncogenic drivers in ~15% of bladder cancer patients. A novel FGFR inhibitor, Erdafitinib, was FDA approved in 2019 based off a Phase II trial with an overall response rate of 40%. However, the majority of patients rapidly relapse at a median of 6 months, requiring combination therapies to prevent drug resistance. We hypothesized that a genome-wide CRISPR screen would identify Erdafitinib-sensitizing co-targets. We screened UM-UC-14, an S249C FGFR3 mutated bladder cancer cell line, and identified Cyclin C as a top hit in addition to known MAPK effectors such as ERK2 and SHP2. Cyclin C is a non-cell cycle related cyclin and part of the CDK8/19 kinase module of Mediator. Pharmacological validation in 4 FGFR3 altered cell lines confirmed a high degree of synergy between Erdafitinib and the CDK8/19 inhibitor Sel120, while no effect was seen in 2 wildtype cell lines, suggesting strong specificity. Moreover, the synergy in FGFR3-mutant cells was superior to that of SHP2 inhibitor SHP099 or the ERK inhibitor BVD-523. Similarly, GATA3 and PPARG also scored highly in our CRISPR screen. These transcription factors have been shown to be sufficient to enforce a luminal identity but not necessarily to maintain it. We validated these 3 genes as sensitizers and show in luminal cells that loss of either GATA3 or PPARG leads to loss of luminal markers and gain of basal markers. In contrast, cells treated with Sel120 showed, on RNAseq, an increase in basal markers without loss of luminal markers. Interestingly, our data suggests that the gain of basal markers, commonly associated with a worse prognosis, is associated with a strong sensitization to FGFR inhibition in FGFR3 alteration driven bladder cancer. We conducted a xenograft experiment confirming that mice responded well to single agent Erdafitinib, but without frank tumor regression. By contrast, Sel120+Erdafitinib induced complete responses in 8/10 mice compared to 6/10 mice for SHP099+Erdafitinib. Remarkably, after 60 days of daily drugging and a 20-day washout, the tumors did not grow back in the complete responders. Importantly, no loss of body weight or other signs of morbidity were found. Our results suggest an immediate and promising benefit in combining either CDK8/19 or SHP2 inhibitors with Erdafitinib in treating patients with FGFR3 alteration driven bladder cancers. Furthermore, we identified a strategy in targeting lineage modulators as sensitizers to existing therapies. Citation Format: Mohamad Karim Koleilat, Yan Zhu, Lawrence Kwong. FGFR3 altered bladder cancer exhibits a lineage-dependent vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4575.

Abstract 160: FGFR1+ cancer associated fibroblasts (CAFs) produce an extracellular matrix (ECM) that curbs infiltration of phagocytic tumor-associated macrophages (TAMs)

Abstract About 40% of diffuse large B-cell lymphoma (DLBCL) pts do not respond to immunochemotherapy. In human and murine models, we and others have shown that the lymphoma microenvironment (LME) changes with disease progression and treatment resistance. This lymphoma cell-LME co-evolutionary process results in expansion of treatment-resistant lymphoma cells as well as reduced infiltration of T cells and reprogramming of CAFs and TAMs into immune suppressive entities. Our analysis of relapse or refractory (RR)-DLBCLs showed not only an immune depleted LME but also an enrichment of CAFs harboring activation of fibroblast growth factor receptor (FGFR1+). Since FGFR1 inhibitors (FGFR1i) are clinically available, we sought to determine its anti-neoplastic effect on DLBCL. We first confirmed that lymphoma cells, including murine and human, and primary and RR-DLBCLs, did not express FGFR1. Accordingly, no anti-tumoral effect was detected when the selective FGFR1i SSR128129E was administered in vitro to a lymphoma cell panel. However, oral administration of FGFR1i to pre-clinical lymphoma models including two RR-DLBCL patient-derived xenografts (PDX) and four syngeneic murine models representing distinct DLBCL genetic subtypes produced a significant anti-lymphoma effect in all cases. In addition to decreased lymphoma cells, we observed increase in the ECM and TAM infiltration, even in the murine models, suggesting that FGFR1i reprogrammed the CAFs. We thus conducted matrisome analysis (i.e., ECM proteomics) in a murine model (p53 mutant) and found a significant up-regulation of proteoglycans and the immune active glycoproteins decorin, lumican and byglican (q-value <0.05 vs. vehicle). To determine whether these ECM changes affect lymphoma cells, we performed 3D co-cultures of lymphoma cells embedded into de-celularized ECM obtained from FGFR1i vs. vehicle mice. We found that FGFR1i-ECM (vs. vehicle-ECM) modifies the secretome of lymphoma cells by upregulating several cytokines including IL6, IL16 and TNF-alpha. To further characterize the functional state of CAFs and TAMs, we conducted single cell RNA-seq in the two RR-DLBCL PDXs treated as before. We found that FGFR1-CAFs in the LME of FGFR1i tumors (vs. vehicle) were reprogrammed into inflammatory-like CAFs characterized by the expression of several monocyte/macrophage chemotactic factors including CCL2, CCL7, CXCL3, and CXCL12. Consequent with these findings we not only observed an increase infiltration of TAMs in the LME of FGFR1i tumors but also that they were reprogrammed in phagocytic phenotypes. In sum, our results suggest that CAF-FGFR1+ produce an immune “silent” ECM that characterizes RR-DLBCL while FGFR1i reverses this ECM phenotype ultimately resulting in the infiltration of antitumoral TAMs, delineating a novel ECM-directed therapeutic approach for this disease. Citation Format: Nicolás Di Siervi, Maria Victoria Revuelta, Nahuel Zamponi, Giorgio Inghirami, Leandro Cerchietti. FGFR1+ cancer associated fibroblasts (CAFs) produce an extracellular matrix (ECM) that curbs infiltration of phagocytic tumor-associated macrophages (TAMs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 160.

Abstract 5947: Discovery of ACE-47228211, a highly potent and selective SOS1 inhibitor

Abstract Dysregulation of receptor tyrosine kinase (RTKs) and KRAS signaling is the most frequent causes of cancer development and progression. SOS1 is one of the major guanine nucleotide exchange factors (GEFs) that facilitates the conversion of the inactive GDP-bound KRAS “off” state to the activated GTP-bound KRAS “on” state. SOS1 is therefore required for dysregulated RTKs, and wildtype and mutant KRAS proteins to drive cancer cells. In addition, SOS1 plays a crucial role in modulating the reactivation of the RAS/MAPK signaling upon the treatment of RAS/MEK/ERK inhibitors. Thus, blocking SOS1 and KRAS protein-protein interactions could be an effective strategy for targeting KRAS-MAPK driven cancers. Here, we report the discovery of a highly potent and orally bioavailable small molecule SOS1 inhibitor, ACE-47228211. This molecule has a unique novel scaffold. It exhibits an IC50 of 8.5 nM in the SOS1-KrasG12D (GDP) exchange assay and 5.0 nM in cellular ERK phosphorylation assay in H1957 cells. ACE-47228211 at 3 mg/kg (BID) demonstrated significant tumor growth inhibition in MiaPaca-2 KRAS G12C tumor model than MRTX0920 at 50 mg/kg (BID). When combined with Sotorasib (5 mg/kg QD), a Kras G12C inhibitor, ACE-47228211 at 3 mg/kg exhibited synergistic effect similar to MRTX0920 at 50 mg/kg. Further, ACE-47228211 demonstrated synergistic effects with EGFR and FGFR2 inhibitors in RTK mutant tumor models. ACE-47228211 has excellent ADME and physical properties with low risks of hERG, CYP inhibition and drug-drug interactions. The evaluation of ACE-47228211 in a safety panel of 87 targets and a kinase panel of >500 targets shows that this molecule has favorable selectivity and safety profiles. The molecule is currently in IND enabling stage of development. Citation Format: Junmei Wang, Wenqian Li, Sanjeev Kumar, Weitao Pan, Zhiming Wen, Tinggui Yin, Genshi Zhao, Kuo-Long Yu. Discovery of ACE-47228211, a highly potent and selective SOS1 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5947.

Abstract 2857: Perspectives from a systematic review of kinase fusion oncogenes and access to corresponding kinase inhibitor clinical trials in pediatric, adolescent, and young adult solid malignancies

Abstract Oncogenic kinase fusions (KFs) have been reported in pediatric, adolescent, and young adult (P-AYA) brain tumors, sarcomas, and rare solid malignancies. Molecularly targeted therapy (MTT) matched to KFs generally produce objective responses even in advanced P-AYA malignancies. KFs occur across a broad age range of P-AYA cancer diagnoses (dx) so data on KF prevalence and the relationship between KFs and clinical characteristics including response and resistance to MTT is distributed across many studies. To address resulting knowledge gaps, we conducted a systematic review of KFs reported in P-AYA cancers and assessed the availability of clinical trials (CTs) for P-AYA patients with KF malignancies. A systematic literature search was conducted with PubMed using publication date 2013-2022, search terms for dx, age, and gene names of interest (BRAF, RAF1, NTRK1-3, ALK, FGFR1-4, MET, RET, ROS1). Known P-AYA cancer sequencing studies were also reviewed. Papers were included if they reported ≥1 patient <18 yo with a KF cancer of any type or ≤30 yo with a KF P-AYA cancer dx. Data abstracted included paper type, KF gene and partner, dx, fusion breakpoints, age, stage, MTT received, response to MTT, and CT enrollment. Clinicaltrials.gov was queried for interventional CTs in the U.S. assessing relevant MTTs. We identified 531 studies (299 case reports/series; 119 P-AYA sequencing studies) reporting 4,329 patients with a KF P-AYA solid tumor. Brain tumors had, in decreasing order of frequency, BRAF, NTRK, FGFR, ALK, ROS1, RAF1, MET, and RET fusions. Dx in which KFs were reported were LGG (n=2451), HGG (n=298), embryonal tumors (n=16), ependymomas (n=8), meningioma (n=1), and other (n=16). Extracranial solid tumors (EST) had, in decreasing order of frequency, NTRK, RET, ALK, BRAF, ROS1, MET, FGFR, and RAF1 fusions. Dx in which KFs were reported were thyroid cancer (n=607), sarcoma (n=546), melanoma and spitz tumor (n=183), renal tumor (n=90), carcinoma (n=57), rhabdomyosarcoma (n=12), neuroblastoma (n=11), osteosarcoma (n=9), and other (n=24). As of Aug 23, 2023, 476 CTs were assessing 65 inhibitors relevant for these KFs, of which only 20 are currently FDA-approved. Patients <18 yo were only eligible for 16% of CTs. RET (47%), RAF/BRAF (33%), and NTRK (32%) inhibitors had the highest percent of CTs enrolling patients <18 yo while ERK (4%) and FGFR (6%) inhibitors had the least. Notably, while 467 P-AYA patients identified in our review were <18 yo and had an ALK- or ROS1-fusion positive tumor, only 24% of ALK or ROS1 inhibitor CTs allowed enrollment of patients <18 yo. To our knowledge, this is the first systematic review of KFs in P-AYA solid tumors. Oncogenic KFs are common and occur across P-AYA solid and brain tumor dx demonstrating a need for CTs of relevant MTTs including patients <18 yo, particularly for drugs targeting ALK, ROS1, and FGFR. Citation Format: Lorena Lazo de la Vega, Sebastian K. Eder, Ellen Sukharevsky, Zachary A. Kahn, Michaela O'Connell, Franziska Wachter, Emily P. Anderson, Maria C. Trissal, Jenna J. LaBelle, Kendall Carpenter, Leo Kager, Mariella G. Filbin, Katherine A. Janeway. Perspectives from a systematic review of kinase fusion oncogenes and access to corresponding kinase inhibitor clinical trials in pediatric, adolescent, and young adult solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2857.

Abstract 727: FGFR4 promotes colon cancer progression through CAF activation via the CXCL10-CXCR3 axis

Abstract Cancer-associated fibroblasts (CAFs) contribute to cancer progression via crosstalk with cancer and immune cells in the tumor microenvironment (TME). Despite the significant role of CAF, the understanding of the trigger mechanisms of cancer-mediated CAF activation remains unclear. We previously reported the mechanism by which FGFR4 in colon cancer secretes amphiregulin (AREG), resulting in anti-EGFR chemoresistance. It motivated us to investigate whether FGFR4 overexpressing tumors may change the TME, so we explored the mechanism of CAF activation under the FGFR4 signaling pathway. CAF was isolated from BALB/C mice after injecting 1 × 106 CT-26/EV or FGFR4 cells using microbeads and conditioned media (CM) was prepared by filtering after 48h cancer cell culture with serum-free medium In mice model, CT-26/FGFR4 showed more significant tumor progression and CAF abundance (α-SMA, FAP, PDGFR, and vimentin) than CT-26/EV. Additionally, CT26/FGFR4-derived CAFs showed significantly increased invasiveness and contractility, indicating the highly activated status of CAFs. In order to verify the effect of cancer secretome from FGFR4 in cancer cells, normal fibroblast (NIH/3T3) were co-cultured with either stable cancer cells or CM. As a result, CT-26/FGFR4 cells robustly induced the expression of CAF markers in NIH/3T3 cells, indicating the promotion of CAF differentiation from resting fibroblasts. Also, CT-26/FGFR4-derived CM significantly promoted further activation of CAFs compared to CT-26/EV-derived CM. Taken together, FGFR4 contributes not only to CAF differentiation but also to CAF activation via secretome. Next, we performed RNA seq of CT-26/EV and CT-26/FGFR4 cells and identified the upregulated genes related with the interferon (IFN) signaling. Among these genes, we identified IFNβ and CXCL10 as targetable molecules via the activation of TLR3-TBK-IRF and IFN-STAT-CXCL10 signaling pathways. These results were confirmed by RT-PCR and western blot assay. Secreted CXCL10 induced gene expression of CAF markers in fibroblast and increased CAF functions such as migration, invasion and contractibility, reflecting CAF activation. Blocking antibodies and knocking down of CXCL10 inhibited the cancer secretome-mediated CAF markers. The blockade of CXCR3, which is the CXCL10 cognate receptor, also showed an inhibitory effect on the FGFR4-overexpressing cancer-educated CAF. In human colon cancer specimens, the expression of FGFR4, CXCL10, and CAF markers showed a positive correlation with each other. Finally, dual inhibition of FGFR4 and CXCR3 in tumor mouse model suppressed the tumor growth, accompanied by CAF suppression. Our findings reveal the novel mechanism of FGFR4 leading CAF differentiation/activation in TME via the CXCL10-CXCR3 axis. This suggests that FGFR4/CXCR3 inhibitor could be used as a potential therapeutic strategy to attenuate CAF in stromal dominant tumors. Citation Format: Sang-Hee Cho, Hyunjin Bang, Eun-Gene Sun, Ji-Na Choi, Mi-Ra Park, Hyun-Jeong Shim, Jun-Eul Hwang, Woo-Kyun Bae, Ik Joo Chung. FGFR4 promotes colon cancer progression through CAF activation via the CXCL10-CXCR3 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 727.

Abstract 5582: Discovery and preclinical characterization of ISM8001, a covalent and selective FGFR2/FGFR3 dual inhibitor with strong monotherapy anti-tumor activity against advanced solid tumors

Abstract Oncogenic alterations in fibroblast growth factor receptors 2 and 3 (FGFR2 and FGFR3) are key drivers in intrahepatic cholangiocarcinoma, urothelial carcinoma and multiple other solid tumors. Currently approved pan-FGFR inhibitor therapy is limited by off-isoform toxicity (FGFR1-mediated hyperphosphatemia and FGFR4-mediated diarrhea) and acquired FGFR2 or FGFR3 kinase domain resistance mutations. The second generation of FGFR inhibitors currently undergoing clinical evaluation such as RLY-4008 and LOXO-435 are primarily focused on achieving selectivity against either the FGFR2 or FGFR3 isoform, which may narrow their clinical potential. To overcome this limitation and to allow a favorable toxicity profile, we designed and evaluated the anti-cancer effects of a novel and selective FGFR2/FGFR3 dual inhibitor, ISM8001. Biochemically, ISM8001 showed superior inhibition against FGFR2 and FGFR3 with >90-fold and >800-fold selectivity of FGFR2 over FGFR1 and FGFR4, respectively. As an irreversible inhibitor, ISM8001 displayed strong affinity and high efficiency of covalent bond formation with FGFR2/3. Additionally, ISM8001 showed broad inhibition against multiple FGFR2/3 mutant isoforms, especially gatekeeper mutation (FGFR2-V564F and FGFR3-V555M). ISM8001 inhibited cell proliferation of multiple human cancer cell lines with FGFR2/3 aberration with IC50 <10 nM while IC50 >1 µM was observed in cell lines without FGFR2/3 alterations. Furthermore, ISM8001 demonstrated robust in vivo anti-tumor efficacy in multiple cell line-derived xenograft mouse models including the SNU-16 gastric cancer model (FGFR2-amplification), the AN3CA endometrial cancer model (FGFR2 N549K mutation) and the RT4 bladder cancer model (FGFR3-fusion), with tumor growth inhibition varying from 80%~120% while no significant serum phosphate increase or body weight loss. ISM8001 also displayed strong anti-tumor effects in xenograft models of Ba/F3-FGFR2/3 engineered cell lines with different gatekeeper mutations. ISM8001 is classified as a BCS II compound with favorable DMPK properties and druggability, high permeability, low efflux ratio and low DDI risk. In vivo PK profiles showed moderate to low clearance and high oral bioavailability across preclinical species. In addition, ISM8001 showed a promising in vitro safety profile with favorable selectivity in 468-kinase and Cerep-44 panel studies, and no hERG concerns. Results of 28-day non-clinical toxicology studies in rat and dog showed a safety window of approximately 2-5 fold based on efficacious exposure in different models. Taken together, these data support the clinical development of ISM8001 as a novel, selective FGFR2/FGFR3 dual inhibitor for the potential tissue-agnostic therapy of advanced solid tumors with FGFR2/3 aberrations. Citation Format: Yihong Zhang, Yazhou Wang, Xin Cai, Jinxin Liu, Hui Cui, Junwen Qiao, Xiaoxia Lin, Xiao Ding, Supriya Bavadekar, Sujata Rao, Man Zhang, Feng Ren, Alex Zhavoronkov. Discovery and preclinical characterization of ISM8001, a covalent and selective FGFR2/FGFR3 dual inhibitor with strong monotherapy anti-tumor activity against advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5582.

Abstract 2884: Resistance to pan-FGFR inhibition is determined by the EMT phenotype associating with clinical outcome in HNSCC

Abstract Receptor tyrosine kinases (RTK) and integrins coalesce at focal adhesions. These cell membrane areas serve as huge signaling hubs critically contributing to central aspects of cancer cell survival and therapy resistance. Despite the fact that these receptors mutually interact, co-dependencies between these receptors and associated therapeutically exploitable vulnerabilities remain largely unclear in HPV-negative head and neck squamous cell carcinoma (HNSCC). Here, we evaluated the cytotoxic and radiochemosensitizing potential of targeting 10 RTK and β1 integrin in up to 20 different 3D matrix grown HNSCC cell models. RNA sequencing and protein-based biochemical assays were performed for molecular and pathway characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts to show clinical relevance. Our observations demonstrate that fibroblast growth factor receptors (FGFR 1-4) present with the strongest cytotoxic and radiosensitizing potential, both as monotherapy and in combination with β1 integrin inhibition, surpassing the efficacy of the other investigated RTK. Pharmacological pan-FGFR inhibition elicited a variable response spectrum ranging from cytotoxicity/radiochemosensitization to resistance/radioprotection. Transcriptomic characterization revealed an association of these contrasting responses to FGFR inhibition with a mesenchymal-to-epithelial transition (MET) for sensitive cell models and a partial epithelial-to-mesenchymal transition (EMT) for resistant cell models. Accordingly, deactivation of EMT associated kinases like EGFR, PKC and PAK proved effective in diminishing the adaptive FGFR-driven resistance. Importantly, the translation of the adaptive resistance profiles to HNSCC patient cohorts showed its prognostic value and provided conclusive validation of the presence of EMT-related vulnerabilities that can be strategically used for therapeutic intervention. In conclusion, our study demonstrates that pan-FGFR inhibition induces both a highly beneficial radiochemosensitizing and a detrimental radioprotective effect in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically. Citation Format: Felix Broghammer, Mahesh Gouda, Irina Korovina, Cornelia Brunner, Robert P. Coppes, Olivier Gires, Michael Seifert, Nils Cordes. Resistance to pan-FGFR inhibition is determined by the EMT phenotype associating with clinical outcome in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2884.

Abstract 220: Thresholding response assessment in biliary tract cancer organoids to inform sensitivity to chemotherapy across molecular subtypes

Abstract Background: Cholangiocarcinoma (CCA) remains a highly morbid cancer for which accurate models. Patient-derived cancer organoid (PCOs) have been generated in CCA, however their utility in clinical prediction has not been validated. Z’ factoring often fails in assessing CCA organoid response. Prior analysis for organoid response has shown an effect size by Glass’s Delta (GΔ) of ≥1.25 in growth inhibition is predictive of clinical response. Here, we present subclonal organoid response across a diverse set of CCA models in response to therapy by growth and organoid level viability response. Methods: PCOs were expanded from the NCI’s Patient-Derived Model Repository (PDMR), tissue biopsy, and rapid autopsy. Organoid response was tracked from growth using Z-stacked high content imaging (Cytation5) with endpoint at 144h and stained using established viability markers, Caspase-3/7 (C3/7) and ToPro3. Treatment groups included media control, positive control of cycloheximide 200uM continuous, gemcitabine (gem) 10uM 24h, cisplatin (cis) 5uM 48h, combination gem+cis, and FGFR2 inhibitor futibatinib (futi) 300nM continuous. Results: Across models, PCO growth directly correlated with Z’ factor thresholding (R= 0.868). NCI PDMR organoids, CK11523 and CK10519 are IDH1mutant (mt), and both showed superior response to gem when compared to cis by growth (gem GΔ 1.18 and 0.44 v. cis GΔ 0.11 and 0.23) and C3/7 (gem GΔ 3.25 and 2.34 v. cis GΔ 1.46 and 0.33). BRCA1mt CCA was found to achieve modest growth inhibition with gem and gem+cis (GΔ 1.09 and 1.17) and yet significant increase in C3/7 (gem GΔ 1.68 and gem+cis GΔ 1.36) and ToPro3 (gem GΔ 1.65 and gem+cis GΔ 1.82). Despite this result, the patient developed early clinical recurrence after neoadjuvant gem+cis chemotherapy (<3 months). FGFR2-HPGDS fusion CCA achieved no meaningful difference in growth response with gem+cis (GΔ 0.94) or futi (GΔ 0.36). These findings were consistent with the clinical outcome of primary chemotherapy resistance and progression on futi. However, we observed increase in C3/7 positivity with both gem+cis (GΔ 2.11) and futi (GΔ 1.37). Conclusions: Primary resistance to both targeted and chemotherapy remains a formidable barrier to therapeutic development in CCA. While growth inhibition remains a validated marker of clinical response, further work is needed to understand thresholds for C3/7 and ToPro3 across physiologic drug dosing. This is the first report of modeling the novel FGFR2-HPGDS fusion CCA in organoids. Ongoing work is evaluating therapeutic sensitivity across prospective specimens of CCA in support of establishing thresholding for response to apoptosis (C3/7) and necrosis (ToPro3). Citation Format: Eleanor Riedl, Austin Stram, Md Shahadat Hossan, Ethan Samuel Lin, Jamie Warner, Luke Koeppel, Jeremy D. Kratz. Thresholding response assessment in biliary tract cancer organoids to inform sensitivity to chemotherapy across molecular subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 220.

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.

FGFR inhibitors in urothelial cancer: Contextualizing THOR within the new treatment landscape

As frontline treatment of advanced urothelial cancer (UC) evolves, optimal sequencing of subsequent therapies remains unclear. The phase 3 THOR trial compared the efficacy of erdafitinib to chemotherapy or immunotherapy in FGFR3/2-altered advanced UC. THOR offers valuable data informing sequencing strategies, reinforcing the need for molecular testing in UC.

FGFR-targeted therapeutics: clinical activity, mechanisms of resistance and new directions.

Fibroblast growth factor (FGF) signalling via FGF receptors (FGFR1-4) orchestrates fetal development and contributes to tissue and whole-body homeostasis, but can also promote tumorigenesis. Various agents, including pan-FGFR inhibitors (erdafitinib and futibatinib), FGFR1/2/3 inhibitors (infigratinib and pemigatinib), as well as a range of more-specific agents, have been developed and several have entered clinical use. Erdafitinib is approved for patients with urothelial carcinoma harbouring FGFR2/3 alterations, and futibatinib and pemigatinib are approved for patients with cholangiocarcinoma harbouring FGFR2 fusions and/or rearrangements. Clinical benefit from these agents is in part limited by hyperphosphataemia owing to off-target inhibition of FGFR1 as well as the emergence of resistance mutations in FGFR genes, activation of bypass signalling pathways, concurrent TP53 alterations and possibly epithelial-mesenchymal transition-related isoform switching. The next generation of small-molecule inhibitors, such as lirafugratinib and LOXO-435, and the FGFR2-specific antibody bemarituzumab are expected to have a reduced risk of hyperphosphataemia and the ability to overcome certain resistance mutations. In this Review, we describe the development and current clinical role of FGFR inhibitors and provide perspective on future research directions including expansion of the therapeutic indications for use of FGFR inhibitors, combination of these agents with immune-checkpoint inhibitors and the application of novel technologies, such as artificial intelligence.

Integration of CD4+ T cells and molecular subtype predicts benefit from PD-L1 blockade in muscle-invasive bladder cancer.

Muscle-invasive bladder cancer (MIBC) is a disease characterized by molecular and clinical heterogeneity, posing challenges in selecting the most appropriate treatment in clinical settings. Considering the significant role of CD4+ T cells, there is an emerging need to integrate CD4+ T cells with molecular subtypes to refine classification. We conducted a comprehensive study involving 895 MIBC patients from four independent cohorts. The Zhongshan Hospital (ZSHS) and The Cancer Genome Atlas (TCGA) cohorts were included to investigate chemotherapeutic response. The IMvigor210 cohort was included to assess the immunotherapeutic response. NCT03179943 was used to evaluate the clinical response to a combination of immune checkpoint blockade (ICB) and chemotherapy. Additionally, we evaluated genomic characteristics and the immune microenvironment to gain deeper insights into the distinctive features of each subtype. We unveiled four immune-molecular subtypes, each exhibiting distinct clinical outcomes and molecular characteristics. These subtypes include luminal CD4+ Thigh, which demonstrated benefits from both immunotherapy and chemotherapy; luminal CD4+ Tlow, characterized by the highest level of fibroblast growth factor receptor 3 (FGFR3) mutation, thus indicating potential responsiveness to FGFR inhibitors; basal CD4+ Thigh, which could benefit from a combination of ICB and chemotherapy; and basal CD4+ Tlow, characterized by an immune suppression microenvironment and likely to benefit from transforming growth factor-β (TGF-β) inhibition. This immune-molecular classification offers new possibilities for optimizing therapeutic interventions in MIBC.

Receptor-based pharmacophore modeling, molecular docking, synthesis and biological evaluation of novel VEGFR-2, FGFR-1, and BRAF multi-kinase inhibitors

A receptor-based pharmacophore model describing the binding features required for the multi-kinase inhibition of the target kinases (VEGFR-2, FGFR-1, and BRAF) were constructed and validated. It showed a good overall quality in discriminating between the active and the inactive in a compiled test set compounds with F1 score of 0.502 and Mathew’s correlation coefficient of 0.513. It described the ligand binding to the hinge region Cys or Ala, the glutamate residue of the Glu-Lys αC helix conserved pair, the DFG motif Asp at the activation loop, and the allosteric back pocket next to the ATP binding site. Moreover, excluded volumes were used to define the steric extent of the binding sites. The application of the developed pharmacophore model in virtual screening of an in-house scaffold dataset resulted in the identification of a benzimidazole-based scaffold as a promising hit within the dataset. Compounds 8a-u were designed through structural optimization of the hit benzimidazole-based scaffold through (un)substituted aryl substitution on 2 and 5 positions of the benzimidazole ring. Molecular docking simulations and ADME properties predictions confirmed the promising characteristics of the designed compounds in terms of binding affinity and pharmacokinetic properties, respectively. The designed compounds 8a-u were synthesized, and they demonstrated moderate to potent VEGFR-2 inhibitory activity at 10 µM. Compound 8u exhibited a potent inhibitory activity against the target kinases (VEGFR-2, FGFR-1, and BRAF) with IC50 values of 0.93, 3.74, 0.25 µM, respectively. The benzimidazole derivatives 8a-u were all selected by the NCI (USA) to conduct their anti-proliferation screening. Compounds 8a and 8d resulted in a potent mean growth inhibition % (GI%) of 97.73% and 92.51%, respectively. Whereas compounds 8h, 8j, 8k, 8o, 8q, 8r, and 8u showed a mean GI% > 100% (lethal effect). The most potent compounds on the NCI panel of 60 different cancer cell lines were progressed further to NCI five-dose testing. The benzimidazole derivatives 8a, 8d, 8h, 8j, 8k, 8o, 8q, 8r and 8u exhibited potent anticancer activity on the tested cell lines reaching sub-micromolar range. Moreover, 8u was found to induce cell cycle arrest of MCF-7 cell line at the G2/M phase and accumulating cells at the sub-G1 phase as a result of cell apoptosis.

Resistance of HNSCC cell models to pan-FGFR inhibition depends on the EMT phenotype associating with clinical outcome

BackgroundFocal adhesion signaling involving receptor tyrosine kinases (RTK) and integrins co-controls cancer cell survival and therapy resistance. However, co-dependencies between these receptors and therapeutically exploitable vulnerabilities remain largely elusive in HPV-negative head and neck squamous cell carcinoma (HNSCC).MethodsThe cytotoxic and radiochemosensitizing potential of targeting 10 RTK and β1 integrin was determined in up to 20 3D matrix-grown HNSCC cell models followed by drug screening and patient-derived organoid validation. RNA sequencing and protein-based biochemical assays were performed for molecular characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts.ResultsFibroblast growth factor receptor (FGFR 1–4) targeting exhibited the strongest cytotoxic and radiosensitizing effects as monotherapy and combined with β1 integrin inhibition, exceeding the efficacy of the other RTK studied. Pharmacological pan-FGFR inhibition elicited responses ranging from cytotoxicity/radiochemosensitization to resistance/radiation protection. RNA sequence analysis revealed a mesenchymal-to-epithelial transition (MET) in sensitive cell models, whereas resistant cell models exhibited a partial epithelial-to-mesenchymal transition (EMT). Accordingly, inhibition of EMT-associated kinases such as EGFR caused reduced adaptive resistance and enhanced (radio)sensitization to FGFR inhibition cell model- and organoid-dependently. Transferring the EMT-associated transcriptomic profiles to HNSCC patient cohorts not only demonstrated their prognostic value but also provided a conclusive validation of the presence of EGFR-related vulnerabilities that can be strategically exploited for therapeutic interventions.ConclusionsThis study demonstrates that pan-FGFR inhibition elicits a beneficial radiochemosensitizing and a detrimental radioprotective potential in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically.