FGFR1 Activation Research Articles

The novel anthraquinone compound Kanglexin prevents endothelial-to-mesenchymal transition in atherosclerosis by activating FGFR1 and suppressing integrin β1/TGFβ signaling.

Endothelial-mesenchymal transition (EndMT) disrupts vascular endothelial integrity and induces atherosclerosis. Active integrin β1 plays a pivotal role in promoting EndMT by facilitating TGFβ/Smad signaling in endothelial cells. Here, we report a novel anthraquinone compound, Kanglexin (KLX), which prevented EndMT and atherosclerosis by activating MAP4K4 and suppressing integrin β1/TGFβ signaling. First, KLX effectively counteracted the EndMT phenotype and mitigated the dysregulation of endothelial and mesenchymal markers induced by TGFβ1. Second, KLX suppressed TGFβ/Smad signaling by inactivating integrin β1 and inhibiting the polymerization of TGFβR1/2. The underlying mechanism involved the activation of FGFR1 by KLX, resulting in the phosphorylation of MAP4K4 and Moesin, which led to integrin β1 inactivation by displacing Talin from its β-tail. Oral administration of KLX effectively stimulated endothelial FGFR1 and inhibited integrin β1, thereby preventing vascular EndMT and attenuating plaque formation and progression in the aorta of atherosclerotic Apoe-/- mice. Notably, KLX (20 mg/kg) exhibited superior efficacy compared with atorvastatin, a clinically approved lipid-regulating drug. In conclusion, KLX exhibited potential in ameliorating EndMT and retarding the formation and progression of atherosclerosis through direct activation of FGFR1. Therefore, KLX is a promising candidate for the treatment of atherosclerosis to mitigate vascular endothelial injury.

Spatial and signaling overlap of growth factor receptor systems at clathrin-coated sites.

Cellular communication is regulated at the plasma membrane by the interactions of receptor, adhesion, signaling, exocytic,and endocytic proteins. Yet, the composition and control of these complexes in response to external cues remain unclear. We use high-resolution and high-throughput fluorescence imaging to map the localization of growth factor receptors and related proteins at single clathrin-coated structures in human squamous HSC3 cells. We find distinct protein signatures between control cells and cells stimulated with growth factors. Clathrin sites at the plasma membrane are preloaded with some receptors but not others. Stimulation with epidermal growth factor induces capture and concentration of epidermal growth factor, fibroblast growth factor1, and low-density lipoprotein receptor (EGFR, FGFR1, and LDLR). Regulatory proteins including ubiquitin ligase Cbl, the scaffold Grb2, and the mechanoenzyme dynamin2 are also recruited. Disrupting FGFR1 or EGFR activity with drugs prevents the recruitment of both EGFR and FGFR1. EGF was able to activate FGFR1 phosphorylation. Our data reveal novel coclustering and activation of receptors and regulatory factors at clathrin-coated sites in response to stimulation by a single growth factor, EGF or FGF. This behavior integrates growth factor signaling and allows for complex responses to extracellular cues and drugs at the plasma membrane of human cells.

Diverse Fgfr1 signaling pathways and endocytic trafficking regulate mesoderm development.

The fibroblast growth factor (FGF) pathway is a conserved signaling pathway required for embryonic development. Activated FGF receptor 1 (FGFR1) drives multiple intracellular signaling cascade pathways, including ERK/MAPK and PI3K/AKT, collectively termed canonical signaling. However, unlike Fgfr1-null embryos, embryos containing hypomorphic mutations in Fgfr1 lacking the ability to activate canonical downstream signals are still able to develop to birth but exhibit severe defects in all mesodermal-derived tissues. The introduction of an additional signaling mutation further reduces the activity of Fgfr1, leading to earlier lethality, reduced somitogenesis, and more severe changes in transcriptional outputs. Genes involved in migration, ECM interaction, and phosphoinositol signaling were significantly downregulated, proteomic analysis identified changes in interactions with endocytic pathway components, and cells expressing mutant receptors show changes in endocytic trafficking. Together, we identified processes regulating early mesoderm development by mechanisms involving both canonical and noncanonical Fgfr1 pathways, including direct interaction with cell adhesion components and endocytic regulation.

Interruption of KLF5 acetylation promotes PTEN-deficient prostate cancer progression by reprogramming cancer-associated fibroblasts.

Inactivation of phosphatase and tensin homolog (PTEN) is prevalent in human prostate cancer and causes high-grade adenocarcinoma with a long latency. Cancer-associated fibroblasts (CAFs) play a pivotal role in tumor progression, but it remains elusive whether and how PTEN-deficient prostate cancers reprogram CAFs to overcome the barriers for tumor progression. Here, we report that PTEN deficiency induced Krüppel-like factor 5 (KLF5) acetylation and that interruption of KLF5 acetylation orchestrated intricate interactions between cancer cells and CAFs that enhance FGF receptor 1 (FGFR1) signaling and promote tumor growth. Deacetylated KLF5 promoted tumor cells to secrete TNF-α, which stimulated inflammatory CAFs to release FGF9. CX3CR1 inhibition blocked FGFR1 activation triggered by FGF9 and sensitized PTEN-deficient prostate cancer to the AKT inhibitor capivasertib. This study reveals the role of KLF5 acetylation in reprogramming CAFs and provides a rationale for combined therapies using inhibitors of AKT and CX3CR1.

Abstract B014: FGFR inhibition upregulates Nectin-4 expression in FGFR3 altered urothelial carcinoma

Abstract Bladder urothelial carcinoma (UC) is a common malignancy and remains a significant cause of mortality; however, in recent years there have been substantial advances in the treatment of metastatic UC (mUC). This includes the approval of the antibody drug conjugate (ADC) enfortumab vedotin, which targets Nectin-4, and the fibroblast growth factor receptor (FGFR) inhibitor erdafitinib (in FGFR2/3 altered tumors). Despite the impressive efficacy of these agents, most patients will ultimately develop progressive disease, underlining the need for further therapeutic development. Previous work has demonstrated that NECTIN4 expression and FGFR3 alterations are enriched in luminal subtypes of UC and NECTIN4 expression is higher in FGFR altered tumors. We validate these findings in scRNAseq from primary tumors as well as bulk RNAseq from murine models. Given the positive correlation between NECTIN4 and FGFR3 alterations we hypothesized that FGFR3 inhibition would downregulate Nectin-4 expression. To our surprise we found in vitro treatment with erdafitinib in cell lines with FGFR3 fusion proteins (RT112, RT4, SW780) lead to an increase in Nectin-4 protein levels. Additionally, in vitro FGFR inhibition in the UMUC-14 cell line which harbors an FGFR3S249C hotspot mutation, upregulates Nectin-4 protein levels. To further investigate this phenomenon, we engineered the FGFR3 WT cell line, 5637 to overexpress FGFR3-TACC3 or FGFR3S249C. We saw that FGFR3-TACC3 and FGFR3S249C overexpression both down regulated Nectin-4 indicating that FGFR3 activity negatively regulates Nectin-4 expression. We have also investigated the association between FGFR inhibition and Nectin-4 expression in two in vivo models: (1) in a xenograft model using the RT112 line we found an overall increase in Nectin-4 expression in tumors treated with a 5-day course of erdafitinib; (2) in a UPFL1 (a cell line derived from a genetically engineered mouse with a S249C mutation) syngeneic model we also saw an increase in Nectin-4 expression after erdafitinib treatment. We have observed that the increase in Nectin-4 expression persists in vitro even after withdrawal of erdafitinib treatment, indicating that intermittent dosing of erdafitinib maybe be sufficient to prime a tumor for Nectin-4 targeted therapy. Lastly, preliminary in vitro investigation of the combination of erdafitinib and enfortumab vedotin in RT112 cells showed synergy, particularly with lower doses of both agents, providing further support of our hypothesis that FGFR3 inhibition may act to sensitize UC to Nectin-4 ADC therapy. A potential synergistic relationship is further supported by the recent results of the phase 1 trial investigating the combination of erdafitinib plus enfortumab vedotin in which the objective response rate was 100% (9/9 patients: 8 PRs & 1CR). Citation Format: Sean Clark-Garvey, Mi Zhou, Michael Sturdivant, Wolfgang Beckabir, Lucia Kim, E. Drew Toomer, Ian McCabe, Akihiro Hamada, Daniel Crona, Jeffrey S. Damrauer, William Y. Kim. FGFR inhibition upregulates Nectin-4 expression in FGFR3 altered urothelial carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B014.

FGFR2-triggered autophagy and activation of Nrf-2 reduce breast cancer cell response to anti-ER drugs

BackgroundGenetic abnormalities in the FGFR signalling occur in 40% of breast cancer (BCa) patients resistant to anti-ER therapy, which emphasizes the potential of FGFR-targeting strategies. Recent findings indicate that not only mutated FGFR is a driver of tumour progression but co-mutational landscapes and other markers should be also investigated. Autophagy has been recognized as one of the major mechanisms underlying the role of tumour microenvironment in promotion of cancer cell survival, and resistance to anti-ER drugs. The selective autophagy receptor p62/SQSTM1 promotes Nrf-2 activation by Keap1/Nrf-2 complex dissociation. Herein, we have analysed whether the negative effect of FGFR2 on BCa cell response to anti-ER treatment involves the autophagy process and/or p62/Keap1/Nrf-2 axis.MethodsThe activity of autophagy in ER-positive MCF7 and T47D BCa cell lines was determined by analysis of expression level of autophagy markers (p62 and LC3B) and monitoring of autophagosomes’ maturation. Western blot, qPCR and proximity ligation assay were used to determine the Keap1/Nrf-2 interaction and Nrf-2 activation. Analysis of 3D cell growth in Matrigel® was used to assess BCa cell response to applied treatments. In silico gene expression analysis was performed to determine FGFR2/Nrf-2 prognostic value.ResultsWe have found that FGFR2 signalling induced autophagy in AMPKα/ULK1-dependent manner. FGFR2 activity promoted dissociation of Keap1/Nrf-2 complex and activation of Nrf-2. Both, FGFR2-dependent autophagy and activation of Nrf-2 were found to counteract the effect of anti-ER drugs on BCa cell growth. Moreover, in silico analysis showed that high expression of NFE2L2 (gene encoding Nrf-2) combined with high FGFR2 expression was associated with poor relapse-free survival (RFS) of ER+ BCa patients.ConclusionsThis study revealed the unknown role of FGFR2 signalling in activation of autophagy and regulation of the p62/Keap1/Nrf-2 interdependence, which has a negative impact on the response of ER+ BCa cells to anti-ER therapies. The data from in silico analyses suggest that expression of Nrf-2 could act as a marker indicating potential benefits of implementation of anti-FGFR therapy in patients with ER+ BCa, in particular, when used in combination with anti-ER drugs.Graphical

Abstract PS17-09: An FGFR1/2 degrader overcomes antiestrogen resistance in ER+/FGFR1-amplified breast cancer

Abstract We previously reported that nuclear FGFR1 promotes antiestrogen resistance in ER+/FGFR1-amplified breast cancer. Nuclear FGFR1 activity was not affected by FGFR tyrosine kinase inhibitors (TKIs). Furthermore, pan-FGFR TKIs are not well tolerated. Therefore, new therapeutic strategies that inhibit nuclear FGFR1 while sparing FGFR3 and FGFR4 would be required to overcome antiestrogen resistance and limit toxicity. For this study, we used the proteolysis-targeting chimera (PROTAC) DGY-09-192, consisting of the FGFR TKI BCJ398 linked with a VHL recruiting ligand, to degrade FGFR1 and FGFR2. We aimed to determine whether selective degradation of FGFR1/2 overcomes endocrine resistance. Treatment with 50-100 nM DGY-09-192 strongly induced FGFR1 degradation and suppressed phosphorylation of FGFR1 and its downstream targets FRS2, AKT, and ERK1/2 in ER+/FGFR1-amplified CAMA1 and MDA-MB-134 breast cancer cells. FGFR1 degradation was evident within 4 h and lasted 48-96 h after drug washout. The proteasome inhibitor MG132 (10 µM) rescued both FGFR degradation and phosphorylation of downstream signaling molecules induced by DGY-09-192, confirming that FGFR1 degradation is dependent on ubiquitination and proteasomal degradation. At equivalent concentrations, DGY-09-192 suppressed phosphorylation of FGFR1 and its downstream targets more potently than BGJ398. Subcellular fractionation showed treatment with DGY-09-192 (100 nM) degraded FGFR1 in membrane, nuclear, and chromatin-bound fractions in both CAMA1 and MDA-MB-134 cells. Treatment of >CAMA1 cells with DGY-09-192 (>100 nM) downregulated nuclear FGFR1 target genes and canonical ERα target genes, such as CCND1, VEGFA, and CDK12 as measured by qRT-PCR whereas treatment with BGJ398 (100 nM) did not. Treatment with DGY-09-192 (500 nM) reversed resistance to the ER degrader fulvestrant in CAMA1 cells as measured by colony formation assays and using the Incucyte Live-Cell Analysis System. Each drug alone showed a modest effect on cell proliferation whereas the combination completed blocked cell growth (p < 0.0001). Finally, treatment of NOD scid gamma (NSG) mice with established ER+/FGFR1-amplified HCI-011 patient-derived xenografts with DGY-09-192 (40 mg/kg daily x2-6) induced FGFR1 degradation and blocked phosphorylation of downstream targets in the tumors. Studies using the combination of DGY-09-192 and fulvestrant in mice bearing established HCI-011 xenografts are in progress. We next investigated the effect of DGY-09-192 on cancer cells harboring FGFR1 and FGFR2 activating mutations. DGY-09-192 treatment induced FGFR2 degradation and blocked downstream signaling in MFE296 endometrial cancer and EFM-19 breast cancer cells that harbor the FGFR2N549K and FGFR2K659E hotspot mutations, respectively. In addition, DGY-09-192 strongly suppressed MFE296 cell proliferation. We are currently investigating whether DGY-09-192 blocks signaling and cell proliferation induced by the N546K and K656E hotspot FGFR1 mutations in ER+ breast cancer cells. Notably, a recent clinical study showed that tumors harboring these FGFR1 activating mutations are refractory to the pan-FGFR TKI pemigatinib. Conclusions: The FGFR1/2 PROTAC DGY-09-192 induced degradation of nuclear and membrane-bound FGFR1 and blocked FGFR1-induced signaling and nuclear activity in ER+/FGFR1-amplified breast cancer cells. The combination of DGY-09-192 and fulvestrant synergistically suppressed proliferation of these cells. Therefore, FGFR1 degradation represents a promising therapeutic strategy to block FGFR1 activity more completely and selectively in ER+ breast cancers harboring activating FGFR1 alterations. Citation Format: Yasuaki Uemoto, Chang-Ching Lin, Alberto Servetto, Bingnan Wang, Dan Ye, Fabiana Napolitano, Hima Milan Patel, Luigi Formisano, M Rosario Chica-Parrado, Emmanuel Bikorimana, Saurabh Mendiratta, Chuo Chen, Ariella Hanker, Carlos Arteaga. An FGFR1/2 degrader overcomes antiestrogen resistance in ER+/FGFR1-amplified breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS17-09.

Targeting FGFR1 by β,β-dimethylacrylalkannin suppresses the proliferation of colorectal cancer in cellular and xenograft models

BackgroundColorectal cancer (CRC) continues to be a major global health challenge, ranking as a top cause of cancer-related mortality. Alarmingly, the five-year survival rate for CRC patients hovers around a mere 10–30 %. The disruption of fibroblast growth factor receptor (FGFRs) signaling pathways is significantly implicated in the onset and advancement of CRC, presenting a promising target for therapeutic intervention in CRC management. Further investigation is essential to comprehensively elucidate FGFR1′s function in CRC and to create potent therapies that specifically target FGFR1. PurposeThis study aims to demonstrate the oncogenic role of FGFR1 in colorectal cancer and to explore the potential of β,β-dimethylacrylalkannin (β,β-DMAA) as a therapeutic option to inhibit FGFR1. MethodsIn this research, we employed a comprehensive suite of techniques including tissue array, kinase profiling, computational docking, knockdown assay to predict and explore the inhibitor of FGFR1. Furthermore, we utilized kinase assay, pull-down, cell proliferation tests, and Patient derived xenograft (PDX) mouse models to further investigate a novel FGFR1 inhibitor and its impact on the growth of CRC. ResultsIn our research, we discovered that FGFR1 protein is markedly upregulated in colorectal cancer tissues, suggesting a significant role in regulating cellular proliferation, particularly in patients with colorectal cancer. Furthermore, we conducted a computational docking, kinase profiling analysis, simulation and identified that β,β-DMAA could directly bind with FGFR1 within ATP binding pocket domain. Cell-based assays confirmed that β,β-DMAA effectively inhibited the proliferation of colon cancer cells and also triggered cell cycle arrest, apoptosis, and altered FGFR1-mediated signaling pathways. Moreover, β,β-DMAA effectively attenuated the development of PDX tumors in mice that were FGFR1-positive, with no notable toxicity observed. In summary, our study highlights the pivotal role of FGFR1 in colorectal cancer, suggesting that inhibiting FGFR1 activity could be a promising strategy for therapeutic intervention. We present strong evidence that targeting FGFR1 with β,β-DMAA is a viable approach for the management of colorectal cancer. Given its low toxicity and high efficacy, β,β-DMAA, as an FGFR1 inhibitor, warrants further investigation in clinical settings for the treatment of FGFR1-positive tumors.

Abstract LB319: Klf5 acetylation remodels tumor microenvironment to constrain PTEN-deficient prostate tumor growth

Abstract Prostate cancer is the most common cancer and the second leading cause of cancer-related death in men in the United States. PTEN deficiency is prevalent in patients with prostate cancer. As a tumor suppressor, PTEN is mutated in approximately 20% of primary prostate cancers, and up to 50% of metastatic castration resistant prostate cancer. PTEN inactivation results in high-grade adenocarcinoma with a long latency. The limited tumor progression induced by PTEN deficiency suggests that additional molecular events are activated to constrain tumor progression, and second hits are required. Understanding these secondary genetic hits will provide the rationale for combined therapies in prostate cancer treatment. In this study, we employed a unique genetically knockin mouse model and found that PTEN deficiency induces a KLF5 K369 acetylation-dependent barrier that attenuates FGFR1 signaling and constrains prostate tumor growth. More importantly, we discovered novel microenvironmental crosstalk between prostate cancer cells and cancer associated fibroblasts (CAFs). As suggested by RNA-seq and bioinformatic analyses and further validated by molecular and cellular experiments, we found that interruption of KLF5 acetylation in cancer cells stimulates CAFs to release FGF9 via TNF-α, and FGF9 activating FGFR1 signaling in cancer cells. In addition to this paracrine crosstalk, deacetylated KLF5 induces CX3CR1 and facilitates FGFR1 activation. Our findings provide a proof of concept for posttranslational modifications as essential molecular events induced by PTEN inactivation to stall prostate cancer progression and demonstrate KLF5 acetylation as a key component in a negative feedback loop by which FGFR1 was suppressed by AKT activation. This study reveals a paracrine reciprocal communication between cancer cells and fibroblasts and provides a clinical rationale for combined therapies using FGFR1 inhibitors (or CX3CR1 inhibitors) and p-AKT inhibitors in PTEN-deficient prostate cancer. Citation Format: Baotong Zhang, Mingcheng Liu, Fengyi Mai, Siyuan Xia, Jin-Tang Dong. Klf5 acetylation remodels tumor microenvironment to constrain PTEN-deficient prostate tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB319.

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 PR-002: Uncovering novel therapeutic avenues for glioma by exploring the functional interplay of FGFR1, p53, and ribosome biogenesis

Abstract PR-002: Uncovering novel therapeutic avenues for glioma by exploring the functional interplay of FGFR1, p53, and ribosome biogenesis

Utilizing bivalent aptamers as first DNA agonist to activate RTKs heterodimer of different families

Heterodimerization in RTKs is of vital importance in the RTK signaling and cell functions. Heterodimerization between RTKs can result in diversity of downstream signals, increasing the ability of cells to respond to external experiments. Traditional RTKs heterodimerization always occur in the same families and is lack of agonists to activate the heterodimeric RTKs signaling pathway. Herein, we developed the DNA agonist based on bivalent aptamers for the heterodimerized RTKs of different families, AF/AM-1, which could simultaneously activate FGFR1 and c-Met signaling. It is the first agonist that realizing the heterodimerization and activation of FGFR1 and c-Met, two different RTK families. The activation of FGFR1/c-Met heterodimer result in the down-stream signals transduction, such as the phosphorylation of Akt and Erk, inducing the cell migration and proliferation. The DNA agonist for RTK heterodimer of different families would have potential applications in the fields of biomedicine.

Activated FGFR2 signalling as a biomarker for selection of intrahepatic cholangiocarcinoma patients candidate to FGFR targeted therapies

FGFR inhibitors have been developed to inhibit FGFR activation and signal transduction; notwithstanding, currently the selection of intrahepatic cholangiocarcinoma (iCCA) patients for these drugs only relies on the detection of FGFR2 genetic alterations (GAs) in tumor tissues or circulating tumor DNAs, without concomitant assessment of FGFR2 signalling status. Accordingly, we performed multi-omic analyses of FGFR2 genes and FGFR2 signalling molecules in the tissue samples from 36 iCCA naïve patients. Gain-of-function FGFR2 GAs were detected in 7 patients, including missense mutations (n = 3; p.F276C, p.C382R and p.Y375C), translocations (n = 1) and copy number gain (n = 4; CNV ≥ 4). In contrast, among 29 patients with wild-type FGFR2, 4 cases showed activation of FGFR2 signalling, as they expressed the FGFR2 ligand FGF10 and phosphorylated FGFR2/FRS2α proteins; the remaining 25 cases resulted negative for activated FGFR2 signalling, as they lacked FGFR2 (n = 8) or phosphorylated FRS2α (n = 17) expression. Overall, we found that activation of FGFR2 signalling occurs not only in iCCA naïve patients with FGFR2 GAs, but also in a subgroup carrying wild-type FGFR2. This last finding entails that also this setting of patients could benefit from FGFR targeted therapies, widening indication of these drugs for iCCA patients beyond current approval. Future clinical studies are therefore encouraged to confirm this hypothesis.

Exploring FGFR3 Mutations in the Male Germline: Implications for Clonal Germline Expansions and Paternal Age-Related Dysplasias.

Delayed fatherhood results in a higher risk of inheriting a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early- or late-onset effects in the offspring. Here, we used digital polymerase chain reaction to assess the frequency and spatial distribution of 10 different FGFR3 missense substitutions in the sexually mature male germline. Our functional assessment of the receptor signaling of the variants with biophysical methods showed that 9 of these variants resulted in a higher activation of the receptor´s downstream signaling, resulting in 2 different expansion behaviors. Variants that form larger subclonal expansions in a dissected postmortem testis also showed a positive correlation of the substitution frequency with the sperm donor's age, and a high and ligand-independent FGFR3 activation. In contrast, variants that measured high FGFR3 signaling and elevated substitution frequencies independent of the donor's age did not result in measurable subclonal expansions in the testis. This suggests that promiscuous signal activation might also result in an accumulation of mutations before the sexual maturation of the male gonad with clones staying relatively constant in size throughout time. Collectively, these results provide novel insights into our understanding of the mutagenesis of driver mutations and their resulting mosaicism in the male germline with important consequences for the transmission and recurrence of associated disorders.

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.

FGFR1 Gene Expression and its Relationship with Sociodemographic and Clinicopathological Indices in Oral Squamous Cell Carcinoma

Introduction: A better prognosis has resulted from the use of molecular techniques in cancer diagnosis. The FGFR family of genes is one of the major gene families involved in the carcinogenic pathways of different malignancies. Tumorigenesis, uncontrolled cell proliferation, and a number of pathologic diseases are linked to improper FGFR activation. The family of genes known as FGFRs encodes cell surface membrane receptors with tyrosine kinases.The development of oral squamous cell carcinoma (OSCC) may be significantly influenced by fibroblast growth factor receptor 1 (FGFR1). Analyzing these elements' expression patterns may provide fresh perspectives on illness management strategies including genetic mediated target therapy. Objectives: In order to improve care and prevent future difficulties from oral squamous cell carcinoma (OSCC), the current study sought to assess the expression of FGFR1 genes in OSCC tumors as a result of the discovery of biomarkers and early diagnosis. Methods:In order to assess the expression of FGFR1 genes in 16 OSCC samples, 16 normal specimens from the same sample, free from cancer margin and 4 control samples from other patient free from cancer, the Oral and Maxillofacial Surgery DepartmentatDental Unit of Rajshahi Medical College Hospital was collected. Trizol and the appropriate kits were used for RNA extraction and cDNA synthesis. Real-time PCR was used to assess the FGFR1 gene's expression following the construction of a specific primer set in order to find and validate molecular biomarkers. The data is analyzed using the ANOVA and independent t-test. Statistics were shown to be significant when P<0.05. Results:According to the findings, there are notable variations in the expression of the FGFR1 gene between tumor and normal or control tissues (P < 0.001). Patients over 60 are more likely to have OSCC, and the majority of them smoke 8. Retromolar trigon 8 is the most frequent location, and the majority of them are in stages III and IV. The FGFR1 gene expression does not significantly differ according on a patient's age, gender, religion and behavior, including whether they smoke, drink, or chew paan. It also does not differ depending on the tumor's location, stages and grade. Conclusions: The FGFR1 gene was often expressed differently in cancerous and control tissues, confirming the gene's involvement in OSCC. The expression of FGFR1 is not correlated with factors such as gender, age, history of smoking, alcohol intake, chewing paan, tumor site, degree of differentiation, or TNM stage. The current investigation demonstrated the critical function of the investigated gene in the diagnosis of OSCC. To verify this, though, more research is required. TAJ 2023; 36: No-2: 155-162

Controlled Plasma Membrane Delivery of FGFR1 and Modulation of Signaling by a Novel Regulated Anterograde RTK Transport Pathway.

How human FGFR1 localizes to the PM is unknown. Currently, it is assumed that newly synthesized FGFR1 is continuously delivered to the PM. However, evidence indicates that FGFR1 is mostly sequestered in intracellular post-Golgi vesicles (PGVs) under normal conditions. In this report, live-cell imaging and total internal reflection fluorescence microscopy (TIRFM) were employed to study the dynamics of these FGFR1-positive vesicles. We designed recombinant proteins to target different transport components to and from the FGFR1 vesicles. Mouse embryoid bodies (mEBs) were used as a 3D model system to confirm major findings. Briefly, we found that Rab2a, Rab6a, Rab8a, RalA and caveolins are integral components of FGFR1-positive vesicles, representing a novel compartment. While intracellular sequestration prevented FGFR1 activation, serum starvation and hypoxia stimulated PM localization of FGFR1. Under these conditions, FGFR1 C-terminus acts as a scaffold to assemble proteins to (i) inactivate Rab2a and release sequestration, and (ii) assemble Rab6a for localized activation of Rab8a and RalA-exocyst to deliver the receptor to the PM. This novel pathway is named Regulated Anterograde RTK Transport (RART). This is the first instance of RTK regulated through control of PM delivery.

Discovery of Potent and Selective Next-Generation Pan-BCR-ABL Inhibitors for the Treatment of CML and Ph+ ALL

Introduction While 6 tyrosine kinase inhibitors (TKIs) are available for patients with chronic myeloid leukemia (CML) that is newly diagnosed (imatinib, dasatinib, nilotinib, bosutinib) or refractory to prior treatments (asciminib, ponatinib), only ponatinib has pan-BCR-ABL activity, i.e., maintains potent activity against all single resistance mutations, including the difficult to inhibit T315I and E255V mutants. BCR-ABL mutations are the major known mechanism of resistance not only in CML, but also in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), where use of ponatinib in newly diagnosed patients is yielding especially encouraging results, presumably due to its ability to suppress emergence of resistance mutations. However, toxicities observed with ponatinib often lead to dose reduction or termination that can limit the benefit of its pan-BCR-ABL profile. The suboptimal safety profile of ponatinib is consistent with its relative lack of kinome selectivity, and its potent activity against KDR, FLT3, and FGFRs (unique amongst the 6 approved BCR-ABL TKIs) could potentially contribute to its unique toxicities. We therefore set out to identify a selective, potent pan-BCR-ABL inhibitor. Methods Compound potency was determined by assessing effects on viability of Ba/F3 cell lines expressing BCR-ABL variants (wild type [WT] and 11 key mutant forms) and off-targets of interest. Preliminary assessment of broader kinome selectivity was performed by calculating the percentage of 88 kinases inhibited by >80% by 1 µM compound (S-score (80)). For efficacy studies, mice implanted with engineered Ba/F3 cell lines were dosed orally once daily. Results Ponatinib potently inhibited BCR-ABL WT, T315I, and E255V with IC50s of 2, 4, and 7 nM respectively, and all 9 other BCR-ABL mutants tested were inhibited with IC50s <4 nM. However, ponatinib also potently inhibited the activity of KDR, FLT3, and FGFR1, with IC50s of 5, 2, and 17 nM, respectively, and had a high kinome S-score (80) of 0.30. Cmpd A is a representative member of a series of compounds we have identified that have a pan-BCR-ABL inhibitory profile and substantially improved selectivity over ponatinib. Cmpd A, potently inhibited BCR-ABL WT, T315I, and E255V, with IC50s of 6, 21, and 55 nM respectively, and 8/9 other BCR-ABL mutants tested were inhibited with IC50s ≤13 nM (Y253H IC50 75 nM). But in contrast to ponatinib, Cmpd A had a high degree of selectivity over FLT3 and FGFR1 (IC50s 1898 and 3323 nM), good selectivity over KDR (IC50 420 nM), and a low kinome S-score (80) of 0.08. We next compared the efficacy and tolerability of ponatinib and Cmpd A in an efficacy model driven by E255V, a key clinical resistance mutant that both compounds inhibited less potently than T315I. At 10 mg/kg, ponatinib inhibited tumor growth by 64% compared to vehicle-treated mice and was well tolerated. At 25 mg/kg, while ponatinib induced strong tumor regression (by 82% compared to baseline), it was poorly tolerated (eg, keratinized skin in 4/6 mice). In contrast 120 mg/kg Cmpd A induced strong tumor regression, by 55%, and was well tolerated (eg, 0/6 mice had keratinized skin). Conclusions We have identified a series of next-generation pan-BCR-ABL inhibitors with substantially improved selectivity over ponatinib. In vitro and in vivo characterization of compounds with further improved selectivity profiles, and the potential to be developed for use in patients with refractory CML and newly diagnosed Ph+ ALL, will be presented.

Light Chain Amyloidosis Plasma Cells Show Specific Chromatin Accessibility and Transcriptome

Immunoglobin light chain amyloidosis (AL) is a monoclonal gammopathy characterized by the accumulation of malignant plasma cells (PC) in the bone marrow. These PCs secrete misfolded immunoglobulin light chains fibrils, which deposit in various organs, leading to organ dysfunction. Unlike other monoclonal gammopathies such as Multiple Myeloma, both its rarity and the lower BM PC burden has limited the deep study of the tumor population. The aim of this study was to analyze the transcriptome and epigenome of the PC from patients with AL amyloidosis to identify the role of chromatin modifications in the pathogenesis of the disease. Highly purified clonal PCs were obtained from bone marrow aspirates samples of 58 AL amyloidosis patients at diagnosis, 19 monoclonal gammopathy of undetermined significance (MGUS), 149 MM and 17 healthy donors. RNA was obtained from PCs and Illumina Stranded Total RNA-seq protocol was performed according to manufacturer's specifications. Accessible chromatin mapping was performed using FAST-ATACseq (Corces et al, 2016) with minor modifications. The data obtained were aligned with the GRCh38 version of the human genome and custom pipelines were used for computational analyses. Expression of immunoglobulin genes was removed for RNAseq analysis. Unsupervised principal component analysis of the RNAseq and ATACseq data showed a clear distinction between PCs from AL patients, MM and HD. To obtain a reliable measure of the magnitude of epigenomic and transcriptome changes in AL, we compared the RNAseq and ATACseq data of AL with MM or HD. The RNAseq analysis showed some clear transcriptional programs (TPs) related to AL and different from MM and HD (Figure 1): TP1 showed 89 genes upregulated and associated with positive regulation of protein kinases and TP2 showed 274 genes downregulated and correlated with immunoregulatory interactions and mitotic regulation. On the other hand, TP3 included 560 downregulated genes associated with glutamate pathway and glycosylation of proteins and TP4 revealed 362 upregulated genes linked with B cell activation. Both patterns were identified in AL patients and HD in comparison with MM patients. Regarding chromatin accessibility, we detected a similar number of ATAC peaks between the AL and MGUS samples and with a significant difference compared to the MM samples (p=0.00014), suggesting that AL and PCs from MGUS have a lower chromatin accessibility (Figure 2). These results provide evidence that the biological perturbations in the chromatin accessibility and transcriptome of PCs from AL patients are highly specific. Although the results depicted in Figure 1 demonstrate that AL patients clustered together by relatively homogenous chromatin accessibility and transcriptome, compared with MM patients, we further investigated whether there were any discernible differences among AL patients themselves. To address this, we conducted an analysis of chromatin accessibility and transcriptome in relation to various factors, including age, gender, organ affected, light chain isotype, percentage of tumoral PCs, presence of t(11,14) or amp1q. Remarkably, we identified differences in the transcriptome between AL patients with and without t(11;14), identifying, 46 upregulated genes linked with T cell and lymphocyte apoptosis and inflammatory cytokines production and 257 downregulated genes linked with FGFR family ligand binding and activation and protein ubiquitination and phosphorylation, in t(11,14) positive AL patients. These results suggest that t(11;14) may exert a significant modulation on the transcriptome of PCs of AL patients. Finally, scRNAseq was performed to address the heterogeneity of PCs. Preliminary analysis has shown a remarkable homogeneity in malignant PCs from AL patients with very limited transcriptional differences between PCs clusters. In summary, in this study we have characterized the PCs of AL patients revealing a homogeneous and specific chromatin accessibility and transcriptome that could contribute to explain the differences between AL and other gammopathies, such as MM. In addition, the identification of these epigenetic and transcriptomic alterations in AL will allow us to better understand the aberrant biology driving this disease, such as those AL with t(11;14). Moreover, these findings open new avenues for the development of innovative therapeutic strategies to improve the outcomes for AL patients.

The importance of escalating molecular diagnostics in patients with low-grade pediatric brain cancer.

Pilocytic astrocytomas are the most common pediatric brain tumors, typically presenting as low-grade neoplasms. We report two cases of pilocytic astrocytoma with atypical tumor progression. Case 1 involves a 12-yr-old boy with an unresectable suprasellar tumor, negative for BRAF rearrangement but harboring a BRAF p.V600E mutation. He experienced tumor size reduction and stable disease following dabrafenib treatment. Case 2 describes a 6-yr-old boy with a thalamic tumor that underwent multiple resections, with no actionable driver detected using targeted next-generation sequencing. Whole-genome and RNA-seq analysis identified an internal tandem duplication in FGFR1 and RAS pathway activation. Future management options include FGFR1 inhibitors. These cases demonstrate the importance of escalating molecular diagnostics for pediatric brain cancer, advocating for early reflexing to integrative whole-genome sequencing and transcriptomic profiling when targeted panels are uninformative. Identifying molecular drivers can significantly impact treatment decisions and improve patient outcomes.