Increased Expression and Altered Cellular Localization of Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) Are Associated with Prostate Cancer Progression

Fibroblast growth factor receptors (FGFRs) 1–4 are involved in prostate cancer (PCa) regulation, but the role of FGFR-like 1 (FGFRL1) in PCa is unclear. FGFRL1 expression was studied by qRT-PCR and immunohistochemistry of patient tissue microarrays (TMAs) and correlated with clinical patient data. The effects of FGFRL1 knockdown (KD) in PC3M were studied in in vitro culture models and in mouse xenograft tumors. Our results showed that FGFRL1 was significantly upregulated in PCa. The level of membranous FGFRL1 was negatively associated with high Gleason scores (GSs) and Ki67, while increased cytoplasmic and nuclear FGFRL1 showed a positive correlation. Cox regression analysis indicated that nuclear FGFRL1 was an independent prognostic marker for biochemical recurrence after radical prostatectomy. Functional studies indicated that FGFRL1-KD in PC3M cells increases FGFR signaling, whereas FGFRL1 overexpression attenuates it, supporting decoy receptor actions of membrane-localized FGFRL1. In accordance with clinical data, FGFRL1-KD markedly suppressed PC3M xenograft growth. Transcriptomics of FGFRL1-KD cells and xenografts revealed major changes in genes regulating differentiation, ECM turnover, and tumor–stromal interactions associated with decreased growth in FGFRL1-KD xenografts. Our results suggest that FGFRL1 upregulation and altered cellular compartmentalization contribute to PCa progression. The nuclear FGFRL1 could serve as a prognostic marker for PCa patients.

Biliary Tract Cancers: Treatment Updates and Future Directions in the Era of Precision Medicine and Immuno-Oncology

The effective management of biliary tract cancers (BTCs) has been hampered by limited options for systemic therapy. In recent years, the focus on precision medicine has made technologies such as next-generation sequencing (NGS) accessible to clinicians to identify targetable mutations in BTCs in tumor tissue (primarily) as well as blood, and to treat them with targeted therapies when possible. It has also expanded our understanding of functional pathways associated with genetic alterations and opened doors for identifying novel targets for treatment. Recent advances in the precision medicine approach allowed us to identify new molecular markers in BTCs, such as epigenetic changes (methylation and histone modification) and non-DNA markers such as messenger RNA, microRNA, and long non-coding RNA. It also made detecting these markers from non-traditional sources such as blood, urine, bile, and cytology (from fine-needle aspiration and biliary brushings) possible. As these tests become more accessible, we can see the integration of different molecular markers from all available sources to aid physicians in diagnosing, assessing prognosis, predicting tumor response, and screening BTCs. Currently, there are a handful of approved targeted therapies and only one class of immunotherapy agents (immune checkpoint inhibitors or ICIs) to treat BTCs. Early success with new targets, vascular endothelial growth factor receptor (VEGFR), HER2, protein kinase receptor, and Dickkopf-1 (DKK1); new drugs for known targets, fibroblast growth factor receptors (FGFRs) such as futabatinib, derazantinib, and erdafitinib; and ICIs such as durvalumab and tremelimumab is encouraging. Novel immunotherapy agents such as bispecific antibodies (bintrafusp alfa), arginase inhibitors, vaccines, and cellular therapy (chimeric antigen receptor—T cell or CAR-T, natural killer cells, tumor-infiltrating lymphocytes) have the potential to improve outcomes of BTCs in the coming years.

Peptibody Based on FGFR1-Binding Peptides From the FGF4 Sequence as a Cancer-Targeting Agent

Targeted therapies are a promising alternative to conventional chemotherapy, with an increasing number of therapeutics targeting specific molecular aberrancies in cancer cells. One of the emerging targets for directed cancer treatments is fibroblast growth factor receptors (FGFRs), which are known to be involved in the pathogenesis and progression of multiple cancer types, specially in lung, bladder, and breast cancers. Here, we are demonstrating the development of the FGFR1-targeting agent based on the interactome screening approach, based on the isolation of binding regions from ligands interacting with the receptor. The parallel analysis by FGFR1 pull-down of chymotryptic peptides coupled with MS analysis, and PepSpot analysis yielded equivalent peptide sequences from FGF4, one of the FGFR1 ligands. Three sequences served as a basis for peptibody (Fc-fusion) generation, to overcome clinical limitations of peptidic agents, and two of them showed favorable FGFR1-binding in vitro and FGFR1-dependent internalization into cells. To validate if developed FGFR1-targeting peptibodies can be used for drug delivery, similar to the well-established concept of antibody–drug conjugates (ADCs), peptibodyF4_1 was successfully conjugated with monomethylauristatin E (MMAE), and has shown significant and specific toxicity toward FGFR1-expressing lung cancer cell lines, with nanomolar EC50 values. Essentially, the development of new effective FGFR1 binders that comprise the naturally occurring FGFR-recognition peptides and Fc region ensuring high plasma stability, and long bloodstream circulation is an interesting strategy expanding targeted anticancer agents’ portfolio. Furthermore, identifying peptides effectively binding the receptor from sequences of its ligands is not limited to FGFRs and is an approach versatile enough to be a basis for a new peptide/peptibodies development strategy.

A designed fusion tag for soluble expression and selective separation of extracellular domains of fibroblast growth factor receptors

Fibroblast growth factor receptors (FGFRs) generate various transduction signals by interaction with fibroblast growth factors (FGFs) and are involved in various biological functions such as cell proliferation, migration, and differentiation. Malfunction of these proteins may lead to the development of various diseases, including cancer. Accordingly, FGFRs are considered an alternative therapeutic target for protein and/or gene therapy. However, the screening of antagonists or agonists of FGFRs is challenging due to their complex structural features associated with protein expression. Herein, we conducted the development of a protease-free cleavable tag (PFCT) for enhancing the solubility of difficult-to express protein by combining maltose-binding protein (MBP) and the C-terminal region of Npu intein. To validate the availability of the resulting tag for the functional production of extracellular domains of FGFRs (Ec_FGFRs), we performed fusion of PFCT with the N-terminus of Ec_FGFRs and analyzed the expression patterns. Almost all PFCT-Ec_FGFR fusion proteins were mainly detected in the soluble fraction except for Ec_FGFR4. Upon addition of the N-terminal region of Npu intein, approximately 85% of the PFCT-Ec_FGFRs was separated into PFCT and Ec_FGFR via intein-mediated cleavage. Additionally, the structural integrity of Ec_FGFR was confirmed by affinity purification using heparin column. Taken together, our study demonstrated that the PFCT could be used for soluble expression and selective separation of Ec_FGFRs.

Modular self-assembly system for development of oligomeric, highly internalizing and potent cytotoxic conjugates targeting fibroblast growth factor receptors

Background Overexpression of FGFR1 is observed in numerous tumors and therefore this receptor constitutes an attractive molecular target for selective cancer treatment with cytotoxic conjugates. The success of cancer therapy with cytotoxic conjugates largely relies on the precise recognition of a cancer-specific marker by a targeting molecule within the conjugate and its subsequent cellular internalization by receptor mediated endocytosis. We have recently demonstrated that efficiency and mechanism of FGFR1 internalization are governed by spatial distribution of the receptor in the plasma membrane, where clustering of FGFR1 into larger oligomers stimulated fast and highly efficient uptake of the receptor by simultaneous engagement of multiple endocytic routes. Based on these findings we aimed to develop a modular, self-assembly system for generation of oligomeric cytotoxic conjugates, capable of FGFR1 clustering, for targeting FGFR1-overproducing cancer cells. Methods Engineered FGF1 was used as FGFR1-recognition molecule and tailored for enhanced stability and site-specific attachment of the cytotoxic drug. Modified streptavidin, allowing for controlled oligomerization of FGF1 variant was used for self-assembly of well-defined FGF1 oligomers of different valency and oligomeric cytotoxic conjugate. Protein biochemistry methods were applied to obtain highly pure FGF1 oligomers and the oligomeric cytotoxic conjugate. Diverse biophysical, biochemical and cell biology tests were used to evaluate FGFR1 binding, internalization and the cytotoxicity of obtained oligomers. Results Developed multivalent FGF1 complexes are characterized by well-defined architecture, enhanced FGFR1 binding and improved cellular uptake. This successful strategy was applied to construct tetrameric cytotoxic conjugate targeting FGFR1-producing cancer cells. We have shown that enhanced affinity for the receptor and improved internalization result in a superior cytotoxicity of the tetrameric conjugate compared to the monomeric one. Conclusions Our data implicate that oligomerization of the targeting molecules constitutes an attractive strategy for improvement of the cytotoxicity of conjugates recognizing cancer-specific biomarkers. Importantly, the presented approach can be easily adapted for other tumor markers.

Targeting PI3K, FGFR, CDK4/6 Signaling Pathways Together With Cytostatics and Radiotherapy in Two Medulloblastoma Cell Lines

ObjectivesMedulloblastoma (MB) is treated with surgery and chemotherapy, with or without irradiation, but unfortunately >20% of the patients are not cured, and treatment comes with serious long-term side effects, so novel treatments are urgently needed. Phosphoinositide 3-kinases (PI3K), fibroblast growth factor receptors (FGFR), and cyclin-D kinases (CDK) play critical roles in cancer, and especially PI3K is crucial in MB, so here targeted therapies against them were explored.MethodsMB cell lines DAOY and UW228-3 were exposed to PI3K (BYL719), FGFR (JNJ-42756493), and CDK4/6 (PD-0332991) inhibitors, as single or combined treatments, and their viability, cell confluence, apoptosis, and cytotoxicity were examined. Moreover, the inhibitors were combined with cisplatin, vincristine, or irradiation.ResultsSingle treatments with FGFR, PI3K, or CDK4/6 inhibitors decreased viability and proliferation slightly; however, when combining two inhibitors, or the inhibitors with irradiation, sensitivity was enhanced and lower doses could be used. A more complex pattern was obtained when combining the inhibitors with cisplatin and vincristine.ConclusionsThe data suggest that combination treatments with PI3K, FGFR, and CDK4/6 inhibitors for MB could be beneficial and their use should be pursued further. Likewise, their combination with irradiation gave positive effects, while the addition of cisplatin and vincristine resulted in more complex patterns, which need to be investigated further.

FGF-Receptors and PD-L1 in Anaplastic and Poorly Differentiated Thyroid Cancer: Evaluation of the Preclinical Rationale

BackgroundTreatment options for poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinoma are unsatisfactory and prognosis is generally poor. Lenvatinib (LEN), a multi-tyrosine kinase inhibitor targeting fibroblast growth factor receptors (FGFR) 1-4 is approved for advanced radioiodine refractory thyroid carcinoma, but response to single agent is poor in ATC. Recent reports of combining LEN with PD-1 inhibitor pembrolizumab (PEM) are promising.Materials and MethodsPrimary ATC (n=93) and PDTC (n=47) tissue samples diagnosed 1997-2019 at five German tertiary care centers were assessed for PD-L1 expression by immunohistochemistry using Tumor Proportion Score (TPS). FGFR 1-4 mRNA was quantified in 31 ATC and 14 PDTC with RNAscope in-situ hybridization. Normal thyroid tissue (NT) and papillary thyroid carcinoma (PTC) served as controls. Disease specific survival (DSS) was the primary outcome variable.ResultsPD-L1 TPS≥50% was observed in 42% of ATC and 26% of PDTC specimens. Mean PD-L1 expression was significantly higher in ATC (TPS 30%) than in PDTC (5%; p<0.01) and NT (0%, p<0.001). 53% of PDTC samples had PD-L1 expression ≤5%. FGFR mRNA expression was generally low in all samples but combined FGFR1-4 expression was significantly higher in PDTC and ATC compared to NT (each p<0.001). No impact of PD-L1 and FGFR 1-4 expression was observed on DSS.ConclusionHigh tumoral expression of PD-L1 in a large proportion of ATCs and a subgroup of PDTCs provides a rationale for immune checkpoint inhibition. FGFR expression is low thyroid tumor cells. The clinically observed synergism of PEM with LEN may be caused by immune modulation.

Novel Driver Strength Index highlights important cancer genes in TCGA PanCanAtlas patients

Elucidating crucial driver genes is paramount for understanding the cancer origins and mechanisms of progression, as well as selecting targets for molecular therapy. Cancer genes are usually ranked by the frequency of mutation, which, however, does not necessarily reflect their driver strength. Here we hypothesize that driver strength is higher for genes that are preferentially mutated in patients with few driver mutations overall, because these few mutations should be strong enough to initiate cancer. We propose a formula to calculate the corresponding Driver Strength Index (DSI), as well as the Normalized Driver Strength Index (NDSI), the latter completely independent of the overall gene mutation frequency. We validate these indices using the largest database of human cancer mutations - TCGA PanCanAtlas, multiple established algorithms for cancer driver prediction (2020plus, CHASMplus, CompositeDriver, dNdScv, DriverNet, HotMAPS, IntOGen Plus, OncodriveCLUSTL, OncodriveFML) and four custom computational pipelines that integrate driver contributions from SNA, CNA and aneuploidy at the patient-level resolution. We demonstrate that NDSI provides substantially different rankings of genes as compared to DSI and frequency approach. For example, NDSI highlighted the importance of guanine nucleotide-binding protein subunits GNAQ, GNA11, GNAI1, GNAZ and GNB3, General Transcription Factor II family members GTF2I and GTF2F2, as well as fibroblast growth factor receptors FGFR2 and FGFR3. Intriguingly, NDSI prioritized CIC, FUBP1, IDH1 and IDH2 mutations, as well as 19q and 1p chromosome arm losses, that comprise characteristic molecular alterations of gliomas. KEGG analysis shows that top NDSI-ranked genes comprise PDGFRA-GRB2-SOS2-HRAS/NRAS-BRAF pathway, GNAQ/GNA11-HRAS/NRAS-BRAF pathway, GNB3-AKT1-IKBKG/GSK3B/CDKN1B pathway and TCEB1-VHL pathway. NDSI does not seem to correlate with the number of protein-protein interactions. We share our software to enable calculation of DSI and NDSI for outputs of any third-party driver prediction algorithms or their combinations.

Role of Klotho in Hyperglycemia: Its Levels and Effects on Fibroblast Growth Factor Receptors, Glycolysis, and Glomerular Filtration

Hyperglycemic conditions (HG), at early stages of diabetic nephropathy (DN), cause a decrease in podocyte numbers and an aberration of their function as key cells for glomerular plasma filtration. Klotho protein was shown to overcome some negative effects of hyperglycemia. Klotho is also a coreceptor for fibroblast growth factor receptors (FGFRs), the signaling of which, together with a proper rate of glycolysis in podocytes, is needed for a proper function of the glomerular filtration barrier. Therefore, we measured levels of Klotho in renal tissue, serum, and urine shortly after DN induction. We investigated whether it influences levels of FGFRs, rates of glycolysis in podocytes, and albumin permeability. During hyperglycemia, the level of membrane-bound Klotho in renal tissue decreased, with an increase in the shedding of soluble Klotho, its higher presence in serum, and lower urinary excretion. The addition of Klotho increased FGFR levels, especially FGFR1/FGFR2, after their HG-induced decrease. Klotho also increased levels of glycolytic parameters of podocytes, and decreased podocytic and glomerular albumin permeability in HG. Thus, we found that the decrease in the urinary excretion of Klotho might be an early biomarker of DN and that Klotho administration may have several beneficial effects on renal function in DN.