Preclinical characterization of CPL304110 as a potent and selective inhibitor of fibroblast growth factor receptors 1, 2, and 3 for gastric, bladder, and squamous cell lung cancer.

Fibroblast Growth Factor Receptors (FGFRs) are a family of receptor tyrosine kinases expressed on a plethora of cell membranes. They play crucial roles in both embryonic development and adult tissue functions. There is an increasing amount of evidence that FGFR-mediated oncogenesis is mainly related to gene amplification, activating mutations, or translocation in tumors of various histological types. Dysregulation of FGFRs has been implicated in a wide variety of neoplasms, such as bladder, gastric, and lung cancers. Given their functional significance, FGFRs emerge as promising targets for cancer therapy. Here, we introduce CPL304100, an innovative and highly potent FGFR1-3 kinase inhibitor demonstrating excellent in vitro biological activity. Comprehensive analyses encompassed kinase assays, cell line evaluations, PK/PD studies surface plasmon resonance studies, molecular docking, and in vivo testing in mouse xenografts. CPL304110 exhibited a distinctive binding profile to FGFR1/2/3 kinase domains, accompanied by a good safety profile and favorable ADMET parameters. Selective inhibition of tumor cell lines featuring active FGFR signaling was observed, distinguishing it from cell lines lacking FGFR aberrations (FGFR1, 2, and 3). CPL304110 demonstrated efficacy in both FGFR-dependent cell lines and patient-derived tumor xenograft (PDTX) in vivo models. Comparative analyses with FDA-approved FGFR inhibitors, erdafitinib and pemigatinib, revealed certain advantages of CPL304110 in both in vitro and in vivo assessments. Encouraging preclinical results led the way for the initiation of a Phase I clinical trial (01FGFR2018; NCT04149691) to further evaluate CPL304110 as a novel anticancer therapy.

Design, Synthesis, and Development of pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part I-Indole Derivatives.

Phosphoinositide 3-kinase δ (PI3Kδ), a member of the class I PI3K family, is an essential signaling biomolecule that regulates the differentiation, proliferation, migration, and survival of immune cells. The overactivity of this protein causes cellular dysfunctions in many human disorders, for example, inflammatory and autoimmune diseases, including asthma or chronic obstructive pulmonary disease (COPD). In this work, we designed and synthesized a new library of small-molecule inhibitors based on indol-4-yl-pyrazolo[1,5-a]pyrimidine with IC50 values in the low nanomolar range and high selectivity against the PI3Kδ isoform. CPL302253 (54), the most potent compound of all the structures obtained, with IC50 = 2.8 nM, is a potential future candidate for clinical development as an inhaled drug to prevent asthma.

Discovery and optimization of novel pyrazole-benzimidazole CPL304110, as a potent and selective inhibitor of fibroblast growth factor receptors FGFR (1-3).

The FGFR family is characterized by four receptors (FGFR 1-4), binding to 18 ligands called fibroblast growth factors (FGFs). Aberrant activation of FGFs and their FGFRs has been implicated in a broad spectrum of human tumors. We employed the scaffolds hybridization approach, scaffold-hopping concept to synthesize a series of novel pyrazole-benzimidazole derivatives 56 (a-x). Compound 56q (CPL304110) was identified as a selective and potent pan-FGFR inhibitor for FGFR1, -2, -3 with IC50s of 0.75 nM, 0.50 nM, 3.05 nM respectively, whereas IC50 of 87.90 nM for FGFR4. Due to its favorable pharmacokinetic profile, low toxicity and potent anti-tumor activity in vivo, compound 56q is currently under evaluation in phase I clinical trial for the treatment of bladder, gastric and squamous cell lung cancers (01FGFR2018; NCT04149691).

Epithelial-mesenchymal transition confers resistance to selective FGFR inhibitors in SNU-16 gastric cancer cells.

BACKGROUND: Up to 10 % of primary gastric cancers are characterized by FGFR2 amplification, and fibroblast growth factor receptor (FGFR) inhibitors may represent therapeutic agents for patients with these malignancies. However, long-term benefits of the treatment might be limited owing to the occurrence of drug resistance. METHODS: To investigate the mechanisms of resistance to selective FGFR inhibitors, we established three FGFR2-amplified SNU-16 gastric cancer cell lines resistant to AZD4547, BGJ398, and PD173074, respectively. RESULTS: The resistant cell lines (SNU-16R) demonstrated changes characteristic of epithelial-to-mesenchymal transition (EMT). In addition, they displayed loss of expression of FGFR2 and other tyrosine kinase receptors concurrent with activation of downstream signaling proteins and upregulation of the transforming growth factor ß (TGF-ß) level. However, treatment of parental SNU-16 cells with TGF-ß1 did not evoke EMT, and pharmacological inhibition of TGF-ß receptor I was not sufficient to reverse EMT changes in the resistant cells. Finally, we showed that the SNU-16R cell lines were sensitive to the human epidermal growth factor receptor 2 inhibitor mubritinib and the heat shock protein 90 inhibitor AUY922. CONCLUSION: In conclusion, we provide experimental evidence that EMT-mediated resistance might emerge in gastric cancer patients following treatment with FGFR inhibitors, and mubritinib or AUY922 treatment may be an alternative therapeutic strategy for these patients.

Differences in gene expression and alterations in cell cycle of acute myeloid leukemia cell lines after treatment with JAK inhibitors.

Janus kinase (JAK) inhibitors are a promising treatment strategy in several hematological malignancies and autoimmune diseases. A number of inhibitors are in clinical development, and two have already reached the market. Unfortunately, all of them are burdened with different toxicity profiles. To check if the JAK inhibitors of different selectivity evoke different responses on JAK2-dependent and independent cells, we have used three acute myeloid leukemia cell lines with confirmed JAK2 mutation status. We have found that JAK inhibitors exert distinct effect on the expression of BCLXL, CCND1 and c-MYC genes, regulated by JAK pathway, in JAK2 wild type cells in comparison to JAK2 V617F-positive cell lines. Moreover, cell cycle analysis showed that inhibitors alter the cycle by arresting cells in different phases. Our results suggest that observed effect of JAK2 inhibitors on transcription and cell cycle level in different cell lines are associated not with activity within JAK family, but presumably with other off-target activities.

Cell-based assay for low- and high-scale screening of the Wnt/ß-catenin signaling modulators.

Deregulation of the Wnt/ß-catenin signaling pathway is associated with many serious disorders, including cancer and Alzheimer's disease. The pivotal player is ß-catenin, which avoids degradation after activation of the pathway and is translocated to the nucleus, where it interacts with TCF/LEF transcription factors and induces expression of genes involved in cell cycle and apoptosis regulation. The identification of small molecules that may affect Wnt/ß-catenin signaling remains an important target during the development of novel therapies. We used the TCF/LEF lentiviral vector and the Wnt-independent H1703 cell line to develop a luciferase reporter-based cell assay for screening of the Wnt/ß-catenin pathway modulators. Following the optimization of cell density, concentration of activator, and stimulation time, the reporter system was validated by demonstrating its specific and dose-dependent response to several established modulators of Wnt/ß-catenin signaling such as Wnt3a, small interfering RNA (siRNA) against ß-catenin, glycogen synthase kinase 3 (GSK-3), and ß-catenin/TCF transcription complex inhibitors. Two pilot screens of inhibitors and activators of Wnt/ß-catenin signaling identified potential novel modulators of this pathway. Our findings suggest that the H1703-7TFP assay constitutes a suitable model of low background and high sensitivity for the low- and high-scale screening of the Wnt/ß-catenin pathway modulators.

Wnt/ß-catenin pathway as a potential prognostic and predictive marker in patients with advanced ovarian cancer.

BACKGROUND: ß-catenin is the key protein in the WNT signalling pathway and it forms adherent junctions together with E-cadherin. In ovarian carcinoma, abnormal expression of ß-catenin, E-cadherin and WNT-1 was observed, but their prognostic and predictive role is unclear. The aim of this study was to clarify the prognostic and predictive role of E-cadherin, ß-catenin and WNT-1 in advanced epithelial ovarian carcinoma (AEOC). METHODS: The expression of E-cadherin, ß-catenin and WNT-1 was determined by immunohistochemistry in AEOC. The correlation between expression of these proteins and progression-free survival (PFS) and overall survival (OS) was evaluated. Statistical analyses included Kaplan-Meier estimation, log-rank test, Spearman correlation and Cox proportional-hazards model. RESULTS: In ovarian cancer, intense expression of E-cadherin, ß-catenin and WNT-1 was found. In multivariate analysis, strong membrane ß-catenin expression was an independent unfavourable predictor for PFS (HR 2.19, 95% CI 1.09-4.39; p = 0.028), while in univariate analysis, strong membrane ß-catenin expression was a prognostic factor for OS in patients with AOC (p = 0.039). In multivariate analysis, only resistance to first-line chemotherapy was an adverse independent prognostic factor for OS (HR 16.84; 95% CI 5.07-55.98; p < 0.0001). Additionally, strong membranous ß-catenin expression was associated with resistance to platinum-based chemotherapy (p = 0.027). CONCLUSIONS: These findings support that WNT/ß-catenin pathway and E-cadherin are important factors in advanced epithelial ovarian cancer.

Activating mutations in ALK kinase domain confer resistance to structurally unrelated ALK inhibitors in NPM-ALK-positive anaplastic large-cell lymphoma.

PURPOSE: Crizotinib, the first FDA-approved ALK inhibitor, showed significant antitumor activity in young patients with anaplastic large-cell lymphoma (ALCL) frequently displaying ALK rearrangement. However, long-term therapeutic benefits of crizotinib are limited due to development of drug resistance. CH5424802--more potent and selective ALK inhibitor--comprises a good candidate for second-line treatment in crizotinib-relapsed patients. The aim of this study was to determine possible mechanisms of resistance to ALK inhibitors that can appear in ALCL patients. METHODS: ALK+ ALCL cell lines resistant to crizotinib (Karpas299CR) and to CH5424802 (Karpas299CHR) were established by long-term exposure of Karpas299 cells to these inhibitors. Next, alterations in their sensitivity to ALK, HSP90 and mTOR inhibitors were investigated by cell viability and BrdU incorporation assays and immunoblot analysis. RESULTS: cDNA sequencing of ALK kinase domain revealed activating mutations-I1171T in Karpas299CR and F1174C in Karpas299CHR. The resistant cells displayed diminished sensitivity to structurally unrelated ALK inhibitors-crizotinib, CH5424802 and TAE684. Nevertheless, CH5424802 and TAE684 were still more potent against the resistant cells than crizotinib. Moreover, Karpas299CR and Karpas299CHR cells remained sensitive to HSP90 or mTOR inhibitors. CONCLUSIONS: Resistance mediated by activating mutations in ALK kinase domain may emerge in ALCL patients during ALK inhibitors treatment. However, more potent second-generation ALK inhibitors, HSP90 or mTOR inhibitors may represent an effective therapy for relapsed ALK+ ALCL patients.

Prognostic significance of Wnt-1, ß-catenin and E-cadherin expression in advanced colorectal carcinoma.

Wnt/ß-catenin pathway plays an important role in initiation and progression of colorectal oncogenesis. The aim of this study was to determine expression and localization of E-cadherin, ß-catenin and Wnt-1 proteins in colorectal tumors. Expression of ß-catenin, E-cadherin and Wnt-1 was determined by immunohistochemistry on advanced colorectal cancers. Abnormal expression of E-cadherin, ß-catenin, Wnt-1 was observed. Additionally, we revealed correlations between levels of studied proteins and histoclinical data. In multivariate analysis nuclear ß-catenin, higher carcinoembryonic antigen serum level before treatment, female sex and tumor localized in colon or rectum were independent unfavorable prognostic factors. These findings support the hypothesis that Wnt/ß-catenin pathway plays an important role in advanced colorectal carcinoma.

Silencing of Wnt-1 by siRNA induces apoptosis of MCF-7 human breast cancer cells.

OBJECTIVE: Wnt family of secreted-type glycoproteins plays key role in carcinogenesis and embryogenesis. Signals of Wnts are transduced through seven-transmembrane-type Wnt receptors encoded by Frizzled (Fzd) genes to the beta-catenin-Tcf pathway, the c-Jun-N-terminal kinase (JNK) pathway or the Ca(2+)-releasing pathway. Aberrant activation of the Wnt/beta-catenin signaling pathway is associated with a variety of human cancers. In human breast cancer, evidence of beta-catenin accumulation implies that the canonical Wnt signaling pathway is active in over 50% of carcinomas. RESULTS: We found that in breast cancer cells overexpressing Wnt-1 siRNA anti-Wnt-1 induced apoptosis and caused changes in downstream proteins levels. Among treated cells there were 71% apoptotic cells in comparison to cells treated with scrambled siRNA (6%) and control cells (6%) after 48 h (p < 0.01). METHODS: To examine if Wnt-1 signal is essential for cancer cell survival, we investigated the effect of Wnt-1 gene silencing in triggering of apoptosis in MCF-7 breast cancer cell line. Light microscopy, viability/cytotoxicity tests, flow cytometry, real-time PCR and western blotting were used for evaluation of the morphological features of cell death, percentage of apoptotic cells, Wnt-1 mRNA and protein level. CONCLUSION: Our results significantly indicate that anti-Wnt-1 siRNA inhibits Wnt-1 signaling, inducing apoptosis in human breast cancer MCF-7 cells and thus may serve as a potential anti-cancer drug.

BID-deficient breast cancer MCF-7 cells as a model for the study of autophagy in cancer therapy.

The BH3-only death factors share just the short BH3 domain with the other Bcl-2 family subclasses. With the exception of BID, which might also bind to BAX, they are thought to act by binding to and neutralizing Bcl-2 like survival factors.(1) Camptothecin (CPT)-induced apoptosis in breast cancer MCF-7 cells is associated with activation of cathepsin B and aggregation of BAX and BID on mitochondria. BID knock down protects cancer cells against apoptosis and induces autophagy, manifested with increased expression of Beclin 1 and MAP1LC3. The compensatory increase in the concentration of Hrk (another member of the BH3-only protein family) and its co-localization with BCL-2 on organelles in BID(-) breast cancer cells has also been observed. Nonetheless, Hrk is not able to substitute for BID in triggering apoptosis. Its role in autophagy induction is also doubtful, since MAP1LC3 expression was equally high in BID(-)Hrk(-) and BID(-)Hrk(+) breast cancer cells exposed to CPT. We conclude that BID can serve as a molecular switch between apoptosis and autophagy. BID(+) and BID(-) breast cancer MCF-7 cells could be considered to be a useful model for the study of the molecular interdependences between apoptosis and autophagy and the role of both processes in cancer therapy.

Minute kinetics of proapoptotic proteins: BAX and Smac/DIABLO in living tumor cells revealed by homeostatic confocal microscopy.

Traditional methods of visualization and analysis based on fixed cell populations treated with the drug for a different time give the limited possibility of time-sequence analysis. In time-lapse microscopy where the whole cell is observed regardless to intracellular structure, precise localization of events and differentiation between colocalization and overlapping of the fluorescence is impossible. Furthermore prolonged experiments with living cells increased the influence of improper environmental conditions. Homeostatic confocal microscopy gives an exceptional insight into minute pattern of changes occurring in the same living cell maintained in stable conditions during whole experimental period. It is built on a confocal system equipped with the homeostatic chamber providing constant, monitored heating and moisturized, CO(2)-enriched atmosphere during long period observations. In the present study 2D/time and 4D homeostatic confocal microscopy were applied for analysis of minute pattern of changes occurring at the mitochondria. The release of Smac/DIABLO from mitochondria in tumor cells under the apoptogenic stimulus, consist of two phases: the first immediately after drug administration, and the major second one after 15 min. Furthermore the time-pattern of BAX translocation to the mitochondria and Smac/DIABLO release coincide, suggesting that the release of Smac/DIABLO is correlated with BAX translocation to the mitochondria.

Colocalization of BAX with BID and VDAC-1 in nimesulide-induced apoptosis of human colon adenocarcinoma COLO 205 cells.

Cyclooxygenase (COX)-2 inhibitors that belong to non-steroid anti-inflammatory drug family have been shown to have an apoptosis-inducing effect on neoplastic cells. In the present study the effect of nimesulide (NIM), a specific COX-2 inhibitor, on apoptosis and interactions between BCL-2 family death promoters BAX and BID and BAX and VDAC-1 were examined in human colon adenocarcinoma COLO 205 cells. Laser scanning cytometry was applied for the measurement of expression and aggregation of apoptosis-related proteins and quantitative analysis of NIM-induced apoptosis. Double-staining immunoconfocal and immunoelectron microscopy were used for subcellular colocalization of examined proteins. NIM induced apoptosis of COLO 205 cells in a dose-dependent manner. This was accompanied by: (1) a decrease in intracellular prostaglandin (PG) E content; (2) subcellular redistribution and aggregation of BAX and BID on organellar membranes and within the nucleus; (3) colocalization of BAX with BID and BAX with VDAC-1 on organelles; and (4) survival of cells with the highest BCL-2 aggregation. A similar pattern of subcellular redistribution and colocalization of BAX with BID and BAX with VDAC-1 suggests that BAX (in association with BID) controls the function of VDAC-1 and its permeability for apoptogenic factors released from mitochondria of COLO 205 cells stimulated to apoptosis with NIM.