ABSTRACT
We previously reported that combined therapy with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) osimertinib and AXL inhibitor ONO-7475 is effective in preventing the survival of drug-tolerant cells in high-AXL-expressing EGFR-mutated non-small cell lung cancer (NSCLC) cells. Nevertheless, certain residual cells are anticipated to eventually develop acquired resistance to this combination therapy. In this study, we attempted to establish a multidrug combination therapy from the first-line setting to overcome resistance to this combination therapy in high-AXL-expressing EGFR-mutated NSCLC. siRNA screening assay showed that fibroblast growth factor receptor 1 (FGFR1) knockdown induced pronounced inhibition of cell viability in the presence of the osimertinib-ONO-7475 combination, which activates FGFR1 by upregulating FGF2 via the c-Myc pathway. Cell-based assays showed that triple therapy with osimertinib, ONO-7475, and the FGFR inhibitor BGJ398 significantly increased apoptosis by increasing expression of proapoptotic factor Bim and reduced cell viability compared with that observed for the osimertinib-ONO-7475 therapy. Xenograft models showed that triple therapy considerably suppressed tumor regrowth. A novel therapeutic strategy of additional initial FGFR1 inhibition may be highly effective in suppressing the emergence of osimertinib- and ONO-7475-resistant cells.
Subject(s)
Acrylamides , Aniline Compounds , Antineoplastic Combined Chemotherapy Protocols , Axl Receptor Tyrosine Kinase , Carcinoma, Non-Small-Cell Lung , ErbB Receptors , Lung Neoplasms , Proto-Oncogene Proteins , Pyrimidines , Receptor Protein-Tyrosine Kinases , Receptor, Fibroblast Growth Factor, Type 1 , Animals , Female , Humans , Mice , Acrylamides/pharmacology , Aniline Compounds/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Benzocycloheptenes , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Survival/drug effects , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Indoles , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice, Inbred BALB C , Mice, Nude , Mutation , Phenylurea Compounds/pharmacology , Phenylurea Compounds/administration & dosage , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Pyrimidines/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Fibroblast Growth Factor, Type 1/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Triazoles , Xenograft Model Antitumor AssaysABSTRACT
Tirabrutinib is a highly selective Bruton's tyrosine kinase (BTK) inhibitor used to treat hematological malignancies. We analyzed the anti-tumor mechanism of tirabrutinib using phosphoproteomic and transcriptomic methods. It is important to check the drug's selectivity against off-target proteins to understand the anti-tumor mechanism based on the on-target drug effect. Tirabrutinib's selectivity was evaluated by biochemical kinase profiling assays, peripheral blood mononuclear cell stimulation assays, and the BioMAP system. Next, in vitro and in vivo analyses of the anti-tumor mechanisms were conducted in activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) cells followed by phosphoproteomic and transcriptomic analyses. In vitro kinase assays showed that, compared with ibrutinib, tirabrutinib and other second-generation BTK inhibitors demonstrated a highly selective kinase profile. Data from in vitro cellular systems showed that tirabrutinib selectively affected B-cells. Tirabrutinib inhibited the cell growth of both TMD8 and U-2932 cells in correlation with the inhibition of BTK autophosphorylation. Phosphoproteomic analysis revealed the downregulation of ERK and AKT pathways in TMD8. In the TMD8 subcutaneous xenograft model, tirabrutinib showed a dose-dependent anti-tumor effect. Transcriptomic analysis indicated that IRF4 gene expression signatures had decreased in the tirabrutinib groups. In conclusion, tirabrutinib exerted an anti-tumor effect by regulating multiple BTK downstream signaling proteins, such as NF-κB, AKT, and ERK, in ABC-DLBCL.
Subject(s)
Lymphoma, Large B-Cell, Diffuse , Humans , Agammaglobulinaemia Tyrosine Kinase , Transcriptome , Proto-Oncogene Proteins c-akt/metabolism , Leukocytes, Mononuclear/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Protein Kinase Inhibitors/pharmacologyABSTRACT
FMS-like Tyrosine Kinase 3 (FLT3) mutation is associated with poor survival in acute myeloid leukemia (AML). The specific Anexelekto/MER Tyrosine Kinase (AXL) inhibitor, ONO-7475, kills FLT3-mutant AML cells with targets including Extracellular- signal Regulated Kinase (ERK) and Myeloid Cell Leukemia 1 (MCL1). ERK and MCL1 are known resistance factors for Venetoclax (ABT-199), a popular drug for AML therapy, prompting the investigation of the efficacy of ONO-7475 in combination with ABT-199 in vitro and in vivo. ONO-7475 synergizes with ABT-199 to potently kill FLT3-mutant acute myeloid leukemia cell lines and primary cells. ONO-7475 is effective against ABT-199-resistant cells including cells that overexpress MCL1. Proteomic analyses revealed that ABT-199-resistant cells expressed elevated levels of pro-growth and anti-apoptotic proteins compared to parental cells, and that ONO-7475 reduced the expression of these proteins in both the parental and ABT-199-resistant cells. ONO-7475 treatment significantly extended survival as a single in vivo agent using acute myeloid leukemia cell lines and PDX models. Compared to ONO-7474 monotherapy, the combination of ONO-7475/ABT-199 was even more potent in reducing leukemic burden and prolonging the survival of mice in both model systems. These results suggest that the ONO-7475/ABT-199 combination may be effective for AML therapy.
Subject(s)
Drug Resistance, Neoplasm , Leukemia, Myeloid, Acute , Protein Kinase Inhibitors , c-Mer Tyrosine Kinase , Animals , Apoptosis , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Mice , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Protein Kinase Inhibitors/pharmacology , Proteomics , Sulfonamides/pharmacology , c-Mer Tyrosine Kinase/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
The present study aims to develop a layered zirconium phosphate/phosphonate (LZP) powder to control the release of therapeutic inorganic ions. Organically modified LZPs were successfully prepared with various contents of phenyl groups via a reflux method in an aqueous solution containing phosphoric and phenylphosphonic acids. Powder X-ray diffraction analysis and Fourier transform infrared spectroscopy revealed that the crystal structure of the synthesized LZP samples was identical to that of α-zirconium phosphate, even after modification. The amount of incorporated organic molecules increased with increasing molar fractions of phenylphosphonic acid in the starting composition, as determined from the thermal analysis. Cobalt ion (Co2+), a type of therapeutic inorganic ion, was incorporated into the organically modified LZP through treatment with an acetonitrile solution containing tetrabutylammonium ions, followed by treatment with an acetonitrile solution containing CoCl2. The amount of incorporated Co2+ depended on the concentration of the phenyl groups. Furthermore, the highest amount of Co2+ was incorporated in the sample (ZP-Ph-0.5) prepared with equimolar phosphoric/phenylphosphonic acid. The ZP-Ph-0.5 sample additionally showed the ability to incorporate copper or iron ions (Cu2+ or Fe3+). The incorporated ion, either Co2+ or Cu2+, was continuously released from the ZP-Ph-0.5 sample in a saline solution over a period of three weeks, whereas the release of Fe3+ was negligible. The quantity of Co2+ released was higher than that of Cu2+. The controlled release of Co2+ from the ZP-Ph-0.5 sample was also observed in a simulated body fluid that mimicked the ionic concentration of human blood plasma. These results confirm that a specific degree of phenyl modification makes LZP a candidate host material for releasing therapeutic inorganic ions.
ABSTRACT
The orally available and novel small molecule ONO-7579 (N-{2-[4-(2-amino-5-chloropyridin-3-yl)phenoxy]pyrimidin-5-yl}-N'-[2-(methanesulfonyl)-5-(trifluoromethyl)phenyl]urea) is a highly potent and selective pan-tropomyosin receptor kinase (TRK) inhibitor. The objective of the present study was to characterize the pharmacokinetic (PK), pharmacodynamic (PD), and antitumor efficacy relationships of ONO-7579 in mice xenografted with a human colorectal cancer cell line, KM12 (harboring the tropomyosin 3 (TPM3) -neurotrophic tyrosine receptor kinase 1 fusion gene), via a PK/PD modeling approach. Plasma and tumor concentrations of ONO-7579, tumor levels of phosphorylated TPM3-TRKA (pTRKA), and tumor volumes in the murine model were measured with a single or multiple dose of ONO-7579 (0.06-0.60 mg/kg) administered once daily. The PK/PD/efficacy models were developed in a sequential manner. Changes in plasma concentrations of ONO-7579 were described with an oral one-compartment model. Tumor concentrations of ONO-7579 were higher than plasma concentrations, and changes in ONO-7579 tumor concentrations were described with an additional tumor compartment that had no influence on plasma concentrations. pTRKA in tumors was described with a direct Emax model, and the tumor ONO-7579 concentration causing 50% of the maximum effect was estimated to be 17.6 ng/g. In addition, a pTRKA-driven tumor growth inhibition model indicated that ONO-7579 started to sharply increase the antitumor effect at pTRKA inhibition rates >60% and required >91.5% to reduce tumors. In conclusion, the developed PK/PD/efficacy models revealed a "switch-like" relationship between pTRKA inhibition rate and antitumor effect in a murine KM12 xenograft model, demonstrating that pTRKA in tumors could serve as an effective biomarker for scheduling the dose regimen in early-stage clinical studies. SIGNIFICANCE STATEMENT: In recent years, clinical development of TRK inhibitors in patients with neurotrophic tyrosine receptor kinase fusion-positive solid tumors has been accelerated. This research found that phosphorylated TRKA was a useful biomarker for explaining the antitumor efficacy of TRK inhibitors using a pharmacokinetic/pharmacodynamic modeling approach in xenograft mice. This finding suggests a rational dosing regimen in early-stage clinical studies for ONO-7579 (N-{2-[4-(2-amino-5-chloropyridin-3-yl)phenoxy]pyrimidin-5-yl}-N'-[2-(methanesulfonyl)-5-(trifluoromethyl)phenyl]urea), a novel pan-TRK inhibitor.
Subject(s)
Organic Chemicals/pharmacology , Organic Chemicals/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacokinetics , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Disease Models, Animal , Female , Heterografts/drug effects , Heterografts/metabolism , Humans , Mice , Mice, Inbred BALB C , Phosphorylation/drug effects , Xenograft Model Antitumor Assays/methodsABSTRACT
PURPOSE: Currently, an optimal therapeutic strategy comprising molecularly targeted agents for treating EGFR-mutated non-small cell lung cancer (NSCLC) patients with acquired resistance to osimertinib is not available. Therefore, the initial therapeutic intervention is crucial for the prolonged survival of these patients. The activation of anexelekto (AXL) signaling is known to be associated with intrinsic and acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). In this study, we investigated the best therapeutic strategy to combat AXL-induced tolerance to EGFR-TKIs using the novel AXL inhibitor ONO-7475. EXPERIMENTAL DESIGN: We examined the efficacy of ONO-7475 in combination with EGFR-TKIs in EGFR-mutated NSCLC cells using in vitro and in vivo experiments. We investigated the correlation between AXL expression in tumors and clinical outcomes with osimertinib for EGFR-mutated NSCLC patients with acquired resistance to initial EGFR-TKIs. RESULTS: ONO-7475 sensitized AXL-overexpressing EGFR-mutant NSCLC cells to the EGFR-TKIs osimertinib and dacomitinib. In addition, ONO-7475 suppressed the emergence and maintenance of EGFR-TKI-tolerant cells. In the cell line-derived xenograft models of AXL-overexpressing EGFR-mutated lung cancer treated with osimertinib, initial combination therapy of ONO-7475 and osimertinib markedly regressed tumors and delayed tumor regrowth compared with osimertinib alone or the combination after acquired resistance to osimertinib. AXL expression in EGFR-TKI refractory tumors did not correlate with the sensitivity of osimertinib. CONCLUSIONS: These results demonstrate that ONO-7475 suppresses the emergence and maintenance of tolerant cells to the initial EGFR-TKIs, osimertinib or dacomitinib, in AXL-overexpressing EGFR-mutated NSCLC cells, suggesting that ONO-7475 and osimertinib is a highly potent combination for initial treatment.
Subject(s)
Acrylamides/pharmacology , Aniline Compounds/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Mutation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Quinolines/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mice , Mice, SCID , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Xenograft Model Antitumor Assays , Axl Receptor Tyrosine KinaseABSTRACT
Bruton's tyrosine kinase (BTK) is a key regulator of the B-cell receptor signaling pathway, and aberrant B-cell receptor (BCR) signaling has been implicated in the survival of malignant B-cells. However, responses of the diffuse large B-cell lymphoma (DLBCL) to inhibitors of BTK (BTKi) are infrequent, highlighting the need to identify mechanisms of resistance to BTKi as well as predictive biomarkers. We investigated the response to the selective BTKi, tirabrutinib, in a panel of 64 hematopoietic cell lines. Notably, only six cell lines were found to be sensitive. Although activated B-cell type DLBCL cells were most sensitive amongst all cell types studied, sensitivity to BTKi did not correlate with the presence of activating mutations in the BCR pathway. To improve efficacy of tirabrutinib, we investigated combination strategies with 43 drugs inhibiting 34 targets in six DLBCL cell lines. Based on the results, an activated B-cell-like (ABC)-DLBCL cell line, TMD8, was the most sensitive cell line to those combinations, as well as tirabrutinib monotherapy. Furthermore, tirabrutinib in combination with idelalisib, palbociclib, or trametinib was more effective in TMD8 with acquired resistance to tirabrutinib than in the parental cells. These targeted agents might be usefully combined with tirabrutinib in the treatment of ABC-DLBCL.
ABSTRACT
The activated B-cell diffuse large B-cell-like lymphoma (ABC-DLBCL) correlates with poor prognosis. The B-cell receptor signaling pathway is known to be dysregulated in NHL/CLL and given BTK is a downstream mediator of BCR signaling, BTK constitutes an interesting and obvious therapeutic target. Given the high potency and selectivity of the BTK inhibitor, ONO/GS-4059, it was hypothesized that, the anti-tumor activity of ONO/GS-4059 could be further enhanced by combining it with the anti-CD20 Abs, rituximab (RTX) or obinutuzumab (GA101). ONO/GS-4059 combined with GA101 or RTX was significantly better than the respective monotherapy with tumor growth inhibition (TGI) of 90% for the GA101 combination and 86% for the RTX combination. In contrast, ibrutinib (PCI-32765) combined with RTX did not result in improved efficacy compared with respective monotherapy. Taken together these data indicate that the combination of ONO/GS-4059 with rituximab and particularly obinutuzumab may be an effective treatment for ABC-DLBCL.