piperidines and crenolanib

The FMS-like tyrosine kinase 3 (FLT3) gene is mutated in approximately one third of patients with acute myeloid leukemia (AML), either by internal tandem duplications (FLT3-ITD), or by a point mutation mainly involving the tyrosine kinase domain (FLT3-TKD). Patients with FLT3-ITD have a high risk of relapse and low cure rates. Several FLT3 tyrosine kinase inhibitors have been developed in the last few years with variable kinase inhibitory properties, pharmacokinetics, and toxicity profiles. FLT3 inhibitors are divided into first generation multi-kinase inhibitors (such as sorafenib, lestaurtinib, midostaurin) and next generation inhibitors (such as quizartinib, crenolanib, gilteritinib) based on their potency and specificity of FLT3 inhibition. These diverse FLT3 inhibitors have been evaluated in myriad clinical trials as monotherapy or in combination with conventional chemotherapy or hypomethylating agents and in various settings, including front-line, relapsed or refractory disease, and maintenance therapy after consolidation chemotherapy or allogeneic stem cell transplantation. In this practical question-and-answer-based review, the main issues faced by the leukemia specialists on the use of FLT3 inhibitors in AML are addressed.

Topics: Aniline Compounds; Antineoplastic Agents; Benzimidazoles; Benzothiazoles; Carbazoles; DNA Methylation; Enzyme Inhibitors; fms-Like Tyrosine Kinase 3; Furans; Humans; Leukemia, Myeloid, Acute; Mutation; Neoplasm Recurrence, Local; Phenylurea Compounds; Piperidines; Prognosis; Pyrazines; Randomized Controlled Trials as Topic; Sorafenib; Staurosporine; Treatment Outcome

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene arise in 25-30% of all acute myeloid leukemia (AML) patients. These mutations lead to constitutive activation of the protein product and are divided in two broad types: internal tandem duplication (ITD) of the juxtamembrane domain (25% of cases) and point mutations in the tyrosine kinase domain (TKD). Patients with FLT3 ITD mutations have a high relapse risk and inferior cure rates, whereas the role of FLT3 TKD mutations still remains to be clarified. Additionally, growing research indicates that FLT3 status evolves through a disease continuum (clonal evolution), where AML cases can acquire FLT3 mutations at relapse - not present in the moment of diagnosis. Several FLT3 inhibitors have been tested in patients with FLT3-mutated AML. These drugs exhibit different kinase inhibitory profiles, pharmacokinetics and adverse events. First-generation multi-kinase inhibitors (sorafenib, midostaurin, lestaurtinib) are characterized by a broad-spectrum of drug targets, whereas second-generation inhibitors (quizartinib, crenolanib, gilteritinib) show more potent and specific FLT3 inhibition, and are thereby accompanied by less toxic effects. Notwithstanding, all FLT3 inhibitors face primary and acquired mechanisms of resistance, and therefore the combinations with other drugs (standard chemotherapy, hypomethylating agents, checkpoint inhibitors) and its application in different clinical settings (upfront therapy, maintenance, relapsed or refractory disease) are under study in a myriad of clinical trials. This review focuses on the role of FLT3 mutations in AML, pharmacological features of FLT3 inhibitors, known mechanisms of drug resistance and accumulated evidence for the use of FLT3 inhibitors in different clinical settings.

Topics: Aniline Compounds; Antineoplastic Agents; Benzimidazoles; Benzothiazoles; Carbazoles; Drug Resistance, Multiple; Drug Resistance, Neoplasm; fms-Like Tyrosine Kinase 3; Forecasting; Furans; Hematopoietic Stem Cell Transplantation; Humans; Imidazoles; Leukemia, Myeloid, Acute; Maintenance Chemotherapy; Mutation; Phenylurea Compounds; Piperidines; Point Mutation; Protein Kinase Inhibitors; Pyrazines; Pyridazines; Recurrence; Sorafenib; Staurosporine

Mutations within the activation loop of members of the class III receptor tyrosine kinase (RTK) subfamily, which includes KIT, PDGFRA, and FLT3, have been observed in multiple human tumor types. These mutations confer constitutive activation as well as resistance to the type II tyrosine kinase inhibitors (TKI) that are currently clinically available, such as imatinib and sunitinib. It is now understood that activation loop mutations in class III RTKs shift the activation state equilibrium away from inactive states, to which type II TKIs bind, to the active state by destabilizing the inactive conformation. Recently, type I TKIs, which can bind to active kinase conformations, have been developed with specificity for class III RTK members. Some type I TKIs, such as crenolanib and avapritinib (BLU-285), have entered clinical studies for patients with activation loop mutations in KIT, PDGFRA, or FLT3. Preliminary results suggest that these type I TKIs show activity in these patient populations that previously lacked effective treatments. This article reviews the inactive and active structures of KIT, PDGFRA, and FLT3, how the mutations seen in human cancers affect kinase structure, and the clinical implications of these mutations in terms of type I vs. type II TKI binding.

Topics: Antineoplastic Agents; Benzimidazoles; Drug Development; fms-Like Tyrosine Kinase 3; Humans; Mutation; Neoplasms; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-kit; Receptor Protein-Tyrosine Kinases; Receptor, Platelet-Derived Growth Factor alpha

The past 15 years have seen major leaps in our understanding of the molecular genetic mutations that act as drivers of acute myeloid leukemia (AML). Clinical trials of agents against specific mutant proteins, such as FLT3-internal tandem duplications (ITDs) and isocitrate dehydrogenase mutations (IDHs) are ongoing. This review discusses agents in clinical trials that target specific gene mutations and/or epigenetic targets.

Topics: Aniline Compounds; Antineoplastic Agents; Benzimidazoles; Benzothiazoles; Clinical Trials as Topic; Epigenesis, Genetic; fms-Like Tyrosine Kinase 3; Humans; Isocitrate Dehydrogenase; Leukemia, Myeloid, Acute; Molecular Targeted Therapy; Mutation; Phenylurea Compounds; Piperidines; Proto-Oncogene Proteins c-bcl-2; Pyrazines

Outcomes for the majority of patients with acute myeloid leukemia (AML) remain poor. Over the past decade, significant progress has been made in the understanding of the cytogenetic and molecular determinants of AML pathogenesis. One such advance is the identification of recurring mutations in the FMS-like tyrosine kinase 3 gene (FLT3). Currently, this marker, which appears in approximately one-third of all AML patients, not only signifies a poorer prognosis but also identifies an important target for therapy. FLT3 inhibitors have now undergone clinical evaluation in Phase I, II and III clinical trials, as both single agents and in combination with chemotherapeutics. Unfortunately, to date, none of the FLT3 inhibitors have gained FDA approval for the treatment of patients with AML. Yet, several promising FLT3 inhibitors are being evaluated in all phases of drug development.. This review aims to highlight the agents furthest along in their development. It also focuses on those FLT3 inhibitors that are being evaluated in combination with other anti-leukemia agents.. The authors believe that the field of research for FLT3 inhibitors remains promising, despite the historically poor prognosis of this subgroup of patients with AML. The most promising areas of research will likely be the elucidation of the mechanisms of resistance to FLT3 inhibitors, and development of potent FLT3 inhibitors alone or in combination with hypomethylating agents, cytotoxic chemotherapy or with other targeted agents.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Benzothiazoles; Drug Resistance, Neoplasm; fms-Like Tyrosine Kinase 3; Humans; Imidazoles; Leukemia, Myeloid, Acute; Niacinamide; Phenylurea Compounds; Piperidines; Protein Kinase Inhibitors; Pyridazines; Sorafenib; Staurosporine

Paclitaxel plus ramucirumab is a standard second-line regimen for patients with advanced gastric adenocarcinoma, but clinical benefit remains modest. One potential resistance mechanism to VEGFR2 inhibition is activation of the PDGF/PDGFR pathway, which can be blocked by the selective inhibitor crenolanib. Therefore, we performed a phase I/Ib study of crenolanib in combination with paclitaxel/ramucirumab.. We enrolled 19 patients in the dose escalation phase and 8 patients in the dose expansion phase at the MTD of crenolanib 100 mg BID. Common grade 3/4 treatment-emergent adverse events included leukopenia (19%), anemia (11%) and neutropenia (11%). In the 14 patients treated at the MTD, 6-month PFS was 43% [95% confidence interval (CI) 23-78%] and the objective response rate (ORR) was 42% (95% CI 15-72%). The trial was terminated early due to withdrawal of crenolanib by the sponsor.. The addition of crenolanib to paclitaxel/ramucirumab is safe and well-tolerated at a dose level up to 100 mg BID.. NCT03193918. June 19, 2017.

Topics: Adenocarcinoma; Adult; Aged; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Dose-Response Relationship, Drug; Esophageal Neoplasms; Female; Humans; Male; Maximum Tolerated Dose; Middle Aged; Paclitaxel; Piperidines; Progression-Free Survival; Ramucirumab; Stomach Neoplasms

FLT3 mutations are prevalent in AML patients and confer poor prognosis. Crenolanib, a potent type I pan-FLT3 inhibitor, is effective against both internal tandem duplications and resistance-conferring tyrosine kinase domain mutations. While crenolanib monotherapy has demonstrated clinical benefit in heavily pretreated relapsed/refractory AML patients, responses are transient and relapse eventually occurs. Here, to investigate the mechanisms of crenolanib resistance, we perform whole exome sequencing of AML patient samples before and after crenolanib treatment. Unlike other FLT3 inhibitors, crenolanib does not induce FLT3 secondary mutations, and mutations of the FLT3 gatekeeper residue are infrequent. Instead, mutations of NRAS and IDH2 arise, mostly as FLT3-independent subclones, while TET2 and IDH1 predominantly co-occur with FLT3-mutant clones and are enriched in crenolanib poor-responders. The remaining patients exhibit post-crenolanib expansion of mutations associated with epigenetic regulators, transcription factors, and cohesion factors, suggesting diverse genetic/epigenetic mechanisms of crenolanib resistance. Drug combinations in experimental models restore crenolanib sensitivity.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Epigenesis, Genetic; Exome Sequencing; Female; fms-Like Tyrosine Kinase 3; GTP Phosphohydrolases; HEK293 Cells; Humans; Inhibitory Concentration 50; Isocitrate Dehydrogenase; Leukemia, Myeloid, Acute; Male; Membrane Proteins; Mice; Middle Aged; Mutation; Piperidines; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Tandem Repeat Sequences; Treatment Outcome

Mutations of the type III receptor tyrosine kinase FLT3 occur in approximately 30% of acute myeloid leukemia patients and lead to constitutive activation. This has made FLT3-activating mutations an attractive drug target because they are probable driver mutations of this disease. As more potent FLT3 inhibitors are developed, a predictable development of resistance-conferring point mutations, commonly at residue D835, has been observed. Crenolanib is a highly selective and potent FLT3 tyrosine kinase inhibitor (TKI) with activity against the internal tandem duplication (FLT3/ITD) mutants and the FLT3/D835 point mutants. We tested crenolanib against a panel of D835 mutant cell lines and primary patient blasts and observed superior cytotoxic effects when compared with other available FLT3 TKIs such as quizartinib and sorafenib. Another potential advantage of crenolanib is its reduced inhibition of c-Kit compared with quizartinib. In progenitor cell assays, crenolanib was less disruptive of erythroid colony growth, which may result in relatively less myelosuppression than quizartinib. Finally, correlative data from an ongoing clinical trial demonstrate that acute myeloid leukemia patients can achieve sufficient levels of crenolanib to inhibit both FLT3/ITD and resistance-conferring FLT3/D835 mutants in vivo. Crenolanib is thus an important next-generation FLT3 TKI.

Topics: Antineoplastic Agents; Benzimidazoles; Bone Marrow; Bone Marrow Cells; Colony-Forming Units Assay; Drug Resistance, Neoplasm; fms-Like Tyrosine Kinase 3; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Piperidines; Point Mutation; Prognosis; Proto-Oncogene Proteins c-kit; Sequence Analysis, DNA; Tetrazolium Salts; Thiazoles; Time Factors

This phase I, first-in-human study evaluated the safety, tolerability, pharmacokinetics, and maximum-tolerated dose (MTD) of an oral platelet-derived growth factor receptor inhibitor, CP-868,596.. Patients with advanced solid tumors were eligible. Dose escalations were performed in three groups with two formulations: uncoated on an empty stomach (UES), uncoated with food (UFED), and film-coated (FC) without food. Initial dose escalation in the UES group was followed by parallel escalations in the UFED and FC groups.. Fifty-nine patients enrolled. CP-868,596 was escalated from 100 mg to 340 mg daily in the UES group, from 60 mg to 100 mg twice daily in the UFED group, and from 100 mg once daily to 140 mg twice daily in the FC group. MTDs were 200 mg daily in the UES group and 100 mg twice daily in the FC group; MTD was not reached at 100 mg twice daily in the UFED group. Dose-limiting toxicities included hematuria, increased gamma-glutamyltransferase or ALT, insomnia, and nausea/vomiting. Most treatment-related AEs were of grades 1 to 2 severity; nausea, vomiting, and diarrhea were reported most frequently. Administration with food generally improved tolerability. CP-868,596 was absorbed slowly; systemic exposure parameters appeared to increase greater than proportionally with dose. Mean serum concentrations exceeded the preclinically predicted minimal efficacious concentration (ie, 16 ng/mL) at all dosages. Food and film coating apparently increased interpatient variability of the maximum observed plasma concentration and the area under the concentration-time curve. No objective responses were reported, and eight patients achieved stable disease (mean duration, 5.7 months).. CP-868,596 potentially demonstrated greater than dose-proportional pharmacokinetics. The recommended dosage of 100 mg twice daily with food was well tolerated. Additional development as a single agent in selected populations or in combination with chemotherapy in broader populations is warranted.

Topics: Administration, Oral; Adolescent; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Benzimidazoles; Dose-Response Relationship, Drug; Female; Humans; Male; Maximum Tolerated Dose; Middle Aged; Neoplasms; Piperidines; Protein Kinase Inhibitors; Receptors, Platelet-Derived Growth Factor; Young Adult

To evaluate the safety, activity, and emergence of FLT3-kinase domain (KD) mutations with combination therapy of crenolanib and sorafenib in acute myeloid leukemia (AML) with FLT3-internal tandem duplication (ITD).. After in vitro and xenograft efficacy studies using AML cell lines that have FLT3-ITD with or without FLT3-KD mutation, a pilot study was performed with crenolanib (67 mg/m2/dose, three times per day on days 1-28) and two dose levels of sorafenib (150 and 200 mg/m2/day on days 8-28) in 9 pediatric patients with refractory/relapsed FLT3-ITD-positive AML. Pharmacokinetic, pharmacodynamic, and FLT3-KD mutation analysis were done in both preclinical and clinical studies.. The combination of crenolanib and sorafenib in preclinical models showed synergy without affecting pharmacokinetics of each agent, inhibited p-STAT5 and p-ERK for up to 8 hours, and led to significantly better leukemia response (P < 0.005) and survival (P < 0.05) compared with single agents. Fewer FLT3-KD mutations emerged with dose-intensive crenolanib (twice daily) and low-intensity sorafenib (three times/week) compared with daily crenolanib or sorafenib (P < 0.05). The crenolanib and sorafenib combination was tolerable without dose-limiting toxicities, and three complete remissions (one with incomplete count recovery) and one partial remission were observed in 8 evaluable patients. Median crenolanib apparent clearance showed a nonsignificant decrease during treatment (45.0, 40.5, and 20.3 L/hour/m2 on days 1, 7, and 14, respectively) without drug-drug interaction. Only 1 patient developed a FLT3-KD mutation (FLT3 F691L).. The combination of crenolanib and sorafenib was tolerable with antileukemic activities and rare emergence of FLT3-TKD mutations, which warrants further investigation.

Topics: Antineoplastic Agents; Benzimidazoles; Child; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Mutation; Phenylurea Compounds; Pilot Projects; Piperidines; Protein Kinase Inhibitors; Sorafenib

The kinase FLT3 internal tandem duplication (FLT3-ITD) is related to poor clinical outcomes of acute myeloid leukemia (AML). FLT3 inhibitors have provided novel strategies for the treatment of FLT3-ITD-positive AML. But they are limited by rapid development of acquired resistance and refractory in monotherapy. Recent evidence shows that inducing the degradation of FLT3-mutated protein is an attractive strategy for the treatment of FLT3-ITD-positive AML, especially those with FLT3 inhibitor resistance. In this study we identified Wu-5 as a novel USP10 inhibitor inducing the degradation of FLT3-mutated protein. We showed that Wu-5 selectively inhibited the viability of FLT3 inhibitor-sensitive (MV4-11, Molm13) and -resistant (MV4-11R) FLT3-ITD-positive AML cells with IC

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents; Apoptosis; Benzimidazoles; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Piperidines; Proteasome Endopeptidase Complex; Proteolysis; Signal Transduction; Thiophenes; Ubiquitin Thiolesterase

Recently developed molecularly targeted therapies such as EGFR inhibitors have notably improved the prognosis of patients with cancer. However, patients with KRAS and BRAF mutations do not currently benefit from these therapies. Here, we aimed to examine potential effects of crenolanib as a new molecularly targeted therapy in colorectal cancer. We used multiple colorectal cancer cell lines to investigate the growth-inhibitory effect of crenolanib and its effect in combination with other cytotoxic agents. Primary cultures of patient-derived organoids (PDO), a model that reflects the heterogeneity of clinical colorectal cancer, were used to further validate the effects of crenolanib. Unlike cetuximab, crenolanib remarkably suppressed ERK and AKT/mTOR pathways in HT29 cells with BRAF mutation and in HCT116 cells with KRAS mutation with corresponding growth-suppressing effects. Additive or synergistic effects were observed in treatments with combination of crenolanib and other cytotoxic drugs. Moreover, crenolanib suppressed the expression of stem cell markers, such as OCT4, NANOG, and SOX2. These observations were substantiated in seven PDOs with KRAS mutation and two PDOs without KRAS/BRAF mutations, with crenolanib suppressing the growth of all PDOs regardless of their KRAS mutation status. Furthermore, crenolanib abrogated PDGF- and TGFβ-induced increase of OCT4-positive cells in PDOs. Together, these findings suggest that crenolanib may have clinical utility for patients with colorectal cancer, especially patients with KRAS/BRAF mutations. IMPLICATIONS: These findings indicate that crenolanib can be a useful target agent for patients with colorectal cancer, especially patients with KRAS/BRAF mutations.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Colorectal Neoplasms; Humans; Mice; Mice, Inbred NOD; Organoids; Piperidines; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Signal Transduction

Chronic liver diseases are associated with excessive deposition of extracellular matrix proteins. This so-called fibrosis can progress to cirrhosis and impair vital functions of the liver. We examined whether the receptor tyrosine kinase (RTK) class III inhibitor Crenolanib affects the behavior of hepatic stellate cells (HSC) involved in fibrogenesis. Rats were treated with thioacetamide (TAA) for 18 weeks to trigger fibrosis. After TAA treatment, the animals received Crenolanib for two weeks, which significantly improved recovery from liver fibrosis. Because Crenolanib predominantly inhibits the RTK platelet-derived growth factor receptor-β, impaired HSC proliferation might be responsible for this beneficial effect. Interestingly, blocking of RTK signaling by Crenolanib not only hindered HSC proliferation but also triggered their specification into hepatic endoderm. Endodermal specification was mediated by p38 mitogen-activated kinase (p38 MAPK) and c-Jun-activated kinase (JNK) signaling; however, this process remained incomplete, and the HSC accumulated lipids. JNK activation was induced by stress response-associated inositol-requiring enzyme-1α (IRE1α) in response to Crenolanib treatment, whereas β-catenin-dependent WNT signaling was able to counteract this process. In conclusion, the Crenolanib-mediated inhibition of RTK impeded HSC proliferation and triggered stress responses, initiating developmental processes in HSC that might have contributed to improved recovery from liver fibrosis in TAA-treated rats.

Topics: Animals; Becaplermin; Benzimidazoles; Biomarkers; Cell Differentiation; Cell Proliferation; Endoderm; Hepatic Stellate Cells; JNK Mitogen-Activated Protein Kinases; Liver Cirrhosis; Male; Models, Biological; p38 Mitogen-Activated Protein Kinases; Piperidines; Rats, Wistar; Thioacetamide; Wnt Signaling Pathway

Recently, several targeted agents have been developed for specific subsets of patients with acute myeloid leukaemia (AML), including midostaurin, the first FDA-approved FLT3 inhibitor for newly diagnosed patients with FLT3 mutations. However, in the initial Phase I/II clinical trials, some patients without FLT3 mutations had transient responses to midostaurin, suggesting that this multi-targeted kinase inhibitor might benefit AML patients more broadly. Here, we demonstrate submicromolar efficacy of midostaurin in vitro and efficacy in vivo against wild-type (wt) FLT3-expressing AML cell lines and primary cells, and we compare its effectiveness with that of other FLT3 inhibitors currently in clinical trials. Midostaurin was found to synergize with standard chemotherapeutic drugs and some targeted agents against AML cells without mutations in FLT3. The mechanism may involve, in part, the unique kinase profile of midostaurin that includes proteins implicated in AML transformation, such as SYK or KIT, or inhibition of ERK pathway or proviability signalling. Our findings support further investigation of midostaurin as a chemosensitizing agent in AML patients without FLT3 mutations.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; Benzimidazoles; Benzothiazoles; Cell Line, Tumor; Cell Proliferation; Drug Synergism; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; Mice; Mutation; Phenylurea Compounds; Piperidines; Protein Kinase Inhibitors; Pyrazines; Sorafenib; Staurosporine; Syk Kinase

To analyze how monocyte and macrophage exposure to CXCL4 induces inflammatory and fibrotic processes observed in Systemic sclerosis (SSc) patients.. In six independent experiments, monocytes of healthy controls (HC) and SSc patients were stimulated with CXCL4, TLR-ligands, IFNɑ or TGFβ and the secretion of cytokines in the supernatant was assessed by multiplex immunoassays. PDGF-BB production by monocyte-derived macrophages was quantified using immunoassays. The number of monocytes and PDGF-BB in circulation was quantified in HC and SSc patients with the Sysmex XT-1800i haematology counter and immunoassays. Intracellular PDGF-BB was quantified in monocytes by Western blot. PDGF-receptor inhibition was achieved using siRNA-mediated knockdown or treatment with Crenolanib. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblasts was analyzed by qPCR, ELISA and ECM deposition assays.. SSc and HC monocytes released PDGF-BB upon stimulation with CXCL4. Conversely, TLR ligands, IFNɑ or TGFβ did not induce PDGF-bb release. PDGF-BB plasma levels were significantly (P = 0.009) higher in diffuse SSc patients (n = 19), compared with HC (n = 21). In healthy dermal fibroblasts, PDGF-BB enhanced TNFɑ-induced expression of inflammatory cytokines and increased ECM production. Comparable results were observed in fibroblasts cultured in supernatant taken from macrophages stimulated with CXCL4. This effect was almost completely abrogated by inhibition of the PDGF-receptor using Crenolanib.. Our findings demonstrate that CXCL4 can drive fibroblast activation indirectly via PDGF-BB production by myeloid cells. Hence, targeting PDGF-BB or CXCL4-induced PDGF-BB release could be clinically beneficial for patients with SSc.

Topics: Adult; Aged; Becaplermin; Benzimidazoles; Cells, Cultured; Cytokines; Female; Fibroblasts; Humans; Inflammation; Macrophages; Male; Middle Aged; Monocytes; Piperidines; Platelet Factor 4; Receptors, Platelet-Derived Growth Factor; Scleroderma, Systemic

Crenolanib (CP-868,596), a potent inhibitor of FLT3 and PDGFRα/β, is currently under phase III clinical investigation for the treatment of acute myeloid leukemia. However, the protein targets of Crenolanib in cancer cells remain obscure, which results in difficulties in understanding the mechanism of actions and side effects. To alleviate this issue, in this study, a photoaffinity probe and two fluorescent probes were created based on Crenolanib, followed by competitive protein profiling and bioimaging studies, with the aim of characterizing the cellular targets. A series of unknown protein hits, such as MAPK1, SHMT2, SLC25A11, and HIGD1A, were successfully identified by means of pull-down/LC-MS/MS; these might provide valuable clues for understanding drug action and potential toxicities. Moreover, the fluorescent probes are suitable for imaging drug distribution at the single-cell level.

Topics: Benzimidazoles; Binding Sites; Cell Line, Tumor; Fluorescent Dyes; fms-Like Tyrosine Kinase 3; Humans; Microscopy, Fluorescence; Molecular Docking Simulation; Photoaffinity Labels; Piperidines; Protein Binding; Protein Kinase Inhibitors; Receptor, Platelet-Derived Growth Factor alpha

Platelet-derived growth factor receptor α (PDGFRα), a receptor tyrosine kinase required for cardiac fibroblast development, is uniquely expressed by fibroblasts in the adult heart. Despite the consensus that PDGFRα is expressed in adult cardiac fibroblasts, we know little about its function when these cells are at rest. Here, we demonstrate that loss of PDGFRα in cardiac fibroblasts resulted in a rapid reduction of resident fibroblasts. Furthermore, we observe that phosphatidylinositol 3-kinase signaling was required for PDGFRα-dependent fibroblast maintenance. Interestingly, this reduced number of fibroblasts was maintained long-term, suggesting that there is no homeostatic mechanism to monitor fibroblast numbers and restore hearts to wild-type levels. Although we did not observe any systolic functional changes in hearts with depleted fibroblasts, the basement membrane and microvasculature of these hearts were perturbed. Through in vitro analyses, we showed that PDGFRα signaling inhibition resulted in an increase in fibroblast cell death, and PDGFRα stimulation led to increased levels of the cell survival factor activating transcription factor 3. Our data reveal a unique role for PDGFRα signaling in fibroblast maintenance and illustrate that a 50% loss in cardiac fibroblasts does not result in lethality.

Topics: Adult; Animals; Apoptosis; Benzimidazoles; Cell Lineage; Cell Survival; Cells, Cultured; Female; Fibroblasts; Heart Ventricles; Humans; Imatinib Mesylate; Male; Mice, Knockout; Middle Aged; Phosphatidylinositol 3-Kinase; Piperidines; Receptor, Platelet-Derived Growth Factor alpha; Signal Transduction

Crenolanib is a tyrosine kinase inhibitor targeting PDGFR-α, PDGFR-β and Fms related tyrosine kinase-3 (FLT3) that is currently evaluated in several clinical trials. Although platelet-derived growth factor receptor (PDGFR) signalling pathway is believed to play an important role in angiogenesis and maintenance of functional vasculature, we here demonstrate a direct angiostatic activity of crenolanib independently of PDGFR signalling.. The activity of crenolanib on cell viability, migration, sprouting, apoptosis and mitosis was assessed in endothelial cells, tumour cells and fibroblasts. Alterations in cell morphology were determined by immunofluorescence experiments. Flow-cytometry analysis and mRNA expression profiles were used to investigate cell differentiation. In vivo efficacy was investigated in human ovarian carcinoma implanted on the chicken chorioallantoic membrane (CAM).. Crenolanib was found to inhibit endothelial cell viability, migration and sprout length, and induced apoptosis independently of PDGFR expression. Treated cells  showed altered actin arrangement and nuclear aberrations. Mitosis was affected at several levels including mitosis entry and centrosome clustering. Crenolanib suppressed human ovarian carcinoma tumour growth and angiogenesis in the CAM model.. The PDGFR/FLT3 inhibitor crenolanib targets angiogenesis and inhibits tumour growth in vivo unrelated to PDGFR expression. Based on our findings, we suggest a broad mechanism of action of crenolanib.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Apoptosis; Benzimidazoles; Cell Movement; Chickens; Female; fms-Like Tyrosine Kinase 3; Human Umbilical Vein Endothelial Cells; Humans; Mitosis Modulators; Ovarian Neoplasms; Piperidines; Receptors, Platelet-Derived Growth Factor

Mutations in two type-3 receptor tyrosine kinases (RTKs), KIT and FLT3, are common in both acute myeloid leukaemia (AML) and systemic mastocytosis (SM) and lead to hyperactivation of key signalling pathways. A large number of tyrosine kinase inhibitors (TKIs) have been developed that target either FLT3 or KIT and significant clinical benefit has been demonstrated in multiple clinical trials. Given the structural similarity of FLT3 and KIT, it is not surprising that some of these TKIs inhibit both of these receptors. This is typified by midostaurin, which has been approved by the US Food and Drug Administration for mutant FLT3-positive AML and for KIT D816V-positive SM. Here, we compare the in vitro activities of the clinically available FLT3 and KIT inhibitors with those of midostaurin against a panel of cells expressing a variety of oncogenic FLT3 or KIT receptors, including wild-type (wt) FLT3, FLT3-internal tandem duplication (ITD), FLT3 D835Y, the resistance mutant FLT3-ITD+ F691L, KIT D816V, and KIT N822K. We also examined the effects of these inhibitors in vitro and in vivo on cells expressing mutations in c-CBL found in AML that result in hypersensitization of RTKs, such as FLT3 and KIT. The results show a wide spectrum of activity of these various mutations to these clinically available TKIs.

Topics: Aniline Compounds; Antineoplastic Agents; Benzimidazoles; Benzothiazoles; Cell Line, Tumor; Drug Screening Assays, Antitumor; fms-Like Tyrosine Kinase 3; Hematologic Neoplasms; Humans; Mutant Proteins; Phenylurea Compounds; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-cbl; Proto-Oncogene Proteins c-kit; Pyrazines; Pyrazoles; Pyrroles; Sorafenib; Staurosporine; Triazines

Fms-like tyrosine kinase 3 (FLT3) belongs to the receptor tyrosine kinase family and expressed in hematopoietic progenitor cells. FLT3 gene mutations are reported in ~30% of acute myeloid leukemia cases. FLT3 kinase domain mutation F691L is one of the common causes of acquired resistance to the FLT3 inhibitors including quizartinib. MZH29 and crenolanib were previously reported to inhibit FLT3 F691L. However, crenolanib was reported for the moderate inhibition. We found that Glu661and Asp829 were the most significant residues to target the FLT3 F691L which contribute most significantly to the binding energy with MZH29 and crenolanib. These interactions were found absent with quizartinib. Further free energy landscape analysis revealed that FLT3 F691L bound to MZH29 and crenolanib was more stable as compared to quizartinib.

Topics: Benzimidazoles; Binding Sites; fms-Like Tyrosine Kinase 3; Humans; Hydrogen Bonding; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Piperidines; Protein Kinase Inhibitors; Protein Structure, Tertiary; Thermodynamics

FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein expressed on normal hematopoietic stem and progenitor cells (HSC) and retained on malignant blasts in acute myeloid leukemia (AML). We engineered CD8

Topics: Benzimidazoles; Cell Line, Tumor; Drug Synergism; fms-Like Tyrosine Kinase 3; Hematopoietic Stem Cell Transplantation; Humans; Immunotherapy, Adoptive; Leukemia, Myeloid, Acute; Piperidines; Receptors, Chimeric Antigen; Tandem Repeat Sequences; Tumor Cells, Cultured

Platelet‑derived growth factor receptor‑β (PDGFR‑β) in epithelial tumors is mainly expressed by stromal cells. High expression of PDGFR‑β has been related to poor prognosis in several cancers, however its significance in colorectal cancer (CRC) remains unknown. The present study aimed to clarify the prognostic impact of PDGFR‑β in CRC patients. The study included 194 patients who underwent surgery for CRC. PDGFR‑β expression was examined by real‑time reverse transcription‑polymerase chain reaction and immunohistochemistry and the expression levels were correlated with various clinical parameters. The biological significance was evaluated by knockdown experiments in CRC cell lines and the specific PDGFR inhibitor, crenolanib. PDGFR‑β mRNA and protein expression levels were positively correlated with each other. Low PDGFR‑β expression was associated with significantly better disease‑free survival after curative surgical resection, than high PDGFR‑β expression, according to univariate and multivariate analyses. The assessment of PDGFR‑β knockdown in two cell lines revealed that small interfering RNA (siRNA) inhibition resulted in statistically significant reductions in cell growth and invasion. PDGFR inhibitor suppressed CRC cell proliferation in vitro in a dose‑dependent manner. In conclusion, PDGFR‑β expression was a risk factor for recurrence in patients with CRC and PDGFR inhibitor may be a useful therapeutic agent for CRC.

Topics: Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Male; Piperidines; Prognosis; Receptor, Platelet-Derived Growth Factor beta; Recurrence; Survival Analysis; Up-Regulation

Gastrointestinal stromal tumors (GIST) carrying the D842V activating mutation in the platelet-derived growth factor receptor alpha (

Topics: Adult; Aged; Benzimidazoles; Drug Resistance, Neoplasm; Electron Transport Complex II; F-Box-WD Repeat-Containing Protein 7; Female; Gastrointestinal Neoplasms; Gastrointestinal Stromal Tumors; Humans; Imatinib Mesylate; Isocitrate Dehydrogenase; Male; Middle Aged; Mutation, Missense; Piperidines; Protein Binding; Protein Kinase Inhibitors; Protein Stability; Receptor, Platelet-Derived Growth Factor alpha; Transcriptome; Tumor Suppressor Protein p53

Phosphotyrosine (pTyr)-regulated protein complexes play critical roles in cancer signaling. The systematic characterization of these protein complexes in tumor samples remains a challenge due to their limited access and the transient nature of pTyr-mediated interactions. We developed a hybrid chemical proteomics approach, termed Photo-pTyr-scaffold, by engineering Src homology 2 (SH2) domains, which specifically bind pTyr proteins, with both trifunctional chemical probes and genetic mutations to overcome these challenges. Dynamic SH2 domain-scaffolding protein complexes were efficiently cross-linked under mild UV light, captured by biotin tag, and identified by mass spectrometry. This approach was successfully used to profile native pTyr protein complexes from breast cancer tissue samples on a proteome scale with high selectivity, achieving about 100 times higher sensitivity for detecting pTyr signaling proteins than that afforded by traditional immunohistochemical methods. Among more than 1,000 identified pTyr proteins, receptor tyrosine kinase PDGFRB expressed on cancer-associated fibroblasts was validated as an important intercellular signaling regulator with poor expression correlation to ERBB2, and blockade of PDGFRB signaling could efficiently suppress tumor growth. The Photo-pTyr-scaffold approach may become a generic tool for readily profiling dynamic pTyr signaling complexes in clinically relevant samples.

Topics: Animals; Benzimidazoles; Biomarkers, Tumor; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Line, Tumor; Female; Humans; Mammary Tumor Virus, Mouse; Mass Spectrometry; Mice, Transgenic; Phosphorylation; Phosphotyrosine; Piperidines; Protein Binding; Protein Engineering; Proteomics; Receptor, ErbB-2; Receptor, Platelet-Derived Growth Factor beta; Signal Transduction; src Homology Domains; Ultraviolet Rays

Systemic sclerosis (SSc) is a multi-organ fibrotic disease with few treatment options. Activated fibroblasts are the key effector cells in SSc responsible for the excessive production of collagen and the development of fibrosis. Platelet-derived growth factor (PDGF), a potent mitogen for cells of mesenchymal origin, has been implicated in the activation of SSc fibroblasts. Our aim was to examine the therapeutic potential of crenolanib, an inhibitor of PDGF receptor signaling, in cultured fibroblasts and in angiotensin II-induced skin and heart fibrosis. Crenolanib effectively inhibited proliferation and migration of SSc and healthy control fibroblasts and attenuated basal and transforming growth factor-β-induced expression of CCN2/CTGF and periostin. In contrast to healthy control fibroblasts, SSc fibroblasts proliferated in response to PDGFAA, whereas a combination of PDGFAA and CCN2 was required to elicit a similar response in healthy control fibroblasts. PDGF receptor α mRNA correlated with CCN2 and other fibrotic markers in the skin of SSc patients. In mice challenged with angiotensin II, PDGF receptor α-positive cells were increased in the skin and heart. These PDGF receptor α-positive cells co-localized with PDGF receptor β, procollagen, and periostin. Treatment with crenolanib attenuated the skin and heart fibrosis. Our data indicate that inhibition of PDGF signaling presents an attractive therapeutic approach for SSc.

Topics: Animals; Benzimidazoles; Cell Adhesion Molecules; Cells, Cultured; Connective Tissue Growth Factor; Disease Models, Animal; Fibroblasts; Gene Expression Regulation; Humans; Immunoblotting; Mice; Mice, Inbred C57BL; Piperidines; Real-Time Polymerase Chain Reaction; Receptors, Platelet-Derived Growth Factor; RNA; RNA, Messenger; Scleroderma, Systemic; Signal Transduction

Inflammatory breast cancer (IBC) is arguably the deadliest form of breast cancer due to its rapid onset and highly invasive nature. IBC carries 5- and 10-year disease-free survival rates of ~45% and <20%, respectively. Multiple studies demonstrate that in comparison with conventional breast cancer, IBC has a unique molecular identity. Here, we have identified platelet-derived growth factor receptor alpha (PDGFRA) as being uniquely expressed and active in IBC patient tumor cells.. Here we focus on characterizing and targeting PDGFRA in IBC. Using gene expression, we analyzed IBC patient samples and compared them with non-IBC patient samples. Further, using IBC cells in culture, we determined the effect of small molecules inhibitors in both in vitro and in vivo assays.. In IBC patients, we show more frequent PDGFRA activation signature than non-IBC samples. In addition, the PDGFRA activation signature is associated with shorter metastasis-free survival in both uni- and multivariate analyses. We also demonstrate that IBC cells express active PDGFRA. Finally, we show that PDGFRA targeting by crenolanib (CP-868-596), but not imatinib (STI571), two small molecule inhibitors, interferes with IBC cell growth and emboli formation in vitro and tumor growth in vivo.. Our data suggest that PDGFRA may be a promising target for therapy in IBC.

Topics: Adult; Aged; Animals; Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Female; Follow-Up Studies; Gene Expression; Humans; Inflammatory Breast Neoplasms; Mice; Middle Aged; Mutation; Neoplasm Grading; Piperidines; Platelet-Derived Growth Factor; Receptor, Platelet-Derived Growth Factor alpha; Xenograft Model Antitumor Assays

Human hepatocellular carcinomas (HCCs) expressing the biliary/hepatic progenitor cell marker keratin 19 (K19) have been linked with a poor prognosis and exhibit an increase in platelet-derived growth factor receptor α (PDGFRα) and laminin beta 1 (LAMB1) expression. PDGFRα has been reported to induce de novo synthesis of LAMB1 protein in a Sjogren syndrome antigen B (La/SSB)-dependent manner in a murine metastasis model. However, the role of this cascade in human HCC remains unclear. This study focused on the functional role of the PDGFRα-La/SSB-LAMB1 pathway and its molecular link to K19 expression in human HCC. In surgical HCC specimens from a cohort of 136 patients, PDGFRα expression correlated with K19 expression, microvascular invasion and metastatic spread. In addition, PDGFRα expression in pre-operative needle biopsy specimens predicted poor overall survival during a 5-year follow-up period. Consecutive histological staining demonstrated that the signaling components of the PDGFRα-La/SSB-LAMB1 pathway were strongly expressed at the invasive front. K19-positive HCC cells displayed high levels of α2β1 integrin (ITG) receptor, both in vitro and in vivo. In vitro activation of PDGFRα signaling triggered the translocation of nuclear La/SSB into the cytoplasm, enhanced the protein synthesis of LAMB1 by activating its internal ribosome entry site, which in turn led to increased secretion of laminin-111. This effect was abrogated by the PDGFRα-specific inhibitor crenolanib. Importantly LAMB1 stimulated ITG-dependent focal adhesion kinase/Src proto-oncogene non-receptor tyrosine kinase signaling. It also promoted the ITG-specific downstream target Rho-associated coiled-coil containing protein kinase 2, induced K19 expression in an autocrine manner, invadopodia formation and cell invasion. Finally, we showed that the knockdown of LAMB1 or K19 in subcutaneous xenograft mouse models resulted in significant loss of cells invading the surrounding stromal tissue and reduced HepG2 colonization into lung and liver after tail vein injection. The PDGFRα-LAMB1 pathway supports tumor progression at the invasive front of human HCC through K19 expression.

Topics: Animals; Autoantigens; Benzimidazoles; Biomarkers, Tumor; Biopsy, Needle; Carcinoma, Hepatocellular; Cohort Studies; Disease Progression; Female; Follow-Up Studies; Gene Knockdown Techniques; Hep G2 Cells; Humans; Immunohistochemistry; Integrin alpha2beta1; Keratin-19; Laminin; Liver Neoplasms; Mice; Neoplasm Invasiveness; Piperidines; Proto-Oncogene Mas; Proto-Oncogenes; Receptor, Platelet-Derived Growth Factor alpha; rho-Associated Kinases; Ribonucleoproteins; RNA, Small Interfering; Signal Transduction; SS-B Antigen; Survival Analysis; Xenograft Model Antitumor Assays

Neurogenesis in the subventricular zone (SVZ) plays a vital role in neurologic recovery after stroke. However, only a small fraction of newly generated neuroblasts from the SVZ will survive long-term. Successful migration and survival of neuroblasts requires angiogenesis, lesion-derived chemo-attractants, and appropriate local microenvironments, which are partly regulated by the platelet-derived growth factor receptor (PDGFR) signaling pathway. In this study, we investigated the effects of PDGFR inhibition in a mouse model of transient middle cerebral artery occlusion (MCAO). We blocked the pathway using a nonselective PDGFR inhibitor, crenolanib, during the acute post-MCAO phase (days 1-3) or during the sub-acute phase (days 7-9). Downregulating the PDGFR signaling pathway with crenolanib from day 1 to day 3 after MCAO significantly decreased the migration of neuroblasts from the SVZ to the peri-infarct region, decreased angiogenesis, and lowered expression of vascular endothelial growth factor, stromal cell-derived factor-1, and monocyte chemotactic protein-1. Downregulation of the PDGFR signaling pathway on days 7-9 with crenolanib significantly increased apoptosis of the neuroblasts that had migrated to the peri-infarct region, increased the number of activated microglia, and decreased the expression of brain-derived neurotrophic factor, neurotrophin-3, and interleukin-10. Crenolanib treatment increased the apoptosis of pericytes and decreased the pericyte/vascular coverage, but had no effects on apoptosis of astrocytes. We conclude that the PDGFR signaling pathway plays a vital role in the SVZ neurogenesis after stroke. It can also affect angiogenesis, lesion-derived chemo-attractants, and the local microenvironment, which are all important to stroke-induced neurogenesis.

Topics: Animals; Benzimidazoles; Disease Models, Animal; Infarction, Middle Cerebral Artery; Lateral Ventricles; Male; Mice; Mice, Inbred C57BL; Neurogenesis; Piperidines; Receptors, Platelet-Derived Growth Factor; Signal Transduction

Activating mutations in the proto-oncogene KRAS are a hallmark of pancreatic ductal adenocarcinoma (PDAC), an aggressive malignancy with few effective therapeutic options. Despite efforts to develop KRAS-targeted drugs, the absolute dependence of PDAC cells on KRAS remains incompletely understood. Here we model complete KRAS inhibition using CRISPR/Cas-mediated genome editing and demonstrate that KRAS is dispensable in a subset of human and mouse PDAC cells. Remarkably, nearly all KRAS deficient cells exhibit phosphoinositide 3-kinase (PI3K)-dependent mitogen-activated protein kinase (MAPK) signaling and induced sensitivity to PI3K inhibitors. Furthermore, comparison of gene expression profiles of PDAC cells retaining or lacking KRAS reveal a role of KRAS in the suppression of metastasis-related genes. Collectively, these data underscore the potential for PDAC resistance to even the very best KRAS inhibitors and provide insights into mechanisms of response and resistance to KRAS inhibition.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Carcinoma, Pancreatic Ductal; DNA Copy Number Variations; Humans; Immunoblotting; Indazoles; Mice; Morpholines; Pancreatic Neoplasms; Phenylurea Compounds; Piperidines; Proto-Oncogene Mas; Proto-Oncogene Proteins p21(ras); Purines; Pyrimidines; Pyrimidinones; Quinazolinones; Sulfonamides; Thiazoles

Topics: Aged; Antineoplastic Agents; Benzimidazoles; Bridged Bicyclo Compounds, Heterocyclic; Clinical Trials as Topic; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Molecular Targeted Therapy; Mutation; Piperidines; Sulfonamides

The invasive and lethal nature of Glioblastoma multiforme (GBM) necessitates the continuous identification of molecular targets and search of efficacious therapies to inhibit GBM growth. The GBM resistance to chemotherapy and radiation it is attributed to the existence of a rare fraction of cancer stem cells (CSC) that we have identified within the tumor core and in peritumor tissue of GBM. Since Notch1 pathway is a potential therapeutic target in brain cancer, earlier we highlighted that pharmacological inhibition of Notch1 signalling by γ-secretase inhibitor-X (GSI-X), reduced cell growth of some c-CSC than to their respective p-CSC, but produced negligible effects on cell cycle distribution, apoptosis and cell invasion. In the current study, we assessed the effects of Hes1-targeted shRNA, a Notch1 gene target, specifically on GBM CSC refractory to GSI-X. Depletion of Hes1 protein induces major changes in cell morphology, cell growth rate and in the invasive ability of shHes1-CSC in response to growth factor EGF. shHes1-CSC show a decrease of the stemness marker Nestin concurrently to a marked increase of neuronal marker MAP2 compared to pLKO.1-CSC. Those effects correlated with repression of EGFR protein and modulation of Stat3 phosphorylation at Y705 and S727 residues. In the last decade Stat3 has gained attention as therapeutic target in cancer but there is not yet any approved Stat3-based glioma therapy. Herein, we report that exposure to a Stat3/5 inhibitor, induced apoptosis either in shHes1-CSC or control cells. Taken together, Hes1 seems to be a favorable target but not sufficient itself to target GBM efficaciously, therefore a possible pharmacological intervention should provide for the use of anti-Stat3/5 drugs either alone or in combination regimen.

Topics: Apoptosis; Benzimidazoles; Brain Neoplasms; Carbamates; Cell Differentiation; Cell Proliferation; Dipeptides; ErbB Receptors; Glioblastoma; Humans; Microtubule-Associated Proteins; Neoplasm Invasiveness; Neoplastic Stem Cells; Phosphorylation; Piperidines; Receptor, Notch1; RNA Interference; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; STAT5 Transcription Factor; Transcription Factor HES-1; Tumor Suppressor Proteins

The novel FMS-like tyrosine kinase 3 (FLT3)-N676K point mutation within the FLT3 kinase domain-1 was recently identified in 6 % of de novo acute myeloid leukemia (AML) patients with inv(16). Because FLT3-N676K was encountered almost exclusively in inv(16) AML, we investigated the transforming potential of FLT3-N676K, the cooperation between FLT3-N676K and core binding factor ß-smooth muscle myosin heavy chain (CBFß-SMMHC) (encoded by the inv(16) chimeric gene CBFB-MYH11) in inducing acute leukemia, and tested the sensitivity of FLT3-N676K-positive leukemic cells to FLT3 inhibitors. Retroviral expression of FLT3-N676K in myeloid 32D cells induced AML in syngeneic C3H/HeJ mice (n = 11/13, median latency 58 days), with a transforming activity similar to FLT3-internal tandem duplication (ITD) (n = 8/8), FLT3-TKD D835Y (n = 8/9), and FLT3-ITD-N676K (n = 9/9) mutations. Three out of 14 (21.4 %) C57BL/6J mice transplanted with FLT3-N676K-transduced primary hematopoietic progenitor cells developed acute leukemia (latency of 68, 77, and 273 days), while no hematological malignancy was observed in the control groups including FLT3-ITD. Moreover, co-expression of FLT3-N676K/CBFß-SMMHC did not promote acute leukemia in three independent experiments (n = 16). In comparison with FLT3-ITD, FLT3-N676K induced much higher activation of FLT3 and tended to trigger stronger phosphorylation of MAPK and AKT. Importantly, leukemic cells carrying the FLT3-N676K mutant in the absence of an ITD mutation were highly sensitive to FLT3 inhibitors AC220 and crenolanib, and crenolanib even retained activity against the AC220-resistant FLT3-ITD-N676K mutant. Taken together, the FLT3-N676K mutant is potent to transform murine hematopoietic stem/progenitor cells in vivo. This is the first report of acute leukemia induced by an activating FLT3 mutation in C57BL/6J mice. Moreover, further experiments investigating molecular mechanisms for leukemogenesis induced by FLT3-N676K mutation and clinical evaluation of FLT3 inhibitors in FLT3-N676K-positive AML seem warranted.

Topics: Amino Acid Substitution; Animals; Antineoplastic Agents; Benzimidazoles; Benzothiazoles; Bone Marrow Transplantation; Cell Transformation, Neoplastic; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Leukemic; Genetic Predisposition to Disease; Genetic Vectors; Humans; Leukemia, Experimental; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mutation, Missense; Neoplasm Transplantation; Neoplastic Stem Cells; Oncogene Proteins, Fusion; Phenylurea Compounds; Piperidines; Point Mutation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Radiation Chimera; Retroviridae; Tandem Repeat Sequences; Transgenes; Tumor Stem Cell Assay

Signaling pathways regulated by mutant Fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD), which mediate resistance to acute myeloid leukemia (AML) cell death, are poorly understood. Here, we reveal that pro-cell death lipid ceramide generation is suppressed by FLT3-ITD signaling. Molecular or pharmacologic inhibition of FLT3-ITD reactivated ceramide synthesis, selectively inducing mitophagy and AML cell death. Mechanistically, FLT3-ITD targeting induced ceramide accumulation on the outer mitochondrial membrane, which then directly bound autophagy-inducing light chain 3 (LC3), involving its I35 and F52 residues, to recruit autophagosomes for execution of lethal mitophagy. Short hairpin RNA (shRNA)-mediated knockdown of LC3 prevented AML cell death in response to FLT3-ITD inhibition by crenolanib, which was restored by wild-type (WT)-LC3, but not mutants of LC3 with altered ceramide binding (I35A-LC3 or F52A-LC3). Mitochondrial ceramide accumulation and lethal mitophagy induction in response to FLT3-ITD targeting was mediated by dynamin-related protein 1 (Drp1) activation via inhibition of protein kinase A-regulated S637 phosphorylation, resulting in mitochondrial fission. Inhibition of Drp1 prevented ceramide-dependent lethal mitophagy, and reconstitution of WT-Drp1 or phospho-null S637A-Drp1 but not its inactive phospho-mimic mutant (S637D-Drp1), restored mitochondrial fission and mitophagy in response to crenolanib in FLT3-ITD

Topics: Animals; Benzimidazoles; Ceramides; Cyclic AMP-Dependent Protein Kinases; Drug Resistance, Neoplasm; Dynamins; fms-Like Tyrosine Kinase 3; GTP Phosphohydrolases; Humans; Leukemia, Myeloid, Acute; Male; Mice; Mice, Inbred NOD; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Mitophagy; Mutation; Phosphorylation; Piperidines; RNA, Small Interfering; Signal Transduction

Fibrosis is a characteristic of Duchenne muscular dystrophy (DMD), yet the cellular and molecular mechanisms responsible for DMD fibrosis are poorly understood. Utilizing the Collagen1a1-GFP transgene to identify cells producing Collagen-I matrix in wild-type mice exposed to toxic injury or those mutated at the dystrophin gene locus (mdx) as a model of DMD, we studied mechanisms of skeletal muscle injury/repair and fibrosis. PDGFRα is restricted to Sca1+, CD45- mesenchymal progenitors. Fate-mapping experiments using inducible CreER/LoxP somatic recombination indicate that these progenitors expand in injury or DMD to become PDGFRα+, Col1a1-GFP+ matrix-forming fibroblasts, whereas muscle fibres do not become fibroblasts but are an important source of the PDGFRα ligand, PDGF-AA. While in toxin injury/repair of muscle PDGFRα, signalling is transiently up-regulated during the regenerative phase in the DMD model and in human DMD it is chronically overactivated. Conditional expression of the constitutively active PDGFRα D842V mutation in Collagen-I+ fibroblasts, during injury/repair, hindered the repair phase and instead promoted fibrosis. In DMD, treatment of mdx mice with crenolanib, a highly selective PDGFRα/β tyrosine kinase inhibitor, reduced fibrosis, improved muscle strength, and was associated with decreased activity of Src, a downstream effector of PDGFRα signalling. These observations are consistent with a model in which PDGFRα activation of mesenchymal progenitors normally regulates repair of the injured muscle, but in DMD persistent and excessive activation of this pathway directly drives fibrosis and hinders repair. The PDGFRα pathway is a potential new target for treatment of progressive DMD. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Topics: Animals; Benzimidazoles; Cells, Cultured; Collagen Type I; Disease Models, Animal; Dystrophin; Enzyme Inhibitors; Fibroblasts; Fibrosis; Male; Mice, Transgenic; Muscle Strength; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Mutation; Piperidines; Protein-Tyrosine Kinases; Receptor, Platelet-Derived Growth Factor alpha; Regeneration; Signal Transduction

Recently platelet derived growth factor receptor-alpha (PDGFRα) was recognized as a potential target to treat aggressive papillary thyroid cancer given its strong association with lymph node metastases. However, it is unclear how PDGFRα potentiates metastases and if it works through the canonical MAPK pathway traditionally linked to PTC oncogenesis. We explored the phenotypic changes driven by PDGFRα activation in human papillary thyroid cancer (PTC) cells and the downstream signalling cascades through which they are effected. We demonstrate that PDGFRα drives an impressive phenotypic change in PTC cell lines as documented by significant cytoskeletal rearrangement, increased migratory potential, and the formation of invadopodia. Cells lacking PDGFRα formed compact and dense spheroids, whereas cells expressing active PDGFRα exhibited invadopodia in three-dimensional culture. To achieve this, active PDGFRα provoked downstream activation of the MAPK/Erk, PI3K/Akt and STAT3 pathways. We further confirmed the role of PDGFRα as a transformative agent promoting the epithelial to mesenchymal transition of PTC cells, through the augmentation of Snail and Slug expression. Crenolanib, a small molecule inhibitor of PDGFRα, suppressed the levels of Snail and Slug and almost completely reversed all the phenotypic changes. We demonstrate that PDGFRα activation is an essential component that drives aggressiveness in PTC cells, and that the signaling pathways are complex, involving not only the MAPK/Erk but also the PI3K/Akt and STAT3 pathways. This argues for upstream targeting of the PDGFRα given the redundancy of oncogenic pathways in PTC, especially in patients whose tumors over-express this tyrosine kinase receptor.

Topics: Antineoplastic Agents; Benzimidazoles; Carcinoma, Papillary; Cell Line, Tumor; Cell Movement; Cytoskeleton; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Humans; Lymphatic Metastasis; Phenotype; Phosphatidylinositol 3-Kinase; Piperidines; Podosomes; Proto-Oncogene Proteins c-akt; Receptor, Platelet-Derived Growth Factor alpha; Signal Transduction; Snail Family Transcription Factors; STAT3 Transcription Factor; Thyroid Cancer, Papillary; Thyroid Neoplasms; Time Factors

The functional mutation of c-kit and platelet-derived growth factor receptor α (PDGFRA) which encode proto-oncogene receptor tyrosine kinase are the crucial pathogeneses of gastrointestinal stromal tumors(GISTs). 80%-85% c-kit gene mutation including exon 11,exon 9,exon 13,exon 17 and 5%-10% PDGFRA gene mutation such as exon 18, exon 12 are examined in GISTs. Neither of c-kit or PDGFRA gene mutation are called wide type GISTs. The pathogeneses of wild type GISTs are not clear. The deficiency of succinate dehydrogenase B(SDHB)-related insulin-like growth factor 1(IGF-1R) activation, BRAF gene mutation and neurofibromatosis type 1 may be related to progression of wild type GISTs. More than half of metastatic GISTs patients receiving imatinib treatment can develop to c-kit secondary mutations, which are responsible for secondary resistance. However, the reasons of imatinib resistance in GISTs without c-kit secondary mutation need to be explored. At present, many clinical trials are ongoing to evaluate new drugs in GISTs treatment, including nilotinib, masitinib, pazopanib, dovitinib, ponatinib, dasatinib, crenolanib, linsitinib and immunotherapy, which may bring resistance GISTs treatment to new hope. Next generation sequencing (NGS) and liquid biopsy will be very important in GISTs research and clinical practice.

Topics: Benzimidazoles; Exons; Gastrointestinal Stromal Tumors; High-Throughput Nucleotide Sequencing; Humans; Imatinib Mesylate; Indazoles; Mutation; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Mas; Proto-Oncogene Proteins c-kit; Pyrimidines; Quinolones; Succinate Dehydrogenase; Sulfonamides

Background Crenolanib (crenolanib besylate, 4-piperidinamine, 1-[2-[5-[(3-methyl-3-oxetanyl)methoxy]-1H-benzimidazol-1-yl]-8-quinolinyl]-, monobenzenesulfonate) is a potent and specific type I inhibitor of fms-like tyrosine kinase 3 (FLT3) that targets the active kinase conformation and is effective against FLT3 with internal tandem duplication (ITD) with point mutations induced by, and conferring resistance to, type II FLT3 inhibitors in acute myeloid leukemia (AML) cells. Crenolanib is also an inhibitor of platelet-derived growth factor receptor alpha and beta and is in clinical trials in both gastrointestinal stromal tumors and gliomas. Methods We tested crenolanib interactions with the multidrug resistance-associated ATP-binding cassette proteins ABCB1 (P-glycoprotein), ABCG2 (breast cancer resistance protein) and ABCC1 (multidrug resistance-associated protein 1), which are expressed on AML cells and other cancer cells and are important components of the blood-brain barrier. Results We found that crenolanib is a substrate of ABCB1, as evidenced by approximate five-fold resistance of ABCB1-overexpressing cells to crenolanib, reversal of this resistance by the ABCB1-specific inhibitor PSC-833 and stimulation of ABCB1 ATPase activity by crenolanib. In contrast, crenolanib was not a substrate of ABCG2 or ABCC1. Additionally, it did not inhibit substrate transport by ABCB1, ABCG2 or ABCC1, at pharmacologically relevant concentrations. Finally, incubation of the FLT3-ITD AML cell lines MV4-11 and MOLM-14 with crenolanib at a pharmacologically relevant concentration of 500 nM did not induce upregulation of ABCB1 cell surface expression. Conclusions Thus ABCB1 expression confers resistance to crenolanib and likely limits crenolanib penetration of the central nervous system, but crenolanib at therapeutic concentrations should not alter cellular exposure to ABC protein substrate chemotherapy drugs.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzimidazoles; Biological Transport; Blood-Brain Barrier; Cyclosporins; Drug Resistance, Neoplasm; fms-Like Tyrosine Kinase 3; Leukemia, Myeloid, Acute; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Piperidines; Platelet-Derived Growth Factor; Tumor Cells, Cultured

In gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-α (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1.. We studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA(+) subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit(+/copGFP), Pdgfra(+/eGFP), and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation.. KIT and PDGFRA were coexpressed in 3%-5% of mouse ICC, 35%-44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT(+)ETV1(+)PDGFRA(+) GIST cells (50% maximal inhibitory concentration = 5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (50% maximal inhibitory concentration >230 nM). Crenolanib inhibited phosphorylation of PDGFRA and PDGFRB, but not KIT. However, Kit overexpression sensitized mouse ICC precursors to crenolanib. ETV1 knockdown reduced KIT expression and GIST proliferation. Crenolanib down-regulated ETV1 by inhibiting extracellular-signal-regulated kinase (ERK)-dependent stabilization of ETV1 protein and also reduced expression of KIT and PDGFRA.. In KIT-mutant GIST, inhibition of PDGFRA disrupts a KIT-ERK-ETV1-KIT signaling loop by inhibiting ERK activation. The PDGFRA inhibitor crenolanib might be used to treat patients with imatinib-resistant, KIT-mutant GIST.

Topics: Animals; Benzamides; Benzimidazoles; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Flow Cytometry; Gastrointestinal Stromal Tumors; Gene Knockdown Techniques; Humans; Imatinib Mesylate; Immunohistochemistry; Mice; Mice, Inbred BALB C; Mutation; Nucleic Acid Precursors; Phosphorylation; Piperazines; Piperidines; Proto-Oncogene Proteins c-kit; Pyrimidines; Receptor, Platelet-Derived Growth Factor alpha; Receptor, Platelet-Derived Growth Factor beta; Signal Transduction; Transcription Factors

Tyrosine kinase inhibitors (TKIs) represent transformative therapies for several malignancies. Two critical features necessary for maximizing TKI tolerability and response duration are kinase selectivity and invulnerability to resistance-conferring kinase domain (KD) mutations in the intended target. No prior TKI has demonstrated both of these properties. Aiming to maximize selectivity, medicinal chemists have largely sought to create TKIs that bind to an inactive (type II) kinase conformation. Here we demonstrate that the investigational type I TKI crenolanib is a potent inhibitor of Fms tyrosine kinase-3 (FLT3) internal tandem duplication, a validated therapeutic target in human acute myeloid leukemia (AML), as well as all secondary KD mutants previously shown to confer resistance to the first highly active FLT3 TKI quizartinib. Moreover, crenolanib is highly selective for FLT3 relative to the closely related protein tyrosine kinase KIT, demonstrating that simultaneous FLT3/KIT inhibition, a prominent feature of other clinically active FLT3 TKIs, is not required for AML cell cytotoxicity in vitro and may contribute to undesirable toxicity in patients. A saturation mutagenesis screen of FLT3-internal tandem duplication failed to recover any resistant colonies in the presence of a crenolanib concentration well below what has been safely achieved in humans, suggesting that crenolanib has the potential to suppress KD mutation-mediated clinical resistance. Crenolanib represents the first TKI to exhibit both kinase selectivity and invulnerability to resistance-conferring KD mutations, which is unexpected of a type I inhibitor. Crenolanib has significant promise for achieving deep and durable responses in FLT3-mutant AML, and may have a profound impact upon future medicinal chemistry efforts in oncology.

Topics: Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Drug Resistance, Neoplasm; fms-Like Tyrosine Kinase 3; Humans; Molecular Docking Simulation; Mutation; Piperidines

Control of BRAF(V600E) metastatic melanoma by BRAF inhibitor (BRAF-I) is limited by intrinsic and acquired resistance. Growth factor receptor up-regulation is among the mechanisms underlying BRAF-I resistance of melanoma cells. Here we demonstrate for the first time that PDGFRα up-regulation causes BRAF-I resistance. PDGFRα inhibition by PDGFRα-specific short hairpin (sh)RNA and by PDGFRα inhibitors restores and increases melanoma cells' sensitivity to BRAF-I in vitro and in vivo. This effect reflects the inhibition of ERK and AKT activation which is associated with BRAF-I resistance of melanoma cells. PDGFRα up-regulation is mediated by Sonic Hedgehog Homolog (Shh) pathway activation which is induced by BRAF-I treatment. Similarly to PDGFRα inhibition, Shh inhibition by LDE225 restores and increases melanoma cells' sensitivity to BRAF-I. These effects are mediated by PDGFRα down-regulation and by ERK and AKT inhibition. The clinical relevance of these data is indicated by the association of PDGFRα up-regulation in melanoma matched biopsies of BRAF-I +/- MEK inhibitor treated patients with shorter time to disease progression and less tumor regression. These findings suggest that monitoring patients for early PDGFRα up-regulation will facilitate the identification of those who may benefit from the treatment with BRAF-I in combination with clinically approved PDGFRα or Shh inhibitors.

Topics: Animals; Antineoplastic Agents; Benzamides; Benzimidazoles; Biphenyl Compounds; Cell Line, Tumor; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Gene Silencing; Hedgehog Proteins; Humans; Imatinib Mesylate; Indoles; MAP Kinase Signaling System; Melanoma; Mice; Piperazines; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Pyrroles; Receptor, Platelet-Derived Growth Factor alpha; RNA, Small Interfering; Sulfonamides; Sunitinib; Up-Regulation; Vemurafenib

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.

Topics: Animals; Azabicyclo Compounds; Benzimidazoles; beta Catenin; Biomarkers, Tumor; Cell Line, Tumor; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; GTP Phosphohydrolases; HSP90 Heat-Shock Proteins; Humans; Indoles; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mass Spectrometry; Melanoma; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, SCID; Neoplasm Transplantation; NF-kappa B; Phosphoinositide-3 Kinase Inhibitors; Phthalic Acids; Piperidines; Proteomics; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Receptor, Platelet-Derived Growth Factor beta; RNA Interference; RNA, Small Interfering; Sulfonamides; Transplantation, Heterologous; Vemurafenib; Wnt Signaling Pathway

A LC-ESI-MS/MS method for the determination of crenolanib (CP-868,596) in human serum was developed and validated employing d4-CP-868,596 as an internal standard (ISTD). In addition to human serum, the method was also partially validated for crenolanib determination in human cerebrospinal fluid (CSF) samples. Sample aliquots (50μl of serum or CSF) were prepared for analysis using liquid-liquid extraction (LLE) with tert-butyl methyl ether. Chromatography was performed using a phenomenex Gemini C18 column (3μm, 100mm×4.6mm I.D.) in a column heater set at 50°C and an isocratic mobile phase (methanol/water/formic acid at a volume ratio of 25/25/0.15, v/v/v). The flow rate was 0.45mL/min, and the retention time for both analyte and ISTD was less than 3.5min. Samples were analyzed with an API-5500 LC-MS/MS system (ESI) in positive ionization mode coupled to a Shimadzu HPLC system. The ion transitions monitored were m/z 444.4→373.1 and m/z 448.2→374.2 for crenolanib and ISTD, respectively. The method was linear over the range of 5-1000ng/mL for serum and 0.5-1000ng/mL for CSF. For human serum, both intra-day and inter-day precision were <4%, while intra-day and inter-day accuracy were within 8% of nominal values. Recovery was greater than 50% for both the analyte and ISTD. For CSF samples, both intra-day and inter-day precision were <9% except at the lower limit of quantification (LLOQ) which was <17%. The intra-day and inter-day accuracy were within 11% of the nominal CSF concentrations. After validation, this method was successfully applied to the analysis of serial pharmacokinetic samples obtained from a child treated with oral crenolanib.

Topics: Benzimidazoles; Chromatography, Liquid; Humans; Piperidines; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

FLT3 kinase internal tandem duplication (ITD) mutations are common in acute myeloid leukemia (AML) and are associated with poor clinical outcomes. Although initial responses to FLT3 tyrosine kinase inhibitors (TKIs) are observed in FLT3-ITD-positive patients, subsequent relapse often occurs upon acquisition of secondary FLT3 kinase domain (KD) mutations, primarily at residues D835 and F691. Using biochemical assays, we determined that crenolanib, a novel TKI, demonstrates type I properties and is active against FLT3 containing ITD and/or D835- or F691-activating mutations. Potent activity was observed in FLT3-ITD-positive AML cell lines. Crenolanib delayed the outgrowth of MV4-11 cells in a xenograft mouse model, whereas in combination with the type II TKI sorafenib, a significant decrease in leukemic burden (P < .001) and prolonged survival (P < .01) was observed compared with either type I or II TKI alone. Crenolanib was active against Ba/F3 cells harboring FLT3-ITD and secondary KD mutations and sorafenib-resistant MOLM-13 cells containing FLT3-ITD/D835Y both in vitro and in vivo. In addition, crenolanib inhibited drug-resistant AML primary blasts with FLT3-ITD and D835H/Y mutations. These preclinical data demonstrate that crenolanib is effective against FLT3-ITD containing secondary KD mutations, suggesting that crenolanib may be a useful therapeutic agent for TKI-naive and drug-resistant FLT3-ITD-positive AML.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Female; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Male; Mice; Mice, Inbred NOD; Mice, SCID; Mutation; Niacinamide; Phenylurea Compounds; Piperidines; Protein Kinase Inhibitors; Sorafenib; Tandem Repeat Sequences; Xenograft Model Antitumor Assays

Platelet-derived growth factor receptor α (PDGFRA) is a target for tyrosine kinase inhibitor (TKI)-based targeted therapy. Dysregulation of PDGFRA has been reported in many cancers. However, PDGFRA mutations in melanomas have not been well studied. We analyzed the genetic mutations of PDGFRA in Chinese patients with melanoma and determined the inhibitory potency of TKIs, such as imatinib and crenolanib, on mutant PDGFRA.. Of note, 351 melanoma tissue samples were examined for genetic mutations in exons 12, 14, and 18 of PDGFRA. Activities of mutations in response to imatinib and crenolanib were analyzed by Western blotting of tyrosine-phosphorylated PDGFRA and cell proliferation assays.. PDGFRA mutations were observed in 4.6% (16 of 351) of melanomas, and these mutations were mainly detected in acral and mucosal melanomas. PDGFRA mutations seem to be mutually exclusive with KIT mutations, but may coexist with BRAF and NRAS mutations. The genetic mutations of PDGFRA were unrelated to the age, thickness, and ulceration status of primary melanomas. Thirteen mutations were not reported before, and five (P577S, V658A, R841K, H845Y, and G853D) of them resulted in strong autophosphorylation of PDGFRA. Crenolanib showed higher potency than imatinib in inhibiting the kinase activity of PDGFRA. Except that V658A mutation was imatinib-resistant, all the other mutations were sensitive to both imatinib and crenolanib.. PDGFRA mutations are detected in a small population of melanoma patients. Our study suggests that patients with melanoma harboring certain PDGFRA mutations may benefit from imatinib and crenolanib treatment.

Topics: Adult; Aged; Benzamides; Benzimidazoles; Cell Proliferation; Drug Resistance, Neoplasm; Female; Humans; Imatinib Mesylate; Male; Melanoma; Middle Aged; Mutation; Piperazines; Piperidines; Protein Kinase Inhibitors; Pyrimidines; Receptor, Platelet-Derived Growth Factor alpha

In this issue of Blood, Zimmerman and colleagues demonstrate that the tyrosine kinase inhibitor (TKI) crenolanib effectively suppresses growth of leukemic cells harboring both FLT3-ITD and FLT3-TKD mutations, the latter of which are increasingly seen to emerge as resistant mutations after FMS-like tyrosine kinase 3 (FLT3) inhibitor therapy.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Female; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Male; Piperidines; Protein Kinase Inhibitors

To determine the potential of crenolanib, a potent inhibitor of PDGFRA, to treat malignancies driven by mutant PDGFRA.. The biochemical activity of crenolanib was compared with imatinib using a panel of PDGFRA-mutant kinases expressed in several different cell line models, including primary gastrointestinal stromal tumors (GIST) cells. The antiproliferative activity of crenolanib was also studied in several cell lines with PDGFRA-dependent growth.. Crenolanib was significantly more potent than imatinib in inhibiting the kinase activity of imatinib-resistant PDGFRA kinases (D842I, D842V, D842Y, DI842-843IM, and deletion I843). For example, crenolanib was 135-fold more potent than imatinib against D842V in our isogenic model system, with an IC(50) of approximately 10 nmol/L. The relative potency of crenolanib was further confirmed in BaF3 and primary GIST cells expressing PDGFRA D842V. In contrast, imatinib was at least 10-fold more potent than crenolanib in inhibiting the V561D mutation. For all other tested PDGFRA mutations, crenolanib and imatinib had comparable potency.. Crenolanib is a potent inhibitor of imatinib-resistant PDGFRA kinases associated with GIST, including the PDGFRA D842V mutation found in approximately 5% of GISTs. The spectrum of activity of crenolanib suggests that this drug is a type I inhibitor (inhibitor of activated conformation of kinase). Based in part on these results, a phase II clinical study of this agent to treat GIST with the PDGFRA D842V mutation has been initiated.

Topics: Animals; Antineoplastic Agents; Benzamides; Benzimidazoles; Cell Line, Tumor; CHO Cells; Cricetinae; Drug Resistance, Neoplasm; Enzyme Activation; Gastrointestinal Stromal Tumors; Humans; Imatinib Mesylate; Inhibitory Concentration 50; Mutation; Piperazines; Piperidines; Protein Kinase Inhibitors; Pyrimidines; Receptor, Platelet-Derived Growth Factor alpha