gilteritinib and Disease-Models--Animal

Patients with relapsed/refractory acute myeloid leukaemia (AML) with FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) have limited treatment options and poor prognosis. Therefore, novel treatment modalities are needed. Since high expression of natural killer group 2 member D ligands (NKG2DLs) can be induced by FLT3 inhibitors, we constructed dual-target FLT3 single-chain fragment variable (scFv)/NKG2D-chimeric antigen receptor (CAR) T cells, and explored whether FLT3 inhibitors combined with FLT3scFv/NKG2D-CAR T cells could have synergistic anti-leukaemia effects.. FLT3scFv and NKG2D expression in CAR T cells, FLT3 and NKG2DL expression in AML cells, and the in vitro cytotoxicity of combining CAR T cells with gilteritinib were assessed by flow cytometry. The therapeutic effect was evaluated in a xenograft mouse model established by injection of MOLM-13 cells. Mechanisms underlying the gilteritinib-induced NKG2DL upregulation were investigated using siRNA, ChIP-QPCR and luciferase assays.. The FLT3scFv/NKG2D-CAR T cells specifically lysed AML cells both in vitro and in the xenograft mouse model. The efficacy of FLT3scFv/NKG2D-CAR T cells was improved by gilteritinib-pretreatment. The noncanonical NF-κB2/Rel B signalling pathway was found to mediate gilteritinib-induced NKG2DL upregulation in AML cells.. Bispecific FLT3scFv/NKG2D-CAR T cells can effectively eradicate AML cells. The FLT3 inhibitor gilteritinib can synergistically improve this effect by upregulating NF-κB2-dependent NKG2DL expression in AML cells.

Topics: Aniline Compounds; Animals; Disease Models, Animal; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Mice; Mutation; NF-kappa B p52 Subunit; NK Cell Lectin-Like Receptor Subfamily K; Protein Kinase Inhibitors; Pyrazines; T-Lymphocytes

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

To investigate the efficacy of the combination of the FLT3 inhibitors midostaurin or gilteritinib with the Bcl-2 inhibitor venetoclax in FLT3-internal tandem duplication (ITD) acute myeloid leukemia (AML) and the underlying molecular mechanism.. Using both FLT3-ITD cell lines and primary patient samples, Annexin V-FITC/propidium iodide staining and flow cytometry analysis were used to quantify cell death induced by midostaurin or gilteritinib, alone or in combination with venetoclax. Western blot analysis was performed to assess changes in protein expression levels of members of the JAK/STAT, MAPK/ERK, and PI3K/AKT pathways, and members of the Bcl-2 family of proteins. The MV4-11-derived xenograft mouse model was used to assess. The combination of midostaurin or gilteritinib with venetoclax potently and synergistically induces apoptosis in FLT3-ITD AML cell lines and primary patient samples. The FLT3 inhibitors induced downregulation of Mcl-1, enhancing venetoclax activity. Phosphorylated-ERK expression is induced by venetoclax but abolished by the combination of venetoclax with midostaurin or gilteritinib. Simultaneous downregulation of Mcl-1 by midostaurin or gilteritinib and inhibition of Bcl-2 by venetoclax results in "free" Bim, leading to synergistic induction of apoptosis.. Inhibition of Bcl-2 via venetoclax synergistically enhances the efficacy of midostaurin and gilteritinib in FLT3-mutated AML.

Topics: Aniline Compounds; Animals; Apoptosis; Biomarkers, Tumor; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Disease Models, Animal; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; fms-Like Tyrosine Kinase 3; Gene Duplication; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Mice; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Staurosporine; Sulfonamides; Xenograft Model Antitumor Assays

Advances in the understanding of the molecular basis for acute myeloid leukemia (AML) have generated new potential targets for treatment. Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in AML and mutations in this gene are associated with poor overall survival. AXL plays a role in the activation of FLT3 and has been implicated in the pathogenesis of AML. The studies reported here evaluated the ability of a novel FLT3/AXL inhibitor, gilteritinib, to block mutated FLT3 in cellular and animal models of AML. Initial kinase studies showed that gilteritinib, a type I tyrosine kinase inhibitor, was highly selective for both FLT3 and AXL while having weak activity against c-KIT. Gilteritinib demonstrated potent inhibitory activity against the internal tandem duplication (FLT3-ITD) and FLT3-D835Y point mutations in cellular assays using MV4-11 and MOLM-13 cells as well as Ba/F3 cells expressing mutated FLT3. Gilteritinib also inhibited FLT3-F691 mutations, although to a lesser degree, in these assays. Furthermore, gilteritinib decreased the phosphorylation levels of FLT3 and its downstream targets in both cellular and animal models. In vivo, gilteritinib was distributed at high levels in xenografted tumors after oral administration. The decreased FLT3 activity and high intratumor distribution of gilteritinib translated to tumor regression and improved survival in xenograft and intra-bone marrow transplantation models of FLT3-driven AML. No overt toxicity was seen in mouse models treated with gilteritinib. These results indicate that gilteritinib may be an important next-generation FLT3 inhibitor for use in the treatment of FLT3 mutation-positive AML.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Axl Receptor Tyrosine Kinase; Cell Line, Tumor; Disease Models, Animal; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Mice; Mice, Nude; Mutation; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Pyrazines; Receptor Protein-Tyrosine Kinases; Xenograft Model Antitumor Assays