Fms-like Tyrosine Kinase-3 Gene Mutations Research Articles

The multi-kinase inhibitor CG-806 exerts anti-cancer activity against acute myeloid leukemia by co-targeting FLT3, BTK, and aurora kinases

Despite the development of several Fms-like tyrosine kinase 3 (FLT3) inhibitors that have improved outcomes in patients with FLT3-mutant acute myeloid leukemia (AML), drug resistance is frequently observed, which may be associated with the activation of additional pro-survival pathways, such as those regulated by BTK, aurora kinases (AuroK), and potentially others, in addition to acquired tyrosine kinase domain (TKD) mutations of FLT3 gene. FLT3 may not always be a driver mutation. We evaluated the anti-leukemia efficacy of the novel multi-kinase inhibitor CG-806, which targets FLT3 and other kinases, to circumvent drug resistance and target FLT3 wild-type (WT) cells. The anti-leukemia activity of CG-806 was investigated by measuring apoptosis induction and analyzing the cell cycle using flow cytometry in vitro. CG-806 demonstrated superior anti-leukemia efficacy compared to commercially available FLT3 inhibitors, both in vitro and in vivo, regardless of FLT3 mutational status. The mechanism of action of CG-806 may involve its broad inhibitory profile against FLT3, BTK, and AuroK. In FLT3 mutant cells, CG-806 induced G1 phase blockage, whereas in FLT3 WT cells, it resulted in G2/M phase arrest. Targeting FLT3 and Bcl-2 and/or Mcl-1 simultaneously results in a synergistic pro-apoptotic effect in FLT3 mutant leukemia cells. The results of this study suggest that CG-806 is a promising multi-kinase inhibitor with anti-leukemic efficacy regardless of FLT3 mutational status. A phase 1 clinical trial of CG-806 for the treatment of AML has been initiated (NCT04477291). Key points The multi-kinase inhibitor CG-806 exerts superior anti-leukemic activity in AML, regardless of its FLT3 status. CG-806 triggered G1 arrest in FLT3 mutated cells and G2/M arrest in FLT3 WT cells through the suppression of FLT3/BTK and aurora kinases. Concomitantly targeting FLT3 and Bcl-2 and/or Mcl-1 exerted synergistic pro-apoptotic effects on both FLT3 WT and mutated AML cells.

Discovery of benzimidazole-indazole derivatives as potent FLT3-tyrosine kinase domain mutant kinase inhibitors for acute myeloid leukemia

The FMS-like tyrosine kinase 3 (FLT3) gene encodes a class III receptor tyrosine kinase that is expressed in hematopoietic stem cells. The mutations of FLT3 gene found in 30% of acute myeloid leukemia (AML), leads to an abnormal constitutive activation of FLT3 kinase of the receptor and results in immature myeloblast cell proliferation. Although small molecule drugs targeting the FLT3 kinase have been approved, new FLT3 inhibitors are needed owing to the side effects and drug resistances arising from kinase domain mutations, such as D835Y and F691L. In this study, we have developed benzimidazole-indazole based novel inhibitors targeting mutant FLT3 kinases through the optimization of diverse chemical moieties substituted around the core skeleton. The most optimized compound 22f exhibited potent inhibitory activities against FLT3 and FLT3/D835Y, with IC50 values of 0.941 and 0.199 nM, respectively. Furthermore, 22f exhibited strong antiproliferative activity against an AML cell line, MV4-11 cells with a GI50 of 0.26 nM. More importantly, 22f showed single-digit nanomolar GI50 values in the mutant FLT kinase expressed Ba/F3 cell lines including FLT-D835Y (GI50 = 0.29 nM) and FLT3-F691L (GI50 = 2.87 nM). Molecular docking studies indicated that the compound exhibits a well-fitted binding mode as a type 1 inhibitor in the homology model of active conformation of FLT3 kinase.

Discover Novel Covalent Inhibitors Targeting FLT3 through Hybrid Virtual Screening Strategy

FMS-like tyrosine kinase 3 (FLT3) plays a very important role in regulating the proliferation, differentiation and survival of normal hematopoietic stem cells. Internal tandem duplications of the FLT3 gene (FLT3-ITD) mutations are present in 25% of all acute myeloid leukemia (AML) patients and are frequently associated with adverse clinical outcomes. Therefore, FLT3-ITD is a promising target for the treatment of AML. The use of covalent virtual screenings has shown that efficient rational approaches for the rapid discovery of new drugs scaffold. Herein, we report a hybrid virtual screening strategy that led to the discovery of FLT3 inhibitors. Using the combination of non-covalent docking and covalent docking, 8 compounds were found to inhibit FLT3, and G856-8335, S346-0154 are also effective against mutant FLT3. These two compounds also show selectivity to receptor tyrosine kinase (C-KIT), which has the potential for optimization. And this work can be extended to the screening of other covalent inhibitors.

Clinical Significance of FLT3 Mutations in a Comprehensive NGS Multicenter Study of AML: HM-Screen-Japan 01

Clinical Significance of FLT3 Mutations in a Comprehensive NGS Multicenter Study of AML: HM-Screen-Japan 01

Abstract 1096: FMS-like tyrosine kinase 3 (FLT3) inhibition by olverembatinib (HQP1351) downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 in preclinical models of FLT3-mutant acute myeloid leukemia (AML)

Abstract AML accounts for 80% of acute leukemias in adults. FLT3 gene mutations are observed in approximately 30% of patients with AML and augur a poor prognosis. Despite antitumor effects of selective FLT3 inhibitors, resistance to these agents continues to pose a formidable clinical challenge. The expression of pro-survival protein BCL-2 is frequently dysregulated, conferring resistance to FLT3 inhibitors, in AML. In this study, we explored the combination of clinical stage multikinase inhibitor HQP1351 (which also targets FLT3) and BCL-2-selective inhibitor APG-2575 in preclinical models of FLT3-mutant AML. Antileukemic activity of HQP1351 was first examined using AML cell lines MV-4-11 and MOLM-13, which harbor FLT3-internal tandem duplication (FLT3/ITD) mutations. In both cell lines, HQP1351 dose-dependently induced cellular apoptosis, APG-2575 also induced apoptosis, and their combination synergistically induced this process. In particular, treatment of subcutaneous MV-4-11 xenograft tumors in nude mice with HQP1351 10 mg/kg significantly suppressed tumor growth, with a tumor growth inhibition (TGI) of 72.4%. This antitumor effect was potentiated when APG-2575 was combined with HQP1351, resulting in a TGI of 97.2%. The combination also improved survival of tumor-bearing mice with a systemic MOLM-13 model compared to either agent alone. Mechanistically, HQP1351 likely inhibited phosphorylation of FLT3 and its downstream signaling pathways, including phosphorylation of AKT, ERK1/2 and STAT5. Taken together, these findings indicate that the antileukemic activity of olverembatinib can be ascribed to on-target inhibition. Single-agent APG-2575 agent triggered cellular apoptosis, as evidenced by cleavage of caspase-3 cleavage and poly(ADP-ribose) polymerase-1 (PARP-1) activation, which are considered hallmarks of apoptosis. Consequently, APG-2575 treatment also increased expression of another antiapoptotic protein (MCL-1), upregulation of which is a pivotal mechanisms of resistance to BCL-2 inhibitors. Notably, HQP1351 substantially decreased cellular MCL-1 expression when combined with APG-2575, further enhancing apoptosis.Taken together, our data suggest that FLT3 inhibition by HQP1351 downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 to potentiate cellular apoptosis in FLT3-mutant AML. The results provide scientific rationale for clinical development of HQP1351 combined with APG-2575 in patients with FLT3-ITD-mutant AML. Citation Format: Douglas D. Fang, Hengrui Zhu, Qiuqiong Tang, Qixin Wang, Na Li, Xu Fang, Ping Min, Guangfeng Wang, Dajun Yang, Yifan Zhai. FMS-like tyrosine kinase 3 (FLT3) inhibition by olverembatinib (HQP1351) downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 in preclinical models of FLT3-mutant acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1096.

Nanopore sequencing sheds a light on the FLT3 gene mutations complexity in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) patients carry in 27% of cases an activating mutation of the fms-like tyrosine kinase-3 (FLT3) gene: internal tandem duplication (ITD) or tyrosine kinase domain (TKD) point mutation. The simultaneous presence of both types of mutations, so-called FLT3 dual mutations, has been reported in 2% of APL, but this circumstance has never been studied. We studied a cohort of 74 APL cases, performing an in-depth analysis of three FLT3 dual mutant cases. Nanopore sequencing (NS) allowed us to characterize their complex mutational profile, showing the occurrence of multiple activating FLT3 mutations on different alleles in the leukemic promyelocytes and suggesting a cumulative impact of these events on the constitutive activation of the FLT3 pathway in APL cells. NS approach not only sheds light on the FLT3 mutational complexity in APL, but may also be useful to better clarify the FLT3 mutations landscape in acute myeloid leukemia.

Analysis of pre-chemotherapy WBC, PLT, monocyte, hemoglobin, and MPV levels in acute myeloid leukemia patients with WT1, FLT3, or NPM gene mutations.

Effects of mutations on AML (acute myeloid leukemia) patients have been an area of clinical interest. The aim of this study was to analyze pre-chemotherapy WBC (white blood cell), platelet, monocyte, hemoglobin, and mean platelet volume (MPV) levels in acute myeloid leukemia patients with Wilms tumor 1 (WT1), FMS-like tyrosine kinase 3 (FLT3), or nucleophosmin (NPM) gene mutations, attempting to detect and compare possible differences in these values.The study included 71 patients with acute myeloid leukemia known to have WT1, FLT3, or NPM gene mutations. The patients were divided into 3 groups: FLT3-mutated AML patients without any accompanying known mutations other than WT1 at the time of diagnosis (Group 1), NPM-mutated AML patients without any accompanying known mutations other than WT1 at the time of diagnosis (Group 2), WT1-mutated AML patients with no other accompanying known mutations at the time of diagnosis (Group 3). We carried out intergroup comparisons of WBC, platelet (PLT), monocyte, hemoglobin, and MPV levels before chemotherapy.There was a statistically significant difference between the groups in terms of WBC parameters (P = .001). There were no statistically significant differences between the groups with respect to hemoglobin, platelet, and monocyte levels.Higher white blood cell counts could be observed in patients with FLT3-mutated AML.

Insight into the inhibitor discrimination by FLT3F691L.

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.

Metabolomic Profiling Defines Distinct Metabolic Signature Associated with FLT3/ITD AML

Abstract AML is a hematological disorder resulting from proliferation and expansion of malignant myeloid cells. Clinical outcome for AML remains dismal despite intensive therapy in part due to the disease heterogeneity with various cytogenetic and molecular lesions. Fms-Like Tyrosine Kinase-3 (FLT3) is a receptor tyrosine kinase expressed hematopoietic stem/progenitor cells. Activating mutations of FLT3 gene due to internal tandem duplication of the juxtamembrane domain coding sequence (FLT3/ITD) causes autonomous cellular proliferations leading to disease progression. Metabolomic profiling has been successfully utilized to identify metabolic alterations in hematological disorders. However, no studies on metabolic alterations associated with pediatric AML have been reported at this time. In this study we propose to establish the metabolomic landscape in pediatric AML patients and identify differential expression of metabolites based on FLT3/ITD status. Cellular and plasma metabolomics profile was generated from 32 matching diagnostic material from 16 patients with and without FLT3/ITD (N=8 for each cohort and each sample was run in duplicate) treated on COG-AAML0531 study. Global metabolomics profiling was performed on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler. All samples were analyzed in duplicate in positive and negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 18-pfp 100 x 2.1 mm, 2 µm column with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. This is a polar embedded stationary phase that provides comprehensive coverage, but does have some limitation is the coverage of very polar species. The flow rate was 350 µL/min with a column temperature of 25¡C. 4 µL was injected for negative ions and 2 µL for positive ions. Statistical analysis was performed using MetaboAnalyst software using all metabolites (known and unknown) as well as only the annotated metabolites. Univariate analysis was performed by volcano plot and Multivariate analysis was performed using PCA, PLS-DA and OPLS-DA. Total of 2966 plasma metabolome (779 negative and 2187 positive) and 1742 (227 negative and 1515 positive) cellular metabolome features were identified. All subsequent data analyses were normalized to the sum of metabolites for each sample. Comparison of the cellular metabolome in patients with and without FLT3/ITD identified 12 known and 135 unknown metabolites that were significantly different between two cohorts (p<0.05). Similar comparison of the plasma metabolome identified19 known and 300 unknown metabolites in the patient with and without FLT3/ITD (top results are shown in Fig.1). Orthogonal partial least squares-discriminant analysis (OPLS-DA) showed clear separation between the 2 groups (Fig.2). Some of the top known plasma metabolites (p<0.01) differentiating patients by FLT3 status include guanine, pyrimidine-2-3dicarboxylate, acetylglycine, acetyl-L-alanine, aminobutonate gaba, L-carnitine, methyl-2 oxovaleric acid, asparagine, acetyl arginine, Hydroxydecanoic acid, cysteic acid and glycocholic acid. Within leukemic cells top metabolites differentiating between FLT3 status included actyly carnitine, adenosine monophosphate, hypoxanthine, diaminohepatanedioate, guanine and sphingosine. Metaboanalyst Pathway analysis module mapped the differentiating metabolites to aminoacyl-tRNA biosynthesis, Glycerophospholipid metabolism, Cysteine and methionine metabolism, Pantothenate and CoA biosynthesis, Purine metabolism. This pilot study defines distinct metabolomics signature associated with genomic subtype of AML (FLT3/ITD). As metabolomics provides an insight into the ultimate metabolic destination of normal and malignant hematopoiesis, it has a potential to provide a unique insight into the altered metabolic pathways in AML and identify pathways and networks that might be shared by varied genomic subtypes of AML. Such data can help merge rare genomic variants based on shared metabolic signatures and more appropriately guide directed therapies. Disclosures No relevant conflicts of interest to declare.

High-resolution melting curve analysis, a rapid and affordable method for mutation analysis in childhood acute myeloid leukemia.

Background: Molecular genetic alterations with prognostic significance have been described in childhood acute myeloid leukemia (AML). The aim of this study was to establish cost-effective techniques to detect mutations of FMS-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1), and a partial tandem duplication within the mixed-lineage leukemia (MLL-PTD) genes in childhood AML.Procedure: Ninety-nine children with newly diagnosed AML were included in this study. We developed a fluorescent dye SYTO-82 based high-resolution melting (HRM) curve analysis to detect FLT3 internal tandem duplication (FLT3-ITD), FLT3 tyrosine kinase domain (FLT3-TKD), and NPM1 mutations. MLL-PTD was screened by real-time quantitative PCR.Results: The HRM methodology correlated well with gold standard Sanger sequencing with less cost. Among the 99 patients studied, the FLT3-ITD mutation was associated with significantly worse event-free survival (EFS). Patients with the NPM1 mutation had significantly better EFS and overall survival. However, HRM was not sensitive enough for minimal residual disease monitoring.Conclusion: High-resolution melting was a rapid and efficient method for screening of FLT3 and NPM1 gene mutations. It was both affordable and accurate, especially in resource underprivileged regions. Our results indicated that HRM could be a useful clinical tool for rapid and cost-effective screening of the FLT3 and NPM1 mutations in AML patients.

Detection of nucleophosmin and FMS-like tyrosine kinase-3 gene mutations in acute myeloid leukemia.

BACKGROUND AND OBJECTIVES:Nucleophosmin gene mutations are frequently reported in acute myeloid leukemia (AML) patients with normal karyotype, which is also frequently associated with internal tandem duplication mutations in the FMS-like tyrosine kinase-3 gene. We sought to detect the nucleophosmin and FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) mutations among Iranian patients with AML and to assess the relationship between these mutations and the subtypes of the disease.DESIGN AND SETTING:Cross-sectional study of patients referred during 2007 through 2009.PATIENTS AND METHODS:Bone marrow and peripheral blood samples of 131 AML patients were randomly collected at the time of diagnosis and prior to treatment and the DNA extracted. After amplifying the nucleophosmin and FLT3 gene regions, positive cases were screened by conformation-sensitive gel electrophoresis and agarose gel electrophoresis techniques.RESULTS:Of 131 patients, 23 (17.5%) (0.95% CI=0.107-0.244) had nucleophosmin gene mutations. The highest frequency of such mutations was found among the subtypes of M4 (30.4%), M3 (21.7%) and M5 (17.4%). There was a high frequency of these mutations in the M3 subtype as well as a high frequency of allele D in all subtypes. Also, 21 (16.0%) samples (0.95% CI=0.092-0.229) had FLT3/ITD mutation, of which 8 samples had mutant nucleophosmin (8 of 23, 35%), and another 13 samples had wild-type nucleophosmin gene (13 of 108, 12%). There was a high degree of association between the occurrence of nucleophosmin and FLT3/ITD mutations (P=.012).CONCLUSION:Our data showed a high frequency of NPM1 mutations in the monocytic subtypes of AML, as well as a high degree of association between the occurrence of NPM1 and FLT3/ITD mutations.

Incidence of FLT3 and nucleophosmin gene mutations in childhood acute myeloid leukemia: Serbian experience and the review of the literature

Mutations in the fms-like tyrosine kinase 3 (FLT3) gene (internal tandem duplication (ITD) and point mutation in the tyrosine kinase domain, FLT3/D835) as well as the nucleophosmin (NPM1) gene are the most common abnormalities in adult acute myeloid leukemia (AML). Their significance in pediatric AML is still unclear. In this study we evaluated the frequency of FLT3 and NPM1 mutations in childhood AML. We also examined clinical features and outcome of these patients. FLT3 and NPM1 mutations were analysed in 42 and 37 childhood AML patients, respectively, using polymerase chain reaction (PCR) and direct sequencing. FLT3 mutations were detected in 4/42 patients (9.5%). The frequencies of FLT3/ITD and FLT3/D835 were the same, 2/42 (4.7%). NMP1 mutations were found in 1/37 patients (2.7%). FLT3 gene mutations were correlated with induction failure. Here we report the results of the study of FLT3 and NPM1 gene mutations in childhood AML patients in Serbia. Low frequencies of these molecular markers point out that these abnormalities are rare in this cohort of patients. Comparative study of data on NPM1 mutations in childhood AML revealed that various NPM1 gene mutation types are associated with childhood AML. Our findings as well as previously reported data, contributes to a hypothesis of different biology and etiology of adult and childhood AML. More extensive studies of NPM1 and FLT3 mutations in childhood AML are needed to determine their biological and clinical importance.

MS-275, a novel histone deacetylase inhibitor with selectivity against HDAC1, induces degradation of FLT3 via inhibition of chaperone function of heat shock protein 90 in AML cells

This study explored the effect of MS-275, a novel histone deacetylase inhibitor (HDACI), against a variety of human leukemia cells with defined genetic alterations. MS-275 profoundly induced growth arrest of acute myelogenous leukemia (AML) MOLM13 and biphenotypic leukemia MV4-11 cells, which possess internal tandem duplication mutation in the fms-like tyrosine kinase 3 ( FLT3) gene ( FLT3-ITD), with IC50s less than 1 μM, as measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay on day two of culture. Exposure of these cells to MS-275 decreased levels of total, as well as, phosphorylated forms of FLT3, resulting in inactivation of its downstream signal pathways, including Akt, ERK, and STAT5. Further studies found that MS-275 induced acetylation of heat shock protein 90 (HSP90) in conjunction with ubiquitination of FLT3, leading to degradation of FLT3 proteins in these cells. This was blunted by treatment with the proteasome inhibitor bortezomib, confirming that FLT was degraded via ubiquitin/proteasome pathway. Moreover, we found that further inhibition of MEK/ERK signaling potentiated the action of MS-275 in leukemia cells. Taken together, MS-275 may be useful for treatment of individuals with leukemia possessing activating mutation of FLT3 gene.

FLT3 Gene Mutations as a Prognostic Factor for Acute Myeloid Leukemia

Two distinct types of fms-like tyrosine kinase 3 (FLT3) gene mutations have been identified in acute myeloid leukemia (AML): D835 and internal tandem duplication (ITD) mutations. These mutations are known to cause the proliferation of leukemic cells and inhibit the apoptosis of leukemic cells due to ligand-independent activation of their receptors. Therefore, the current study attempted to investigate the frequency of FLT3 gene mutations and their prognostic implications for AML in terms of treatment response, survival, and relapse. Polymerase chain reaction (PCR) was performed to detect D835 and ITD mutations in 84 newly diagnosed AML patients from February 2001 to October 2004. Restriction fragment length polymorphism (RFLP) and direct sequencing were performed to analyze the D835 mutations. The results were examined based on a comparison with previously known prognostic factors, and the treatment outcomes analyzed according to the existence of the mutations in relation to the event free survival (EFS), overall survival (OS), and complete remission (CR) rates. D835 and IDT mutations were detected in 4.7% (4/84) and 19.0% (16/84), respectively, of the AML patients. The FLT3 gene mutations were not found to be associated with previously known prognostic factors, such as the WBC count, age, and cytogenetic risk group, but were associated with the lactate dehydrogenase levels. The EFS and OS rates were also significantly lower in the FLT3 gene mutation group, especially in AML with normal karyotypes. FLT3 gene mutations were observed in 23.8% of AML patients and appeared to have a prognostic implication on patient survival. Accordingly, the presence of FLT3 gene mutations, which could be tested easily by using PCR/RFLP methods, should be investigated routinely at the time of diagnosis.