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Acute myeloid leukemia (AML) is the most common hematological cancer in the adult population worldwide. Approximately 35% of patients with AML present internal tandem duplication (ITD) mutations in the FMS‑like tyrosine kinase 3 (FLT3) receptor associated with poor prognosis, and thus, this receptor is a relevant target for potential therapeutics. Tyrosine kinase inhibitors (TKIs) are used to treat AML; however, their molecular interactions and effects on leukemic cells are poorly understood. The present study aimed to gain insights into the molecular interactions and affinity forces of four TKI drugs (sorafenib, midostaurin, gilteritinib and quizartinib) with the wild‑type (WT)‑FLT3 and ITD‑mutated (ITD‑FLT3) structural models of FLT3, in its inactive aspartic acid‑phenylalanine‑glycine motif (DFG‑out) and active aspartic acid‑phenylalanine‑glycine motif (DFG‑in) conformations. Furthermore, the present study evaluated the effects of the second‑generation TKIs gilteritinib and quizartinib on cancer cell viability, apoptosis and proliferation in the MV4‑11 (ITD‑FLT3) and HL60 (WT‑FLT3) AML cell lines. Peripheral blood mononuclear cells (PBMCs) from a healthy volunteer were included as an FLT3‑negative group. Molecular docking analysis indicated higher affinities of second‑generation TKIs for WT‑FLT3/DFG‑out and WT‑FLT3/DFG‑in compared with those of the first‑generation TKIs. However, the ITD mutation changed the affinity of all TKIs. The in vitro data supported the in silico predictions: MV4‑11 cells presented high selective sensibility to gilteritinib and quizartinib compared with the HL60 cells, whereas the drugs had no effect on PBMCs. Thus, the current study presented novel information about molecular interactions between the FLT3 receptors (WT or ITD‑mutated) and some of their inhibitors. It also paves the way for the search for novel inhibitory molecules with potential use against AML.

FMS-like tyrosine kinase (FLT3) has become the legitimate molecular therapeutic target for acute myeloid leukemia therapy. Though FLT3 inhibitors have impact on disease progression, drug resistance induced by secondary point mutations is the primary mechanism and urgent to overcome. Herein, we sought to decipher the mechanism of HM43239 inhibiting the mutant F691L resistant to gilteritinib in FLT3. A series of molecular modeling studies, including molecular dynamics (MD) simulation, dynamic cross-correlation (DCC) analysis, binding free energy (MM-GBSA) and docking study were explored to elucidate the differential tolerance mechanisms of two inhibitors to the same mutant. The F691L mutation had relatively larger effect on gilteritinib than HM43239, which showed as the changed and fixed conformation, respectively. These observations rationalized that the binding affinity of gilteritinib decreased more than that of HM43239 in the F691L mutant.Communicated by Ramaswamy H. Sarma.

Overexpression and frequent mutations in FMS-like tyrosine kinase-3 (FLT3) are considered risk factors for severe acute myeloid leukemia (AML). Hyperactive FLT3 induces premature activation of multiple intracellular signaling pathways, resulting in cell proliferation and anti-apoptosis. We conducted the computational modeling studies of 40 pyrimidine-4,6-diamine-based compounds by integrating docking, molecular dynamics, and three-dimensional structure-activity relationship (3D-QSAR). Molecular docking showed that K644, C694, F691, E692, N701, D829, and F830 are critical residues for the binding of ligands at the hydrophobic active site. Molecular dynamics (MD), together with Molecular Mechanics Poison-Boltzmann/Generalized Born Surface Area, i.e., MM-PB(GB)SA, and linear interaction energy (LIE) estimation, provided critical information on the stability and binding affinity of the selected docked compounds. The MD study suggested that the mutation in the gatekeeper residue F691 exhibited a lower binding affinity to the ligand. Although, the mutation in D835 in the activation loop did not exhibit any significant change in the binding energy to the most active compound. We developed the ligand-based comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) models. CoMFA ( q 2 = 0.802, r 2 = 0.983, and QF32 = 0.698) and CoMSIA ( q 2 = 0.725, r 2 = 0.965 and QF32 = 0.668) established the structure-activity relationship (SAR) and showed a reasonable external predictive power. The contour maps from the CoMFA and CoMSIA models could explain valuable information about the favorable and unfavorable positions for chemical group substitution, which can increase or decrease the inhibitory activity of the compounds. In addition, we designed 30 novel compounds, and their predicted pIC 50 values were assessed with the CoMSIA model, followed by the assessment of their physicochemical properties, bioavailability, and free energy calculation. The overall outcome could provide valuable information for designing and synthesizing more potent FLT3 inhibitors.

The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease., (© 2021. The Author(s).)

Objectives: Acute myeloid leukemia (AML) is still challenging in predicting the prognosis due to its high heterogeneity. Molecular aberrations and abnormalities play a significant prognostic role in AML patients. Our aim of the study was to investigate the prognostic role of TNFRSF4 gene expression in AML patients and its potential effect on treatment protocols., Methods: Bone marrow mononuclear cells were analyzed for TNFRSF4 expression by real-time quantitative PCR as well as of FLT3/ITD and NPM1 mutations in 80 newly diagnosed AML patients and 80 control subjects., Results: TNFRSF4 was significantly overexpressed in the AML patients (p < 0.001). TNFRSF4 expression was associated with unfavorable clinical outcomes including treatment response, relapse free survival, and overall survival. On multivariate testing, TNFRSF4 high expression proved to be an independent prognostic marker for clinical remission and relapse free survival but not overall survival., Conclusion: TNFRSF4 expression was revealed as an unfavorable prognostic marker and might be a target for immunotherapy in the future., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Activating mutations in the Fms-like tyrosine kinase 3 (FLT3) are among the most prevalent oncogenic mutations in acute myeloid leukaemia. Inhibitors selectively targeting FLT3 kinase have shown promising clinical activity; their success in the clinic, however, has been limited due to the emergence of acquired resistance., Methods: CCT245718 was identified and characterised as a dual Aurora A/FLT3 inhibitor through cell-based and biochemical assays. The ability of CCT245718 to overcome TKD-mediated resistance was evaluated in a cell line-based model of drug resistance to FLT3 inhibitors., Results: CCT245718 exhibits potent antiproliferative activity towards FLT3-ITD + AML cell lines and strongly binds to FLT3-ITD and TKD (D835Y) mutants in vitro. Activities of both FLT3-ITD and Aurora A are also inhibited in cells. Inhibition of FLT3 results in reduced phosphorylation of STAT5, downregulation of survivin and induction of apoptotic cell death. Moreover, CCT245718 overcomes TKD-mediated resistance in a MOLM-13-derived cell line containing FLT3 with both ITD and D835Y mutations. It also inhibits FLT3 signalling in both parental and resistant cell lines compared to FLT3-specific inhibitor MLN518, which is only active in the parental cell line., Conclusions: Our results demonstrate that CCT245718 is a potent dual FLT3/Aurora A inhibitor that can overcome TKD-mediated acquired resistance., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The t(1;11)(p32;q23) translocation is a rare but recurrent cytogenetic aberration in acute myeloid leukemia (AML) and B-cell acute lymphoblastic leukemia (B-ALL). This translocation was initially shown to form a fusion gene between KMT2A exon 8 at 11q23 and EPS15 exon 2 at 1p32 in AML. Activating mutations of FLT3 are frequently found in AML but are very rare in ALL. Here, we describe a 75-year-old woman who was diagnosed with B-ALL since her bone marrow was made up of 98.2% lymphoblasts. These blasts were positive for CD19, CD22, CD79a, CD13, and CD33 but negative for CD10 and myeloperoxidase. The karyotype by G-banding and spectral karyotyping was 46,XX,t(1;11)(p32;q23). Expression of KMT2A/EPS15 and reciprocal EPS15/KMT2A fusion transcripts were shown: KMT2A exon 8 was in-frame fused to EPS15 exon 12, indicating that this fusion transcript was a novel type. Considering three reported B-ALL cases, EPS15 breakpoints were markedly different between AML (exon 2) and B-ALL (exons 10-12). Furthermore, an uncommon type of FLT3 mutation in the juxtamembrane domain was detected: in-frame 4-bp deletion and 10-bp insertion. Accordingly, our results indicate that the novel type of KMT2A/EPS15 fusion transcript and FLT3 mutation may cooperate in the pathogenesis of adult B-ALL as class II and class I mutations, respectively., Competing Interests: Declaration of Competing Interest All authors have no conflict of interest., (Copyright © 2021. Published by Elsevier Inc.)

The overarching paradigm for the activation of class III and V receptor tyrosine kinases (RTKs) prescribes cytokine-mediated dimerization of the receptor ectodomains and homotypic receptor-receptor interactions. However, structural studies have shown that the hematopoietic receptor FLT3, a class III RTK, does not appear to engage in such receptor-receptor contacts, despite its efficient dimerization by dimeric FLT3 ligand (FL). As part of efforts to better understand the intricacies of FLT3 activation, we sought to engineer a monomeric FL. It was found that a Leu27Asp substitution at the dimer interface of the cytokine led to a stable monomeric cytokine (FL L27D ) without abrogation of receptor binding. The crystal structure of FL L27D at 1.65 Å resolution revealed that the introduced point mutation led to shielding of the hydrophobic footprint of the dimerization interface in wild-type FL without affecting the conformation of the FLT3 binding site. Thus, FL L27D can serve as a monomeric FL variant to further interrogate the assembly mechanism of extracellular complexes of FLT3 in physiology and disease., (open access.)

Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis and a high degree of relapse seen in patients. Overexpression of FMS-like tyrosine kinase 3 (FLT3) is associated with up to 70% of AML patients. Wild-type FLT3 induces proliferation and inhibits apoptosis in AML cells, while uncontrolled proliferation of FLT3 kinase activity is also associated with FLT3 mutations. Therefore, inhibiting FLT3 activity is a promising AML therapy. Flavonoids are a group of phytochemicals that can target protein kinases, suggesting their potential antitumor activities. In this study, several plant-derived flavonoids have been identified with FLT3 inhibitory activity. Among these compounds, compound 40 (5,7,4'-trihydroxy-6-methoxyflavone) exhibited the most potent inhibition against not only FLT3 (IC 50 = 0.44 μM) but also FLT3-D835Y and FLT3-ITD mutants (IC 50 = 0.23 and 0.39 μM, respectively). The critical interactions between the FLT3 binding site and the compounds were identified by performing a structure-activity relationship analysis. Furthermore, the results of cellular assays revealed that compounds 28 , 31 , 32 , and 40 exhibited significant cytotoxicity against two human AML cell lines (MOLM-13 and MV-4-11), and compounds 31 , 32 , and 40 resulted in cell apoptosis and G0/G1 cell cycle arrest. Collectively, these flavonoids have the potential to be further optimized as FLT3 inhibitors and provide valuable chemical information for the development of new AML drugs.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by several gene mutations and cytogenetic abnormalities affecting differentiation and proliferation of myeloid lineage cells. FLT3 is a receptor tyrosine kinase commonly overexpressed or mutated, and its mutations are associated with poor prognosis in AML. Although aggressive chemotherapy often followed by hematopoietic stem cell transplant is the current standard of care, the recent approval of FLT3-targeted drugs is revolutionizing AML treatment that had remained unchanged since the 1970s. However, despite the dramatic clinical response to targeted agents, such as FLT3 inhibitors, remission is almost invariably short-lived and ensued by relapse and drug resistance. Hence, there is an urgent need to understand the molecular mechanisms driving drug resistance in order to prevent relapse. In this review, we discuss FLT3 as a target and highlight current understanding of FLT3 inhibitor resistance.

FMS-like tyrosine kinase 3 (FLT3) has been found to be mutated in ~ 30% of acute myeloid leukaemia patients. Small-molecule inhibitors targeting FLT3 that are currently approved or still undergoing clinical trials are subject to drug resistance due to FLT3 mutations. How these mutations lead to drug resistance is hitherto poorly understood. Herein, we studied the molecular mechanism of the drug resistance mutations D835N, Y842S and M664I, which confer resistance against the most advanced inhibitors, quizartinib and PLX3397 (pexidartinib), using enzyme kinetics and computer simulations. In vitro kinase assays were performed to measure the comparative catalytic activity of the native protein and the mutants, using a bacterial expression system developed to this aim. Our results reveal that the differential drug sensitivity profiles can be rationalised by the dynamics of the protein-drug interactions and perturbation of the intraprotein contacts upon mutations. Drug binding induced a single conformation in the native protein, whereas multiple conformations were observed otherwise (in the mutants or in the absence of drugs). The end-point kinetics measurements indicated that the three resistant mutants conferred catalytic activity that is at least as high as that of the reference without such mutations. Overall, our calculations and measurements suggest that the structural dynamics of the drug-resistant mutants that affect the active state and the increased conformational freedom of the remaining inactive drug-bound population are the two major factors that contribute to drug resistance in FLT3 harbouring cancer cells. Our results explain the mechanism of drug resistance mutations and can aid to the design of more effective tyrosine kinase inhibitors., (© 2020 Federation of European Biochemical Societies.)

FMS-like receptor tyrosine kinase-3 (FLT3) is expressed on acute leukemia cells and is implicated in the survival, proliferation and differentiation of hematopoietic cells in most acute myeloid leukemia (AML) patients. Despite recent achievements in the development of FLT3-targeted small-molecule drugs, there are still unmet medical needs related to kinase selectivity and the progression of some mutant forms of FLT3. Herein, we describe the discovery of novel orally available type 1 FLT3 inhibitors from structure-activity relationship (SAR) studies for the optimization of indirubin derivatives with biological and pharmacokinetic profiles as potential therapeutic agents for AML. The SAR exploration provided important structural insights into the key substituents for potent inhibitory activities of FLT3 and in MV4-11 cells. The profile of the most optimized inhibitor (36) showed IC 50 values of 0.87 and 0.32 nM against FLT3 and FLT3/D835Y, respectively, along with potent inhibition against MV4-11 and FLT3/D835Y expressed MOLM14 cells with a GI 50 value of 1.0 and 1.87 nM, respectively. With the high oral bioavailability of 42.6%, compound 36 displayed significant in vivo antitumor activity by oral administration of 20 mg/kg once daily dosing schedule for 21 days in a mouse xenograft model. The molecular docking study of 36 in the homology model of the DFG-in conformation of FLT3 resulted in a reasonable binding mode in type 1 kinases similar to the reported type 1 FLT3 inhibitors Crenolanib and Gilteritinib., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

FLT3 and dual Aurora B/FLT3 inhibitors have shown relevance in the search for promising new anticancer compounds, mainly for acute myeloid leukemia (AML). This study was designed to investigate the interactions between human FLT3 in the kinase domain with several indolin-2-one derivatives, structurally similar to Sunitinib. Molegro Virtual Docker (MVD) software was utilized in docking analyses. The predicted model of the training group, considering nineteen amino acid residues, performed in Chemoface, achieved an R 2 of 0.82, suggesting that the binding conformations of the ligands with FLT3 are reasonable, and the data can be used to predict the interaction energy of other FLT3 inhibitors with similar molecular patterns. The MolDock Score for energy for compound 1 showed more stable interaction energy (-233.25 kcal mol -1 ) than the other inhibitors studied, while Sunitinib presented as one of the least stable (-160.94 kcal mol -1 ). Compounds IAF70, IAF72, IAF75, IAF80, IAF84, and IAF88 can be highlighted as promising derivatives for synthesis and biological evaluation against FLT3. Furthermore, IAF79 can be considered to be a promising dual Aurora B/FLT3 inhibitor, and its molecular pattern can be exploited synthetically to search for new indolin-2-one derivatives that may become drugs used in the treatment of cancers, including AML.

FLT3 (FMS-like tyrosine kinase 3), expressed on the surface of acute myeloid leukemia (AML) blasts, is a promising AML target, given its role in the development and progression of leukemia, and its limited expression in tissues outside the hematopoietic system. Small molecule FLT3 kinase inhibitors have been developed, but despite having clinical efficacy, they are effective only on a subset of patients and associated with high risk of relapse. A durable therapy that can target a wider population of AML patients is needed. Here, we developed an anti-FLT3-CD3 immunoglobulin G (IgG)-based bispecific antibody (7370) with a high affinity for FLT3 and a long half-life, to target FLT3-expressing AML blasts, irrespective of FLT3 mutational status. We demonstrated that 7370 has picomolar potency against AML cell lines in vitro and in vivo. 7370 was also capable of activating T cells from AML patients, redirecting their cytotoxic activity against autologous blasts at low effector-to-target (E:T) ratio. Additionally, under our dosing regimen, 7370 was well tolerated and exhibited potent efficacy in cynomolgus monkeys by inducing complete but reversible depletion of peripheral FLT3 + dendritic cells (DCs) and bone marrow FLT3 + stem cells and progenitors. Overall, our results support further clinical development of 7370 to broadly target AML patients., (Copyright © 2020. Published by Elsevier Inc.)

FMS-like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase that plays an important role in hematopoietic cell survival, proliferation and differentiation. The most clinically important point is that mutation of the FLT3 gene is the most frequent genetic alteration and a poor prognostic factor in acute myeloid leukemia (AML) patients. There are two major types of FLT3 mutations: internal tandem duplication mutations in the juxtamembrane domain (FLT3-ITD) and point mutations or deletion in the tyrosine kinase domain (FLT3-TKD). Both mutant FLT3 molecules are activated through ligand-independent dimerization and trans-phosphorylation. Mutant FLT3 induces the activation of multiple intracellular signaling pathways, mainly STAT5, MAPK and AKT signals, leading to cell proliferation and anti-apoptosis. Because high-dose chemotherapy and allogeneic hematopoietic stem cell transplantation cannot sufficiently improve the prognosis, clinical development of FLT3 kinase inhibitors expected. Although several FLT3 inhibitors have been developed, it takes more than 20 years from the first identification of FLT3 mutations until FLT3 inhibitors become clinically available for AML patients with FLT3 mutations. To date, three FLT3 inhibitors have been clinically approved as monotherapy or combination therapy with conventional chemotherapeutic agents in Japan and/or Europe and United states. However, several mechanisms of resistance to FLT3 inhibitors have already become apparent during their clinical trials. The resistance mechanisms are complex and emerging resistant clones are heterogenous. Further basic and clinical studies are required to establish the best therapeutic strategy for AML patients with FLT3 mutations., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Fms-like tyrosine kinase 3 (FLT3) is a glycoprotein, that is a member of the class III receptor tyrosine kinase family. Approximately one-third of acute myeloid leukemia (AML) patients have mutations of this gene, and activation of the FLT3 downstream pathway plays an important role in both normal and malignant hematopoiesis. However, the role of N-glycosylation for FLT3 activation remains unclear. In this study, we showed that the N-glycan structures on wild type (WT), internal tandem duplication (ITD), and tyrosine kinase domain (TKD) mutants of FLT3 were different. Interestingly, expression of either WT or mutant FLT3 in Ba/F3 cells, an interleukin-3 (IL-3)-dependent hematopoietic progenitor cell, greatly induced core fucosylation. To elucidate the function of core fucosylation in FLT3-mediated signaling, we used a CRISPR/Cas9 system to establish α1,6-fucosyltransferase (Fut8) knockout (KO) cells. Surprisingly, the Fut8KO resulted in cell proliferation in an IL-3-independent manner in FLT3-WT cells, which was not observed in the parental cells, and suggested that this proliferation is dependent on FLT3 expression. Fut8KO greatly increased cellular tyrosine phosphorylation levels, together with an activation of STAT5, AKT, and ERK signaling, which could be completely neutralized by restoration with Fut8 in the KO cells. Consistently, a tyrosine kinase inhibitor efficiently inhibited cell proliferation induced by Fut8KO or specific fucosylation inhibitor. Additionally, immunostaining with FLT3 showed that the proteins were mainly expressed on the cell surface in the KO cells, which is similar to FLT3-WT cells, but different from the ITD mutant. Finally, we found that Fut8KO could induce dimer-formation in FLT3 without ligand-stimulation. Taken together, the present study clearly defines the regulatory function of core fucosylation in FLT3, which could provide a valuable direction for development of drugs could be effective in the treatment of AML., (© 2020 Federation of American Societies for Experimental Biology.)

Oxidative modification of cysteine residues has been shown to regulate the activity of several protein-tyrosine kinases. We explored the possibility that Fms-like tyrosine kinase 3 (FLT3), a hematopoietic receptor-tyrosine kinase, is subject to this type of regulation. An underlying rationale was that the FLT3 gene is frequently mutated in Acute Myeloid Leukemia patients, and resulting oncogenic variants of FLT3 with 'internal tandem duplications (FLT3ITD)' drive production of reactive oxygen in leukemic cells. FLT3 was moderately activated by treatment of intact cells with hydrogen peroxide. Conversely, FLT3ITD signaling was attenuated by cell treatments with agents inhibiting formation of reactive oxygen species. FLT3 and FLT3ITD incorporated DCP-Bio1, a reagent specifically reacting with sulfenic acid residues. Mutation of FLT3ITD cysteines 695 and 790 reduced DCP-Bio1 incorporation, suggesting that these sites are subject to oxidative modification. Functional characterization of individual FLT3ITD cysteine-to-serine mutants of all 8 cytoplasmic cysteines revealed phenotypes in kinase activity, signal transduction and cell transformation. Replacement of cysteines 681, 694, 695, 807, 925, and 945 attenuated signaling and blocked FLT3ITD-mediated cell transformation, whereas mutation of cysteine 790 enhanced activity of both FLT3ITD and wild-type FLT3. These effects were not related to altered FLT3ITD dimerization, but likely caused by changed intramolecular interactions. The findings identify the functional relevance of all cytoplasmic FLT3ITD cysteines, and indicate the potential for redox regulation of this clinically important oncoprotein., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Herein, we describe the design, synthesis, and structure-activity relationships of a series of unique 4-(1 H -pyrazol-4-yl)-pyrimidin-2-amine derivatives that selectively inhibit Janus kinase 2 (JAK2) and FLT3 kinases. These screening cascades revealed that 18e was a preferred compound, with IC 50 values of 0.7 and 4 nM for JAK2 and FLT3, respectively. Moreover, 18e was a potent JAK2 inhibitor with 37-fold and 56-fold selectivity over JAK1 and JAK3, respectively, and possessed an excellent selectivity profile over the other 100 representative kinases. In a series of cytokine-stimulated cell-based assays, 18e exhibited a higher JAK2 selectivity over other JAK isoforms. The oral administration of 60 mg/kg of 18e could significantly inhibit tumor growth, with a tumor growth inhibition rate of 93 and 85% in MV4-11 and SET-2 xenograft models, respectively. Additionally, 18e showed an excellent bioavailability ( F = 58%), a suitable half-life time ( T 1/2 = 4.1 h), a satisfactory metabolic stability, and a weak CYP3A4 inhibitory activity, suggesting that 18e might be a potential drug candidate for JAK2-driven myeloproliferative neoplasms and FLT3-internal tandem duplication-driven acute myelogenous leukemia.

The aberrant expression of FLT3 tyrosine kinase is associated primarily with acute myeloid leukaemia. This blood malignancy is often related to the onset of internal tandem duplications (ITDs) in the native sequence of the protein. The ITDs occur mainly in the juxtamembrane domain of the protein and alter the normal activity of the enzyme. In this work, we have studied the native form of FLT3 and six mutants by molecular dynamics simulations. The catalytic activity of FLT3 is exerted by the tyrosine kinase domain (KD) and regulated by the juxtamembrane (JM) domain. Analysis of the dynamics of these two domains have shown that the introduction of ITDs in the JM domain alters both structural and dynamic parameters. The presence of ITDs allowed the protein to span a larger portion of the conformational space, particularly in the JM domain and the activation loop. The FLT3 mutants were found to adopt more stable configurations than the native enzyme. This was due to the different arrangements assumed by the JM domain. Larger fluctuations of the activation loop were found in four of the six mutants. In the native FLT3, the key residue Tyr572 is involved in a strong and stable interaction with an ion pair. This interaction, which is thought to keep the JM in place hence regulating the activity of the enzyme, was found to break in all FLT3 mutants.

FLT3 mutation is among the most common genetic mutations in acute myeloid leukemia (AML), which is also related with poor overall survival and refractory in AML patients. Recently, FLT3 inhibitors have been approved for AML therapy. Herein, a series of new compounds with pyrazole amine scaffold was discovered, which showed potent inhibitory activity against FLT3-ITD and significant selectivity against both FLT3-ITD and AML cells expressing FLT3-ITD. Compound 46, possessing the most promising cellular activity, blocked the autophosphorylation of FLT3 pathway in MV4-11 cell line. Furthermore, the apoptosis and downregulation of P-STAT5 were also observed in tumor cells extracted from the MV4-11 cell xenografts model upon compound 46 treatment. Compound 46 was also metabolically stable in vitro and suppressed tumor growth significantly in MV4-11 xenografts model in vivo. Compound 46 showed no toxicity to the viscera of mice and caused no decrease in body weight of mice. In conclusion, the results of this study could provide valuable insights into discovery of new FLT3 inhibitors, and compound 46 was worthy of further development as potential drug candidate to treat AML., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

The presence of FMS-like receptor tyrosine kinase-3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate primitive FLT3-ITD + leukemia cells, which are potential sources of relapse. Thus, understanding the mechanisms underlying FLT3-ITD + AML cell persistence is essential to devise future AML therapies. Here, we show that expression of protein arginine methyltransferase 1 (PRMT1), the primary type I arginine methyltransferase, is increased significantly in AML cells relative to normal hematopoietic cells. Genome-wide analysis, coimmunoprecipitation assay, and PRMT1 -knockout mouse studies indicate that PRMT1 preferentially cooperates with FLT3-ITD, contributing to AML maintenance. Genetic or pharmacological inhibition of PRMT1 markedly blocked FLT3-ITD + AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginine 972/973, and PRMT1 promoted leukemia cell growth in an FLT3 methylation-dependent manner. Moreover, the effects of FLT3-ITD methylation in AML cells were partially due to cross talk with FLT3-ITD phosphorylation at tyrosine 969. Importantly, FLT3 methylation persisted in FLT3-ITD + AML cells following kinase inhibition, indicating that methylation occurs independently of kinase activity. Finally, in patient-derived xenograft and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD + AML cells relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes AML maintenance and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to eliminate FLT3-ITD + AML cells., (© 2019 by The American Society of Hematology.)

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aberrant expression of kinases is often associated with pathologies such as cancer and autoimmune diseases. Like other types of enzymes, kinases can adopt active and inactive states, where a shift toward more stable active state often leads to disease. Dozens of kinase inhibitors are, therefore, used as drugs. Most of these bind to either the inactive or active state. In this work, we study the transitions between these two states in FLT3, an important drug target in leukemias. Kinases are composed of two lobes (N- and C-terminal lobes) with the catalytic site in-between. Through projection of the largest motions obtained through molecular dynamics (MD) simulations, we show that each of the end-states (active or inactive) already possess the ability for transition as the two lobes rotate which initiates the transition. A targeted simulation approach known as essential dynamics sampling (EDS) was used to speed up the transition between the two protein states. Coupling the EDS to implicit-solvent MD was performed to estimate the free energy barriers of the transitions. The activation energies were found in good agreement with previous estimates obtained for other kinases. Finally, we identified FLT3 intermediates that assumed configurations that resemble that of the c-Src nonreceptor tyrosine kinase. The intermediates show better binding to the drug ponatinib than c-Src and the inactive state of FLT3. This suggests that targeting intermediate states can be used to explain the drug-binding patterns of kinases and for rational drug design.

Flt3 is an oncogenic kinase involved in different types of leukemia. It is most prominently associated with acute myeloid leukemia (AML). Flt3-specific inhibitors have shown promising results in interfering with AML prompting us to model this interesting target. We implemented ligand-based, QSAR-guided, pharmacophore exploration combined with novel structure-based computational workflow based on docking-based comparative intermolecular contacts analysis (db-CICA) combined with homology modelling to explore the pharmacophoric features of 93 diverse cyclic Flt3 inhibitors. The resulting pharmacophore models were used as virtual search queries to scan the National Cancer Institute (NCI) database for novel Flt3 inhibitory leads. Ten hits of novel scaffolds were captured showing anti-Flt3 IC 50 values ranging from 1.2 to 14.7 μM. Interestingly, six of them illustrated low micromolar and submicromolar potencies against the mutated active form of Flt3 (Flt3D835Y) and the related vascular endothelial growth factor receptor 2 (VEGFR2)., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The interpretation of high-dimensional structure-activity data sets in drug discovery to predict ligand-protein interaction landscapes is a challenging task. Here we present Drug Discovery Maps (DDM), a machine learning model that maps the activity profile of compounds across an entire protein family, as illustrated here for the kinase family. DDM is based on the t-distributed stochastic neighbor embedding (t-SNE) algorithm to generate a visualization of molecular and biological similarity. DDM maps chemical and target space and predicts the activities of novel kinase inhibitors across the kinome. The model was validated using independent data sets and in a prospective experimental setting, where DDM predicted new inhibitors for FMS-like tyrosine kinase 3 (FLT3), a therapeutic target for the treatment of acute myeloid leukemia. Compounds were resynthesized, yielding highly potent, cellularly active FLT3 inhibitors. Biochemical assays confirmed most of the predicted off-targets. DDM is further unique in that it is completely open-source and available as a ready-to-use executable to facilitate broad and easy adoption.

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Outcome of patients with mutation of the FLT3 tyrosine kinase domain (FLT3-TKD) in acute myeloid leukemia (AML) remains controversial., Patients and Methods: Herein, we present a retrospective study of 126 newly diagnosed patients with AML performed in 2 French centers., Results: FLT3-TKD mutations represented 12.7% of patients, whereas FLT3-internal tandem duplication (ITD) mutation was observed in 20.6% of AML cases and 1.6% of patients harbored both anomalies. At diagnosis, FLT3-TKD and FLT3-ITD were associated with higher peripheral leukocytes count and a higher blast count in bone marrow (P < 10 -4 ). Mutations of the NPM1 gene were frequently associated to FLT3-TKD (68.7%) and FLT3-ITD (61.5%) mutations compared with FLT3 wild type (WT) patients (P < 10 -4 ). Patients with both FLT3-TKD and NPM1 mutations (n = 12; 9.5%) showed a favorable outcome. Interestingly, mutations in NPM1 gene lost their favorable prognostic when not associated with FLT3-TKD both in univariate and multivariate analyses., Conclusion: Our data suggest that FLT3-TKD mutations should be routinely determined at the time of diagnosis. In association with NPM1 mutations, patients should follow the therapeutic schedule of favorable-risk patients with AML., (Copyright © 2018 Elsevier Inc. All rights reserved.)

FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in hematological malignancies. FLT3 internal tandem duplication (FLT3-ITD) mutations located in juxtamembrane domain (JMD) and tyrosine kinase domain 1 (TKD1) regions account for two-thirds of all FLT3 mutations. The outcome of patients remains unsatisfactory, with low survival rates. It is not yet known whether the different mutations within the FLT3 gene are all associated with patient outcome. In addition, the cause of FLT3-ITD in-frame duplication events remains unknown. Although there are some published studies investigating the FLT3-ITD mutation and its clinical implications in Chinese acute myeloid leukemia (AML) patients, sample sizes tend to be small and detailed molecular profiles of FLT3 mutations are lacking in these studies. In our study, 227 FLT3-ITD sequences were analyzed from 227 Chinese de novo AML patients. ITD were next classified into 3 types based on molecular profiles of insertion DNA sequences: DNA complete duplication (type I), DNA partial duplication (type II) and complete random sequence (type III). From the 154 patients, we confirmed that high ITD allelic ratio (≥.5) and allogeneic stem cell transplant treatment under CR1 are independent prognostic factors. We also presented evidence that ITD integration sites in the hinge region or beta1-sheet region are an unfavorable prognostic factor in adult AML patients with FLT3-ITD mutations. These findings may help to decipher the mechanisms of FLT3-ITD in-frame duplication events and stratify patients when considering different therapeutic combinations., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The identification of recurrent driver mutations in genes encoding tyrosine kinases has resulted in the development of molecularly-targeted treatment strategies designed to improve outcomes for patients diagnosed with acute myeloid leukemia (AML). The receptor tyrosine kinase FLT3 is the most commonly mutated gene in AML, with internal tandem duplications within the juxtamembrane domain (FLT3-ITD) or missense mutations in the tyrosine kinase domain (FLT3-TKD) present in 30⁻35% of AML patients at diagnosis. An established driver mutation and marker of poor prognosis, the FLT3 tyrosine kinase has emerged as an attractive therapeutic target, and thus, encouraged the development of FLT3 tyrosine kinase inhibitors (TKIs). However, the therapeutic benefit of FLT3 inhibition, particularly as a monotherapy, frequently results in the development of treatment resistance and disease relapse. Commonly, FLT3 inhibitor resistance occurs by the emergence of secondary lesions in the FLT3 gene, particularly in the second tyrosine kinase domain (TKD) at residue Asp835 (D835) to form a 'dual mutation' (ITD-D835). Individual FLT3-ITD and FLT3-TKD mutations influence independent signaling cascades; however, little is known about which divergent signaling pathways are controlled by each of the FLT3 specific mutations, particularly in the context of patients harboring dual ITD-D835 mutations. This review provides a comprehensive analysis of the known discrete and cooperative signaling pathways deregulated by each of the FLT3 specific mutations, as well as the therapeutic approaches that hold the most promise of more durable and personalized therapeutic approaches to improve treatments of FLT3 mutant AML.

FLT3 tyrosine kinase is a potential drug target in acute myeloid leukemia (AML) because patients with FLT3-ITD mutations respond poorly to standard cytotoxic agents and there is a clear link between the disease and the oncogenic properties of FLT3. We present novel 2,6,9-trisubstituted purine derivatives with potent FLT3 inhibitory activity. The lead compound 7d displays nanomolar activity in biochemical assays and selectively blocks proliferation of AML cell lines harboring FLT3-ITD mutations, whereas other transformed and normal human cells are several orders of magnitude less sensitive. The MV4-11 cells treated with 7d suppressed the phosphorylation of FLT3 and its downstream signaling pathways, with subsequent G1 cell cycle arrest and apoptosis. Additionally, a single dose of 7d in mice with subcutaneous MV4-11 xenografts caused sustained inhibition of FLT3 and STAT5 phosphorylation over 48 h, in contrast to the shorter effect observed after administration of the reference FLT3 inhibitor quizartinib.

An activating mutation of Fms-like tyrosine kinase 3 ( FLT3 ) is the most frequent genetic alteration associated with poor prognosis in acute myeloid leukemia (AML). Although many FLT3 inhibitors have been clinically developed, no first-generation inhibitors have demonstrated clinical efficacy by monotherapy, due to poor pharmacokinetics or unfavorable safety profiles possibly associated with low selectivity against FLT3 kinase. Recently, a selective FLT3 inhibitor, quizartinib, demonstrated favorable outcomes in clinical studies. However, several resistant mutations emerged during the disease progression. To overcome these problems, we developed a novel FLT3 inhibitor, FF-10101, designed to possess selective and irreversible FLT3 inhibition. The co-crystal structure of FLT3 protein bound to FF-10101 revealed the formation of a covalent bond between FF-10101 and the cysteine residue at 695 of FLT3. The unique binding brought high selectivity and inhibitory activity against FLT3 kinase. FF-10101 showed potent growth inhibitory effects on human AML cell lines harboring FLT3 internal tandem duplication ( FLT3 -ITD), MOLM-13, MOLM-14, and MV4-11, and all tested types of mutant FLT3-expressing 32D cells including quizartinib-resistant mutations at D835, Y842, and F691 residues in the FLT3 kinase domain. In mouse subcutaneous implantation models, orally administered FF-10101 showed significant growth inhibitory effect on FLT3-ITD-D835Y- and FLT3-ITD-F691L-expressing 32D cells. Furthermore, FF-10101 potently inhibited growth of primary AML cells harboring either FLT3 -ITD or FLT3 -D835 mutation in vitro and in vivo. These results indicate that FF-10101 is a promising agent for the treatment of patients with AML with FLT3 mutations, including the activation loop mutations clinically identified as quizartinib-resistant mutations., (© 2018 by The American Society of Hematology.)

Background: Cabozantinib, a tyrosine kinase inhibitor of FMS-like tyrosine kinase 3 (FLT3), MET, AXL, vascular endothelial growth factor receptor, and KIT, is approved for use in multiple malignancies. We assessed the safety and tolerability of cabozantinib in AML, given up-regulation of multiple relevant pathways., Methods: Adults were eligible if they were 18 years old or older with relapsed/refractory AML or if they were 70 years old or older with newly diagnosed AML but were ineligible for conventional therapy. Cabozantinib was administered in 28-day cycles, and dose escalation occurred via cohorts. A pharmacodynamic evaluation of serial plasma samples via a plasma inhibitory assay (PIA) was used to assess FLT3-inhibitory activity in FLT3-mutant cell lines., Results: Among 18 patients enrolled, 5 were found to harbor FLT3/ITD mutations. Sixteen patients (89%) had relapsed/refractory AML, and most were treated with 2 or more lines of prior treatment. No dose-limiting toxicities (DLTs) were detected at the first dose level (40 mg daily), but 2 patients experienced DLTs at the next level (60 mg daily). The remaining patients were then dosed at 40 mg daily, the maximum tolerated dose (MTD). Additional grade 2 or higher toxicities, possibly/probably related to cabozantinib, included fatigue, nausea, transaminitis, and electrolyte imbalance. No patients had a marrow response according to formal criteria, but 4 had peripheral blast reductions; 2 of these 4 patients transiently cleared circulating blasts. One patient experienced a reduction in marrow blasts, and 1 had stable disease. The FLT3-inhibitory activity of plasma samples, as assessed with the PIA, revealed potent and sustained inhibition in FLT3/ITD and, notably, F691 tyrosine kinase domain (TKD)-mutant cells., Conclusions: Cabozantinib is well tolerated in AML patients at an MTD of 40 mg daily and is a potent inhibitor of FLT3/ITD- and F691 TKD-altered tyrosine kinases. Cancer 2018;124:306-14. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.

The receptor tyrosine kinase fms -like tyrosine kinase 3 (FLT3), involved in regulating survival, proliferation, and differentiation of hematopoietic stem/progenitor cells, is expressed on acute myeloid leukemia (AML) cells in most patients. Mutations of FLT3 resulting in constitutive signaling are common in AML, including internal tandem duplication (ITD) in the juxtamembrane domain in 25% of patients and point mutations in the tyrosine kinase domain in 5%. Patients with AML with FLT3-ITD have a high relapse rate and short relapse-free and overall survival after chemotherapy and after transplant. A number of inhibitors of FLT3 signaling have been identified and are in clinical trials, both alone and with chemotherapy, with the goal of improving clinical outcomes in patients with AML with FLT3 mutations. While inhibitor monotherapy produces clinical responses, they are usually incomplete and transient, and resistance develops rapidly. Diverse combination therapies have been suggested to potentiate the efficacy of FLT3 inhibitors and to prevent development of resistance or overcome resistance. Combinations with epigenetic therapies, proteasome inhibitors, downstream kinase inhibitors, phosphatase activators, and other drugs that alter signaling are being explored. This review summarizes the current status of translational and clinical research on FLT3 inhibitors in AML, and discusses novel combination approaches. Mol Cancer Ther; 16(6); 991-1001. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Acute myeloid leukemia (AML) is characterized by an increase in the number of myeloid cells in the marrow and an arrest in their maturation. Various genetic mutations are associated with AML. FMS-like tyrosine kinase 3 (FLT3), a member of the class III receptor tyrosine kinase family, plays an important role in stem cell survival, and the development of dendritic and natural killer cells. FLT3/TKD mutations are generally missense mutations or in-frame alterations of residues D835 and I836 within the activation loop of the FLT3 protein. D835 mutations have been reported to occur in ≈ 7% of AML patients. Mutations have also been reported in exon 4 of isocitrate dehydrogenase 1 (IDH1) in ≈9% of AML patients. Mutations in FLT3/TKD and IDH1 genes were studied in AML patients from Pakistan and correlated with the laboratory findings. FLT3/TKD mutations were found in 7%, while IDH1 mutations were found in 10% Pakistani AML patients. Neither of these mutations was significantly correlated with age and sex, although the incidence of these mutations was higher in female patients. These mutations were found to be positively associated with each other. IDH1 mutations were positively associated with FAB type M1 and negatively associated with FAB type M2. In conclusion, the overall incidence of all these mutations in Pakistani patients was within the globally reported ranges. J. Cell. Biochem. 118: 1174-1181, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

More than one-third of patients with acute myeloid leukemia (AML) harbor aberrant mutations in Fms-like tyrosine kinase 3 (FLT3). Among them, the internal tandem duplication (ITD) mutation predicts poor prognosis. MZH29 is a novel FLT3 inhibitor synthesized in our laboratory that showed that cellular and kinase assays sustained inhibitory effects on wild-type and mutant FLT3, including the FLT3-ITD, FLT3-D835H/Y/V and FLT3-K663Q mutants. More importantly, MZH29 retained its potent inhibitory effect against the FLT3-ITD/F691L mutation, a drug resistance mutation against the well-known FLT3 inhibitor, AC220. MZH29 is a type II FLT3 inhibitor that tolerated the F691L mutation in molecular docking studies. Oral administration of 10 mg/kg MZH29 caused complete tumor regression and extended survival in a mouse model of AML with less toxicity. Subsequent proteomics study revealed less proteome perturbation in the MZH29-treated group than in the AC220-treated group. MZH29 demonstrates potential and potent novel FLT3 inhibitory effects for the treatment of AML.

An in vitro drug-screening platform on patient samples was developed and validated to design personalized treatment for relapsed/refractory acute myeloid leukemia (AML). Unbiased clustering and correlation showed that homoharringtonine (HHT), also known as omacetaxine mepesuccinate, exhibited preferential antileukemia effect against AML carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD). It worked synergistically with FLT3 inhibitors to suppress leukemia growth in vitro and in xenograft mouse models. Mechanistically, the effect was mediated by protein synthesis inhibition and reduction of short-lived proteins, including total and phosphorylated forms of FLT3 and its downstream signaling proteins. A phase 2 clinical trial of sorafenib and HHT combination treatment in FLT3-ITD AML patients resulted in complete remission (true or with insufficient hematological recovery) in 20 of 24 patients (83.3%), reduction of ITD allelic burden, and median leukemia-free and overall survivals of 12 and 33 weeks. The regimen has successfully bridged five patients to allogeneic hematopoietic stem cell transplantation and was well tolerated in patients unfit for conventional chemotherapy, including elderly and heavily pretreated patients. This study validated the principle and clinical relevance of in vitro drug testing and identified an improved treatment for FLT3-ITD AML. The results provided the foundation for phase 2/3 clinical trials to ascertain the clinical efficacy of FLT3 inhibitors and HHT in combination., (Copyright © 2016, American Association for the Advancement of Science.)

Mutations in Fetal Liver Tyrosine Kinase 3 (FLT3) genes are implicated in the constitutive activation and development of Acute Myeloid Leukaemia (AML). They are involved in signalling pathway of autonomous proliferation and block differentiation in leukaemia cells. FLT3 is considered as a promising target for the therapeutic intervention of AML. There are a few missense mutations associated with FLT3 that are found in AML patients. The D835N mutation is the most frequently observed and the aspartic acid in this position acts as a key residue for the receptor activation. The present study aims to understand the structural effect of D835N mutation in FLT3. We carried out the molecular dynamics (MD) simulation for a period of 120 ns at 300 K. Root-mean square deviation, root-mean square fluctuations, surface accessibility, radius of gyration, hydrogen bond, eigenvector projection analysis, trace of covariance matrix, and density analysis revealed the instability of mutant (D835N) protein. Our study provides new insights on the conformational changes in the mutant (D835N) structure of FLT3 protein. Our observations will be useful for researchers exploring AML and for the development of FLT3 inhibitors., (© 2015 Wiley Periodicals, Inc.)

Anticancer properties of chemically synthesized compounds have continuously been optimized for better efficacy and selectivity. Derivatives of heterocyclic compounds are well known to have selective antiproliferative effect against many types of cancer. In this study, we investigated the ability of an indigenously synthesized anticancer molecule, G-11 [1-(2",3",4",6"-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-trifluoromethylphenylhydrazono)-3-trifluoromethyl-1,4-dihydropyrazol-5-one], to cause selective cytotoxicity and induce differentiation in the acute myeloid leukemia HL-60 cells. G-11 was able to exert cytotoxic effect on hematological (Jurkat, U937, K562, HL-60, CCRF-SB) and solid tumor (MCF-7, HepG2, HeLa, Caco-2) cell lines, with IC50 values significantly lower than noncancerous cells (HEK-293, BJ and Vero) and normal peripheral blood mononuclear cells. G-11 induced differentiation of HL-60 cells to granulocytes and monocytes/macrophages by inhibiting the activation of FLT3 (CD135 tyrosine kinase). ITD-FLT3 mutation found in many acute myeloid leukemia patients could also be targeted by G-11 as exhibited by its inhibitory effect on MOLM-13 and MV4-11 cell lines. Molecular docking studies suggest the involvement of Leu616, Asp698, Cys694 and Cys828 residues in binding of G-11 to FLT3. The ability of G-11 to cause selective cytotoxicity and induce differentiation in cancer cells could be clinically relevant for therapeutic gains., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) occur in 25% of acute lymphoid and 30% of acute myeloid leukaemia cases. Therefore, FLT3 is a potential therapeutic target for small molecule kinase inhibitors. In this study, protein-ligand interactions between FLT3 and kinase inhibitors (CEP701, PKC412, sunitinib, imatinib and dasatinib) were obtained through homology modelling and molecular docking. A cellular system for experimental testing of the inhibitors was also established by expressing wildtype and internal tandem duplication mutant FLT3 (FLT3-WT and FLT3-ITD) in FDC-P1 cells. Imatinib and dasatinib could not be docked into any of the FLT3 models, consistent with their lack of activity in the experimental assays. CEP701, PKC412 and sunitinib interacted with the ATP-binding pocket of FLT3, forming H-bonds with Cys694 and Glu692. Based on the EC50 values in the cell proliferation assay, CEP701 was the most potent inhibitor; sunitinib and PKC412 were ranked second and third, respectively. Sunitinib was the most selective inhibitor, followed by PKC421 and CEP701. The potency of sunitinib and to a lesser extent CEP701 in inhibition of FLT3 autophosphorylation was lower than the cell proliferation inhibition, indicating that inhibition of FLT3 downstream proteins may contribute to the cellular effects. It was shown in this study that the docking procedure was able to differentiate FLT3 inhibitors from ineffective compounds. Additionally, interaction with the phosphate binding region in the ATP-binding pocket increased potency at the cost of selectivity. These findings can be applied in designing highly effective and selective inhibitors for FLT3 and other related kinases., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Activating mutations in FLT3 occur in ~30% of adult acute myeloid leukemia, primarily consisting of internal tandem duplication (ITD) mutations (~25%) and point mutations in the tyrosine kinase domain (~5%), commonly at the activation loop residue D835. Secondary kinase domain mutations in FLT3-ITD, particularly at the D835 residue are frequently associated with acquired clinical resistance to effective FLT3 tyrosine kinase inhibitors (TKIs). Molecular docking studies have suggested that D835 mutations primarily confer resistance by stabilizing an active Asp-Phe-Gly in ('DFG-in') kinase conformation unfavorable to the binding of type II FLT3 TKIs, which target a 'DFG-out' inactive conformation. We profiled the activity of active type II FLT3 TKIs against D835 kinase domain mutants that have been clinically detected to date. We found that type II inhibitors (quizartinib, sorafenib, ponatinib and PLX3397) retain activity against specific D835 substitutions. Modeling studies suggest that bulky hydrophobic substitutions (D835Y/V/I/F) at this residue are particularly resistant, whereas mutations that preserve interactions between D835 and S838 are relatively sensitive (D835E/N). All mutants retain sensitivity to the type I inhibitor crenolanib. These results suggest that patients with relatively sensitive D835 mutations should be included in clinical trials of type II FLT3 TKIs.

Minimal Residual Disease (MRD) detection can be used for early intervention in relapse, risk stratification, and treatment guidance. FLT3 ITD is the most common mutation found in AML patients with normal karyotype. We evaluated the feasibility of NGS with high coverage (up to 2.4.10(6) PE fragments) for MRD monitoring on FLT3 ITD. We sequenced 37 adult patients at diagnosis and various times of their disease (64 samples) and compared the results with FLT3 ITD ratios measured by fragment analysis. We found that NGS could detect variable insertion sites and lengths in a single test for several patients. We also showed mutational shifts between diagnosis and relapse, with the outgrowth of a clone at relapse different from that dominant at diagnosis. Since NGS is scalable, we were able to adapt sensitivity by increasing the number of reads obtained for follow-up samples, compared to diagnosis samples. This technique could be applied to detect biological relapse before its clinical consequences and to better tailor treatments through the use of FLT3 inhibitors. Larger cohorts should be assessed in order to validate this approach.

Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton's tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML.

The inhibition of FMS-like tyrosine kinase 3 (FLT3) activity using small-molecule inhibitors has emerged as a target-based alternative to traditional chemotherapy for the treatment of acute myeloid leukemia (AML). In this study, we report the use of structure-based virtual screening (SBVS), a computer-aided drug design technique for the identification of new chemotypes for FLT3 inhibition. For this purpose, homology modeling (HM) of the DFG-in FLT3 structure was carried using two template structures, including PDB ID: 1RJB (DFG-out FLT3 kinase domain) and PDB ID: 3LCD (DFG-in CSF-1 kinase domain). The modeled structure was able to correctly identify known DFG-in (SU11248, CEP-701, and PKC-412) and DFG-out (sorafenib, ABT-869 and AC220) FLT3 inhibitors, in docking studies. The modeled structure was then used to carry out SBVS of an HTS library of 125,000 compounds. The top scoring 97 compounds were tested for FLT3 kinase inhibition, and two hits (BPR056, IC50 = 2.3 and BPR080, IC50 = 10.7 μM) were identified. Molecular dynamics simulation and density functional theory calculation suggest that BPR056 (MW: 325.32; cLogP: 2.48) interacted with FLT3 in a stable manner and could be chemically optimized to realize a drug-like lead in the future.

Unlabelled: Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase "gatekeeper" residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position., Significance: We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop., (©2015 American Association for Cancer Research.)

The FLT3 kinase represents an attractive target to effectively treat AML. Unfortunately, no FLT3 targeted therapeutic is currently approved. In line with our continued interests in treating kinase related disease for anti-FLT3 mutant activity, we utilized pioneering synthetic methodology in combination with computer aided drug discovery and identified low molecular weight, highly ligand efficient, FLT3 kinase inhibitors. Compounds were analyzed for biochemical inhibition, their ability to selectively inhibit cell proliferation, for FLT3 mutant activity, and preliminary aqueous solubility. Validated hits were discovered that can serve as starting platforms for lead candidates., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an "Abl-like" inactive conformation with the activation loop stabilized in the "DFG-out" orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain of FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.