The structural basis for autoinhibition of FLT3 by the juxtamembrane domain

FLT3 is a type III receptor tyrosine kinase that is thought to play a key role in hematopoiesis. Certain classes of FLT3 mutations cause constitutively activated forms of the receptor that are found in significant numbers of patients with acute myelogenous leukemia (AML). The mutations occur either in the activation loop, for example, as point mutations of Asp835 or as internal tandem duplication (ITD) sequences in the juxtamembrane (JM) domain. To further understand the nature of FLT3 autoinhibition and regulation, we have determined the crystal structure of the autoinhibited form of FLT3. This structure shows the autoinhibitory conformation of a complete JM domain in this class of receptor tyrosine kinases. The detailed inhibitory mechanism of the JM domain is revealed, which is likely utilized by other members of type III receptor tyrosine kinases.     

The molecular mechanism behind resistance of the kinase FLT3 to the inhibitor quizartinib

Fms‐like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is a drug target for leukemias. Several potent inhibitors of FLT3 exist, and bind to the inactive form of the enzyme. Unfortunately, resistance due to mutations in the kinase domain of FLT3 limits the therapeutic effects of these inhibitors. As in many other cases, it is not straightforward to explain why certain mutations lead to drug resistance. Extensive fully atomistic molecular dynamics (MD) simulations of FLT3 were carried out with an inhibited form (FLT‐quizartinib complex), a free (apo) form, and an active conformation. In all cases, both the wild type (wt) proteins and two resistant mutants (D835F and Y842H) were studied. Analysis of the simulations revealed that impairment of protein‐drug interactions cannot explain the resistance mutations in question. Rather, it appears that the active state of the mutant forms is perturbed by the mutations. It is therefore likely that perturbation of deactivation of the protein (which is necessary for drug binding) is responsible for the reduced affinity of the drug to the mutants. Importantly, this study suggests that it is possible to explain the source of resistance by mutations in FLT3 by an analysis of unbiased MD simulations.     

Novel Oncogenic Mutations of CBL in Human Acute Myeloid Leukemia That Activate Growth and Survival Pathways Depend on Increased Metabolism

Acute myeloid leukemia (AML) is characterized by multiple mutagenic events that affect proliferation, survival, as well as differentiation. Recently, gain-of-function mutations in the α helical structure within the linker sequence of the E3 ubiquitin ligase CBL have been associated with AML. We identified four novel CBL mutations, including a point mutation (Y371H) and a putative splice site mutation in AML specimens. Characterization of these two CBL mutants revealed that coexpression with the receptor tyrosine kinases FLT3 (Fms-like tyrosine kinase 3) or KIT-induced ligand independent growth or ligand hyperresponsiveness, respectively. Growth of cells expressing mutant CBL required expression and kinase activity of FLT3. In addition to the CBL-dependent phosphorylation of FLT3 and CBL itself, transformation was associated with activation of Akt and STAT5 and required functional expression of the small GTPases Rho, Rac, and Cdc42. Furthermore, the mutations led to constitutively elevated intracellular reactive oxygen species levels, which is commonly linked to increased glucose metabolism in cancer cells. Inhibition of hexokinase with 2-deoxyglucose blocked the transforming activity of CBL mutants and reduced activation of signaling mechanisms. Overall, our data demonstrate that mutations of CBL alter cellular biology at multiple levels and require not only the activation of receptor proximal signaling events but also an increase in cellular glucose metabolism. Pathways that are activated by CBL gain-of-function mutations can be efficiently targeted by small molecule drugs.     

Over-expression of FoxM1 is associated with adverse prognosis and FLT3-ITD in acute myeloid leukemia

Forkhead box M1 (FoxM1) drives cell cycle progression and the prevention of growth arrest and is over-expressed in many human malignancies. However, the characteristics of FoxM1 in acute myeloid leukemia (AML) are not clearly understood. We investigated the expression level of FoxM1 and analyzed the correlation of FoxM1 expression with AML patient characteristics and prognoses. Changes in FoxM1 expression were detected after MV4–11 cells, which have an internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 gene (FLT3-ITD), and control THP1 cells (encoding wild-type FLT3) were treated with the FLT3 receptor tyrosine kinase inhibitor AC220 (quizartinib) or FLT3 ligand (FL). Finally, we determined the apoptosis rates after the addition of the FoxM1 inhibitor thiostrepton (TST) to AML cells with or without FLT3-ITD. The expression of FoxM1 in AML patients was correlated with the presence of FLT3-ITD, genetic groups, and possibly overall survival. Inhibition of FLT3-ITD by AC220 down-regulated FoxM1 expression in MV4–11 cells, and stimulation of FLT3 by FL up-regulated FoxM1 expression in MV4–11 and THP1 cells. TST induced the apoptosis of MV4–11 and THP1 cells in a dose-dependent manner. Thus, FoxM1 is a potential prognostic marker and a promising therapeutic target in AML.     

Profound ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia type II.

Thanatophoric dysplasia type II (TDII) is a neonatal lethal skeletal dysplasia caused by a recurrent Lys-650-->Glu mutation within the highly conserved activation loop of the kinase domain of fibroblast growth factor receptor 3 (FGFR3). We demonstrate here that this mutation results in profound constitutive activation of the FGFR3 tyrosine kinase, approximately 100-fold above that of wild-type FGFR3. The mechanism of FGFR3 activation in TDII was probed by constructing various point mutations in the activation loop. Substitutions at position 650 indicated that not only Glu but also Asp and, to a lesser extent, Gln and Leu result in pronounced constitutive activation of FGFR3. Additional mutagenesis within the beta10-beta11 loop region (amino acids Tyr-647 to Leu-656) demonstrated that amino acid 650 is the only residue which can activate the receptor when changed to a Glu, indicating a specificity of position as well as charge for mutations which can give rise to kinase activation. Furthermore, when predicted sites of autophosphorylation at Tyr-647 and Tyr-648 were mutated to Phe, either singly or in combination, constitutive kinase activity was still observed in response to the Lys-650-->Glu mutation, although the effect of these mutations on downstream signalling was not investigated. Our data suggest that the molecular effect of the TDII activation loop mutation is to mimic the conformational changes that activate the tyrosine kinase domain, which are normally initiated by ligand binding and autophosphorylation. These results have broad implications for understanding the molecular basis of other human developmental syndromes that involve mutations in members of the FGFR family. Moreover, these findings are relevant to the study of kinase regulation and the design of activating mutations in related tyrosine kinases.     

SU11652 Inhibits tyrosine kinase activity of FLT3 and growth of MV-4-11 cells

FLT3-ITD and FLT3-TKD mutations are frequently found in acute myeloid leukemia (AML). This makes tyrosine kinase FLT3 a highly attractive target for therapeutic drug development. However, effective drugs have not yet emerged. This study is intended to identify and to characterize new FLT3 inhibitors. By using the protein substrate GST-FLT3S to analyze kinase activity of recombinant proteins carrying the catalytic domain of wild type and mutant forms of FLT3, we screened a chemical library containing 80 known protein kinase inhibitors. We identified SU11652 as a potent FLT3 inhibitor and further employed FLT3-ITD-positive MV- 4–11 cells to study its effects on cell growth, apoptosis, cell cycles, and cell signaling. SU11652 strongly inhibited the activity of wild type, D835Y, and D835H mutant forms of FLT3 with IC50 values of 1.5, 16, and 32 nM, respectively. It effectively blocked the growth of FLT3-ITD -positive MV-4-11 cells at nanomolar concentrations but exhibited much less effects on several other cells which do not carry mutations of FLT3. SU11652 inhibited growth of MV-4-11 cells by inducing apoptosis, causing cell cycle arrest, and blocking activation of the ERK, Akt, and STAT signaling pathways. SU11652 is a potent FLT3 inhibitor which selectively targets FLT3-ITD-positive cells. It should serve as a good candidate for development of therapeutic drugs to treat AML.     

Crystal structure of bisphosphorylated IGF-1 receptor kinase: insight into domain movements upon kinase activation

BACKGROUND: The insulin-like growth-factor-1 (IGF-1) receptor, which is widely expressed in cells that have undergone oncogenic transformation, is emerging as a novel target in cancer therapy. IGF-1-induced receptor activation results in autophosphorylation of cytoplasmic kinase domains and enhances their capability to phosphorylate downstream substrates. Structures of the homologous insulin receptor kinase (IRK) exist in an open, unphosphorylated form and a closed, trisphosphorylated form. RESULTS: We have determined the 2.1 A crystal structure of the IGF-1 receptor protein tyrosine kinase domain phosphorylated at two tyrosine residues within the activation loop (IGF-1RK2P) and bound to an ATP analog. The ligand is not in a conformation compatible with phosphoryl transfer, and the activation loop is partially disordered. Compared to the homologous insulin receptor kinase, IGF-1RK2P is trapped in a half-closed, previously unobserved conformation. Observed domain movements can be dissected into two orthogonal rotational components. CONCLUSIONS: Conformational changes upon kinase activation are triggered by the degree of phosphorylation and are crucially dependent on the conformation of the proximal end of the kinase activation loop. This IGF-1RK structure will provide a molecular basis for the design of selective antioncogenic therapeutic agents.     

Crystal Structure of the Src Family Kinase Hck SH3-SH2 Linker Regulatory Region Supports an SH3-dominant Activation Mechanism

Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the polyproline type II helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near full-length, down-regulated Hck. However, the SH2 kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these noncatalytic regions work together as a “conformational switch” that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3 domain binding proteins and implicate the existence of innate conformational states unique to individual Src family members that “fine-tune” their sensitivities to activation by SH3-based ligands.     

Functional characterization of FLT3 receptor signaling deregulation in acute myeloid leukemia by single cell network profiling (SCNP)

Background

Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative.

Methodology/Principal Findings

Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management.

Conclusions/Significance

These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations.     

FLT3 inhibitors in acute myeloid leukemia

FLT3 mutations are one of the most common findings in acute myeloid leukemia (AML). FLT3 inhibitors have been in active clinical development. Midostaurin as the first-in-class FLT3 inhibitor has been approved for treatment of patients with FLT3-mutated AML. In this review, we summarized the preclinical and clinical studies on new FLT3 inhibitors, including sorafenib, lestaurtinib, sunitinib, tandutinib, quizartinib, midostaurin, gilteritinib, crenolanib, cabozantinib, Sel24-B489, G-749, AMG 925, TTT-3002, and FF-10101. New generation FLT3 inhibitors and combination therapies may overcome resistance to first-generation agents.     

Structure of the Parainfluenza Virus 5 (PIV5) Hemagglutinin-Neuraminidase (HN) Ectodomain

Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.     

MiR-424 and miR-155 deregulated expression in cytogenetically normal acute myeloid leukaemia: correlation with NPM1 and FLT3 mutation status

Background

Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in acute myeloid leukemia (AML). This has made FLT3 an important marker for disease diagnosis and a highly attractive target for therapeutic drug development. This study is intended to generate a sensitive substrate for assays of the FLT3 enzymatic activity.

Methods

We expressed in Escherichia coli cells a glutathione S-transferase (GST) fusion protein designated GST-FLT3S, which contains a peptide sequence derived from an autophosphorylation site of FLT3. The protein was used to analyze tyrosine kinase activity of baculovirus-expressed FLT3 and crude cell extracts of bone marrow cells from AML patients. It was also employed to perform FLT3 kinase assays for FLT3 inhibitor screening.

Results

GST-FLT3S in solution or on beads was strongly phosphorylated by recombinant proteins carrying the catalytic domain of wild type FLT3 and FLT3D835 mutants, with the latter exhibiting much higher activity and efficiency. GST-FLT3S was also able to detect elevated tyrosine kinase activity in bone marrow cell extracts from AML patients. A small-scale inhibitor screening led to identification of several potent inhibitors of wild type and mutant forms of FLT3.

Conclusions

GST-FLT3S is a sensitive protein substrate for FLT3 assays. It may find applications in diagnosis of diseases related to abnormal FLT3 activity and in inhibitor screening for drug development.     

Structure of the Tie2 RTK domain: self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail

BACKGROUND: Angiogenesis, the formation of new vessels from the existing vasculature, is a critical process during early development as well as in a number of disease processes. Tie2 (also known as Tek) is an endothelium-specific receptor tyrosine kinase involved in both angiogenesis and vasculature maintenance. RESULTS: We have determined the crystal structure of the Tie2 kinase domain to 2.2 A resolution. The structure contains the catalytic core, the kinase insert domain (KID), and the C-terminal tail. The overall fold is similar to that observed in other serine/threonine and tyrosine kinase structures; however, several unique features distinguish the Tie2 structure from those of other kinases. The Tie2 nucleotide binding loop is in an inhibitory conformation, which is not seen in other kinase structures, while its activation loop adopts an "activated-like" conformation in the absence of phosphorylation. Tyr-897, located in the N-terminal domain, may negatively regulate the activity of Tie2 by preventing dimerization of the kinase domains or by recruiting phosphatases when it is phosphorylated. CONCLUSION: Regulation of the kinase activity of Tie2 is a complex process. Conformational changes in the nucleotide binding loop, activation loop, C helix, and the C-terminal tail are required for ATP and substrate binding.     

Comprehensive structure-activity-relationship of azaindoles as highly potent FLT3 inhibitors

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.     

ATP and MO25α Regulate the Conformational State of the STRADα Pseudokinase and Activation of the LKB1 Tumour Suppressor

Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADα in complex with MO25α. The structure reveals an intricate web of interactions between STRADα and MO25α involving the αC-helix of STRADα, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADα binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADα for MO25α, and conversely, binding of MO25α promotes interaction of STRADα with ATP. Mutagenesis studies reveal that association of STRADα with either ATP or MO25α is essential for LKB1 activation. We conclude that ATP and MO25α cooperate to maintain STRADα in an “active” closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADα that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE) syndrome. We demonstrate this mutation destabilizes STRADα and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADα to activate LKB1 is dependent on a closed “active” conformation, aided by ATP and MO25α binding. Thus, the function of STRADα is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations.     

Atomistic Insights into Regulatory Mechanisms of the HER2 Tyrosine Kinase Domain: A Molecular Dynamics Study

HER2 (ErbB2/Neu) is a receptor tyrosine kinase belonging to the epidermal growth factor receptor (EGFR)/ErbB family and is overexpressed in 20-30% of human breast cancers. Although several crystal structures of ErbB kinases have been solved, the precise mechanism of HER2 activation remains unknown, and it has been suggested that HER2 is unique in its requirement for phosphorylation of Y877, a key tyrosine residue located in the activation loop. To elucidate mechanistic details of kinase domain regulation, we performed molecular dynamics simulations of a homology-modeled HER2 kinase structure in active and inactive conformations. Principal component analysis of the atomistic fluctuations reveals a tight coupling between the activation loop and catalytic loop that may contribute to alignment of residues required for catalysis in the active kinase. The free energy perturbation method is also employed to predict a role for phosphorylated Y877 in stabilizing the kinase conformations. Finally, simulation results are presented for a HER2/EGFR heterodimer and reveal that the dimeric interface induces a rearrangement of the αC helix toward the active conformation. Elucidation of the molecular regulatory mechanisms in HER2 will help establish structure-function relationships in the wild-type kinase, as well as predict mutations with a propensity for constitutive activation in HER2-mediated cancers.     

Functional Pathway Analysis Using SCNP of FLT3 Receptor Pathway Deregulation in AML Provides Prognostic Information Independent from Mutational Status

FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.     

Insights into the conformational flexibility of Bruton's tyrosine kinase from multiple ligand complex structures

Bruton's tyrosine kinase (BTK) plays a key role in B cell receptor signaling and is considered a promising drug target for lymphoma and inflammatory diseases. We have determined the X-ray crystal structures of BTK kinase domain in complex with six inhibitors from distinct chemical classes. Five different BTK protein conformations are stabilized by the bound inhibitors, providing insights into the structural flexibility of the Gly-rich loop, helix C, the DFG sequence, and activation loop. The conformational changes occur independent of activation loop phosphorylation and do not correlate with the structurally unchanged WEI motif in the Src homology 2-kinase domain linker. Two novel activation loop conformations and an atypical DFG conformation are observed representing unique inactive states of BTK. Two regions within the activation loop are shown to structurally transform between 3(10)- and α-helices, one of which collapses into the adenosine-5'-triphosphate binding pocket. The first crystal structure of a Tec kinase family member in the pharmacologically important DFG-out conformation and bound to a type II kinase inhibitor is described. The different protein conformations observed provide insights into the structural flexibility of BTK, the molecular basis of its regulation, and the structure-based design of specific inhibitors.     

The combination of FLT3 and SYK kinase inhibitors is toxic to leukaemia cells with CBL mutations

Mutations in the E3 ubiquitin ligase CBL, found in several myeloid neoplasms, lead to decreased ubiquitin ligase activity. In murine systems, these mutations are associated with cytokine‐independent proliferation, thought to result from the activation of hematopoietic growth receptors, including FLT3 and KIT. Using cell lines and primary patient cells, we compared the activity of a panel of FLT3 inhibitors currently being used or tested in AML patients and also evaluated the effects of inhibition of the non‐receptor tyrosine kinase, SYK. We show that FLT3 inhibitors ranging from promiscuous to highly targeted are potent inhibitors of growth of leukaemia cells expressing mutant CBL in vitro, and we demonstrate in vivo efficacy of midostaurin using mouse models of mutant CBL. Potentiation of effects of targeted FLT3 inhibition by SYK inhibition has been demonstrated in models of mutant FLT3‐positive AML and AML characterized by hyperactivated SYK. Here, we show that targeted SYK inhibition similarly enhances the effects of midostaurin and other FLT3 inhibitors against mutant CBL‐positive leukaemia. Taken together, our results support the notion that mutant CBL‐expressing myeloid leukaemias are highly sensitive to available FLT3 inhibitors and that this effect can be significantly augmented by optimum inhibition of SYK kinase.