midostaurin and Leukemia--Myeloid

The management of acute myeloid leukemia (AML) presents significant challenges, and there remains a need for new therapies with greater efficacy and better tolerability than existing treatments. An improved understanding of the genetic and molecular changes underlying AML can help both to guide treatment strategies and to predict clinical outcomes, thereby enabling more precise decision-making regarding the optimal treatment strategy for individual patients.. The tyrosine kinase receptor FLT3 plays an important role in the survival and proliferation of blasts, and approximately 25% of patients with AML have mutations in the FLT3 gene. This protein is therefore an obvious therapeutic target in AML. Amongst recently developed tyrosine kinase inhibitors of FLT3, lestaurtinib and midostaurin are two orally bioavailable agents that have shown encouraging activity, both preclinically and in relapsed AML, and are now in phase III clinical trials. These agents are also being tested in combination with conventional chemotherapy.. Oral FLT3 inhibitors offer a hope of improved treatment outcomes for patients with relapsed and newly diagnosed AML.

Topics: Acute Disease; Animals; Carbazoles; Drug Evaluation, Preclinical; fms-Like Tyrosine Kinase 3; Furans; Humans; Leukemia, Myeloid; Molecular Structure; Mutation; Staurosporine

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.

Topics: Animals; Cell Line, Tumor; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid; Mice; Mutation; Proto-Oncogene Proteins c-cbl; Signal Transduction; Staurosporine; Syk Kinase

The addition of midostaurin to induction chemotherapy improves survival in younger patients with newly diagnosed, FLT3-mutated acute myeloid leukemia (AML). Sorafenib is a potent multikinase inhibitor with efficacy when given as monotherapy. The authors investigated whether the addition of sorafenib to intensive induction chemotherapy improves outcomes in patients with FLT3-internal tandem duplication (ITD)-mutated AML.. In total, 183 patients who were newly diagnosed with FLT3-ITD-mutated AML between February 2001 and December 2017 were identified. Of these, 79 patients (43%) underwent intensive chemotherapy with the addition of sorafenib, and 104 (57%) received intensive chemotherapy alone. Propensity score matching identified 42 patients in each cohort.. The overall response rate was 98% in the sorafenib cohort and 83% in the intensive chemotherapy cohort (P = .057). The median follow-up was 54 months. The median event-free survival was 35 months in the sorafenib cohort and 8 months in the intensive chemotherapy cohort (P = .019), and the median overall survival was 42 and 13 months, respectively (P = .026). With censoring at the time of allogeneic stem cell transplantation, the median event-free survival was 31 and 8 months in the sorafenib and intensive therapy cohorts, respectively (P = .031), and the median overall survival was not reached and 10 months, respectively (P = .001). Multivariate Cox proportional hazards models confirmed that treatment with sorafenib was a favorable prognostic factor (P = .009; hazard ratio, 0.558; 95% CI, 0.360-0.865).. The addition of sorafenib improves survival in patients with FLT3-ITD-mutated AML regardless of whether they undergo allogeneic stem cell transplantation.

Topics: Acute Disease; Adult; Antineoplastic Combined Chemotherapy Protocols; Cohort Studies; Combined Modality Therapy; Female; fms-Like Tyrosine Kinase 3; Hematopoietic Stem Cell Transplantation; Humans; Induction Chemotherapy; Kaplan-Meier Estimate; Leukemia, Myeloid; Male; Middle Aged; Mutation; Remission Induction; Sorafenib; Staurosporine; Tandem Repeat Sequences; Transplantation, Homologous; Young Adult

Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22(phox), a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22(phox) and p22(phox)-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22(phox), and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H2O2)-specific dye, peroxy orange 1 (PO1), and nuclear H2O2, as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H2O2 following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22(phox) localize to the nuclear membrane in MV4-11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22(phox) mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability.

Topics: Acute Disease; Animals; Blotting, Western; Cell Line; DNA Damage; fms-Like Tyrosine Kinase 3; Genomic Instability; HL-60 Cells; Humans; Hydrogen Peroxide; Leukemia, Myeloid; Mice; Microscopy, Confocal; Mutation; NADPH Oxidase 4; NADPH Oxidases; Protein Kinase Inhibitors; RNA Interference; Signal Transduction; Staurosporine; Tandem Repeat Sequences

Leukemias harboring MLL translocations are frequent in children and adults, and respond poorly to therapies. The receptor tyrosine kinase FLT3 is highly expressed in these leukemias. In vitro studies have shown that pediatric MLL-rearranged ALL cells are sensitive to FLT3 inhibitors and clinical trials are ongoing to measure their therapeutic efficacy. We sought to determine the contribution of Flt3 in the pathogenesis of MLL-rearranged leukemias using a myeloid leukemia mouse model. Bone marrow from Flt3 null mice transduced with MLL-ENL or MLL-CBP was transplanted into host mice and Flt3 (-/-) leukemias were compared to their Flt3 wild type counterparts. Flt3 deficiency did not delay disease onset and had minimal impact on leukemia characteristics. To determine the anti-leukemic effect of FLT3 inhibition we studied the sensitivity of MLL-ENL leukemia cells to the FLT3 inhibitor PKC412 ex vivo. As previously reported for human MLL-rearranged leukemias, murine MLL-ENL leukemia cells with higher Flt3 levels were more sensitive to the cytotoxicity of PKC412. Interestingly, Flt3 deficient leukemia samples also displayed some sensitivity to PKC412. Our findings demonstrate that myeloid leukemias induced by MLL-rearranged genes are not dependent upon Flt3 signaling. They also highlight the discrepancy between the sensitivity of cells to Flt3 inhibition in vitro and the lack of contribution of Flt3 to the pathogenesis of MLL-rearranged leukemias in vivo.

Topics: Animals; Bone Marrow; Bone Marrow Transplantation; Cells, Cultured; Disease Models, Animal; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Leukemic; Histone-Lysine N-Methyltransferase; Leukemia, Myeloid; Mice; Mice, Knockout; Myeloid Progenitor Cells; Myeloid-Lymphoid Leukemia Protein; Oncogene Proteins, Fusion; Protein Kinase Inhibitors; Signal Transduction; Staurosporine

Autotaxin (ATX) has been reported to act as a motility and growth factor in a variety of cancer cells. The ATX protein acts as a secreted lysophospholipase D by converting lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which signals via G-protein-coupled receptors and has important functions in cell migration and proliferation. This study demonstrates that ATX expression is specifically upregulated and functionally active in acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) mutation of the FLT3 receptor gene. Moreover, ATX expression was also found in normal human CD34+ progenitor cells and selected myeloid and lymphoid subpopulations. Enforced expression of mutant FLT3-ITD by retroviral vector transduction increased ATX mRNA in selected cell lines, whereas inhibition of FLT3-ITD signaling by sublethal doses of PKC412 or SU5614 led to a significant downregulation of ATX mRNA and protein levels. In the presence of LPC, ATX expression significantly increased proliferation. LPA induced proliferation, regardless of ATX expression, and induced chemotaxis in all tested human leukemic cell lines and human CD34(+) progenitors. LPC increased chemotaxis only in cells with high expression of endogenous ATX by at least 80%, demonstrating the autocrine action of ATX. Inhibition of ATX using a small molecule inhibitor selectively induced killing of ATX-expressing cell lines and reduced motility in these cells. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration during hematopoiesis and that deregulation of ATX contributes to the pathogenesis of AML.

Topics: Acute Disease; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; fms-Like Tyrosine Kinase 3; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Hematopoietic Stem Cells; Humans; Indoles; K562 Cells; Leukemia, Myeloid; Lysophosphatidylcholines; Lysophospholipids; Mutation; Oligonucleotide Array Sequence Analysis; Phosphoric Diester Hydrolases; Protein Kinase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Staurosporine; Tandem Repeat Sequences

Deregulated accumulation of nuclear beta-catenin enhances transcription of beta-catenin target genes and promotes malignant transformation. Recently, acute myeloid leukemia (AML) cells with activating mutations of FMS-like tyrosine kinase-3 (FLT3) were reported to display elevated beta-catenin-dependent nuclear signaling. Tyrosine phosphorylation of beta-catenin has been shown to promote its nuclear localization. Here, we examined the causal relationship between FLT3 activity and beta-catenin nuclear localization. Compared to cells with wild-type FLT3 (FLT3-WT), cells with the FLT3 internal tandem duplication (FLT3-ITD) and tyrosine kinase domain mutation (FLT3-TKD) had elevated levels of tyrosine-phosphorylated beta-catenin. Although beta-catenin was localized mainly in the cytoplasm in FLT3-WT cells, it was primarily nuclear in FLT3-ITD cells. Treatment with FLT3 kinase inhibitors or FLT3 silencing with RNAi decreased beta-catenin tyrosine phosphorylation and nuclear localization. Conversely, treatment of FLT3-WT cells with FLT3 ligand increased tyrosine phosphorylation and nuclear accumulation of beta-catenin. Endogenous beta-catenin co-immunoprecipitated with endogenous activated FLT3, and recombinant activated FLT3 directly phosphorylated recombinant beta-catenin. Finally, FLT3 inhibitor decreased tyrosine phosphorylation of beta-catenin in leukemia cells obtained from FLT3-ITD-positive AML patients. These data demonstrate that FLT3 activation induces beta-catenin tyrosine phosphorylation and nuclear localization, and thus suggest a mechanism for the association of FLT3 activation and beta-catenin oncogeneic signaling in AML.

Topics: Active Transport, Cell Nucleus; Acute Disease; Animals; beta Catenin; Cell Line, Tumor; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Leukemic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Interleukin-3; Leukemia, Myeloid; Membrane Proteins; Mice; Neoplasm Proteins; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Recombinant Proteins; RNA, Small Interfering; Staurosporine; Transcription, Genetic; Tyrphostins

Activating mutations in the FLT3 tyrosine kinase (TK) occur in approximately 35% of patients with acute myeloid leukemia (AML). Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3 TK inhibitors (TKI) showed measurable clinical responses. Most of these responses were transient; however, in a subset of patients blast recurrence was preceded by an interval of prolonged remission. The etiology of clinical resistance to FLT3-TKI in AML is unclear but is of major significance for the development of future therapeutic strategies. We searched for mechanisms of resistance in 6 patients with AML who had relapses upon PKC412 treatment. In an index AML patient, an algorithm of analyses was applied using clinical material. In vivo and in vitro investigation of primary blasts at relapse revealed persistent TK phosphorylation of FLT3 despite sufficient PKC412 serum levels. Through additional molecular analyses, we identified a single amino acid substitution at position 676 (N676K) within the FLT3 kinase domain as the sole cause of resistance to PKC412 in this patient. Reconstitution experiments expressing the N676K mutant in 32D cells demonstrated that FLT3-ITD-N676K was sufficient to confer an intermediate level of resistance to PKC412 in vitro. These studies point out that a genetically complex malignancy such as AML may retain dependence on a single oncogenic signal.

Topics: Acute Disease; Drug Resistance, Neoplasm; Enzyme Activation; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid; Mutation, Missense; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Recurrence; Staurosporine

The receptor tyrosine kinase FLT3 is a promising molecular therapeutic target in acute myeloid leukemia (AML). Activating mutations of FLT3 are present in approximately one-third of patients, while many nonmutants show evidence of FLT3 activation, which appears to play a significant role in leukemogenesis. We studied the effects of lestaurtinib (CEP701) and PKC412, 2 small molecule inhibitors of FLT3, on 65 diagnostic AML blast samples. Both agents induced concentration-dependent cytotoxicity in most cases, although responses to PKC412 required higher drug concentrations. Cytotoxic responses were highly heterogeneous and were only weakly associated with FLT3 mutation status and FLT3 expression. Importantly, lestaurtinib induced cytotoxicity in a synergistic fashion with cytarabine, particularly in FLT3 mutant samples. Both lestaurtinib and PKC412 caused inhibition of FLT3 phosphorylation in all samples. Translation of FLT3 inhibition into cytotoxicity was influenced by the degree of residual FLT3 phosphorylation remaining and correlated with deactivation of STAT5 and MAP kinase. FLT3 mutant and wild-type cases both varied considerably in their dependence on FLT3 signaling for survival. These findings support the continued clinical assessment of FLT3 inhibitors in combination with cytotoxic chemotherapy: Entry to future clinical trials should include FLT3 wild-type patients and should remain unrestricted by FLT3 expression level.

Topics: Acute Disease; Adolescent; Adult; Aged; Apoptosis; Blast Crisis; Carbazoles; Drug Screening Assays, Antitumor; Female; fms-Like Tyrosine Kinase 3; Furans; Humans; Indoles; Leukemia, Myeloid; Male; Middle Aged; Mutation; Protein Kinase C; Protein-Tyrosine Kinases; Signal Transduction; Staurosporine; Tumor Cells, Cultured

Activating mutations of Flt3 are found in approximately one third of patients with acute myeloid leukemia (AML) and are an attractive drug target. Two classes of Flt3 mutations occur: internal tandem duplications (ITDs) in the juxtamembrane and point mutations in the tyrosine kinase domain (TKD). We and others have shown that Flt3-ITD induced aberrant signaling including strong activation of signal transducer and activator of transcription 5 (STAT5) and repression of CCAAT/estradiol-binding protein alpha (c/EBPalpha) and Pu.1. Here, we compared the signaling properties of Flt3-ITD versus Flt3-TKD in myeloid progenitor cells. We demonstrate that Flt3-TKD mutations induced autonomous growth of 32D cells in suspension cultures. However, in contrast to Flt3-ITD and similar to wild-type Flt3 (Flt3-WT), Flt3-TKD cannot support colony formation in semisolid media. Also, in contrast to Flt3-ITD, neither Flt3-WT nor Flt3-TKD induced activation or induction of STAT5 target genes. Flt3-TKD also failed to repress c/EBPalpha and Pu.1. No significant differences were observed in receptor autophosphorylation and the phosphorylation of Erk-1 and -2, Akt, and Shc. Importantly, TKD but not ITD mutations were a log power more sensitive toward the tyrosine kinase inhibitor protein kinase C 412 (PKC412) than Flt3-WT. In conclusion, Flt3-ITD and Flt3-TKD mutations display differences in their signaling properties that could have important implications for their transforming capacity and for the design of mutation-specific therapeutic approaches.

Topics: Acute Disease; Animals; Apoptosis; Cell Line; DNA-Binding Proteins; fms-Like Tyrosine Kinase 3; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid; Milk Proteins; Muridae; Mutagenesis, Site-Directed; Myeloid Cells; Phosphorylation; Point Mutation; Protein Structure, Tertiary; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Signal Transduction; STAT5 Transcription Factor; Staurosporine; Tandem Repeat Sequences; Trans-Activators; Transcription Factors

Activating mutations in the juxtamembrane domain (FLT3-length mutations, FLT3-LM) and in the protein tyrosine kinase domain (TKD) of FLT3 (FLT3-TKD) represent the most frequent genetic alterations in acute myeloid leukemia (AML) and define a molecular target for therapeutic interventions by protein tyrosine kinase (PTK) inhibitors. We could show that distinct activating FLT3-TKD mutations at position D835 mediate primary resistance to FLT3 PTK inhibitors in FLT3-transformed cell lines. In the presence of increasing concentrations of the FLT3 PTK inhibitor SU5614, we generated inhibitor resistant Ba/F3 FLT3-internal tandem duplication (ITD) cell lines (Ba/F3 FLT3-ITD-R1-R4) that were characterized by a 7- to 26-fold higher IC50 (concentration that inhibits 50%) to SU5614 compared with the parental ITD cells. The molecular characterization of ITD-R1-4 cells demonstrated that specific TKD mutations (D835N and Y842H) on the ITD background were acquired during selection with SU5614. Introduction of these dual ITD-TKD, but not single D835N or Y842H FLT3 mutants, in Ba/F3 cells restored the FLT3 inhibitor resistant phenotype. Our data show that preexisting or acquired mutations in the PTK domain of FLT3 can induce drug resistance to FLT3 PTK inhibitors in vitro. These findings provide a molecular basis for the evaluation of clinical resistance to FLT3 PTK inhibitors in patients with AML.

Topics: Acute Disease; Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; Cell Division; Cell Line, Transformed; Cytarabine; DNA-Binding Proteins; Drug Resistance, Neoplasm; Enzyme Inhibitors; fms-Like Tyrosine Kinase 3; Genistein; Humans; Indoles; Leukemia, Myeloid; MAP Kinase Signaling System; Milk Proteins; Mutagenesis; Phenotype; Phosphorylation; Protein Structure, Tertiary; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; STAT5 Transcription Factor; Staurosporine; Trans-Activators; Tyrphostins

Mutations in the receptor tyrosine kinase FLT3 occur frequently in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Small molecules that selectively inhibit FLT3 kinase activity induce apoptosis in blasts from AML patients with FLT3 mutations and prolong survival in animal models of FLT3-induced myeloproliferative disease. A spectrum of structurally different small molecules with activity against FLT3 have been described, and their efficacy for treatment of AML and ALL is now being investigated in clinical trials. Here, we describe the results of an in vitro screen designed to identify mutations in the ATP-binding pocket of FLT3 that confer resistance to tyrosine kinase inhibitors. Mutations at four different positions (Ala-627, Asn-676, Phe-691, and Gly-697) were identified that confer varying degrees of resistance to PKC412, SU5614, or K-252a. FLT3 proteins mutated at Ala-627, Asn-676, or Phe-691 remained sensitive to higher concentrations of the inhibitors, but the G697R mutation conferred high-level resistance to each of these inhibitors as well as to six additional experimental inhibitors. These data provide insights into potential mechanisms of acquired resistance of FLT3 to small molecule inhibitors and indicate that the G697R mutation may be a clinically problematic resistance mutation that warrants proactive screening for additional inhibitors.

Topics: Acute Disease; Amino Acid Sequence; Animals; Drug Resistance, Neoplasm; Enzyme Inhibitors; fms-Like Tyrosine Kinase 3; Leukemia, Myeloid; Mice; Models, Molecular; Molecular Sequence Data; Point Mutation; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Structure, Tertiary; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Staurosporine; Structure-Activity Relationship

Activating mutations of FLT3 have been detected in patients with acute myeloid leukemia (AML). Two distinct types of FLT3 mutations are most common: internal tandem duplication (ITD) of sequences coding for the juxtamembrane domain and point mutations at codon 835 (Asp835) within the kinase domain. Both types of mutations constitutively activate the tyrosine kinase activity of FLT3 in experimental systems and result in factor-independent proliferation of Ba/F3 and 32D cells. Recently, novel mutations within the activation loop were identified in patients with AML: deletion of isoleucine 836 (Ile836del) and an exchange of isoleucine 836 to methionine plus an arginine insertion (Ile836Met+Arg). To examine whether the Ile836 mutations result in constitutive activation of the FLT3 receptor, we introduced both mutant FLT3 cDNAs transiently into HEK 293 cells. Both mutant FLT3 receptors were constitutively autophosphorylated in the absence of ligand and kinase activity led to constitutive activation of downstream signaling cascades as determined by activation of the STAT5 (signal transducer and activator of transcription 5) pathway. When stably expressed in the growth factor-dependent cell lines Ba/F3 and 32D, both deletion and insertion mutants led to factor-independent proliferation, indicating that both mutants have transforming capabilities. We then examined the sensitivity of the FLT3 ITD, FLT3 Asp835Tyr, and the novel FLT3 receptor mutants toward the kinase inhibitors AG1296, PKC412, and SU5614. We show that these FLT3 kinase inhibitors have distinct inhibitory potencies against different activating FLT3 receptor mutants. These results suggest that it may be useful to determine the exact kind of FLT3 mutation when applying receptor kinase inhibitors in clinical trials.

Topics: Acute Disease; Amino Acid Substitution; Animals; Cell Division; Cell Line; Codon; DNA-Binding Proteins; Drug Resistance; Enzyme Activation; Enzyme Inhibitors; fms-Like Tyrosine Kinase 3; Hematopoietic Stem Cells; Humans; Indoles; Leukemia, Myeloid; Membrane Proteins; Mice; Milk Proteins; Mutagenesis, Insertional; Mutation, Missense; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Recombinant Proteins; Sequence Deletion; STAT5 Transcription Factor; Staurosporine; Structure-Activity Relationship; Trans-Activators; Transfection; Tyrphostins