FMS-like Tyrosine Kinase 3 Kinase Research Articles

Phase 1 Study of PKC412, an Oral FLT3 Kinase Inhibitor, in Sequential and Concomitant Combinations with Daunorubicin and Cytarabine (DA) Induction and High-Dose Cytarabine (HDAra-C) Consolidation in Newly Diagnosed Patients with AML.

The Fms-like tyrosine kinase 3 (FLT3), a receptor expressed in 80% of AML patients, is constitutively activated in 25–35% of adult AML patients due to mutations, in the form of either ITDs or activating loop point mutations, most commonly at D835Y. PKC412 is an oral multi-targeted tyrosine kinase inhibitor known to potently inhibit both activated wild type (WT) and mutated (mut) FLT3 kinase. PKC412 (100 mg po bid) was tested in a phase IB study in newly diagnosed AML patients between 18 and 60 years of age (both WT and mut FLT3) in combinations with DA for induction therapy (up to 2 cycles) and HDAra-C for consolidation (up to 3 cycles).Study design: PKC412 was given daily beginning on Day 8 (Arm 1) or Day 1 (Arm 2) of induction chemoRX in cycle 1. Primary endpoints were PK, tolerability, and safety as defined by fewer than 2 drug-related deaths in the initial cohort of 6 pts per arm (stage 1) and fewer than 4 drug-related deaths among the total 20 pts per arm (stage 2) during induction.Results: 15 pts were enrolled (median age 50; 1 FLT3 ITD+, 14 WT; ): 7 in Arm 1 and 8 in Arm 2. Cytogenetic prognostic categories of the 15 patients were: 0 (good), 9 (intermediate) and 6 (bad). Of the 15 pts, 14 were evaluable for toxicity; 1 pt did not receive PKC412; no drug-related deaths were observed during induction therapy. The most common G3 drug-related toxicities included: nausea (46%), vomiting (32%), transaminitis (18%), and hyperbilirubinemia (18%), all were transient and/or reversible. 13/15 patients were evaluable for response: 2 pts were not evaluated: one pt never received PKC412 and one pt did not complete the full-course of induction chemoRx; 3/7 (43%) achieved a CR in Arm 1 and 3/6 (50%) achieved a CR in Arm 2. 3 pts have completed the study (Days 143, 141, 132) and one pt is ongoing (Day 127). Because of the high rate of G3 nausea and vomiting, the study was amended, (5 patients are ongoing) such that PKC412 is given with each cycle of chemoRx on either Days 1–7 and 15–21 (Arm 1), or on Days 8–21 (Arm 2).Conclusions: PKC412 can be given safely in newly diagnosed AML patients in combinations with DA and HDAra-C chemoRx. An update of this study, along with PK data of PKC412, daunorubicin, and Ara-C levels will be presented.

CHIR258: A Multitargeted Growth Factor Receptor Tyrosine Kinase (RTK) Inhibitor Induces Significant Apoptosis of Human FLT3 ITD Acute Myelogenous Leukemia Cells In Vitro and in Vivo.

Fms-like tyrosine kinase 3 (FLT3) encodes a receptor tyrosine kinase (RTK) for which activating mutations have been identified in a proportion of acute myelogenous leukemia (AML) patients and associated with poor clinical prognosis. CHIR258, a novel, orally active, small molecule exhibits potent activity against FLT3 kinase and other RTKs involved in myeloid leukemias as well as endothelial and solid tumor cell proliferation (biochemical IC50 for FLT3 = 1 nM; cKIT = 2 nM; VEGFR1/2/3 ~ 8 nM, FGFR1/3 ~ 10nM, PDGFRβ = 27 nM, CSF-R1 = 36 nM). CHIR258 was tested in two human leukemic cell lines with differing FLT3 mutational status (MV4;11, FLT3 ITD vs. RS4;11, FLT3 WT). Cell cycle analysis of leukemic cells incubated with CHIR258 (0.1 μM, 48 h) showed a significant increase in the sub-Go/G1 cell population in MV4;11cells compared to the RS4;11 cells. The pro-apoptotic activity of CHIR258 against MV4;11 cells in culture was confirmed by AnnexinV staining, and appearance of cleaved poly ADP-ribose polymerase (PARP) after 24 h. Interestingly, cleaved PARP fragments were detected with either pulsed (1 h) or continuous incubations of MV4;11 cells with CHIR258. In addition, cell cycle arrest and apoptosis was consistent with degradation of survivin and loss of phosphorylation of Retinoblastoma (Rb) protein observed at CHIR258 concentrations ≥ 0.1 μM. The mechanism of action of CHIR258 was examined in a subcutaneous (s.c.) xenograft model of leukemia in SCID-NOD mice. In s.c. xenografts, daily oral doses of CHIR258 resulted in dose-dependent increased efficacy against FLT3-ITD MV4;11 tumors compared to RS4;11 WT tumors. Modulation of FLT3 downstream signaling molecules (p-FLT3, p-ERK) was achieved in MV4;11 tumors at biologically active doses in vivo, confirming molecular mechanism of action. Target modulation (p-FLT3) was maintained for up to 24 h post-dose. MV4;11 tumor responses to CHIR258 were evidenced within 1–2 days of initiating drug treatment. Immunohistochemical analyses of MV4;11 tumors revealed that early responses to CHIR258 correlated well with decreased tumor cellularity, and proliferation. Tumors stained positive for active caspase-3 and cleaved PARP suggesting cell death was mainly mediated via apoptosis. Efficacy was also demonstrated in an intravenous MV4;11 bone marrow (BM) engraftment model, wherein daily CHIR258 treatments substantially increased the life span of mice, with 33% long term survivors or “cures”. Strikingly, CHIR258-treated mice were devoid of MV4;11 tumor cells in BM as confirmed by flow cytometry and immunohistochemical analyses. In conclusion, our data indicates that CHIR258 might become a highly effective therapy in AML associated with FLT3 expression and warrants clinical trials with AML patients.

Effects of MLN518, a dual FLT3 and KIT inhibitor, on normal and malignant hematopoiesis

AbstractInternal tandem duplications (ITDs) of the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase are found in approximately 30% of patients with acute myelogenous leukemia (AML) and are associated with a poor prognosis. FLT3 ITD mutations result in constitutive kinase activation and are thought to be pathogenetically relevant, implicating FLT3 as a plausible therapeutic target. MLN518 (formerly CT53518) is a small molecule inhibitor of the FLT3, KIT, and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases with significant activity in murine models of FLT3 ITD-positive leukemia. Given the importance of FLT3 and KIT for normal hematopoietic progenitor cells, we analyzed the effect of MLN518 on murine hematopoiesis under steady-state conditions, after chemotherapy-induced myelosuppression, and during bone marrow transplantation. In these assays, we show that MLN518 has mild toxicity toward normal hematopoiesis at concentrations that are effective in treating FLT3 ITD-positive leukemia in mice. We also demonstrate that MLN518 preferentially inhibits the growth of blast colonies from FLT3 ITD-positive compared with ITD-negative patients with AML, at concentrations that do not significantly affect colony formation by normal human progenitor cells. In analogy to imatinib mesylate in BCR-ABL–positive acute leukemia, MLN518-induced remissions may not be durable. Our studies provide the basis for integrating this compound into chemotherapy and transplantation protocols.

Cotreatment with 17-Allylamino-Demethoxygeldanamycin and FLT-3 Kinase Inhibitor PKC412 Is Highly Effective against Human Acute Myelogenous Leukemia Cells with Mutant FLT-3

Presence of the activating length mutation (LM) in the juxtamembrane domain or point mutation in the kinase domain of FMS-like tyrosine kinase-3 (FLT-3) mediates ligand-independent progrowth and prosurvival signaling in approximately one-third of acute myelogenous leukemia (AML). PKC412, an inhibitor of FLT-3 kinase activity, is being clinically evaluated in AML. Present studies demonstrate that treatment of human acute leukemia MV4-11 cells (containing a FLT-3 LM) with the heat shock protein 90 inhibitor 17-allylamino-demethoxy geldanamycin (17-AAG) attenuated the levels of FLT-3 by inhibiting its chaperone association with heat shock protein 90, which induced the poly-ubiquitylation and proteasomal degradation of FLT-3. Treatment with 17-AAG induced cell cycle G(1) phase accumulation and apoptosis of MV4-11 cells. 17-AAG-mediated attenuation of FLT-3 and p-FLT-3 in MV4-11 cells was associated with decrease in the levels of p-AKT, p-ERK1/2, and p-STAT5, as well as attenuation of the DNA binding activity of STAT-5. Treatment with 17-AAG, downstream of STAT5, reduced the levels of c-Myc and oncostatin M, which are transactivated by STAT5. Cotreatment with 17-AAG and PKC412 markedly down-regulated the levels of FLT-3, p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5, as well as induced more apoptosis of MV4-11 cells than either agent alone. Furthermore, the combination of 17-AAG and PKC412 exerted synergistic cytotoxic effects against MV4-11 cells. Importantly, 17-AAG and PKC412 induced more loss of cell viability of primary AML blasts containing FLT-3 LM, as compared with those that contained wild-type FLT-3. Collectively, these in vitro findings indicate that the combination of 17-AAG and PKC412 has high level of activity against AML cells with FLT-3 mutations.

SU5416 and SU5614 inhibit kinase activity of wild-type and mutant FLT3 receptor tyrosine kinase

Internal tandem duplication (ITD) in the juxtamembrane portion of Fms-like tyrosine kinase 3 (FLT3), a type III receptor tyrosine kinase (RTK), is the most common molecular defect associated with acute myeloid leukemia (AML). The high prevalence of this activating mutation makes it a potential target for molecularly based therapy. Indolinone tyrosine kinase inhibitors have known activity against KIT, another member of the type III RTK family. Given the conserved homology between members of this family, we postulated that the activity of some KIT inhibitors would extend to FLT3. We used various leukemic cell lines (BaF3, MV 4-11, RS 4;11) to test the activity of indolinone compounds against the FLT3 kinase activity of both wild-type (WT) and ITD isoforms. Both SU5416 and SU5614 were capable of inhibiting autophosphorylation of ITD and WT FLT3 (SU5416 concentration that inhibits 50% [IC(50)], 100 nM; and SU5614 IC(50) 10 nM). FLT3-dependent activation of the downstream signaling proteins mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 5 (STAT5) was also inhibited by treatment in the same concentration ranges. FLT3 inhibition by SU5416 and SU5614 resulted in reduced proliferation (IC(50), 250 nM and 100 nM, respectively) and induction of apoptosis of FLT3 ITD-positive leukemic cell lines. Treatment of these cells with an alternative growth factor (granulocyte-macrophage colony-stimulating factor [GM-CSF]) restored MAPK signaling and cellular proliferation, demonstrating specificity of the observed inhibitory effects. We conclude that SU5416 and SU5614 are potent inhibitors of FLT3. Our finding that inhibition of FLT3 induces apoptosis of leukemic cells supports the feasibility of targeting FLT3 as a novel treatment strategy for AML.