FMS-like Tyrosine Kinase 3 Kinase Research Articles

FLT3 Inhibitor Treatment in FLT3-Mutated AML Is Associated with Development of Secondary FLT3-TKD Mutations

Abstract 1493 Background:The two most frequent mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor are internal tandem duplication (ITD) of the juxtamembrane region and mutations involving the D835/I836 residues of the tyrosine kinase domain (TKD). They are present in approximately 30% and 7% of AML cases respectively, and have been associated with higher relapse rate and worse overall survival. ITD and TKD mutations produce constitutive activation of the FLT3 receptor; however they appear to have different downstream effectors and different clinical significance. Currently, several FLT3 kinase inhibitors are in development and although some have shown promising clinical activity, responses tend to be transient and insufficient to induce a durable response. Sensitivity of different mutants to diverse FLT3 inhibitors is variable (Zhang et al. JNCI 2008; 100: 184). Aim: To determine the change in mutation status of patients with FLT3 ITD treated with FLT3 inhibitors. Methods: We analyzed 67 patients with FLT3 mutation positive AML, treated in our institution in clinical trials with different FLT3 inhibitors from October 2002 to July 2011 and in whom we obtained mutational assessment before and after treatment. Results: At baseline 58 (87%) patients had an ITD mutation, 5 (7%) had a D835/I836 mutation and 4 (6%) had combined ITD and codon D835/I836 mutations. Thirty three patients were male, median age was 55 years (range 18–87), and the median number of prior leukemia treatments was 2 (range 1–6). Karyotype was diploid in 36%, miscellaneous in 17%, complex in 14%, not done in 23%, and insufficient in 9% of patients. Concomitant NPM1, RAS, and CEBPA mutations were observed in 21%, 13%, and 4% of the patients, respectively. Patients remained on therapy for a median time of 50 days (range 18–561) before progression, 45 (67%) did not achieve response, 13 (19%) cleared their bone marrow blasts, 6 (9%) achieved a partial response, 4 (6%) a complete response and 1 (1%) patient died during treatment. None of the patients with D835/I836 or ITD+D835/I836 achieved a response. Treatment was always discontinued due to progressive disease, except in 9 (13%) patients, 6 who cleared bone marrow blasts and proceeded to hematopoietic stem cell transplantation and 3 who discontinued due to toxicity. One of them was in complete response but progressed within 4 months, and 2 who cleared their bone marrow blasts and were also bridged with other treatments to transplant. All patients in this cohort had assessment of their FLT3 mutation status at the time FLT3 inhibitor was discontinued, 14 (21%) patients progressed from a single ITD mutation to have combined ITD+D835/I836 mutations, 7 (10%) patients (6 ITD and 1 D835/I836) were negative for FLT3 mutation, and 46 (69%) patients had unchanged mutation status. At the time of this analysis, 10 patients were alive, 1 was lost to follow and 56 had died. The median survival in the entire group from the time of FLT3 inhibitor start was 5.3 months, and from time of treatment discontinuation 3.2 months. In those patients in whom the FLT3 mutation became negative the median survival was 7 months, in those with unchanged mutation 4.5 months, and in those with ITD-D835/I836 mutations the median survival was 6.1 months. Further therapy with another FLT3 inhibitor was attempted in 6 out of 14 patients with ITD-D835/I836 mutations, 5 failed to respond and 1 had a transient decrease in bone marrow blasts and peripheral white blood cell count (2 months) with a combination of a FLT3 inhibitor and a hypomethylating agent. Conclusion: There is evidence that a secondary TKD mutations arise after the use of FLT3 inhibitors in patients with single FLT3-ITD mutated AML, a phenomenon that is associated with a poor prognosis. Disclosures:Ravandi:Bayer: Research Funding; Onyx: Research Funding. Cortes:Ambit: Research Funding; Novartis: Research Funding.

Pacritinib (SB1518), a JAK2/FLT3 inhibitor for the treatment of acute myeloid leukemia

FMS-like tyrosine kinase 3 (FLT3) is the most commonly mutated gene found in acute myeloid leukemia (AML) patients and its activating mutations have been proven to be a negative prognostic marker for clinical outcome. Pacritinib (SB1518) is a tyrosine kinase inhibitor (TKI) with equipotent activity against FLT3 (IC50=22 n) and Janus kinase 2 (JAK2, IC50=23 n). Pacritinib inhibits FLT3 phosphorylation and downstream STAT, MAPK and PI3 K signaling in FLT3-internal-tandem duplication (ITD), FLT3-wt cells and primary AML blast cells. Oral administration of pacritinib in murine models of FLT3-ITD-driven AML led to significant inhibition of primary tumor growth and lung metastasis. Upregulation of JAK2 in FLT3-TKI-resistant AML cells was identified as a potential mechanism of resistance to selective FLT3 inhibition. This resistance could be overcome by the combined FLT3 and JAK2 activities of pacritinib in this cellular model. Our findings provide a rationale for the clinical evaluation of pacritinib in AML including patients resistant to FLT3-TKI therapy.

FLT3-regulated antigens as targets for leukemia-reactive cytotoxic T lymphocytes

The FMS-like tyrosine kinase 3 (FLT3) is highly expressed in acute myeloid leukemia (AML). Internal tandem duplications (ITD) of the juxtamembrane domain lead to the constitutive activation of the FLT3 kinase inducing the activation of multiple genes, which may result in the expression of leukemia-associated antigens (LAAs). We analyzed the regulation of LAA in FLT3-wild-type (WT)- and FLT3-ITD+ myeloid cells to identify potential targets for antigen-specific immunotherapy for AML patients. Antigens, such as PR-3, RHAMM, Survivin, WT-1 and PRAME, were upregulated by constitutively active FLT3-ITD as well as FLT3-WT activated by FLT3 ligand (FL). Cytotoxic T-cell (CTL) clones against PR-3, RHAMM, Survivin and an AML-directed CTL clone recognized AML cell lines and primary AML blasts expressing FLT3-ITD, as well as FLT3-WT+ myeloid dendritic cells in the presence of FL. Downregulation of FLT3 led to the abolishment of CTL recognition. Comparing our findings concerning LAA upregulation by the FLT3 kinase with those already made for the Bcr-Abl kinase, we found analogies in the LAA expression pattern. Antigens upregulated by both FLT3 and Bcr-Abl may be promising targets for the development of immunotherapeutical approaches against myeloid leukemia of different origin.

Inhibition of signal transducer and activator of transcription 5 by the inhibitor of janus kinases stimulates dormant human leukemia CD34+/CD38− cells and sensitizes them to antileukemia agents

To verify molecular mechanisms by which leukemia stem cells (LSCs) maintain a dormant state, we explored the activity of the major prosurvival signal pathways in CD34(+) /CD38(-) compartment, supposed to contain LSCs, and CD34(+) /CD38(+) counterparts from patients with acute myelogenous leukemia (AML, n = 11) by fluorescence-activated cell sorting (FACS). CD34(+) /CD38(-) cells expressed a greater amount of p-janus kinase 2 (JAK2) and p-signal transducer and activator of transcription 5 (STAT5) than CD34(+) /CD38(+) counterparts in all patients except for one case. In addition, we found that CD34(+) /CD38(-) cells were relatively resistant to cytarabine- and the inhibitor of the fms-like tyrosine kinase 3 (FLT3)-mediated growth inhibition, as measured by the clonogenic assay. Interestingly, blockade of JAK2/STAT5 signaling by the specific JAK2 inhibitor AZ960 stimulated cell cycling in CD34(+) /CD38(-) cells in conjunction with downregulation of cyclin-dependent kinase inhibitor p21(waf1) and sensitized these cells to the growth inhibition mediated by cytarabine and the FLT3 kinase inhibitor. Moreover, exposure of CD34(+) /CD38(-) cells to AZ960 potently induced apoptosis in parallel with downregulation of antiapoptotic protein Bcl-xL, as measured by Western blot analysis. Taken together, JAK2/STAT5 signaling may be a promising molecular target to eradicate CD34(+) /CD38(-) leukemia cells in individuals with AML.

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.

A substrate peptide for the FLT3 receptor tyrosine kinase

FLT3 (fms-like tyrosine kinase 3) is frequently activated by mutation in acute myeloid leukemia, and is therefore under study as a drug target. Testing and characterization of tyrosine kinase inhibitors is facilitated by the availability of efficient peptide substrates. Searching for FLT3 peptide substrates using phosphorylation experiments on peptide arrays and in solution revealed that the peptide F-T-D-R-L-Q-Q-Y(8)-I-S-T-R-G-L-G is efficiently phosphorylated (apparent Km 10 micromol/l), with Y8 as the phosphorylated site. This peptide presents a novel tool for identifying and characterizing FLT3 kinase inhibitors.

A Phase I Dose Escalation Study of KW-2449, An Oral Multi-Kinase Inhibitor against FLT3, Abl, FGFR1 and Aurora in Patients with Relapsed/Refractory AML, ALL and MDS or Resistant/Intolerant CML

Background: Activating mutations of FMS-like tyrosine kinase 3 (FLT3) occur in 30% of patients with de novo AML and confer a poor prognosis. KW-2449 is an oral multi-kinase inhibitor which is highly potent against mutant FLT3 (IC50=0 1–7 nmol/L) and other tyrosine kinases including FGFR1, TrkA, Abl (including T315I) and Aurora A serine threonine kinase. Based on the activity of KW-2449 and its metabolite (M1) in both in vitro and in vivo preclinical leukemia models, KW-2449 was evaluated in patients with leukemia and MDS in this first-in-man study. M1 is formed via monoamine oxidase-B and aldehyde oxidase mediated oxidation of KW-2449.Methods: The objectives were to assess the safety, tolerability, pharmacokinetic (PK) and pharmacodynamic effects of KW-2449 in patients with refractory/relapsed AML, ALL and MDS, or resistant/intolerant CML. A range of daily doses of KW-2449 (12.5–250 mg twice daily, i.e. 25–500 mg/day) on 2 treatment schedules (14 or 28 days) with a recovery period of 7–28 days between cycles were evaluated. Dose limiting toxicity (DLT) and maximum tolerated dose (MTD) were assessed for the 1st cycle. The 28-day schedule was later eliminated. The plasma concentration of KW-2449 and M1 were analyzed by LC-MS/MS method. A plasma inhibitory activity (PIA) assay [Blood 108(10) 3477–83] for P-FLT3 and P-STAT5 was used to measure FLT3 inhibition.Results: 37 patients aged 26–88 years (16 male) were treated at 7 dose levels: 25, 50, 100, 200, 300, 400, and 500 mg daily. Thirty-one patients had AML, 5 CML and 1 ALL. The mean duration of therapy was 2 cycles in AML patients and 4 cycles in CML patients. KW-2449 was rapidly absorbed and metabolized to M1. The half-lives for KW-2449 and M1 were not dose-related and ranged from 2.4 to 4.9 hours and 2.6 to 6.6 hours, respectively. Administration of KW-2449 in a BID regimen led to minor accumulation and was consistent with the short half-life. The PIA assay demonstrated the near complete down-regulation of P-FLT3 and P-STAT5 2 hours post-dose at a dosing level of 400 mg daily. The extent of inhibition was lower at 8 hours and generally absent at 12 hours post-dose. The most frequently reported adverse events (AEs; any grade, regardless of causality) were nausea (70.3%), vomiting (48.6%), fatigue (45.9%), diarrhea (32.4%), dyspnea (29.7%), febrile neutropenia (29.7%), pain in extremity (29.7%), and arthralgia (27.0%). Febrile neutropenia (24.3%), pneumonia (10.8%), and thrombocytopenia (10.8%) were the most frequently reported Grade 3/4 AEs. DLTs occurred in 2 patients: Grade 3 atrial fibrillation (100 mg daily) and Grade 3 nausea and vomiting (500 mg daily). A total of 70 SAEs were reported in 27 patients including 11 on-study deaths; only atrial fibrillation and pleural effusion were considered possibly related to KW-2449. Eight of 31 patients with AML (26%) (FLT3 mutation: 5 positive and 3 negative) and 1 of 5 patients with CML (20%) exhibited a ³ 50% reduction in peripheral blasts and/or bone marrow blasts from baseline to the end of Cycle 1. One patient (500 mg daily) with AML exhibited a > 50% decrease in peripheral blasts, increased platelets, and ANC, and decreased WBC count. A patient with CML (Bcr-Abl T315I +) lost the mutant clone while on KW-2449 treatment.Conclusions: KW-2449 was safe and well tolerated at the dose levels evaluated. There were no complete or partial responses, but transient decreases in peripheral blood and bone marrow blasts were observed, justifying continued investigation of this agent. Sustained inhibition of P-STAT5 and P-FLT3 was not achieved at trough at the highest BID dose evaluated. TID and QID dosing schedules should be evaluated to accommodate the short t1/2 and to achieve sufficient target inhibition.

Novel Transforming Mutations of CBL in Human Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a genetically heterogeneous disease initiated within the bone marrow. It is characterized by multiple mutagenic events that affect proliferation and/or viability as well as differentiation. Deregulation of tyrosine kinase activity is frequently associated with AML and required for transformation. In particular, mutational activation of the FLT3 (Fms-like tyrosine kinase 3) receptor tyrosine kinase occurs in about one third of AML and FLT3 is expressed at high levels in more than 80% of AML patient cells. Recently, it has been suggested that FLT3, Kit or other receptor tyrosine kinases (RTKs) can be active in cells containing inactivating mutations within the linker region of CBL. CBL proteins function as E3 ligases that ubiquitinate and negatively regulate activated RTKs such as FLT3. Nevertheless, the in vivo role and frequency of these CBL mutations in AML remains unknown. In order to identify novel CBL mutations, we prepared cDNA from 43 AML patients and 10 healthy individuals for sequencing of the linker region. We identified two novel CBL mutations, which included a point mutation leading to a substitution in position 371 of the coding sequence and a splice site mutation (intron7/exon8) resulting in a six base pair insertion. Both mutations were located in the alpha helical structure within the linker sequence known to play an important role in promoting receptor ubiquitination. These mutations were inserted into vectors containing the c-CBL cDNA by site-directed mutagenesis. We demonstrate that the co-expression of these mutations with FLT3 induced factor independent growth and survival of BaF3 cells, similar to control cells expressing CBL with a deletion of Y371 in the linker region This region is upstream of the RING finger, which is required for its E3 ligase function. Whereas cells expressing wild-type CBL did not grow in a three day culture, the cell number increased 13.7-fold for the CBL point mutant and 14.2-fold for the splice site mutant. This result is comparable to CBL Y731 deletion containing cells (12.8-fold increase). Also, growth of cells expressing mutant CBL required expression of FLT3 and could be inhibited by the FLT3 inhibitor midostaurin. Further, both CBL mutations lead to an increase in apoptosis with increased caspase3/7 enzyme activity. The point mutation at Y371 also led to an increase in intracellular reactive oxygen species (ROS), similar to cells containing the Y371 deletion or cells transformed by FLT3-ITD. Previously, chronically elevated ROS levels have been implicated in the induction of DNA damage and therapy related drug resistance as well as activation of signaling pathways required for transformation. Overall, our in vitro data demonstrate that mutations of CBL in the linker region are sufficient to support growth and viability of BaF3 cells and that the transforming activity can be targeted by small molecule drugs against RTKs. Thus, an abnormal increase in RTK activity in AML may occur indirectly through inactivating mutations within CBL or other protein degradation pathway proteins, independent of RTK mutations. It will now be of interest to further evaluate the role of CBL in AML in the context of disease phenotype and mutational status of RTKs.

Symadex™, a FLT3 kinase inhibitor, is metabolized by aldehyde oxidase

Symadex™ (C‐1311) is a potent inhibitor of FMS‐like tyrosine kinase 3 (FLT3). This imidazoacridinone class compound has started development for oncology indications and is being investigated as a therapy for autoimmune diseases. A major circulating metabolite is an oxygenated species (+16 amu) that is found in plasma and urine after oral and intravenous administration.Aldehyde oxidase (AO) is a molybdenum‐containing enzyme present in liver that catalyzes the oxidation of a wide variety of nitrogen containing heterocycles. Data are presented from in vitro incubations with microsomes, liver cytosol, inhibitors specific for AO, and an isotopic labelling experiment to confirm the role of AO in the oxidation of Symadex™.The proposed metabolite was synthesized and its structure verified by NMR. It was shown to have similar chromatography, spectroscopy, and MS/MS fragmentation compared to the in vitro and in vivo derived metabolite. The structure was consistent with an oxidation catalyzed by AO.This non‐CYP mediated transformation is important for the metabolism of imidazoacridinones and likely plays a significant role in the metabolism of other tyrosine kinase inhibitors with imidazole and related susceptible heterocyclic scaffolds. This work presents a comprehensive investigation into this novel route of drug metabolism.

FLT3 Kinase Inhibitors in the Management of Acute Myeloid Leukemia

FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase (TK) expressed by immature hematopoietic cells and is important for the normal development of stem cells and the immune system. Mutations of the juxtamembranous and TK domain of the gene are described in 30%-35% of patients with acute myeloid leukemia (AML). These mutations alter the biologic properties of AML and are associated with prognosis. In recent years, there has been an enormous development of potential inhibitors of FLT3 mutations. These substances are now being studied in clinical protocols. The initial trials reveal that, unlike in patients with chronic myeloid leukemia, TK inhibitor (TKI) therapy in AML is more complex. To date, most FLT3 TKIs investigated in clinical studies show a favorable toxicity profile with considerable biologic activity. However, refractory disease and/or the rapid development of resistance toward these new drugs remain major challenges. Strategies to circumvent this unsatisfactory clinical potential of FLT3 TKIs are mainly based on the combination with cytotoxic chemotherapy. Herein, we summarize results from studies using FLT3 TKIs as single agents and report on the first clinical trials investigating FLT3 TKIs in combination with chemotherapy.

A Phase I Dose Escalation Study of KW-2449, an Oral Multi-Kinase Inhibitor Against FLT3, Abl, FGFR1 and Aurora in Patients with Relapsed/Refractory AML, Treatment Resistant/Intolerant CML, ALL and MDS.

Background: Activating mutations of FMS-like tyrosine kinase 3 (FLT3) occur in 30% of patients (pts) with de novo AML and confer a worse prognosis. KW-2449 is an oral multi-kinase inhibitor highly potent against mutant FLT3 (IC50= 1–7 nmol/L) and other tyrosine kinases including FGFR1, TrkA, Abl (including T315I), JAK2, c-KIT, and c-SRC and Aurora A serine tyrosine kinase. Based on the anti-leukemia activity of KW-2449 demonstrated both in vitro and in vivo preclinical leukemia models, KW-2449 is being evaluated in hematologic conditions in the first-in-man study.Methods: The study objectives were to assess the safety, tolerability, pharmacokinetic (PK) and pharmacodynamic effects of KW-2449 in pts with refractory/relapsed AML or treatment resistant/intolerant CML/ALL and MDS. A range of daily doses of KW-2449 (25–500 mg/day) divided into q 12h dosing on 2 treatment schedules (14 days vs. 28 days) with a recovery period of 7–28 days between cycles. Dose limiting toxicity (DLT) and maximally tolerated dose were assessed for the 1st cycle. Serial samples for PK analysis were collected immediately before and after KW-2449 on treatment days 1, 14 and 28. The 28-day schedule was later eliminated. Plasma concentrations of KW-2449 and its active metabolite (M1) were analyzed by LC-MS/MS. A plasma inhibitory activity (PIA) assay [Blood 108(10) 3477–83] for P-FLT3 and P-STAT5 was used to measure FLT3 inhibition.Results: To date, 29 pts (15 female) have been enrolled (median age 60 years; range 25–82) and treated at 5 dose levels: 25, 50, 100, 200, and 300 mg daily. Twenty-five pts had AML and 4 CML (3 with T315I mutation); 24 pts completed at least 1 cycle. KW-2449 was rapidly absorbed and metabolized to M1. Elimination half-lives were 2.8–3.9 h for KW-2449 and 3.8–5.5 h for M1. Plasma levels of M1 were lower on Days 14 and 28 compared to Day 1, suggesting inhibition of this pathway upon multiple dosing. A single DLT of grade 3 pneumonia was reported on 100 mg but no further DLTs were seen in the expanded cohort at that dose or at 200 or 300 mg daily. A total of 33 SAEs were reported of which 6 were considered possibly related (by the Investigator) to KW-2449: dyspnea, pneumonia, atrial fibrillation, cardiac ischemia, ventricular arrhythmia, and pleural effusion. There have been 5 deaths on study (none drug-related): disease progression (2), neutropenic sepsis, infection with renal failure, and pneumonia. Seven pts had stable disease after 1 cycle. Three pts with AML (2 FLT3+) had ≥ 50% reduction in peripheral and/or bone marrow blasts in the 1st cycle. In vivo FLT3 inhibition, as measured directly in patient blasts, correlated with blast reduction. The extent and duration of FLT3 inhibition increased with increases in dose.Conclusions: KW-2449 appears safe and well tolerated at the dose levels evaluated. Transient decreases in peripheral blood and bone marrow blasts have been observed in a few patients justifying continued investigation with this agent. Accrual is ongoing and a different schedule to accommodate the short t1/2 will be explored.

Overactivation of STAT Pathways and Overexpression of Survivin Confer Resistance to FLT3 Inhibitors and Could Be Therapeutic Targets in AML.

Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) receptor plays an important role in the pathogenesis of acute myeloid leukemia (AML). A number of small molecule kinase inhibitors are currently proceeding in different phases of clinical trials. As with imatinib in CML, leukemic cells could develop resistance to these RTK inhibitors when used as monotherapy. Mutations in the ATP-binding pocket have been identified through PCR-based mutagenesis screening in Ba/F3-FLT3-ITD cells and selected for growth in the presence of PKC412, or in a resistant Ba/F3-FLT3-ITD cell line developed by cocultured with SU5416. Resistance to PKC412 resulting from the N676K point mutation in the FLT3 kinase domain has been described in a clinical trial patient. Selection of activating Ras mutations has been found in 2 out of 6 FLT3 inhibitor resistant cell lines, but no point mutation of FLT3 kinase domain was found in all 6 resistant cell lines. To further investigate other potential mechanisms of resistance to FLT3 inhibitors, we developed a resistant cell line by long-term culture of MV4-11 cells with ABT-869, designated as MV4-11-R (IC50: 52 vs 6 nM for the parental MV4-11 cell line), which is also cross resistant to other structurally unrelated inhibitors including SU5416, AG1296 and a FLT3 inhibitor III (MERCK). No point mutation in the kinase domain of FLT3 was found in MV4-11-R cells. Western blot and FACS analysis excluded overexpression of p-FLT3, FLT3 or 3 multidrug resistance related proteins (MDR, MRP1 and LRP) in this resistant line. Gene expression profiling revealed up-regulation of FLT3 ligand (FLT3LG) (2.4 fold) and Survivin (2 fold), while down-regulation of SOCS1, SOCS2, and SOCS3 was observed in MV4-11-R compared to MV4-11 parental cells. Overexpression of FLT3LG and Survivin was also demonstrated at the protein level. Survivin is a unique member of the inhibitor of apoptosis proteins (IAP) family and a known target of the STAT3 pathway. Down-regulation of suppressor of cytokine signaling (SOCS) proteins (negative regulators of STAT pathways) was observed even in the presence of overactivation of the STAT1, STAT3 and STAT5 pathways in the MV4-11-R line. We screened a panel of small molecule inhibitors including a STAT inhibitor (indirubin derivative IDR E804), 3 JAK inhbitors (Tyrene CR4, AG490, and JAK3 Inhibitor II), and a CDK/survivin inhibitor (NU6140). We found that MV4-11-R is most sensitive to IDR E804 (an inhibitor of CDKs and the SRC-STAT3 pathway). The IC50 value of ABT-869 in MV4-11-R was decreased from 52 to 6.2 nM in the presence of 2 nM of IDR E804. Further validation of the therapeutic effect of IDR E804 in combination with ABT-869 in the MV4-11-R mouse xenograft model is ongoing. Targeting Survivin by shRNA and a dominant-negative vector (survivin-T34A) induced MV4-11-R to undergo apoptosis. Taken together, these results demonstrate that overactivation of STAT pathways and overexpression of survivin are the main mechanism of resistance to ABT-869; suggesting that the STAT pathways and survivin could be potential targets for the treatment of patients who develop resistance to FLT3 inhibitors.

Blockade of MEK/ERK signaling enhances sunitinib-induced growth inhibition and apoptosis of leukemia cells possessing activating mutations of the FLT3 gene

The FMS-like tyrosine kinase 3 (FLT3) is a cell surface receptor tyrosine kinase. Activating mutations of this gene occur in nearly 30% of acute myelogenous leukemia (AML) patients. These mutations, in part, result in activation of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. In this study, we found that AZD6244 (ARRY-142886), a novel inhibitor of MEK1/2 kinases, effectively inhibited the proliferation of acute biphenotypic leukemia MV4-11 and acute monocytic leukemia MOLM13 cells. The concentrations that inhibited 50% growth were approximately 0.3 and 1.2 μM, respectively, as measured by thymidine uptake on day 2 of culture. AZD6244 potently down-regulated the levels of phospho-ERK1/2 and its downstream effector, p-p70S6K, in the MV4-11 and MOLM13 cells as measured by Western blot analysis. Interestingly, when AZD6244 was combined with sunitinib, a FLT3 kinase inhibitor, growth inhibition and apoptosis of both MV4-11 and MOLM13 cells were synergistically enhanced in association with further down-regulation of phospho-ERK1/2 and p-p70S6K in these cells. Taken together, concomitant blockade of FLT3 and MEK signaling represents a promising treatment strategy for individuals with leukemia who possess activating mutations of FLT3.

FLT3 regulates β-catenin tyrosine phosphorylation, nuclear localization, and transcriptional activity in acute myeloid leukemia cells

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.

Effect of FLT3 Inhibition on Normal Hematopoietic Progenitor Cells

Ligand-mediated activation of the FMS-like tyrosine kinase-3 (FLT3) receptor is important for normal proliferation of primitive hematopoietic cells. FLT3 expression in the bone marrow is restricted to CD34(+) cells and a subset of dendritic precursors. FLT3, as a member of the type III RTK subfamily, is closely related to c-kit, c-FMS, and PDGFalpha/beta and is an unspecific target of tyrosine kinase inhibitors, such as imatinib. Activating mutations of FLT3 play an important role in leukemogenesis and their presence is associated with poor prognosis in acute myeloid leukemia (AML). Targeting the mutation by inhibiting the tyrosine kinase activity of FLT3 is a promising therapeutic option in the treatment of AML patients. CEP-701 (Lestaurtinib), an indocarbazole derivate, is an FLT3 tyrosine kinase inhibitor. In this study, we investigated the effect of FLT3 kinase inhibition on normal hematopoietic stem and progenitor cells in vitro. FLT3 inhibition in normal CD34(+) cells resulted in a dose-dependent inhibitory effect in cell expansion. In contrast, progenitor cell function remained nearly unaffected. Blocking the FLT3 ligand by a neutralizing antibody partially restored the effects of FLT3 inhibition. These findings might explain hematotoxicity of tyrosine kinase inhibitors such as imatinib.

AS602868, a dual inhibitor of IKK2 and FLT3 to target AML cells

Acute myeloid leukemia (AML) cells carry molecular defects that promote their leukemic proliferation, resistance to apoptosis and defect in differentiation. Pharmacological targeting of the nuclear factor kappaB (NF-kappaB) pathway has been shown to promote apoptosis of primary AML cells and to sensitize blasts to neoplastic drugs (Frelin, Blood 2005, 105, 804). The Fms-like tyrosine kinase 3 (FLT3), which sustains proliferation of normal hematopoietic progenitors is frequently overexpressed or mutated in AML patients. Using Ba/F3 murine pre-B cells transfected with various mutants of FLT3 (ITD, D835V, D835Y) and the MV4-11 human AML line, we show that normal or oncogenic stimulation of FLT3 led to activation of NF-kappaB. Pharmacological inhibition of either FLT3 with AG1296 or NF-kappaB with the small molecule inhibitor of IkappaB kinase-2 AS602868 reduced viability and triggered cell death. Moreover, AS602868 was also found to interfere directly with FLT3 kinase activation. AS602868 thus appears to target two different kinases that play a crucial role in the pathogenesis of AML, making it particularly attractive as a new therapeutical approach for AML.

Crystal Structure of the Tyrosine Kinase Domain of Colony-stimulating Factor-1 Receptor (cFMS) in Complex with Two Inhibitors

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.

Selective FLT3 Inhibition Uncovers the Role of FL and FLT3 in Normal CD34+ Hematopoietic Stem Cells.

Several cytokines in the bone marrow act through stem cell or progenitor cell specific receptors to regulate the capacity of immature hematopoietic cells to potentiate downstream multilineage expansion. Ligand-mediated activation of the FMS-like tyrosine kinase-3 (FLT3) receptor is important for normal proliferation of primitive hematopoietic cells. FLT3 expression in the bone marrow is restricted to CD34+ cells and a subset of dendritic precursors. FLT3 as a member of the type III RTK subfamily is closely related to c-KIT, c-FMS and PDGFRα/β. Activating mutations of FLT3 play an important role in leukemogenesis and their presence is associated with poor prognosis in acute myeloid leukemia (AML). Targeting the mutation by inhibiting the tyrosine kinase activity of FLT3 is a promising therapeutic option in treatment of AML patients. CEP-701, a potent FLT3 tyrosine kinase inhibitor is known to be cytotoxic to cell lines and primary AML cells harbouring FLT3 mutations and shows biological and clinical activity in patients with relapsed or refractory AML. In this study, we investigated the effect of FLT3 kinase inhibition in normal hematopoietic stem and progenitor cells in-vitro. Peripheral blood mobilized CD34+ cells were cultured in serum-free conditions supplemented with various cytokine combinations. Flt-3 inhibition was performed with addition of CEP-701 in different concentrations. FLT3 inhibition resulted in dose-dependent growth inhibition of CD34+ cells in in-vitro culture. This effect was independent of cytokine combinations chosen. A tetrazolium-based MTT assay was used to quantify 50% growth inhibition after 48h of exposure to CEP-701. The mean IC50 (± SD) for CEP-701 on normal CD34+ progenitor cells was 56 ± 19 nM. Immunophenotypic analysis of cell cultures showed a markedly decrease of CD34+ expressing cells under FLT3 inhibiting conditions. Surprisingly, inhibition of cell growth was even present, when cell culture was performed in absence of FL. In addition, the effect of FLT3 inhibition could be restored by addition of a neutralizing FL-antibody to cell culture even in conditions without FL substitution. Expression of FLT3 and FL under cytokine-supplemented culture conditions and FLT-3 inhibition were monitored by Western Blot analysis. In order to evaluate the effect of FLT-3 inhibition on the progenitor cell function, colony formation was analysed. Addition of CEP-701 into cell culture resulted in decrease of absolute colony production, however, relative colony formation per input cells was not significantly decreased showing that progenitor cell function of surviving cells was not markedly affected. These data demonstrate a significant inhibitory effect of CEP-701 on normal CD34+ cells. We could demonstrate for the first time, that FL might act as an autocrine mediator activating FLT3 in CD34+ cells as CD34+ cells similarily express both, FLT3 and FL. Finally, these findings might also explain hematotoxicity of various tyrosine kinase inhibitors, e.g. imatinib, which also show unspecific targeting of FLT3.

28 ORAL Imidazole derivatives with vascular disrupting activity

The FMS-like tyrosine kinase 3 (FLT3) is a cell surface receptor tyrosine kinase. Activating mutations of this gene occur in nearly 30% of acute myelogenous leukemia (AML) patients. These mutations, in part, result in activation of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. In this study, we found that AZD6244 (ARRY-142886), a novel inhibitor of MEK1/2 kinases, effectively inhibited the proliferation of acute biphenotypic leukemia MV4-11 and acute monocytic leukemia MOLM13 cells. The concentrations that inhibited 50% growth were approximately 0.3 and 1.2 μM, respectively, as measured by thymidine uptake on day 2 of culture. AZD6244 potently down-regulated the levels of phospho-ERK1/2 and its downstream effector, p-p70S6K, in the MV4-11 and MOLM13 cells as measured by Western blot analysis. Interestingly, when AZD6244 was combined with sunitinib, a FLT3 kinase inhibitor, growth inhibition and apoptosis of both MV4-11 and MOLM13 cells were synergistically enhanced in association with further down-regulation of phospho-ERK1/2 and p-p70S6K in these cells. Taken together, concomitant blockade of FLT3 and MEK signaling represents a promising treatment strategy for individuals with leukemia who possess activating mutations of FLT3.

LS104, a Novel Kinase Inhibitor, Induces Apoptosis, Synergizes with Cytostatic Drugs and Is Targeting the Receptor Tyrosine Kinase FLT3.

Fms-like tyrosine kinase 3 (FLT3), a member of the class III tyrosine kinase receptor family, is expressed in up to 90% of acute myeloid leukemia (AML). Activating mutations like internal tandem duplication (ITD) of the juxtamembrane domain and kinase domain point mutations are found in approximately 35% of AML-cases and are considered to represent an attractive therapeutic target. In this study, we report that the novel hydroxystyryl-acrylonitrile compound LS104 induces potent cytotoxic effects in FLT3 ITD-positive leukemic cells. As a cellular model to investigate FLT3-ITD specific effects we used 32D myeloid cells stably transfected with FLT3-ITD and wt-FLT3, respectively. In MTS assays, pronounced inhibition of cell growth was seen at nanomolar concentration (IC50=50nM) which could be partially rescued by addition of IL3. LS104 at a concentration ranging from 3–10μM readily induced apoptosis as evaluated by cell cycle analysis, annexin-V assays and PARP cleavage. Throughout the experiments, FLT3-ITD expressing cells showed significantly higher sensitivity towards LS104 than cells expressing wt-FLT3. Similar results were observed evaluating the cytotoxic effects of LS104 in the human myeloid-leukemia derived FLT3-ITD harbouring MV4;11 and the wt-FLT3 expressing lymphoid-leukemia derived RS4;11 cell line. Immunoblot analysis demonstrated that LS104 inhibits tyrosine phosphorylation of FLT3-ITD and of its downstream target STAT5. This points out that FLT3-ITD is a molecular target of LS104. Interestingly, efficacy of LS104 to induce apoptosis was significantly reduced in 32D cells transfected with a FLT3-ITD isoform (N676K) previously reported to be associated with clinical resistance of FLT3-ITD to the kinase inhibitor PKC412 (Heidel et al., ASH 2004). This result strongly suggests that the mechanism of action of LS104 is indeed inhibition of FLT3 kinase activity rather than inhibition of other kinases targeted by LS104. As clinical development of FLT3 kinase inhibitors in AML likely will be in combination with chemotherapy, we evaluated the in vitro effects of combining LS104 with the cytostatic drugs cytosine arabinosid (Ara-C) and daunorubicin (DNR). In 32D FLT3-ITD cells, cell cycle analysis showed strong synergy in induction of apoptosis upon incubation with LS104 (1–3μM) plus Ara-C (1μM) and DNR (10nM), respectively. Using primary blasts from AML patients, we further evaluated cytotoxicity of LS104 at various concentrations and in combination with Ara-C and DNR, respectively. LS104 as a single agent was shown to induce apoptosis in primary AML blasts and combination of LS104 with cytostatic drugs resulted in additive and synergistic effects in leukemic blasts from 4 out of 6 patients investigated. Western-blotting of primary AML blasts revealed inhibition of FLT3-ITD and STAT5 tyrosine phosphorylation by LS104 in a dose-dependent way (3–50μM). Interestingly, LS104 showed intrinsic fluorescence activity. Using FACS analysis and fluorescence microscopy we could demonstrate a rapid and dose-dependent cellular uptake of LS104 in leukemic cell lines and in primary blasts. After removal of LS104 from medium, enhanced fluorescence corresponding to intracellular LS104 was detectable up to 24 hours. This property could be used to monitor drug uptake of primary AML blasts in vivo. In conclusion, our data provide a preclinical framework for clinical trials of LS104 in FLT3-ITD positive leukemia.