FLT3 (fms-related tyrosine kinase/Flk2/ Stk-2) is a receptor tyrosine kinase (RTK) primarily expressed on hematopoietic cells. In blasts from acute myelogenous leukemia (AML) patients, 2 classes of FLT3 activating mutations have been identified: internal tandem duplication (ITD) mutations in the juxtamembrane domain (25%-30% of patients) and point mutations in the kinase domain activation loop (7%-8% of patients). FLT3-ITD mutations are the most common molecular defect identified in AML and have been shown to be an independent prognostic factor for decreased survival. FLT3-ITD is therefore an attractive molecular target for therapy. SU11248 is a recently described selective inhibitor with selectivity for split kinase domain RTKs, including platelet-derived growth factor receptors, vascular endothelial growth factor receptors, and KIT. We show that SU11248 also has potent activity against wild-type FLT3 (

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IntroductionAcute myeloid leukemia (AML) remains a difficult disease to treat. Patients typically respond to initial treatment with anthracycline and cytarabine-based induction chemotherapy, but most patients ultimately relapse and die of their disease. Internal tandem duplications (ITDs) of the juxtamembrane domain of the fetal liver tyrosine kinase 3 (FLT3) receptor tyrosine kinase are found in 20% to 30% of cases of de novo AML. FLT3 ITD mutations are the most common molecular abnormality associated with AML and presence of such a mutation predicts for a worse outcome for patients, 1-3 particularly those with a high mutant-to-normal FLT3 allelic ratio. 4 An additional 5% to 7% of AML patients have an activating mutation of the FLT3 kinase activation loop. 5 There is substantial experimental and clinical evidence to support the hypothesis that FLT3 mutations are important in the initiation or maintenance of AML in some patients. Activating mutations of FLT3 result in constitutive activation of FLT3 tyrosine kinase activity and can transform factor-dependent hematopoietic cells as evidenced by conversion to factor-independent growth and formation of tumors in immunodeficient mice. [6][7][8] In addition, retroviral transduction of primary murine bone marrow with an AML patient-derived FLT3 ITD cDNA results in a lethal myeloproliferative syndrome. 9 Finally, retroviral transduction of bone marrow derived from promyelocytic leukemia/retinoic acid receptor ␣ (PML-RAR␣) transgenic mice with FLT3 ITD results in a marked increase in the incidence of acute progranulocytic (APL)-like leukemia in such mice when compared with mice that received a transplant of mock-transduced bone marrow. 10 These findings suggest that FLT3 may be a target for molecular therapy, analogous to that of BCR-ABL in chronic myeloid leukemia (CML). In CML, the use of single-agent imatinib (STI571, Gleevec) is associated with impressive response rates and is now considered the standard frontline nontransplantation medical therapy. 11,12 Agents capable of inhibiting FLT3 kinase have been developed and display promising preclinical activity. [13][14][15][16][17][18][19] These agents are capable of halting leukemic cell proliferation and inducing apoptosis in vitro and in different animal models. Several of these FLT3 inhibitors are currently being studied in phase-1 and -2 clinical trials of patients with AML but thus far have demonstrated only modest single-agent activity. [20][21][22] These results are somewhat analogous to those seen using imatinib to treat patients with CML blast crisis, where the rate of complete hematologic or cytogenetic response, as well as the duration of such responses, were markedly inferior to the results obtained in treating chronicphase patients. This experience suggests that FLT3 inhibitors may need to be combined with other antileukemic treatments to obtain the best clinical results. Theoretically, it would be advantageous to add chemotherapy agents that act synergistically with FLT3 We sought to profile the activit...

IntroductionKIT is a receptor tyrosine kinase (RTK) of the type III subgroup, characterized by 5 extracellular immunoglobulin-like domains and a split tyrosine kinase domain. 1 Stem cell factor (SCF) binding induces dimerization and transphosphorylation, activating multiple signaling pathways, including mitogen-activated protein (MAP) kinases, Src kinases, Janus kinases/signal transducers and activators of transcription (Jaks/Stats), and phosphatidylinositol-3 (PI3) kinase. 1 Oncogenic mutations of KIT occur in diverse malignancies including gastrointestinal stromal tumors (GISTs), sinonasal natural killer (NK)/T-cell lymphoma, seminomas/dysgerminomas, acute myelogenous leukemia (AML), and systemic mastocytosis (SM). [2][3][4] The site of mutation tends to be cell-type specific. For example, 57% to 70% of GISTs have activating point mutations or small deletions in the juxtamembrane region of KIT. 3 In contrast, nearly all patients with SM exhibit a mutation in codon 816, typically an aspartate to valine substitution (D816V). 2,3,5 The D816V mutation, within the activation loop of the kinase, produces ligandindependent activation, increases the catalytic activity of KIT, and alters substrate specificity leading to aberrant signaling. [6][7][8] Codon 816 mutations were also reported in AML, as were small deletions in exon 8 in the extracellular region of KIT, typically concurrent with core binding factor mutations. [9][10][11] Treatment of SM is primarily symptomatic, although cladribine and interferon-␣ have some effect. 12,13 AML with core binding factor mutations generally carries a good prognosis, but KIT mutations in this group confer a higher risk of relapse. 9 Therefore, specifically targeting D816 mutant KIT may improve treatment of these malignancies.Various small molecule inhibitors of KIT have been developed, including FLT3 tyrosine kinase inhibitors [14][15][16] and imatinib mesylate, initially designed to target Bcr-Abl. 17 The sensitivity of KIT mutants to small molecule inhibitors depends on the nature of the mutation. Imatinib, for example, potently inhibits KIT juxtamembrane mutants and induced partial responses in 54% of GIST patients in phase 2 clinical trials. 3,18 In contrast, imatinib does not target D816 mutant KIT and is therefore ineffective in mastocytosis patients harboring these mutations. [19][20][21][22] In this study we assess the inhibitory potency of the tyrosine kinase inhibitors MLN518 (formerly CT53518) 14 and PD180970 23,24 against 2 classes of KIT mutations to determine whether these compounds might be useful for treatment of SM and other KIT-driven malignancies. Study design Cell linesThe Ba/F3 mouse pro-B-cell line and P815, a mouse mastocytoma cell line expressing murine D814Y KIT, were purchased from American Type Culture Collection (ATCC; Manassas, VA). The HMC-1 human mast cell line containing an activating juxtamembrane domain point mutation (V560G) 25 and One of the authors (C.L.R.) has declared a financial interest in a company (Millennium Pharmaceuticals) whose produ...

Somatic mutations of FLT3 resulting in constitutive kinase activation are the most common acquired genomic abnormality found in acute myeloid leukemia (AML). The majority of these mutations are internal tandem duplications (ITD) of the juxtamembrane region (JM). In addition, a minority of cases of AML are associated with mutation of the FLT3 activation loop (AL), typically involving codons D835 and/or I836. We hypothesized that other novel mutations of FLT3 could also contribute to leukemogenesis. We genotyped 109 cases of AML and identified two novel gain-of-function mutations. The first mutation, N841 H, is similar to previously described mutations involving amino-acid substitutions of codon 841. The other novel mutation, FLT3 K663Q, is the first AML-associated gain-of-function mutation located outside the JM and AL domains. Of note, this mutation was potently inhibited by Sunitinib (SU11248), a previously described FLT3 kinase inhibitor. Sunitinib reduced the proliferation and induced apoptosis of transformed Ba/F3 cells expressing FLT3 K663Q. The potency of Sunitinib against FLT3 K663Q was similar to its potency against FLT3 ITD mutations. We conclude that FLT3 mutations in AML can involve novel regions of the TK1. Future studies are needed to define the incidence and prognostic significance of FLT3 mutations outside the well-established JM and AL regions.