Extracellular KIT receptor mutants, commonly found in core binding factor AML, are constitutively active and respond to imatinib mesylate

Abstract

Multiple genetic alterations are required to induce acute myelogenous leukemia (AML). Mutations in the extracellular domain of the KIT receptor are almost exclusively found in patients with AML carrying translocations or inversions affecting members of the core binding factor (CBF) gene family and correlate with a high risk of relapse. We demonstrate that these complex insertion and deletion mutations lead to constitutive activation of the KIT receptor, which induces factorindependent growth of interleukin-3 (IL-3)-dependent cells. Mutation of the evolutionary conserved amino acid D419 within the extracellular domain was sufficient to constitutively activate the KIT receptor, although high expression levels were required. Dose-dependent growth inhibition and apoptosis were observed using either the protein tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec) or by blocking the phosphoinositide-3-kinase (PI3K) - IntroductionAcute myelogenous leukemia (AML) is a complex disease that is classified by histochemical, cytochemical, and genetic markers. Genetic alterations affecting members of the core binding factor (CBF), a heterodimeric transcription factor, are some of the most common events in AML and define a large group commonly designated CBF-AML, 1 which includes patients with either t(8;21) or inv(16). Evidence from experimental studies supports the hypothesis that these fusion proteins impair myeloid differentiation and/or expand a hematopoietic stem/progenitor cell pool, but are insufficient to cause leukemia. [2][3][4][5][6] Secondary genetic alterations, such as mutations in receptor tyrosine kinases (RTKs), are thus required for induction of an overt AML. Although the KIT receptor is expressed on the majority of AML blasts, mutations in the KIT gene are exclusively associated with CBF-AML. [7][8][9][10][11] In addition to mutations affecting D816 within the KIT tyrosine kinase domain 2 (TK2), complex insertion/deletion mutations in exon 8 affecting the extracellular (EC) domain are also observed in CBF-AML, with an especially high prevalence (26%) in inv(16) patients, and correlate with a higher risk of relapse. 8,9 In addition to EC mutations, a mutation in the transmembrane (TM) domain (V530I) has also been described in CBF-AML. 9,11 Previous studies have shown that the KIT D816V mutation induces growth factorindependent growth of myeloid and mast cells and is insensitive to the RTK inhibitor imatinib mesylate (STI571, Gleevec [manufactured by Novartis, Basel, Switzerland]). [12][13][14] The purpose of this study was to test the functional activity of the EC and TM KIT mutations associated with CBF-AML, to determine their sensitivity to imatinib, and to delineate the activated signaling pathways. Study designSite-directed mutagenesis was used to create KIT mutants from either murine or human KIT cDNAs, as indicated in Table 1. KIT mutants were cloned into the SF91 retroviral vector coexpressing the yellow fluorescence protein (YFP), and were used to infect FDC-P1 cells in the presence of inter...

KIT oncogenic signaling mechanisms in imatinib-resistant gastrointestinal stromal tumor: PI3-kinase/AKT is a crucial survival pathway

“…The PI3-K/AKT pathway is essential to oncogenic signaling in KIT-mutant acute myeloid leukemia (Cammenga et al, 2005), KIT-mutant mast cell disease (Shivakrupa et al, 2003), and GIST murine models (Rossi et al, 2006) and our studies indicate a key role, as well, in imatinib-resistant GIST. mTOR, which is strongly activated by PI3-K pathways, has been evaluated as a clinical target, because the mTOR inhibitors rapamycin, CCI779 and RAD001 have an acceptable toxicity profile in vivo, compared to highly toxic PI3-K inhibitors.…”

Targeting receptor tyrosine kinase signaling in acute myeloid leukemia

“…Nevertheless, a pre-clinical study analyzing cells expressing c-Kit mutants cloned from AML patients reported a dose-dependent increase in apoptosis upon treatment with the inhibitor (Cammenga et al, 2005). In a first clinical phase II trial of patients with refractory or recurrent AML, Imatinib as single agent did not induce any significant beneficial responses .…”

Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition

“…Mutations found in D5 are clustered in two spots that are located in the D5-D5 contact interface (6). One set of mutations, found primarily in AML patients, involves either substitution of D419 or its loss together with one or two flanking amino acids, like the T417IΔ418-419 mutant in which T417 is substituted with Ile and Y418 and D419 are deleted (21,22). D419 is located in a region connecting strand A with the AB loop of Ig-like D5 (Fig.…”