High-Throughput Sequence Analysis of the Tyrosine Kinome in Acute Myeloid Leukemia.

Mutations that result in constitutive tyrosine kinase activation occur in a wide spectrum of human malignancies, including acute myeloid leukemia (AML). Although activating mutations in the tyrosine kinases FLT3 and c-KIT occur in a significant proportion of patients with AML, the genomic events responsible for oncogenic signaling in most patients with AML have not been identified. To determine whether other aberrantly activated tyrosine kinases contribute to the pathogenesis of AML, we employed high throughput (HT) DNA sequence analysis to screen the exons encoding domains implicated in kinase activation (activation loop), and autoinhibition (juxtamembrane domains) in 85 tyrosine kinase genes in 192 AML patients without FLT3 or c-KIT mutations. The screen identified 34 non-synonymous sequence variations in 25 different kinases that had not been reported in single-nucleotide polymorphism (SNP) databases. These included a novel activating allele in FLT3, and a previously described activating mutation in MET (METT1010I). The majority of the novel sequence variants were cloned into their respective tyrosine kinase cDNA, and stably expressed in the factor-dependent Ba/F3 hematopoietic cell line. Apart from one FLT3 allele, none of the other novel variants showed evidence of constitutive phosphorylation by immunoblot analysis, and none transformed Ba/F3 cell lines to factor independent growth. These findings indicate that the majority of these alleles are not potent activators of tyrosine kinase activity in this cellular context, and that a significant proportion of non-synonymous sequence variants identified in HT DNA sequencing screens may not have functional significance. Although several explanations are possible for this observation, these data are consistent with recent reports that a significant fraction of such sequence variants are “passenger” rather than “driver” alleles, in cancer, and underscore the importance of functional assessment of candidate disease alleles.