Somatic mutations and germline sequence variants in the expressed tyrosine kinase genes of patients with de novo acute myeloid leukemia

Abstract

Activating mutations in tyrosine kinase (TK) genes (eg, FLT3 and KIT) are found in more than 30% of patients with de novo acute myeloid leukemia (AML); many groups have speculated that mutations in other TK genes may be present in the remaining 70%. We performed high-throughput resequencing of the kinase domains of 26 TK genes (11 receptor TK; 15 cytoplasmic TK) expressed in most AML patients using genomic DNA from the bone marrow (tumor) and matched skin biopsy samples ("germline") from 94 patients with de novo AML; sequence variants were validated in an additional 94 AML tumor samples (14.3 million base pairs of sequence were obtained and analyzed). We identified known somatic mutations in FLT3, KIT, and JAK2 TK genes at the expected frequencies and found 4 novel somatic mutations, JAK1(V623A), JAK1(T478S), DDR1(A803V), and NTRK1(S677N), once each in 4 respective patients of 188 tested. We also identified novel germline sequence changes encoding amino acid substitutions (ie, nonsynonymous changes) in 14 TK genes, including TYK2, which had the largest number of nonsynonymous sequence variants (11 total detected). Additional studies will be required to define the roles that these somatic and germline TK gene variants play in AML pathogenesis.

Figure 1

Ranking of tyrosine kinase (TK) genes based on expression in AML. Expression microarray data were obtained from 92 de novo AML patient bone marrow samples from the WU Discovery set samples. Normalized absolute expression values are shown for all annotated RTKs (A) and CTKs (B) on the Affymetrix U133 Plus 2 array platform. Genes that were sequenced in this study are indicated by the black bars.

Figure 2

Nonsynonymous base changes identified by exonic resequencing of DNAs from 94 AML patients. Unique patient numbers (UPNs) are shown in the rows, and the names of sequenced genes are shown in the columns. T indicates tumor (sequence variants in the AML tumor sample); G, germline (sequence variants in the “germline” (skin) sample from the same patient). Green indicates that no nonsynonymous sequence variants were identified in that sample; red identifies samples with nonsynonymous variants; the predicted consequences of all sequence variants detected are listed; yellow indicates the presence of a homozygous sequence variant; white boxes indicate that no sequence was obtained. All somatic mutations were confirmed by automated resequencing and/or hand validation. No nonsynonymous sequence variants were detected in FES, LYN, YES1, BTK, PTK2B, IGF1R, SYK, RYK, or CSK (data not shown). Mutations found in FLP3, KIT, N-RAS, KRAS, and PTPN11 are exactly the same as described previously. The nonsynonymous sequence variants shown for FGR (D230V) and FYN (D502E) are known to be SNPs and therefore were not sequenced in the skin samples.

Figure 3

DDR1 and NTRK1 somatic mutations map onto activation loop of a prototypical kinase domain. The kinase model was based on the alignment of the DDR1 and NTRK1 sequences onto the structure of the Hck kinase in complex with Src kinase inhibitor (PDB ID 1qcf). The N-terminal domain (N-lobe) is in blue, the C-terminal domain (C-lobe) is in magenta, and the activation segment is in yellow. The catalytic Tyr is in ball-and-stick representation, whereas the mutations are represented by red spheres.

Figure 4

Nonsynonymous germline sequence variants in the TYK2 gene. (A) Diagram of the TYK2 protein showing nineteen (19) nonsynonymous changes. Four novel germline SNPs (*) were detected in the 94 Discovery set samples. Previously identified SNPs are also shown. Percentages indicate the frequency of the indicated sequence variant in the 94 Discovery set samples. †The TYK2^G363S sequence change is found at a significantly different frequency between AML patients and normal controls. (B) Autophosphorylation of variant TYK2 alleles in response to stimulation by interferon-α (IFN). The human TYK2-deficient cell line U1A was transduced with cDNAs encoding patient-derived variant TYK2 alleles. An artificial allele, V678F, containing an amino acid substitution homologous to the V617F activating mutation in JAK2, was used as a positive control. Most patient-derived alleles were indistinguishable from wild-type. In contrast, TYK2^I684S consistently demonstrated reduced total TYK2 protein levels, whereas the absolute level of phosphorylated TYK2^I684S appeared no different from wild-type (lane 8). Autophosphorylation of the TYK2^P1104V was reduced after IFN stimulation, suggesting a decreased level of kinase activity (lane 12). TYK2^V362F phosphorylation appears slightly increased (lane 5), but this finding was not reproduced in replicate experiments. This experiment was performed 3 times, and a representative blot is shown.

Figure 5

Cytoplasmic tyrosine kinase (CTK) gene expression in AML and normal myeloid cells. RNA isolated from unfractionated AML bone marrow samples or from normal human bone marrow CD34⁺ cells, flow-sorted promyelocytes, and flow-sorted polymorphonuclear leukocytes was hybridized to Affymetrix U133 Plus 2 microarrays. Expression of CTK genes in our discovery set AML samples is rank-ordered from the highest to lowest levels of mean expression (92 of 94 samples had successful array studies performed; the 2 samples without array data are shown as white columns). AML samples are arranged by FAB classification. Expression levels are based on scaled signal intensity values for each probe set (mean value for all probe sets on each array was scaled to a value of 1500 using the MAS 5.0 algorithm), with a value of 0 represented in green and 10 000 or greater as red. Black squares in the right-most column indicate the CTK genes that were resequenced in this study. Below the expression data, the cytogenetic findings and somatic mutation analysis for each sample are shown. Blue boxes indicate the presence of the indicated cytogenetic abnormalities and/or somatic mutations. The bottom-most row indicates the percentage of bone marrow blasts in the AML samples, scaled from 0% (green) to 100% (red); the average blast count for the 94 samples analyzed was 70.5% (range, 30%-100%).

Figure 6

Heat map of receptor tyrosine kinase gene expression in AML and normal myeloid development. The same expression arrays were used to plot data for the RTK genes, using the same approach outlined in Figure 5. The bottom half of the figure is identical to that of Figure 5 and is shown to allow for direct comparisons of the data within samples.