Inversion inv(16) or translocation t(16;16) and translocation t(8;21) are recurring rearrangements in acute myeloid leukemia (AML), which result in the fusion genes CBFB/MYH11 or RUNX1/RUNX1T1, respectively. These rearrangements are found in 15–20% of adult de novo AML cases and are associated with favourable prognosis. CBFB and RUNX1 form the core binding factor (CBF), which is a transcription factor essential for normal hematopoiesis and myeloid development. By disrupting the physiological transcription factor activity of CBF the fusion proteins causes repression of the CBF-target genes resulting in a block of differentiation. Since expression of CBFB/MYH11 or RUNX1/RUNX1T1 on their own is not sufficient to cause leukemia it is likely that additional mutations are required for malignant transformation.
To systematically identify mutations which may collaborate with CBFB/MYH11 during leukemogenesis we performed exome sequencing of an AML sample with an inv(16). The sample was selected based on availability and absence of known additional genetic alterations. By comparing the AML exome sequence with the exome sequence of a remission sample from the same patient we were able to identify leukemia-specific sequence variants as described previously (Greif et al., 2012, Blood). Using this approach we found an N676K mutation in the ATP-binding domain (TKD1) of the fms-related tyrosine kinase 3 (FLT3) gene. Mutations affecting N676 resulting in variable amino acid changes (N676D or N676S) were initially discovered in a screen for resistance to tyrosine kinase inhibitors (TKI) in FLT3 internal tandem duplication (ITD) expressing Ba/F3 cells (Cools et al., 2004, Cancer Res). An N676K point mutation has been reported in a cytogenetically normal (CN) AML patient with FLT3-ITD and TKI-resistance (Heidel et al., 2006, Blood). In contrast, our patient with inv(16) and the FLT3 N676K did not carry an additional FLT3-ITD. In a cohort of 69 patients with inv(16) we found a total of 4 patients with FLT3 N676K mutations (4/69, 6%). In 14 patients with t(16;16) and in 36 patients with t(8;21) we identified one patient each with FLT3 N676K (1/14, 7% and 1/36, 3%). Thus, the overall mutation frequency for patients with CBFB/MYH11 rearrangement was 6% (5/83). None of the CBF AML patients with FLT3 N676K mutation had an additional FLT3-ITD. In 90 CN-AML patients we detected only a single FLT3 N676K mutation and the affected patient had a concurrent FLT3-ITD. We are currently testing the frequency of FLT3 N676K mutations in independent CBF-AML cohorts.
To test the transforming potential we expressed the FLT3 N676K mutant in Ba/F3 cells. As controls we expressed FLT3 wild type (WT), FLT3 mutants D835Y or ITD in parallel. Cell surface expression of N676K was similar to WT, but increased compared to D835Y and ITD. Cell proliferation assays were done in presence and absence of IL-3 or FLT3 ligand (FL). FLT3 N676K leads to IL-3 and FL independent cell growth reaching 25% of IL-3 mediated growth. FLT3 inhibition by AC220 or PKC412 abrogates this proliferation, but N676K is slightly more resistant to inhibition than ITD. In contrast to ITD expression that results in STAT5 phosphorylation, N676K expression leads to phosphorylation of MAPK and AKT.
Gene expression profiling of patients with inv(16) revealed that patients with additional FLT3 N676K mutation show a significant enrichment of gene sets including ubiquitin mediated proteolysis, adherens junction and JAK/STAT signaling pathway.
According to a structural model, the N676K mutation stabilizes the fold of the kinase domain between the juxtamembrane domain (JMD) and a hydrophobic pocket that is the target of FLT3 inhibitors. N676K mutations could therefore reduce the autoinhibition of FLT3 by the JMD and also negatively affect the binding affinity of FLT3 inhibitors.
Ours is the first report of recurring FLT3 N676 mutations in the absence of FLT3-ITD. Our findings point towards a specific association of FLT3 N676K mutations with CBF leukemia. Based on our functional assays N676K acts as a gain-of-function mutation. Although FLT3 has been known for more than a decade to be mutated in one third of AML patients, it appears that the spectrum of FLT3 mutations is still not fully understood, in particular, in defined cytogenetic subgroups of AML. Unbiased mutation screening by exome sequencing allows the detection of novel sequence variations even in extensively studied genes.
Krebs:Illumina: Honoraria. Greif:Illumina: Honoraria.
Asterisk with author names denotes non-ASH members.