Key Points

  • FLT3-ITDs unexpectedly show junctional N-nucleotides with properties consistent with synthesis by terminal deoxyucleotidyl transferase.

  • Off-target terminal deoxyucleotidyl transferase activity in AML is proposed to promote FLT3-ITD formation by priming replication slippage.

FLT3-ITDs (FLT3-internal tandem duplications) are prognostic driver mutations found in acute myeloid leukemia (AML). Although these short duplications occur in 25% of AML patients, little is known about the molecular mechanism underlying their formation. Understanding the origin of FLT3-ITDs would advance our understanding of the genesis of AML. We analysed the sequence and molecular anatomy of 300 FLT3-ITDs to address this issue, including 114 ITDs with additional nucleotides of unknown origin located between the two copies of the repeat. We observed anatomy consistent with replication slippage, but could only identify the germline microhomology (1-6 bp) anticipated to prime such slippage in a third of FLT3-ITDs. We explain the paradox of the "missing" microhomology in the majority of FLT3-ITDs through occult microhomology: specifically, by priming through use of non-templated nucleotides (N-nucleotides) added by terminal deoxynucleotidyl transferase (TdT). We suggest that TdT-mediated nucleotide addition in excess of that required for priming creates N-regions at the duplication junctions, explaining the additional nucleotides observed at this position. FLT3-ITD N-regions have a G/C content (66.9%), dinucleotide composition (P < 0.001) and length characteristics consistent with synthesis by TdT. AML types with high TdT show an increased incidence of FLT3-ITDs (M0, P = 0.0017). These results point to an unexpected role for the lymphoid enzyme TdT in priming FLT3-ITDs. While the physiological role of TdT is to increase antigenic diversity through N-nucleotide addition during V(D)J recombination of IG/TCR genes, here we propose that illegitimate TdT activity makes a significant contribution to the genesis of AML.

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