Key Points

  • FLT3-ITDs unexpectedly show junctional N-nucleotides with properties consistent with synthesis by TdT.

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

Abstract

FLT3–internal tandem duplications (FLT3-ITDs) 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 analyzed 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 2 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 one-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 nontemplated 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 < .001), and length characteristics consistent with synthesis by TdT. AML types with high TdT show an increased incidence of FLT3-ITDs (M0; P = .0017). These results point to an unexpected role for the lymphoid enzyme TdT in priming FLT3-ITDs. Although 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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