IM identifies known and novel collaborators with Flt3-ITD. (A) Genome-wide distribution of transposon integration site densities in 38 FTD-IM leukemic samples (only insertions reported by ≥20 reads were included), showing previously reported local hopping around the GrOnc transposon donor site on chromosome 19 (top)¹²  and smaller peaks at genes such as Notch1, Ash1l, Lincpint, Ets1, Ikzf1, Runx1, Erg, and Setbp1 (bottom). (B) Grid displaying transposon CISs and associated leukemia types in FTD-IM leukemia samples (n = 38). CIS lists were generated separately by TAPDANCE and kernel convolution analyses and combined. Read counts were normalized for each leukemia and ranked 0 to 1 (see key and “Methods”), to make use of the quantitative nature of QiSeq mapping and depict whether a particular insertion is in the main leukemic clone or in a subclone. (C) Overlap of CISs in different IM cohorts using the GrOnc transposon: FTD, Npm1^cA/+,¹²  and WT (with the WT-IM cohort combined with the equivalent Npm1^+/+ control cohort). (D) RT-PCR validation of transposon insertions in intron 1 of Setbp1 from 3 different leukemias (including sample 11 with multiple insertions) and location of all Setbp1 insertions in our FTD-IM cohort (n = 38). A chromatogram shows the transposon-initiated transcript splices into exon 2 of Setbp1 and upstream of its native ATG initiation codon, predicting overexpression of the full-length SETBP1 protein. (E) Significantly increased Setbp1 mRNA expression in Flt3^ITD/+/Setbp1^IM+ leukemias compared with WT lineage-negative cells. ****P < .0001. (F) RT-PCR validation of transposon insertions in exon 5 of the Runx1 gene and location of all Runx1 insertions in our FTD-IM cohort (n = 38). The predicted RUNX1 protein truncation closely mimics the effect of hotspot mutations in the runt homology domain (RHD) seen in human myeloid cancers (mouse and human RUNX1 amino acid sequences are identical between amino acids 60 and 240). TAD, transactivating domain. (G) Comparison of SETBP1 expression between 3 FLT3-ITD⁺ AML subgroups showing that, compared with AMLs with class-defining mutations, SETBP1 expression is higher in cases lacking such mutations and also those harboring a mutant RUNX1. Mean ± standard deviation. *P < .05, ****P < .00005; 1-way analysis of variance. (H) SETBP1 expression in AMLs without (green bars) and with (other color bars) class-defining mutations in the Leucegene cohort. Within the latter group, SETBP1 expression was higher in RUNX1-mutant and lower in NPM1-mutant AMLs.