Advances in cancer genomics have revealed genomic classes of acute myeloid 
leukemia (AML) characterized by class-defining mutations, such as chimeric 
fusion genes or in genes such as NPM1, MLL, and CEBPA. These class-defining 
mutations frequently synergize with internal tandem duplications in FLT3 
(FLT3-ITDs) to drive leukemogenesis. However, ∼20% of FLT3-ITD-positive AMLs 
bare no class-defining mutations, and mechanisms of leukemic transformation in 
these cases are unknown. To identify pathways that drive FLT3-ITD mutant AML in 
the absence of class-defining mutations, we performed an insertional mutagenesis 
(IM) screening in Flt3-ITD mice, using Sleeping Beauty transposons. All mice 
developed acute leukemia (predominantly AML) after a median of 73 days. Analysis 
of transposon insertions in 38 samples from Flt3-ITD/IM leukemic mice identified 
recurrent integrations at 22 loci, including Setbp1 (20/38), Ets1 (11/38), Ash1l 
(8/38), Notch1 (8/38), Erg (7/38), and Runx1 (5/38). Insertions at Setbp1 led 
exclusively to AML and activated a transcriptional program similar, but not 
identical, to those of NPM1-mutant and MLL-rearranged AMLs. Guide RNA targeting 
of Setbp1 was highly detrimental to Flt3ITD/+/Setbp1IM+, but not to 
Flt3ITD/+/Npm1cA/+, AMLs. Also, analysis of RNA-sequencing data from hundreds of 
human AMLs revealed that SETBP1 expression is significantly higher in FLT3-ITD 
AMLs lacking class-defining mutations. These findings propose that SETBP1 
overexpression collaborates with FLT3-ITD to drive a subtype of human AML. To 
identify genetic vulnerabilities of these AMLs, we performed genome-wide 
CRISPR-Cas9 screening in Flt3ITD/+/Setbp1IM+ AMLs and identified potential 
therapeutic targets, including Kdm1a, Brd3, Ezh2, and Hmgcr. Our study gives new 
insights into epigenetic pathways that can drive AMLs lacking class-defining 
mutations and proposes therapeutic approaches against such cases.