Identification of mutations that cooperate with defects in B cell transcription factors to initiate leukemia [variation]

The transcription factors EBF1 and PAX5 are frequently mutated in B cell acute lymphoblastic leukemia (B-ALL). We demonstrate that Pax5+/- x Ebf1+/- compound heterozygous mice develop leukemia with high penetrance. Similar results were seen in Pax5+/- x Ikzf1+/- and Ebf1+/- x Ikzf1+/- mice for B-ALL, or in Tcf7+/- x Ikzf1+/- mice for T cell leukemia. To identify genetic defects that cooperate with Pax5 and Ebf1 compound heterozygosity to initiate leukemia, we carried out a Sleeping Beauty transposon screen. This screen identified a number of cooperating partners including gain-of-function mutations in Stat5 (~65%) and Jak1 (~68%), as well as loss-of-function mutations in Cblb (61%) and Myb (32%). These findings highlight the key role of JAK/STAT5 signaling in cooperating with Pax5 and Ebf1 compound haploinsufficiency to drive B cell transformation. Moreover, these studies pointed to unexpected roles for loss of function mutations in Cblb and Myb in B cell transformation. Subsequent RNA-Seq studies on WT, Pax5+/-, Ebf1+/-, Pax5+/- x Ebf1+/- pre-leukemic, Pax5+/- x Ebf1+/- leukemic cells and Pax5+/- x Ebf1+/- Sleeping Beauty leukemic cells demonstrated upregulation of a PDK1>SGK3>MYC pathway; treatment of Pax5+/- x Ebf1+/- leukemias with PDK1 specific inhibitors blocked their proliferation in vitro. Finally, we identified conserved transcriptional variation in a subset of genes between human leukemias and our mouse B-ALL models. Thus, compound haploinsufficiency for B cell transcription factors likely plays a critical role in transformation of human B cells and suggest that newly developed PDK1 inhibitors may be effective for treating patients characterized by such defects. Overall design: Progenitor B cells were extracted from three groups of wild-type mice and pooled (three WT replicates). Leukemic B cells were extracted from Pax5 x Ebf1 mice with leukemia (five replicates from five individual mice).