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. 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).

转录因子EBF1与PAX5在B细胞急性淋巴细胞白血病（B cell acute lymphoblastic leukemia, B-ALL）中频发突变。本研究证实，Pax5+/- × Ebf1+/-复合杂合小鼠可高外显率地发生白血病。类似的结果在Pax5+/- × Ikzf1+/-与Ebf1+/- × Ikzf1+/-小鼠的B-ALL模型，以及Tcf7+/- × Ikzf1+/-小鼠的T细胞白血病模型中均被观察到。
为鉴定可协同Pax5与Ebf1复合杂合性引发白血病的遗传缺陷，本研究开展了睡美人转座子（Sleeping Beauty transposon）筛选实验。该筛选鉴定出多个协同致病靶点，包括Stat5（约65%）与Jak1（约68%）的功能获得性突变（gain-of-function mutation），以及Cblb（61%）与Myb（32%）的功能丧失性突变（loss-of-function mutation）。上述研究结果凸显了JAK/STAT5信号通路（JAK/STAT5 signaling pathway）在协同Pax5与Ebf1复合单倍体不足（compound haploinsufficiency）以驱动B细胞转化中的关键作用。此外，本研究还揭示了Cblb与Myb的功能丧失性突变在B细胞转化中此前未被关注的意外作用。
后续针对野生型（wild-type, WT）、Pax5+/-、Ebf1+/-、Pax5+/- × Ebf1+/-白血病前期细胞、Pax5+/- × Ebf1+/-白血病细胞以及Pax5+/- × Ebf1+/-睡美人转座子白血病细胞的RNA测序（RNA-Sequencing, RNA-Seq）分析显示，PDK1>SGK3>MYC通路存在上调现象；使用PDK1特异性抑制剂处理Pax5+/- × Ebf1+/-白血病细胞，可在体外阻断其增殖。
最后，我们在人类白血病与本研究小鼠B-ALL模型的部分基因中鉴定出了保守的转录变异。综上，B细胞转录因子的复合单倍体不足可能在人类B细胞转化过程中发挥关键作用，同时提示新型PDK1抑制剂或可有效治疗存在此类缺陷的患者。
我们从三组野生型小鼠中分离祖B细胞并混合，共设置3份野生型生物学重复；从5只罹患白血病的Pax5+/- × Ebf1+/-小鼠中分离白血病B细胞，包含5份来自单只个体小鼠的生物学重复。