Retroviral Integration Mutagenesis in Mice and Comparative Analysis in Human AML Identify Reduced PTP4A3 Expression as a Prognostic Indicator

Acute myeloid leukemia (AML) results from multiple genetic and epigenetic aberrations, many of which remain unidentified. Frequent loss of large chromosomal regions marks haplo-insufficiency as one of the major mechanisms contributing to leukemogenesis. However, which haplo-insufficient genes (HIGs) are involved in leukemogenesis is largely unknown and powerful experimental strategies aimed at their identification are currently lacking. Here, we present a new approach to discover HIGs, using retroviral integration mutagenesis in mice in which methylated viral integration sites and neighbouring genes were identified. In total we mapped 6 genes which are flanked by methylated viral integration sites (mVIS). Three of these, i.e., Lrmp, Hcls1 and Prkrir, were up regulated and one, i.e., Ptp4a3, was down regulated in the affected tumor. Next, we investigated the role of PTP4A3 in human AML and we show that PTP4A3 expression is a negative prognostic indicator, independent of other prognostic parameters. In conclusion, our novel strategy has identified PTP4A3 to potentially have a role in AML, on one hand as a candidate HIG contributing to leukemogenesis in mice and on the other hand as a prognostic indicator in human AML. We designed a new strategy to specifically identify DNA methylated proviral integrations on a large scale by combining methylated DNA immunoprecipitation (MeDIP), inverse PCR and promoter array hybridization. We used 6 murine leukemia samples from a previous screen that were induced by injecting Gr 1.4 MLV into newborn mice.

急性髓系白血病（Acute myeloid leukemia, AML）由多种遗传与表观遗传畸变引发，其中多数畸变的分子机制尚未明确。大片段染色体区域的频繁缺失提示单倍体不足（haplo-insufficiency）是促成白血病发生（leukemogenesis）的主要机制之一。然而，具体哪些单倍体不足基因（haplo-insufficient genes, HIGs）参与了白血病发生过程，目前仍知之甚少，且目前缺乏用于高效鉴定这类基因的实验策略。本研究提出一种全新的鉴定HIGs的方法：利用小鼠体内的逆转录病毒整合诱变（retroviral integration mutagenesis）技术，对甲基化病毒整合位点及其邻近基因进行鉴定。最终我们共定位到6个受甲基化病毒整合位点（methylated viral integration sites, mVIS）侧翼锚定的基因，其中Lrmp、Hcls1与Prkrir在受影响的肿瘤组织中呈上调表达，而Ptp4a3则呈下调表达。随后，我们针对人类AML中PTP4A3的功能展开研究，结果证实PTP4A3的表达水平可作为独立于其他预后参数的阴性预后标志物。综上，本研究的新型策略成功鉴定出PTP4A3可能在AML中发挥双重作用：一方面可作为小鼠白血病发生过程中的候选单倍体不足基因，另一方面可作为人类AML的预后标志物。我们通过结合甲基化DNA免疫沉淀（methylated DNA immunoprecipitation, MeDIP）、反向PCR（inverse PCR）与启动子芯片杂交（promoter array hybridization）技术，设计出一套可大规模特异性鉴定DNA甲基化前病毒整合位点的全新方案。本研究使用了此前通过向新生小鼠注射Gr 1.4型小鼠白血病病毒（Gr 1.4 MLV）诱导获得的6份小鼠白血病样本。