Identification of genes cooperating with PML/RAR in leukemogenesis. Mus musculus

The oncogenic fusion protein PML/RAR is expressed in most cases of acute promyelocytic leukemia (APL). In transgenic models, PML/RAR is able to induce a preleukemic state. However, it is not sufficient to induce leukemia, which develops with low frequency (~50%) and long latency (about 1 year). These data indicate that secondary genetic cooperating alterations are required in order to induce leukemia. In an effort to identify the genes that cooperate in the leukemogenesis PML/RAR-dependent, we performed an insertional mutagenesis screening infecting the PML/RAR KI mice with Moloney Murine Leukemia Virus (MLV). The infection of the PML/RAR KI mice significantly accelerated the onset (~6 months) and increased the penetrance (~75% of infected animals) of the leukemia. To identify genes mutated by the proviral integrations, flanking sequences of the retrovirus insertions were cloned by IPCR and analysed by parallel sequencing. We sequenced DNAs from the spleen of 48 independent leukemic mice. After filtering, ~133,000 reads were univocally mapped onto the mouse genome. Of them, ~131,000 were present more than once in the DNA from the same mouse and produced a total of 1,707 independent genomic insertions (retroviral insertion sites - RISs). To identify chromosomal regions that are frequently mutated by retrovirus insertions (common insertion sites; CISs), we evaluated the density of RISs distribution over the mouse genome, using statistical approaches that calculate the significance of RISs occurrence (p<0.05) within specific genomic regions (Monte Carlo simulation or Kernel Convolution). The two different approaches identified the same set of 221 CISs. Finally, we identified 271 genes putatively targeted by retroviral insertions in the 221 CISs. These putative targets appeared to be enriched in genes identified as relevant cancer-genes in different tumor types. Moreover, from a biological point of view, our screening was performed in saturating conditions. Therefore, to our knowledge, our is the first report of an exhaustive list of virtually all the mouse genes that functionally cooperate with PML/RAR in the induction of APL. Overall design: Insertional mutagenesis screen by infection with MLV of PML/RAR KI transgenic mice (analysis of 48 independent leukemias). PML/RAR KI newborn mice were injected i.p. with viral supernatant containing high titer MLV. At signs of disease mice were sacrificed and underwent necropsy. Infiltrated organs were collected and frozen.

致癌融合蛋白PML/RAR在大多数急性早幼粒细胞白血病（acute promyelocytic leukemia, APL）病例中表达。在转基因模型中，PML/RAR能够诱导前白血病状态，但不足以直接诱发白血病：白血病的发生频率极低（约50%）且潜伏期长达约1年。上述结果表明，诱发白血病需要继发的遗传学协同改变。

为鉴定与PML/RAR依赖性白血病发生协同作用的基因，我们通过莫洛尼鼠白血病病毒（Moloney Murine Leukemia Virus, MLV）感染PML/RAR基因敲入（knock-in, KI）小鼠，开展了插入诱变筛选。对该类小鼠的感染显著加速了白血病发病（潜伏期约6个月），并提高了白血病的外显率（受感染动物中约75%发病）。

为鉴定被病毒整合诱变的基因，我们通过反向PCR（inverse PCR, IPCR）克隆了逆转录病毒整合位点的侧翼序列，并通过平行测序进行分析。我们对48只独立白血病小鼠的脾脏DNA进行了测序。过滤后，约133,000条读段被唯一比对至小鼠基因组。其中约131,000条读段在同一只小鼠的DNA中重复出现，共产生1,707个独立的基因组整合位点（retroviral insertion sites, RISs）。

为鉴定被逆转录病毒整合高频靶向的染色体区域（即常见整合位点，common insertion sites, CISs），我们采用统计方法评估了RISs在小鼠基因组中的分布密度，通过蒙特卡洛模拟（Monte Carlo simulation）或核卷积（Kernel Convolution）计算特定基因组区域内RISs出现的显著性（p<0.05）。两种不同的方法均鉴定出221个CISs。最终，我们在这221个CISs中鉴定出271个推测被逆转录病毒整合靶向的基因。

这些推测的靶基因显著富集于不同肿瘤类型中被鉴定为相关癌基因的基因集。此外，从生物学角度来看，本研究的筛选是在饱和条件下完成的。因此，据我们所知，本研究首次报道了在APL诱发过程中与PML/RAR存在功能协同作用的几乎全部小鼠基因的详尽列表。

整体实验设计：通过莫洛尼鼠白血病病毒（MLV）感染PML/RAR基因敲入（KI）转基因小鼠开展插入诱变筛选（分析48例独立白血病样本）。将PML/RAR基因敲入（KI）新生小鼠腹腔注射高滴度MLV病毒上清液。当小鼠出现疾病症状时，将其处死并进行尸检，收集受浸润的器官并冻存。