小儿急性淋巴细胞性白血病（ALL）是最常见的小儿血液恶性肿瘤，其中ETV6::RUNX1是最常见的易位，其确切发病机制尚不清楚。IGF2BP1（胰岛素样生长因子2结合蛋白1）是一个发现在ETV6::RUNX1阳性B-ALL中特异性过表达的胎儿肿瘤RNA结合蛋白。在本研究中，我们研究了IGF2BP1在白血病发生中的机制角色以及其与ETV6::RUNX1融合蛋白之间的协同作用。使用实时逆转录定量PCR（RT-qPCR）分析了患者骨髓RNA中的基因表达。使用CRISPR-Cas9基于慢病毒载体创建了敲除细胞系。使用Illumina平台进行了RNA-Seq和RNA免疫共沉淀测序（RIP-Seq）分析IGF2BP1沉降后的结果。通过转播病毒超表达供体造血干细胞，并使用骨髓移植模型对受体进行致死性辐射，进行了小鼠实验。我们观察到在印度小儿ALL队列（n=167）中ETV6::RUNX1阳性患者中IGF2BP1的特异性过表达，与泼尼松抵抗力呈正相关。在多个ETV6::RUNX1阳性B-ALL细胞系中，IGF2BP1表达对肿瘤细胞存活至关重要。对Reh细胞系中IGF2BP1敲除后的转录组测序和IGF2BP1沉降后的RIP-Seq结果进行综合分析表明，IGF2BP1靶向多种促肿瘤信号通路，包括TNFα/NFκB和PI3K-Akt信号通路。这些信号通路在我们中心的原发性ETV6::RUNX1阳性B-ALL患者样本以及公共B-ALL患者数据集中也发生了失调。IGF2BP1与ETV6::RUNX1融合转录本结合并稳定其自身。这种正反馈环路导致多个致癌信号通路的持续紊乱。在小鼠骨髓中强制共表达ETV6::RUNX1和IGF2BP1导致骨髓细胞过度增殖，其特征是多系前体细胞扩张和强烈的Ki67阳性。这种白血前体病变表型证实了它们在体内的协同作用。明显观察到过表达ETV6::RUNX1和IGF2BP1的细胞克隆扩张。这些小鼠还出现脾肿大，表明发生了骨髓外造血。我们的数据表明，ETV6::RUNX1融合蛋白和RNA结合蛋白IGF2BP1在B-ALL中激活多个致癌信号通路，使IGF2BP1和这些信号通路成为有吸引力的治疗靶点和生物标志物。© 2023. 意大利国家癌症研究所“Regina Elena”。

Acute lymphoblastic leukemia (ALL) is the most common pediatric hematological malignancy, with ETV6::RUNX1 being the most prevalent translocation whose exact pathogenesis remains unclear. IGF2BP1 (Insulin-like Growth Factor 2 Binding Protein 1) is an oncofetal RNA binding protein seen to be specifically overexpressed in ETV6::RUNX1 positive B-ALL. In this study, we have studied the mechanistic role of IGF2BP1 in leukemogenesis and its synergism with the ETV6::RUNX1 fusion protein.Gene expression was analyzed from patient bone marrow RNA using Real Time RT-qPCR. Knockout cell lines were created using CRISPR-Cas9 based lentiviral vectors. RNA-Seq and RNA Immunoprecipitation sequencing (RIP-Seq) after IGF2BP1 pulldown were performed using the Illumina platform. Mouse experiments were done by retroviral overexpression of donor HSCs followed by lethal irradiation of recipients using a bone marrow transplant model.We observed specific overexpression of IGF2BP1 in ETV6::RUNX1 positive patients in an Indian cohort of pediatric ALL (n=167) with a positive correlation with prednisolone resistance. IGF2BP1 expression was essential for tumor cell survival in multiple ETV6::RUNX1 positive B-ALL cell lines. Integrated analysis of transcriptome sequencing after IGF2BP1 knockout and RIP-Seq after IGF2BP1 pulldown in Reh cell line revealed that IGF2BP1 targets encompass multiple pro-oncogenic signalling pathways including TNFα/NFκB and PI3K-Akt pathways. These pathways were also dysregulated in primary ETV6::RUNX1 positive B-ALL patient samples from our center as well as in public B-ALL patient datasets. IGF2BP1 showed binding and stabilization of the ETV6::RUNX1 fusion transcript itself. This positive feedback loop led to constitutive dysregulation of several oncogenic pathways. Enforced co-expression of ETV6::RUNX1 and IGF2BP1 in mouse bone marrow resulted in marrow hypercellularity which was characterized by multi-lineage progenitor expansion and strong Ki67 positivity. This pre-leukemic phenotype confirmed their synergism in-vivo. Clonal expansion of cells overexpressing both ETV6::RUNX1 and IGF2BP1 was clearly observed. These mice also developed splenomegaly indicating extramedullary hematopoiesis.Our data suggest a combined impact of the ETV6::RUNX1 fusion protein and RNA binding protein, IGF2BP1 in activating multiple oncogenic pathways in B-ALL which makes IGF2BP1 and these pathways as attractive therapeutic targets and biomarkers.© 2023. Italian National Cancer Institute ‘Regina Elena’.