Pathogenesis of ETV6-Related Acute Lymphoblastic Leukemia

项目总结 作为肿瘤发生的始动遗传事件，癌症易感基因中的生殖系变异扰乱细胞 生长和分化，并为恶变奠定了基础。因此，对癌症的研究 易感基因及其相关遗传综合征提供了对正常生理学的关键见解 和癌症生物学。通过调查常染色体显性遗传的血小板减少症和B- 急性淋巴细胞白血病(B-ALL)，我们和其他人发现了影响ETV6的致病胚系变异， 编码Ets变异体6转录抑制因子的基因。随后，我们对生殖系样本进行了测序 来自4,405名患有B-ALL的儿童，在0.5%的患者中检测到类似的变异。进一步的关联研究 显示与134,187相比，致病性ETV6变异在所有队列中显著增加了22.94倍 GnomAD中非全部对照(P=2.2×10-16)。这些数据有力地支持了我们的总体假设 生殖系ETV6变异体易患儿童ALL。为了更好地了解生殖系ETV6变异株如何 促进白血病的发生，我们使用体外试验来询问它们对编码的功能的影响 ETV6蛋白。值得注意的是，所研究的每一种致病性ETV6变异体都显著降低了ETV6的转录 抑制子活性，ETV6 DNA结合能力受损，ETV6错误定位于细胞质。同时，我们 产生了一种新的小鼠模型，该模型含有一个与B-ALL相关的反复出现的ETV6变异体(ETV6R355X)。初步 对Etw6R355X/+小鼠的研究表明，在早期B细胞发育和造血方面存在显著的干扰 干细胞和祖细胞(HSPC)的数量和功能。最后，我们获得了同源诱导的多能干细胞。 含有致病ETV6变异的细胞(IPSC)，可产生发育不良的巨核细胞，类似于 ETV6变异阳性患者。基于这些结果，我们假设ETV6变异株容易患上 这一切都是通过扰乱损害造血发育的关键转录程序来实现的。在这份提案中， 我们将利用我们独特的模型系统来严格检验这一假设。在目标1中，我们将描述 携带或不携带ETV6致病胚系的人和小鼠的造血室 变种。我们将检测WT和ETV6变异阳性的IPSCs或小鼠造血祖细胞 沿着不同的血统适当地区分。确定生殖系ETV6变异如何影响基因表达 在培养造血祖细胞方面，在目标2中，我们将使用RNA测序和ATAC测序来探索 小鼠和IPSC来源的B祖细胞的转录景观和鉴定可能的ETV6靶基因 以及携带WT或变种ETV6的HSPC。最后，在目标3中，我们将进行全面的全基因组和 对B-ALL样本进行RNA测序以阐明与生殖系一致的体细胞遗传损伤 ETV6变异体驱动B细胞白血病的发生。我们将使用体外和体内方法来评估白血病- 促进了这些第二次命中的效果。这个项目将为生殖系的影响提供新的见解。 最常见的儿童癌症B-ALL的造血和发育的遗传变异。

PROJECT SUMMARY As the initiating genetic events in tumorigenesis, germline variants in cancer predisposing genes perturb cell growth and differentiation and set the stage for malignant transformation. Accordingly, the study of cancer predisposing genes and their associated hereditary syndromes provides critical insights into normal physiology and cancer biology. By investigating families with autosomal dominant transmission of thrombocytopenia and B- acute lymphoblastic leukemia (B-ALL), we and others identified pathogenic germline variants affecting ETV6, the gene encoding the ETS variant 6 transcriptional repressor. Subsequently, we sequenced germline samples from 4,405 children with B-ALL and detected similar variants in 0.5% of patients. Further association studies revealed a significant 22.94-fold enrichment of pathogenic ETV6 variants in this ALL cohort compared to 134,187 non-ALL controls in gnomAD (P= 2.2 × 10-16). These data firmly support our overall premise that pathogenic germline ETV6 variants predispose to childhood ALL. To better understand how germline ETV6 variants promote leukemogenesis, we used in vitro assays to interrogate their effects on the functions of the encoded ETV6 protein. Notably, each of the pathogenic ETV6 variants examined significantly reduced ETV6 transcription repressor activity, impaired ETV6 DNA binding capacity and mis-localized ETV6 to the cytoplasm. In parallel, we generated a novel mouse model harboring a recurrent B-ALL-associated Etv6 variant (Etv6R355X). Preliminary studies of Etv6R355X/+ mice reveal significant perturbations in early B cell development, as well as hematopoietic stem and progenitor cell (HSPC) number and function. Finally, we generated isogenic induced pluripotent stem cell (iPSC) lines harboring pathogenic ETV6 variants, which generate dysplastic megakaryocytes, similar to ETV6 variant positive patients. Based on these results, we hypothesize that ETV6 variants predispose to ALL by perturbing key transcriptional programs that impair hematopoietic development. In this proposal, we will make use of our unique model systems to rigorously test this hypothesis. In Aim 1, we will characterize the hematopoietic compartments of humans and mice that do or do not harbor pathogenic germline ETV6 variants. We will examine whether WT and ETV6 variant-positive iPSCs or mouse hematopoietic progenitors properly differentiate along various lineages. To establish how germline ETV6 variants influence gene expression in developing hematopoietic progenitors, in Aim 2 we will use RNA-sequencing and ATAC-sequencing to explore transcriptional landscapes and identify putative ETV6 target genes in mouse and iPSC-derived B progenitors and HSPC harboring WT or variant ETV6. Finally, in Aim 3 we will perform comprehensive whole genome and RNA-sequencing of B-ALL samples to elucidate the somatic genetic lesions that function in concert with germline ETV6 variants to drive B-leukemogenesis. We will use in vitro and in vivo approaches to assess the leukemia- promoting effects of these second hits. This project will provide new insights into the influence of germline genetic variation on hematopoiesis and development of B-ALL, the most common childhood cancer.