Rewiring leukemogenic transformation of GATA1s-mediated CH by KANSL1 loss-of-function mutations (Project B4)

通过 KANSL1 功能丧失突变重新连接 GATA1s 介导的 CH 的白血病转化（项目 B4）

• 批准号: 533776703
• 负责人: Professor Dr. Jan-Henning Cornelius Klusmann
• 金额: --
• 依托单位: Klinik für Kinder- und Jugendmedizin
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/533776703?language=en
• 关键词: Rewiring; leukemogenic; transformation; GATA1s; mediated

Clonal hematopoiesis (CH), often associated with age-related somatic mutations, is typically the subject of adult-centric studies. Nevertheless, a similar manifestation of clonal cell expansion is identifiable in children with Down syndrome (DS). Trisomy 21 and in utero acquired GATA1 mutations collectively confer a significant predisposition to myeloid malignancies. Initial phase of this condition mirrors CH as GATA1s mutated cells display a proliferative superiority, leading to preleukemia known as transient abnormal myelopoiesis (TAM). Although the preleukemia commonly goes into remission, approximately 30% of cases progress to myeloid leukemia (ML) triggered by additional, secondary mutations. To study the stepwise evolution of TAM to leukemia (ML-DS), we have recently established a platform to mimic this process using CRISPR mediated gene editing in primary human fetal liver derived stem and progenitor cells. By introducing the GATA1s mutation, alone or in combination with other ML-DS related mutations, cells with one or two hits were generated in vitro. These cells were then xeno-transplanted into humanized immunodeficient mice, providing an opportunity to determine if the secondary mutations were transformative. We assessed a selection of epigenetic factors commonly mutated in ML-DS, and discovered that while factors like CTCF, a transcription factor associated with chromatin looping, were unable to transform GATA1s cells, mutation of either cohesin or KANSL1 did result in the engraftment of human cells within the bone marrow of these mice, which ultimately led to development of leukemia. In this proposal, we will direct our focus to studying the molecular mechanisms by which KANSL1, an essential component of the NSL complex, drives oncogenic transformation of preleukemic GATA1s mutated cells. Through acetylation of H4K8ac and H4K5ac at gene promoters, the NSL complex regulates gene transcription. We will investigate the proteins KANSL1 interacts with in normal and malignant hematopoiesis using mass-spectrometry. Simultaneously, we will generate degron lines by endogenously tagging KANSL1 with a dTAG domain to evaluate changes in the acetylome and affected genes immediately post-protein depletion. Moreover, we will delineate the transcriptome and epigenomic landscape of patients with TAM (sole GATA1s mutation) and ML-DS (concurrent GATA1s and KANSL1 mutations) to shed light on the epigenomic profile in human subjects. Lastly, we will explore whether these changes are reversible and if the employment of HDAC inhibitors could offset the diminished acetylation resulting from KANSL1 mutations. This comprehensive, mechanistic examination will enhance our understanding of how this protein/complex drives evolution of TAM (CH) to ML-DS. These investigations will offer critical insights into the broader understanding of CH in adults, highlighting potential similarities in molecular mechanisms and opening up new avenues for therapeutic interventions

克隆性造血（CH），通常与年龄相关的体细胞突变，通常是以成人为中心的研究的主题。然而，克隆细胞扩增的类似表现在唐氏综合征（DS）儿童中是可识别的。21三体和子宫内获得性GATA 1突变共同赋予骨髓恶性肿瘤的显著易感性。这种情况的初始阶段反映了CH，因为GATA 1突变的细胞表现出增殖优势，导致称为短暂异常骨髓生成（TAM）的白血病前期。虽然白血病前期通常会缓解，但大约30%的病例进展为由额外的继发性突变引发的髓性白血病（ML）。为了研究TAM向白血病（ML-DS）的逐步进化，我们最近建立了一个平台，在原代人胎肝源性干细胞和祖细胞中使用CRISPR介导的基因编辑来模拟这一过程。通过单独或与其他ML-DS相关突变组合引入GATA 1 s突变，在体外产生具有一个或两个命中的细胞。然后将这些细胞异种移植到人源化免疫缺陷小鼠中，提供了确定继发突变是否具有转化性的机会。我们评估了ML-DS中常见突变的表观遗传因子的选择，并发现虽然CTCF（一种与染色质循环相关的转录因子）等因子无法转化GATA 1 s细胞，但粘附素或KANSL 1的突变确实导致人类细胞在这些小鼠的骨髓中植入，最终导致白血病的发展。在这个提议中，我们将把我们的重点放在研究KANSL 1，NSL复合物的一个重要组成部分，驱动白血病前GATA 1 s突变细胞的致癌转化的分子机制。通过在基因启动子处的H4 K8 ac和H4 K5 ac的乙酰化，NSL复合物调节基因转录。我们将研究蛋白质KANSL 1相互作用，在正常和恶性造血使用质谱。同时，我们将通过用dTAG结构域内源性标记KANSL 1来产生降解决定子系，以评估蛋白质耗尽后乙酰组和受影响基因的变化。此外，我们将描绘TAM（唯一GATA 1 s突变）和ML-DS（同时GATA 1 s和KANSL 1突变）患者的转录组和表观基因组景观，以阐明人类受试者的表观基因组特征。最后，我们将探讨这些变化是否是可逆的，以及HDAC抑制剂的使用是否可以抵消KANSL 1突变导致的乙酰化减少。这种全面的机制研究将增强我们对这种蛋白质/复合物如何驱动TAM（CH）进化为ML-DS的理解。这些研究将为更广泛地了解成人CH提供重要的见解，突出分子机制的潜在相似性，并为治疗干预开辟新途径