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

克隆造血（CH）通常与年龄相关的体细胞突变有关，通常是成人中心研究的主题。然而，在唐氏综合症（DS）的儿童中，可以识别出克隆细胞膨胀的类似表现。 Trisome 21和子宫中获得的GATA1突变统称为髓样恶性肿瘤有重大倾向。这种情况的初始阶段反映了CH，因为GATA1S突变的细胞表现出增殖优势，导致核血症被称为瞬时异常骨髓骨髓菌（TAM）。尽管普雷亚血症通常会缓解，但大约30％的病例发展为髓样白血病（ML），这些病例是由额外的次要突变触发的。为了研究TAM到白血病（ML-DS）的逐步演变，我们最近建立了一个平台，使用CRISPR介导的基因编辑在原代人胎儿肝脏衍生的茎和祖细胞中模仿该过程。通过单独或与其他ML-DS相关突变结合引入GATA1S突变，在体外产生了一个或两个命中的细胞。然后将这些细胞的异种转移到人性化的免疫缺陷小鼠中，提供了确定次级突变是否转化的机会。我们评估了通常在ML-DS中突变的表观遗传因子的选择，并发现，尽管CTCF（与染色质循环相关的转录因子）之类的因子无法转化GATA1S细胞，但粘蛋白或KANSL1的突变确实导致了这些小鼠在这些小鼠中的植入，最终导致了Leukemia的骨髓中的骨骼。在此提案中，我们将把重点引向研究KANSL1（NSL复合物的重要组成部分）的分子机制，驱动了Proleukemic GATA1S突变细胞的致癌转化。通过基因启动子的H4K8AC和H4K5AC的乙酰化，NSL复合物调节基因转录。我们将使用质量光谱法研究蛋白质KANSL1与正常和恶性造血相互作用。同时，我们将通过用DTAG结构域对kansl1进行内源标记来评估乙酰基团的变化和受影响的基因立即产生dtag域，从而生成DEGRON线。此外，我们将描述TAM（唯一GATA1S突变）和ML-DS（同时GATA1S和KANSL1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可逆，以及HDAC抑制剂的使用是否可以抵消KANSL1突变导致的乙酰化降低。这种全面的机械检查将增强我们对这种蛋白质/复合物如何驱动TAM（CH）向ML-DS进化的理解。这些调查将为成人对CH的更广泛理解提供关键的见解，突出了分子机制的潜在相似之处，并为治疗干预开辟了新的途径