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

克隆造血（CH）通常与年龄相关的体细胞突变有关，是典型的以成人为中心的研究主题。然而，克隆细胞扩增在唐氏综合症（DS）儿童中也有类似的表现。21三体和子宫内获得的GATA1突变共同赋予髓系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GATA1s突变的细胞表现出增殖优势，导致称为短暂性异常骨髓生成（TAM）的白血病前期。虽然白血病前期通常会进入缓解期，但大约30%的病例会发展为髓性白血病（ML），这是由额外的继发性突变引发的。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，利用CRISPR介导的基因编辑在原代人胎儿肝源性干细胞和祖细胞中模拟这一过程。通过引入GATA1s突变，单独或与其他ML-DS相关突变结合，在体外产生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的免疫缺陷小鼠中，为确定继发性突变是否具有变革性提供了机会。我们评估了ML-DS中常见突变的表观遗传因子的选择，发现虽然CTCF（一种与染色质环相关的转录因子）等因子不能转化GATA1s细胞，但内聚素或KANSL1的突变确实会导致这些小鼠骨髓内的人类细胞植入，最终导致白血病的发展。在本课题中，我们将重点研究NSL复合体的重要组成部分KANSL1驱动白血病前期GATA1s突变细胞的致癌转化的分子机制。NSL复合体通过在基因启动子处乙酰化H4K8ac和H4K5ac，调控基因转录。我们将使用质谱法研究KANSL1在正常和恶性造血过程中与之相互作用的蛋白。同时，我们将通过内源性标记带有dTAG结构域的KANSL1来产生退化系，以评估蛋白质耗尽后乙酰酶和受影响基因的变化。此外，我们将描述TAM（单一GATA1s突变）和ML-DS （GATA1s和KANSL1并发突变）患者的转录组和表观基因组图谱，以阐明人类受试者的表观基因组图谱。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突变导致的乙酰化减少。这项全面的、机制的研究将增强我们对这种蛋白质/复合物如何驱动TAM （CH）向ML-DS的进化的理解。这些研究将为更广泛地了解成人CH提供关键见解，突出分子机制的潜在相似性，并为治疗干预开辟新的途径

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