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

克隆性造血(CH)通常与年龄相关的体细胞突变有关，通常是以成人为中心的研究的主题。然而，在唐氏综合征(DS)的儿童中也可以发现类似的克隆性细胞扩张的表现。21三体和宫内获得性GATA1突变共同赋予了髓系恶性肿瘤的显著易感性。这种情况的初始阶段反映了CHAS GATA1突变细胞显示出增殖优势，导致白血病前期称为一过性异常骨髓生成()。虽然白血病前期通常处于缓解状态，但大约30%的病例进展为髓系白血病(ML)，这是由额外的继发性突变引发的。为了研究向白血病(ML-DS)的逐步演变，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肝来源的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，在体外产生一到两次命中的细胞。这些细胞然后被异种移植到人源化免疫缺陷小鼠中，提供了一个确定次级突变是否具有变革性的机会。我们评估了一组在ML-DS中常见的表观遗传因子突变，发现虽然像CTCF这样的与染色质循环相关的转录因子无法转化GATA1细胞，但粘附素或KANSL1的突变确实会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。在这项提案中，我们将把重点放在研究NSL复合体的重要组成部分KANSL1驱动白血病前期GATA1突变细胞的致癌转化的分子机制上。通过H4K8ac和H4K5ac在基因启动子上的乙酰化，NSL复合体调节基因转录。我们将用质谱仪研究KANSL1在正常和恶性造血中相互作用的蛋白。同时，我们将通过用Dtag结构域内源标记KANSL1来产生降解线，以评估蛋白质耗尽后立即发生的乙酰体和受影响基因的变化。此外，我们将描绘(单一GATA1突变)和ML-DS(同时存在GATA1和KANSL1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。这一全面的、机械性的检查将加强我们对这种蛋白质/复合体如何推动(CH)向ML-DS进化的理解。这些研究将为更广泛地了解成年人的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