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

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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