A Genetic Disorder Reveals a Hematopoietic Stem Cell Regulatory Network Co-Opted in Leukemia

The molecular regulation of human hematopoietic stem cell (HSC) self-renewal and maintenance is of substantial interest, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. To better discern in vivo HSC function in humans, we have studied a rare genetic disorder due to MECOM haploinsufficiency that is characterized by neonatal aplastic anemia with an early-onset absence of HSCs in vivo.

To establish a faithful model of MECOM haploinsufficiency, we performed CRISPR/Cas9 editing of MECOM in human hematopoietic stem and progenitor cells (HSPCs) and achieved predominantly heterozygous editing in phenotypic long-term (LT)-HSCs, as substantiated through both bulk and single cell assessments. Following MECOM editing, HSPCs showed a significant reduction in the number of phenotypic LT-HSCs as well as multipotential progenitor colonies in vitro, and had impaired engraftment following xenotransplantation into immunodeficient and Kit mutant mice. Next, we sought to use this model of MECOM haploinsufficiency to define the regulatory networks driven by MECOM that are critical for HSC maintenance. We therefore performed single-cell RNA sequencing on several thousand phenotypic LT-HSCs following MECOM editing and identified a list of 724 subtly but significantly differentially expressed genes compared to controls, including 322 genes that are downregulated after MECOM perturbation. Given the profound phenotypic effects associated with loss of MECOM and dysregulation of these gene sets, we sought to identify other cooperating factors that control the MECOM dependent gene network which underlies HSC self-renewal. To do so, we used integrative genomic approaches involving accessible chromatin and gene expression correlations, as well as long range chromatin interaction data across human hematopoiesis, to comprehensively define associations between genes and putative regulatory elements. Inspection of the nominated cis-regulatory elements controlling genes impacted by MECOM perturbation revealed significant enrichment for motifs and chromatin occupancy by several key cooperating transcription factors, including RUNX1, FLI1, and GATA2.

In addition to the identification of cooperating transcription factors, we also discovered a strong binding motif enrichment for, and chromatin occupancy by, CTCF. CTCF is crucial to enable differentiation of LT-HSCs, and we found that MECOM regulated genes frequently had associated cis-regulatory elements that were occupied by CTCF. Moreover, these occupied cis-elements became more highly enriched for binding during hematopoietic differentiation. Chromatin conformation analysis revealed that the MECOM regulated genes with cis-elements bound by CTCF underwent chromatin reorganization and became more highly looped as LT-HSCs underwent differentiation, suggesting opposing functions of MECOM and CTCF in the regulation of the MECOM gene network. In light of these findings, we performed tandem perturbation of MECOM and CTCF and demonstrated rescue of LT-HSC loss with the dual perturbation, thereby illuminating a key role for MECOM in constraining CTCF-dependent chromatin reorganization that occurs as HSCs undergo differentiation.

Finally, based on the observation that elevated MECOM expression is associated with high-risk myeloid malignancies, we investigated the role of the MECOM-regulated HSC gene network in acute myeloid leukemias (AML). Across three independent AML datasets, we found that the MECOM regulated gene network had an independent and strong prognostic prediction ability that enabled risk stratification beyond currently used approaches, including a variety of molecular criteria and the previously described LSC17 signature. To validate these correlative observations, we performed CRISPR/Cas9 editing of MECOM in the MUTZ-3 AML cell line that is characterized by MECOM overexpression. We found that MECOM editing results in a loss of CD34 ⁺ leukemia progenitors and that the same transcriptional network that we identified in LT-HSCs is similarly altered upon MECOM perturbation in these AML cells.

Collectively, we use the study of a rare experiment of nature due to MECOM haploinsufficiency resulting in neonatal aplastic anemia to illuminate a gene regulatory network necessary for HSC self-renewal and maintenance that is co-opted in high-risk forms of AML.