Immune thrombocytopenia (ITP) is an autoimmune disorder, in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia, although the underlying mechanisms remain unclear. Here, we performed single-cell transcriptome profiling of bone marrow CD34+ hematopoietic stem and progenitor cells (HSPCs) to determine defects in megakaryopoiesis in ITP. Gene expression, cell-cell interactions, and transcriptional regulatory networks varied in HSPCs of ITP, particularly in immune cell progenitors. Differentially expressed gene (DEG) analysis indicated that there was an impaired megakaryopoiesis of ITP. Flow cytometry confirmed that the number of CD9+ and HES1+ cells from Lin-CD34+CD45RA- HSPCs decreased in ITP. Liquid culture assays demonstrated that CD9+Lin-CD34+CD45RA- HSPCs tended to differentiate into megakaryocytes; however, this tendency was not observed in ITP patients and more erythrocytes were produced. The percentage of megakaryocytes differentiated from CD9+Lin-CD34+CD45RA- HSPCs was 3-fold higher than that of the CD9- counterparts from healthy controls (HCs), whereas, in ITP patients, the percentage decreased to only 1/4th of that in the HCs and was comparable to that from the CD9- HSPCs. Additionally, when co-cultured with pre-B cells from ITP patients, the differentiation of CD9+Lin-CD34+CD45RA- HSPCs toward the megakaryopoietic lineage was impaired. Further analysis revealed that megakaryocytic progenitors (MkP) can be divided into seven subclusters with different gene expression patterns and functions. The ITP-associated DEGs were MkP subtype-specific, with most DEGs concentrated in the subcluster possessing dual functions of immunomodulation and platelet generation. This study comprehensively dissects defective hematopoiesis and provides novel insights regarding the pathogenesis of ITP.
© 2022. The Author(s).

ZNF384-rearranged fusion oncoproteins (FO) define a subset of lineage ambiguous leukemias, but their mechanistic role in leukemogenesis and lineage ambiguity is poorly understood. Using viral expression in mouse and human hematopoietic stem and progenitor cells (HSPC) and a Ep300::Znf384 knockin mouse model, we show that ZNF384 FO promote hematopoietic expansion, myeloid lineage skewing, and self-renewal. In mouse HSPCs, concomitant lesions, such as NRASG12D, were required for fully penetrant leukemia, whereas in human HSPCs, expression of ZNF384 FO drove B/myeloid leukemia, with sensitivity of a ZNF384-rearranged xenograft to FLT3 inhibition in vivo. Mechanistically, ZNF384 FO occupy a subset of predominantly intragenic/enhancer regions with increased histone 3 lysine acetylation and deregulate expression of hematopoietic stem cell transcription factors. These data define a paradigm for FO-driven lineage ambiguous leukemia, in which expression in HSPCs results in deregulation of lineage-specific genes and hematopoietic skewing, progressing to full leukemia in the context of proliferative stress.
Expression of ZNF384 FO early in hematopoiesis results in binding and deregulation of key hematopoietic regulators, skewing of hematopoiesis, and priming for leukemic transformation. These results reveal the interplay between cell of origin and expression of ZNF384 FO to mediate lineage ambiguity and leukemia development. This article is highlighted in the In This Issue feature, p. 171.
©2022 American Association for Cancer Research.

It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination.Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.

In vitro differentiation of human pluripotent stem cells (hPSCs) offers a genetically tractable system to examine the physiology and pathology of human tissue development and differentiation. We have used this approach to model the earliest stages of human B lineage development and characterize potential target cells for the in utero initiation of childhood B acute lymphoblastic leukemia. Herein, we detail critical aspects of the protocol including reagent validation, controls, and examples of surface markers used for analysis and cell sorting. For complete details on the use and execution of this protocol, please refer to Boiers et al. (2018).
© 2021 The Author(s).

Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics.
Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.