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Fetal and adult hematopoiesis are regulated by largely distinct sets of cell-intrinsic gene regulatory networks as well as extracellular cues in their respective microenvironment. These ontogeny-specific programs drive hematopoietic stem and progenitor cells (HSPCs) in fetus and adult to divergent susceptibility to initiation and progression of hematological malignancies, such as leukemia. Elucidating how leukemogenic hits disturb the intra- and extracellular programs in HSPCs along ontogeny will provide a better understanding of the causes for age-associated differences in malignant hematopoiesis and facilitate the improvement of strategies for prevention and treatment of pediatric and adult acute leukemia. Here, we review current knowledge of the intrinsic and extrinsic programs regulating normal and malignant hematopoiesis, with a particular focus on the differences between infant and adult acute leukemia. We discuss the recent advances in mass spectrometry-based proteomics and its opportunity for resolving the interplay of cell-intrinsic and niche-associated factors in regulating malignant hematopoiesis.

The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4 , one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4 -null phenotype in vivo. We demonstrated that the Ypel4 -null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4 -null cells was diminished. Furthermore, Ypel4 -null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4 -null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4 -null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity.

Infant and adult MLL1/KMT2A -rearranged (MLLr) leukemia represents a disease with a dismal prognosis. Here, we present a functional and proteomic characterization of in utero -initiated and adult-onset MLLr leukemia. We reveal that fetal MLL::ENL -expressing lymphomyeloid multipotent progenitors (LMPPs) are intrinsically programmed towards a lymphoid fate but give rise to myeloid leukemia in vivo, highlighting a complex interplay of intra- and extracellular factors in determining disease subtype. We characterize early proteomic events of MLL::ENL -mediated transformation in fetal and adult blood progenitors and reveal that whereas adult pre-leukemic cells are mainly characterized by retained myeloid features and downregulation of ribosomal and metabolic proteins, expression of MLL::ENL in fetal LMPPs leads to enrichment of translation-associated and histone deacetylases signaling proteins, and decreased expression of inflammation and myeloid differentiation proteins. Integrating the proteome of pre-leukemic cells with their secretome and the proteomic composition of the extracellular environment of normal progenitors highlights differential regulation of Igf2 bioavailability, as well as of VLA-4 dimer and its ligandome, upon initiation of fetal- and adult-origin leukemia, with implications for human MLLr leukemia cells’ ability to communicate with their environment through granule proteins. Our study has uncovered opportunities for targeting ontogeny-specific proteomic vulnerabilities in in utero -initiated and adult-onset MLLr leukemia.

Despite rapid advances in mapping genetic drivers and gene expression changes in hematopoietic stem cells (HSCs), there is a relative paucity of studies at the protein level. Here, we perform a deep, multi-omic characterization (epigenome, transcriptome and proteome) of HSCs carrying a loss-of-function mutation in Tet2, a key driver of increased self-renewal in blood cancers. Using state-of-the-art, multiplexed, low-input mass spectrometry (MS)-based proteomics, we profile wildtype (WT) and TET2-deficient (Tet2-/-) HSCs and show that the proteome captures previously unrecognized molecular processes which define the pre-leukemic HSC molecular landscape. Specifically, we obtain more accurate stratification of WT and Tet2-/- HSCs than transcriptomic approaches and identify extracellular matrix (ECM) molecules as novel points of dysregulation upon TET2 loss. HSC expansion assays using ECM-functionalized hydrogels confirm a selective effect on the expansion of Tet2-mutant HSCs. Taken together, our study represents a comprehensive molecular characterization of Tet2-mutant HSCs and identifies a previously unanticipated role of ECM molecules in regulating self-renewal of disease-driving HSCs.Competing Interest StatementThe DGK lab has received research funding from STEMBOND Inc. (Cambridge, UK) to conduct experiments using hematopoietic cells that were unrelated to this manuscript.Footnotes* Section \"Resource availability\" has been removed while the manuscript undergoes peer-review.

The process of hematopoiesis is subject to extensive ontogenic remodeling that is accompanied by alterations in cellular fate both during normal development and upon malignant transformation. Although the functional differences between fetal and adult hematopoiesis are well established, the responsible molecular mechanisms have long remained largely unexplored at the proteomic level. Here, we have applied state-of-the-art mass spectrometry to gain deep coverage of the proteome of 100,000 fetal and adult lympho-myeloid multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and granulocyte-monocyte progenitors (GMPs). Our analysis resulted in the identification and quantification of 4189 proteins, with over 200 proteins per cell type displaying differential expression between the fetus and the adult. The proteomic data demonstrate that features traditionally attributed to adult hematopoiesis are conserved across lymphoid and myeloid lineages, while generic fetal features are considerably more prominent in LMPPs and CLPs than in GMPs. Furthermore, we reveal molecular and functional evidence for a diminished granulocyte differentiation capacity in fetal LMPPs and GMPs relative to their adult counterparts, and show indications of a differential requirement of myosin activity for granulopoiesis in fetal and adult LMPPs. We have additionally identified the transcription factor Irf8 as significantly lower expressed in fetal relative to adult GMPs, and shown that its expression pattern correlates with an altered capacity for monocytic differentiation in the fetal cells. Collectively, our work represents a significant advancement in the understanding of the molecular programs that govern ontogenic differences in early hematopoiesis and mature blood cell production. Competing Interest Statement The authors have declared no competing interest.

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells being non-HSCs and single cell-initiated cultures displaying substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. Linking single clone functional transplantation data with single clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to actively cycling fetal liver HSCs and shares a gene expression signature with functional HSCs from all sources, including Prdm16, Fstl1 and Palld. This new tool can now be applied to a wide-range of functional screening and molecular experiments previously not possible due to limited HSC numbers. Competing Interest Statement The authors have declared no competing interest.