Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation

The regulated lysosomal degradation pathway of autophagy prevents cellular damage and thus protects from malignant transformation. Autophagy is also required for the maturation of various hematopoietic lineages, namely the erythroid and lymphoid ones, yet its role in adult hematopoietic stem cells (HSCs) remained unexplored. While normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs or early progenitors leads to leukemia. Mechanisms protecting HSCs from cellular damage are therefore essential to prevent hematopoietic malignancies. By conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system, we found that autophagy is required for the maintenance of true HSCs and therefore also of downstream hematopoietic progenitors. Loss of autophagy in HSCs leads to the expansion of a progenitor cell population in the bone marrow, giving rise to a severe, invasive myeloproliferation, which strongly resembles human acute myeloid leukemia (AML).     

Evi-1 is a critical regulator for hematopoietic stem cells and transformed leukemic cells

Evi-1 has been recognized as one of the dominant oncogenes associated with murine and human myeloid leukemia. Here, we show that hematopoietic stem cells (HSCs) in Evi-1-deficient embryos are severely reduced in number with defective proliferative and repopulating capacity. Selective ablation of Evi-1 in Tie2(+) cells mimics Evi-1 deficiency, suggesting that Evi-1 function is required in Tie2(+) hematopoietic stem/progenitors. Conditional deletion of Evi-1 in the adult hematopoietic system revealed that Evi-1-deficient bone marrow HSCs cannot maintain hematopoiesis and lose their repopulating ability. In contrast, Evi-1 is dispensable for blood cell lineage commitment. Evi-1(+/-) mice exhibit the intermediate phenotype for HSC activity, suggesting a gene dosage requirement for Evi-1. We further demonstrate that disruption of Evi-1 in transformed leukemic cells leads to significant loss of their proliferative activity both in vitro and in vivo. Thus, Evi-1 is a common and critical regulator essential for proliferation of embryonic/adult HSCs and transformed leukemic cells.     

Distinct patterns of hematopoietic stem cell involvement in acute lymphoblastic leukemia

The cellular targets of primary mutations and malignant transformation remain elusive in most cancers. Here, we show that clinically and genetically different subtypes of acute lymphoblastic leukemia (ALL) originate and transform at distinct stages of hematopoietic development. Primary ETV6-RUNX1 (also known as TEL-AML1) fusions and subsequent leukemic transformations were targeted to committed B-cell progenitors. Major breakpoint BCR-ABL1 fusions (encoding P210 BCR-ABL1) originated in hematopoietic stem cells (HSCs), whereas minor BCR-ABL1 fusions (encoding P190 BCR-ABL1) had a B-cell progenitor origin, suggesting that P190 and P210 BCR-ABL1 ALLs represent largely distinct tumor biological and clinical entities. The transformed leukemia-initiating stem cells in both P190 and P210 BCR-ABL1 ALLs had, as in ETV6-RUNX1 ALLs, a committed B progenitor phenotype. In all patients, normal and leukemic repopulating stem cells could successfully be separated prospectively, and notably, the size of the normal HSC compartment in ETV6-RUNX1 and P190 BCR-ABL1 ALLs was found to be unaffected by the expansive leukemic stem cell population.     

Autophagy in the pathogenesis of myelodysplastic syndrome and acute myeloid leukemia

Autophagy is a conserved cellular pathway responsible for the sequestration of spent organelles and protein aggregates from the cytoplasm and their delivery into lysosomes for degradation. Autophagy plays an important role in adaptation to starvation, in cell survival, immunity, development and cancer. Recent evidence in mice suggests that autophagic defects in hematopoietic stem cells (HSCs) may be implicated in leukemia. Indeed, mice lacking Atg7 in HSCs develop an atypical myeloproliferation resembling human myelodysplastic syndrome (MDS) progressing to acute myeloid leukemia (AML). Our studies suggest that accumulation of damaged mitochondria and reactive oxygen species result in cell death of the majority of progenitor cells and, possibly, concomitant transformation of some surviving ones. Interestingly, bone marrow cells from MDS patients are characterized by mitochondrial abnormalities and increased cell death. A role for autophagy in the transformation to cancer has been proposed in other cancer types. This review focuses on autophagy in human MDS development and progression to AML within the context of the role of mitochondria, apoptosis and reactive oxygen species (ROS) in its pathogenesis.Key words: autophagy, mitophagy, Atg7, hematopoiesis, HSCs, myelodysplastic syndrome, acute myeloid leukemia     

FIP200, an essential component of mammalian autophagy is indispensible for fetal hematopoiesis

Autophagy, an evolutionarily conserved cellular process for bulk protein degradation through lysosomes, plays important roles in various physiological and pathological processes. Recent studies suggest that autophagy also participates in erythroid development. However, to what extent autophagy is involved in hematopoiesis is largely unknown. FIP200 (focal adhesion kinase family interacting protein of 200 kD) is a newly identified essential autophagy gene and a component of the ULK-Atg13-FIP200 complex. We show that mice lacking FIP200 in hematopoietic cells (CKO mice) experience perinatal lethality associated with severe erythroblastic anemia. FIP200 is cell-autonomously required for the maintenance and function of fetal hematopoietic stem cells (HSCs). FIP200 deletion in HSCs does not result in increased apoptosis. However, aberrantly increased HSC proliferation and myeloid expansion are found in CKO embryos, which may be responsible for the depletion of fetal HSCs. Consistent with an essential role of FIP200 in autophagy, FIP200-null fetal HSCs as well as other hematopoietic cells exhibit increased mitochondria mass and reactive oxygen species (ROS). Together, our data identify FIP200 as a key intrinsic regulator of fetal HSCs and suggest a role of autophagy in fetal hematopoiesis and the maintenance of fetal HSCs.     

FIP200, an essential component of mammalian autophagy is indispensible for fetal hematopoiesis

Autophagy, an evolutionarily conserved cellular process for bulk protein degradation through lysosomes, plays important roles in various physiological and pathological processes. Recent studies suggest that autophagy also participates in erythroid development. However, to what extent autophagy is involved in hematopoiesis is largely unknown. FIP200 (focal adhesion kinase family interacting protein of 200 kD) is a newly identified essential autophagy gene and a component of the ULK-Atg13-FIP200 complex. We show that mice lacking FIP200 in hematopoietic cells (CKO mice) experience perinatal lethality associated with severe erythroblastic anemia. FIP200 is cell-autonomously required for the maintenance and function of fetal hematopoietic stem cells (HSCs). FIP200 deletion in HSCs does not result in increased apoptosis. However, aberrantly increased HSC proliferation and myeloid expansion are found in CKO embryos, which may be responsible for the depletion of fetal HSCs. Consistent with an essential role of FIP200 in autophagy, FIP200-null fetal HSCs as well as other hematopoietic cells exhibit increased mitochondria mass and reactive oxygen species (ROS). Together, our data identify FIP200 as a key intrinsic regulator of fetal HSCs and suggest a role of autophagy in fetal hematopoiesis and the maintenance of fetal HSCs.     

Identification of novel genes and networks governing hematopoietic stem cell development

Hematopoietic stem cells (HSCs) are capable of giving rise to all blood cell lineages throughout adulthood, and the generation of engraftable HSCs from human pluripotent stem cells is a major goal for regenerative medicine. Here, we describe a functional genome‐wide RNAi screen to identify genes required for the differentiation of embryonic stem cell (ESC) into hematopoietic stem/progenitor cells (HSPCs) in vitro. We report the discovery of novel genes important for the endothelial‐to‐hematopoietic transition and subsequently for HSPC specification. High‐throughput sequencing and bioinformatic analyses identified twelve groups of genes, including a set of 351 novel genes required for HSPC specification. As in vivo proof of concept, four of these genes, Ap2a1, Mettl22, Lrsam1, and Hal, are selected for validation, confirmed to be essential for HSPC development in zebrafish and for maintenance of human HSCs. Taken together, our results not only identify a number of novel regulatory genes and pathways essential for HSPC development but also serve as valuable resource for directed differentiation of therapy grade HSPCs using human pluripotent stem cells.     

Cell-autonomous regulation of hematopoietic stem cell cycling activity by ATP

Extracellular nucleotides regulate many cellular functions through activation of purinergic receptors in the plasma membrane. Here, we show that in hematopoietic stem cell (HSC), ATP is stored in vesicles and released in a calcium-sensitive manner. HSC expresses ATP responsive P2X receptors and in vitro pharmacological P2X antagonism restrained hematopoietic progenitors proliferation, but not myeloid differentiation. In mice suffering from chronic inflammation, HSCs were significantly expanded and their cycling activity was sensitive to treatment with the P2X antagonist periodate-oxidized 2,3-dialdehyde ATP. Our results indicate that ATP acts as an autocrine stimulus in regulating HSCs pool size.     

An Mll-dependent Hox program drives hematopoietic progenitor expansion

Chromosomal translocations disrupting the Mixed lineage leukemia (Mll) gene result in leukemia, with aberrant expression of some native Mll target genes (reviewed in). The Mll gene encodes a Trithorax-group chromatin regulator that is essential for the development of hematopoietic stem cells (HSCs) during embryogenesis. Like Trithorax, MLL positively regulates clustered homeodomain or Hox genes, yet the role of Hox genes collectively in the development of the mammalian hematopoietic system has been difficult to ascertain because of redundancy among Hox paralogs. Here, we show that in the absence of MLL, early hematopoietic progenitors develop despite reduced expression of HoxA, HoxB, and HoxC genes. However, these progenitors exhibit a marked reduction in their ability to generate hematopoietic colonies, a subsequent process requiring cell division and differentiation. Reactivation of a subset of Hox genes or, remarkably, reexpression of a single Hox gene in Mll-deficient progenitors rescued hematopoietic-colony frequency and growth. In contrast, expression of other MLL target genes such as Pitx2 or expression of anti-apoptotic BCL-2 failed to rescue hematopoietic-colony frequency. Furthermore, our results highlight a shared function of Hox proteins at this point in the development of the hematopoietic system.     

Stop the dicing in hematopoiesis: What have we learned?

MicroRNAs (miRNAs) belong to an abundant class of highly conserved small (22nt) non-coding RNAs. MiRNA profiling studies indicate that their expression is highly cell type-dependent. DICER1 is an essential RNase III endoribonuclease for miRNA processing. Hematopoietic cell type- and developmental stage-specific Dicer1 deletion models show that miRNAs are essential regulators of cellular survival, differentiation and function. For instance, miRNA deficiency in hematopoietic stem cells and progenitors of different origins results in decreased cell survival, dramatic developmental aberrations or dysfunctions in mice. We recently found that homozygous Dicer1 deletion in myeloid-committed progenitors results in an aberrant expression of stem cell genes and induces a regained self-renewal capacity. Moreover, Dicer1 deletion causes a block in macrophage development and myeloid dysplasia, a cellular condition that may be considered as a preleukemic state. However, Dicer1-null cells do not develop leukemia in mice, indicating that depletion of miRNAs is not enough for tumorigenesis. Surprisingly, we found that heterozygous Dicer1 deletion in myeloid-committed progenitors, but not Dicer1 knockout, collaborates with p53 deletion in leukemic progression and results in various types of leukemia. Our data indicate that Dicer1 is a haploinsufficient tumorsuppressor in hematopoietic neoplasms, which is consistent with the observed downregulation of miRNA expression in human leukemia samples. Here, we review the various hematopoietic specific Dicer1 deletion mouse models and the phenotypes observed within the different hematopoietic lineages and cell developmental stages. Finally, we discuss the role for DICER1 in mouse and human malignant hematopoiesis.     

Unique and independent roles for MLL in adult hematopoietic stem cells and progenitors

The Mixed Lineage Leukemia (MLL) gene is essential for embryonic hematopoietic stem cell (HSC) development, but its role during adult hematopoiesis is unknown. Using an inducible knockout model, we demonstrate that Mll is essential for the maintenance of adult HSCs and progenitors, with fatal bone marrow failure occurring within 3 weeks of Mll deletion. Mll-deficient cells are selectively lost from mixed bone marrow chimeras, demonstrating their failure to self-renew even in an intact bone marrow environment. Surprisingly, HSCs lacking Mll exhibit ectopic cell-cycle entry, resulting in the depletion of quiescent HSCs. In contrast, Mll deletion in myelo-erythroid progenitors results in reduced proliferation and reduced response to cytokine-induced cell-cycle entry. Committed lymphoid and myeloid cells no longer require Mll, defining the early multipotent stages of hematopoiesis as Mll dependent. These studies demonstrate that Mll plays selective and independent roles within the hematopoietic system, maintaining quiescence in HSCs and promoting proliferation in progenitors.     

Functional ramifications for the loss of P-selectin expression on hematopoietic and leukemic stem cells

Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between blood cells and their microenvironment to preserve the homeostatic balance of long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitors (MPPs), and differentiated cells. Adhesion molecules like P-selectin (encoded by the Selp gene) are essential to hematopoiesis, and their dysregulation has been linked to leukemogenesis. Like HSCs, leukemic stem cells (LSCs) depend upon their microenvironments for survival and propagation. P-selectin plays a crucial role in Philadelphia chromosome-positive (Ph(+)) chronic myeloid leukemia (CML). In this paper, we show that cells deficient in P-selectin expression can repopulate the marrow more efficiently than wild type controls. This results from an increase in HSC self-renewal rather than alternative possibilities like increased homing velocity or cell cycle defects. We also show that P-selectin expression on LT-HSCs, but not ST-HSCs and MPPs, increases with aging. In the absence of P-selectin expression, mice at 6 months of age possess increased levels of short-term HSCs and multipotent progenitors. By 11 months of age, there is a shift towards increased levels of long-term HSCs. Recipients of BCR-ABL-transduced bone marrow cells from P-selectin-deficient donors develop a more aggressive CML, with increased percentages of LSCs and progenitors. Taken together, our data reveal that P-selectin expression on HSCs and LSCs has important functional ramifications for both hematopoiesis and leukemogenesis, which is most likely attributable to an intrinsic effect on stem cell self-renewal.     

Autophagy in the pathogenesis of myelodysplastic syndrome and acute myeloid leukemia

Autophagy is a conserved cellular pathway responsible for the sequestration of spent organelles and protein aggregates from the cytoplasm and their delivery into lysosomes for degradation. Autophagy plays an important role in adaptation to starvation, in cell survival, immunity, development and cancer. Recent evidence in mice suggests that autophagic defects in hematopoietic stem cells (HSCs) may be implicated in leukemia. Indeed, mice lacking Atg7 in HSCs develop an atypical myeloproliferation resembling human myelodysplastic syndrome (MDS) progressing to acute myeloid leukemia (AML). Studies suggest that accumulation of damaged mitochondria and reactive oxygen species result in cell death of the majority of progenitor cells and, possibly, concomitant transformation of some surviving ones. Interestingly, bone marrow cells from MDS patients are characterized by mitochondrial abnormalities and increased cell death. A role for autophagy in the transformation to cancer has been proposed in other cancer types. This review focuses on autophagy in human MDS development and progression to AML within the context of the role of mitochondria, apoptosis and reactive oxygen species (ROS) in its pathogenesis.     

New relationships of human hematopoietic lineages facilitate detection of multipotent hematopoietic stem and progenitor cells

Three important goals of hematopoietic stem cell research are to understand of how hematopoietic stem cells (HSCs) self-renew, how lineage commitment takes place, and how HSCs can be expanded ex vivo. Research in this area requires a reliable model of hematopoiesis. Performing detailed functional analyses of human hematopoietic progenitor subsets, we recently gained evidence for new hematopoietic lineage relationships.¹ According to our data, neutrophils belong to the same branch of the hematopoietic tree as lymphocytes. In contrast, eosinophils and basophils derive from another branch, the erythro-myeloid branch. Here, after introducing the newly proposed hematopoietic model, we discuss its consequences for the identification and expansion of human multipotent progenitors and suggest a fast and reliable method to screen for multipotent hematopoietic cells in vitro.     

The effect of inflammatory factors and their inhibitors on the hematopoietic stem cells fate

Inflammatory cytokines exert different effects on hematopoietic stem cells (HSCs), lead to the development of various cell lineages in bone marrow (BM) and are thus a differentiation axis for HSCs. The content used in this article has been obtained by searching PubMed database and Google Scholar search engine of English-language articles (1995–2020) using “Hematopoietic stem cell,” “Inflammatory cytokine,” “Homeostasis,” and “Myelopoiesis.” Inflammatory cytokines are involved in the differentiation and proliferation of hematopoietic progenitors to compensate for cellular death due to inflammation. Since each of these cytokines differentiates HSCs into a specific cell line, the difference in the effect of these cytokines on the fate of HSC progenitors can be predicted. Inhibitors of these cytokines can also control the inflammatory process as well as the cells involved in leukemic conditions. In general, inflammatory signaling can specify the dominant cell line in BM to counteract inflammation and leukemic condition via stimulating or inhibiting hematopoietic progenitors. Therefore, detection of the effects of inflammatory cytokines on the differentiation of HSCs can be an appropriate approach to check inflammatory and leukemic conditions and the suppression of these cytokines by their inhibitors allows for control of homeostasis in stressful conditions.