The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision. The validity of this model in the mouse has been questioned; however, ...little is known about the lineage potential of human progenitors. Here we provide a comprehensive analysis of the human hematopoietic hierarchy by clonally mapping the developmental potential of seven progenitor classes from neonatal cord blood and adult bone marrow. Human multilymphoid progenitors, identified as a distinct population of Thy-1(neg-lo)CD45RA(+) cells in the CD34(+)CD38(-) stem cell compartment, gave rise to all lymphoid cell types, as well as monocytes, macrophages and dendritic cells, which indicated that these myeloid lineages arise in early lymphoid lineage specification. Thus, as in the mouse, human hematopoiesis does not follow a rigid model of myeloid-lymphoid segregation.
The nonobese diabetic/severe combined immune deficiency (NOD-scid) xenotransplantation model is the “gold standard” for assaying human hematopoietic stem cell activity. Systematic advancements, such ...as depletion of natural killer cell activity with anti-CD122 antibody, direct intrafemoral injection, and deletion or truncation of IL2Rγ, have improved human cell engraftment; however, questions remain whether these mouse models are equivalent or, if not, which model is superior for assaying hematopoietic stem cell activity. To address this, we compared overall engraftment and multilineage differentiation of near-limiting doses of lineage-depleted human umbilical cord blood cells by direct intrafemoral injection into NOD/Lt-scid, NOD/Shi-scid, NOD/Lt-scid/IL2Rγnull (NSG), and NOD/Shi-scid/IL2Rγnull mice. Transplantation into NSG mice generated moderately higher human engraftment levels in bone marrow compared with other strains. At limiting doses, NSG mice of both sexes were 3.6-fold more sensitive in detecting SCID-repopulating cells compared with NOD/Lt-scid mice. However, NSG females exhibited higher engraftment at limiting cell doses, resulting in an overall increase in SCID-repopulating cell detection of 9-fold. Both NSG and NOD/Shi-scid/IL2Rγnull support significantly improved engraftment in peripheral tissues compared with NOD/Lt-scid and NOD/Shi-scid mice, whereas NSG mice provide greater human engraftment in bone marrow than all other strains, especially at limiting doses.
Highly regenerative tissues such as blood must possess effective DNA damage responses (DDR) that balance long-term regeneration with protection from leukemogenesis. Hematopoietic stem cells (HSCs) ...sustain life-long blood production, yet their response to DNA damage remains largely unexplored. We report that human HSCs exhibit delayed DNA double-strand break rejoining, persistent γH2AX foci, and enhanced p53- and ASPP1-dependent apoptosis after γ-radiation compared to progenitors. p53 inactivation or Bcl-2 overexpression reduced radiation-induced apoptosis and preserved in vivo repopulating HSC function. Despite similar protection from irradiation-induced apoptosis, only Bcl-2-overexpressing HSCs showed higher self-renewal capacity, establishing that intact p53 positively regulates self-renewal independently from apoptosis. The reduced self-renewal of HSCs with inactivated p53 was associated with increased spontaneous γH2AX foci in secondary transplants of HSCs. Our data reveal distinct physiological roles of p53 that together ensure optimal HSC function: apoptosis regulation and prevention of γH2AX foci accumulation upon HSC self-renewal.
► Irradiation induces a distinct DNA damage response in HSCs compared to progenitors ► Irradiation-induced p53-, ASPP1-, and Bcl-2-mediated apoptosis inhibits HSC activity ► p53 and Bcl-2 regulate HSC self-renewal potential ► Apoptosis-independent functions of p53 are required for optimal HSC self-renewal
Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated ...cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients.
Our ability to manage acute myeloid leukemia (AML) is limited by our incomplete understanding of the epigenetic disruption central to leukemogenesis, including improper histone methylation. Here we ...examine 16 histone H3 genes in 434 primary AML samples and identify Q69H, A26P, R2Q, R8H and K27M/I mutations (1.6%), with higher incidence in secondary AML (9%). These mutations occur in pre-leukemic hematopoietic stem cells (HSCs) and exist in the major leukemic clones in patients. They increase the frequency of functional HSCs, alter differentiation, and amplify leukemic aggressiveness. These effects are dependent on the specific mutation. H3K27 mutation increases the expression of genes involved in erythrocyte and myeloid differentiation with altered H3K27 tri-methylation and K27 acetylation. The functional impact of histone mutations is independent of RUNX1 mutation, although they at times co-occur. This study establishes that H3 mutations are drivers of human pre-cancerous stem cell expansion and important early events in leukemogenesis.
The hematopoietic system sustains regeneration throughout life by balancing self-renewal and differentiation. To stay poised for mature blood production, hematopoietic stem cells (HSCs) maintain ...low-level expression of lineage-associated genes, a process termed lineage priming. Here, we modulated expression levels of Inhibitor of DNA binding (ID) proteins to ask whether lineage priming affects self-renewal of human HSCs. We found that lentiviral overexpression of ID proteins in cord blood HSCs biases myeloerythroid commitment at the expense of lymphoid differentiation. Conversely, reducing ID2 expression levels increases lymphoid potential. Mechanistically, ID2 inhibits the transcription factor E47 to attenuate B-lymphoid priming in HSCs and progenitors. Strikingly, ID2 overexpression also results in a 10-fold expansion of HSCs in serial limiting dilution assays, indicating that early lymphoid transcription factors antagonize human HSC self-renewal. The relationship between lineage priming and self-renewal can be exploited to increase expansion of transplantable human HSCs and points to broader implications for other stem cell populations.
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•ID2-mediated inhibition of E47 activity controls human HSC lineage commitment•ID2 OE reduces lymphoid priming and increases myeloid and stemness programs in HSC•Attenuation of lymphoid differentiation genes in HSC increases self-renewal
Hematopoietic stem cells express differentiation genes at low levels, a mechanism termed lineage priming. Here, van Galen et al. report that ID2 overexpression attenuates lymphoid genes and causes a 10-fold expansion of human HSC, demonstrating how lineage priming can be exploited to boost stem cell self-renewal.
Cardiac glycosides are approved for the treatment of heart failure as Na
/K
pump inhibitors. Their repurposing in oncology is currently investigated in preclinical and clinical studies. However, the ...identification of a specific cancer type defined by a molecular signature to design targeted clinical trials with cardiac glycosides remains to be characterized. Here, we demonstrate that cardiac glycoside proscillaridin A specifically targets MYC overexpressing leukemia cells and leukemia stem cells by causing MYC degradation, epigenetic reprogramming and leukemia differentiation through loss of lysine acetylation.
Proscillaridin A anticancer activity was investigated against a panel of human leukemia and solid tumor cell lines with different MYC expression levels, overexpression in vitro systems and leukemia stem cells. RNA-sequencing and differentiation studies were used to characterize transcriptional and phenotypic changes. Drug-induced epigenetic changes were studied by chromatin post-translational modification analysis, expression of chromatin regulators, chromatin immunoprecipitation, and mass-spectrometry.
At a clinically relevant dose, proscillaridin A rapidly altered MYC protein half-life causing MYC degradation and growth inhibition. Transcriptomic profile of leukemic cells after treatment showed a downregulation of genes involved in MYC pathways, cell replication and an upregulation of hematopoietic differentiation genes. Functional studies confirmed cell cycle inhibition and the onset of leukemia differentiation even after drug removal. Proscillaridin A induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27) and in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferases. Importantly, proscillaridin A demonstrated anticancer activity against lymphoid and myeloid stem cell populations characterized by MYC overexpression.
Overall, these results strongly support the repurposing of proscillaridin A in MYC overexpressing leukemia.
The leukemia stem cell (LSC) populations of acute myeloid leukemia (AML) exhibit phenotypic, genetic, and functional heterogeneity that contribute to therapy failure and relapse. Progress toward ...understanding the mechanistic basis for therapy resistance in LSCs has been hampered by difficulties in isolating cell fractions that enrich for the entire heterogeneous population of LSCs within individual AML samples. We previously reported that CD200 gene expression is upregulated in LSC-containing AML fractions. Here, we show that CD200 is present on a greater proportion of CD45dim blasts compared with more differentiated CD45high cells in AML patient samples. In 75% (49 of 65) of AML cases we examined, CD200 was expressed on ≥10% of CD45dim blasts; of these, CD200 identified LSCs within the blast population in 9 of 10 (90%) samples tested in xenotransplantation assays. CD200+ LSCs could be isolated from CD200+ normal HSCs with the use of additional markers. Notably, CD200 expression captured both CD34– and CD34+ LSCs within individual AML samples. Analysis of highly purified CD200+ LSC-containing fractions from NPM1-mutated AMLs, which are commonly CD34–, exhibited an enrichment of primitive gene expression signatures compared with unfractionated cells. Overall, our findings support CD200 as a novel LSC marker that is able to capture the entire LSC compartment from AML patient samples, including those with NPM1 mutation.
•CD200 marks LSCs in AML patient samples expressing CD200 on ≥10% of blasts.•CD200 captures both CD34– and CD34+ LSCs from individual AML patient samples.
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A number of genes are implicated in the initiation and progression of osteosarcoma; however, cytogenetic and comparative genomic hybridization studies indicate the involvement of additional ...unidentified genes. An examination of gene expression profiles in 22 high-grade osteosarcoma tumor specimens from 15 patients (including paired primary and metastatic samples from five patients) indicated that von Willebrand factor (vWF) mRNA expression may increase during tumor progression. vWF, a large glycoprotein previously considered to be expressed exclusively by endothelial cells and megakaryocytes, is involved in platelet aggregation and adhesion to the subendothelial matrix, processes critical to hematogenous tumor cell metastasis to the lung. Analysis of paired primary and metastatic osteosarcoma tumor samples from 10 patients revealed an increase in vWF gene expression in metastases (P=0.005). Immunohistochemistry showed that, in addition to the endothelial cells, vWF protein was also detected in osteosarcoma cells in vivo in 13 of 29 tumor specimens as well as in SAOS2, an osteosarcoma cell line. The tumor cell staining correlated positively with high vWF expression in the sample (P=0.006). Although vascular endothelial cells contribute to the vWF mRNA detected in the tumor samples, there was neither any correlation between vascular density (VD) and vWF mRNA expression nor between VD and clinical outcome. These findings suggest that vWF expression is deregulated in osteosarcoma tumors, potentially contributing to metastasis.
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