Abstract 9: Histone Deacetylases 1 And 2 Regulate Six2 Function To Maintain Nephron Progenitor Cells During Nephrogenesis

Low nephron endowment is strongly associated with cardiovascular disease, especially hypertension. Sine oculis homeobox 2 (Six2) is the master transcriptional regulator in balancing self-renewal and differentiation of nephron progenitor cells (NPCs) for appropriate nephron endowment. Loss of Six2 in mice causes early-onset loss of self-renewal and premature differentiation of NPCs. However, it is unclear how Six2 is functionally regulated during nephrogenesis. In vivo interaction of histone deacetylase1 and 2 (Hdac1/2) to Six2 was detected in developing kidney by co-immunoprecipitation and proximity ligation assay. Chromatin immunoprecipitation and DNA sequencing experiments in isolated E16.5 NPCs revealed 1,180 (84.58%) of the Six2 peaks overlapped with Hdac2 peaks, implying the involvement of Hdac1/2 in Six2 DNA binding and its function in NPCs. To test whether Hdac1/2 are required for Six2 function to regulate nephron formation, we employed Six2 GC mouse line, in which the eGFP and Cre fusion gene (GC) replaces and fully recapitulates the endogenous Six2 gene expression pattern. Analysis of kidneys at embryonic day (E) 19.5 and newborn (P0) showed that Six2 heterozygous (Six2 GC ) together with three alleles knockout of Hdac1/2 resulted in severely hypoplastic kidneys, while three alleles knockout of Hdac1/2 by transgenic Six2-Cre only led to very subtle phenotypes. Immunostaining at E 19.5 and P0 revealed about 50% reduction of Six2 level in the kidney of Six2 GC only and Six2 GC together with three alleles knockout of Hdac1/2 mice. In the kidneys of Six2 heterozygous mice, no change was observed for most of the NPC identity makers for self-renewal, including the three Six2 targets, Pax2, Sall1 and WT1. However, sequential removal of three alleles of Hdac1/2 of Six2 GC mice did not change Six2 protein level but significantly decreased the expression of Pax2, Sall1 and WT1, suggesting the requirement of Hdac1/2 for Six2’s function to transcriptionally activate the expression of its target genes. We also observed the premature differentiation and decreased nephron formation in mutant kidneys. Therefore, we conclude that Hdac1/2 are required for Six2’s function to promote NPC self-renewal and repress premature differentiation during nephrogenesis.

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Progenitor translatome changes coordinated by Tsc1 increase perception of Wnt signals to end nephrogenesis

Nature Communications ◽

10.1038/s41467-021-26626-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Alison E. Jarmas ◽

Eric W. Brunskill ◽

Praneet Chaturvedi ◽

Nathan Salomonis ◽

Raphael Kopan

Keyword(s):

Progenitor Cells ◽

Gradual Increase ◽

Tipping Point ◽

Ureteric Bud ◽

Self Renewal ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Nephron Progenitor

AbstractMammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that translatome analysis in Tsc1+/− nephron progenitor cells from mice with elevated nephron numbers reveals how differential translation of Wnt antagonists over agonists tips the balance between self-renewal and differentiation. Wnt agonists are poorly translated in young niches, resulting in an environment with low R-spondin and high Fgf20 promoting self-renewal. In older niches we find increased translation of Wnt agonists, including R-spondin and the signalosome-promoting Tmem59, and low Fgf20, promoting differentiation. This suggests that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and possibly clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving synchronized differentiation. As predicted by these findings, removing one Rspo3 allele in nephron progenitors delays cessation and increases nephron numbers in vivo.

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Age-Dependent Changes in the Progenitor Translatome Coordinated in part by Tsc1 Increase Perception of Signaling Inputs to End Nephrogenesis

10.1101/2020.03.12.989343 ◽

2020 ◽

Author(s):

Eric Brunskill ◽

Alison Jarmas ◽

Praneet Chaturvedi ◽

Raphael Kopan

Keyword(s):

Gradual Increase ◽

Tipping Point ◽

Ureteric Bud ◽

Self Renewal ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Age Dependent ◽

Cellular Translation ◽

Nephron Progenitor

AbstractMammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that in young niches, cellular Wnt agonists are poorly translated, Fgf20 levels are high and R-spondin levels are low, resulting in a pro self-renewal environment. By contrast, older niches are low in Fgf20 and high in R-spondin, with increased cellular translation of Wnt agonists, including the signalosome-promoting Tmem59. This suggests a hypothesis that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving differentiation. We show Tsc1 hemizygosity differentially promoted translation of Wnt antagonists over agonists, expanding a transitional (Six2+, Cited1+, Wnt4+) state and delaying the tipping point. As predicted by these findings, reducing Rspo3 dosage in nephron progenitors or Tmem59 globally increased nephron numbers in vivo.

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Drug Induced MLL Fusion Degradation Via Hsp90 Inhibition

Blood ◽

10.1182/blood.v126.23.4850.4850 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4850-4850

Author(s):

Sandra Cantilena ◽

Nicholas Goulden ◽

Owen Williams ◽

Jasper de Boer

Keyword(s):

Stem Cell ◽

Fusion Protein ◽

Target Genes ◽

Fusion Gene ◽

Leukemic Stem Cell ◽

Down Regulation ◽

Drug Induced ◽

Acute Leukemias ◽

Self Renewal

Abstract The survival rate for infants is less than 50%. The fast majority of infant acute leukemias are characterized cytogenetically by balanced chromosomal translocations involving the mixed lineage leukemia (MLL) gene. Leukemic therapies that degrade the driver oncogene are associated with loss of cancer cell self-renewal and excellent cure rates. Therefore, therapy that degrades the MLL fusion gene would offer new hope to these patients. Recently it was shown that the Drosophila Trithorax gene, an analogue of the human MLL, is degraded by Radicicol. Radicicol is a natural compound and a well-known Hsp90 inhibitor. Here, we show that Radicicol is able to induce a dose dependent degradation of the MLL-fusion protein in a panel of human MLL rearranged cell lines and in human cord blood-derived MLL-AF9 immortalised myeloid cells. This drug induced degradation of the MLL-fusion gene results in down-regulation of the expression of MLL target genes, including HOXA9, MEIS1 and c-MYB. Functionally, this results in a loss of self-renewal of the leukemic stem cells, as shown by methylcellulose colony forming assays. Radicicol proved ineffective and too toxic for in vivo use. One of the best tolerated Hsp90 inhibitors is Ganetespib. It is currently in phase II/III clinical trials. We extended our Radicicol data to Ganetespib. Like Radicicol, Ganetespib induces MLL-fusion protein degradation and downregulation of MLL target genes. Treatment of MLL rearranged leukemia with Ganetespib results in a loss of leukemic stem cell activity. In conclusion, we show how the inactivation of the MLL-fusion and down-regulation of MLL target genes results in a block of leukemic stem cell self- renewal. We will validate these findings in a pre-clinical in vivo model in the near future. Disclosures No relevant conflicts of interest to declare.

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MLL1 is required for PAX7 expression and satellite cell self-renewal in mice

Nature Communications ◽

10.1038/s41467-019-12086-9 ◽

2019 ◽

Vol 10 (1) ◽

Author(s):

Gregory C. Addicks ◽

Caroline E. Brun ◽

Marie-Claude Sincennes ◽

John Saber ◽

Christopher J. Porter ◽

...

Keyword(s):

Satellite Cell ◽

Satellite Cells ◽

Transcriptional Activation ◽

Cell Activation ◽

Target Genes ◽

Cell Function ◽

Skeletal Muscle Regeneration ◽

Differentiation Potential ◽

Self Renewal

Abstract PAX7 is a paired-homeobox transcription factor that specifies the myogenic identity of muscle stem cells and acts as a nodal factor by stimulating proliferation while inhibiting differentiation. We previously found that PAX7 recruits the H3K4 methyltransferases MLL1/2 to epigenetically activate target genes. Here we report that in the absence of Mll1, myoblasts exhibit reduced H3K4me3 at both Pax7 and Myf5 promoters and reduced Pax7 and Myf5 expression. Mll1-deficient myoblasts fail to proliferate but retain their differentiation potential, while deletion of Mll2 had no discernable effect. Re-expression of PAX7 in committed Mll1 cKO myoblasts restored H3K4me3 enrichment at the Myf5 promoter and Myf5 expression. Deletion of Mll1 in satellite cells reduced satellite cell proliferation and self-renewal, and significantly impaired skeletal muscle regeneration. Pax7 expression was unaffected in quiescent satellite cells but was markedly downregulated following satellite cell activation. Therefore, MLL1 is required for PAX7 expression and satellite cell function in vivo. Furthermore, PAX7, but not MLL1, is required for Myf5 transcriptional activation in committed myoblasts.

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Differential Expression of Mll-AF9 Up-Regulated Genes Correlates with Enhanced Self-Renewal of Hematopoietic Progenitors.

Blood ◽

10.1182/blood.v108.11.733.733 ◽

2006 ◽

Vol 108 (11) ◽

pp. 733-733 ◽

Cited By ~ 1

Author(s):

Ashish R. Kumar ◽

Wendy A. Hudson ◽

Weili Chen ◽

Rodney A. Staggs ◽

Anne-Francoise Lamblin ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Fusion Gene ◽

Expression Profiles ◽

Cell Types ◽

Third Generation ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

The Third

Abstract In order to understand the pathophysiology of leukemia, we need to study the effects of leukemic oncogenes on the rare hematopoietic stem and progenitor cells. We investigated the self-renewal capabilities of the various hematopoietic cell types derived from Mll-AF9 knock-in mice. We used the murine knock-in model since it offers the advantage of a single copy of the Mll-fusion gene under the control of the endogenous promoter present in every hematopoietic stem/progenitor cell. In methylcellulose cultures, we compared myeloid colony formation of Mll-AF9 cells to wild type progenitor populations over three generations of plating. In the first generation of plating, the Mll-AF9 common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) formed more colonies than the hematopoietic stem cells (HSCs) and common lymphoid progenitors (CLPs). However, at the third generation of plating, colony numbers formed by Mll-AF9 HSCs and CLPs were significantly greater than those formed by CMPs and GMPs. By the third generation only occasional colonies were found in the wild type groups. These results demonstrate that while Mll-AF9 led to an increase in self-renewal of all 4 cell types studied, these effects were more pronounced in HSCs and CLPs. To identify the downstream genes that mediate the growth deregulatory effects of Mll-AF9, we compared gene expression profiles of Mll-AF9 derived cells to their wild type counterparts. To assess gene expression levels, we extracted RNA from wild type and Mll-AF9 HSCs, CLPs, CMPs and GMPs. We then amplified and labeled the RNA for analysis by Affymetrix murine 430 2.0 genome arrays. In an unsupervised analysis, the various Mll-AF9 cells clustered with their corresponding wild type counterparts, indicating that the expression of most genes was not significantly altered by Mll-AF9. To identify the genes that are differentially expressed in the Mll-AF9 derived cells, we performed a two-way ANOVA (with the genotype and cell type as the two variables) allowing for a false discovery rate of 10%. In this analysis, we found that 76 genes were up-regulated in all Mll-AF9 progenitor cells compared to their wild-type counterparts. This list included known targets of Mll-fusion proteins Hoxa5, Hoxa7, Hoxa9 and Hoxa10. Also included were Evi1 and Mef2c, two genes that have been implicated in promoting enhanced self-renewal of murine hematopoietic cells. Importantly, in wild type mice, these 6 genes were expressed at higher levels in HSCs and CLPs compared to CMPs and GMPs (average 3–25 fold). While we observed an average 2–10 fold increase in expression of these genes in all Mll-AF9 cell types compared to their respective wild type controls, the expression level was 3–8 fold higher in Mll-AF9 HSCs and CLPs compared to CMPs and GMPs. Thus, the expression of genes known to be intrinsically related to self-renewal is further enhanced as a result of the Mll-AF9 fusion gene. In conclusion, while activation of the Mll-AF9 genetic program and the resulting enhanced self-renewal occurs in all 4 cell types studied, these effects are greatest in HSCs and CLPs. Thus, HSCs and CLPs are likely to be more efficient than CMPs and GMPs in producing cellular expansion and targets for cooperating mutations resulting in leukemia.

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Pre-Clinical Efficacy of Combined Therapy with LSD1 Antagonist SP-2509 and Pan-Histone Deacetylase Inhibitor Against AML Blast Progenitor Cells

Blood ◽

10.1182/blood.v120.21.868.868 ◽

2012 ◽

Vol 120 (21) ◽

pp. 868-868 ◽

Cited By ~ 2

Author(s):

Warren Fiskus ◽

Sunil Sharma ◽

Sunil Abhyankar ◽

Joseph McGuirk ◽

David J. Bearss ◽

...

Keyword(s):

Progenitor Cells ◽

Histone Deacetylases ◽

Combined Therapy ◽

Colony Growth ◽

Clinical Activity ◽

Gene Promoters ◽

Chromatin Mark ◽

Tail Vein ◽

Nsg Mice

Abstract Abstract 868 LSD1 (KDM1A) is an FAD-dependent histone demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K) 4 on histone H3, reducing the permissive H3K4Me3 chromatin mark for gene expression. LSD1 forms a complex with the histone deacetylases (HDAC) 1 and 2 and with the co-repressor CoREST, which stimulates the activity of LSD1 toward nucleosomes. While high LSD1 expression may be an effector of blocked differentiation and confers poor prognosis in AML, LSD1 inhibition induces the expression of myeloid–differentiation associated genes and attenuates growth of AML blast progenitor cells (BPCs). Recently, LSD1 was shown to sustain the in vivo leukemogenic potential of MLL-AF9 expressing leukemia stem cells. Also, co-treatment with the LSD1 inhibitor tranylcypromine (TCP) and all-trans retinoic acid (ATRA) was shown to diminish the engraftment of primary AML BPCs in vivo in NOD-SCID-γIL-2 receptor deficient (NSG) mice. Previous studies have shown that HDAC inhibitors attenuate the levels of LSD1 through Sp1 inhibition. SP-2509 is a potent and selective FAD-binding pocket, non-MAOA and MAOB, inhibitor with an IC50 of 13 nM for LSD1. In the present studies, we determined the chromatin effects and anti-AML efficacy of SP-2509 alone and in combination with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharmaceuticals) in cultured (HL-60, OCI-AML3, MV4-11, MOLM13, THP1 and SKM1 cells) and primary human AML BPCs. Treatment with SP-2509 (250 to 1000 nM) dose-dependently increased the levels of H3K4Me2 & Me3 chromatin mark, and chromatin immunoprecipitation followed by QPCR analyses showed an increase in the H3K4Me3 mark on the gene promoters of KLF4, HMOX1, p57 and p21 in AML BPCs. SP-2509 treatment attenuated the binding of LSD1 with CoREST, accompanied with increased levels of p16, p21 and p27 in AML BPCs. Consistent with this, treatment with SP-2509 inhibited the suspension and colony growth of AML BPCs regardless of whether they expressed MLL fusion oncoproteins. Knockdown of LSD1 by shRNA also inhibited the suspension and colony growth of AML blast progenitor cells. SP-2509 also induced C/EBPα expression and features of morphologic differentiation in the cultured and primary AML BPCs. Following tail vein infusion and establishment of AML by OCI-AML3 or MOLM13 cells in NOD/SCID mice, treatment with SP-2509 (25 mg/kg b.i.w. via IP injection) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). We have previously reported that treatment with PS depleted polycomb repressive complex proteins EZH2, SUZ12 and BMI1 but also reduced LSD1 expression in AML cells. Co-treatment with PS enhanced SP-2509-induced chromatin effects and differentiation of AML cells. Also, PS and SP-2509 synergistically induced apoptosis of the cultured AML OCI-AML3, MOLM13 and MV4-11cells (combination indices, CI <1.0). Additionally, co-treatment with SP-2509 sensitized AML cells to ATRA-induced differentiation. Notably, co-treatment with SP-2509 and PS also induced significantly greater loss of viability of primary AML BPCs but not of normal CD34+ cells. SP-2509 treatment (15 mg/kg b.i.w. IP) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts. Survival was further significantly improved upon co-treatment with SP-2509 and PS (5 mg/kg IP, MWF) (p < 0.001). Mice did not experience any toxicity or weight loss. Taken together, these findings demonstrate promising pre-clinical activity of combined therapy with SP-2509 and PS, warranting further in vivo development and testing of SP-2509 against human AML. Disclosures: Sharma: Salarius Pharmaceuticals: Equity Ownership.

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Replication Factor C3 Is a Direct Target Of CREB, Promotes G1/S Transition Of Acute Myeloid Leukemia Cells, and Increases Hematopoietic Stem/Progenitor Cell Self-Renewal

Blood ◽

10.1182/blood.v122.21.3754.3754 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3754-3754

Author(s):

Hee-Don Chae ◽

Kathleen Sakamoto

Keyword(s):

Mrna Expression ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Target Genes ◽

S Phase ◽

Self Renewal ◽

Hematopoietic Stem ◽

Expression Levels ◽

Creb Activation ◽

Kg1 Cells

Abstract CREB (cAMP Response Element Binding protein) promotes cellular transformation of hematopoietic cells and proliferation of myeloid leukemia cells. However, the underlying mechanisms of CREB function in leukemic transformation and hematopoiesis are not fully understood. To address this, we have investigated the downstream pathways of CREB activation in proliferation using a human acute myeloid leukemia (AML) cell line KG1 cells knocked-down for CREB with specific shRNAs. The CREB-knockdown KG1 cells were significantly defective in proliferative capability compared to control cells [cell number after 4d (X105), seeding (1X105), control vs. CREB-knockdown: 34.18 +/– 1.27 vs. 14.52 +/– 0.46, n=3, p< 0.01, mean +/– SEM]. In order to characterize the specific role of CREB in cell proliferation, we analyzed cell cycle progression patterns of CREB-knockdown and control KG1 cells after release from mitotic arrest. Our results indicated that G1 to S phase transition as assessed by % S phase was impeded by CREB-knockdown [S phase (%), control vs. CREB-knockdown cells, 8h after release: 53.29 +/– 0.54 vs. 23.57 +/– 1.69; 12h: 66.92 +/– 0.63 vs. 45.16 +/– 0.50, n=3, p< 0.01, mean +/– SEM]. To identify potential CREB target genes, we chose several cell cycle related genes such as CCNE1, CCNA1, CCNB1 and PCNA and compared their RNA expression levels in the CREB-knockdown with those in control KG1 cells after release from mitotic arrest. To our surprise, we failed to detect any noticeable differences in the mRNA expression levels of those genes between CREB-knockdown and control KG1 cells. In an effort to search for CREB responsive target genes, we analyzed additional CREB targets previously identified from microarray data (Pellegrini et al BMC Cancer 2008). We found that expression of replication factor C3 (RFC3), a 38kDa subunit of the RFC complex involved in DNA replication and repair processes, was significantly reduced in CREB-knockdown cells compared to control cells [38 +/– 1% of control, n=3, p<0.01]. CREB-knockdown also inhibited RFC3 mRNA expression in U937 and HL60 AML cell lines. Consistent with these results, mRNA expression levels of RFC3 appeared to be closely correlated with those of CREB when we examined bone marrow samples obtained from AML patients [n = 16, Pearson coefficient = 0.6366, p = 0.0008]. Moreover, we found that CREB directly interacted with the CRE site in the RFC3 promoter region in vivo, as assessed by chromatin immunoprecipitation assays. Exogenous overexpression of RFC3 in CREB-knockdown KG1 cells restored the defective G1/S progression [S phase (%), CREB-knockdown vs. CREB-knockdown with RFC3 overexpression, 9h after release: 38.97 +/– 0.45 vs. 62.24 +/– 1.06; 12h: 48.12 +/– 0.60 vs. 67.70 +/– 1.15, n=3, p< 0.01, mean +/– SEM]. Taken together, these results suggest that RFC3 may act as a novel downstream oncogenic target of activated CREB in AML cells. We previously reported that CREB is a critical regulator of normal myelopoiesis (Cheng et al Blood 2008). To determine whether RFC3 could exert similar effects on normal hematopoiesis, we compared human umbilical cord blood derived CD34-positive cells with and without RFC3 overexpression for the capacity to form hematopoietic colonies. Overexpression of RFC3 in the CD34-positive cells resulted in significant increases of multi-potential CFU-GEMM colony numbers [without vs. with overexpression of RFC3 (per 1000 cells): 3.2 +/– 1.3 vs. 22.3 +/– 3.3, n=3, p< 0.01, mean +/– SEM]. The RFC3 effect on stimulating colony formation was magnified in secondary colony forming assays [without vs. with overexpression of RFC3 (per 100,000 cells): 10.7 +/– 3.5 vs. 180.2 +/– 44.4, n=3, p< 0.05, mean +/– SEM]. Since the formation of secondary colonies was derived mainly from residual stem/progenitor cell populations after long-term culture, RFC3 overexpression may enhance self-renewal of stem/progenitor cells. In conclusion, our results suggest that RFC3 is able to promote G1/S transition in a human AML cell line downstream of CREB activation. In addition, we provide evidence that RFC3 is involved in normal hematopoiesis and contributes to increased self-renewal potential of hematopoietic stem/progenitor cells. Our data demonstrate that RFC3 plays multiple roles in promoting AML cells proliferation as well as normal myelopoiesis through increasing the self-renewal potential of hematopoietic stem/progenitor cells in response to CREB activation. Disclosures: No relevant conflicts of interest to declare.

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ATG5 and ATG7 Fulfill Essential and Non-Redundant Roles in Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v124.21.4316.4316 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4316-4316

Author(s):

Hendrik Folkerts ◽

Maria Catalina Gomez Puerto ◽

Albertus T.J. Wierenga ◽

Koen Schepers ◽

Jan Jacob Schuringa ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Target Genes ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Human Hscs

Abstract Macroautophagy is a catabolic process by which intracellular contents are delivered to lysosomes for degradation. ATG5 and ATG7 play an essential role in this process. Recent studies have shown that mouse hematopoietic stem cells (HSCs) lacking ATG7 were unable to survive in vivo, however, the role of macroautophagy in proliferation and survival of human HSCs has not yet been defined. Here, we demonstrate that autophagy is functional in human hematopoietic stem/progenitor cells. Robust accumulation of the autophagy markers LC3 and p62 were observed in cord blood (CB)-derived CD34+ cells treated with bafilomycin-A1 (BAF) or hydroxychloroquine (HCQ), as defined by Western blotting. When these cells were subsequently differentiated towards the myeloid or erythroid lineage, a decreased accumulation of LC3 was observed. In addition, CB CD34+CD38- cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BAF or HCQ treatment. In line with these results, also more mature CB CD33+ and CD14+ myeloid cells or CD71+CD235+ erythroid cells showed reduced levels of cyto-ID accumulation upon BAF or HCQ treatment. These findings indicate that human hematopoietic stem and progenitor cells (HSPCs) have a higher basal autophagy flux compared to more differentiated cells. To study the functional consequences of autophagy in human HSCs and their progeny, ATG5 and ATG7 were downregulated in CB-derived CD34+ cells, using a lentiviral shRNA approach which resulted in 80% and 70% reduced expression, respectively. Downmodulation of ATG5 or ATG7 in CB CD34+ cells resulted in a significant reduction of erythroid progenitor frequencies, as assessed by colony forming cell (CFC) assays (shATG5 2.2 fold, p<0.05 or shATG7 1.4 fold p<0.05). Additionally, a strong reduction in expansion was observed when transduced cells were cultured under myeloid (shATG5 17.9 fold, p<0.05 or shATG7 12.3 fold, p<0.05) or erythroid permissive conditions (shATG5 6.7 fold, p<0.05 or shATG7 1.7 fold, p<0.05), whereby differentiation was not affected. The phenotype upon knockdown of ATG5 or ATG7 could not be reversed by culturing the cells on a MS5 stromal layer. In addition to progenitor cells, HSCs were also affected since long term culture-initiating cell (LTC-IC) assays in limiting dilution revealed a 3-fold reduction in stem cell frequency after ATG5 and ATG7 knockdown. The inhibitory effects of shATG5 and shATG7 in cultured CD34+ cells were at least in part due to a decline in the percentage of cells in S phase and (shATG5 1.4 fold, p<0.01 and shATG7 1.3 fold, p<0.01) and an increase of Annexin V positive cells. The changes in cell cycle and apoptosis coincided with a marked increase in expression of the cell cycle-dependent kinase inhibitor p21, an increase in p53 levels, and an increase in proapoptotic downstream target genes BAX, PUMA and PHLDA3. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

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