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• W2899451374 abstract "•ISR pathway activity in human HSC/MPPs is maintained by low eIF2 and high ATF4•ATF4 upregulation following amino acid deprivation promotes HSC survival•Functional HSCs can be purified using an ATF4 reporter that measures ISR activity•ISR activity marks primitive cells in normal and malignant hematopoietic hierarchies Lifelong maintenance of the blood system requires equilibrium between clearance of damaged hematopoietic stem cells (HSCs) and long-term survival of the HSC pool. Severe perturbations of cellular homeostasis result in rapid HSC loss to maintain clonal purity. However, normal homeostatic processes can also generate lower-level stress; how HSCs survive these conditions remains unknown. Here we show that the integrated stress response (ISR) is uniquely active in HSCs and facilitates their persistence. Activating transcription factor 4 (ATF4) mediates the ISR and is highly expressed in HSCs due to scarcity of the eIF2 translation initiation complex. Amino acid deprivation results in eIF2α phosphorylation-dependent upregulation of ATF4, promoting HSC survival. Primitive acute myeloid leukemia (AML) cells also display eIF2 scarcity and ISR activity marks leukemia stem cells (LSCs) in primary AML samples. These findings identify a link between the ISR and stem cell survival in the normal and leukemic contexts. Lifelong maintenance of the blood system requires equilibrium between clearance of damaged hematopoietic stem cells (HSCs) and long-term survival of the HSC pool. Severe perturbations of cellular homeostasis result in rapid HSC loss to maintain clonal purity. However, normal homeostatic processes can also generate lower-level stress; how HSCs survive these conditions remains unknown. Here we show that the integrated stress response (ISR) is uniquely active in HSCs and facilitates their persistence. Activating transcription factor 4 (ATF4) mediates the ISR and is highly expressed in HSCs due to scarcity of the eIF2 translation initiation complex. Amino acid deprivation results in eIF2α phosphorylation-dependent upregulation of ATF4, promoting HSC survival. Primitive acute myeloid leukemia (AML) cells also display eIF2 scarcity and ISR activity marks leukemia stem cells (LSCs) in primary AML samples. These findings identify a link between the ISR and stem cell survival in the normal and leukemic contexts. Replenishment of the blood system throughout the lifetime of an organism requires longevity and integrity of the stem cell pool. However, human HSCs are sensitive to perturbations of cellular homeostasis and prone to undergo apoptosis. The induction of reactive oxygen species (ROS) and accumulation of DNA damage leads to heightened apoptosis of HSCs compared to downstream progenitor cells (Milyavsky et al., 2010Milyavsky M. Gan O.I. Trottier M. Komosa M. Tabach O. Notta F. Lechman E. Hermans K.G. Eppert K. Konovalova Z. et al.A distinctive DNA damage response in human hematopoietic stem cells reveals an apoptosis-independent role for p53 in self-renewal.Cell Stem Cell. 2010; 7: 186-197Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, Yahata et al., 2011Yahata T. Takanashi T. Muguruma Y. Ibrahim A.A. Matsuzawa H. Uno T. Sheng Y. Onizuka M. Ito M. Kato S. Ando K. Accumulation of oxidative DNA damage restricts the self-renewal capacity of human hematopoietic stem cells.Blood. 2011; 118: 2941-2950Crossref PubMed Scopus (229) Google Scholar). Likewise, endoplasmic reticulum (ER) stress causes strong activation of the unfolded protein response in HSCs, resulting in increased apoptosis compared to progenitor cells (Miharada et al., 2014Miharada K. Sigurdsson V. Karlsson S. Dppa5 improves hematopoietic stem cell activity by reducing endoplasmic reticulum stress.Cell Rep. 2014; 7: 1381-1392Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, van Galen et al., 2014avan Galen P. Kreso A. Mbong N. Kent D.G. Fitzmaurice T. Chambers J.E. Xie S. Laurenti E. Hermans K. Eppert K. et al.The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.Nature. 2014; 510: 268-272Crossref PubMed Scopus (237) Google Scholar). These responses promote stem cell pool integrity by purging damaged stem cells, but to ensure longevity, HSCs also must be able to survive perturbations that regularly occur during homeostasis such as DNA damage and calorie restriction (Ho et al., 2017Ho T.T. Warr M.R. Adelman E.R. Lansinger O.M. Flach J. Verovskaya E.V. Figueroa M.E. Passegué E. Autophagy maintains the metabolism and function of young and old stem cells.Nature. 2017; 543: 205-210Crossref PubMed Scopus (493) Google Scholar, Warr et al., 2013Warr M.R. Binnewies M. Flach J. Reynaud D. Garg T. Malhotra R. Debnath J. Passegué E. FOXO3A directs a protective autophagy program in haematopoietic stem cells.Nature. 2013; 494: 323-327Crossref PubMed Scopus (428) Google Scholar). The signals that ensure persistence of HSCs in the context of lower-level stress caused by metabolic processes during normal homeostasis are unknown. Many stressors converge on the ISR pathway. This pathway serves to balance stress signals that activate cell death pathways with those that protect the cell to enable restoration of cellular homeostasis (Pakos-Zebrucka et al., 2016Pakos-Zebrucka K. Koryga I. Mnich K. Ljujic M. Samali A. Gorman A.M. The integrated stress response.EMBO Rep. 2016; 17: 1374-1395Crossref PubMed Scopus (1045) Google Scholar). In low-stress conditions, the ISR favors survival as a consequence of the intrinsic stability of adaptive mRNAs and proteins, whereas high stress levels tip the balance toward distal ISR targets that promote cell death (Han et al., 2013Han J. Back S.H. Hur J. Lin Y.H. Gildersleeve R. Shan J. Yuan C.L. Krokowski D. Wang S. Hatzoglou M. et al.ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death.Nat. Cell Biol. 2013; 15: 481-490Crossref PubMed Scopus (1049) Google Scholar, Rutkowski et al., 2006Rutkowski D.T. Arnold S.M. Miller C.N. Wu J. Li J. Gunnison K.M. Mori K. Sadighi Akha A.A. Raden D. Kaufman R.J. Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and proteins.PLoS Biol. 2006; 4: e374Crossref PubMed Scopus (625) Google Scholar). This survival-death equilibrium is maintained by four stress-inducible kinases: GCN2, PKR, HRI, and PERK (Figure 1A). These kinases phosphorylate eIF2α, a subunit of the eIF2 complex (consisting of eIF2α, β, and γ), thereby preventing formation of the ternary complex (eIF2, GTP, and Met-tRNAi) (Aitken and Lorsch, 2012Aitken C.E. Lorsch J.R. A mechanistic overview of translation initiation in eukaryotes.Nat. Struct. Mol. Biol. 2012; 19: 568-576Crossref PubMed Scopus (265) Google Scholar). This leads to attenuation of global translation initiation, which conserves amino acid pools for essential cellular functions, relieves chaperones of their load, and lowers metabolic demands associated with protein synthesis (Wek et al., 2006Wek R.C. Jiang H.Y. Anthony T.G. Coping with stress: eIF2 kinases and translational control.Biochem. Soc. Trans. 2006; 34: 7-11Crossref PubMed Scopus (1014) Google Scholar). Thus, the ISR integrates many different stressors and initiates global translational attenuation as a protective mechanism. Highly regulated translation dynamics are critical for HSCs, and recent studies revealed the importance of ribosomal proteins and translation factors to maintain stem cell self-renewal and lineage commitment (Blanco et al., 2016Blanco S. Bandiera R. Popis M. Hussain S. Lombard P. Aleksic J. Sajini A. Tanna H. Cortés-Garrido R. Gkatza N. et al.Stem cell function and stress response are controlled by protein synthesis.Nature. 2016; 534: 335-340Crossref PubMed Scopus (245) Google Scholar, Cai et al., 2015Cai X. Gao L. Teng L. Ge J. Oo Z.M. Kumar A.R. Gilliland D.G. Mason P.J. Tan K. Speck N.A. Runx1 deficiency decreases ribosome biogenesis and confers stress resistance to hematopoietic stem and progenitor cells.Cell Stem Cell. 2015; 17: 165-177Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, Khajuria et al., 2018Khajuria R.K. Munschauer M. Ulirsch J.C. Fiorini C. Ludwig L.S. McFarland S.K. Abdulhay N.J. Specht H. Keshishian H. Mani D.R. et al.Ribosome levels selectively regulate translation and lineage commitment in human hematopoiesis.Cell. 2018; 173: 90-103.e19Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, Signer et al., 2014Signer R.A. Magee J.A. Salic A. Morrison S.J. Haematopoietic stem cells require a highly regulated protein synthesis rate.Nature. 2014; 509: 49-54Crossref PubMed Scopus (373) Google Scholar, Signer et al., 2016Signer R.A. Qi L. Zhao Z. Thompson D. Sigova A.A. Fan Z.P. DeMartino G.N. Young R.A. Sonenberg N. Morrison S.J. The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs.Genes Dev. 2016; 30: 1698-1703Crossref PubMed Scopus (55) Google Scholar). The ISR delays translation initiation through eIF2α phosphorylation. Paradoxically, ISR-induced eIF2α phosphorylation increases translation of specific transcription factors including activating transcription factor 4 (ATF4), ATF5, and CHOP (DDIT3), with ATF4 being the most direct ISR effector (Pakos-Zebrucka et al., 2016Pakos-Zebrucka K. Koryga I. Mnich K. Ljujic M. Samali A. Gorman A.M. The integrated stress response.EMBO Rep. 2016; 17: 1374-1395Crossref PubMed Scopus (1045) Google Scholar). Mechanistically, ternary complex scarcity leads to ribosomal bypass of an inhibitory upstream open reading frame (uORF) in the ATF4 mRNA, leading to efficient ATF4 translation and protein upregulation (Lu et al., 2004Lu P.D. Harding H.P. Ron D. Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response.J. Cell Biol. 2004; 167: 27-33Crossref PubMed Scopus (655) Google Scholar). Transcriptional targets of ATF4 include regulators of amino acid metabolism, redox balance, autophagy, and protein synthesis (B’chir et al., 2013B’chir W. Maurin A.C. Carraro V. Averous J. Jousse C. Muranishi Y. Parry L. Stepien G. Fafournoux P. Bruhat A. The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression.Nucleic Acids Res. 2013; 41: 7683-7699Crossref PubMed Scopus (668) Google Scholar, Han et al., 2013Han J. Back S.H. Hur J. Lin Y.H. Gildersleeve R. Shan J. Yuan C.L. Krokowski D. Wang S. Hatzoglou M. et al.ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death.Nat. Cell Biol. 2013; 15: 481-490Crossref PubMed Scopus (1049) Google Scholar, Harding et al., 2003Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. et al.An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2364) Google Scholar). Thus, ISR activation results in efficient translation of ATF4, which in turn activates gene networks to facilitate restoration of cellular homeostasis. The ISR regulates cellular homeostasis in various tissues and cancer cells, but it has typically been studied in bulk populations. Whether the ISR plays distinct roles within the individual cells that make up a tissue hierarchy like the hematopoietic system is unknown. By monitoring ATF4 activity in normal and malignant blood cells, we show that the pro-survival ISR pathway is integral to modulating stem cell stress responses. To assess the expression levels of key ISR pathway components, we analyzed the proteome of purified human HSCs and progenitor cells. Quantitative label-free mass spectrometry revealed lower protein levels of eIF2α, eIF2β, and eIF2γ in HSC/MPPs (multipotent progenitors) compared to downstream progenitors (Figure 1B; Table S1) (E.M.S., S. Xie, A. Mitchell, K.B. Kaufmann, Y. Ge, E. Lechman, T. Kislinger, B.T. Porse, J.E.D., unpublished data). Analysis of more highly purified HSC and progenitor populations showed that eIF2 subunits are similarly expressed in HSCs, MPPs, and multilymphoid progenitors (MLPs), but upregulated in downstream granulocyte-macrophage progenitors (GMPs) (Figure 1C), which is also seen in mouse hematopoiesis (Klimmeck et al., 2012Klimmeck D. Hansson J. Raffel S. Vakhrushev S.Y. Trumpp A. Krijgsveld J. Proteomic cornerstones of hematopoietic stem cell differentiation: distinct signatures of multipotent progenitors and myeloid committed cells.Mol. Cell. Proteomics. 2012; 11: 286-302Crossref PubMed Scopus (50) Google Scholar, Signer et al., 2014Signer R.A. Magee J.A. Salic A. Morrison S.J. Haematopoietic stem cells require a highly regulated protein synthesis rate.Nature. 2014; 509: 49-54Crossref PubMed Scopus (373) Google Scholar). To assess levels of total and phosphorylated eIF2α (P-eIF2α), we used immunohistochemistry and intracellular flow cytometry. Within the progenitor compartment, we observed heterogeneity with low and high expression of eIF2α, which may represent different levels within committed progenitor cells of the erythroid, myeloid, and lymphoid lineages. In HSC/MPPs, levels of eIF2α and P-eIF2α were lower on average than in progenitors (Figure 1D; Figures S1A–S1C). The eIF2α kinase PKR was lower in HSC/MPPs compared to progenitors, whereas GCN2 showed an opposite trend (Figure S1D). These data show that key ISR components are distinctly regulated within the hematopoietic hierarchy and that eIF2α, β, and γ protein levels are low in HSC/MPPs. Low eIF2-GTP-Met-tRNAi ternary complex leads to efficient translation of ATF4, the principal transcription factor that activates ISR target genes. To gain more insight into ATF4 regulation and activity in HSCs and progenitor cells, we used gene expression data from sorted stem and progenitor cell populations from lineage-depleted cord blood (lin– CB) (Laurenti et al., 2013Laurenti E. Doulatov S. Zandi S. Plumb I. Chen J. April C. Fan J.B. Dick J.E. The transcriptional architecture of early human hematopoiesis identifies multilevel control of lymphoid commitment.Nat. Immunol. 2013; 14: 756-763Crossref PubMed Scopus (146) Google Scholar). ATF4 mRNA is highly expressed in CB progenitor cells (mean detection of three ATF4 probes is in the top 20% of 29,331 probes). Furthermore, ATF4 expression is higher in HSC/MPPs compared to progenitors, consistent with previous qPCR results (van Galen et al., 2014avan Galen P. Kreso A. Mbong N. Kent D.G. Fitzmaurice T. Chambers J.E. Xie S. Laurenti E. Hermans K. Eppert K. et al.The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.Nature. 2014; 510: 268-272Crossref PubMed Scopus (237) Google Scholar) (Figure 1E). Of the 225 recently identified ATF4 target genes (Han et al., 2013Han J. Back S.H. Hur J. Lin Y.H. Gildersleeve R. Shan J. Yuan C.L. Krokowski D. Wang S. Hatzoglou M. et al.ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death.Nat. Cell Biol. 2013; 15: 481-490Crossref PubMed Scopus (1049) Google Scholar), 54 were differentially expressed between HSC/MPP and myeloid progenitor cells (Figure 1F; STAR Methods). Gene set enrichment analysis showed that this set of 54 ATF4 target genes is significantly enriched in HSC/MPPs compared to progenitor cells (false discovery rate [FDR] < 0.001; Figure 1G), suggesting that ATF4 is active in HSCs and contributes to the transcriptional activation of ISR target genes. To monitor ATF4 translation, we utilized a lentiviral ATF4 reporter (ATF4rep) that increases translation of an ATF4-GFP fusion gene under conditions of stress that result in eIF2α phosphorylation (Figure S2A) (van Galen et al., 2014avan Galen P. Kreso A. Mbong N. Kent D.G. Fitzmaurice T. Chambers J.E. Xie S. Laurenti E. Hermans K. Eppert K. et al.The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.Nature. 2014; 510: 268-272Crossref PubMed Scopus (237) Google Scholar). To determine the specificity of the ATF4rep to different kinds of stress, we transduced TLS-ERG-immortalized cord blood cells (TEX cells) with the ATF4rep and treated them with various stressors. As expected, the ER stress agent thapsigargin, which is commonly used to induce eIF2α phosphorylation, increased ATF4rep 2.1-fold, as measured by the transgene ratio (TGR) between GFP and TagBFP (Figure 2A; STAR Methods). In contrast, bortezomib, which disrupts proteostasis but is not known to function through the ISR, did not cause a notable increase of the ATF4rep TGR. Similarly, the mTOR inhibitor temsirolimus, the translation elongation inhibitor cycloheximide, and the histone deacetylase inhibitor valproic acid did not induce ATF4rep, despite having an impact on cell viability (Figure S2B). Negative and positive control vectors showed no change of the ATF4rep TGR (Figures S2C and S2D). Collectively, these results indicate that the ATF4rep is efficiently induced by ISR activation but not by stressors that are independent of the ISR. To assess the functionality of the ATF4rep in primary human blood stem and progenitor cells, we transduced lin– CB cells with the ATF4rep and assessed ATF4 protein levels in sorted GFP-high and GFP-low cells (Figure 2B). ATF4 protein levels were higher in GFP-high cells, indicating that GFP provides a measure for the level of endogenous ATF4. We previously showed that severe tunicamycin-induced ER stress activates the ATF4rep in lin– CB cells (van Galen et al., 2014avan Galen P. Kreso A. Mbong N. Kent D.G. Fitzmaurice T. Chambers J.E. Xie S. Laurenti E. Hermans K. Eppert K. et al.The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.Nature. 2014; 510: 268-272Crossref PubMed Scopus (237) Google Scholar). Other conditions, such as oxidative stress or amino acid deprivation, may lead to less severe stress in the native environment of HSCs. We subjected ATF4rep-transduced CD34+ CB cells to hypoxia and valine depletion, the latter of which strongly induced ATF4rep activation (Figure 2C). More broadly, depletion of 13 amino acids induced the ATF4rep in lin– CB cells (Figure 2D). Thus, amino acid deprivation strongly induces ISR activation and ATF4 translation in primitive human CB cells. Phosphorylation of eIF2α reduces ternary complex availability and increases ATF4 levels because slow translation reinitiation favors the ATF4 ORF instead of the inhibitory uORF2 (Figure S2A) (Lu et al., 2004Lu P.D. Harding H.P. Ron D. Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response.J. Cell Biol. 2004; 167: 27-33Crossref PubMed Scopus (655) Google Scholar). To investigate whether ATF4rep activation following valine depletion depends on eIF2α phosphorylation, we transduced CD34+ CB cells with overexpression vectors of wild-type eIF2α and eIF2αS52A, a mutant protein that cannot be inactivated by phosphorylation. Following overexpression of wild-type eIF2α, valine depletion resulted in strong ATF4rep activation in CD34+ CB cells (10-fold TGR increase; Figure 2E). In contrast, overexpression of eIF2αS52A abolished valine depletion-induced activation of the ATF4rep, indicating that eIF2α phosphorylation is responsible for ATF4 upregulation following valine depletion. Knockdown of eIF2α, eIF2β, or eIF2γ increased ATF4rep activity (Figure S2E; Figure 2F), indicating that eIF2 availability regulates ATF4 translation. These data establish that valine depletion leads to eIF2α phosphorylation, which reduces ternary complex availability resulting in efficient ATF4 translation. ATF4 upregulation can induce an adaptive response to diverse stimuli such as oxidative stress, whereas the same pathway can induce apoptosis following severe or prolonged stress (Pakos-Zebrucka et al., 2016Pakos-Zebrucka K. Koryga I. Mnich K. Ljujic M. Samali A. Gorman A.M. The integrated stress response.EMBO Rep. 2016; 17: 1374-1395Crossref PubMed Scopus (1045) Google Scholar). To assess the effect of ATF4 upregulation on survival of CB cells undergoing valine deprivation, we used short hairpin RNA (shRNA) to knock down ATF4 transcript levels (Figure S2F). Following knockdown of ATF4 in CD34+ CB cells, the cells were incubated in valine deficient media to induce translational upregulation of ATF4. Valine depletion of non-targeting short hairpin RNA (shCTRL)-transduced cells for 2 days did not affect proliferation or apoptosis, as measured by EdU incorporation and Annexin-V (Figures 2G and 2H). In contrast, valine depletion of shATF4-transduced cells resulted in decreased proliferation (2-fold, p = 0.0004; Figure 2G) and increased apoptosis (4-fold, p < 0.0001; Figure 2H). Thus, ATF4 promotes cell survival of primitive CD34+ CB cells undergoing valine depletion. To assess ISR activity across the human hematopoietic hierarchy, we sorted CD34+CD38– HSC/MPPs and CD34+CD38+ progenitors from lin– CB and transduced them with the ATF4rep. After 4 days in culture, ATF4rep TGR was 1.6-fold higher in HSC/MPPs compared to progenitors (Figure 3A; Figure S3A), indicating that HSC/MPPs have high ISR activity compared to committed progenitors in vitro. Since in vitro culture could induce stress, we sought to undertake in vivo xenograft studies to examine HSC/MPP and progenitors in the best available setting to approximate homeostatic conditions for human cells. Transplantation of ATF4rep-transduced lin– CB cells showed that human HSC/MPPs in the mouse bone marrow (BM) maintained a 2.4-fold higher ATF4rep TGR compared to downstream progenitors (Figure 3B). Furthermore, HSC/MPPs displayed a 2.3-fold higher ATF4rep TGR in comparison to closely related MLPs (Figure 3C). ATF4rep TGR further declined in downstream common myeloid progenitor and megakaryocyte-erythroid progenitor (CMP and MEP) populations and, compared to HSC/MPPs, was 13-fold lower within mature monocytes, granulocytes, pro-B, and B cells (Figure 3C). These results indicate that HSCs display higher ISR activity than progenitors when assessed in basal conditions, which may contribute to HSC endurance. To determine if the high ISR activity in HSCs is associated with improved survival, we transplanted lin– CB cells expressing high ATF4rep activity (GFP-high) and low ATF4rep activity (GFP-low) into mice (Figure S3B). The level of engraftment from GFP-high cells was increased compared to GFP-low cells (Figure 3D). Furthermore, the number of engrafted mice from GFP-high cells was increased compared to GFP-low cells. Using limiting dilution analysis, we calculated that the frequency of engrafting cells was 1 in 19,804 cells in the GFP-high population and 1 in 71,922 cells in the GFP-low population; bulk TagBFP+ cells possessed an intermediate HSC frequency of 1 in 41,046 cells (Figure 3E; Figure S3C). Thus, cells that efficiently translate ATF4 are robust and survive transplantation, indicating that high ISR activity marks functional HSCs. The hierarchical structure of normal hematopoiesis is partially maintained in acute myeloid leukemia (AML) and leukemia stem cells (LSCs) that share numerous stemness properties with normal HSCs are responsible for disease propagation (Dick, 2008Dick J.E. Stem cell concepts renew cancer research.Blood. 2008; 112: 4793-4807Crossref PubMed Scopus (811) Google Scholar). We examined the expression of central ISR components in AML cell populations. Similar to our findings in normal CB cells, mass spectrometry revealed lower protein levels of eIF2α, eIF2β, and eIF2γ in phenotypically primitive (CD34+CD38–) compared to differentiated (CD34+CD38+) AML cell populations (Figure 4A). Gene set enrichment analysis of 54 ATF4 target genes showed enrichment in primitive AML cells (Figure 4B). Similar trends were observed when we compared functionally validated LSC+ versus LSC– populations for eIF2β and eIF2γ protein detection (Figure S3D) and ATF4 target mRNA expression (Figure S3E). Low eIF2 levels and high ATF4 target gene expression suggest that the high ISR activity we observed in normal HSC/MPPs is conserved in LSCs. To determine if ISR activity could be used to separate functionally distinct AML cell populations, we examined ATF4rep expression in human AML cells. Using the 8227 AML cell culture system, which maintains hierarchical organization in vitro (Lechman et al., 2016Lechman E.R. Gentner B. Ng S.W. Schoof E.M. van Galen P. Kennedy J.A. Nucera S. Ciceri F. Kaufmann K.B. Takayama N. et al.miR-126 regulates distinct self-renewal outcomes in normal and malignant hematopoietic stem cells.Cancer Cell. 2016; 29: 214-228Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), we found that primitive CD34+CD38– cells displayed the highest ATF4rep TGR, which progressively declined in downstream progenitors (Figure 4C; Figure S3F). We sorted GFP-high and GFP-low populations from ATF4rep-transduced 8227 AML cells and assessed culture-initiating capacity, a surrogate assay for LSC function. Only GFP-high cells were able to expand in culture for over 3 weeks (Figure 4D), indicating that ATF4rep activity can be used to prospectively isolate cells that display features of primitive LSCs. We wanted to evaluate ATF4rep expression in the malignant hierarchy using primary AML patient samples. Since in vitro expansion of primary AML cells is challenging and does not maintain the developmental hierarchy, we transduced AML cells with the ATF4rep and established AML in immunodeficient mice (Figure 4E; Table S2). After 6–8 weeks, mice were sacrificed and human AML cells were assessed for CD34/CD38 expression and ATF4rep TGR by flow cytometry. In four out of five samples, the ATF4rep TGR was higher in CD34+ compared to CD34– cells (Figure 4F; Figure S4A). AML09191 was an exception, showing higher ATF4rep TGR in CD34– compared to CD34+ cells. Taken together, these ATF4rep measurements suggest that most primary AML samples have high ISR activity in phenotypically primitive cells. Although CD34 can enrich for LSCs in most AML cases, the surface phenotype of AML cells can be decoupled from LSC activity. To test whether primary AML cells that possess high ISR activity are enriched for LSC function, we sorted TagBFP+ (bulk), GFP-low and GFP-high populations and transplanted cells into secondary recipient mice (Figure 4E). GFP-high cells showed higher engraftment in three out of eight samples, with two additional samples showing the same trend (Figure S4B). In AML09191, GFP-high cells showed higher engraftment, indicating that ATF4rep activity maintained its correlation with LSC activity despite its negative correlation with CD34 expression. On average, GFP-high cells showed 8.5-fold higher secondary engraftment than GFP-low cells (p < 0.0001; Figure 4G). These data indicate that high ISR activity is associated with normal and malignant stem cells. Our data establish that the ISR-mediated pro-survival program is uniquely wired in HSCs where it plays a role in governing HSC function. The tendency of HSCs to undergo apoptosis following stress and damage (Milyavsky et al., 2010Milyavsky M. Gan O.I. Trottier M. Komosa M. Tabach O. Notta F. Lechman E. Hermans K.G. Eppert K. Konovalova Z. et al.A distinctive DNA damage response in human hematopoietic stem cells reveals an apoptosis-independent role for p53 in self-renewal.Cell Stem Cell. 2010; 7: 186-197Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, van Galen et al., 2014avan Galen P. Kreso A. Mbong N. Kent D.G. Fitzmaurice T. Chambers J.E. Xie S. Laurenti E. Hermans K. Eppert K. et al.The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.Nature. 2014; 510: 268-272Crossref PubMed Scopus (237) Google Scholar, Yahata et al., 2011Yahata T. Takanashi T. Muguruma Y. Ibrahim A.A. Matsuzawa H. Uno T. Sheng Y. Onizuka M. Ito M. Kato S. Ando K. Accumulation of oxidative DNA damage restricts the self-renewal capacity of human hematopoietic stem cells.Blood. 2011; 118: 2941-2950Crossref PubMed Scopus (229) Google Scholar) needs to be balanced with alleviation of low-level perturbations that occur under homeostasis to ensure HSC functionality. Our results show that amino acid deprivation is one such perturbation where ATF4-dependent pro-survival signals play a protective role. We show that high expression of the ISR effector ATF4 is contingent on eIF2 scarcity, representing a mechanism of ISR activity that may also safeguard stem cells in other tissues (Blanco et al., 2016Blanco S. Bandiera R. Popis M. Hussain S. Lombard P. Aleksic J. Sajini A. Tanna H. Cortés-Garrido R. Gkatza N. et al.Stem cell function and stress response are controlled by protein synthesis.Nature. 2016; 534: 335-340Crossref PubMed Scopus (245) Google Scholar, Zismanov et al., 2016Zismanov V. Chichkov V. Colangelo V. Jamet S. Wang S. Syme A. Koromilas A.E. Crist C. Phosphorylation of eIF2α is a translational control mechanism regulating muscle stem cell quiescence and self-renewal.Cell Stem Cell. 2016; 18: 79-90Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). ATF4 is modulated by additional signals including mTOR and post-translational modifications (Ben-Sahra et al., 2016Ben-Sahra I. Hoxhaj G. Ricoult S.J.H. Asara J.M. Manning B.D. mTORC1 induces purine synthesis through control of the mitochondrial tetrahydrofolate cycle.Science. 2016; 351: 728-733Crossref PubMed Scopus (444) Google Scholar, Wortel et al., 2017Wortel I.M.N. van der Meer L.T. Kilberg M.S. van Leeuwen F.N. Surviving Stress: Modulation of ATF4-Mediated Stress Responses in Normal and Malignant Cells.Trends Endocrinol. Metab. 2017; 28: 794-806Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Several processes that are important for HSC maintenance are regulated by ATF4, including autophagy, amino acid metabolism and oxidative stress resistance (B’chir et al.," @default.
• W2899451374 created "2018-11-09" @default.
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• W2899451374 title "Integrated Stress Response Activity Marks Stem Cells in Normal Hematopoiesis and Leukemia" @default.
• W2899451374 cites W1736968472 @default.
• W2899451374 cites W1773238988 @default.
• W2899451374 cites W1968261414 @default.
• W2899451374 cites W1972482541 @default.
• W2899451374 cites W1982460924 @default.
• W2899451374 cites W1999203943 @default.
• W2899451374 cites W2000630389 @default.
• W2899451374 cites W2019199359 @default.
• W2899451374 cites W2019425923 @default.
• W2899451374 cites W2027821655 @default.
• W2899451374 cites W2031759349 @default.
• W2899451374 cites W2048098925 @default.
• W2899451374 cites W2048551040 @default.
• W2899451374 cites W2055807959 @default.
• W2899451374 cites W2073692681 @default.
• W2899451374 cites W2074471441 @default.
• W2899451374 cites W2074670317 @default.
• W2899451374 cites W2080752012 @default.
• W2899451374 cites W2084528616 @default.
• W2899451374 cites W2089238459 @default.
• W2899451374 cites W2097879198 @default.
• W2899451374 cites W2116826477 @default.
• W2899451374 cites W2116999641 @default.
• W2899451374 cites W2119873438 @default.
• W2899451374 cites W2131632095 @default.
• W2899451374 cites W2135925682 @default.
• W2899451374 cites W2147260787 @default.
• W2899451374 cites W2154816100 @default.
• W2899451374 cites W2166063624 @default.
• W2899451374 cites W2170776626 @default.
• W2899451374 cites W2197815519 @default.
• W2899451374 cites W2255229856 @default.
• W2899451374 cites W2258318060 @default.
• W2899451374 cites W2430983120 @default.
• W2899451374 cites W2491651701 @default.
• W2899451374 cites W2518959324 @default.
• W2899451374 cites W2533191544 @default.
• W2899451374 cites W2560012305 @default.
• W2899451374 cites W2592615787 @default.
• W2899451374 cites W2742971656 @default.
• W2899451374 cites W2766910695 @default.
• W2899451374 cites W2793800931 @default.
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