The Integrated Stress Response Activity Marks Stem Cells in Normal Hematopoiesis and Leukemia
Bibliographic record
Abstract
Abstract Lifelong maintenance of the blood system requires equilibrium between clearance of damaged hematopoietic stem cells (HSCs) and long-term survival of the HSC pool. Perturbations of cellular homeostasis such as nutrient deprivation, irradiation, and endoplasmic reticulum stress can result in HSC loss. However, HSCs must survive low-level stressors in order to sustain lifelong replenishment of the hematopoietic system. It is poorly understood how human HSCs balance apoptosis with survival in the context of basal stress, and how adaptive signalling is regulated in leukemia stem cells (LSCs). The Integrated Stress Response (ISR) is an adaptive pathway that can protect cells against stressors such as ROS, nutrient deprivation and misfolded proteins. To assess the expression levels of key ISR pathway components, we analyzed the proteome of purified human HSCs and progenitor cells from cord blood (CB). Quantitative label-free mass spectrometry revealed lower expression of eIF2α, eIF2β and eIF2γ subunits in HSCs compared to downstream progenitors. Furthermore, activated-transcription factor 4 (ATF4) mRNA is highly expressed in HSCs compared to progenitors. Similar to our findings in normal CB cells, analysis of acute myeloid leukemia (AML) patient samples revealed lower protein levels of eIF2α, eIF2β and eIF2γ in phenotypically primitive (CD34+CD38-) compared to differentiated (CD34+CD38+) AML cell populations. These results suggest that primitive cells in normal hematopoiesis and AML are primed for ISR activation.To assess ISR activity in human HSPCs, we used an ATF4 lentiviral reporter (ATF4rep) that measures ISR-induced ATF4 translation. We subjected ATF4rep-transduced CD34+ CB cells to hypoxia and amino acid deprivation, and found that valine depletion strongly induced ATF4rep activity. ATF4rep upregulation was abolished in the presence of an eIF2αS52A mutant that cannot be phosphorylated. Furthermore, knockdown of eIF2α, eIF2β or eIF2γ subunits in CD34+ CB cells increased ATF4rep activity. Thus, low levels of eIF2α, eIF2β or eIF2γ result in efficient ATF4 translation, and nutrient deprivation upregulates ATF4 through eIF2α phosphorylation. We assessed the effect of ATF4 upregulation on CB cell proliferation and survival. Following knockdown of ATF4 mRNA in CD34+ CB cells, the cells were incubated in valine deficient media to induce translational upregulation of ATF4. Valine depletion of shCTRL-transduced cells for 2 days did not affect proliferation or apoptosis, as measured by EdU incorporation or Annexin-V. In contrast, valine depletion of shATF4-transduced cells resulted in decreased proliferation (2-fold, P = 0.0004) and increased apoptosis (4-fold, P < 0.0001,). Thus, ATF4 promotes survival of primitive CD34+ CB cells undergoing valine depletion.We performed in vivo xenograft studies to examine the ISR activity in the best available setting to approximate homeostatic conditions for human HSPCs. Transplantation of ATF4rep-transduced CB cells showed that human HSPCs in the mouse bone marrow maintained a 2.4-fold higher ATF4rep activity compared to downstream progenitors (P = 0.0002). ATF4rep activity further declined in mature monocytes, granulocytes and B-cells (13-fold, P < 0.0001). To determine if high ISR activity is associated with improved HSC function, we transplanted lin- CB cells expressing high ATF4rep activity (GFP-high) and low ATF4rep activity (GFP-low) into mice. The level of engraftment as well as the number of engrafted mice was increased from GFP-high cells compared to GFP-low cells (P = 0.001). The hierarchical structure of normal hematopoiesis is partially maintained in AML. We evaluated ATF4rep expression in the malignant hierarchy and found that 4/5 patient samples had higher ATF4rep expression in CD34+ cells compared to CD34- cells. Furthermore, serial transplantation of ATF4rep-transduced cells showed higher engraftment from GFP-high compared to GFP-low cells ( P < 0.0001). Thus, primary human AML cells that possess high ISR activity are enriched for LSC function.Our data establish that the adaptive ISR pathway plays a key role in maintaining homeostasis of normal and malignant stem cells. We show that Amino acid deprivation activates the ISR in human HSPCs resulting in ATF4-dependent pro-survival signals. In an unperturbed state, HSCs are in a state of primed ISR activity, mechanistically maintained by eIF2 scarcity and high ATF4 levels. Disclosures No relevant conflicts of interest to declare.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".