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The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells 1 . This process requires major molecular adaptations to allow HSCs to meet the regulatory and metabolic requirements for cell division 2 – 4 . The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA) 5 , a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation. Haematopoietic stem cells show progressive functional decline with age that can be reversed by stimulation of chaperone-mediated autophagy in old mice and aged humans.

Myelodysplastic syndromes (MDS) frequently progress to acute myeloid leukemia (AML); however, the cells leading to malignant transformation have not been directly elucidated. As progression of MDS to AML in humans provides a biological system to determine the cellular origins and mechanisms of neoplastic transformation, we studied highly fractionated stem cell populations in longitudinal samples of patients with MDS who progressed to AML. Targeted deep sequencing combined with single-cell sequencing of sorted cell populations revealed that stem cells at the MDS stage, including immunophenotypically and functionally defined pre-MDS stem cells (pre-MDS-SC), had a significantly higher subclonal complexity compared to blast cells and contained a large number of aging-related variants. Single-cell targeted resequencing of highly fractionated stem cells revealed a pattern of nonlinear, parallel clonal evolution, with distinct subclones within pre-MDS-SC and MDS-SC contributing to generation of MDS blasts or progression to AML, respectively. Furthermore, phenotypically aberrant stem cell clones expanded during transformation and stem cell subclones that were not detectable in MDS blasts became dominant upon AML progression. These results reveal a crucial role of diverse stem cell compartments during MDS progression to AML and have implications for current bulk cell–focused precision oncology approaches, both in MDS and possibly other cancers that evolve from premalignant conditions, that may miss pre-existing rare aberrant stem cells that drive disease progression and leukemic transformation. High-resolution sequencing of longitudinal patient samples reveals subclonal mutational diversity in the stem cell compartment driving parallel evolution patterns in acute myeloid leukemia progression.

Gain of function mutations in isocitrate dehydrogenase 1 (IDH1) have been detected in cases of acute myeloid leukemia (AML). The application of an allosteric IDH1 inhibitor in AML cells promotes blast differentiation and restores DNA cytosine methylation patterns. Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo . Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

During aging, hematopoietic stem cell (HSC) function progressively declines which can lead to reduced blood cell production and regeneration. This work uncovered that cell surface presentation of P-selectin (CD62P, encoded by Selp ) increases in a large fraction of aging HSCs driven by a proinflammatory milieu in mice. Notably, expression of P-selectin molecularly and functionally dichotomized the aging HSC pool; stem cells presenting with highly abundant P-selectin were hallmarked by aging-associated gene expression programs and reduced repopulation capacity upon regenerative stress. Ectopic expression of Selp in young HSCs was sufficient to impair long-term reconstitution potential and impair erythropoiesis. Mechanistically, we uncovered that P-selectin receptor activation by its primary ligand, P-selectin glycoprotein ligand-1, suppressed aging-associated gene expression, and, reversely, lack of P-selectin signaling led to HSC premature aging. Collectively, our study uncovered a functional role of P-selectin engagement in regulating HSC regeneration and driving stem cell aging when perturbed. P-selectin has been considered as a biomarker of hematopoietic stem cell (HSC) aging. Here, Yang et al. uncovered a new functional role of P-selectin engagement in regulating HSC regeneration and driving stem cell aging when perturbed.

During aging, hematopoietic stem cell (HSC) function progressively declines which can lead to reduced blood cell production and regeneration. In this study, we uncovered that during aging the cell surface presentation of P-selectin (CD62P, encoded by Selp) increases in a large fraction of HSCs. Notably, expression of P-selectin molecularly and functionally dichotomized the aging HSC pool; stem cells presenting with high abundance of P-selectin were hallmarked by aging-associated gene expression programs and reduced repopulation upon regenerative stress. Overexpression of Selp in young HSCs was sufficient to impair long-term reconstitution potential and repress erythropoiesis. Moreover, IL-1β triggered Selp expression in HSCs. The aged transcriptome, including Selp, was largely restored when aged HSCs were transplanted to young mice. Mechanistically, we uncovered that appropriate stimulation of P-selectin by its primary ligand, P-selectin glycoprotein ligand-1 (PSGL-1), suppressed aging-associated gene expression and reversely, lack of P-selectin signaling led to HSC premature aging. Collectively, our study uncovered a functional role of P-selectin engagement in regulating HSC regeneration and driving stem cell aging when perturbed.

In the version of this article originally published, Ulrich Steidl’s name was listed as “and Ulrich Steidl.” His name has been updated to “Ulrich Steidl.” The error has been fixed in the print, PDF and HTML versions of this article.

Iron homeostasis-regulatory pathways mediate hematopoietic stem cell fate Hematopoiesis is a highly regulated, step-wise process in which hematopoietic stem cells (HSCs) residing at the top of the hematopoietic hierarchy are capable of self-renewing to maintain the stem cell pool, and differentiating to give rise to blood cells of all lineages. Inefficient hematopoiesis is a frequent and critical clinical problem in aplastic anemia, myelodysplastic syndromes (MDS), immune thrombocytopenia, as well as chemotherapy-induced pancytopenia. Eltrombopag (EP), a small molecule initially designed as a thrombopoietin receptor (TPO-R) agonist, has emerged as a potent platelet-stimulating agent and has also shown remarkable efficacy in stimulating sustained multilineage hematopoiesis, suggesting an effect at the level of primitive HSCs. Apart from stimulating TPO signaling, EP has been reported to trigger TPO-R independent pathways involving iron chelation. Nevertheless, it remains to be determined whether EP exerts its effect at the HSC level, and whether the iron-chelating property is functionally relevant to the HSC stimulation by EP. We found that EP significantly enhanced not only multilineage differentiation, but also serial replating capacity of purified human HSCs. In addition, comparative analysis of stem cells in the bone marrow of patients receiving EP showed a marked increase in the number of functional stem cells compared to patients treated with romiplostim, another TPO-R agonist lacking iron-chelating ability. Microarray analysis of human HSCs also confirmed iron-associated molecular changes in EP-treated HSCs that were absent in TPO-treated HSCs. This cellular and molecular evidence strongly suggests a role of iron-mediated pathways in regulating HSC function that is distinct from TPO stimulation. Therefore, we utilized separation-of-function mouse models, including wild type and TPO receptor (TPOR) knockout models, to examine TPO-R independent effects of EP on HSC function ex vivo and in vivo. In both mouse models, we observed a significant increase of HSC self-renewal upon EP treatment, which was also consistently observed with two other clinically available iron chelators, Deferoxamine (DFO) and Deferasirox (DFX). Importantly, the increase of HSC self-renewal upon iron chelation was abrogated by preloading with ferric ammonium citrate (FAC), demonstrating the causative role of intracellular iron levels in the modulation of HSC self-renewal. Gene expression profiling of mouse HSCs treated ex vivo with DFO or EP revealed alterations in molecular pathways that are consistent with reduction of intracellular labile iron pools (LIP), including the activation of transferrin receptor (TfrciCD71) and Nuclear receptor coactivator 4 (encoded by Ncoa4). Intriguingly, simultaneous inhibition of CD71 and NOCA4 abrogated the increase of HSC self-renewal by iron chelators, suggesting the activation of iron-regulatory pathways following iron reduction mediated the HSC stimulatory effects. Further gene expression and metabolite profiling of cells exposed to iron chelators also revealed alterations in metabolic pathways associated with fatty acid oxidation (FAO), which was validated by Seahorse, an assay that directly measures the extracellular fluxes of oxygen consumption. Furthermore, iron chelation-mediated increase in HSC number was rescued by pharmacologic inhibition of CPT-1, a mitochondrial enzyme involved in the conjugation of fatty acids to carnitine for subsequent transfer inside mitochondria. Together, our data demonstrates the integral role of FAO in governing HSC fate transitions following reduction of LIP. Further molecular interrogation revealed an increase in free arachidonic acid (AA) following iron chelator treatment ex vivo, which we hypothesized could be partially regulated by NCOA4-mediated ferritinophagy. We designed short hairpin RNA (shRNA) constructs to knockdown Acsl4 , a member of the long-chain acyl-CoA synthetases that preferentially utilizes AA as substrates, to selectively inhibit the increase in FAO contributed by AA. We found that Acsl4 knockdown abrogated the increase in FAO rate stimulated by DFO, indicating that iron chelation increases the rate of FAO through the mobilization of intracellular AA stores. It has been previously described that upon nutrient deprivation, fatty acids packaged in lipid droplets mobilize to mitochondria and induce β-oxidation of the fatty acids. Inhibition of lipolysis by diethylumbelliferyl phosphate (DEUP) abrogated FAO stimulation upon iron chelation, suggesting the contribution of lipid droplets in fueling mitochondrial oxidation. Interestingly, simultaneous inactivation of AA and inhibition of lipolysis did not further decrease FAO. These findings indicate that AA fuels FAO by a mechanism that is predominantly dependent on lipid droplet mobilization and lipolysis. In conclusion, our data has provided proof-of-concept that experimental reduction of the intracellular labile iron pool, the most readily chelatable form of iron within cells, leads to an array of metabolic reprogramming and an increase in HSC numbers. In-depth investigation on the molecular underpinnings demonstrate that the intracellular labile iron pool reinforces stem cell-maintaining metabolic programs and acts as a rheostat in dividing HSCs. *Please refer to dissertation for diagrams.

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in AML patients’ cells. Our study provides proof-of-concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Bone marrow resident and rarely dividing haematopoietic stem cells (HSC) harbour an extensive self-renewal capacity to sustain life-long blood formation; albeit their function declines during ageing. Various molecular mechanisms confer stem cell identity, ensure long-term maintenance and are known to be deregulated in aged stem cells. How these programs are coordinated, particularly during cell division, and what triggers their ageing-associated dysfunction has been unknown. Here, we demonstrate that HSC, containing the lowest amount of cytoplasmic chelatable iron (labile iron pool) among hematopoietic cells, activate a limited iron response during mitosis. Engagement of this iron homeostasis pathway elicits mobilization and β-oxidation of arachidonic acid and enhances stem cell-defining transcriptional programs governed by histone acetyl transferase Tip60/KAT5. We further find an age-associated expansion of the labile iron pool, along with loss of Tip60/KAT5-dependent gene regulation to contribute to the functional decline of ageing HSC, which can be mitigated by iron chelation. Together, our work reveals cytoplasmic redox active iron as a novel rheostat in adult stem cells; it demonstrates a role for the intracellular labile iron pool in coordinating a cascade of molecular events which reinforces HSC identity during cell division and to drive stem cell ageing when perturbed. As loss of iron homeostasis is commonly observed in the elderly, we anticipate these findings to trigger further studies into understanding and therapeutic mitigation of labile iron pool-dependent stem cell dysfunction in a wide range of degenerative and malignant pathologies. Competing Interest Statement B.W. has received funds for research projects and serving on advisory boards from Novartis Pharmaceuticals.