Search

The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers. Significance: We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation. See related commentary by Doulatov.

Target identification for chimeric antigen receptor (CAR) T-cell therapies remains challenging due to the limited repertoire of tumor-specific surface proteins. Intracellular proteins presented in the context of cell surface HLA provide a wide pool of potential antigens targetable through T-cell receptor mimic antibodies. Mass spectrometry (MS) of HLA ligands from 8 hematologic and nonhematologic cancer cell lines identified a shared, non-immunogenic, HLA-A*02–restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene. CAR T cells directed against the ALNEQIARL:HLA-A*02 complex exhibited high sensitivity and specificity for recognition and killing of multiple cancer types, especially those of hematologic origin, and were efficacious in mouse models against a human leukemia and a solid tumor. In contrast, no toxicities toward resting or activated healthy leukocytes as well as hematopoietic stem cells were observed. This shows how MS can inform the design of broadly reactive therapeutic T-cell receptor mimic CAR T-cell therapies that can target multiple cancer types currently not druggable by small molecules, conventional CAR T cells, T cells, or antibodies.

To gain insight into how ERG translocations cause prostate cancer, we performed single cell transcriptional profiling of an autochthonous mouse model at an early stage of disease initiation. Despite broad expression of ERG in prostate epithelial cells, we observed enhanced proliferation primarily in an intermediate subpopulation with a basal identity based on transcriptomics but with luminal morphology and cytokeratin expression. Through a series of lineage tracing and primary prostate epithelial transplantation experiments, we find that tumor initiating activity resides in a subpopulation of basal cells that also express luminal genes such asTmprss2andNkx3.1, but not in the larger population of classical luminal cells. Upon ERG activation, these basal-luminal hybrid cells expand into a highly proliferative population of intermediate basal-luminal identity then transition to fully differentiated luminal cells that reflect the lineage of ERG-positive cancers. These findings help resolve a preexisting debate about the cell of origin of ERG-driven prostate cancer and narrow the focus for future mechanistic studies to a basal-luminal subpopulation of tumor initiating cells.

Supplemental Figure 1. Generation of a panel of iPSCs from patients with AML. Supplemental Figure 2. Reprogramming aids reconstruction of the evolutionary history and clonal composition of AML. Supplemental Figure 3. Transplantation of AML-iPSCs into immunodeficient mice. Supplemental Figure 4. Developmental block in a subset of AML-iPSC lines. Supplemental Figure 5. Transplantation of primary AML cells and patient-matched AMLiPSC lines. Supplemental Figure 6. Single-cell RNA-sequencing analyses of matched primary and iPSC-derived leukemia cells from patient AML-47. Supplemental Figure 7. Cell cycle and pseudotime analyses. Supplemental Figure 8. Comparison of scRNA-Seq data integration and clustering methods and pseudobulk differential gene expression analyses.

Table S1. Patient characteristics. AML: acute myeloid leukemia; MDS: myelodysplastic syndrome; MPN: myeloproliferative neoplasm; ET: essential thrombocythemia; PBMCs: peripheral blood mononuclear cells; BMMCs: bone marrow mononuclear cells; PDX: patient-derived xenografts Table S2. All patient samples used in this study with genetic characterization and reprogramming outcomes. Blue font denotes partially reprogrammed (as opposed to bona fide iPSC) colonies and clones. Table S3. All AML-iPSC lines phenotypically characterized. Table S4. Top 50 upregulated genes (highest log2 fold change) in each cluster. Table S5. Primers used for genotyping. Table S6. Primers used for qRT-PCR analyses.

The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers.Significance:We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation.See related commentary by Doulatov.

SUMMARYGlutathione (GSH) is a small molecule thiol abundantly present in all eukaryotes with key roles in oxidative metabolism1. Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions2. GSH is exclusively synthesized in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remain elusive. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, to regulate GSH transport into mitochondria. SLC25A39 loss reduces mitochondrial GSH import and abundance without impacting whole cell GSH levels. Cells lacking both SLC25A39 and its paralog SLC25A40 exhibit defects in the activity and stability of ironsulfur cluster containing proteins. Moreover, mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Remarkably, the heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enabled mitochondrial GSH production and ameliorated the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH import machinery.

Significance Modeling cancer formation requires introduction of relevant oncogenic perturbations into normal cells in a tissue/cell-type–specific manner. Genetically engineered mouse models are powerful but require significant time and cost to generate and maintain. The ability to edit primary epithelial cells ex vivo followed by orthotopic transplantation provides an alternative strategy for cancer modeling but requires efficient gene editing, typically in a multiplex fashion. Here we successfully engineer multigenic perturbations or chromosomal rearrangements in primary prostate organoids through single-step Cas9–sgRNA ribonucleoprotein electroporation. This approach can also address cell-of-origin questions by directly editing and transplanting freshly isolated subpopulations without the intermediate step of organoid culture, providing a rapid complement to traditional lineage tracing approaches., The increasing complexity of different cell types revealed by single-cell analysis of tissues presents challenges in efficiently elucidating their functions. Here we show, using prostate as a model tissue, that primary organoids and freshly isolated epithelial cells can be CRISPR edited ex vivo using Cas9–sgRNA (guide RNA) ribotnucleoprotein complex technology, then orthotopically transferred in vivo into immunocompetent or immunodeficient mice to generate cancer models with phenotypes resembling those seen in traditional genetically engineered mouse models. Large intrachromosomal (∼2 Mb) or multigenic deletions can be engineered efficiently without the need for selection, including in isolated subpopulations to address cell-of-origin questions.

In this issue of Cell Stem Cell, Shen et al. (2020) and Wang et al. (2020) independently identify the essential function of m(6)A demethylase ALKBH5 in maintaining myeloid leukemia stem cells. These studies expand the regulators of the epitranscriptome that are required for acute myeloid leukemia (AML) development.

Glutathione (GSH) is a small-molecule thiol that is abundant in all eukaryotes and has key roles in oxidative metabolism

Context Target identification for CAR-T cell therapies, especially shared targets among different malignancies, remains challenging due to the limited repertoire of tumor-specific surface proteins. Intracellular proteins presented in the context of cell surface HLA provide a wide pool of potential antigens targetable through TCR-mimic antibodies. Objective and Design We hypothesized that mass spectrometry (MS)-based analysis of the presented HLA ligands of multiple cancer cell lines can be utilized to identify a shared, tumor-associated HLA ligand, which could then be targeted by TCR-mimic CAR-T cells specific for this particular HLA ligand. Results MS of HLA ligands from eight hematological and non-hematological cancer cell lines identified a shared, non-immunogenic, HLA-A*02 restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene, which was later identified in >90% (20/22) of all A*02-positive cell lines tested. A TCR-mimic scFv was prepared against this epitope, termed “NDC80-C.” CAR-T cells transduced with the NDC80-C, directed against the ALNEQIARL:HLA-A*02 complex on cancer cells, demonstrated high sensitivity and specificity for recognizing and killing multiple cancer types with a high preference for hematological malignancies (e.g., AML, ALL, DLBCL, and ALCL). In contrast, healthy leukocytes, activated B and T-cells, or hematopoietic stem cells from A*02-positive and -negative donors were not lysed. NDC80-C CAR-T cells suppressed colony formation of primary AML cells but not of healthy stem cells. Additionally, NDC80-C CAR-T cells were efficacious in mouse models against human mesothelioma and leukemia. Conclusions Our study demonstrates how MS can inform the design of tumor-agnostic TCR-mimic therapeutic platforms that target structures that are currently not druggable by small molecules, conventional CAR-T cells, T-cells, or antibodies. This strategy lays the groundwork for a potential antibody platform therapy or CAR-T cell with efficacy against highly proliferative A*02-positive cancer cells, independent of the respective cancer type.

N(6)-methyladenosine (m(6)A) on mRNAs mediates different biological processes and its dysregulation contributes to tumorigenesis. How m(6)A dictates its diverse molecular and cellular effects in leukemias remains unknown. We found that YTHDC1 is the essential m(6)A reader in myeloid leukemia from a genome-wide CRISPR screen and m(6)A is required for YTHDC1 to undergo liquid-liquid phase separation and form nuclear YTHDC1-m(6)A Condensates (nYACs). The number of nYACs increases in acute myeloid leukemia (AML) cells compared to normal hematopoietic stem and progenitor cells. AML cells require the nYACs to maintain cell survival and the undifferentiated state that is critical for leukemia maintenance. Furthermore, nYACs enable YTHDC1 to protect m(6)A-mRNAs from the PAXT-complex and exosome-associated RNA degradation. Collectively, m(6)A is required for the formation of a nuclear body mediated by phase separation that maintains mRNA stability and control cancer cell survival and differentiation.

Summary: Leukemia stem cells (LSCs) are believed to have more distinct vulnerabilities than the bulk acute myeloid leukemia (AML) cells, but their rarity and the lack of universal markers for their prospective isolation hamper their study. We report that genetically clonal induced pluripotent stem cells (iPSCs) derived from an AML patient and characterized by exceptionally high engraftment potential give rise, upon hematopoietic differentiation, to a phenotypic hierarchy. Through fate-tracking experiments, xenotransplantation, and single-cell transcriptomics, we identify a cell fraction (iLSC) that can be isolated prospectively by means of adherent in vitro growth that resides on the apex of this hierarchy and fulfills the hallmark features of LSCs. Through integrative genomic studies of the iLSC transcriptome and chromatin landscape, we derive an LSC gene signature that predicts patient survival and uncovers a dependency of LSCs, across AML genotypes, on the RUNX1 transcription factor. These findings can empower efforts to therapeutically target AML LSCs.

RNA binding proteins (RBPs) have been increasingly recognized as an important class of regulators of normal and malignant hematopoiesis. However, the exact function and underpinning mechanisms of the RBPs that govern hematopoietic stem cells (HSCs) remains poorly characterized. We had previously identified SYNCRIP as a critical RBP that controls leukemia stem cell program in myeloid leukemia. Here, using the novel murine genetic conditional knockout (cKO) model, we delineated the role of SYNCRIP in regulating the low-output HSC. We developed a Syncrip cKO allele and crossed Syncripf/f mice to the interferon (IFN) -a-inducible Mx-1-Cre mice to create Syncripf/f Mx-1-Cre+. We consistently obtained near complete depletion of SYNCRIP 3 weeks after two consecutive Poly(I:C) injections. We observed that SYNCRIP is dispensable for static hematopoiesis and Syncrip KO animals showed equivalent number and frequencies of stem and progenitor cells (Lin-Sca+cKit+ (LSK)- LT-HSC (CD48-CD150+); MPP1 (CD48-CD150-); MPP2 (CD48+CD150+); MPP4 (CD48-CD150-)). However, KO SyncripD/D deficient cells were outcompeted by WT Syncripf/f cells in the transplantation setting (bone marrow (BM) chimerism WT (n=9) 38% ± 7.8% vs. KO (n=9) 2.7% ± 0.8%, p Disclosures No relevant conflicts of interest to declare.

SUMMARY Stem cells balance cellular fates through asymmetric and symmetric divisions in order to self-renew or to generate downstream progenitors. Symmetric commitment divisions in stem cells are required for rapid regeneration during tissue damage and stress. The control of symmetric commitment remains poorly defined. Using single-cell RNA sequencing (scRNA-seq) in combination with transcriptomic profiling of HSPCs (hematopoietic stem and progenitor cells) from control and m6A methyltransferase Mettl3 conditional knockout mice, we found that m6A-deficient hematopoietic stem cells (HSCs) fail to symmetrically differentiate. Dividing HSCs are expanded and are blocked in an intermediate state that molecularly and functionally resembles multipotent progenitors. Mechanistically, RNA methylation controls Myc mRNA abundance in differentiating HSCs. We identified MYC as a marker for HSC asymmetric and symmetric commitment. Overall, our results indicate that RNA methylation controls symmetric commitment and cell identity of HSCs and may provide a general mechanism for how stem cells regulate differentiation fate choice., In Brief Cheng et al. uncover RNA methylation as a guardian in hematopoietic stem cell (HSC) fate decisions. m6A maintains hematopoietic stem cell symmetric commitment and identity. This study may provide a general mechanism for how RNA methylation controls cellular fate., Graphical Abstract

Aging-associated defects in hematopoietic stem cells (HSCs) can manifest in their progeny, leading to aberrant activation of the NLRP3 inflammasome in macrophages and affecting distant tissues and organismal health span. Whether the NLRP3 inflammasome is aberrantly activated in HSCs during physiological aging is unknown. We show here that SIRT2, a cytosolic NAD+-dependent deacetylase, is required for HSC maintenance and regenerative capacity at an old age by repressing the activation of the NLRP3 inflammasome in HSCs cell autonomously. With age, reduced SIRT2 expression and increased mitochondrial stress lead to aberrant activation of the NLRP3 inflammasome in HSCs. SIRT2 overexpression, NLRP3 inactivation, or caspase 1 inactivation improves the maintenance and regenerative capacity of aged HSCs. These results suggest that mitochondrial stress-initiated aberrant activation of the NLRP3 inflammasome is a reversible driver of the functional decline of HSC aging and highlight the importance of inflammatory signaling in regulating HSC aging., Cell Reports, 26 (4), ISSN:2666-3864, ISSN:2211-1247

It is well documented that the rate of aging can be slowed, but it remains unclear to which extent aging-associated conditions can be reversed. How the interface of immunity and metabolism impinges upon the diabetes pandemic is largely unknown. Here we show that NLRP3, a pattern recognition receptor, is modified by acetylation in macrophages and is deacetylated by SIRT2, an NAD(+)-dependent deacetylase and a metabolic sensor. We have developed a cell-based system that models aging-associated inflammation, a defined co-culture system that simulates the effects of inflammatory milieu on insulin resistance in metabolic tissues during aging, and aging mouse models, and demonstrate that SIRT2 and NLRP3 deacetylation prevent, and can be targeted to reverse, aging-associated inflammation and insulin resistance. These results establish the dysregulation of the acetylation switch of the NLRP3 inflammasome as an origin of aging-associated chronic inflammation and highlight the reversibility of aging-associated chronic inflammation and insulin resistance.

Summary The mitochondrial unfolded protein response (UPR mt), a cellular protective program that ensures proteostasis in the mitochondria, has recently emerged as a regulatory mechanism for adult stem cell maintenance that is conserved across tissues. Despite the emerging genetic evidence implicating the UPR mt in stem cell maintenance, the underlying molecular mechanism is unknown. While it has been speculated that the UPR mt is activated upon stem cell transition from quiescence to proliferation, the direct evidence is lacking. In this study, we devised three experimental approaches that enable us to monitor quiescent and proliferating hematopoietic stem cells (HSCs) and provided the direct evidence that the UPR mt is activated upon HSC transition from quiescence to proliferation, and more broadly, mitochondrial integrity is actively monitored at the restriction point to ensure metabolic fitness before stem cells are committed to proliferation.

Keeping stem cells in tip top condition Stem cells are important in the maintenance and growth of tissues. For the health of the organism as a whole, it is important that only healthy stem cells be used. Mohrin et al. elucidated a regulatory branch of the mitochondrial unfolded protein response that is coupled to cellular energy metabolism and proliferation in stem cells (see the Perspective by Ocampo and Belmonte). Mitochondrial protein folding stress triggered a metabolic checkpoint that regulates the cell cycle. Deregulation of this pathway interfered with stem cell quiescence and compromised regenerative function. Science , this issue p. 1374 ; see also p. 1319

N-6-methyladenosine (m6A) is one of the most abundant posttranscriptional modifications in eukaryotic mRNAs and long noncoding RNAs. We previously found a critical role for m6A in promoting human myeloid leukemia (Vu et al. Nature Medicine 2017). Targeting the RNA methylation program in leukemias has been suggested as a potential novel therapeutic strategy. However, it is unknown whether the m6A modification controls normal adult hematopoiesis and hematopoietic stem cells (HSC) function. To investigate the role of m6A in adult hematopoiesis, we crossed with the Mettl3 conditional knockout (cKO) mouse model with the interferon inducible Mx1-Cre system to abolish m6A in the hematopoietic compartment. Deletion of Mettl3 (3 weeks post pIpC injections) resulted in pancytopenia (white blood count 9.44 k/ul in WT versus 4.35 k/ul cKO) and a 55% reduction in red blood cell counts and nearly 70% loss in platelet counts. Most remarkably, METTL3 depletion resulted in a 5-fold increase in the number of overall hematopoietic stem and progenitors HSPCs (LSKs; Lin-c-kit+Sca1+). Within the HSPC compartment there was a about a 10-fold expansion in immunophenotypic long term hematopoietic stem cells LT-HSCs (Lin-c-kit+Sca1+CD150+CD48-), multipotent progenitors (MPP-2s), (Lin-c-kit+Sca1+CD150+CD48+) and MPP4s (Lin-c-kit+Sca1+CD150-CD48+). In contrast to this general increase in HSPCs, we observed a decrease in common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) populations by 70% and 60% respectively. Altogether, these results suggest that loss of METTL3 results in a partial blockage in hematopoietic stem and progenitor cell differentiation, and an accumulation of LT-HSC and MPPs. Despite this phenotypic expansion of LT-HSCs and MPPs, their function was impaired as demonstrated by a reduction in long-term chimerism in non-competitive transplants into congenic mice compared to the remaining wildtype host cells (66% in WT vs 26% in cKO). Interestingly, the relative differentiation block and accumulation of HSCs remained after transplantation. Furthermore, Mettl3 cKO HSCs are less quiescent (79% WT vs. 44% cKO) and more proliferative based on cell cycle profiling with pyronin Y/Hoechst or Ki67/Hoechst. RNA-seq in sorted LT-HSC, MPP1, MPP2 and MPP4s from WT and Mettl3 cKO mice demonstrated that Mettl3 cKO HSCs lose the HSC signature while MPP1 and MPP2 cells exhibited gene signatures resembling WT HSCs. Interestingly, genes uniquely upregulated in Mettl3 cKO LT-HSC showed a significant enrichment of the Mettl3 KO mouse embryonic stem cells (mESCs) expression signature. To decipher METTL3 regulation of transcriptional states in individual cells along the hematopoietic hierarchy, we performed single-cell RNA-seq using sorted WT and Mettl3 cKO cKit+ cells. Surprisingly, tSNE analysis of scRNA-seq data uncovered the loss of the normal HSC cluster and the emergence of three unique HSC-like populations in Mettl3 cKO mice. Single cell analysis identified a reduction in c-MYC levels in the HSPCs that was then confirmed by immunofluorescence in sorted HSCs. More importantly, when forced into division, Mettl3 cKO HSCs exhibited increased symmetric c-MYC low division pattern compared to control (60% to 48%) based on c-MYC IF staining in paired daughter cells. We further confirmed that sorted HSCs underwent increased symmetric renewal divisions in the Mettl3 cKO compared to controls (65% to 49%) without any effect on asymmetric division based on NUMB IF staining in paired daughter cells. These data suggest that without METTL3, HSCs are partially blocked in a self-renewing state albeit with reduced fitness compared to wildtype HSCs. Overall, our studies uncovered a novel role for METTL3 and RNA methylation to maintain normal HSC identity and progenitor differentiation. Also, these studies suggest that inhibiting METTL3 could result in significant hematopoietic defects. Disclosures No relevant conflicts of interest to declare.

Leukemia stem cells (LSCs) are believed to be a prominent source of relapse in acute myeloid leukemia (AML). Human AML LSCs are classically studied via xenotransplantation and defined as cells with (a) self-renewal ability, giving rise to leukemic engraftment that can be maintained over serial transplantation; and (b) ability to give rise to more differentiated progeny that are unable to engraft. LSCs are chemoresistant presumably because they have properties distinct from those of the bulk AML cells. A better understanding of the properties of LSCs can ultimately allow us to develop new therapies specifically targeting these cells to achieve lasting responses or even cures. We recently reported that induced pluripotent stem cells (iPSCs) generated from AML patients (AML-iPSCs) exhibit leukemic features upon hematopoietic differentiation, including extensive proliferation, maintenance of hematopoietic stem cell (HSC) markers, and serial engraftment of a lethal leukemia in immunodeficient mice (Kotini et al. Cell Stem Cell 2017). More recently, we found that the hematopoietic stem/progenitor cells (HSPCs) derived from AML-iPSCs exhibit phenotypic and functional heterogeneity. We first observed the presence of two cell populations with distinct growth characteristics and morphology: a population exhibiting adherent growth (adherent, A) and a population growing in suspension (S). The A cell population contained cells with immature morphology and HSC immunophenotype (CD34+/CD38-/CD90+/CD45RA-/ CD49f+), while the S fraction contained more differentiated cells (CD34-/low/CD38+/CD90-/CD45RA+). Experiments utilizing serial replating, single-cell plating of GFP-labeled cells and mathematical modeling revealed a hierarchical organization, whereby the A cells continuously give rise to the S cell fraction. Time-lapse imaging showed that A cells divide both symmetrically and asymmetrically with the majority (~60%) of cell divisions giving rise to two A cells and less frequently generating either one A and one S (~15%) or two S cells (~15%). Serial transplantation experiments in NSG mice revealed that the engraftment potential was largely contained within the A cell fraction. Cell cycle analysis showed that adherent cells contained a lower S and higher G0/G1 phase fraction than suspension cells. Thus, these AML-iPSC-derived hematopoietic cells exhibit hallmarks of an LSC model, namely phenotypic and functional heterogeneity and hierarchical organization, with the A fraction containing LSCs that serially transplant leukemia and give rise to more differentiated cells (S fraction) without engraftment potential. Transcriptome analyses showed that the A to S transition was predominantly mediated by gene upregulation with only 2 out of 418 differentially expressed genes being downregulated and revealed enrichment for HSC and LSC gene sets in the A fraction. Consistent with a general transcriptional upregulation characterizing the A to S transition, differentially accessible regions by ATAC-seq analysis were found predominantly in intragenic regions and enhancers in A cells, while they mostly localized in gene promoters in S cells. Time-course single cell-RNA-seq analyses and their integration with bulk RNA-seq and ATAC-seq data revealed cell clusters specific to the A cell fraction, which were enriched in HSC, LSC and hESC genes. These integrated analyses revealed candidate genes with a role in maintaining LSC properties. The transcription factor RUNX1 was particularly prominent, with RUNX1 motifs found highly accessible in A compared to S cells. Overexpression of RUNX1 in S cells conferred a more LSC-like immunophenotype. RUNX1 is a key transcriptional regulator of hematopoiesis. It has a well-characterized role during development of the hematopoietic system and is often mutated or translocated in AML. A role of RUNX1 in LSC maintenance was unanticipated in view of its known tumor-suppressor function. In further support for a role for RUNX1 in LSCs, we found RUNX1 expression to significantly correlate with survival in AML patient cohorts. In summary, we developed a new model that enables us to prospectively isolate large numbers of genetically clonal human AML LSCs and perform genome-wide integrative molecular studies, with which we obtained new insights into the biology of AML LSCs. Disclosures No relevant conflicts of interest to declare.

Formaldehyde (FA) exposure is known to be toxic to central nervous system of mammals. In this paper, we evaluated the aggressive behavior after repetitive inhalative FA exposure to male SD rats, and explored the potential mechanism. The rats, ranging from 160 to 180 g, were randomly designated into the orchiectomized (ORX) group, the control and the inhalative FA treatment groups. Eight rats underwent orchiectomy surgery. Three days after the orchiectomy surgery, the inhalative FA (monitored to be 13.5 ± 1.5 ppm) treatment began. We found that the male SD rats, those were exposed to FA showed more aggressive behavior compared to the control. And the ORX rats exhibited less aggressive behavior than the control. Furthermore, the dopamine increased and 5-HT decreased significantly in frontal cortex synaptosome after inhalative FA treatment. It is the first to evaluate aggressive behavior and identified monoamines disturbances in the frontal cortex synaptosome after the repetitive inhalative FA exposure to rodents. © 2009 Elsevier Ireland Ltd. All rights reserved.

Deterioration of adult stem cells accounts for much of aging-associated compromised tissue maintenance. How stem cells maintain metabolic homeostasis remains elusive. Here, we identified a regulatory branch of the mitochondrial unfolded protein response (UPR(mt)), which is mediated by the interplay of SIRT7 and NRF1 and is coupled to cellular energy metabolism and proliferation. SIRT7 inactivation caused reduced quiescence, increased mitochondrial protein folding stress (PFS(mt)), and compromised regenerative capacity of hematopoietic stem cells (HSCs). SIRT7 expression was reduced in aged HSCs, and SIRT7 up-regulation improved the regenerative capacity of aged HSCs. These findings define the deregulation of a UPR(mt)-mediated metabolic checkpoint as a reversible contributing factor for HSC aging.

Summary Nonalcoholic fatty liver disease is the most common chronic liver disorder in developed countries. Its pathogenesis is poorly understood, and therapeutic options are limited. Here, we show that SIRT7, an NAD + -dependent H3K18Ac deacetylase, functions at chromatin to suppress ER stress and prevent the development of fatty liver disease. SIRT7 is induced upon ER stress and is stabilized at the promoters of ribosomal proteins through its interaction with the transcription factor Myc to silence gene expression and to relieve ER stress. SIRT7-deficient mice develop chronic hepatosteatosis resembling human fatty liver disease. Myc inactivation or pharmacological suppression of ER stress alleviates fatty liver caused by SIRT7 deficiency. Importantly, SIRT7 suppresses ER stress and reverts the fatty liver disease in diet-induced obese mice. Our study identifies SIRT7 as a cofactor of Myc for transcriptional repression and delineates a druggable regulatory branch of the ER stress response that prevents and reverts fatty liver disease.