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Thomas, Egan et al. report that hexokinase 2 localizes to the nucleus of leukaemic and normal haematopoietic cells to maintain stemness by interacting with nuclear proteins and modulating chromatin accessibility independently of its kinase activity.Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function.

In acute myeloid leukemia (AML), molecular heterogeneity across patients constitutes a major challenge for prognosis and therapy. AML with NPM1 mutation is a distinct genetic entity in the revised World Health Organization classification. However, differing patterns of co-mutation and response to therapy within this group necessitate further stratification. Here we report two distinct subtypes within NPM1 mutated AML patients, which we label as primitive and committed based on the respective presence or absence of a stem cell signature. Using gene expression (RNA-seq), epigenomic (ATAC-seq) and immunophenotyping (CyToF) analysis, we associate each subtype with specific molecular characteristics, disease differentiation state and patient survival. Using ex vivo drug sensitivity profiling, we show a differential drug response of the subtypes to specific kinase inhibitors, irrespective of the FLT3-ITD status. Differential drug responses of the primitive and committed subtype are validated in an independent AML cohort. Our results highlight heterogeneity among NPM1 mutated AML patient samples based on stemness and suggest that the addition of kinase inhibitors to the treatment of cases with the primitive signature, lacking FLT3-ITD, could have therapeutic benefit., Molecular heterogeneity of acute myeloid leukaemia (AML) across patients is a major challenge for prognosis and therapy. Here, the authors show that NPM1 mutated AML is a heterogeneous class, consisting of two subtypes which exhibit distinct molecular characteristics, differentiation state, patient survival and drug response.

It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination., Cell Stem Cell, 28 (10), ISSN:1934-5909, ISSN:1875-9777

Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSCs by activating stress myelopoiesis, such roles in LSCs are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator that drives myeloid differentiation and activates inflammatory programs in both HSCs and LSCs. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across cohorts of patients with AML, each with distinct phenotypic and clinical properties. S1PR3 was high in LSCs and blasts of mature myeloid samples with linkages to chemosensitivity, whereas S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset. Significance: S1PR3 is a novel regulator of myeloid fate in normal hematopoiesis that is heterogeneously expressed in AML. S1PR3 marks a subset of less primitive AML cases with a distinct inflammatory signature and therefore has clinical implications as both a therapeutic target and a biomarker to distinguish primitive from mature AML. See related commentary by Yang et al., p. 3. This article is highlighted in the In This Issue feature, p. 1

Espresso efficiently distinguishes true mutations from background noise common in deep sequencing data., Sensitive mutation detection by next-generation sequencing is critical for early cancer detection, monitoring minimal/measurable residual disease (MRD), and guiding precision oncology. Nevertheless, because of artifacts introduced during library preparation and sequencing, the detection of low-frequency variants at high specificity is problematic. Here, we present Espresso, an error suppression method that considers local sequence features to accurately detect single-nucleotide variants (SNVs). Compared to other advanced error suppression techniques, Espresso consistently demonstrated lower numbers of false-positive mutation calls and greater sensitivity. We demonstrated Espresso’s superior performance in detecting MRD in the peripheral blood of patients with acute myeloid leukemia (AML) throughout their treatment course. Furthermore, we showed that accurate mutation calling in a small number of informative genomic loci might provide a cost-efficient strategy for pragmatic risk prediction of AML development in healthy individuals. More broadly, we aim for Espresso to aid with accurate mutation detection in many other research and clinical settings.

Inherited bone marrow failure syndromes (IBMFSs) such as Fanconi Anemia (FA) and Shwachman-Diamond syndrome (SDS) feature progressive cytopenia and a risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Using deep immunophenotypic analysis of hematopoietic stem cells (HSCs) and early progenitors in bone marrow samples from 13 FA/SDS patients without MDS/AML and 6 healthy controls, we revealed relative survival of progenitors that phenotypically resembled granulocyte-monocyte progenitors (GMP) in the patients. In contrast, HSCs, multipotent progenitors, common myeloid progenitors and megakaryocyte-erythroid progenitors were remarkably reduced in FA/SDS patients compared to controls. Whole exome and targeted sequencing of GMP-like cells in leukemia-free patients revealed a prominently higher mutation load in FA/SDS patient samples than in healthy controls. Patient samples also manifested molecular changes that are characteristic of AML: increased G>A/C>T variants, decreased A>G/T>C variants, increased trinucleotide mutations at Xp(C>T)pT, decreased lower mutation rates at Xp(C>T)pG sites compared to other Xp(C>T)pX sites and enrichment for cancer signature 1. AML cells have previously been reported as commonly harboring this cancer signature. Potential pre-leukemic targets in the GMP-like cells from FA/SDS patients included SYNE1, DST, HUWE1, LRP2, NOTCH2 and TP53. Serial analysis of GMPs from a SDS patient, who progressed to AML revealed a gradual increase in mutational burden, enrichment of G>A/C>T signature and emergence of new clones. Interestingly, the molecular signature of marrow cells from two FA/SDS patients with AML was similar to that of FA/SDS without transformation. The predicted founding clones in SDS-AML harbored mutations in several genes including TP53, while in FA-AML the mutated genes included ARID1B and SFPQ. In conclusion, we described an architectural change in the hematopoietic hierarchy of FA/SDS with remarkable preservation of GMP-like populations harboring unique mutation signatures. GMP-like cells might represent a cellular reservoir for clonal evolution. Disclosures No relevant conflicts of interest to declare.

A deep proteomics analysis was conducted on a primary acute myeloid leukemia culture system to identify potential protein targets regulated by miR-126. Leukemia cells were transduced either with an empty control lentivirus or one containing the sequence for miR-126, and resulting cells were analyzed using ultra-high performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD001994. The proteomics data and statistical analysis described in this article is associated with a research article, “miR-126 regulates distinct self-renewal outcomes in normal and malignant hematopoietic stem cells” (Lechman et al., 2016) [1], and serves as a resource for researchers working in the field of microRNAs and their regulation of protein levels.

The incidence of acute myeloid leukaemia (AML) increases with age and mortality exceeds 90% when diagnosed after age 65. Most cases arise without any detectable early symptoms and patients usually present with the acute complications of bone marrow failure(1). The onset of such de novo AML cases is typically preceded by the accumulation of somatic mutations in preleukaemic haematopoietic stem and progenitor cells (HSPCs) that undergo clonal expansion(2,3). However, recurrent AML mutations also accumulate in HSPCs during ageing of healthy individuals who do not develop AML, a phenomenon referred to as age-related clonal haematopoiesis (ARCH)(4-8). Here we use deep sequencing to analyse genes that are recurrently mutated in AML to distinguish between individuals who have a high risk of developing AML and those with benign ARCH. We analysed peripheral blood cells from 95 individuals that were obtained on average 6.3 years before AML diagnosis (pre-AML group), together with 414 unselected age- and gender-matched individuals (control group). Pre-AML cases were distinct from controls and had more mutations per sample, higher variant allele frequencies, indicating greater clonal expansion, and showed enrichment of mutations in specific genes. Genetic parameters were used to derive a model that accurately predicted AML-free survival; this model was validated in an independent cohort of 29 pre-AML cases and 262 controls. Because AML is rare, we also developed an AML predictive model using a large electronic health record database that identified individuals at greater risk. Collectively our findings provide proof-of-concept that it is possible to discriminate ARCH from pre-AML many years before malignant transformation. This could in future enable earlier detection and monitoring, and may help to inform intervention.

A locus on human chromosome 11q23 tagged by marker rs3802842 was associated with colorectal cancer (CRC) in a genome-wide association study; this finding has been replicated in case-control studies worldwide. In order to identify biologic factors at this locus that are related to the etiopathology of CRC, we used microarray-based target selection methods, coupled to next-generation sequencing, to study 103 kb at the 11q23 locus. We genotyped 369 putative variants from 1,030 patients with CRC (cases) and 1,061 individuals without CRC (controls) from the Ontario Familial Colorectal Cancer Registry. Two previously uncharacterized genes, COLCA1 and COLCA2, were found to be co-regulated genes that are transcribed from opposite strands. Expression levels of COLCA1 and COLCA2 transcripts correlate with rs3802842 genotypes. In colon tissues, COLCA1 co-localizes with crystalloid granules of eosinophils and granular organelles of mast cells, neutrophils, macrophages, dendritic cells and differentiated myeloid-derived cell lines. COLCA2 is present in the cytoplasm of normal epithelial, immune and other cell lineages, as well as tumor cells. Tissue microarray analysis demonstrates the association of rs3802842 with lymphocyte density in the lamina propria (p = 0.014) and levels of COLCA1 in the lamina propria (p = 0.00016) and COLCA2 (tumor cells, p = 0.0041 and lamina propria, p = 6 × 10(-5)). In conclusion, genetic, expression and immunohistochemical data implicate COLCA1 and COLCA2 in the pathogenesis of colon cancer. Histologic analyses indicate the involvement of immune pathways.

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the selfrenewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL invivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL.

CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody–drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent monomethyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34+CD38− leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML. Mol Cancer Ther; 14(7); 1650–60. ©2015 AACR.

Effective immunotherapy for human leukemia based on infusions of T lymphocytes requires the identification of effector T cells that target the leukemic stem cell. The transplantation of human acute myeloid leukemia into nonobese diabetic/severe combined immune deficient (SCID) mice has identified a rare leukemic progenitor termed the SCID leukemia-initiating cell, which is present in low frequency in the leukemic population and is essential for establishing leukemic hematopoiesis. Thus, this transplant model may be ideally suited to identify effector T cells with antileukemic activity. We report that CD8 + cytotoxic T lymphocyte (CTL) clones specific for minor histocompatibility antigens inhibit the engraftment of human acute myeloid leukemia cells in nonobese diabetic/SCID mice and demonstrate that this inhibition is mediated by direct CTL recognition of SCID leukemia-initiating cells. These results indicate that CD8 + minor histocompatibility antigen-specific CTL may be mediators of the graft-versus-leukemia effect associated with allogeneic hematopoietic cell transplantation and provide an experimental model to identify and select T cell clones for immunotherapy to prevent or treat relapse after allogeneic hematopoietic cell transplantation.

The purification of primitive human hematopoietic stem cells has been impaired by the absence of repopulation assays. By using a stringent two-step strategy involving depletion of lineage-positive cells followed by fluorescence-activated cell sorting, we have purified a cell population that is highly enriched for cells capable of multilineage repopulation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) recipients. TheseSCID-repopulatingcells (SRCs) were exclusively found in a cell fraction that expressed high levels of CD34 and no CD38. Through limiting dilution analysis using Poisson statistics, we calculated a frequency of 1 SRC in 617 CD34+CD38−cells. The highly purified SRC were capable of extensive proliferation in NOD/SCID mice. Mice transplanted with 1 SRC (at limiting cell doses) were able to produce approximately 400,000 progeny 6 weeks after the transplant. Detailed flow cytometric analysis of the marrow of highly engrafted mice demonstrated both lymphoid and myeloid differentiation, as well as the retention of a significant fraction of CD34+CD38−cells. These highly purified fractions should be useful for identification of the cellular and molecular mechanisms that regulate primitive human hematopoietic cells. Moreover, the ability to detect and purify primitive cells provides a means to develop conditions for maintaining and/or expanding these cells duringin vitroculture.

Highly regenerative tissues such as blood must possess effective DNA damage responses (DDR) that balance long-term regeneration with protection from leukemogenesis. Hematopoietic stem cells (HSCs) sustain life-long blood production, yet their response to DNA damage remains largely unexplored. We report that human HSCs exhibit delayed DNA double-strand break rejoining, persistent gammaH2AX foci, and enhanced p53- and ASPP1-dependent apoptosis after gamma-radiation compared to progenitors. p53 inactivation or Bcl-2 overexpression reduced radiation-induced apoptosis and preserved in vivo repopulating HSC function. Despite similar protection from irradiation-induced apoptosis, only Bcl-2-overexpressing HSCs showed higher self-renewal capacity, establishing that intact p53 positively regulates self-renewal independently from apoptosis. The reduced self-renewal of HSCs with inactivated p53 was associated with increased spontaneous gammaH2AX foci in secondary transplants of HSCs. Our data reveal distinct physiological roles of p53 that together ensure optimal HSC function: apoptosis regulation and prevention of gammaH2AX foci accumulation upon HSC self-renewal.

SummaryTo identify FDA-approved agents targeting leukemic cells, we performed a chemical screen on two human leukemic cell lines and identified the antimicrobial tigecycline. A genome-wide screen in yeast identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated lethality. Tigecycline selectively killed leukemia stem and progenitor cells compared to their normal counterparts and also showed antileukemic activity in mouse models of human leukemia. ShRNA-mediated knockdown of EF-Tu mitochondrial translation factor in leukemic cells reproduced the antileukemia activity of tigecycline. These effects were derivative of mitochondrial biogenesis that, together with an increased basal oxygen consumption, proved to be enhanced in AML versus normal hematopoietic cells and were also important for their difference in tigecycline sensitivity.

SummaryThe hematopoietic system sustains regeneration throughout life by balancing self-renewal and differentiation. To stay poised for mature blood production, hematopoietic stem cells (HSCs) maintain low-level expression of lineage-associated genes, a process termed lineage priming. Here, we modulated expression levels of Inhibitor of DNA binding (ID) proteins to ask whether lineage priming affects self-renewal of human HSCs. We found that lentiviral overexpression of ID proteins in cord blood HSCs biases myeloerythroid commitment at the expense of lymphoid differentiation. Conversely, reducing ID2 expression levels increases lymphoid potential. Mechanistically, ID2 inhibits the transcription factor E47 to attenuate B-lymphoid priming in HSCs and progenitors. Strikingly, ID2 overexpression also results in a 10-fold expansion of HSCs in serial limiting dilution assays, indicating that early lymphoid transcription factors antagonize human HSC self-renewal. The relationship between lineage priming and self-renewal can be exploited to increase expansion of transplantable human HSCs and points to broader implications for other stem cell populations.

Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor α chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing to bone marrow (BM) and activating innate immunity of nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival. Mice with pre-established disease showed reduced AML burden in the BM and periphery and impaired secondary transplantation upon treatment, establishing that AML-LSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34+CD38− cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application. Refereed/Peer-reviewed

SummaryFrom an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.

Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)-based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD.