KDM6 demethylases integrate DNA repair gene regulation and loss of KDM6A sensitizes human acute myeloid leukemia to PARP and BCL2 inhibition.

Acute myeloid leukemia (AML) is a heterogeneous, aggressive malignancy with dismal prognosis and with limited availability of targeted therapies. Epigenetic deregulation contributes to AML pathogenesis. KDM6 proteins are histone-3-lysine-27-demethylases that play context-dependent roles in AML. We inform that KDM6-demethylase function critically regulates DNA-damage-repair-(DDR) gene expression in AML. Mechanistically, KDM6 expression is regulated by genotoxic stress, with deficiency of KDM6A-(UTX) and KDM6B-(JMJD3) impairing DDR transcriptional activation and compromising repair potential. Acquired KDM6A loss-of-function mutations are implicated in chemoresistance, although a significant percentage of relapsed-AML has upregulated KDM6A. Olaparib treatment reduced engraftment of KDM6A-mutant-AML-patient-derived xenografts, highlighting synthetic lethality using Poly-(ADP-ribose)-polymerase-(PARP)-inhibition. Crucially, a higher KDM6A expression is correlated with venetoclax tolerance. Loss of KDM6A increased mitochondrial activity, BCL2 expression, and sensitized AML cells to venetoclax. Additionally, BCL2A1 associates with venetoclax resistance, and KDM6A loss was accompanied with a downregulated BCL2A1. Corroborating these results, dual targeting of PARP and BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing AML apoptosis, and primary AML cells carrying KDM6A-domain mutations were even more sensitive to the combination. Together, our study illustrates a mechanistic rationale in support of a novel combination therapy for AML based on subtype-heterogeneity, and establishes KDM6A as a molecular regulator for determining therapeutic efficacy.

PLAG1 dampens protein synthesis to promote human hematopoietic stem cell self-renewal.

Hematopoietic stem cell (HSC) dormancy is understood as supportive of HSC function and its long-term integrity. Although regulation of stress responses incurred as a result of HSC activation is recognized as important in maintaining stem cell function, little is understood of the preventive machinery present in human HSCs that may serve to resist their activation and promote HSC self-renewal. We demonstrate that the transcription factor PLAG1 is essential for long-term HSC function and, when overexpressed, endows a 15.6-fold enhancement in the frequency of functional HSCs in stimulatory conditions. Genome-wide measures of chromatin occupancy and PLAG1-directed gene expression changes combined with functional measures reveal that PLAG1 dampens protein synthesis, restrains cell growth and division, and enhances survival, with the primitive cell advantages it imparts being attenuated by addition of the potent translation activator, c-MYC. We find PLAG1 capitalizes on multiple regulatory factors to ensure protective diminished protein synthesis including 4EBP1 and translation-targeting miR-127 and does so independently of stress response signaling. Overall, our study identifies PLAG1 as an enforcer of human HSC dormancy and self-renewal through its highly context-specific regulation of protein biosynthesis and classifies PLAG1 among a rare set of bona fide regulators of messenger RNA translation in these cells. Our findings showcase the importance of regulated translation control underlying human HSC physiology, its dysregulation under activating demands, and the potential if its targeting for therapeutic benefit.

Identification of the global miR-130a targetome reveals a role for TBL1XR1 in hematopoietic stem cell self-renewal and t(8;21) AML.

Gene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSCs) point to shared core stemness properties. However, discordance between mRNA and protein signatures highlights an important role for post-transcriptional regulation by microRNAs (miRNAs) in governing this critical nexus. Here, we identify miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impairs B lymphoid differentiation and expands long-term HSCs. Integration of protein mass spectrometry and chimeric AGO2 crosslinking and immunoprecipitation (CLIP) identifies TBL1XR1 as a primary miR-130a target, whose loss of function phenocopies miR-130a overexpression. Moreover, we report that miR-130a is highly expressed in t(8;21) acute myeloid leukemia (AML), where it is critical for maintaining the oncogenic molecular program mediated by the AML1-ETO complex. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of genes and molecular networks underpinning stemness properties of normal and leukemic cells.

Nicotinamide phosphoribosyltransferase inhibitors selectively induce apoptosis of AML stem cells by disrupting lipid homeostasis.

Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Here, we performed a metabolic drug screen to identify LSC-specific vulnerabilities and found that nicotinamide phosphoribosyltransferase (NAMPT) inhibitors selectively killed LSCs, while sparing normal hematopoietic stem and progenitor cells. Treatment with KPT-9274, a NAMPT inhibitor, suppressed the conversion of saturated fatty acids to monounsaturated fatty acids, a reaction catalyzed by the stearoyl-CoA desaturase (SCD) enzyme, resulting in apoptosis of AML cells. Transcriptomic analysis of LSCs treated with KPT-9274 revealed an upregulation of sterol regulatory-element binding protein (SREBP)-regulated genes, including SCD, which conferred partial protection against NAMPT inhibitors. Inhibition of SREBP signaling with dipyridamole enhanced the cytotoxicity of KPT-9274 on LSCs in vivo. Our work demonstrates that altered lipid homeostasis plays a key role in NAMPT inhibitor-induced apoptosis and identifies NAMPT inhibition as a therapeutic strategy for targeting LSCs in AML.

Mapping the cellular origin and early evolution of leukemia in Down syndrome.

Children with Down syndrome have a 150-fold increased risk of developing myeloid leukemia, but the mechanism of predisposition is unclear. Because Down syndrome leukemogenesis initiates during fetal development, we characterized the cellular and developmental context of preleukemic initiation and leukemic progression using gene editing in human disomic and trisomic fetal hematopoietic cells and xenotransplantation. GATA binding protein 1 (GATA1) mutations caused transient preleukemia when introduced into trisomy 21 long-term hematopoietic stem cells, where a subset of chromosome 21 microRNAs affected predisposition to preleukemia. By contrast, progression to leukemia was independent of trisomy 21 and originated in various stem and progenitor cells through additional mutations in cohesin genes. CD117+/KIT proto-oncogene (KIT) cells mediated the propagation of preleukemia and leukemia, and KIT inhibition targeted preleukemic stem cells.

Quantitative single-cell proteomics as a tool to characterize cellular hierarchies.

Large-scale single-cell analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems, but have largely been limited to RNA-based technologies. Here we present a comprehensive benchmarked experimental and computational workflow, which establishes global single-cell mass spectrometry-based proteomics as a tool for large-scale single-cell analyses. By exploiting a primary leukemia model system, we demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within this aberrant developmental hierarchy. Our approach is capable of consistently quantifying ~1000 proteins per cell across thousands of individual cells using limited instrument time. Furthermore, we develop a computational workflow (SCeptre) that effectively normalizes the data, integrates available FACS data and facilitates downstream analysis. The approach presented here lays a foundation for implementing global single-cell proteomics studies across the world.

Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells.

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.

An ERG Enhancer-Based Reporter Identifies Leukemia Cells with Elevated Leukemogenic Potential Driven by ERG-USP9X Feed-Forward Regulation.

Acute leukemia is a rapidly progressing blood cancer with low survival rates. Unfavorable prognosis is attributed to insufficiently characterized subpopulations of leukemia stem cells (LSC) that drive chemoresistance and leukemia relapse. Here we utilized a genetic reporter that assesses stemness to enrich and functionally characterize LSCs. We observed heterogeneous activity of the ERG+85 enhancer-based fluorescent reporter in human leukemias. Cells with high reporter activity (tagBFPHigh) exhibited elevated expression of stemness and chemoresistance genes and demonstrated increased clonogenicity and resistance to chemo- and radiotherapy as compared with their tagBFPNeg counterparts. The tagBFPHigh fraction was capable of regenerating the original cellular heterogeneity and demonstrated increased invasive ability. Moreover, the tagBFPHigh fraction was enriched for leukemia-initiating cells in a xenograft assay. We identified the ubiquitin hydrolase USP9X as a novel ERG transcriptional target that sustains ERG+85-positive cells by controlling ERG ubiquitination. Therapeutic targeting of USP9X led to preferential inhibition of the ERG-dependent leukemias. Collectively, these results characterize human leukemia cell functional heterogeneity and suggest that targeting ERG via USP9X inhibition may be a potential treatment strategy in patients with leukemia. SIGNIFICANCE: This study couples a novel experimental tool with state-of-the-art approaches to delineate molecular mechanisms underlying stem cell-related characteristics in leukemia cells.

A stemness screen reveals C3orf54/INKA1 as a promoter of human leukemia stem cell latency.

There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.

A novel method for detecting the cellular stemness state in normal and leukemic human hematopoietic cells can predict disease outcome and drug sensitivity.

Acute leukemia is an aggressive blood malignancy with low survival rates. A high expression of stem-like programs in leukemias predicts poor prognosis and is assumed to act in an aberrant fashion in the phenotypically heterogeneous leukemia stem cell (LSC) population. A lack of suitable genome engineering tools that can isolate LSCs based on their stemness precludes their comprehensive examination and full characterization. We hypothesized that tagging endogenous stemness-regulatory regions could generate a genome reporter for the putative leukemia stemness-state. Our analysis revealed that the ERG + 85 enhancer region can serve as a marker for stemness-state and a fluorescent lentiviral reporter was developed that can accurately recapitulate the endogenous activity. Using our novel reporter, we revealed cellular heterogeneity in several leukemia cell lines and patient-derived samples. Alterations in reporter activity were associated with transcriptomic and functional changes that were closely related to the hematopoietic stem cell (HSC) identity. Notably, the differentiation potential was skewed towards the erythro-megakaryocytic lineage. Moreover, an ERG + 85High fraction of AML cells could regenerate the original cellular heterogeneity and was enriched for LSCs. RNA-seq analysis coupled with in silico drug-screen analysis identified 4HPR as an effective inhibitor of ERG + 85High leukemia growth. We propose that further utilization of our novel molecular tool will identify crucial determinants of LSCs, thus providing a rationale for their therapeutic targeting.

Global proteomics dataset of miR-126 overexpression in acute myeloid leukemia.

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.

Ectopic miR-125a Expression Induces Long-Term Repopulating Stem Cell Capacity in Mouse and Human Hematopoietic Progenitors.

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 self-renewal 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.

miRNA-126 Orchestrates an Oncogenic Program in B Cell Precursor Acute Lymphoblastic Leukemia.

MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden.

miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells.

To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance.

Reduced lymphoid lineage priming promotes human hematopoietic stem cell expansion.

The 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.

Attenuation of miR-126 activity expands HSC in vivo without exhaustion.

Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3ß pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.

Stem cell gene expression programs influence clinical outcome in human leukemia.

Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarchies sustained by cancer stem cells (CSCs). Despite the promise of the CSC model, its relevance in humans remains uncertain. Here we show that acute myeloid leukemia (AML) follows a CSC model on the basis of sorting multiple populations from each of 16 primary human AML samples and identifying which contain leukemia stem cells (LSCs) using a sensitive xenograft assay. Analysis of gene expression from all functionally validated populations yielded an LSC-specific signature. Similarly, a hematopoietic stem cell (HSC) gene signature was established. Bioinformatic analysis identified a core transcriptional program shared by LSCs and HSCs, revealing the molecular machinery underlying 'stemness' properties. Both stem cell programs were highly significant independent predictors of patient survival and were found in existing prognostic signatures. Thus, determinants of stemness influence the clinical outcome of AML, establishing that LSCs are clinically relevant and not artifacts of xenotransplantation.

Identification of hematopoietic stem cell-specific miRNAs enables gene therapy of globoid cell leukodystrophy.

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.

A distinctive DNA damage response in human hematopoietic stem cells reveals an apoptosis-independent role for p53 in self-renewal.

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.