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Loss of long-term haematopoietic stem cell function (LT-HSC) hampers the success ofex vivoHSC gene therapy and expansion procedures, but the kinetics and the mechanisms by which this occurs remain incompletely characterized. Here through time-resolved scRNA-Seq, matchedin vivofunctional analysis and the use of a reversiblein vitrosystem of early G1arrest, we define the sequence of transcriptional and functional events occurring during the firstex vivodivision of human LT-HSCs. We demonstrate that contrary to current assumptions, loss of long-term repopulation capacity during culture is independent of cell cycle progression. Instead it is a rapid event that follows an early period of adaptation to culture, characterised by transient gene expression dynamics and constrained global variability in gene expression. Cell cycle progression however contributes to the establishment of differentiation programmes in culture. Our data have important implications for improving HSC gene therapy and expansion protocols.

Acute myeloid leukemia (AML) is a heterogeneous, aggressive malignancy with dismal prognosis and with limited availability of targeted therapies. AML exhibits epigenetic deregulation and transcriptional plasticity that contributes to pathogenesis. KDM6 proteins are histone-3 lysine-27 demethylases that play major context dependent roles in AML evolution and therapy resistance. Here, we demonstrate that KDM6 demethylase function critically regulates DNA damage repair (DDR) gene expression programs in AML. Mechanistically, KDM6 family protein 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 have been implicated in chemoresistance, although a significant percentage of relapsed AML have upregulated KDM6A. Based on these mechanistic findings, olaparib treatment significantly reduced engraftment of patient-derived xenografts. Thus KDM6A-mutant human primary AML samples have increased susceptibility to Poly-(ADP-ribose)-polymerase (PARP) inhibition in vivo. 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, KDM6A loss was accompanied with a downregulated BCL2A1, which is commonly associated with venetoclax resistance. 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 acquired KDM6A-domain mutations were even more sensitive to the combination. Together, our study illustrates a mechanistic rationale in support for a novel combination targeted therapy for human AML based on subtype heterogeneity, and establishes KDM6A as an important molecular regulator for determining therapeutic efficacy.

SUMMARYAcute myeloid leukemia (AML) progression and relapse is fueled by self-renewing leukemic stem cells (LSCs) whose molecular determinants have been difficult to discern from normal hematopoietic stem cells (HSCs) or to uncover in screening approaches focused on general AML cell properties. We have identified a unique set of RNA binding proteins (RBPs) that are enriched in human AML LSCs but repressed in HSCs. Using an in vivo two step CRISPR-Cas9-mediated screening approach to specifically score for cancer stem cell functionality, we found 32 RBPs essential for LSC propagation and self-renewal in MLL-AF9 translocated AML. Using knockdown or small molecule approaches we show that targeting key hit RBP ELAVL1 impaired LSC-driven in vivo leukemic reconstitution and selectively depleted primitive AML cells vs. normal hematopoietic stem and progenitors. Importantly, knockdown of Elavl1 spared HSCs while significantly reducing LSC numbers across genetically diverse leukemias. Integrative RNA-seq and eCLIP-seq profiling revealed hematopoietic differentiation, RNA splicing and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with the mitochondrial import protein TOMM34 being a direct ELAVL1-stabilized target whose inhibition impairs AML propagation. Altogether, through the use of a stem cell-adapted in vivo CRISPR dropout screening strategy, this work demonstrates that a wide variety of post-transcriptionally acting RBPs are important regulators of LSC-survival and self-renewal and, as exemplified by ELAVL1, highlights their potential as therapeutic targets in AML.

The ability of leukemic stem cells (LSC) to evade therapy and fuel leukemic progression causing relapse impedes therapeutic success in acute myeloid leukemia (AML). The LSC pool within a patient sample is not homogenous but comprises distinct LSC subsets that vary in self-renewal and propagation properties. The stemness programs that underlie LSC types are poorly understood since human LSC studies require primary patient samples where LSC numbers are low and isolation methods impure. To overcome these challenges, we developed a patient-derived AML model system (OCI-AML22) displaying a functionally, transcriptionally and epigenetically defined cellular hierarchy driven by functional LSCs that can be immunophenotypically identified and isolated. Through single cell and functional approaches, the OCI-AML22 LSC fraction was found to contain distinct LSCs that vary in proliferative and differentiation properties. OCI-AML22 represents a valuable resource to decipher mechanisms driving stemness and the multiple layers of heterogeneity within LSCs.

The treatment landscape of AML is evolving with promising therapies entering clinical translation, yet patient responses remain heterogeneous and biomarkers for tailoring treatment are lacking. To understand how disease heterogeneity links with therapy response, we determined the leukemia cell hierarchy make-up from bulk transcriptomes of over 1000 patients through deconvolution using single-cell reference profiles of leukemia stem, progenitor, and mature cell types. Leukemia hierarchy composition was associated with functional, genomic, and clinical properties and converged into four overall classes, spanning Primitive, Mature, GMP, and Intermediate. Critically, variation in hierarchy composition along the Primitive vs GMP or Primitive vs Mature axes were associated with response to chemotherapy or drug sensitivity profiles of targeted therapies, respectively. A 7-gene biomarker derived from the Primitive vs Mature axis was predictive of patient response to 105 investigational drugs. Thus, hierarchy composition constitutes a novel framework for understanding disease biology and advancing precision medicine in AML.

SUMMARYGene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSC) point to shared core stemness properties. However, discordance between mRNA and protein signatures underscores an important role for post-transcriptional regulation by miRNAs in governing this critical nexus. Here, we identified miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impaired B lymphoid differentiation and expanded long-term HSC. Integration of protein mass spectrometry and chimeric AGO2 eCLIP-seq identified TBL1XR1 as a primary miR-130a target, whose loss of function phenocopied miR-130a overexpression. Moreover, we found that miR-130a is highly expressed in t(8;21) AML where it is critical for maintaining the oncogenic molecular program mediated by AML1-ETO. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of novel genes and molecular networks underpinning stemness properties of normal and leukemic cells.HIGHLIGHTSmiR-130a is a regulator of HSC self-renewal and lineage commitmentTBL1XR1 is a principal target of miR-130aTBL1XR1 loss of function in HSPC phenocopies enforced expression of miR-130aElevated miR-130a levels maintain the AML1-ETO repressive program in t(8;21) AML

Single-molecule molecular inversion probes (smMIPs) provides a modular and cost-effective platform for high-multiplex targeted next-generation sequencing (NGS). Nevertheless, translating the raw smMIP-derived sequencing data into accurate and meaningful information currently requires proficient computational skills and a large amount of computational work, prohibiting wide-scale adoption of smMIP-based technologies. To enable easy, efficient, and accurate interrogation of smMIP-derived data, we developed SmMIP-tools, a computational toolset that combines the critical analytic steps for smMIP data interpretation into a single computational pipeline. Here, we describe in detail two of the software’s major components. The first is a read processing tool that performs quality control steps, generates read-smMIP linkages and retrieves molecular tags. The second is an error-aware variant caller capable of detecting single nucleotide variants (SNVs) and short insertions and deletions (indels). Using a cell-line DNA dilution series and a cohort of blood cancer patients, we benchmarked SmMIP-tools and evaluated its performance against clinical sequencing reports. We anticipate that SmMIP-tools will increase accessibility to smMIP-technology, enabling cost-effective genetic research to push personalized medicine forward.

Signaling pathways that control vital features of leukemic stem cells including multipotency, self-renewal, clonal expansion and quiescence remain unclear. Emerging studies illustrate critical roles for lysosomes in hematopoietic and leukemic stem cell fate. By investigating consequences of INPP4B alterations in AML, we have discovered its role in driving leukemic ‘stemness’. We observed that INPP4B is highly expressed leukemic stem cell populations and Inpp4b-deficeint leukemias demonstrate increased disease latency, reduced leukemia initiating potential which is associated with a differentiated leukemic phenotype. Molecular analyses show that Inpp4b-deficient leukemias have compromised lysosomal gene expression, lysosomal content, and lysosomal activity. Our discovery of a novel pathway linking INPP4B, lysosomal biogenesis and leukemic stemness, provides a mechanism to explain the association of high INPP4B expression with poor AML prognosis, and highlights novel patient stratification strategies and LSC-specific leukemic therapies.Key PointsOur findings highlight a novel pathway linking INPP4B, lysosomal function and leukemic stemness that explains the prognostic role of INPP4B in AML.Our data reveal the utility of INPP4B as a biomarker of aggressive AML and provide a rationale to explore INPP4B and its associated function in lysosome biology as novel strategies to target LSC and AML

SummaryIt is critical to understand how quiescent long-term hematopoietic stem cells (LT-HSC) sense demand from daily and stress-mediated cues and transition into bioenergetically active progeny to differentiate and meet these cellular needs. Here, we show that lysosomes, which are sophisticated nutrient sensing and signaling centers, are dichotomously regulated by the Transcription Factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSC and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, which promotes quiescence, self-renewal and governs erythroid-myeloid commitment. By contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism to drive LT-HSC activation. Collectively, our study identifies lysosomes as a central regulatory hub for proper and coordinated stem cell fate determination.

Despite most acute myeloid leukemia (AML) patients achieving complete remission after induction chemotherapy, two thirds of patients will relapse with fatal disease within 5 years. AML is organized as a cellular hierarchy sustained by leukemia stem cells (LSC) at the apex, with LSC properties directly linked to tumor progression, therapy failure and disease relapse 1–5. Despite the central role of LSC in poor patient outcomes, little is known of the genetic determinants of their stemness properties 6–8. Although much AML research focuses on mutational processes and their impact on gene expression programs, the genetic determinants of cell state properties including stemness expand beyond mutations, relying on the genetic architecture captured in the chromatin of each cell 9–11. As LSCs share many functional and molecular properties with normal hematopoietic stem cells (HSC), we identified genetic determinants of primitive populations enriched for LSCs and HSCs in comparison with their downstream mature progeny by investigating their chromatin accessibility. Our work reveals how distinct transposable element (TE) subfamilies are used in primitive versus mature populations, functioning as docking sites for stem cell-associated regulators of genome topology, including CTCF, or lineage-specific transcription regulators in primitive and mature populations, respectively. We further show how TE subfamilies accessible in LSCs define docking sites for several oncogenic drivers in AML, namely FLI1, LYL1 and MEIS1. Using chromatin accessibility profiles from a cohort of AML patients, we further show the clinical utility of our TE accessibility-based LSCTE121 scoring scheme to identify patients with high rates of relapse. Collectively, our work reveals how different accessible TE subfamilies serve as genetic determinants of stemness properties in normal and leukemic hematopoietic stem cells.

Children with Down syndrome have a 150-fold increased risk of developing myeloid leukemia, but the mechanism of predisposition is unclear. As Down syndrome leukemogenesis initiates during fetal development, we characterized the cellular context of preleukemic initiation and leukemic progression using gene editing in human disomic and trisomic fetal liver hematopoietic cells and xenotransplantation. GATA1 mutations caused transient preleukemia only when introduced into trisomy 21 long-term hematopoietic stem cells, where a subset of chromosome 21 miRNAs triggers 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 cells mediated the propagation of preleukemia and leukemia, and functional KIT inhibition targeted preleukemic stem cells, blocking progression to leukemia.

1 Abstract Cancers are composed of genetically distinct subpopulations of malignant cells. By sequencing DNA from cancer tissue samples, we can characterize the somatic mutations specific to each population and build clone trees describing the evolutionary ancestry of populations relative to one another. These trees reveal critical points in disease development and inform treatment. Pairtree is a new method for constructing clone trees using DNA sequencing data from one or more bulk samples of an individual cancer. It uses Bayesian inference to compute posterior distributions over the evolutionary relationships between every pair of identified subpopulations, then uses these distributions in a Markov Chain Monte Carlo algorithm to perform efficient inference of the posterior distribution over clone trees. Unlike existing methods, Pairtree can perform clone tree reconstructions using as many as 100 samples per cancer that reveal 30 or more cell subpopulations. On simulated data, Pairtree is the only method whose performance reliably improves when provided with additional bulk samples from a cancer. This suggests a shortcoming of existing methods, as more samples provide more information, and should always make clone tree reconstruction easier. On 14 B-progenitor acute lymphoblastic leukemias with up to 90 samples from each cancer, Pairtree was the only method that could reproduce or improve upon expert-derived clone tree reconstructions. By scaling to more challenging problems, Pairtree supports new biomedical research applications that can improve our understanding of the natural history of cancer, as well as better illustrate the interplay between cancer, host, and therapeutic interventions. The Pairtree method, along with an interactive visual interface for exploring the clone tree posterior, is available at https://github.com/morrislab/pairtree.

Myeloproliferative neoplasms (MPNs) are blood cancers characterized by excessive production of mature myeloid cells that result from the acquisition of somatic driver mutations in hematopoietic stem cells (HSCs)1. While substantial progress has been made to define the causal somatic mutation profile for MPNs2, epidemiologic studies indicate a significant heritable component for the disease that is among the highest known for all cancers3. However, only a limited set of genetic risk loci have been identified, and the underlying biological mechanisms leading to MPN acquisition remain unexplained. Here, to define the inherited risk profile, we conducted the largest genome-wide association study of MPNs to date (978,913 individuals with 3,224 cases) and identified 14 genome-wide significant loci, as well as a polygenic signature that increases the odds for disease acquisition by nearly 3-fold between the top and median deciles. Interestingly, we find a shared genetic architecture between MPN risk and several hematopoietic traits spanning distinct lineages, as well as an association between increased MPN risk and longer leukocyte telomere length, collectively implicating HSC function and self-renewal. Strikingly, we find a significant enrichment for risk variants mapping to accessible chromatin in HSCs compared with other hematopoietic populations. Finally, gene mapping identifies modulators of HSC biology and targeted variant-to-function analyses suggest likely roles for CHEK2 and GFI1B in altering HSC function to confer disease risk. Overall, we demonstrate the power of human genetic studies to illuminate a previously unappreciated mechanism for MPN risk through modulation of HSC function.

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., Single-cell proteomics can provide insights into the molecular basis for cellular heterogeneity. Here, the authors develop a multiplexed single-cell proteomics and computational workflow, and show that their strategy captures the cellular hierarchies in an Acute Myeloid Leukemia culture model.

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., Previous gene editing in haematopoietic stem cells (HSCs) has focussed on a heterogeneous CD34+ population. Here, the authors demonstrate high efficiency CRISPR/Cas9-based editing of purified long-term HSCs using non-homologous end joining and homology-directed repair, by directing isoform-specific expression of GATA1.