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Many cancers are organized as cellular hierarchies sustained by cancer stem cells (CSC), whose eradication is crucial for achieving long-term remission. Difficulties to isolate and undertake in vitro and in vivo experimental studies of rare CSC under conditions that preserve their original properties currently constitute a bottleneck for identifying molecular mechanisms involving coding and non-coding genomic regions that govern stemness. We focussed on acute myeloid leukemia (AML) as a paradigm of the CSC model and developed a patient-derived system termed OCI-AML22 that recapitulates the cellular hierarchy driven by leukemia stem cells (LSC). Through classical flow sorting and functional analyses, we established that a single phenotypic population is highly enriched for LSC. The LSC fraction can be easily isolated and serially expanded in culture or in xenografts while faithfully recapitulating functional, transcriptional and epigenetic features of primary LSCs. A novel non-coding regulatory element was identified with a new computational approach using functionally validated primary AML LSC fractions and its role in LSC stemness validated through efficient CRISPR editing using methods optimized for OCI-AML22 LSC. Collectively, OCI-AML22 constitutes a valuable resource to uncover mechanisms governing CSC driven malignancies.

In embryonic stem cells, promoters of key lineage-specific differentiation genes are found in a bivalent state, having both activating H3K4me3 and repressive H3K27me3 histone marks, making them poised for transcription upon loss of H3K27me3. Whether cancer-initiating cells (C-ICs) have similar epigenetic mechanisms that prevent lineage commitment is unknown. Here we show that colorectal C-ICs (CC-ICs) are maintained in a stem-like state through a bivalent epigenetic mechanism. Disruption of the bivalent state through inhibition of the H3K27 methyltransferase EZH2, resulted in decreased self-renewal of patient-derived C-ICs. Epigenomic analyses revealed that the promoter of Indian Hedgehog ( IHH ), a canonical driver of normal colonocyte differentiation, exists in a bivalent chromatin state. Inhibition of EZH2 resulted in de-repression of IHH, decreased self-renewal, and increased sensitivity to chemotherapy in vivo. Our results reveal an epigenetic block to differentiation in CC-ICs and demonstrate the potential for epigenetic differentiation therapy of a solid tumour through EZH2 inhibition. The presence of bivalent epigenetic active and repressive histone marks control lineage-specific differentiation in embryonic stem cells. Here, the authors reveal that bivalent marks repress the differentiation gene IHH in colorectal cancer-initiating cells, and can be targeted by EZH2 inhibition

The imbalance between Th17 and regulatory T cells in inflammatory bowel diseases (IBD) promotes intestinal epithelial cell damage. In this scenario, T helper cell lineage commitment is accompanied by dynamic changes to the chromatin that facilitate or repress gene expression. Here, we characterized the chromatin landscape and heterogeneity of intestinal and peripheral CD4 T cellsfrom IBD patients using in house ATAC-Seq and single cell RNA-Seq libraries. We show that chromatin accessibility profiles of CD4 T cells from inflamed intestinal biopsies relate to genes associated with a network of inflammatory processes. After integrating the chromatin profiles of tissue-derived CD4 T cells and in-vitro polarized CD4 T cell subpopulations, we found that the chromatin accessibility changes of CD4 T cells were associated with a higher predominance of pathogenic Th17 cells (pTh17 cells) in inflamed biopsies. In addition, IBD risk loci in CD4 T cells were colocalized with accessible chromatin changes near pTh17-related genes, as shown in intronic STAT3 and IL23R regions enriched in areas of active intestinal inflammation. Moreover, single cell RNA-Seq analysis revealed a population of pTh17 cells that co-expresses Th1 and cytotoxic transcriptional programs associated with IBD severity. Altogether, we show that cytotoxic pTh17 cells were specifically associated with IBD genetic variants and linked to intestinal inflammation of IBD patients.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by high rate of therapy resistance. Since the cell of origin can impact response to therapy, it is crucial to understand the lineage composition of AML cells at time of therapy resistance. Here we leverage single-cell chromatin accessibility profiling of 22 AML bone marrow aspirates from eight patients at time of therapy resistance and following subsequent therapy to characterize their lineage landscape. Our findings reveal a complex lineage architecture of therapy-resistant AML cells that are primed for stem and progenitor lineages and spanning quiescent, activated and late stem cell/progenitor states. Remarkably, therapy-resistant AML cells are also composed of cells primed for differentiated myeloid, erythroid and even lymphoid lineages. The heterogeneous lineage composition persists following subsequent therapy, with early progenitor-driven features marking unfavorable prognosis in The Cancer Genome Atlas AML cohort. Pseudotime analysis further confirms the vast degree of heterogeneity driven by the dynamic changes in chromatin accessibility. Our findings suggest that therapy-resistant AML cells are characterized not only by stem and progenitor states, but also by a continuum of differentiated cellular lineages. The heterogeneity in lineages likely contributes to their therapy resistance by harboring different degrees of lineage-specific susceptibilities to therapy. Single cell ATAC-sequencing of human acute myeloid leukemia cells at the time of therapy resistance reveals a mix of stem cells, progenitors and differentiated erythroid, myeloid and lymphoid cells, a heterogeneity that may contribute to therapy resistance.

We have sequenced 463 presenting cases of myeloma entered into the UK Myeloma XI study using whole exome sequencing. Here we identify mutations induced as a consequence of misdirected AID in the partner oncogenes of IGH translocations, which are activating and associated with impaired clinical outcome. An APOBEC mutational signature is seen in 3.8% of cases and is linked to the translocation-mediated deregulation of MAF and MAFB , a known poor prognostic factor. Patients with this signature have an increased mutational load and a poor prognosis. Loss of MAF or MAFB expression results in decreased APOBEC3B and APOBEC4 expression, indicating a transcriptional control mechanism. Kataegis, a further mutational pattern associated with APOBEC deregulation, is seen at the sites of the MYC translocation. The APOBEC mutational signature seen in myeloma is, therefore, associated with poor prognosis primary and secondary translocations and the molecular mechanisms involved in generating them. Rearrangements of the Ig loci are essential for generating antibody diversity but abnormal translocations can be a driving event for myeloma. Here Walker et al . perform whole exome sequencing on myeloma patients to capture the diversity of mutational changes.

The leukemia stem cell (LSC) compartment is a complex reservoir fueling disease progression in acute myeloid leukemia (AML). The existence of heterogeneity within this compartment is well documented but prior studies have focused on genetic heterogeneity without being able to address functional heterogeneity. Understanding this heterogeneity is critical for the informed design of therapies targeting LSC, but has been hampered by LSC scarcity and the lack of reliable cell surface markers for viable LSC isolation. To overcome these challenges, we turned to the patient-derived OCI-AML22 cell model. This model includes functionally, transcriptionally and epigenetically characterized LSC broadly representative of LSC found in primary AML samples. Focusing on the pool of LSC, we used an integrated approach combining xenograft assays with single-cell analysis to identify two LSC subtypes with distinct transcriptional, epigenetic and functional properties. These LSC subtypes differed in depth of quiescence, differentiation potential, repopulation capacity, sensitivity to chemotherapy and could be isolated based on CD112 expression. A majority of AML patient samples had transcriptional signatures reflective of either LSC subtype, and some even showed coexistence within an individual sample. This work provides a framework for investigating the LSC compartment and designing combinatorial therapeutic strategies in AML.

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112 lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112 hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir. Dick and colleagues identify human LT-HSC subsets with distinct quiescent states. They link these differences to INKA1-mediated downregulation of the transmembrane protein CD112 and its interaction with the protein deacetylase SIRT1. INKA1 is inversely correlated with the histone H4K16Ac mark, which then distinguishes ‘latent’ CD112 lo LT-HSCs from CD112 hi LT-HSCs that are more readily activated in response to hematopoietic stress.

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.

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.

Transition between activation and quiescence states in hematopoietic stem and progenitor cells (HSPCs) is tightly governed by cell-intrinsic means and microenvironmental co-adaptation. Although this balance is fundamental for lifelong hematopoiesis and immunity, the underlying molecular mechanisms remain poorly defined. Multimodal analysis divulging differential transcriptional activity between distinct HSPC states indicates the presence of Fli-1 transcription factor binding motif in activated hematopoietic stem cells. We reveal that Fli-1 activity is essential during regenerative hematopoiesis in mice. Fli-1 directs activation programs while priming cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche through propagation of niche-derived angiocrine Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional hematopoietic stem cells impairments in the absence of Fli-1, without leukemic transformation. Applying FLI-1 transient modified-mRNA transduction into latent adult human mobilized HSPCs, enables their niche-mediated expansion and superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immunological regenerative medicine. Itkin et al. identify a role for Fli-1 in hematopoietic stem cell activation during regenerative hematopoiesis. They show that Fli-1 coordinates hematopoietic stem cells to stimulate niche-derived Notch1 feedback signals for demand-needed hematopoietic cell output.