Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Jak2V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms.

Gain-of-function mutations activating JAK/STAT signaling are seen in the majority of patients with myeloproliferative neoplasms (MPN), most commonly JAK2V617F. Although clinically approved JAK inhibitors improve symptoms and outcomes in MPNs, remissions are rare, and mutant allele burden does not substantively change with chronic therapy. We hypothesized this is due to limitations of current JAK inhibitors to potently and specifically abrogate mutant JAK2 signaling. We therefore developed a conditionally inducible mouse model allowing for sequential activation, and then inactivation, of Jak2V617F from its endogenous locus using a combined Dre-rox/Cre-lox dual-recombinase system. Jak2V617F deletion abrogates MPN features, induces depletion of mutant-specific hematopoietic stem/progenitor cells, and extends overall survival to an extent not observed with pharmacologic JAK inhibition, including when cooccurring with somatic Tet2 loss. Our data suggest JAK2V617F represents the best therapeutic target in MPNs and demonstrate the therapeutic relevance of a dual-recombinase system to assess mutant-specific oncogenic dependencies in vivo. SIGNIFICANCE: Current JAK inhibitors to treat myeloproliferative neoplasms are ineffective at eradicating mutant cells. We developed an endogenously expressed Jak2V617F dual-recombinase knock-in/knock-out model to investigate Jak2V617F oncogenic reversion in vivo. Jak2V617F deletion abrogates MPN features and depletes disease-sustaining MPN stem cells, suggesting improved Jak2V617F targeting offers the potential for greater therapeutic efficacy. See related commentary by Celik and Challen, p. 701. This article is featured in Selected Articles from This Issue, p. 695.

MAVS signaling is required for preventing persistent chikungunya heart infection and chronic vascular tissue inflammation.

Chikungunya virus (CHIKV) infection has been associated with severe cardiac manifestations, yet, how CHIKV infection leads to heart disease remains unknown. Here, we leveraged both mouse models and human primary cardiac cells to define the mechanisms of CHIKV heart infection. Using an immunocompetent mouse model of CHIKV infection as well as human primary cardiac cells, we demonstrate that CHIKV directly infects and actively replicates in cardiac fibroblasts. In immunocompetent mice, CHIKV is cleared from cardiac tissue without significant damage through the induction of a local type I interferon response from both infected and non-infected cardiac cells. Using mice deficient in major innate immunity signaling components, we found that signaling through the mitochondrial antiviral-signaling protein (MAVS) is required for viral clearance from the heart. In the absence of MAVS signaling, persistent infection leads to focal myocarditis and vasculitis of the large vessels attached to the base of the heart. Large vessel vasculitis was observed for up to 60 days post infection, suggesting CHIKV can lead to vascular inflammation and potential long-lasting cardiovascular complications. This study provides a model of CHIKV cardiac infection and mechanistic insight into CHIKV-induced heart disease, underscoring the importance of monitoring cardiac function in patients with CHIKV infections.

Single-cell multi-omics of human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation.

Somatic mutations in cancer genes have been detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, because mutated and wild-type cells are admixed, we have limited ability to link genotypes with phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing genotype, transcriptomes and methylomes in progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations result in myeloid over lymphoid bias, and an expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate, with dysregulated expression of lineage and leukemia stem cell markers. Mutated DNMT3A leads to preferential hypomethylation of polycomb repressive complex 2 targets and a specific CpG flanking motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics paves the road to defining the downstream consequences of mutations that drive clonal mosaicism.

Halting ErbB-2 isoforms retrograde transport to the nucleus as a new theragnostic approach for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is clinically defined by the absence of estrogen and progesterone receptors and the lack of membrane overexpression or gene amplification of receptor tyrosine kinase ErbB-2/HER2. Due to TNBC heterogeneity, clinical biomarkers and targeted therapies for this disease remain elusive. We demonstrated that ErbB-2 is localized in the nucleus (NErbB-2) of TNBC cells and primary tumors, from where it drives growth. We also discovered that TNBC expresses both wild-type ErbB-2 (WTErbB-2) and alternative ErbB-2 isoform c (ErbB-2c). Here, we revealed that the inhibitors of the retrograde transport Retro-2 and its cyclic derivative Retro-2.1 evict both WTErbB-2 and ErbB-2c from the nucleus of BC cells and tumors. Using BC cells from several molecular subtypes, as well as normal breast cells, we demonstrated that Retro-2 specifically blocks proliferation of BC cells expressing NErbB-2. Importantly, Retro-2 eviction of both ErbB-2 isoforms from the nucleus resulted in a striking growth abrogation in multiple TNBC preclinical models, including tumor explants and xenografts. Our mechanistic studies in TNBC cells revealed that Retro-2 induces a differential accumulation of WTErbB-2 at the early endosomes and the plasma membrane, and of ErbB-2c at the Golgi, shedding new light both on Retro-2 action on endogenous protein cargoes undergoing retrograde transport, and on the biology of ErbB-2 splicing variants. In addition, we revealed that the presence of a functional signal peptide and a nuclear export signal (NES), both located at the N-terminus of WTErbB-2, and absent in ErbB-2c, accounts for the differential subcellular distribution of ErbB-2 isoforms upon Retro-2 treatment. Our present discoveries provide evidence for the rational repurposing of Retro-2 as a novel therapeutic agent for TNBC.

Specification of fetal liver endothelial progenitors to functional zonated adult sinusoids requires c-Maf induction.

The liver vascular network is patterned by sinusoidal and hepatocyte co-zonation. How intra-liver vessels acquire their hierarchical specialized functions is unknown. We study heterogeneity of hepatic vascular cells during mouse development through functional and single-cell RNA-sequencing. The acquisition of sinusoidal endothelial cell identity is initiated during early development and completed postnatally, originating from a pool of undifferentiated vascular progenitors at E12. The peri-natal induction of the transcription factor c-Maf is a critical switch for the sinusoidal identity determination. Endothelium-restricted deletion of c-Maf disrupts liver sinusoidal development, aberrantly expands postnatal liver hematopoiesis, promotes excessive postnatal sinusoidal proliferation, and aggravates liver pro-fibrotic sensitivity to chemical insult. Enforced c-Maf overexpression in generic human endothelial cells switches on a liver sinusoidal transcriptional program that maintains hepatocyte function. c-Maf represents an inducible intra-organotypic and niche-responsive molecular determinant of hepatic sinusoidal cell identity and lays the foundation for the strategies for vasculature-driven liver repair.

Epigenetic encoding, heritability and plasticity of glioma transcriptional cell states.

Single-cell RNA sequencing has revealed extensive transcriptional cell state diversity in cancer, often observed independently of genetic heterogeneity, raising the central question of how malignant cell states are encoded epigenetically. To address this, here we performed multiomics single-cell profiling-integrating DNA methylation, transcriptome and genotype within the same cells-of diffuse gliomas, tumors characterized by defined transcriptional cell state diversity. Direct comparison of the epigenetic profiles of distinct cell states revealed key switches for state transitions recapitulating neurodevelopmental trajectories and highlighted dysregulated epigenetic mechanisms underlying gliomagenesis. We further developed a quantitative framework to directly measure cell state heritability and transition dynamics based on high-resolution lineage trees in human samples. We demonstrated heritability of malignant cell states, with key differences in hierarchal and plastic cell state architectures in IDH-mutant glioma versus IDH-wild-type glioblastoma, respectively. This work provides a framework anchoring transcriptional cancer cell states in their epigenetic encoding, inheritance and transition dynamics.

Canonical ErbB-2 isoform and ErbB-2 variant c located in the nucleus drive triple negative breast cancer growth.

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2/HER2, a receptor tyrosine kinase. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Clinical biomarkers and targeted therapies for this disease remain elusive, so chemotherapy has been the standard of care for early and metastatic TNBC. Our present findings placed ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing events, using a PCR-sequencing approach combined with an RNA interference strategy, revealed that TNBC cells express either the canonical (wild-type) ErbB-2, encoded by transcript variant 1, or the non-canonical ErbB-2 isoform c, encoded by alternative variant 3 (RefSeq), or both. These ErbB-2 isoforms function in the nucleus as transcription factors. Evicting both from the nucleus or silencing isoform c only, blocks TN cell and tumor growth. This reveals not only NErbB-2 canonical and alternative isoforms role as targets of therapy in TNBC, but also isoform c dominant oncogenic potential. Furthermore, we validated our findings in the clinic and observed that NErbB-2 correlates with poor prognosis in primary TN tumors, disclosing NErbB-2 as a novel biomarker for TNBC. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type RefSeq proteins, which conserve the canonical domains and are located in their classical cellular compartments.

Nuclear PDCD4 Expression Defines a Subset of Luminal B-Like Breast Cancers with Good Prognosis.

The hormone receptor-positive (estrogen and/or progesterone receptor (PR)-positive) and HER2-negative breast cancer (BC) subtype is a biologically heterogeneous entity that includes luminal A-like (LumA-like) and luminal B-like (LumB-like) subtypes. Decreased PR levels is a distinctive biological feature of LumB-like tumors. These tumors also show reduced sensitivity to endocrine therapies and poorer prognosis than LumA-like tumors. Identification of biomarkers to accurately predict disease relapse in these subtypes is crucial in order to select effective therapies. We identified the tumor suppressor PDCD4 (programmed cell death 4), located in the nucleus (NPDCD4), as an independent prognostic factor of good clinical outcome in LumA-like and LumB-like subtypes. NPDCD4-positive LumB-like tumors presented overall and disease-free survival rates comparable to those of NPDCD4-positive LumA-like tumors, indicating that NPDCD4 improves the outcome of LumB-like patients. In contrast, NPDCD4 loss increased the risk of disease recurrence and death in LumB-like compared with LumA-like tumors. This, along with our results showing that LumB-like tumors present lower NPDCD4 positivity than LumA-like tumors, suggests that NPDCD4 loss contributes to endocrine therapy resistance in LumB-like BCs. We also revealed that PR induces PDCD4 transcription in LumB-like BC, providing a mechanistic explanation to the low PDCD4 levels in LumB-like BCs lacking PR. Finally, PDCD4 silencing enhanced BC cell survival in a patient-derived explant model of LumB-like disease. Our discoveries highlight NPDCD4 as a novel biomarker in LumA- and LumB-like subtypes, which could be included in the panel of immunohistochemical markers used in the clinic to accurately predict the prognosis of LumB-like tumors.

Acute Myeloid Leukemia iPSCs Reveal a Role for RUNX1 in the Maintenance of Human Leukemia Stem Cells.

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.

DNA methylation disruption reshapes the hematopoietic differentiation landscape.

Mutations in genes involved in DNA methylation (DNAme; for example, TET2 and DNMT3A) are frequently observed in hematological malignancies1-3 and clonal hematopoiesis4,5. Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts in the frequencies of erythroid versus myelomonocytic progenitors following Tet2 or Dnmt3a loss. Notably, these shifts trace back to transcriptional priming skews in uncommitted hematopoietic stem cells. To reconcile genome-wide DNAme changes with specific erythroid versus myelomonocytic skews, we provide evidence in support of differential sensitivity of transcription factors due to biases in CpG enrichment in their binding motif. Single-cell transcriptomes with targeted genotyping showed similar skews in transcriptional priming of DNMT3A-mutated human clonal hematopoiesis bone marrow progenitors. These data show that DNAme shapes the topography of hematopoietic differentiation, and support a model in which genome-wide methylation changes are transduced to differentiation skews through biases in CpG enrichment of the transcription factor binding motif.

Inhibition of EZH2 Catalytic Activity Selectively Targets a Metastatic Subpopulation in Triple-Negative Breast Cancer.

Epigenetic changes are increasingly being appreciated as key events in breast cancer progression. However, breast cancer subtype-specific epigenetic regulation remains poorly investigated. Here we report that EZH2 is a leading candidate of epigenetic modulators associated with the TNBC subtype and that it predicts poor overall survival in TNBC patients. We demonstrate that specific pharmacological or genetic inhibition of EZH2 catalytic activity impairs distant metastasis. We further define a specific EZH2high population with enhanced invasion, mammosphere formation, and metastatic potential that exhibits marked sensitivity to EZH2 inhibition. Mechanistically, EZH2 inhibition differentiates EZH2high basal cells to a luminal-like phenotype by derepressing GATA3 and renders them sensitive to endocrine therapy. Furthermore, dissection of human TNBC heterogeneity shows that EZH2high basal-like 1 and mesenchymal subtypes have exquisite sensitivity to EZH2 inhibition compared with the EZH2low luminal androgen receptor subtype. These preclinical findings provide a rationale for clinical development of EZH2 as a targeted therapy against TNBC metastasis.

EpiMethylTag: simultaneous detection of ATAC-seq or ChIP-seq signals with DNA methylation.

Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low-input, low sequencing depth method, EpiMethylTag, that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites.

m6A RNA Methylation Maintains Hematopoietic Stem Cell Identity and Symmetric Commitment.

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.

Somatic mutations and cell identity linked by Genotyping of Transcriptomes.

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.

Genetic and epigenetic determinants of B-cell lymphoma evolution.

PURPOSE OF REVIEW: The success of targeted therapies fostered the development of increasingly specific and effective therapeutics for B-cell malignancies. However, cancer plasticity facilitates disease relapse, whereby intratumoral heterogeneity fuels tumor evolution into a more aggressive and resistant form. Understanding cancer heterogeneity and the evolutionary processes underlying disease relapse is key for overcoming this limitation of current treatment strategies. In the present review, we delineate the current understanding of cancer evolution and the advances in both genetic and epigenetic fields, with a focus on non-Hodgkin B-cell lymphomas. RECENT FINDINGS: The use of massively parallel sequencing has provided insights into tumor heterogeneity, allowing determination of intratumoral genetic and epigenetic variability and identification of cancer driver mutations and (epi-)mutations. Increased heterogeneity prior to treatment results in faster disease relapse, and in many cases studying pretreatment clonal admixtures predicts the future evolutionary trajectory of relapsed disease. SUMMARY: Understanding the mechanisms underlying tumor heterogeneity and evolution provides valuable tools for the design of therapy within an evolutionary framework. This framework will ultimately aid in accurately predicting the evolutionary paths of B-cell malignancies, thereby guiding therapeutic strategies geared at directly anticipating and addressing cancer evolution.

Heregulin Co-opts PR Transcriptional Action Via Stat3 Role As a Coregulator to Drive Cancer Growth.

Accumulated findings have demonstrated the presence of bidirectional interactions between progesterone receptor (PR) and the ErbB family of receptor tyrosine kinases signaling pathways in breast cancer. We previously revealed signal transducer and activator of transcription 3 (Stat3) as a nodal convergence point between said signaling pathways proving that Stat3 is activated by one of the ErbBs' ligands, heregulin (HRG)ß1 via ErbB2 and through the co-option of PR as a signaling molecule. Here, we found that HRGß1 induced Stat3 recruitment to the promoters of the progestin-regulated cell cycle modulators Bcl-XL and p21(CIP1) and also stimulated Stat3 binding to the mouse mammary tumor virus promoter, which carries consensus progesterone response elements. Interestingly, HRGß1-activated Stat3 displayed differential functions on PR activity depending on the promoter bound. Indeed, Stat3 was required for PR binding in bcl-X, p21(CIP1), and c-myc promoters while exerting a PR coactivator function on the mouse mammary tumor virus promoter. Stat3 also proved to be necessary for HRGß1-induced in vivo tumor growth. Our results endow Stat3 a novel function as a coregulator of HRGß1-activated PR to promote breast cancer growth. These findings underscore the importance of understanding the complex interactions between PR and other regulatory factors, such as Stat3, that contribute to determine the context-dependent transcriptional actions of PR.

Progesterone receptor activation downregulates GATA3 by transcriptional repression and increased protein turnover promoting breast tumor growth.

INTRODUCTION: The transcription factor GATA3 is involved in mammary gland development and is crucial for the maintenance of the differentiated status of luminal epithelial cells. The role of GATA3 in breast cancer as a tumor suppressor has been established, although insights into the mechanism of GATA3 expression loss are still required. METHODS: Chromatin immunoprecipitation assays were conducted to study progestin modulation of recruitment of transcription factors to GATA3 promoter. We performed western blot and reverse RT-qPCR experiments to explore progestin regulation of GATA3 protein and mRNA expression respectively. Confocal microscopy and in vitro phosphorylation studies were conducted to examine progestin capacity to induce GATA3 serine phosphorylation in its 308 residue. GATA3 participation in progestin-induced breast cancer growth was addressed in in vitro proliferation and in vivo tumor growth experiments. RESULTS: In this study, we demonstrate that progestin-activated progesterone receptor (PR) reduces GATA3 expression through regulation at the transcriptional and post-translational levels in breast cancer cells. In the former mechanism, the histone methyltransferase enhancer of zeste homolog 2 is co-recruited with activated PR to a putative progesterone response element in the GATA3 proximal promoter, increasing H3K27me3 levels and inducing chromatin compaction, resulting in decreased GATA3 mRNA levels. This transcriptional regulation is coupled with increased GATA3 protein turnover through progestin-induced GATA3 phosphorylation at serine 308 followed by 26S proteasome-mediated degradation. Both molecular mechanisms converge to accomplish decreased GATA3 expression levels in breast cancer cells upon PR activation. In addition, we demonstrated that decreased GATA3 levels are required for progestin-induced upregulation of cyclin A2, which mediates the G1 to S phase transition of the cell cycle and was reported to be associated with poor prognosis in breast cancer. Finally, we showed that downregulation of GATA3 is required for progestin stimulation of both in vitro cell proliferation and in vivo tumor growth. CONCLUSIONS: In the present study, we reveal that progestin-induced PR activation leads to loss of GATA3 expression in breast cancer cells through transcriptional and post-translational regulation. Importantly, we demonstrate that GATA3 downregulation is required for progestin-induced upregulation of cyclin A2 and for progestin-induced in vitro and in vivo breast cancer cell growth.