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.

Mutant IDH inhibitors induce lineage differentiation in IDH-mutant oligodendroglioma.

A subset of patients with IDH-mutant glioma respond to inhibitors of mutant IDH (IDHi), yet the molecular underpinnings of such responses are not understood. Here, we profiled by single-cell or single-nucleus RNA-sequencing three IDH-mutant oligodendrogliomas from patients who derived clinical benefit from IDHi. Importantly, the tissues were sampled on-drug, four weeks from treatment initiation. We further integrate our findings with analysis of single-cell and bulk transcriptomes from independent cohorts and experimental models. We find that IDHi treatment induces a robust differentiation toward the astrocytic lineage, accompanied by a depletion of stem-like cells and a reduction of cell proliferation. Furthermore, mutations in NOTCH1 are associated with decreased astrocytic differentiation and may limit the response to IDHi. Our study highlights the differentiating potential of IDHi on the cellular hierarchies that drive oligodendrogliomas and suggests a genetic modifier that may improve patient stratification.

GoT-Splice protocol for multi-omics profiling of gene expression, cell-surface proteins, mutational status, and RNA splicing in human cells.

Studying RNA splicing factor mutations is challenging due to difficulties in distinguishing wild-type and mutant cells within complex human tissues and inaccuracies associated with reconstructing splicing signals from short-read sequencing data. Here, we present Genotyping of Transcriptomes (GoT)-Splice, a protocol that overcomes these limitations by combining GoT with enhanced long-read single-cell transcriptome and cell-surface proteomics profiling. We describe steps for long-read library preparation and analysis, followed by cDNA re-amplification, enrichment of mutation of interest, sample indexing, and GoT library preparation. For complete details on the use and execution of this protocol, please refer to Cortés-López et al.1.

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.

Cancer Evolution: A Multifaceted Affair.

Cancer cells adapt and survive through the acquisition and selection of molecular modifications. This process defines cancer evolution. Building on a theoretical framework based on heritable genetic changes has provided insights into the mechanisms supporting cancer evolution. However, cancer hallmarks also emerge via heritable nongenetic mechanisms, including epigenetic and chromatin topological changes, and interactions between tumor cells and the tumor microenvironment. Recent findings on tumor evolutionary mechanisms draw a multifaceted picture where heterogeneous forces interact and influence each other while shaping tumor progression. A comprehensive characterization of the cancer evolutionary toolkit is required to improve personalized medicine and biomarker discovery. SIGNIFICANCE: Tumor evolution is fueled by multiple enabling mechanisms. Importantly, genetic instability, epigenetic reprogramming, and interactions with the tumor microenvironment are neither alternative nor independent evolutionary mechanisms. As demonstrated by findings highlighted in this perspective, experimental and theoretical approaches must account for multiple evolutionary mechanisms and their interactions to ultimately understand, predict, and steer tumor evolution.

SON is an essential m6A target for hematopoietic stem cell fate.

Stem cells regulate their self-renewal and differentiation fate outcomes through both symmetric and asymmetric divisions. m6A RNA methylation controls symmetric commitment and inflammation of hematopoietic stem cells (HSCs) through unknown mechanisms. Here, we demonstrate that the nuclear speckle protein SON is an essential m6A target required for murine HSC self-renewal, symmetric commitment, and inflammation control. Global profiling of m6A identified that m6A mRNA methylation of Son increases during HSC commitment. Upon m6A depletion, Son mRNA increases, but its protein is depleted. Reintroduction of SON rescues defects in HSC symmetric commitment divisions and engraftment. Conversely, Son deletion results in a loss of HSC fitness, while overexpression of SON improves mouse and human HSC engraftment potential by increasing quiescence. Mechanistically, we found that SON rescues MYC and suppresses the METTL3-HSC inflammatory gene expression program, including CCL5, through transcriptional regulation. Thus, our findings define a m6A-SON-CCL5 axis that controls inflammation and HSC fate.

Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization.

Tumor-infiltrating macrophages support critical steps in tumor progression, and their accumulation in the tumor microenvironment (TME) is associated with adverse outcomes and therapeutic resistance across human cancers. In the TME, macrophages adopt diverse phenotypic alterations, giving rise to heterogeneous immune activation states and induction of cell cycle. While the transcriptional profiles of these activation states are well-annotated across human cancers, the underlying signals that regulate macrophage heterogeneity and accumulation remain incompletely understood. Here, we leveraged a novel ex vivo organotypic TME (oTME) model of breast cancer, in vivo murine models, and human samples to map the determinants of functional heterogeneity of TME macrophages. We identified a subset of F4/80highSca-1+ self-renewing macrophages maintained by type-I interferon (IFN) signaling and requiring physical contact with cancer-associated fibroblasts. We discovered that the contact-dependent self-renewal of TME macrophages is mediated via Notch4, and its inhibition abrogated tumor growth of breast and ovarian carcinomas in vivo, as well as lung dissemination in a PDX model of triple-negative breast cancer (TNBC). Through spatial multi-omic profiling of protein markers and transcriptomes, we found that the localization of macrophages further dictates functionally distinct but reversible phenotypes, regardless of their ontogeny. Whereas immune-stimulatory macrophages (CD11C+CD86+) populated the tumor epithelial nests, the stroma-associated macrophages (SAMs) were proliferative, immunosuppressive (Sca-1+CD206+PD-L1+), resistant to CSF-1R depletion, and associated with worse patient outcomes. Notably, following cessation of CSF-1R depletion, macrophages rebounded primarily to the SAM phenotype, which was associated with accelerated growth of mammary tumors. Our work reveals the spatial determinants of macrophage heterogeneity in breast cancer and highlights the disruption of macrophage self-renewal as a potential new therapeutic strategy.

Single-cell multi-omics defines the cell-type-specific impact of splicing aberrations in human hematopoietic clonal outgrowths.

RNA splicing factors are recurrently mutated in clonal blood disorders, but the impact of dysregulated splicing in hematopoiesis remains unclear. To overcome technical limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteogenomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to hematopoietic progenitors from myelodysplastic syndrome (MDS) patients with mutations in the core splicing factor SF3B1. SF3B1mut cells were enriched in the megakaryocytic-erythroid lineage, with expansion of SF3B1mut erythroid progenitor cells. We uncovered distinct cryptic 3' splice site usage in different progenitor populations and stage-specific aberrant splicing during erythroid differentiation. Profiling SF3B1-mutated clonal hematopoiesis samples revealed that erythroid bias and cell-type-specific cryptic 3' splice site usage in SF3B1mut cells precede overt MDS. Collectively, GoT-Splice defines the cell-type-specific impact of somatic mutations on RNA splicing, from early clonal outgrowths to overt neoplasia, directly in human samples.

Integration of whole transcriptome spatial profiling with protein markers.

Spatial transcriptomics and proteomics provide complementary information that independently transformed our understanding of complex biological processes. However, experimental integration of these modalities is limited. To overcome this, we developed Spatial PrOtein and Transcriptome Sequencing (SPOTS) for high-throughput simultaneous spatial transcriptomics and protein profiling. Compared with unimodal measurements, SPOTS substantially improves signal resolution and cell clustering and enhances the discovery power in differential gene expression analysis across tissue regions.

Nanobody-tethered transposition enables multifactorial chromatin profiling at single-cell resolution.

Chromatin states are functionally defined by a complex combination of histone modifications, transcription factor binding, DNA accessibility and other factors. Current methods for defining chromatin states cannot measure more than one aspect in a single experiment at single-cell resolution. Here we introduce nanobody-tethered transposition followed by sequencing (NTT-seq), an assay capable of measuring the genome-wide presence of up to three histone modifications and protein-DNA binding sites at single-cell resolution. NTT-seq uses recombinant Tn5 transposase fused to a set of secondary nanobodies (nb). Each nb-Tn5 fusion protein specifically binds to different immunoglobulin-G antibodies, enabling a mixture of primary antibodies binding different epitopes to be used in a single experiment. We apply bulk-cell and single-cell NTT-seq to generate high-resolution multimodal maps of chromatin states in cell culture and in human immune cells. We also extend NTT-seq to enable simultaneous profiling of cell surface protein expression and multimodal chromatin states to study cells of the immune system.

CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue.

Pooled genetic screens are powerful tools to study gene function in a high-throughput manner. Typically, sequencing-based screens require cell lysis, which limits the examination of critical phenotypes such as cell morphology, protein subcellular localization, and cell-cell/tissue interactions. In contrast, emerging optical pooled screening methods enable the investigation of these spatial phenotypes in response to targeted CRISPR perturbations. In this study, we report a multi-omic optical pooled CRISPR screening method, which we have named CRISPRmap. Our method combines a novel in situ CRISPR guide identifying barcode readout approach with concurrent multiplexed immunofluorescence and in situ RNA detection. CRISPRmap barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency, while reducing both dependency on third party proprietary sequencing reagents and assay cost. Notably, we conducted a multi-omic base-editing screen in a breast cancer cell line on core DNA damage repair genes involved in the homologous recombination and Fanconi anemia pathways investigating how nucleotide variants in those genes influence DNA damage signaling and cell cycle regulation following treatment with ionizing radiation or DNA damaging agents commonly used for cancer therapy. Approximately a million cells were profiled with our multi-omic approach, providing a comprehensive phenotypic assessment of the functional consequences of the studied variants. CRISPRmap enabled us to pinpoint likely-pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance. Furthermore, our approach effectively distinguished barcodes of a pooled library in tumor tissue, and we coupled it with cell-type and molecular phenotyping by cyclic immunofluorescence. Multi-omic spatial analysis of how CRISPR-perturbed cells respond to various environmental cues in the tissue context offers the potential to significantly expand our understanding of tissue biology in both health and disease.

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.

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.

Multi-platform profiling characterizes molecular subgroups and resistance networks in chronic lymphocytic leukemia.

Knowledge of the genomic landscape of chronic lymphocytic leukemia (CLL) grows increasingly detailed, providing challenges in contextualizing the accumulated information. To define the underlying networks, we here perform a multi-platform molecular characterization. We identify major subgroups characterized by genomic instability (GI) or activation of epithelial-mesenchymal-transition (EMT)-like programs, which subdivide into non-inflammatory and inflammatory subtypes. GI CLL exhibit disruption of genome integrity, DNA-damage response and are associated with mutagenesis mediated through activation-induced cytidine deaminase or defective mismatch repair. TP53 wild-type and mutated/deleted cases constitute a transcriptionally uniform entity in GI CLL and show similarly poor progression-free survival at relapse. EMT-like CLL exhibit high genomic stability, reduced benefit from the addition of rituximab and EMT-like differentiation is inhibited by induction of DNA damage. This work extends the perspective on CLL biology and risk categories in TP53 wild-type CLL. Furthermore, molecular targets identified within each subgroup provide opportunities for new treatment approaches.

Smart-RRBS for single-cell methylome and transcriptome analysis.

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system.

Discovery of Candidate DNA Methylation Cancer Driver Genes.

Epigenetic alterations, such as promoter hypermethylation, may drive cancer through tumor suppressor gene inactivation. However, we have limited ability to differentiate driver DNA methylation (DNAme) changes from passenger events. We developed DNAme driver inference-MethSig-accounting for the varying stochastic hypermethylation rate across the genome and between samples. We applied MethSig to bisulfite sequencing data of chronic lymphocytic leukemia (CLL), multiple myeloma, ductal carcinoma in situ, glioblastoma, and to methylation array data across 18 tumor types in TCGA. MethSig resulted in well-calibrated quantile-quantile plots and reproducible inference of likely DNAme drivers with increased sensitivity/specificity compared with benchmarked methods. CRISPR/Cas9 knockout of selected candidate CLL DNAme drivers provided a fitness advantage with and without therapeutic intervention. Notably, DNAme driver risk score was closely associated with adverse outcome in independent CLL cohorts. Collectively, MethSig represents a novel inference framework for DNAme driver discovery to chart the role of aberrant DNAme in cancer. SIGNIFICANCE: MethSig provides a novel statistical framework for the analysis of DNA methylation changes in cancer, to specifically identify candidate DNA methylation driver genes of cancer progression and relapse, empowering the discovery of epigenetic mechanisms that enhance cancer cell fitness.This article is highlighted in the In This Issue feature, p. 2113.

Targeting histone acetylation dynamics and oncogenic transcription by catalytic P300/CBP inhibition.

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.