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Although melanoma is notorious for its high degree of heterogeneity and plasticity1,2, the origin and magnitude of cell-state diversity remains poorly understood. Equally, it is unclear whether growth and metastatic dissemination are supported by overlapping or distinct melanoma subpopulations. Here, by combining mouse genetics, single-cell and spatial transcriptomics, lineage tracing and quantitative modelling, we provide evidence of a hierarchical model of tumour growth that mirrors the cellular and molecular logic underlying the cell-fate specification and differentiation of the embryonic neural crest. We show that tumorigenic competence is associated with a spatially localized perivascular niche, a phenotype acquired through an intercellular communication pathway established by endothelial cells. Consistent with a model in which only a fraction of cells are fated to fuel growth, temporal single-cell tracing of a population of melanoma cells with a mesenchymal-like state revealed that these cells do not contribute to primary tumour growth but, instead, constitute a pool of metastatic initiating cells that switch cell identity while disseminating to secondary organs. Our data provide a spatially and temporally resolved map of the diversity and trajectories of melanoma cell states and suggest that the ability to support growth and metastasis are limited to distinct pools of cells. The observation that these phenotypic competencies can be dynamically acquired after exposure to specific niche signals warrant the development of therapeutic strategies that interfere with the cancer cell reprogramming activity of such microenvironmental cues.

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Primary resistance drastically limits the clinical success of immune checkpoint blockade (ICB) in melanoma. Resistance to ICB may also develop when tumours relapse after targeted therapy. To identify cancer cell-intrinsic mechanisms driving resistance to ICB, we generated single-cell RNA-sequencing (scRNA-seq) data from a prospective longitudinal cohort of patients on ICB therapy, including an early time point obtained after only one cycle of treatment. Comparing these data with murine scRNA-seq datasets, we established a comprehensive view of the cellular architecture of the treatment-naïve melanoma ecosystem, and defined 6 evolutionarily conserved melanoma transcriptional metaprograms (Melanocytic or MEL, Mesenchymal-like or MES, Neural Crest-like, Antigen Presentation, Stress (hypoxia response) and Stress (p53 response)). Spatial multi-omics revealed a non-random geographic distribution of cell states that is, at least partly, driven by the tumour microenvironment. The single-cell data allowed unambiguous discrimination between melanoma MES cells and cancer-associated fibroblasts both in silico and in situ, a long-standing challenge in the field. Importantly, two of the melanoma transcriptional metaprograms were associated with divergent clinical responses to ICB. While the Antigen Presentation cell population was more abundant in tumours from patients who exhibited a clinical response to ICB, MES cells were significantly enriched in early on-treatment biopsies from non-responders, and their presence significantly predicted lack of response. Critically, we identified TCF4 (E2-2) as a master regulator of the MES program and suppressor of both MEL and Antigen Presentation programs. Targeting TCF4 expression in MES cells either genetically or pharmacologically using a bromodomain inhibitor increased immunogenicity and sensitivity to targeted therapy. This study describes an increasingly complex melanoma transcriptional landscape and its rapid evolution under ICB. It also identifies a putative biomarker of early response to ICB and an epigenetic therapeutic strategy that increases both immunogenicity of ICB-refractory melanoma and their sensitivity to targeted therapy.

Therapy resistance arises from heterogeneous drug-tolerant persister cells or minimal residual disease (MRD) through genetic and nongenetic mechanisms. A key question is whether specific molecular features of the MRD ecosystem determine which of these two distinct trajectories will eventually prevail. We show that, in melanoma exposed to mitogen-activated protein kinase therapeutics, emergence of a transient neural crest stem cell (NCSC) population in MRD concurs with the development of nongenetic resistance. This increase relies on a glial cell line-derived neurotrophic factor-dependent signaling cascade, which activates the AKT survival pathway in a focal adhesion kinase (FAK)-dependent manner. Ablation of the NCSC population through FAK inhibition delays relapse in patient-derived tumor xenografts. Strikingly, all tumors that ultimately escape this treatment exhibit resistance-conferring genetic alterations and increased sensitivity to extracellular signal-regulated kinase inhibition. These findings identify an approach that abrogates the nongenetic resistance trajectory in melanoma and demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths. ispartof: CANCER CELL vol:39 issue:8 pages:1135-+ ispartof: location:United States status: published

Induction and reversal of chromatin silencing is critical for successful development, tissue homeostasis, and the derivation of induced pluripotent stem cells (iPSCs). X-Chromosome inactivation (XCI) and reactivation (XCR) in female cells represent chromosome-wide transitions between active and inactive chromatin states. Although XCI has long been studied, providing important insights into gene regulation, the dynamics and mechanisms underlying the reversal of stable chromatin silencing of X-linked genes are much less understood. Here, we use allele-specific transcriptomics to study XCR during mouse iPSC reprogramming in order to elucidate the timing and mechanisms of chromosome-wide reversal of gene silencing. We show that XCR is hierarchical, with subsets of genes reactivating early, late, and very late during reprogramming. Early genes are activated before the onset of late pluripotency genes activation. Early genes are located genomically closer to genes that escape XCI, unlike genes reactivating late. Early genes also show increased pluripotency transcription factor (TF) binding. We also reveal that histone deacetylases (HDACs) restrict XCR in reprogramming intermediates and that the severe hypoacetylation state of the inactive X Chromosome (Xi) persists until late reprogramming stages. Altogether, these results reveal the timing of transcriptional activation of monoallelically repressed genes during iPSC reprogramming, and suggest that allelic activation involves the combined action of chromatin topology, pluripotency TFs, and chromatin regulators. These findings are important for our understanding of gene silencing, maintenance of cell identity, reprogramming, and disease. ispartof: Genome Research vol:29 issue:10 pages:1659-1672 ispartof: location:United States status: published

Background Precise gene dosage of the X chromosomes is critical for normal development and cellular function. In mice, XX female somatic cells show transcriptional X chromosome upregulation of their single active X chromosome, while the other X chromosome is inactive. Moreover, the inactive X chromosome is reactivated during development in the inner cell mass and in germ cells through X chromosome reactivation, which can be studied in vitro by reprogramming of somatic cells to pluripotency. How chromatin processes and gene regulatory networks evolved to regulate X chromosome dosage in the somatic state and during X chromosome reactivation remains unclear. Results Using genome-wide approaches, allele-specific ATAC-seq and single-cell RNA-seq, in female embryonic fibroblasts and during reprogramming to pluripotency, we show that chromatin accessibility on the upregulated mammalian active X chromosome is increased compared to autosomes. We further show that increased accessibility on the active X chromosome is erased by reprogramming, accompanied by erasure of transcriptional X chromosome upregulation and the loss of increased transcriptional burst frequency. In addition, we characterize gene regulatory networks during reprogramming and X chromosome reactivation, revealing changes in regulatory states. Our data show that ZFP42/REX1, a pluripotency-associated gene that evolved specifically in placental mammals, targets multiple X-linked genes, suggesting an evolutionary link between ZFP42/REX1, X chromosome reactivation, and pluripotency. Conclusions Our data reveal the existence of intrinsic compensatory mechanisms that involve modulation of chromatin accessibility to counteract X-to-Autosome gene dosage imbalances caused by evolutionary or in vitro X chromosome loss and X chromosome inactivation in mammalian cells.

T cells are critical in cancer immune surveillance but they can also shape tumor immunogenicity, described as cancer immunoediting. Melanoma patients commonly harbor T cells recognizing melanocyte differentiation antigens (MDAs). However, the roles of MDA-specific T cells in shaping melanoma immunogenicity and the response to immune checkpoint inhibition remain elusive. Here, we prospectively profiled peripheral CD8+ T cells from 27 stage IV patients before initiation of checkpoint inhibitor therapy. Clinical failure was associated with increased MDA-specific CD8+ T cells and reduced tumor MDA expression pretreatment. In nonresponders, decreased tumor MDA expression was concomitant with a dedifferentiated melanoma phenotype. We confirmed in 30 stage III patients that individuals with relapse disease during adjuvant anti-PD-1 therapy demonstrated a significantly higher incidence of dedifferentiated tumors pretreatment than individuals without recurrence. Thus, MDA-directed CD8+ T cells are associated with a dedifferentiated phenotype and reduced clinical response to checkpoint inhibitor therapy suggesting immunoediting as an important resistance mechanism.

SummaryThe ability to predict the future behaviour of an individual cancer is crucial for precision cancer medicine and, in particular, for the development of strategies that prevent acquisition of resistance to anti-cancer drugs. Therapy resistance, which often develops from a heterogeneous pool of drug-tolerant cells known as minimal residual disease (MRD), is thought to mainly occur through acquisition of genetic alterations. Increasing evidence, however, indicates that drug resistance might also be acquired though nongenetic mechanisms. A key emerging question is therefore whether specific molecular and/or cellular features of the MRD ecosystem determine which of these two distinct resistance trajectories will eventually prevail. We show herein that, in melanoma exposed to MAPK-therapeutics, the presence of a neural crest stem cell (NCSC) subpopulation in MRD concurred with the rapid development of resistance through nongenetic mechanisms. Emergence of this drug-tolerant population in MRD relies on a GDNF-dependent autocrine and paracrine signalling cascade, which activates the AKT survival pathway in a Focal-adhesion kinase-(FAK) dependent manner. Ablation of this subpopulation through inhibition of FAK/SRC-signalling delayed relapse in patient-derived tumour xenografts. Strikingly, all tumours that eventually escaped this treatment exhibited resistance-conferring genetic alterations and increased sensitivity to ERK-inhibition. These findings firmly establish that nongenetic reprogramming events contribute to therapy resistance in melanoma and identify a clinically-compatible approach that abrogates such a trajectory. Importantly, these data demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths and highlight key principles that may permit more effective pre-emptive therapeutic interventions.

NEAT1 is one of the most studied lncRNAs, in part because its silencing in mice causes defects in mammary gland development and corpus luteum formation and protects them from skin cancer development. Moreover, depleting NEAT1 in established cancer cell lines reduces growth and sensitizes cells to DNA damaging agents. However, NEAT1 produces two isoforms and because the short isoform, NEAT1_1, completely overlaps the 5′ part of the long NEAT1_2 isoform; the respective contributions of each of the isoforms to these phenotypes has remained unclear. Whereas NEAT1_1 is highly expressed in most tissues, NEAT1_2 is the central architectural component of paraspeckles, which are nuclear bodies that assemble in specific tissues and cells exposed to various forms of stress. Using dual RNA-FISH to detect both NEAT1_1 outside of the paraspeckles and NEAT1_2/NEAT1 inside this nuclear body, we report herein that NEAT1_1 levels are dynamically regulated during the cell cycle and targeted for degradation by the nuclear RNA exosome. Unexpectedly, however, cancer cells engineered to lack NEAT1_1, but not NEAT1_2, do not exhibit cell cycle defects. Moreover, Neat1_1-specific knockout mice do not exhibit the phenotypes observed in Neat1-deficient mice. We propose that NEAT1 functions are mainly, if not exclusively, attributable to NEAT1_2 and, by extension, to paraspeckles.

NEAT1 is one of the most studied lncRNAs, in part because its silencing in mice causes defects in mammary gland development and corpus luteum formation, and protects them from skin cancer development. Moreover, depleting NEAT1 in established cancer cell lines reduces growth and sensitizes cells to DNA damaging agents. However, NEAT1 produces two isoforms and because the short isoform, NEAT1_1, completely overlaps the 5’ part of the long NEAT1_2 isoform, the respective contributions of each of the isoforms to these phenotypes has remained unclear. Whereas NEAT1_1 is highly expressed in most tissues, NEAT1_2 is the central architectural component of paraspeckles, which are nuclear bodies that assemble in specific tissues and cells exposed to various forms of stress. Using dual RNA-FISH to detect both NEAT1_1 outside of paraspeckles and NEAT1_2/NEAT1 inside this nuclear body, we report herein that NEAT1_1 levels are dynamically regulated during the cell cycle and targeted for degradation by the nuclear RNA exosome. Unexpectedly, however, cancer cells engineered to lack NEAT1_1, but not NEAT1_2, do not exhibit cell cycle defects. Moreover, Neat1_1-specific knockout mice do not exhibit the phenotypes observed in Neat1-deficient mice. We propose that NEAT1 functions are mainly, if not exclusively, attributable to NEAT1_2 and, by extension, to paraspeckles. Graphical abstract

BackgroundInduction and reversal of chromatin silencing is critical for successful development, tissue homeostasis and the derivation of induced pluripotent stem cells (iPSCs). X-chromosome inactivation (XCI) and reactivation (XCR) in female cells represent chromosome-wide transitions between active and inactive chromatin states. While XCI has long been studied and provided important insights into gene regulation, the dynamics and mechanisms underlying the reversal of stable chromatin silencing of X-linked genes are much less understood. Here, we use allele-specific transcriptomic approaches to study XCR during mouse iPSC reprogramming in order to elucidate the timing and mechanisms of chromosome-wide reversal of gene silencing.ResultsWe show that XCR is hierarchical, with subsets of genes reactivating early, late and very late. Early genes are activated before the onset of late pluripotency genes activation and the complete silencing of the long non-coding RNA (lncRNA) Xist. These genes are located genomically closer to genes that escape XCI, unlike those reactivating late. Interestingly, early genes also show increased pluripotency transcription factor (TF) binding. We also reveal that histone deacetylases (HDACs) restrict XCR in reprogramming intermediates and that the severe hypoacetylation state of the Xi persists until late reprogramming stages.ConclusionsAltogether, these results reveal the timing of transcriptional activation of mono-allelically repressed genes during iPSC reprogramming, and suggest that allelic activation involves the combined action of chromatin topology, pluripotency transcription factors and chromatin regulators. These findings are important for our understanding of gene silencing, maintenance of cell identity, reprogramming and disease.