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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.

Melanoma cells can switch between a melanocytic and a mesenchymal-like state. Scattered evidence indicates that additional intermediate state(s) may exist. Here, to search for such states and decipher their underlying gene regulatory network (GRN), we studied 10 melanoma cultures using single-cell RNA sequencing (RNA-seq) as well as 26 additional cultures using bulk RNA-seq. Although each culture exhibited a unique transcriptome, we identified shared GRNs that underlie the extreme melanocytic and mesenchymal states and the intermediate state. This intermediate state is corroborated by a distinct chromatin landscape and is governed by the transcription factors SOX6, NFATC2, EGR3, ELF1 and ETV4. Single-cell migration assays confirmed the intermediate migratory phenotype of this state. Using time-series sampling of single cells after knockdown of SOX10, we unravelled the sequential and recurrent arrangement of GRNs during phenotype switching. Taken together, these analyses indicate that an intermediate state exists and is driven by a distinct and stable 'mixed' GRN rather than being a symbiotic heterogeneous mix of cells. ispartof: NATURE CELL BIOLOGY vol:22 issue:8 pages:986-+ ispartof: location:England status: published

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

Epithelial-to-mesenchymal transition (EMT), a process through which epithelial tumor cells acquire mesenchymal phenotypic properties, contributes to both metastatic dissemination and therapy resistance in cancer. Accumulating evidence indicates that nonepithelial tumors, including melanoma, can also gain mesenchymal-like properties that increase their metastatic propensity and decrease their sensitivity to therapy. In this review, we discuss recent findings, illustrating the striking similarities-but also knowledge gaps-between the biology of mesenchymal-like state(s) in melanoma and mesenchymal state(s) from epithelial cancers. Based on this comparative analysis, we suggest hypothesis-driven experimental approaches to further deepen our understanding of the EMT-like process in melanoma and how such investigations may pave the way towards the identification of clinically relevant biomarkers for prognosis and new therapeutic strategies. ispartof: FEBS JOURNAL vol:289 issue:5 pages:1352-1368 ispartof: location:England status: published

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.

Tumor microenvironment (TME) plays a critical role in cancer progression and response to therapies. Therapeutic intervention strategies for cancer treatment can induce inflammation, resulting in changes in the TME. To investigate the response of cancer cells to chemotherapy in the context of an inflammatory microenvironment, we studied the effects driven by the chemotherapeutic drug doxorubicin, and the inflammatory cytokine TNFá. We previously demonstrated using expression microarrays experiments (validated also through qPCR) that the Doxo+TNFá combined treatment determined a strong up-regulation of migration-related genes as well as increased motility in breast cancer cells (MCF7). We confirmed the synergistic activation of a group of 6 different genes upon Doxo+TNFá combined treatment in different cell line models coming from different cancer types (A549-lung, U2OS-osteosarcoma). Moreover, we demonstrated that this effect was only partially p53-depenedent but strongly Doxo-dependent using p53 mutated MDA-MB-231 breast cancer cells or knocking-out p53 in MCF7 or U2OS cells through the cutting edge technology CRISPR/Cas9. We also demonstrated that the combined Doxo+TNFá treatment was not only able to disrupt the 3D architecture of mammary acini observed with MCF10A primary cells grown within matrigel, but also to stimulate the tube-forming potential of Human Umbilical Vein Endothelial Cells (HUVEC). Furthermore, a signature of Doxo+TNFá highly synergistic genes (DT-29) was shown to exhibit prognostic value for luminal breast cancer patients, with adverse outcome correlating with higher relative expression (based on Kaplan-Meier plotter tool). We further used available experimental data sets (RNA-seq measurements from Gene Expression Omnibus) both from breast cancer cell lines (luminal-like vs basal-like breast cancer cells) and breast cancer patients (ER positive vs Triple Negative Breast Cancer, TNBC and vs healthy adjacent tissues). The expression of DT-29 gene signature was analyzed and compared among the different groups of samples. The most statistically relevant genes differentially expressed among the different groups were involved in the STAT3-driven pathways and were prevalently highly expressed in TNBC patients and cell lines. In order to demonstrate a STAT3-dependent regulation of up-regulated genes upon Doxo+TNFá combined treatment, we pharmacologically inhibited STAT3 using Stattic or we knocked-out STAT3 using CRISPR/Cas9 in MCF7, MDA-MB-231 and U2OS cells. Results demonstrated a STAT3-dependent up-regulation for some of the selected genes and STAT3 inhibition resulted also in a reduced migration potential particularly in U2OS cells. We propose that the combined treatment with Doxo+TNFá can lead to the activation of specific gene expression programs that may impact on cancer phenotypes and potentially modify the efficacy of cancer therapy. Citation Format: Federica Alessandrini, Laura Pezze, Francesca Precazzini, Silvia D’Ambrosi, Dennis Pedri, Lia Pinto, Yari Ciribilli. Chemotherapy and inflammation show a p65- and STAT3-dependent pro-tumorigenic potential. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 706.