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Metastasis arises from disseminated tumour cells (DTCs) that are characterized by intrinsic phenotypic plasticity and the capability of seeding to secondary organs. DTCs can remain latent for years before giving rise to symptomatic overt metastasis. In this context, DTCs fluctuate between a quiescent and proliferative state in response to systemic and microenvironmental signals including immune-mediated surveillance. Despite its relevance, how intrinsic mechanisms sustain DTCs plasticity has not been addressed. By interrogating the epigenetic state of metastatic cells, we find that tumour progression is coupled with the activation of oncogenic enhancers that are organized in variable interconnected chromatin domains. This spatial chromatin context leads to the activation of a robust transcriptional response upon repeated exposure to retinoic acid (RA). We show that this adaptive mechanism sustains the quiescence of DTCs through the activation of the master regulator SOX9. Finally, we determine that RA-stimulated transcriptional memory increases the fitness of metastatic cells by supporting the escape of quiescent DTCs from NK-mediated immune surveillance. Overall, these findings highlight the contribution of oncogenic enhancers in establishing transcriptional memories as an adaptive mechanism to reinforce cancer dormancy and immune escape, thus amenable for therapeutic intervention., (© 2024. The Author(s).)

Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.

Cancer is characterized by pervasive epigenetic alterations with enhancer dysfunction orchestrating the aberrant cancer transcriptional programs and transcriptional dependencies. Here, we epigenetically characterize human colorectal cancer (CRC) using de novo chromatin state discovery on a library of different patient-derived organoids. By exploring this resource, we unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic and independent of interpatient heterogeneity. We show that the transcriptional coactivators YAP/TAZ act as key regulators of the conserved CRC gained enhancers. The same YAP/TAZ-bound enhancers display active chromatin profiles across diverse human tumors, highlighting a pan-cancer epigenetic rewiring which at single-cell level distinguishes malignant from normal cell populations. YAP/TAZ inhibition in established tumor organoids causes extensive cell death unveiling their essential role in tumor maintenance. This work indicates a common layer of YAP/TAZ-fueled enhancer reprogramming that is key for the cancer cell state and can be exploited for the development of improved therapeutic avenues.

Immune checkpoint inhibitors are used for treating patients with metastatic melanoma. Since the response to treatment is variable, biomarkers are urgently needed to identify patients who may benefit from such therapy. Here, we combine single-cell RNA-sequencing and multiparameter flow cytometry to assess changes in circulating CD8 + T cells in 28 patients with metastatic melanoma starting anti-PD-1 therapy, followed for 6 months: 17 responded to therapy, whilst 11 did not. Proportions of activated and proliferating CD8 + T cells and of mucosal-associated invariant T (MAIT) cells are significantly higher in responders, prior to and throughout therapy duration. MAIT cells from responders express higher level of CXCR4 and produce more granzyme B. In silico analysis support MAIT presence in the tumor microenvironment. Finally, patients with >1.7% of MAIT among peripheral CD8 + population show a better response to treatment. Our results thus suggest that MAIT cells may be considered a biomarker for patients responding to anti-PD-1 therapy.

Human pluripotent stem cells (hPSCs) have the capacity to give rise to all differentiated cells of the adult. TGF-beta is used routinely for expansion of conventional hPSCs as flat epithelial colonies expressing the transcription factors POU5F1/OCT4, NANOG, SOX2. Here we report a global analysis of the transcriptional programme controlled by TGF-beta followed by an unbiased gain-of-function screening in multiple hPSC lines to identify factors mediating TGF-beta activity. We identify a quartet of transcriptional regulators promoting hPSC self-renewal including ZNF398, a human-specific mediator of pluripotency and epithelial character in hPSCs. Mechanistically, ZNF398 binds active promoters and enhancers together with SMAD3 and the histone acetyltransferase EP300, enabling transcription of TGF-beta targets. In the context of somatic cell reprogramming, inhibition of ZNF398 abolishes activation of pluripotency and epithelial genes and colony formation. Our findings have clear implications for the generation of bona fide hPSCs for regenerative medicine.

In Drosophila melanogaster the single male chromosome X undergoes an average twofold transcriptional upregulation for balancing the transcriptional output between sexes. Previous literature hypothesised that a global change in chromosome structure may accompany this process. However, recent studies based on Hi-C failed to detect these differences. Here we show that global conformational differences are specifically present in the male chromosome X and detectable using Hi-C data on sex-sorted embryos, as well as male and female cell lines, by leveraging custom data analysis solutions. We find the male chromosome X has more mid-/long-range interactions. We also identify differences at structural domain boundaries containing BEAF-32 in conjunction with CP190 or Chromator. Weakening of these domain boundaries in male chromosome X co-localizes with the binding of the dosage compensation complex and its co-factor CLAMP, reported to enhance chromatin accessibility. Together, our data strongly indicate that chromosome X dosage compensation affects global chromosome structure.

The Hippo pathway is an oncosuppressor signalling cascade that plays a major role in the control of cell growth, tissue homoeostasis and organ size. Dysregulation of the Hippo pathway leads to aberrant activation of the transcription co-activator YAP (Yes-associated protein) that contributes to tumorigenesis in several tissues. Here we identify glucocorticoids (GCs) as hormonal activators of YAP. Stimulation of glucocorticoid receptor (GR) leads to increase of YAP protein levels, nuclear accumulation and transcriptional activity in vitro and in vivo. Mechanistically, we find that GCs increase expression and deposition of fibronectin leading to the focal adhesion-Src pathway stimulation, cytoskeleton-dependent YAP activation and expansion of chemoresistant cancer stem cells. GR activation correlates with YAP activity in human breast cancer and predicts bad prognosis in the basal-like subtype. Our results unveil a novel mechanism of YAP activation in cancer and open the possibility to target GR to prevent cancer stem cells self-renewal and chemoresistance.

The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia.