DNA promoter hypermethylation of melanocyte lineage genes determines melanoma phenotype.

Cellular stress contributes to the capacity of melanoma cells to undergo phenotype switching into highly migratory and drug-tolerant dedifferentiated states. Such dedifferentiated melanoma cell states are marked by loss of melanocyte-specific gene expression and increase of mesenchymal markers. Two crucial transcription factors, microphthalmia-associated transcription factor (MITF) and SRY-box transcription factor 10 (SOX10), important in melanoma development and progression, have been implicated in this process. In this study we describe that loss of MITF is associated with a distinct transcriptional program, MITF promoter hypermethylation, and poor patient survival in metastatic melanoma. From a comprehensive collection of melanoma cell lines, we observed that MITF-methylated cultures were subdivided in 2 distinct subtypes. Examining mRNA levels of neural crest-associated genes, we found that 1 subtype had lost the expression of several lineage genes, including SOX10. Intriguingly, SOX10 loss was associated with SOX10 gene promoter hypermethylation and distinct phenotypic and metastatic properties. Depletion of SOX10 in MITF-methylated melanoma cells using CRISPR/Cas9 supported these findings. In conclusion, this study describes the significance of melanoma state and the underlying functional properties explaining the aggressiveness of such states.

Author Correction: Tertiary lymphoid structures improve immunotherapy and survival in melanoma.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Role of PTEN Loss in Immune Escape, Melanoma Prognosis and Therapy Response.

Checkpoint blockade therapies have changed the clinical management of metastatic melanoma patients considerably, showing survival benefits. Despite the clinical success, not all patients respond to treatment or they develop resistance. Although there are several treatment predictive biomarkers, understanding therapy resistance and the mechanisms of tumor immune evasion is crucial to increase the frequency of patients benefiting from treatment. The PTEN gene is thought to promote immune evasion and is frequently mutated in cancer and melanoma. Another feature of melanoma tumors that may affect the capacity of escaping T-cell recognition is melanoma cell dedifferentiation characterized by decreased expression of the microphtalmia-associated transcription factor (MITF) gene. In this study, we have explored the role of PTEN in prognosis, therapy response, and immune escape in the context of MITF expression using immunostaining and genomic data from a large cohort of metastatic melanoma. We confirmed in our cohort that PTEN alterations promote immune evasion highlighted by decreased frequency of T-cell infiltration in such tumors, resulting in a worse patient survival. More importantly, our results suggest that dedifferentiated PTEN negative melanoma tumors have poor patient outcome, no T-cell infiltration, and transcriptional properties rendering them resistant to targeted- and immuno-therapy.

Analysis of DNA methylation patterns in the tumor immune microenvironment of metastatic melanoma.

The presence of immune cells in the tumor microenvironment has been associated with response to immunotherapies across several cancer types, including melanoma. Despite its therapeutic relevance, characterization of the melanoma immune microenvironments remains insufficiently explored. To distinguish the immune microenvironment in a cohort of 180 metastatic melanoma clinical specimens, we developed a method using promoter CpG methylation of immune cell type-specific genes extracted from genome-wide methylation arrays. Unsupervised clustering identified three immune methylation clusters with varying levels of immune CpG methylation that are related to patient survival. Matching protein and gene expression data further corroborated the identified epigenetic characterization. Exploration of the possible immune exclusion mechanisms at play revealed likely dependency on MITF protein level and PTEN loss-of-function events for melanomas unresponsive to immunotherapies (immune-low). To understand whether melanoma tumors resemble other solid tumors in terms of immune methylation characteristics, we explored 15 different solid tumor cohorts from TCGA. Low-dimensional projection based on immune cell type-specific methylation revealed grouping of the solid tumors in line with melanoma immune methylation clusters rather than tumor types. Association of survival outcome with immune cell type-specific methylation differed across tumor and cell types. However, in melanomas immune cell type-specific methylation was associated with inferior patient survival. Exploration of the immune methylation patterns in a pan-cancer context suggested that specific immune microenvironments might occur across the cancer spectrum. Together, our findings underscore the existence of diverse immune microenvironments, which may be informative for future immunotherapeutic applications.

Tertiary lymphoid structures improve immunotherapy and survival in melanoma.

Checkpoint blockade therapies that reactivate tumour-associated T cells can induce durable tumour control and result in the long-term survival of patients with advanced cancers1. Current predictive biomarkers for therapy response include high levels of intratumour immunological activity, a high tumour mutational burden and specific characteristics of the gut microbiota2,3. Although the role of T cells in antitumour responses has thoroughly been studied, other immune cells remain insufficiently explored. Here we use clinical samples of metastatic melanomas to investigate the role of B cells in antitumour responses, and find that the co-occurrence of tumour-associated CD8+ T cells and CD20+ B cells is associated with improved survival, independently of other clinical variables. Immunofluorescence staining of CXCR5 and CXCL13 in combination with CD20 reveals the formation of tertiary lymphoid structures in these CD8+CD20+ tumours. We derived a gene signature associated with tertiary lymphoid structures, which predicted clinical outcomes in cohorts of patients treated with immune checkpoint blockade. Furthermore, B-cell-rich tumours were accompanied by increased levels of TCF7+ naive and/or memory T cells. This was corroborated by digital spatial-profiling data, in which T cells in tumours without tertiary lymphoid structures had a dysfunctional molecular phenotype. Our results indicate that tertiary lymphoid structures have a key role in the immune microenvironment in melanoma, by conferring distinct T cell phenotypes. Therapeutic strategies to induce the formation of tertiary lymphoid structures should be explored to improve responses to cancer immunotherapy.

NRARP displays either pro- or anti-tumoral roles in T-cell acute lymphoblastic leukemia depending on Notch and Wnt signaling.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a dismal prognosis in patients with resistant or relapsed disease. Although NOTCH is a known driver in T-ALL, its clinical inhibition has significant limitations. Our previous studies suggested that NRARP, a negative regulator of Notch signaling, could have a suppressive role in T-ALL. Here, we report that NRARP levels are significantly increased in primary T-ALL cells suggesting that NRARP is not sufficient to block NOTCH oncogenic signals. Interestingly, although NRARP overexpression blocks NOTCH1 signaling and delays the proliferation of T-ALL cells that display high levels of Notch1 signaling, it promotes the expansion of T-ALL cells with lower levels of Notch1 activity. We found that NRARP interacts with lymphoid enhancer-binding factor 1 (LEF1) and potentiates Wnt signaling in T-ALL cells with low levels of Notch. Together these results indicate that NRARP plays a dual role in T-ALL pathogenesis, regulating both Notch and Wnt pathways, with opposite functional effects depending on Notch activity. Consistent with this hypothesis, mice transplanted with T-cells co-expressing NOTCH1 and NRARP develop leukemia later than mice transplanted with T-NOTCH1 cells. Importantly, mice transplanted with T-cells overexpressing NRARP alone developed leukemia with similar kinetics to those transplanted with T-NOTCH1 cells. Our findings uncover a role for NRARP in T-ALL pathogenesis and indicate that Notch inhibition may be detrimental for patients with low levels of Notch signaling, which would likely benefit from the use of Wnt signaling inhibitors. Importantly, our findings may extend to other cancers where Notch and Wnt play a role.

The X-Linked DDX3X RNA Helicase Dictates Translation Reprogramming and Metastasis in Melanoma.

The X-linked DDX3X gene encodes an ATP-dependent DEAD-box RNA helicase frequently altered in various human cancers, including melanomas. Despite its important roles in translation and splicing, how DDX3X dysfunction specifically rewires gene expression in melanoma remains completely unknown. Here, we uncover a DDX3X-driven post-transcriptional program that dictates melanoma phenotype and poor disease prognosis. Through an unbiased analysis of translating ribosomes, we identified the microphthalmia-associated transcription factor, MITF, as a key DDX3X translational target that directs a proliferative-to-metastatic phenotypic switch in melanoma cells. Mechanistically, DDX3X controls MITF mRNA translation via an internal ribosome entry site (IRES) embedded within the 5' UTR. Through this exquisite translation-based regulatory mechanism, DDX3X steers MITF protein levels dictating melanoma metastatic potential in vivo and response to targeted therapy. Together, these findings unravel a post-transcriptional layer of gene regulation that may provide a unique therapeutic vulnerability in aggressive male melanomas.