Long non-coding RNA GRASLND links melanoma differentiation and interferon-gamma response.

Melanoma is a highly malignant tumor, that stands as the most lethal form of skin cancer and is characterized by notable phenotypic plasticity and intratumoral heterogeneity. Melanoma plasticity is involved in tumor growth, metastasis and therapy resistance. Long non-coding RNAs (lncRNAs) could influence plasticity due to their regulatory function. However, their role and mode of action are poorly studied. Here, we show a relevance of lncRNA GRASLND in melanoma differentiation and IFNγ signaling. GRASLND knockdown revealed switching of differentiated, melanocytic melanoma cells towards a dedifferentiated, slow-proliferating and highly-invasive cell state. Interestingly, GRASLND is overexpressed in differentiated melanomas and associated with poor prognosis. Accordingly, we found GRASLND expressed in immunological "cold" tumors and it negatively correlates with gene signatures of immune response activation. In line, silencing of GRASLND under IFNγ enhanced the expression of IFNγ-stimulated genes, including HLA-I antigen presentation, demonstrating suppressive activity of GRASLND on IFNγ signaling. Our findings demonstrate that in differentiated melanomas elevated expression of GRASLND interferes with anti-tumor effects of IFNγ, suggesting a role of GRASLND in tumor immune evasion.

HLA Class II Loss and JAK1/2 Deficiency Coevolve in Melanoma Leading to CD4 T-cell and IFNγ Cross-Resistance.

PURPOSE: Recent studies have demonstrated HLA class II (HLA-II)-dependent killing of melanoma cells by cytotoxic CD4 T cells. We investigated evolution of HLA-II-loss tumors that escape cytotoxic CD4 T-cell activity and contribute to immunotherapy resistance. EXPERIMENTAL DESIGN: Melanoma cells from longitudinal metastases were studied for constitutive and IFN-inducible HLA-II expression, sensitivity towards autologous CD4 T cells, and immune evasion by HLA-II loss. Clinical significance of HLA-II-low tumors was determined by analysis of transcriptomic data sets from patients with immune checkpoint blockade (ICB). RESULTS: Analysis of longitudinal samples revealed strong intermetastatic heterogeneity in melanoma cell-intrinsic HLA-II expression and subclonal HLA-II loss. Tumor cells from early lesions either constitutively expressed HLA-II, sensitizing to cytotoxic CD4 T cells, or induced HLA-II and gained CD4 T-cell sensitivity in the presence of IFNγ. In contrast, late outgrowing subclones displayed a stable CD4 T-cell-resistant HLA-II-loss phenotype. These cells lacked not only constitutive but also IFNγ-inducible HLA-II due to JAK1/2-STAT1 pathway inactivation. Coevolution of JAK1/2 deficiency and HLA-II loss established melanoma cross-resistance to IFNγ and CD4 T cells, as detected in distinct stage IV metastases. In line with their immune-evasive phenotype, HLA-II-low melanomas showed reduced CD4 T-cell infiltrates and correlated with disease progression under ICB. CONCLUSIONS: Our study links melanoma resistance to CD4 T cells, IFNγ, and ICB at the level of HLA-II, highlighting the significance of tumor cell-intrinsic HLA-II antigen presentation in disease control and calling for strategies to overcome its downregulation for improvement of patient outcome.

Innate immune receptor signaling induces transient melanoma dedifferentiation while preserving immunogenicity.

BACKGROUND: Immune-stimulatory agents, like agonists of the innate immune receptor RIG-I, are currently tested in clinical trials as an intratumoral treatment option for patients with unresectable melanoma, aiming to enhance anti-tumor T cell responses. Switching of melanoma toward a dedifferentiated cell state has recently been linked to T cell and therapy resistance. It remains to be determined whether RIG-I agonists affect melanoma differentiation, potentially leading to T cell resistance. METHODS: Patient metastases-derived melanoma cell lines were treated with the synthetic RIG-I agonist 3pRNA, and effects on tumor cell survival, phenotype and differentiation were determined. Transcriptomic data sets from cell lines and metastases were analyzed for associations between RIG-I (DDX58) and melanoma differentiation markers and used to define signaling pathways involved in RIG-I-driven dedifferentiation. The impact of 3pRNA-induced melanoma dedifferentiation on CD8 T cell activation was studied in autologous tumor T cell models. RESULTS: RIG-I activation by 3pRNA induced apoptosis in a subpopulation of melanoma cells, while the majority of tumor cells switched into a non-proliferative cell state. Those persisters displayed a dedifferentiated cell phenotype, marked by downregulation of the melanocytic lineage transcription factor MITF and its target genes, including melanoma differentiation antigens (MDA). Transition into the MITFlow/MDAlow cell state was JAK-dependent, with some cells acquiring nerve growth factor receptor expression. MITFlow/MDAlow persisters switched back to the proliferative differentiated cell state when RIG-I signaling declined. Consistent with our in vitro findings, an association between melanoma dedifferentiation and high RIG-I (DDX58) levels was detected in transcriptomic data from patient metastases. Notably, despite their dedifferentiated cell phenotype, 3pRNA-induced MITFlow/MDAlow persisters were still efficiently targeted by autologous CD8 tumor-infiltrating T lymphocytes (TILs). CONCLUSIONS: Our results demonstrate that RIG-I signaling in melanoma cells drives a transient phenotypic switch toward a non-proliferative dedifferentiated persister cell state. Despite their dedifferentiation, those persisters are highly immunogenic and sensitive toward autologous TILs, challenging the concept of melanoma dedifferentiation as a general indicator of T cell resistance. In sum, our findings support the application of RIG-I agonists as a therapeutic tool for the generation of long-term clinical benefit in non-resectable melanoma.

Melanoma Differentiation Trajectories Determine Sensitivity toward Pre-Existing CD8+ Tumor-Infiltrating Lymphocytes.

The highly plastic nature of melanoma enables its transition among diverse cell states to survive hostile conditions. However, the interplay between specific tumor cell states and intratumoral T cells remains poorly defined. With MAPK inhibitor‒treated BRAFV600-mutant tumors as models, we linked human melanoma state transition to CD8+ T cell responses. Repeatedly, we observed that isogenic melanoma cells could evolve along distinct differentiation trajectories on single BRAF inhibitor (BRAFi) treatment or dual BRAFi/MEKi treatment, resulting in BRAFi‒induced hyperdifferentiated and BRAFi/MEKi‒induced dedifferentiated resistant subtypes. Taking advantage of patient-derived autologous CD8+ tumor-infiltrating lymphocytes (TILs), we demonstrate that progressive melanoma cell state transition profoundly affects TIL function. Tumor cells along the hyperdifferentiation trajectory continuously gained sensitivity toward tumor-reactive CD8+ TILs, whereas those in the dedifferentiation trajectory acquired T cell resistance in part owing to the loss of differentiation antigens. Overall, our data reveal the tight connection of MAPKi‒induced temporary (drug-tolerant transition state) and stable (resistant state) phenotype alterations with T cell function and further broaden the current knowledge on melanoma plasticity in terms of sculpting local antitumor immune responses, with implications for guiding the optimal combination of targeted therapy and immunotherapy.

Innate RIG-I signaling restores antigen presentation in tumors and overcomes T cell resistance.

In recent years, therapy with immune modulating antibodies, termed immune checkpoint blockade (ICB), has revolutionized the treatment of advanced metastatic melanoma, yielding long-lasting clinical responses in a subgroup of patients. But despite this remarkable progress, resistance to therapy represents a major clinical challenge. ICB efficacy is critically dependent on cytotoxic CD8+ T cells targeting tumor cells in an HLA class I (HLA-I) antigen-dependent manner. Transcriptional suppression of the HLA-I antigen processing and presentation machinery (HLA-I APM) in melanoma cells leads to HLA-I-low/-negative tumor cell phenotypes escaping CD8+ T cell recognition and contributing to ICB resistance. In general, HLA-I-low/-negative tumor cells can be re-sensitized to T cells by interferons (IFN), augmenting HLA-I APM expression. However, this mechanism fails when melanoma cells acquire resistance to IFN, which recently turned out as a key resistance mechanism in ICB, besides HLA-I APM suppression. Seeking for a strategy to overcome these barriers, we identified a novel mechanism that restores HLA-I antigen presentation in tumor cells independent of IFN (Such et al. (2020) J Clin Invest, doi: 10.1172/JCI131572). We demonstrated that tumor cell-intrinsic activation of the cytosolic innate immunoreceptor RIG-I by its synthetic ligand 3pRNA overcomes transcriptional HLA-I APM suppression in patient-derived IFN-resistant melanoma cells. De novo HLA-I APM expression is IRF1/IRF3-dependent and re-sensitizes melanoma cells to autologous cytotoxic CD8+ T cells. Notably, synthetic RIG-I ligands and ICB synergize in T cell activation, suggesting combinational therapy could be an efficient strategy to improve patient outcomes in melanoma.

Arenavirus Induced CCL5 Expression Causes NK Cell-Mediated Melanoma Regression.

Immune activation within the tumor microenvironment is one promising approach to induce tumor regression. Certain viruses including oncolytic viruses such as the herpes simplex virus (HSV) and non-oncolytic viruses such as the lymphocytic choriomeningitis virus (LCMV) are potent tools to induce tumor-specific immune activation. However, not all tumor types respond to viro- and/or immunotherapy and mechanisms accounting for such differences remain to be defined. In our current investigation, we used the non-cytopathic LCMV in different human melanoma models and found that melanoma cell lines produced high levels of CCL5 in response to immunotherapy. In vivo, robust CCL5 production in LCMV infected Ma-Mel-86a tumor bearing mice led to recruitment of NK cells and fast tumor regression. Lack of NK cells or CCL5 abolished the anti-tumoral effects of immunotherapy. In conclusion, we identified CCL5 and NK cell-mediated cytotoxicity as new factors influencing melanoma regression during virotherapy.

Targeting the innate immunoreceptor RIG-I overcomes melanoma-intrinsic resistance to T cell immunotherapy.

Understanding tumor resistance to T cell immunotherapies is critical to improve patient outcomes. Our study revealed a role for transcriptional suppression of the tumor-intrinsic HLA class I (HLA-I) antigen processing and presentation machinery (APM) in therapy resistance. Low HLA-I APM mRNA levels in melanoma metastases before immune checkpoint blockade (ICB) correlated with nonresponsiveness to therapy and poor clinical outcome. Patient-derived melanoma cells with silenced HLA-I APM escaped recognition by autologous CD8+ T cells. However, targeted activation of the innate immunoreceptor RIG-I initiated de novo HLA-I APM transcription, thereby overcoming T cell resistance. Antigen presentation was restored in interferon-sensitive (IFN-sensitive) but also immunoedited IFN-resistant melanoma models through RIG-I-dependent stimulation of an IFN-independent salvage pathway involving IRF1 and IRF3. Likewise, enhanced HLA-I APM expression was detected in RIG-Ihi (DDX58hi) melanoma biopsies, correlating with improved patient survival. Induction of HLA-I APM by RIG-I synergized with antibodies blocking PD-1 and TIGIT inhibitory checkpoints in boosting the antitumor T cell activity of ICB nonresponders. Overall, the herein-identified IFN-independent effect of RIG-I on tumor antigen presentation and T cell recognition proposes innate immunoreceptor targeting as a strategy to overcome intrinsic T cell resistance of IFN-sensitive and IFN-resistant melanomas and improve clinical outcomes in immunotherapy.