Identification of a miRNA-based non-invasive predictive biomarker of response to target therapy in BRAF-mutant melanoma.

Rationale: Metastatic melanoma is the most aggressive and dangerous form of skin cancer. The introduction of immunotherapy with Immune checkpoint Inhibitors (ICI) and of targeted therapy with BRAF and MEK inhibitors for BRAF mutated melanoma, has greatly improved the clinical outcome of these patients. Nevertheless, response to therapy remains highly variable and the development of drug resistance continues to be a daunting challenge. Within this context there is a need to develop diagnostic tools capable of predicting response or resistance to therapy in order to select the best therapeutic approach. Over the years, accumulating evidence brought to light the role of microRNAs (miRNAs) as disease biomarkers. Methods: In particular, the detection of miRNAs in whole blood or specific blood components such as serum or plasma, allows these molecules to be good candidates for diagnosis, prognosis and for monitoring response to anticancer therapy. In this paper, we evaluated circulating basal levels of 6 previously identified miRNAs in serum samples of 70 BRAF-mutant melanoma patients before starting targeted therapy. Results: Results show that the circulating levels of the oncosuppressor miR-579-3p and of the oncomiR miR-4488 are able to predict progression free survival (PFS) but not overall survival (OS). Most importantly, we observed that the best predictor of disease outcome is represented by the ratio of circulating miR-4488 vs. miR-579-3p (miRatio). Finally, the combination of the Lactate dehydrogenase (LDH) blood levels with the two circulating miRNAs alone or together did not produce any improvement in predicting PFS indicating that miR-579-3p and miR-4488 are independent predictors of PFS as compared to LDH. Conclusions: All together these data underscored the relevance of circulating miRNAs as suitable tools to predict therapy response in melanoma and maybe further developed as companion diagnostics in the clinic.

The Promise of Liquid Biopsy to Predict Response to Immunotherapy in Metastatic Melanoma.

Metastatic melanoma is the deadliest form of skin cancer whose incidence has been rising dramatically over the last few decades. Nowadays, the most successful approach in treating advanced melanoma is immunotherapy which encompasses the use of immune checkpoint blockers able to unleash the immune system's activity against tumor cells. Immunotherapy has dramatically changed clinical practice by contributing to increasing long term overall survival. Despite these striking therapeutic effects, the clinical benefits are strongly mitigated by innate or acquired resistance. In this context, it is of utmost importance to develop methods capable of predicting patient response to immunotherapy. To this purpose, one major step forward may be provided by measuring non-invasive biomarkers in human fluids, namely Liquid Biopsies (LBs). Several LB approaches have been developed over the last few years thanks to technological breakthroughs that have allowed to evaluate circulating components also when they are present in low abundance. The elements of this so-called "circulome" mostly encompass: tumor DNA, tumor and immune cells, soluble factors and non-coding RNAs. Here, we review the current knowledge of these molecules as predictors of response to immunotherapy in metastatic melanoma and predict that LB will soon enter into routine practice in order to guide clinical decisions for cancer immunotherapy.

In Vitro Biophysical and Biological Characterization of Lipid Nanoparticles Co-Encapsulating Oncosuppressors miR-199b-5p and miR-204-5p as Potentiators of Target Therapy in Metastatic Melanoma.

Uncontrolled MAPK signaling is the main oncogenic driver in metastatic melanomas bearing mutations in BRAF kinase. These tumors are currently treated with the combination of BRAF/MEK inhibitors (MAPKi), but this therapy is plagued by drug resistance. In this context we recently discovered that several microRNAs are involved in the development of drug resistance. In particular miR-204-5p and miR-199b-5p were found to function as antagonists of resistance because their enforced overexpression is able to inhibit melanoma cell growth in vitro either alone or in combination with MAPKi. However, the use of miRNAs in therapy is hampered by their rapid degradation in serum and biological fluids, as well as by the poor intracellular uptake. Here, we developed lipid nanoparticles (LNPs) encapsulating miR-204-5p, miR-199b-5p individually or in combination. We obtained LNPs with mean diameters < 200 nm and high miRNA encapsulation efficiency. These formulations were tested in vitro on several melanoma cell lines sensitive to MAPKi or rendered drug resistant. Our results show that LNPs encapsulating combinations of the two oncosuppressor miRNAs are highly efficient in impairing melanoma cell proliferation and viability, affect key signaling pathways involved in melanoma cell survival, and potentiate the efficacy of drugs inhibiting BRAF and MEK. These results warrant further assessment of the anti-tumor efficacy of oncosuppressor miRNAs encapsulating LNPs in in vivo tumor models.

Single cell analysis to dissect molecular heterogeneity and disease evolution in metastatic melanoma.

Originally described as interpatient variability, tumour heterogeneity has now been demonstrated to occur intrapatiently, within the same lesion, or in different lesions of the same patient. Tumour heterogeneity involves both genetic and epigenetic changes. Intrapatient heterogeneity is responsible for generating subpopulations of cancer cells which undergo clonal evolution with time. Tumour heterogeneity develops also as a consequence of the selective pressure imposed by the immune system. It has been demonstrated that tumour heterogeneity and different spatiotemporal interactions between all the cellular compontents within the tumour microenvironment lead to cancer adaptation and to therapeutic pressure. In this context, the recent advent of single cell analysis approaches which are able to better study tumour heterogeneity from the genomic, transcriptomic and proteomic standpoint represent a major technological breakthrough. In this review, using metastatic melanoma as a prototypical example, we will focus on applying single cell analyses to the study of clonal trajectories which guide the evolution of drug resistance to targeted therapy.

ErbB3 Phosphorylation as Central Event in Adaptive Resistance to Targeted Therapy in Metastatic Melanoma: Early Detection in CTCs during Therapy and Insights into Regulation by Autocrine Neuregulin.

In recent years the introduction of target therapies with BRAF and MEK inhibitors (MAPKi) and of immunotherapy with anti-CTLA-4 and anti-PD-1 monoclonal antibodies have dramatically improved survival of metastatic melanoma patients. Despite these changes drug resistance remains a major hurdle. Several mechanisms are at the basis of drug resistance. Particular attention has been devoted over the last years to unravel mechanisms at the basis of adaptive/non genetic resistance occurring in BRAF mutated melanomas upon treatment with to MAPKi. In this paper we focus on the involvement of activation of ErbB3 receptor following early exposure of melanoma cells to BRAF or MEK inhibitors, and the following induction of PI3K/AKT pathway. Although different mechanisms have been invoked in the past at the basis of this activation we show here with a combination of approaches that autocrine production of neuregulin by melanoma cells is a major factor responsible for ErbB3 phosphorylation and downstream AKT activation. Interestingly the kinetic of neuregulin production and of the ensuing ErbB3 phosphorylation is different in different melanoma cell lines which underscores the high degree of tumor heterogeneity. Moreover, heterogeneity is further highlighted by the evidence that in different cell lines neuregulin upregulation can occur at the transcriptional or at the post-transcritpional level. Finally we complement our study by showing with a liquid biopsy assay that circulating tumor cells (CTCs) from melanoma patients undergo upregulation of ErbB3 phosphorylation in vivo shortly after initiation of therapy.

Reprogramming miRNAs global expression orchestrates development of drug resistance in BRAF mutated melanoma.

Drug resistance imposes severe limitations to the efficacy of targeted therapy in BRAF-mutated metastatic melanoma. Although this issue has been mitigated by the development of combination therapies with BRAF plus MEK inhibitors, drug resistance inevitably occurs with time and results in clinical recurrences and untreatable disease. Hence, there is strong need of developing new combination therapies and non-invasive diagnostics for the early identification of drug-resistant patients. We report here that the development of drug resistance to BRAFi is dominated by a dynamic deregulation of a large population of miRNAs, leading to the alteration of cell intrinsic proliferation and survival pathways, as well as of proinflammatory and proangiogenic cues, where a prominent role is played by the miR-199b-5p/VEGF axis. Significant alterations of miRNA expression levels are detectable in tumor biopsies and plasma from patients after disease recurrence. Targeting these alterations blunts the development of drug resistance.

MicroRNAs in melanoma development and resistance to target therapy.

microRNAs constitute a complex class of pleiotropic post-transcriptional regulators of gene expression involved in the control of several physiologic and pathologic processes. Their mechanism of action is primarily based on the imperfect matching of a seed region located at the 5' end of a 21-23 nt sequence with a partially complementary sequence located in the 3' untranslated region of target mRNAs. This leads to inhibition of mRNA translation and eventually to its degradation. Individual miRNAs are capable of binding to several mRNAs and several miRNAs are capable of influencing the function of the same mRNAs. In recent years networks of miRNAs are emerging as capable of controlling key signaling pathways responsible for the growth and propagation of cancer cells. Furthermore several examples have been provided which highlight the involvement of miRNAs in the development of resistance to targeted drug therapies. In this review we provide an updated overview of the role of miRNAs in the development of melanoma and the identification of the main downstream pathways controlled by these miRNAs. Furthermore we discuss a group of miRNAs capable to influence through their respective up- or down-modulation the development of resistance to BRAF and MEK inhibitors.