Genomic and non-genomic alterations in melanoma contributing to disease progression and therapy resistance

Abstract

Melanoma is an aggressive cancer that arises from transformed melanocytes of the skin and the ocular region, the uvea or conjunctiva. Two main therapies are currently applied to patients with cutaneous melanoma, small molecule inhibitors blocking dysregulated oncogenic pathways (in case of BRAFV600E mutant melanoma), and antibody-based immunotherapy, boosting cytotoxic T cell responses against cancer cells. So far, none of these therapies has been approved for conjunctival melanoma. Given the rarity of this disease, it has been difficult in the past to thoroughly study the molecular mechanisms and genetic alterations driving this type of melanoma. By analyzing a large cohort of 67 tumor samples, we aimed to elucidate recurrent genetic alterations in conjunctival melanoma. Targeted next-generation sequencing was performed covering genes recurrently mutated in cutaneous and uveal melanoma. This led to the identification of NF1 mutations in conjunctival melanoma, frequently detected in tumors lacking BRAF and RAS mutations. Based on these results, we proposed the classification of conjunctival melanoma into BRAF-mutated (25%), RAS-mutated (19%), NF1-mutated (33%) and triple-WT tumors, similar to cutaneous melanoma (paper Scholz et al., British J Cancer, 2018, in this thesis). Overall, our study provides insights into the mutational make-up of conjunctival melanoma and reveals the genetic similarities between cutaneous and conjunctival melanomas. Given the therapeutic success of inhibitor therapy and immunotherapy in skin melanoma, we propose both therapies can be considered as a valid option for patients suffering from conjunctival melanoma. As indicated above, T cell-based immunotherapy of cutaneous melanoma has achieved great success over the past few years. But approximately half of the treated patients are intrinsically resistant or acquire resistance to therapy, indicating the urgent need to elucidate resistance mechanisms. The T cell-derived cytokine IFNγ plays a fundamental role in tumor cell killing under immunotherapy. Inactivation of the kinase JAK2, an IFNγ signaling pathway component, is known to contribute to immunotherapy resistance in tumor cells. In Horn, Leonardelli et al., we provide insight into the evolution of JAK2 deficiency. The JAK2 locus maps closely to the CDKN2A locus, encoding the tumor suppressor p16 on chromosome 9p. Allelic CDKN2A loss is an important step in melanoma development and caused by large deletions on chromosome 9p. Analyzing SNP array data of 46 melanoma cell lines and copy number data of the TCGA melanoma cohort (n= 367), we found CDKN2A-associated JAK2 losses in the majority of samples analyzed (> 60%). Thus, allelic JAK2 loss occurs early in development of the disease, followed by inactivating gene mutations in single tumor clones finally establishing JAK2 deficiency (paper Horn, Leonardelli et al., JNCI, 2018, in this thesis). Our results suggest that screening of tumor lesions for genetic JAK2 alterations could be a valuable strategy to identify patients with an enhanced risk of developing immunotherapy resistance. While the contribution of genetic alterations to IFNγ resistance is now well accepted, the role of non-genetic mechanisms is poorly defined. We postulated that non- genomic mechanisms could protect melanoma cells from IFNγ-induced apoptosis thereby interfering with the efficacy of immunotherapy. To study this, we established IFNγ-resistant variants from IFNγ-sensitive parental tumor cells. The sensitive and resistant tumor cell pairs were analyzed for transcriptional signatures associated with resistance to cytokine-induced apoptosis and phenotypic alterations were confirmed at the protein level. This led to the identification of an EMT (epithelial to mesenchymal transition)-like phenotypic switch in cells with acquired resistance to IFNγ going along with the downregulation of IFNγ signaling pathway genes (paper Leonardelli et al., unpublished, in this thesis). Interestingly, the EMT signature has previously been reported to be associated with unresponsiveness to both treatment with targeted inhibitors and immune-modulating antibodies, suggesting a broader relevance of our findings for melanoma therapy. In summary, the different studies of this thesis provide further insight into the genetic and phenotypic heterogeneity of melanoma cells and its role in resistance to immunotherapy, which should guide therapeutic decisions to improve the clinical outcome of patients.