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Abstract Background The weight that gene copy number plays in transcription remains controversial; although in specific cases gene expression correlates with copy number, the relationship cannot be inferred at the global level. We hypothesized that genes steadily expressed by 15 melanoma cell lines (CMs) and their parental tissues (TMs) should be critical for oncogenesis and their expression most frequently influenced by their respective copy number. Results Functional interpretation of 3,030 transcripts concordantly expressed (Pearson's correlation coefficient p-value < 0.05) by CMs and TMs confirmed an enrichment of functions crucial to oncogenesis. Among them, 968 were expressed according to the transcriptional efficiency predicted by copy number analysis (Pearson's correlation coefficient p-value < 0.05). We named these genes, "genomic delegates" as they represent at the transcriptional level the genetic footprint of individual cancers. We then tested whether the genes could categorize 112 melanoma metastases. Two divergent phenotypes were observed: one with prevalent expression of cancer testis antigens, enhanced cyclin activity, WNT signaling, and a Th17 immune phenotype (Class A). This phenotype expressed, therefore, transcripts previously associated to more aggressive cancer. The second class (B) prevalently expressed genes associated with melanoma signaling including MITF, melanoma differentiation antigens, and displayed a Th1 immune phenotype associated with better prognosis and likelihood to respond to immunotherapy. An intermediate third class (C) was further identified. The three phenotypes were confirmed by unsupervised principal component analysis. Conclusions This study suggests that clinically relevant phenotypes of melanoma can be retraced to stable oncogenic properties of cancer cells linked to their genetic back bone, and offers a roadmap for uncovering novel targets for tailored anti-cancer therapy.

Adoptive T cell therapy has shown clinical potential for patients with cancer, though effective treatment is dependent on longevity and potency of the exploited tumor-reactive T cells. Previously, we showed that ex vivo inhibition of AKT using the research compound Akt-inhibitor VIII retained differentiation and improved functionality of minor histocompatibility antigen (MiHA)-specific CD8+ T cells. Here, we compared a panel of clinically applicable AKT-inhibitors with an allosteric or adenosine triphosphate-competitive mode of action. We analyzed phenotype, functionality, metabolism and transcriptome of AKT-inhibited CD8+ T cells using different T cell activation models. Most inhibitors facilitated T cell expansion while preserving an early memory phenotype, reflected by maintenance of CD62L, CCR7 and CXCR4 expression. Moreover, transcriptome profiling revealed that AKT-inhibited CD8+ T cells clustered closely to naturally occurring stem cell-memory CD8+ T cells, while control T cells resembled effector-memory T cells. Interestingly, AKT-inhibited CD8+ T cells showed enrichment of hypoxia-associated genes, which was consistent with enhanced glycolytic function. Notably, AKT-inhibition during MiHA-specific CD8+ T cell priming uncoupled preservation of early memory differentiation from ex vivo expansion. Furthermore, AKT-inhibited MiHA-specific CD8+ T cells showed increased polyfunctionality with co-secretion of IFN-γ and IL-2 upon antigen recall. Together, these data demonstrate that AKT-inhibitors with different modality of action promote the ex vivo generation of stem cell memory-like CD8+ T cells with a unique metabolic profile and retained polyfunctionality. Akt-inhibitor VIII and GDC-0068 outperformed other inhibitors, and are therefore promising candidates for ex vivo generation of superior tumor-reactive T cells for adoptive immunotherapy in cancer patients. [ABSTRACT FROM AUTHOR]

Background In the recent years there have been major advances in the field of cancer immunology and the existence of a dichotomy between immunologically active and quiescent tumor phenotypes has been recognized in several cancers. The activation of a Th1 immune signature has been shown to confer better prognosis and likelihood to respond to immunotherapy. However, whether such dichotomy depends on the genetic make-up of individual cancers is not known yet. In melanoma, BRAF and NRAS mutations are commonly acquired during tumor progression. Although the oncogenic potential of BRAF and NRAS alterations has been attributed to reduced apoptosis, increased invasiveness and increased metastatic behavior, the role of BRAF and NRAS in the immunological landscape of cutaneous melanoma has been poorly investigated and the effects of BRAF and NRAS mutations on global gene expression remain to be understood. We explored the role of BRAF and NRAS mutations at the transcriptome level and in influencing the immune phenotype (based on a classification previously identified by our group). Materials and methods One-hundred-thirteen pre-treatment snap frozen tumor biopsies were collected from patients treated at the Surgery Branch, NCI (Bethesda, Maryland) and processed for DNA and RNA isolation. Each sample underwent microarray analysis and BRAF and NRAS genotyping. Allele-specific PCR was also performed in order to exclude low-frequency mutations. Fifteen melanoma cell lines were also tested for BRAF and NRAS mutation by Sanger sequencing and RNA-sequencing. Results Comparison between BRAF and NRAS mutant versus wild type samples identified mostly constituents or regulators of MAPK and related pathways. Initially, we postulated that there might be a common MAPK activation signature resulting from either BRAF or NRAS mutation; however, we found no overabundance of discriminatory genes for the combined group of samples displaying either BRAF or NRAS mutations. This suggests that the transcriptional consequences resulting from mutations of BRAF or NRAS might be different, although there was overlapping of some genes, presumably due to their differential capacity to receive input signals and transduce them through different effectors. When testing gene lists discriminative of BRAF, NRAS and MAPK alterations, we found that 112 BRAF-specific transcripts were able to distinguish the two immunerelated phenotypes already described in melanoma, with the poor phenotype associated mostly with BRAF mutation. Noteworthy, such association was stronger in samples displaying low BRAF mRNA expression. However, when testing NRAS mutation, we were not able to find the same association. Class comparison between BRAF mutant samples with high and low expression of the same gene identified 6296 transcripts. Functional interpretation analysis showed that these 6296 transcripts were associated to IL-2 and JAK/Stat signaling pathways, supporting the immunoregulatory role of BRAF. Additionally, fifteen melanoma cell lines were also tested by BRAF and NRAS DNA genotyping and RNA-sequencing. Interestingly, we found that among 8 cell lines BRAF mutated (V600E), three of them expressed BRAF at low level and may have preferential wild type allele selection at the RNA level. Conclusion In conclusion we provide novel insights into the effect of BRAF and NRAS mutations on gene expression according to the immune classification. However, further deeper analyses are warranted to understand the mechanisms behind the association of BRAF mutation with a poor immune phenotype and also behind BRAF low expression and wild type allele selection at the RNA level. [ABSTRACT FROM AUTHOR]