Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases1, while rare variants in CDK4, BRCA2, BAP1, and the promoter of TERT, have also been linked to the disease2-5. Here we set out to identify novel high-penetrance susceptibility genes in unexplained cases by sequencing 184 melanoma patients from 105 pedigrees recruited in the United Kingdom, the Netherlands, and Australia that were negative for variants in known predisposition genes. We identify families where melanoma co-segregates with loss-of-function variants in the protection of telomeres 1 (POT1) gene, a proportion of members presenting with an early age of onset and multiple primaries. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide-/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding, leading to increased telomere length. Thus, POT1 variants predispose to melanoma formation via a direct effect on telomeres.

L-Type amino acid transporters such as LAT1 and LAT3 mediate the uptake of essential amino acids. Here, we report that prostate cancer cells coordinate the expression of LAT1 and LAT3 to maintain sufficient levels of leucine needed for mTORC1 signaling and cell growth. Inhibiting LAT function was sufficient to decrease cell growth and mTORC1 signaling in prostate cancer cells. These cells maintained levels of amino acid influx through androgen receptor-mediated regulation of LAT3 expression and ATF4 regulation of LAT1 expression after amino acid deprivation. These responses remained intact in primary prostate cancer, as indicated by high levels of LAT3 in primary disease, and by increased levels of LAT1 after hormone ablation and in metastatic lesions. Taken together, our results show how prostate cancer cells respond to demands for increased essential amino acids by coordinately activating amino acid transporter pathways vital for tumor outgrowth. Cancer Res; 71(24); 7525-36. Ó2011 AACR.

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAF WT (C8161 and WM852) and BRAF V600E mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2-mediated glutamine transport is a potential therapeutic target for both BRAF WT and BRAF V600E melanoma.

Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

Monoclonal antibodies against immune checkpoint blockade have proven to be a major success in the treatment of melanoma. The programmed death receptor-1 ligand-1 (PD-L1) expression on melanoma cells is believed to have an inhibitory effect on T cell responses and to be an important escape mechanism from immune attack. Previous studies have shown that PD-L1 can be expressed constitutively or can be induced by IFN-γ secreted by infiltrating lymphocytes. In the present study we have investigated the mechanism underlying these two modes of PD-L1 expression in melanoma cells including cells that had acquired resistance to the BRAF inhibitor vemurafenib. PD-L1 expression was examined by flow cytometry and immunoblotting. Specific inhibitors and siRNA knockdown approaches were used to examine the roles of the RAF/ MEK, PI3K, NF-κB, STAT3 and AP1/ c-Jun pathways. IFN-γ inducible expression of PD-L1 was dependent on NF-κB as shown by inhibition with BMS-345541, an inhibitor of IκB and the BET protein inhibitor I-BET151, as well as by siRNA knockdown of NF-κB subunits. We were unable to implicate the BRAF/MEK pathway as major regulators in PD-L1 expression on vemurafenib resistant cells. Similarly the PI3K/AKT pathway and the transcription factors STAT3 and c-Jun had only minor roles in IFN-γ induced expression of PD-L1. The mechanism underlying constitutive expression remains unresolved. We suggest these results have significance in selection of treatments that can be used in combination with monoclonal antibodies against PD1, to enhance their effectiveness and to reduce inhibitory effects melanoma cells have against cytotoxic T cell activity.

Methylation of DNA at CpG sites is the most common and stable of epigenetic changes in cancer. Hypermethylation acts to limit immune checkpoint blockade immunotherapy by inhibiting endogenous interferon responses needed for recognition of cancer cells. By contrast, global hypomethylation results in the expression of programmed death ligand 1 (PD-L1) and inhibitory cytokines, accompanied by epithelial-mesenchymal changes that can contribute to immunosuppression. The drivers of these contrasting methylation states are not well understood. DNA methylation also plays a key role in cytotoxic T cell 'exhaustion' associated with tumor progression. We present an updated exploratory analysis of how DNA methylation may define patient subgroups and can be targeted to develop tailored treatment combinations to help improve patient outcomes. DNA Methylation, ICB, and Cancer DNA methylation can have major effects on gene expression and is the most commonly studied type of epigenetic modification (see Glossary). It comprises the covalent modification of the nucleotide cytosine at the 5ʹ position at sites preceding guanine (CpG) [1]. During mammalian cell division, it is replicated by the maintenance enzyme DNA methyltransferase 1 (DNMT1) on the daughter strand cytosine at the complementary CpG, usually resulting in gene silencing. New sites of DNA methylation, known as de novo methylation, can be introduced by DNMT3a or DNMT3b (Box 1). Conversely, methyl groups can be erased by ten-eleven translocation (TET) family proteins followed by glycosylation and replacement with an unmethylated cytosine. DNMT1 recruitment to replicating chromatin is facilitated by the 'ubiquitin-like with PHD and ring-finger domains' (UHRF) E3 ubiquitin ligase UHRF1 [2,3]. Methylation at CpG sites can also be recognized by methyl CpG binding 'reader' proteins such as methyl-CpG binding domain protein 1 (MBD1) and methyl-CpG binding protein 2 (MeCP2) harboring transcriptionally repressive activity. The latter may bind histone deacetylases (HDACs), which can contribute to the repression of certain genes [4]. Highlights Genome-wide DNA methylation is a relatively stable epigenetic characteristic of cells, which can be dysregulated in cancer cells by oncogenic signals.

A major challenge for cancer genetics is to determine which low frequency somatic mutations are drivers of tumorigenesis. Here we interrogate the genomes of 7,651 diverse human cancers to identify novel drivers and find inactivating mutations in the homeodomain transcription factor CUX1 (cut-like homeobox 1) in ~1-5% of tumors. Meta-analysis of CUX1 mutational status in 2,519 cases of myeloid malignancies reveals disruptive mutations associated with poor survival, highlighting the clinical significance of CUX1 loss. In parallel, we validate CUX1 as a bona fide tumor suppressor using mouse transposon-mediated insertional mutagenesis and Drosophila cancer models. We demonstrate that CUX1 deficiency activates phosphoinositide 3-kinase (PI3K) signaling through direct transcriptional downregulation of the PI3K inhibitor PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), leading to increased tumor growth, while exposing susceptibility to PI3K-AKT inhibition. Thus, our complementary approaches identify CUX1 as a new pan-driver of tumorigenesis and uncover a potential strategy for treating CUX1-mutant tumors.

BORIS and CTCF are paralogous, multivalent 11-zinc finger transcription factors that play important roles in organizing higherorder chromatin architecture. BORIS is a cancer-testis antigen with a poorly defined function in cancer, although it has been hypothesized to exhibit oncogenic properties. CTCF, however, has been postulated as a candidate tumor suppressor. We collated the genetic lesions in BORIS and CTCF from multiple cancers identified using high-throughput genomics. In BORIS, nonsense and missense mutations are evenly distributed. In CTCF, recurrent mutations are mostly clustered in the conserved zinc finger domain and at residues critical for contacting DNA and zinc ion co-ordination. Three missense mutations are common to both proteins. We used an inducible lentivector to express wildtype BORIS or CTCF in primary cells and cancer cell lines in order to define their functional differences. Both BORIS and CTCF caused a significant decrease in cell proliferation and clonogenic capacity, without alteration of specific cell cycle phases. Both BORIS and CTCF conferred protective effects in primary cells and some cancer cells during UV damage-induced apoptosis. Using a bioluminescent MCF-7 orthotopic breast cancer model in vivo, we demonstrated that CTCF and BORIS suppressed breast cancer growth. These findings provide further evidence that CTCF behaves as a tumor suppressor, and show BORIS has a similar growth inhibitory effect in vitro and in vivo. Hence, acquired zinc finger mutations may disrupt these functions, thereby contributing to tumor growth and development.