Jennifer Lo

An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background

People with pale skin, red hair, freckles and an inability to tan—the ‘red hair/fair skin’ phenotype—are at highest risk of developing melanoma, compared to all other pigmentation types. Genetically, this phenotype is frequently the product of inactivating polymorphisms in the melanocortin 1 receptor (MC1R) gene. MC1R encodes a cyclic AMP-stimulating G-protein-coupled receptor that controls pigment production. Minimal receptor activity, as in red hair/fair skin polymorphisms, produces the red/yellow pheomelanin pigment, whereas increasing MC1R activity stimulates the production of black/brown eumelanin. Pheomelanin has weak shielding capacity against ultraviolet radiation relative to eumelanin, and has been shown to amplify ultraviolet-A-induced reactive oxygen species. Several observations, however, complicate the assumption that melanoma risk is completely ultraviolet-radiation-dependent. For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and ultraviolet radiation signature mutations are infrequently oncogenic drivers. Although linkage of melanoma risk to ultraviolet radiation exposure is beyond doubt, ultraviolet-radiation-independent events are likely to have a significant role. Here we introduce a conditional, melanocyte-targeted allele of the most common melanoma oncoprotein, BRAFV600E, into mice carrying an inactivating mutation in the Mc1r gene (these mice have a phenotype analogous to red hair/fair skin humans). We observed a high incidence of invasive melanomas without providing additional gene aberrations or ultraviolet radiation exposure. To investigate the mechanism of ultraviolet-radiation-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1re/e background. Selective absence of pheomelanin synthesis was protective against melanoma development. In addition, normal Mc1re/e mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1re/e mouse skin. These data suggest that the pheomelanin pigment pathway produces ultraviolet-radiation-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage. Although protection from ultraviolet radiation remains important, additional strategies may be required for optimal melanoma prevention.

The melanoma revolution: from UV carcinogenesis to a new era in therapeutics.

Melanoma, the deadliest form of skin cancer, is an aggressive disease that is rising in incidence. Although melanoma is a historically treatment-resistant malignancy, in recent years unprecedented breakthroughs in targeted therapies and immunotherapies have revolutionized the standard of care for patients with advanced disease. Here, we provide an overview of recent developments in our understanding of melanoma risk factors, genomics, and molecular pathogenesis and how these insights have driven advances in melanoma treatment. In addition, we review benefits and limitations of current therapies and look ahead to continued progress in melanoma prevention and therapy. Remarkable achievements in the field have already produced a paradigm shift in melanoma treatment: Metastatic melanoma, once considered incurable, can now be treated with potentially curative rather than palliative intent.

Response to BRAF Inhibition in Melanoma Is Enhanced When Combined with Immune Checkpoint Blockade

Cooper, Juneja, Sage, and colleagues show that combining BRAF and PD-1/PD-L1 blockade slowed tumor growth and prolonged survival in a melanoma mouse model, with increased number and activity of tumor-infiltrating lymphocytes similar to that in a human melanoma patient treated with this regimen. BRAF-targeted therapy results in objective responses in the majority of patients; however, the responses are short lived (∼6 months). In contrast, treatment with immune checkpoint inhibitors results in a lower response rate, but the responses tend to be more durable. BRAF inhibition results in a more favorable tumor microenvironment in patients, with an increase in CD8+ T-cell infiltrate and a decrease in immunosuppressive cytokines. There is also increased expression of the immunomodulatory molecule PDL1, which may contribute to the resistance. On the basis of these findings, we hypothesized that BRAF-targeted therapy may synergize with the PD1 pathway blockade to enhance antitumor immunity. To test this hypothesis, we developed a BRAF(V600E)/Pten−/− syngeneic tumor graft immunocompetent mouse model in which BRAF inhibition leads to a significant increase in the intratumoral CD8+ T-cell density and cytokine production, similar to the effects of BRAF inhibition in patients. In this model, CD8+ T cells were found to play a critical role in the therapeutic effect of BRAF inhibition. Administration of anti-PD1 or anti-PDL1 together with a BRAF inhibitor led to an enhanced response, significantly prolonging survival and slowing tumor growth, as well as significantly increasing the number and activity of tumor-infiltrating lymphocytes. These results demonstrate synergy between combined BRAF-targeted therapy and immune checkpoint blockade. Although clinical trials combining these two strategies are ongoing, important questions still remain unanswered. Further studies using this new melanoma mouse model may provide therapeutic insights, including optimal timing and sequence of therapy. Cancer Immunol Res; 2(7); 643–54. ©2014 AACR.

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR–Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

How does pheomelanin synthesis contribute to melanomagenesis?: Two distinct mechanisms could explain the carcinogenicity of pheomelanin synthesis.

Recently, we reported that melanoma risk in redheads is linked not only to pale skin, but also to the synthesis of the pigment – called pheomelanin – that gives red hair its color. We demonstrated that pheomelanin synthesis is associated with increased oxidative stress in the skin, yet we have not uncovered the chemical pathway between the molecule pheomelanin and the DNA damage that drives melanoma formation. Here, we hypothesize two possible pathways. On one hand, pheomelanin might generate reactive oxygen species (ROS) that directly or indirectly cause oxidative DNA damage. On the other hand, pheomelanin synthesis might consume cellular antioxidant stores and make the cell nucleus more vulnerable to other endogenous ROS. Uncovering the mechanistic pathway between pheomelanin and oxidative DNA damage will be an important step in developing strategies to lower melanoma risk in redheads.

Prognostic Significance of Cutaneous Adverse Events Associated With Pembrolizumab Therapy.

The goal of cancer immunotherapy is to harness the immune system to recognize and destroy tumor cells, with the potential to produce durable responses that may translate into curative outcomes in patients with metastatic cancers. Results from multiple randomized clinical trials have established immune checkpoint inhibitors as the most successful class of immunotherapies to date. These include monoclonal antibodies that reinvigorate T cell responses by blocking cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1), two coinhibitory receptors that regulate T cell activation.

A structure-based mechanism for tRNA and retroviral RNA remodelling during primer annealing

To prime reverse transcription, retroviruses require annealing of a transfer RNA molecule to the U5 primer binding site (U5-PBS) region of the viral genome. The residues essential for primer annealing are initially locked in intramolecular interactions; hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements. Here we show that, unlike classical chaperones, the Moloney murine leukaemia virus NC uses a unique mechanism for remodelling: it specifically targets multiple structured regions in both the U5-PBS and tRNAPro primer that otherwise sequester residues necessary for annealing. This high-specificity and high-affinity binding by NC consequently liberates these sequestered residues—which are exactly complementary—for intermolecular interactions. Furthermore, NC utilizes a step-wise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release. Our structures of NC bound to U5-PBS and tRNAPro reveal the structure-based mechanism for retroviral primer annealing and provide insights as to how ATP-independent chaperones can target specific RNAs amidst the cellular milieu of non-target RNAs.

Melanoma Immunotherapy: Mechanisms and Opportunities

Immune checkpoint blockade via inhibition of Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) and Programmed Cell Death 1 Receptor (PD-1) has demonstrated significant clinical benefits in treating melanoma and other types of cancers and has since become a very progressive field in cancer research. Despite durable tumor regression observed in some patients, response rates to CTLA-4 and PD-1 still have room for improvement. There are many additional immune modulatory pathways, including inhibitory molecules expressed on tumor cells and secretion of pro-inflammatory cytokines by lymphatic cells that could potentially be targeted to enhance the anti-tumor responses to PD-1 and CTLA-4. Here, we review the current status of CTLA-4 and PD-1 inhibitors in the treatment of melanoma and several therapeutic targets and strategies that may synergize with checkpoint blockades. *Corresponding Author: David E. Fisher, MD, PhD, Department of Dermatology, East Cutaneous Biology Research Center, Massachusetts General Hospital, Building 149, 3rd Floor, 13th Street Charlestown, MA 02129, USA. Tel: (+1) 617-643-5428; E-mail: dfisher3@partners.org Citation: Fisher, D.E., et al. Melanoma Immunotherapy: Mechanisms and Opportunities. (2015) Invest Dermatol Venereol Res 1(2): 1-7. Melanoma Immunotherapy: Mechanisms and Opportunities Yu Xu1,3, Anita Van Der Sande1, Jennifer A. Lo1, David E. Fisher1,2* Received date: September 29, 2015 Accepted date: November 20, 2015 Published date: November 26, 2015 DOI: 10.15436/2381-0858.15.010 Invest Dermatol Venereol Res | Volume 1: Issue 2 Fisher, D.E., et al. 2 and PD-1 and PD-L1 inhibitors have demonstrated comparable clinical efficacy[9]. In summary, CTLA-4 primarily suppresses the activation of naive T-cells by APCs while PD-1 inhibits previously-activated effector T-cells[10,11]. This is thought to explain why PD-1 inhibitors have a lower toxicity profile compared to CTLA-4 inhibitors. Although CTLA-4 and PD-1 inhibitors are currently the most promising cancer immunotherapy treatment options, most melanoma patients still do not respond to these therapies. In this review, we discuss progress in the use of CTLA-4 and PD-1 inhibitors for the treatment of melanoma, and different mechanisms and therapeutic methods that could potentially improve the efficacy of CTLA-4 and PD-1 inhibitors. Current Status of CTLA-4 and PD-1 Inhibitors There are three checkpoint blockade agents that have been approved by the FDA for the treatment of advanced melanoma: Ipilimumab (antibody against CTLA-4), Pembrolizumab and Nivolumab (antibodies against PD-1). Compared to traditional targeted therapies, such as BRAF inhibitors, checkpoint blockade offers more durable responses but with lower response rates. A recently published meta-analysis of survival data following Ipilimumab therapy reported that Ipilimumab extended overall survival from approximately 8 months to 11.4 months in patients with metastatic melanoma. A plateau survival rate of 21% was reached around year 3, with follow-up of up to 10 years[12]. Compared to Ipilimumab, PD-1 inhibitors elicit higher response rates with fewer side effects[13,14]. In a recently published phase III study comparing Pembrolizumab versus Ipilimumab in the treatment of advanced melanoma[13], the estimated 12-month survival rates were 74.1% for Pembrolizumab administered every 2 weeks at the dose of 10 mg/kg, 68.4% for Pembrolizumab administered every 3 weeks at the dose of 10mg/kg, and 58.2% for Ipilimumab administered every 3 weeks at the dose of 3mg/ kg. The response rates for Pembrolizumab administered every 2 weeks and 3 weeks were 33.7% and 32.9% respectively, significantly higher than the response rate for Ipilimumab administered every 3 weeks (11.9%). Rates of grade 3 5 adverse events in the two Pembrolizumab groups (13.3% in the 2-week group and 10.1% in the 3-week group) were lower than the rate in the Ipilimumab group (19.9%). It should be noted that clinical trials for Pembrolizumab and Nivolumab started 6 years later than the trials for Ipilimumab and clinical studies with longer follow-up are needed to fully evaluate the efficacy of PD-1 inhibitors. The non-redundant mechanisms of CTLA-4 and PD-1 provide a rationale for combination of CTLA-4 and PD-1 inhibitors. Several clinical studies have shown superior response rates and progression free survival with combined Ipilimumab and Nivolumab treatment[15-17]. For example, the phase III study testing Nivolumab and Ipilimumab combined therapy in patients with metastatic melanoma reported an objective response rate of 43.7% in the Nivolumab group (3mg/kg administered every 2 weeks), 19.0% in Ipilimumab group (3mg/kg administered every 3 weeks) and 53.6% in the combined Nivolumab Ipilimumab group (4 doses of 1 mg/kg Nivolumab plus 3 mg/kg Ipilimumab administered every 3 weeks, followed by 3 mg/kg Nivolumab administered every two weeks)[17]. The median progression-free survival for the combined therapy was 11.5 months, compared to 2.9 months for Ipilimumab, and 6.9 months for Nivolumab. It should be noted that in patients with high PD-L1 expression on tumor cells, the median progression-free survival was the same in the combined therapy group and in the Nivolumab group (14 months), but in patients with tumors that expressed a low level of PD-L1, progression-free survival was longer in the combined therapy group (11.2 months vs. 5.3 months). This suggests that PD-L1 expression in the tumor is associated with but does not www.ommegaonline.org Melanoma Immunotherapy Invest Dermatol Venereol Res | Volume 1: Issue 2 Figure 1: Anti-tumor immunity. The immune response against melanoma begins with the uptake of tumor-associated antigens by APCs, which then migrate to lymphatic tissues to activate naive T-cells. Two signals are required for T-cell activation: antigen presentation and co-stimulatory signaling. In this process, CTLA-4 competes with the co-stimulatory molecule CD28 for ligands CD80 and CD86, inhibiting naive T-cell activation. Blocking monoclonal antibodies against CTLA-4 prevent binding of CTLA-4 to its ligands, allowing T-cell activation. Activated T-cells secrete type I interferons that can activate other immune cells including natural killer cells and macrophages. Effector T-cells subsequently traffic to and infiltrate tumors, destroying cancer cells. During this stage, PD-L1 expressed on tumor and stromal cells bind PD-1 receptors on T-cells. PD-1 signaling inhibits T-cells. In addition, interferon-γ secreted by tumor infiltrating lymphocytes upregulates PD-L1 expression on tumor cells, suppressing T-cell responses. PD-1 inhibitors reinvigorate exhausted T-cells by blocking the interaction between PD-L1 and PD-1. perfectly predict PD-1 inhibitor efficacy. Grade 3 or 4 adverse events (grade 3 or 4) were much more frequent in the combined therapy group than in the two monotherapy groups (55.0% in the combined group, 16.3% in the Nivolumab group and 27.3% in the Ipilimumab group). Higher rates of adverse events resulting from the combined CTLA-4 and PD-1 therapies were observed in all studies, raising safety concerns regarding combined therapy[15-18]. Mechanisms and Therapeutic Strategies to Enhance Immune Checkpoint Blockade Efficacy The potential for highly durable responses has led to great interest in developing synergistic combinatorial approaches with other treatment modalities that could expand the proportion of responders to checkpoint blockade. Overview of anti-tumor immune response The immune response against tumor cells can be conceptualized in four steps (Figure 1): 1) APCs are activated by tumor-associated antigens and present antigens to T-cells in the lymphatic system; 2) APCs activate antigen-specific T-cells; 3) activated T-cells traffic and infiltrate into the tumor; 4) Cytotoxic T-cells recognize and attack cancer cells[19]. In this process, CTLA-4 and PD-1 inhibitors are known to enhance T-cell activation and Subsequent cancer cell recognition and killing[20], but APC activation and T-cell trafficking must be addressed by other therapeutic methods. Role of the inflammatory tumor microenvironment in immunotherapy Density of tumor infiltrating cytotoxic CD8+ T-cells is one of the best predictors of response to current checkpoint blockade therapies[21-23]. Characterization of the tumor microenvironment reveals two immunologic phenotypes: inflamed and non-inflamed tumors[24]. Many studies have shown that inflamed tumors, with dense T-cell infiltration and high concentrations of type I interferons, are more likely to respond to checkpoint blockade. Non-inflamed tumors lack T-cell infiltrate and may require additional interventions to achieve optimal inflammation and innate immune activation in the tumor microenvironment. However, the role of the inflammation is complex. Previous studies have shown a higher incidence of cancer in tissues that have experienced chronic inflammation, suggesting a pro-tumorigenic effect in some inflammatory contexts[25-28]. Chronic inflammation in the tumor microenvironment may contribute to tumor growth by promoting angiogenesis, cancer cell proliferation, tissue invasion and metastasis[29]. Thus, there is a delicate balance between pro and anti-tumor immunity that is determined by the relative activation of different cell types and expression of various immune mediators in the tumor microenvironment[25,30,31]. Further studies are needed to improve our understanding of optimal targets and mechanisms in the inflammatory tumor microenvironment in order to improve immunotherapy. Innate immunity is critical in the anti-tumor immune response Innate immunity represents the first line of immune defense. One major part of the innate immune system that is important for anti-tumor immune responses is the activation of antigen-specific T-cells by APCs. As discussed earlier, T-cell 3 priming requires two signals: the interactions between antigens and the T-cell receptors, and the binding of