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Abstract The circadian clock regulator Bmal1 modulates tumorigenesis, but its reported effects are inconsistent. Here, we show that Bmal1 has a context-dependent role in mouse melanoma tumor growth. Loss of Bmal1 in YUMM2.1 or B16-F10 melanoma cells eliminates clock function and diminishes hypoxic gene expression and tumorigenesis, which could be rescued by ectopic expression of HIF1α in YUMM2.1 cells. By contrast, over-expressed wild-type or a transcriptionally inactive mutant Bmal1 non-canonically sequester myosin heavy chain 9 (Myh9) to increase MRTF-SRF activity and AP-1 transcriptional signature, and shift YUMM2.1 cells from a Sox10high to a Sox9high immune resistant, mesenchymal cell state that is found in human melanomas. Our work describes a link between Bmal1, Myh9, mouse melanoma cell plasticity, and tumor immunity. This connection may underlie cancer therapeutic resistance and underpin the link between the circadian clock, MRTF-SRF and the cytoskeleton.

Summary: By sorting receptor tyrosine kinases into endolysosomes, the endosomal sorting complexes required for transport (ESCRTs) are thought to attenuate oncogenic signaling in tumor cells. Paradoxically, ESCRT members are upregulated in tumors. Here, we show that disruption of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a pivotal ESCRT component, inhibited tumor growth by promoting CD8+ T cell infiltration in melanoma and colon cancer mouse models. HRS ablation led to misfolded protein accumulation and triggered endoplasmic reticulum (ER) stress, resulting in the activation of the type I interferon pathway in an inositol-requiring enzyme-1α (IRE1α)/X-box binding protein 1 (XBP1)-dependent manner. HRS was upregulated in tumor cells with high tumor mutational burden (TMB). HRS expression associates with the response to PD-L1/PD-1 blockade therapy in melanoma patients with high TMB tumors. HRS ablation sensitized anti-PD-1 treatment in mouse melanoma models. Our study shows a mechanism by which tumor cells with high TMB evade immune surveillance and suggests HRS as a promising target to improve immunotherapy.

Summary: Background: Only a minority of melanoma patients experience durable responses to immunotherapies due to inter- and intra-tumoral heterogeneity in melanoma. As a result, there is a pressing need for suitable preclinical models to investigate resistance mechanisms and enhance treatment efficacy. Methods: Here, we report two different methods for generating melanoma patient-derived organoids (MPDOs), one is embedded in collagen gel, and the other is inlaid in Matrigel. MPDOs in Matrigel are used for assessing the therapeutic effects of anti-PD-1 antibodies (αPD-1), autochthonous tumor infiltrating lymphocytes (TILs), and small molecule compounds. MPDOs in collagen gel are used for evaluating the chemotaxis and migratory capacity of TILs. Finding: The MPDOs in collagen gel and Matrigel have similar morphology and immune cell composition to their parental melanoma tissues. MPDOs show inter- and intra-tumoral heterogeneity and contain diverse immune cells such as CD4+, CD8+ T, Treg, CD14+ monocytic, CD15+, and CD11b+ myeloid cells. The tumor microenvironment (TME) in MPDOs is highly immunosuppressive, and the lymphoid and myeloid lineages express similar levels of PD-1, PD-L1, and CTLA-4 as their parental melanoma tissues. Anti-PD-1 antibodies (αPD-1) reinvigorate CD8+ T cells and induce melanoma cell death in the MPDOs. TILs expanded by IL-2 and αPD-1 show significantly lower expression of TIM-3, better migratory capacity and infiltration of autochthonous MPDOs, and more effective killing of melanoma cells than TILs expanded by IL-2 alone or IL-2 with αCD3. A small molecule screen discovers that Navitoclax increases the cytotoxicity of TIL therapy. Interpretation: MPDOs may be used to test immune checkpoint inhibitors and cellular and targeted therapies. Funding: This work was supported by the NIH grants CA114046, CA261608, CA258113, and the Tara Miller Melanoma Foundation.

Lack of CD8+ T-cell infiltration into solid tumors is associated with poor responsiveness to immune checkpoint therapy (ICT). Here, the authors show that blocking the phosphorylation of HRS to reduce the induction of immunosuppressive exosomes promotes CD8+ T-cell infiltration into tumors and enhances the efficacy of ICT in mouse melanoma models.

Slow-cycling melanoma persister cells are characterised by a high, reversible expression of H3K4 demethylase KDM5B. Here, the authors use genetic and chemical methods to enforce a permanent high expression of KDM5B and show that these cells transit to a melanocytic differentiated state and undergo cell cycle arrest.

Mechanisms underlying tumor cell plasticity remain poorly understood. Here, the authors show the presence of a dormant-invasive SOX10- subpopulation in cutaneous melanoma that can be targeted by cIAP1/2 inhibitors.

Gene signatures that predict response to immune checkpoint blockade (ICB) therapies in melanoma have been based on preclinical models and pre-treatment samples. Here the authors develop pathway-based signatures to predict ICB response in melanoma using on-treatment samples, leading to improved performance.

Patient-derived xenograft models (PDX) have been extensively used to study the molecular and clinical features of cancers. Here the authors present a cohort of 536 PDX models from 25 cancers, as well as their genomic and evolutionary profiles and their suitability for clinical trials.

Abstract Background Gamma delta (γδ) T lymphocytes are promising candidate for adoptive T cell therapy, however, their treatment efficacy is not satisfactory. Vδ2 T cells are unique to primates and few suitable models are available to assay their anti‐tumour function. Methods We tested human γδ T cell activation, tumour infiltration, and tumour‐killing in four three‐dimensional (3D) models, including unicellular, bicellular and multicellular melanoma spheroids, and patient‐derived melanoma organoids. We studied the effects of checkpoint inhibitors on γδ T cells and performed a small molecule screen using these platforms. Results γδ T cells rapidly responded to melanoma cells and infiltrated melanoma spheroids better than αβ T cells in PBMCs. Cancer‐associated fibroblasts (CAFs) in bicellular spheroids, stroma cells in multicellular melanoma spheroids and inhibitory immune cells in organoids significantly inhibited immune cell infiltrates including γδ T cells and lessened their cytotoxicity to tumour cells. Tumour‐infiltrating γδ T cells showed exhausted immunophenotypes with high checkpoints expression (CTLA‐4, PD‐1 and PD‐L1). Immune checkpoint inhibitors increased γδ T cell infiltration of 3D models and killing of melanoma cells in all four 3D models. Our small molecule screen assay and subsequent mechanistic studies demonstrated that epigenetic modifiers enhanced the chemotaxis and cytotoxicity of γδ T cells through upregulating MICA/B, inhibiting HDAC6/7 pathway and downregulating the levels of PD‐L1 and PD‐L2 in CAFs and tumour cells. These compounds increased CXCR4 and CD107a expression, IFN‐γ production and decreased PD‐1 expression of γδ T cells. Conclusions Tumour‐infiltrating γδ T cells show exhausted immunophenotypes and limited anti‐tumour capacity in melanoma 3D models. Checkpoint inhibitors and epigenetic modifiers enhance anti‐tumour functions of γδ T cells. These four 3D models provided valuable preclinical platforms to test γδ T cell functions for immunotherapy.

Immune checkpoint therapies (ICT) are promising for treating various cancers, but response rates vary. Here the authors show, in mouse models, that tumor-infiltrating mast cells colocalize with regulatory T cells, coincide with local reduction of MHC-I and CD8 T cells, and is associated with resistance to ICT, which can be reversed by c-kit inhibitor treatment.

Despite the new targeted and immunotherapies for metastatic melanoma, several patients show therapeutic plateau. Here, the authors review the current pre-clinical models of cutaneous melanoma and discuss their strengths and limitations that may help with overcoming therapeutic plateau.

The regulation of tumor inflammation is incompletely understood and the role of B cells is unclear. Here, the authors show that a specific subtype of B cells is induced in melanoma and required to recruit T lymphocytes and elicit inflammation.

Abstract Most patients with advanced cancer eventually acquire resistance to targeted therapies, spurring extensive efforts to identify molecular events mediating therapy resistance. Many of these events involve synthetic rescue (SR) interactions, where the reduction in cancer cell viability caused by targeted gene inactivation is rescued by an adaptive alteration of another gene (the rescuer). Here, we perform a genome‐wide in silico prediction of SR rescuer genes by analyzing tumor transcriptomics and survival data of 10,000 TCGA cancer patients. Predicted SR interactions are validated in new experimental screens. We show that SR interactions can successfully predict cancer patients’ response and emerging resistance. Inhibiting predicted rescuer genes sensitizes resistant cancer cells to therapies synergistically, providing initial leads for developing combinatorial approaches to overcome resistance proactively. Finally, we show that the SR analysis of melanoma patients successfully identifies known mediators of resistance to immunotherapy and predicts novel rescuers.

Prolyl hydroxylase domain protein 2 (PHD2) regulates cellular response to hypoxia. Here the authors show that PHD2 is downregulated in melanoma and that PHD2 depletion, in a mouse model, promotes the progression of benign melanocytic lesions into melanoma, via activation of the Akt/mTOR signaling cascade.

Abstract Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS‐mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that BET proteins are overexpressed in NRAS‐mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS‐mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis. Our studies demonstrate that co‐targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment‐resistant tumor types.

Summary: Diverse pathways drive resistance to BRAF/MEK inhibitors in BRAF-mutant melanoma, suggesting that durable control of resistance will be a challenge. By combining statistical modeling of genomic data from matched pre-treatment and post-relapse patient tumors with functional interrogation of >20 in vitro and in vivo resistance models, we discovered that major pathways of resistance converge to activate the transcription factor, c-MYC (MYC). MYC expression and pathway gene signatures were suppressed following drug treatment, and then rebounded during progression. Critically, MYC activation was necessary and sufficient for resistance, and suppression of MYC activity using genetic approaches or BET bromodomain inhibition was sufficient to resensitize cells and delay BRAFi resistance. Finally, MYC-driven, BRAFi-resistant cells are hypersensitive to the inhibition of MYC synthetic lethal partners, including SRC family and c-KIT tyrosine kinases, as well as glucose, glutamine, and serine metabolic pathways. These insights enable the design of combination therapies that select against resistance evolution. : Diverse pathways drive resistance to BRAF/MEK inhibitors in BRAF-mutant melanoma, but by combining statistical modeling of tumor data with functional interrogation of resistance models, Singleton et al. show that these pathways converge to activate MYC. BRAFi-resistant cells are hypersensitive to the inhibition of MYC synthetic lethal partners, informing therapies that select against resistance. Keywords: melanoma, cancer therapeutics, therapeutic resistance, signaling networks, MYC, metabolism, synthetic lethality

Summary: Therapy of advanced melanoma is changing dramatically. Following mutational and biological subclassification of this heterogeneous cancer, several targeted and immune therapies were approved and increased survival significantly. To facilitate further advancements through pre-clinical in vivo modeling, we have established 459 patient-derived xenografts (PDX) and live tissue samples from 384 patients representing the full spectrum of clinical, therapeutic, mutational, and biological heterogeneity of melanoma. PDX have been characterized using targeted sequencing and protein arrays and are clinically annotated. This exhaustive live tissue resource includes PDX from 57 samples resistant to targeted therapy, 61 samples from responders and non-responders to immune checkpoint blockade, and 31 samples from brain metastasis. Uveal, mucosal, and acral subtypes are represented as well. We show examples of pre-clinical trials that highlight how the PDX collection can be used to develop and optimize precision therapies, biomarkers of response, and the targeting of rare genetic subgroups. : Krepler et al. have established a collection of melanoma patient-derived xenografts (PDX). Melanoma is a very heterogeneous cancer, and this large collection includes even rare subtypes and genetic aberrations in sufficient numbers. Multiple PDX from therapy-resistant patients are characterized and tested in pre-clinical trials for second line therapies. Keywords: melanoma, patient-derived xenografts, targeted therapy, immune checkpoint blockade, melanoma brain metastasis, in vivo models, BRAF inhibitor resistance, ERK inhibitor, MDM2 inhibitor, PI3K beta inhibitor

Summary: Tumor-sequencing studies have revealed the widespread genetic diversity of melanoma. Sequencing of 108 genes previously implicated in melanomagenesis was performed on 462 patient-derived xenografts (PDXs), cell lines, and tumors to identify mutational and copy number aberrations. Samples came from 371 unique individuals: 263 were naive to treatment, and 108 were previously treated with targeted therapy (34), immunotherapy (54), or both (20). Models of all previously reported major melanoma subtypes (BRAF, NRAS, NF1, KIT, and WT/WT/WT) were identified. Multiple minor melanoma subtypes were also recapitulated, including melanomas with multiple activating mutations in the MAPK-signaling pathway and chromatin-remodeling gene mutations. These well-characterized melanoma PDXs and cell lines can be used not only as reagents for a large array of biological studies but also as pre-clinical models to facilitate drug development. : Garman et al. have characterized melanoma PDXs and cell lines described in Krepler et al. (see the related paper in this issue of Cell Reports), identifying major and minor subtypes, some of which were previously not well defined, targeted and immunotherapy resistance, and tumor heterogeneity, creating a set of reagents for future drug discovery and biological studies. Keywords: melanoma, patient-derived xenografts, massively parallel sequencing, cell lines

Resistance to BRAFV600E inhibitors often occurs in melanoma patients. Here, the authors describe a potential mechanism of acquired drug resistance mediated by tumor-associated B cells-derived IGF-1.

Summary: BRAF fusions are detected in numerous neoplasms, but their clinical management remains unresolved. We identified six melanoma lines harboring BRAF fusions representative of the clinical cases reported in the literature. Their unexpected heterogeneous responses to RAF and MEK inhibitors could be categorized upon specific features of the fusion kinases. Higher expression level correlated with resistance, and fusion partners containing a dimerization domain promoted paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway and hyperproliferation in response to first- and second-generation RAF inhibitors. By contrast, next-generation αC-IN/DFG-OUT RAF inhibitors blunted paradoxical activation across all lines and had their therapeutic efficacy further increased in vitro and in vivo by combination with MEK inhibitors, opening perspectives in the clinical management of tumors harboring BRAF fusions. : Botton et al. shed light on the heterogeneity of BRAF fusions encountered in melanocytic tumors and characterize features influencing their signaling and drug response. These findings unveil the singularities of BRAF fusions and establish general principles to guide their clinical management in melanoma and other malignancies. Keywords: BRAF fusion, melanoma, paradoxical activation, RAF inhibitor, MEK inhibitor, sequencing, rearrangement, translocation, kinase, pre-clinical

Summary: Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurs by activation of mTORC1 signaling, which directly mediates increased translation of the mRNA encoding ribose-5-phosphate isomerase A (RPIA), a pentose phosphate pathway enzyme. p16 loss correlates with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibits proliferation only in p16-low cells by inducing senescence both in vitro and in vivo. These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells. : Senescence bypass through p16 loss predisposes to transformation and tumorigenesis. Buj et al. found that the loss of p16 upregulates nucleotide metabolism through increased mTORC1-mediated translation of RPIA to bypass senescence in an RB-independent manner. Thus, the mTORC1-RPIA axis is a metabolic vulnerability for p16-null cancers. Keywords: cancer metabolism, ribonucleotide reductase M2, BRAF, cell cycle, senescence, melanoma, pancreatic cancer, nevi, pentose phosphate pathway, ribose-5-phosphate isomerase A

Melanoma patients with oncogenic BRAFV600E mutation have poor prognoses. While the role of BRAFV600E in tumorigenesis is well established, its involvement in metastasis that is clinically observed in melanoma patients remains a topic of debate. Here, we show that BRAFV600E melanoma cells have extensive invasion activity as assayed by the generation of F-actin and cortactin foci that mediate membrane protrusion, and degradation of the extracellular matrix (ECM). Inhibition of BRAFV600E blocks melanoma cell invasion. In a BRAFV600E-driven murine melanoma model or in patients’ tumor biopsies, cortactin foci decrease upon inhibitor treatment. In addition, genome-wide expression analysis shows that a number of invadopodia-related genes are downregulated after BRAFV600E inhibition. Mechanistically, BRAFV600E induces phosphorylation of cortactin and the exocyst subunit Exo70 through ERK, which regulates actin dynamics and matrix metalloprotease secretion, respectively. Our results provide support for the role of BRAFV600E in metastasis and suggest that inhibiting invasion is a potential therapeutic strategy against melanoma.

Merkel cell carcinoma (MCC) is a highly lethal skin cancer. MCC tumors rapidly develop resistance to the chemotherapies tested to date. While PD-1/PD-L1 immune checkpoint blockade has demonstrated success in MCC treatment, a significant portion of MCC patients are nonresponsive. Therefore, the pressing need for effective MCC chemotherapies remains. We screened a library of natural products and discovered that one compound, glaucarubin, potently reduced the viability of Merkel cell polyomavirus (MCPyV)-positive MCCs, while remaining nontoxic to primary human fibroblasts and MCPyV-negative MCC cell lines tested. Protein array and Western blot analyses revealed that glaucarubin induces DNA damage and PARP-1 cleavage that correlates with the loss of viability in MCC cells. However, high basal expression of the antiapoptotic factor BCL-2 allowed a subpopulation of cells to survive glaucarubin treatment. Previous studies have shown that, while targeting BCL-2 family proteins significantly decreases MCC cell viability, BCL-2 antisense therapy alone was insufficient to inhibit tumor growth in patients with advanced MCC. We discovered that treatment with an FDA-approved BCL-2 inhibitor in the context of glaucarubin-induced DNA damage led to near complete killing in multiple MCPyV-positive MCC cell lines that express high levels of BCL-2. The combination of DNA damage-induced apoptosis and BCL-2 inhibition thus represents a novel therapeutic strategy for MCPyV-positive MCCs.

The unfolded protein response (UPR) regulates cell fate following exposure of cells to endoplasmic reticulum stresses. PERK, a UPR protein kinase, regulates protein synthesis and while linked with cell survival, exhibits activities associated with both tumor progression and tumor suppression. For example, while cells lacking PERK are sensitive to UPR-dependent cell death, acute activation of PERK triggers both apoptosis and cell cycle arrest, which would be expected to contribute tumor suppressive activity. We have evaluated these activities in the BRAF-dependent melanoma and provide evidence revealing a complex role for PERK in melanoma where a 50% reduction is permissive for BrafV600E-dependent transformation, while complete inhibition is tumor suppressive. Consistently, PERK mutants identified in human melanoma are hypomorphic with dominant inhibitory function. Strikingly, we demonstrate that small molecule PERK inhibitors exhibit single agent efficacy against BrafV600E-dependent tumors highlighting the clinical value of targeting PERK.

The original version of this Article contained an error in the spelling of the author Brett L. Ecker, which was incorrectly given as Brett Ecker. This has now been corrected in both the PDF and HTML versions of the Article.

Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.

Oncogene-induced senescence is characterized by a stable cell growth arrest, thus providing a tumor suppression mechanism. However, the underlying mechanisms for this phenomenon remain unknown. Here, we show that a decrease in deoxyribonucleotide triphosphate (dNTP) levels underlies oncogene-induced stable senescence-associated cell growth arrest. The decrease in dNTP levels is caused by oncogene-induced repression of ribonucleotide reductase subunit M2 (RRM2), a rate-limiting protein in dNTP synthesis. This precedes the senescence-associated cell-cycle exit and coincides with the DNA damage response. Consistently, RRM2 downregulation is both necessary and sufficient for senescence. Strikingly, suppression of nucleotide metabolism by RRM2 repression is also necessary for maintenance of the stable senescence-associated cell growth arrest. Furthermore, RRM2 repression correlates with senescence status in benign nevi and melanoma, and its knockdown drives senescence of melanoma cells. These data reveal the molecular basis whereby the stable growth arrest of oncogene-induced senescence is established and maintained through suppression of nucleotide metabolism.

Summary Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare disorder characterized by segmental accelerated aging and early death from coronary artery disease or stroke. Nearly 90% of HGPS sufferers carry a G608G mutation within exon 11 of LMNA, producing a truncated form of prelamin A, referred to as “progerin”. Here, we report the isolation of naïve multipotent skin-derived precursor (SKP) cells from dermal fibroblast cultures from HGPS donors. These cells form spheres and express the neural crest marker, nestin, in addition to the multipotent markers, OCT4, Sox2, Nanog and TG30; these cells can self-renew and differentiate into smooth muscle cells (SMCs) and fibroblasts. The SMCs derived from the HGPS-SKPs accumulate nuclear progerin with increasing passages. A subset of the HGPS-naïve SKPs express progerin in vitro and in situ in HGPS skin sections. This is the first in vivo evidence that progerin is produced in adult stem cells, and implies that this protein could induce stem cells exhaustion as a mechanism contributing to aging. Our study provides a basis on which to explore therapeutic applications for HGPS stem cells and opens avenues for investigating the pathogenesis of other genetic diseases.

Recently, integrin alpha10 was described as a collagen type II-binding integrin expressed mainly in chondrocytes. However, by array studies we detected integrin alpha10 also to be upregulated in malignant melanoma compared to primary melanocytes. Subsequent analysis of melanoma cell lines and melanoma tumor samples confirmed this finding. Further, we demonstrated that expression of integrin alpha10 is controlled by AP-2 and Ets-1, two transcription factors known to be involved in melanoma development and progression. To investigate the functional relevance of integrin alpha10, expression was downregulated via stable antisense transfection. Proliferation assays and colony forming assays revealed no differences comparing antisense integrin alpha10 cell clones with control and wild type melanoma cells, respectively. However, antisense integrin alpha10 cell clones and Mel Im cells treated with an inhibitory antibody against integrin alpha10 showed a reduced migratory potential.

Noncoding regions of the genome play an important role in tumorigenesis of cancer. Using expression cloning, we have identified a cytotoxic T lymphocyte (CTL)–defined antigen that recognizes a protein sequence derived from an open reading frame transcribed from the reverse strand in the 3′ untranslated region of tRNA isopentenyltransferase 1 (TRIT1). A peptide derived from this open reading frame (ORF) sequence and predicted to bind to HLA-B57, sensitized HLA-B57+ tumor cells to lysis by CTL793. The peptide also induced a CTL response in peripheral blood mononuclear cells (PBMC) of patient 793 and in two other melanoma patients. The CTL lysed peptide-pulsed HLA-B57+ target cells and melanoma cells with endogenous antigen expression. The recognition of this antigen is not limited to HLA-B57-restricted CTLs. An HLA-A2 peptide derived from the ORF was able to induce CTLs in PBMC of 2 HLA-A2+ patients. This study describes for the first time a CTL-defined melanoma antigen that is derived from an ORF on the reverse strand of the putative tumor suppressor gene TRIT1. This antigen has potential use as a vaccine or its ability to induce CTLs in vitro could be used as a predictive biomarker.

Although cyclin dependent kinase (CDK)-2 is known to be dispensable for the growth of most tumors, it is thought to be important for the proliferation of melanoma cells, where its expression is controlled by the melanocyte-lineage specific transcription factor MITF. Treatment of a panel of melanoma cells with the CDK inhibitor dinaciclib led to a concentration-dependent inhibition of growth under both 2D adherent and 3D organotypic cell culture conditions. Dinaciclib targeted melanoma cell lines regardless of cdk2 or MITF levels. Inhibition of growth was associated with a rapid induction of G2/M cell arrest and apoptosis. Treatment of human melanoma mouse xenografts with dinaciclib led to tumor regression associated with reduced retinoblastoma protein phosphorylation and Bcl-2 expression. Further mechanistic studies revealed that dinaciclib induces p53 expression whilst simultaneously downregulating the expression of the anti-apoptotic factors Mcl-1 and XIAP. To clarify the role of p53 activation in the dinaciclib-induced cell death, we generated melanoma cell lines in which p53 expression was knocked down using a shRNA lentiviral vector. Knockdown of p53 completely abolished the induction of apoptosis seen following dinaciclib treatment as shown by a lack of annexin-V staining and caspase-3 cleavage. Altogether, these data show that dinaciclib induces apoptosis in a large panel of melanoma cell lines through a mechanism requiring p53 expression.

This proof-of-concept study was designed to determine if functional, pharmacodynamic profiles relevant to targeted therapy could be derived from live human melanoma samples using a novel automated platform.A series of 13 melanoma cell lines was briefly exposed to a BRAF inhibitor (PLX-4720) on a platform employing automated fluidics for sample processing. Levels of the phosphoprotein p-ERK in the mitogen-activated protein kinase (MAPK) pathway from treated and untreated sample aliquots were determined using a bead-based immunoassay. Comparison of these levels provided a determination of the pharmacodynamic effect of the drug on the MAPK pathway. A similar ex vivo analysis was performed on fine needle aspiration (FNA) biopsy samples from four murine xenograft models of metastatic melanoma, as well as 12 FNA samples from patients with metastatic melanoma.Melanoma cell lines with known sensitivity to BRAF inhibitors displayed marked suppression of the MAPK pathway in this system, while most BRAF inhibitor-resistant cell lines showed intact MAPK pathway activity despite exposure to a BRAF inhibitor (PLX-4720). FNA samples from melanoma xenografts showed comparable ex vivo MAPK activity as their respective cell lines in this system. FNA samples from patients with metastatic melanoma successfully yielded three categories of functional profiles including: MAPK pathway suppression; MAPK pathway reactivation; MAPK pathway stimulation. These profiles correlated with the anticipated MAPK activity, based on the known BRAF mutation status, as well as observed clinical responses to BRAF inhibitor therapy.Pharmacodynamic information regarding the ex vivo effect of BRAF inhibitors on the MAPK pathway in live human melanoma samples can be reproducibly determined using a novel automated platform. Such information may be useful in preclinical and clinical drug development, as well as predicting response to targeted therapy in individual patients.