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Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho-S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho-S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance. Significance: Mutant BRAF and NRAS melanomas acquire resistance to combined MEK and CDK4/6 inhibition via upregulation of mTOR pathway signaling. This resistance mechanism provides the preclinical basis to utilize mTORC1/2 inhibitors to improve MEKi plus CDK4/6i drug regimens. Cancer Discov; 8(5); 568–81. ©2018 AACR. See related commentary by Sullivan, p. 532. See related article by Romano et al., p. 556. This article is highlighted in the In This Issue feature, p. 517

Therapeutic agonists targeting CD40 have shown encouraging signs of anti-tumor efficacy in patients. Despite their initial promise however, optimal dosing of systemically active agents has been limited by toxicity and their full therapeutic potential not achieved. We have previously demonstrated in vitro the ability of MP0317, a novel multi-specific DARPin® therapeutic, to selectively activate CD40 bearing B-cells, dendritic cells and macrophages in the presence of fibroblast activation protein (FAP). FAP is highly expressed in the stroma of many solid tumors and is found only at lower levels in other tissues. MP0317 utilizes FAP and CD40 binding domains to selectively cross-link and activate CD40 in the presence of FAP and thus avoid systemic CD40 activation classically associated with toxicity. Here we present new data which confirm the unique therapeutic potential of MP0317 in ex vivo model systems, demonstrate modulation of macrophage phenotype and reveal a release of T-cells from macrophage-mediated suppression. Ex vivo functional assays performed with MP0317 on dissociated human tumors demonstrated FAP-dependent activation of CD40-expressing B-cell and myeloid cell populations. Immunohistochemical analysis of tissue micro-arrays demonstrated FAP levels consistent with those required for immune cell activation in a broad range of solid tumor indications. Furthermore, by mining publicly available data, we classified tumor indications based on their immune cell content, pathway activation and MP0317 ex vivo mode of action (T cell/macrophage ratio, FAP and CD40 expression) and proposed patient populations and indications for the upcoming clinical trials. To further investigate the functional impact of MP0317, macrophages were differentiated in vitro using canonical polarising cytokines. Suppressive tumor-associated macrophage (TAM)-like cells, were repolarized by MP0317 to an anti-tumor-like phenotype. Furthermore, we explored whether modulation of macrophage phenotype by MP0317 could relieve their suppressive effect on T-cells. TAM-like macrophages potently supressed anti-CD3/28 driven T-cell activation and indeed the addition of MP0317 was found to restore T-cell response. Together these data further support MP0317's FAP targeted multimodal mechanism of action in human tumors, provide evidence for an improved therapeutic window over existing CD40 agonists and support progression to the clinic with an informed target patient profile. Citation Format: Kyriaki Ioannou, Simone Ragusa, Joanna Roquette, Ana Florescu, Mariam Gachechiladze, Eliane Müller, Julia M. Martinez-Gomez, Sophie Barsin, Sarah Jetzer, Nicolo Rigamonti, Clara Domke, Tamara Lekishvili, Karolin Rommel, Ivana Tosevski, Anne Goubier, Philippe Legenne, Vladimir Kirkin, Mitch Levesque, Hong Ji, Rupert Kenefeck. MP0317, a CD40xFAP targeting multi-specific DARPin® therapeutic, drives immune activation and reverts myeloid-mediated T-cell suppression in vitro and ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1733.

Trials utilizing cyclin-dependent kinases 4/6 (CDK4/6) inhibitor alone or in combination with BRAF and MEK inhibitors are under way in melanoma, but their use needs to be optimized. MEK plus CDK4/6i combinations lead to tumor regressions in vivo; however, residual disease and acquired resistance frequently persisted. Previously, we showed that amplification or mutation of NRAS was associated with acquired resistance in in vivo mutant NRAS models and mutant BRAF patient samples, respectively. These mechanisms led to upregulation of mTOR-S6 pathway. Here, we observed upregulation of dormancy signature and increased NR2F1 expression in residual disease. To further identify targetable pathway alterations associated with these resistance mechanisms, we have established longitudinal cell lines from multiple metastases from a single patient on the LOGIC2 (BRAF-MEK-CDK4/6i) trial. RPPA analysis highlights differences between the lines that we are further analyzing in functional assays. There is also increasing evidence that CDK4/6 inhibitors (CDK4/6i) may regulate the tumor immune microenvironment. We show that MEK plus CD4/6i led to better control of tumor growth in syngeneic models compared to immune-deficient NSG mice. The response to MEK plus CDK4/6i was associated with increased infiltration of CD8 positive T cells and T-cell activation markers. Together, we aim to identify mechanisms underlying resistance/tolerance to MEK plus CDK4/6i and to determine effects on the tumor immune microenvironment to inform potential new treatment strategies. Citation Format: Jessica L.F. Teh, Phil F. Cheng, Dan E. Erkes, Timothy J. Purwin, Conroy Field, Connor Hollingworth, Julio Aguirre-Ghiso, Mitch P. Levesque, Reinhard Dummer, Andrew E. Aplin. Effects of CDK4/6/MAPK targeting combinations on melanoma and the tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A22.

A deepened understanding of the cellular and molecular processes in the tumor microenvironment is necessary for the development of precision immunotherapy (IT). We simultaneously investigated CD3, PDL1, and IDO by immunohistochemistry in paired biopsies from various organs of 43 metastatic melanoma patients treated with IT and targeted therapy (TT). Intraindividual biopsies taken after a period of weeks to months demonstrate discordant results in 30% of the cases. Overlap of IDO and PDL1 increased after therapy. IT only marginally impacted PDL1 expression over time in contrast to TT. Standardized repeated assessments of multiple immune markers in repeated biopsies will generate detailed insights in melanoma's immune evolution and adaption during therapies and might be used to support treatment decisions.

There is good evidence that an immune response to melanoma predicts better survival. We report a bioinformatic study designed to identify immune subgroups of primary melanomas in order to better understand the biology of immune cell/tumor interaction, and to identify candidate predictive biomarkers for immunotherapy. We applied an approach described by Angelova et al. to the inference of tumor immune cell infiltrates using differential expression of signature genes, in 703 primary melanoma transcriptomes from the Leeds Melanoma Cohort (LMC). Consensus clustering of immune cell scores identified 3 subgroups in LMC and replication was sought in The Cancer Genome Atlas (TCGA) data set, (primarily metastatic samples). We then used the whole transcriptomic data to explore associated biological pathways: a protein-protein interaction network analysis of genes was performed in Cytoscape plugin Reactome FIVIZ. Nodal genes were identified and the cellular origin of the gene expression signature further explored using immunohistochemistry (IHC) and in patient-derived melanoma cell culture transcriptomes. Clinicopathological and environmental factors, and immune cell scores associated with survival within immune subgroups, were then analysed using a multivariate Cox proportional hazard model. We identified three subgroups with transcriptomic patterns indicative of: low, intermediate and high immune cell infiltration. The groups were concordant with histological evidence of tumor infiltrating lymphocytes. Genes enriched in the low, compared with the high immune group, were in cell cycle, metabolic and Beta-catenin pathways. MYC was the nodal (the most influential) gene of the protein-protein interaction network in this group. In the high immune group, genes were enriched in immune pathways, including antigen processing and presentation, with the nodal gene - NFKB1. Based on the literature and our results, we hypothesised that MYC downregulates HLA-class I, hence we tested MYC correlations in the melanoma cell line transcriptomes (in which immune cells were absent). Indeed, MYC was negatively correlated with many HLA class I genes, with HLA-B having the strongest result: R=-0.57. IHC of primary tumors showed tumor nuclear expression of MYC. Reported smoking independently predicted a worse outcome for patients classified as the high immune group (HR=3.52, P=0.008). The transcriptomic analysis showed that smoking correlated with an increased Th2 cell score (P=0.03) and reduced NK56 bright score (P=0.01) in that group. We report evidence that MYC plays an important role in immune evasion in primary melanoma. The transcriptomic data support in vitro findings that MYC downregulates HLA class I expression. Furthermore, we present important results that smoking status modifies immune response in melanoma and is an independent risk factor for melanoma death. We hypothesise that smoking may impact on immunotherapy efficacy. Citation Format: Joanna Pozniak, Jeremie Nsengimana, Jon Laye, Sally O'Shea, Sabrina Schindler, Mitch Levesque, Tim Bishop, Julia Newton-Bishop. MYC expression and smoking as drivers of failure of immune response to melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5204.

With incidence of melanoma growing worldwide and new therapies prolonging the survival of patients with advanced disease, complex medical care is needed.Best care of complicated melanoma cases is achieved in specialized referral centers. Aims to provide optimized melanoma therapy, best patient-reported treatment outcome, and successful clinical and translational research, necessitate a dedicated interdisciplinary team.We report on critical aspects of the interaction between patients, medical care givers, clinical trial and biobanking teams, and emphasize the importance of interdisciplinary tumor boards. Specialized skin cancer nurses and local patient advocacy groups should be involved in patient care and could be the binding link between the patients and the treatment team.

In the original version of [this article][1] ([1][2]), an error in Fig. 6C was introduced by the compositor during the production of the paper. Specifically, the Raptor Western blot was improperly shifted. The figure has been corrected in the latest online HTML and PDF versions of the article. The

Tumor initiation and metastasis formation in many cancers have been associated with emergence of a gene expression program normally active in embryonic or organ-specific stem cells. In particular, the stem cell transcription factor Sox2 is not only expressed in a variety of tumors, but is also required for their formation. Melanoma, the most aggressive skin tumor, derives from melanocytes that during development originate from neural crest stem cells. While neural crest stem cells do not express Sox2, expression of this transcription factor has been reported in melanoma. However, the role of Sox2 in melanoma is controversial. To study the requirement of Sox2 for melanoma formation, we therefore performed CRISPR-Cas9-mediated gene inactivation in human melanoma cells. In addition, we conditionally inactivated Sox2 in a genetically engineered mouse model, in which melanoma spontaneously develops in the context of an intact stroma and immune system. Surprisingly, in both models, loss of Sox2 did neither affect melanoma initiation, nor growth, nor metastasis formation. The lack of a tumorigenic role of Sox2 in melanoma might reflect a distinct stem cell program active in neural crest stem cells and during melanoma formation.Oncogene advance online publication, 3 April 2017; doi:10.1038/onc.2017.55.

SummaryBackground There is a medical need for new drugs in patients with BRAF wild-type metastatic melanoma. Pazopanib is a multitarget tyrosine kinase inhibitor with antitumour and antiangiogenic activity. Objectives The primary aim was to investigate the metabolic response to pazopanib monotherapy and pazopanib plus paclitaxel in patients with BRAF wild-type melanoma. Secondary end points were the early cytokine and chemokine profiles and histological findings. Methods Pazopanib (400 mg twice daily) was administered orally from days 1 to 10 and from days 14 to 70. An intravenous infusion with paclitaxel (150 mg m−2 body surface) was administered on days 14, 35 and 56. Metabolic response evaluation was performed before treatment, after treatment with pazopanib (day 10) and after treatment with pazopanib and paclitaxel (day 70). Skin biopsy of metastatic tissue for chemokine and cytokine expression analysis and histology and immunohistochemistry (CD68, CD163) evaluation, and blood samples were taken at the same time points. Results Two patients failed screening and 17 were dosed. Of 67 adverse events, nine (13%) were grade 3 or 4. Five of 14 evaluable patients had a partial metabolic response at day 10 under pazopanib monotherapy. The response rate at day 70 under combined pazopanib–paclitaxel treatment was 0%. Immunohistochemistry revealed an increase of M2-like macrophages in nonresponders compared with responders. We observed a significant upregulation of five cytokines (CXCL1, CXCL2, CXCL13, CCL22 and SPP1) in responding vs. nonresponding lesions. Overall, the median progression-free survival was 70 days (range 5–331), which did not differ significantly between responders (148 days) and nonresponders (70 days, P = 0·17). Conclusions In this patient population pazopanib efficacy was limited. Response is associated with low M2-like macrophage density and increased expression of several chemokines.

Targeting cyclin dependent kinases 4 and 6 (CDK4/6) represents a viable therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor in different genetic subsets of melanoma. However, continuous dosing of MEK inhibitor (MEKi) plus CDK4/6 inhibitor (CDK4/6i) elicits toxicities in patients. Utilizing an in vivo E2F reporter system, we analyzed the efficacy of MEKi plus CDK4/6i schedules in a quantitative and temporal manner. Continuous MEKi with intermittent CDK4/6i led to more complete responses as compared to either continuous CDK4/6i with intermittent MEKi or intermittent dosing of both drugs. Nevertheless, some tumors acquired resistance. Phospho-proteomic analysis of resistant tumors revealed increased phosphorylation of ribosomal S6 protein (RPS6). These data were supported by staining of patient biopsies from clinical trials, which also indicated that high phospho S6 levels may serve as a predictor of response to CDK4/6i plus targeted inhibitors. Enhanced phospho S6 provided a therapeutic window for the mTORC1/2 inhibitor, AZD2014, in MEKi plus CDK4/6i-resistant tumors. Mechanistically, aberrant regulation of NRAS was associated with resistance and sufficient to mediate resistance. This study highlights the use of an in vivo reporter model for optimization of schedules and supports targeting the mTORC1/2-S6 pathway as a salvage option to overcome MEKi plus CDK4/6i resistance. Citation Format: Jessica Teh, Phil F. Cheng, Timothy J. Purwin, Neda Nikbakht, Prem Patel, Inna Chervoneva, Ines Keibler, HooKim Kim, Michael A. Davies, Lawrence N. Kwong, Mitch P. Levesque, Reinhard Dummer, Andrew E. Aplin. In vivo E2F reporting reveals efficacious schedules of MEK1/2-CDK4/6 targeting and mTOR-S6 resistance mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2304.

Introduction The transcription factor Sall4 is a well-known developmental regulator involved in embryonic patterning and stem cell maintenance. More recently Sall4 has been suggested to be an oncofetal gene due to being expressed in many fetal tissues and malignant tumours but only rarely in normal adult tissues. The re-expression of such genes in cancer might reflect crucial processes during development, which are reactivated in neoplasias such as increased migratory and invasive capacities. Sall4 is discussed as novel marker and target in some solid tumours and haematological malignancies, however its role in melanoma is unknown. Material and methods To address a putative role of Sall4 in melanoma, we used the Tyr::Nras Q61K INK4a −/− murine melanoma model, which spontaneously develops metastatic melanoma. Those mice were crossed with Tyr::Cre ERT2 Sall4 lox/lox R26R::GFP mice, allowing us to conditionally ablate Sall4 in melanocytes and to trace Cre activity via GFP expression. We further used human melanoma patient-derived primary cell cultures for in vitro experiments and the same cell lines as xenographs in immunocompromised mice. Results and discussions Mice lacking Sall4 did not form primary tumours compared to the control animals however strikingly had more GFP +melanoma metastases in the lungs. Sall4 lox/wt animals formed tumours like control animals but also showed more lung metastases. Through RNA Sequencing on siSALL4-treated human melanoma cell lines we found that SALL4 decrease correlates with upregulation of melanoma invasiveness genes and functionally increased invasiveness in vitro . We further found by Co-IP that SALL4 binds to members of the histone deacetylase family (HDACs) and by running a ChIP-Seq for H3K27ac, we could indeed verify that knocking down SALL4 lead to increased histone acetylation of enhancer regions of the mentioned invasiveness genes. Interestingly, HDAC inhibitor (HDACi) treatment in vitro resulted in an invasiveness gene expression pattern very similar to SALL4 knock down and HDACi treatment of xenograph tumours also lead to an expression profile hinting towards increased invasiveness in vivo . Conclusion All in all we found that downregulation of Sall4 in melanoma leads to increased acetylation and expression of invasiveness genes in vitro and increased metastasis in vivo, with both phenotypes being closely mimicked by HDAC inhibitor application.

Background: Folate hydrolase-1 (FOLH1; NAALADase; PSMA) is a type II transmembrane protein that binds substrates with terminal glutamates. The MXXXL motif on the cytoplasmic N-terminal domain of FOLH1 interacts with clathrin and caveolin-1 to facilitate constitutive internalization upon substrate binding. J591, an established monoclonal antibody (AB) to FOLH1, is highly specific to and is effectively endocytosed after binding to the extracellular domain of FOLH1; J591 is presently being developed in the clinical trial setting, as a vehicle for AB-based brachytherapy in cancers that express FOLH1. Physiological FOLH1 is expressed in cellular regions that are protected by tight-junctions or the blood-brain-barrier, and are therefore not targeted by circulating J591. Functionally, FOLH1 is responsible for cerebral glutamate production. In the oncological setting, FOLH1 is upregulated throughout prostate cancer cellular membranes, and is luminally expressed by cancer neovessels. We have characterized neovascular FOLH1 expression in malignant melanoma (MM), a solid tumor of neural crest (NC) origin. Comparative RTqPCR analysis of normal skin, primary (p), and metastatic (m) MM revealed respective 10.64 and 18.21 -fold increases in FOLH1 gene-expression in pMM (p=0.0041) and mMM (p=0.042) samples as compared to normal skin. Immunohistochemistry of paraffin-embedded MM tumors revealed FOLH1 protein expression in the neovasculature of 4/11 (36%) of pMM and 9/14 (64%) of mMM cases; we noted a subset of keratinocytic and melanocytic FOLH1 positive cells. These results, and the known functional role of glutamate production in a subset of NC-derived tissue, suggest that MM cells may also express cellular FOLH1. Methods: Six (3 BRAF+; 3 BRAF-) MM cell lines were evaluated for cellular FOLH1 and Oct4 expression with J591-based and anti-Oct4 AB immunofluorescence (IF). Oct4 is a homeodomain binding protein encoded by POU5f1, widely accepted as a stem cell marker. FOLH1+ cell lines with permeablized and non-permeablized membranes were incubated with J591. The effect of γ-irradiation (0, 4.5, or 9 Gy) was also tested in FOLH1+ cell lines with harvesting 6 or 24 hours post-irradiation. Results: Ten to 40% of MM cells demonstrated intracellular FOLH1 expression in 2 BRAF-/6 MM cell lines, with a predominant perinuclear and terminal dendritic distribution (e.g., axonal morphology). IF co-labeling with J591 (cytoplasmic) and antibodies to Oct4 (nuclear) identified possible stemness of FOLH1+ / Oct4+ MM cells. RT-PCR analysis of the irradiated cell lines demonstrated ~2-fold increased FOLH1 mRNA levels in 1/2 MM cell lines. Conclusions: Herein is the first demonstration that MM cells express FOLH1. Given that FOLH1 glutamate production is closely linked to NC-originating glial cells, it can be reasonably postulated that this newly identified subset of FOLH1+/Oct4+ MM cells could be NC precursors, or cancer stem cells, for MM. Further, radiation-induced FOLH1 upregulation may increase the therapeutic ratio of AB-based brachytherapy. Citation Format: Marigdalia K. Ramirez-Fort, He Liu, Vicente Navarro, Barbara Meier, Mitch Levesque, Jonathan Moy, Jessica Vissicchio, Emmanuel Contassot, Sae Kim, Talal Syed, Michael Zhang, Vincent Tem, Paul J. Christos, Scott T. Tagawa, Neil H. Bander, Christopher S. Lange, Lars E. French. Possible cancer stem cells: Folate hydrolase-1 is expressed in a subset of Oct4-positive melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1905. doi:10.1158/1538-7445.AM2017-1905