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Acknowledgements We wish to thank M. Canney, V. Unterkircher, R. Laricchia and M. Salomé for excellent technical assistance, and M. Acuña Villa and M. W. Bennett for editorial and secretarial work. We also thank S. Campello for critical reading of the manuscript. We are indebted to R. Sears (Portland, Oregon, USA), A. C. Gingras (Toronto, Canada) and A. Teleman and K. Dimitriadis (Heidelberg, Germany) for providing us with V5–Flag–c-Myc and Flag–PR65A constructs and Tsc2 MEFs, respectively, and to S. Cannata (Rome) for his advice on histopathology. This work was supported by grants from KBVU (R72-A4408), Lundbeck Foundation (R167-2013-16100), Novo Nordisk Foundation (7559), The Bjarne Saxhof Foundation, AIRC (IG2010 and IG2012 to both F.C. and M.P.), and in part from FISM (2009), the Telethon Foundation (GGP10225), the Italian Ministry of University and Research (PRIN 2009 and FIRB Accordi di Programma 2011) and the Italian Ministry of Health (RF 2009). V.C. is supported by the Lundbeck Foundation (R165-2013-15982). Also, we are grateful to the Spanish Ministry of Economy and Competitiveness (MINECO) (PS09/01401; PI12/02248, FR2009-0052 and IT2009-0053) and to Fundación Mutua Madrileña (AP101042012) for funding the laboratory of G.V., Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

BACKGROUND: Loss of Ambra1 (autophagy and beclin 1 regulator 1), a multifunctional scaffold protein, promotes the formation of nevi and contributes to several phases of melanoma development. The suppressive functions of Ambra1 in melanoma are mediated by negative regulation of cell proliferation and invasion; however, evidence suggests that loss of Ambra1 may also affect the melanoma microenvironment. Here, we investigate the possible impact of Ambra1 on antitumor immunity and response to immunotherapy. METHODS: This study was performed using an Ambra1-depleted Braf(V600E) /Pten(-/) (-) genetically engineered mouse (GEM) model of melanoma, as well as GEM-derived allografts of Braf(V600E) /Pten(-/) (-) and Braf(V600E) /Pten(-/) (-)/Cdkn2a(-/) (-) tumors with Ambra1 knockdown. The effects of Ambra1 loss on the tumor immune microenvironment (TIME) were analyzed using NanoString technology, multiplex immunohistochemistry, and flow cytometry. Transcriptome and CIBERSORT digital cytometry analyses of murine melanoma samples and human melanoma patients (The Cancer Genome Atlas) were applied to determine the immune cell populations in null or low-expressing AMBRA1 melanoma. The contribution of Ambra1 on T-cell migration was evaluated using a cytokine array and flow cytometry. Tumor growth kinetics and overall survival analysis in Braf(V600E) /Pten(-/) (-)/Cdkn2a(-/) (-) mice with Ambra1 knockdown were evaluated prior to and after administration of a programmed cell death protein-1 (PD-1) inhibitor. RESULTS: Loss of Ambra1 was associated with altered expression of a wide range of cytokines and chemokines as well as decreased infiltration of tumors by regulatory T cells, a subpopulation of T cells with potent immune-suppressive properties. These changes in TIME composition were associated with the autophagic function of Ambra1. In the Braf(V600E) /Pten(-/) (-)/Cdkn2a(-/) (-) model inherently resistant to immune checkpoint blockade, knockdown of Ambra1 led to accelerated tumor

Immune-checkpoint inhibitors (ICI) are highly effective in reinvigorating T cells to attack cancer. Nevertheless, a large subset of patients fails to benefit from ICI, partly due to lack of the cancer neoepitopes necessary to trigger an immune response. In this study, we used the thiopurine 6-thioguanine (6TG) to induce random mutations and thus increase the level of neoepitopes presented by tumor cells. Thiopurines are prodrugs which are converted into thioguanine nucleotides that are incorporated into DNA (DNA-TG), where they can induce mutation through single nucleotide mismatching. In a pre-clinical mouse model of a mutation-low melanoma cell line, we demonstrated that 6TG induced clinical-grade DNA-TG integration resulting in an improved tumor control that was strongly T cell dependent. 6TG exposure increased the tumor mutational burden, without affecting tumor cell proliferation and cell death. Moreover, 6TG treatment re-shaped the tumor microenvironment by increasing T and NK immune cells, making the tumors more responsive to immune-checkpoint blockade. We further validated that 6TG exposure improved tumor control in additional mouse models of melanoma. These findings have paved the way for a phase I/II clinical trial that explores whether treatment with thiopurines can increase the proportion of otherwise treatment-resistant cancer patients who may benefit from ICI therapy (NCT05276284).

Cancer genomics and cancer mutation databases have made an available wealth of information about missense mutations found in cancer patient samples. Contextualizing by means of annotation and predicting the effect of amino acid change help identify which ones are more likely to have a pathogenic impact. Those can be validated by means of experimental approaches that assess the impact of protein mutations on the cellular functions or their tumorigenic potential. Here, we propose the integrative bioinformatic approach Cancermuts, implemented as a Python package. Cancermuts is able to gather known missense cancer mutations from databases such as cBioPortal and COSMIC, and annotate them with the pathogenicity score REVEL as well as information on their source. It is also able to add annotations about the protein context these mutations are found in, such as post-translational modification sites, structured/unstructured regions, presence of short linear motifs, and more. We applied Cancermuts to the intrinsically disordered protein AMBRA1, a key regulator of many cellular processes frequently deregulated in cancer. By these means, we classified mutations of AMBRA1 in melanoma, where AMBRA1 is highly mutated and displays a tumor-suppressive role. Next, based on REVEL score, position along the sequence, and their local context, we applied cellular and molecular approaches to validate the predicted pathogenicity of a subset of mutations in an in vitro melanoma model. By doing so, we have identified two AMBRA1 mutations which show enhanced tumorigenic potential and are worth further investigation, highlighting the usefulness of the tool. Cancermuts can be used on any protein targets starting from minimal information, and it is available at https://www.github.com/ELELAB/cancermuts as free software.