AMBRA1 levels predict resistance to MAPK inhibitors in melanoma.

Intrinsic and acquired resistance to mitogen-activated protein kinase inhibitors (MAPKi) in melanoma remains a major therapeutic challenge. Here, we show that the clinical development of resistance to MAPKi is associated with reduced tumor expression of the melanoma suppressor Autophagy and Beclin 1 Regulator 1 (AMBRA1) and that lower expression levels of AMBRA1 predict a poor response to MAPKi treatment. Functional analyses show that loss of AMBRA1 induces phenotype switching and orchestrates an extracellular signal-regulated kinase (ERK)-independent resistance mechanism by activating focal adhesion kinase 1 (FAK1). In both in vitro and in vivo settings, melanomas with low AMBRA1 expression exhibit intrinsic resistance to MAPKi therapy but higher sensitivity to FAK1 inhibition. Finally, we show that the rapid development of resistance in initially MAPKi-sensitive melanomas can be attributed to preexisting subclones characterized by low AMBRA1 expression and that cotreatment with MAPKi and FAK1 inhibitors (FAKi) effectively prevents the development of resistance in these tumors. In summary, our findings underscore the value of AMBRA1 expression for predicting melanoma response to MAPKi and supporting the therapeutic efficacy of FAKi to overcome MAPKi-induced resistance.

An analysis pipeline for understanding 6-thioguanine effects on a mouse tumour genome.

Mouse tumour models are extensively used as a pre-clinical research tool in the field of oncology, playing an important role in anticancer drugs discovery. Accordingly, in cancer genomics research, the demand for next-generation sequencing (NGS) is increasing, and consequently, the need for data analysis pipelines is likewise growing. Most NGS data analysis solutions to date do not support mouse data or require highly specific configuration for their use. Here, we present a genome analysis pipeline for mouse tumour NGS data including the whole-genome sequence (WGS) data analysis flow for somatic variant discovery, and the RNA-seq data flow for differential expression, functional analysis and neoantigen prediction. The pipeline is based on standards and best practices and integrates mouse genome references and annotations. In a recent study, the pipeline was applied to demonstrate the efficacy of low dose 6-thioguanine (6TG) treatment on low-mutation melanoma in a pre-clinical mouse model. Here, we further this study and describe in detail the pipeline and the results obtained in terms of tumour mutational burden (TMB) and number of predicted neoantigens, and correlate these with 6TG effects on tumour volume. Our pipeline was expanded to include a neoantigen analysis, resulting in neopeptide prediction and MHC class I antigen presentation evaluation. We observed that the number of predicted neoepitopes were more accurate indicators of tumour immune control than TMB. In conclusion, this study demonstrates the usability of the proposed pipeline, and suggests it could be an essential robust genome analysis platform for future mouse genomic analysis.

AMBRA1 phosphorylation by CDK1 and PLK1 regulates mitotic spindle orientation.

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.

Metabolic modelling-based in silico drug target prediction identifies six novel repurposable drugs for melanoma.

Despite high initial response rates to targeted kinase inhibitors, the majority of patients suffering from metastatic melanoma present with high relapse rates, demanding for alternative therapeutic options. We have previously developed a drug repurposing workflow to identify metabolic drug targets that, if depleted, inhibit the growth of cancer cells without harming healthy tissues. In the current study, we have applied a refined version of the workflow to specifically predict both, common essential genes across various cancer types, and melanoma-specific essential genes that could potentially be used as drug targets for melanoma treatment. The in silico single gene deletion step was adapted to simulate the knock-out of all targets of a drug on an objective function such as growth or energy balance. Based on publicly available, and in-house, large-scale transcriptomic data metabolic models for melanoma were reconstructed enabling the prediction of 28 candidate drugs and estimating their respective efficacy. Twelve highly efficacious drugs with low half-maximal inhibitory concentration values for the treatment of other cancers, which are not yet approved for melanoma treatment, were used for in vitro validation using melanoma cell lines. Combination of the top 4 out of 6 promising candidate drugs with BRAF or MEK inhibitors, partially showed synergistic growth inhibition compared to individual BRAF/MEK inhibition. Hence, the repurposing of drugs may enable an increase in therapeutic options e.g., for non-responders or upon acquired resistance to conventional melanoma treatments.

Ambra1 modulates the tumor immune microenvironment and response to PD-1 blockade in melanoma.

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 BrafV600E /Pten-/ - genetically engineered mouse (GEM) model of melanoma, as well as GEM-derived allografts of BrafV600E /Pten-/ - and BrafV600E /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 BrafV600E /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 BrafV600E /Pten-/ -/Cdkn2a-/ - model inherently resistant to immune checkpoint blockade, knockdown of Ambra1 led to accelerated tumor growth and reduced overall survival, but at the same time conferred sensitivity to anti-PD-1 treatment. CONCLUSIONS: This study shows that loss of Ambra1 affects the TIME and the antitumor immune response in melanoma, highlighting new functions of Ambra1 in the regulation of melanoma biology.

GSNOR deficiency promotes tumor growth via FAK1 S-nitrosylation.

Nitric oxide (NO) production in the tumor microenvironment is a common element in cancer. S-nitrosylation, the post-translational modification of cysteines by NO, is emerging as a key transduction mechanism sustaining tumorigenesis. However, most oncoproteins that are regulated by S-nitrosylation are still unknown. Here we show that S-nitrosoglutathione reductase (GSNOR), the enzyme that deactivates S-nitrosylation, is hypo-expressed in several human malignancies. Using multiple tumor models, we demonstrate that GSNOR deficiency induces S-nitrosylation of focal adhesion kinase 1 (FAK1) at C658. This event enhances FAK1 autophosphorylation and sustains tumorigenicity by providing cancer cells with the ability to survive in suspension (evade anoikis). In line with these results, GSNOR-deficient tumor models are highly susceptible to treatment with FAK1 inhibitors. Altogether, our findings advance our understanding of the oncogenic role of S-nitrosylation, define GSNOR as a tumor suppressor, and point to GSNOR hypo-expression as a therapeutically exploitable vulnerability in cancer.

Thiopurine 6TG treatment increases tumor immunogenicity and response to immune checkpoint blockade.

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).

New Insights into the Phenotype Switching of Melanoma.

Melanoma is considered one of the deadliest skin cancers, partly because of acquired resistance to standard therapies. The most recognized driver of resistance relies on acquired melanoma cell plasticity, or the ability to dynamically switch among differentiation phenotypes. This confers the tumor noticeable advantages. During the last year, two new features have been included in the hallmarks of cancer, namely "Unlocking phenotypic plasticity" and "Non-mutational epigenetic reprogramming". Such are inextricably intertwined as, most of the time, plasticity is not discernable at the genetic level, as it rather consists of epigenetic reprogramming heavily influenced by external factors. By analyzing current literature, this review provides reasoning about the origin of plasticity and clarifies whether such features already exist among tumors or are acquired by selection. Moreover, markers of plasticity, molecular effectors, and related tumor advantages in melanoma will be explored. Ultimately, as this new branch of tumor biology opened a wide landscape of therapeutic possibilities, in the final paragraph of this review, we will focus on newly characterized drugs targeting melanoma plasticity.

The Cancermuts software package for the prioritization of missense cancer variants: a case study of AMBRA1 in melanoma.

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.

AMBRA1 and FAK1: crosstalking for improved targeted therapy in melanoma.

Through genetically engineered mouse models of melanoma, we identified Autophagy/beclin 1 regulator 1 (Ambra1) as novel tumor-suppressor in melanoma. In these settings, loss of Ambra1 associated with the hyperactivation of focal adhesion kinase 1 (Fak1) signaling, the inhibition of which resulted in reduced tumor growth and invasiveness. We therefore propose FAK1 inhibition for current melanoma therapy in AMBRA1-low tumors. ABBREVIATIONS: AKT, serine/threonine kinase 1; AMBRA1, autophagy/beclin 1 regulator 1; BRAF, v-raf murine sarcoma viral oncogene homolog; BRAFi, BRAF inhibitor; CCLE, Cancer Cell Line Encyclopedia;g ESTDAB, European Searchable Tumor Line Database; FAK1, focal adhesion kinase 1; FAKi, FAK1 inhibitor; LMC, Leeds Melanoma Cohort; MEK, MAPK/ERK kinase; PP2A, protein phosphatase 2A; PTEN, phosphatase and tensin homolog; TCGA-SKCM, The Cancer Genome Atlas - Skin Cutaneous Melanoma; YAP, yes-associated protein 1.

AMBRA1 has an impact on melanoma development beyond autophagy.

AMBRA1 (autophagy/beclin 1 regulator 1) is a multifunctional scaffold protein involved in several cellular processes spanning from cell proliferation to apoptosis and to regulation of macroautophagy/autophagy. Our recent publication revealed that Ambra1 has an antitumorigenic role in melanoma, the most aggressive and deadly skin cancer. We have indeed collected data indicating that the increased proliferative and invasive/metastatic features that we observed in ambra1-ablated melanomas are related to a remarkable regulation by Ambra1 on cellular processes which are beyond autophagy. Our study therefore sheds light on intriguing processes affected by Ambra1 which can be exploited as therapeutic targets in AMBRA1 low-expressing melanoma.

Loss of Ambra1 promotes melanoma growth and invasion.

Melanoma is the deadliest skin cancer. Despite improvements in the understanding of the molecular mechanisms underlying melanoma biology and in defining new curative strategies, the therapeutic needs for this disease have not yet been fulfilled. Herein, we provide evidence that the Activating Molecule in Beclin-1-Regulated Autophagy (Ambra1) contributes to melanoma development. Indeed, we show that Ambra1 deficiency confers accelerated tumor growth and decreased overall survival in Braf/Pten-mutated mouse models of melanoma. Also, we demonstrate that Ambra1 deletion promotes melanoma aggressiveness and metastasis by increasing cell motility/invasion and activating an EMT-like process. Moreover, we show that Ambra1 deficiency in melanoma impacts extracellular matrix remodeling and induces hyperactivation of the focal adhesion kinase 1 (FAK1) signaling, whose inhibition is able to reduce cell invasion and melanoma growth. Overall, our findings identify a function for AMBRA1 as tumor suppressor in melanoma, proposing FAK1 inhibition as a therapeutic strategy for AMBRA1 low-expressing melanoma.

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

Altered Tregs Differentiation and Impaired Autophagy Correlate to Atherosclerotic Disease.

Atherosclerosis is a progressive vascular disease representing the primary cause of morbidity and mortality in developed countries. Formerly, atherosclerosis was considered as a mere passive accumulation of lipids in blood vessels. However, it is now clear that atherosclerosis is a complex and multifactorial disease, in which the involvement of immune cells and inflammation play a key role. A variety of studies have shown that autophagy-a cellular catalytic mechanism able to remove injured cytoplasmic components in response to cellular stress-may be proatherogenic. So far, in this context, its role has been investigated in smooth muscle cells, macrophages, and endothelial cells, while the function of this catabolic protective process in lymphocyte functionality has been overlooked. The few studies carried out so far, however, suggested that autophagy modulation in lymphocyte subsets may be functionally related to plaque formation and development. Therefore, in this research, we aimed at better clarifying the role of lymphocyte subsets, mainly regulatory T cells (Tregs), in human atherosclerotic plaques and in animal models of atherosclerosis investigating the contribution of autophagy on immune cell homeostasis. Here, we investigate basal autophagy in a mouse model of atherosclerosis, apolipoprotein E (ApoE)-knockout (KO) mice, and we analyze the role of autophagy in driving Tregs polarization. We observed defective maturation of Tregs from ApoE-KO mice in response to tumor growth factor-ß (TGFß). TGFß is a well-known autophagy inducer, and Tregs maturation defects in ApoE-KO mice seem to be related to autophagy impairment. In this work, we propose that autophagy underlies Tregs maturation, advocating that the study of this process in atherosclerosis may open new therapeutic strategies.

Mitophagy contributes to alpha-tocopheryl succinate toxicity in GSNOR-deficient hepatocellular carcinoma.

The downregulation of the denitrosylating enzyme S-nitrosoglutathione reductase (GSNOR, EC:1.1.1.284), is a feature of hepatocellular carcinoma (HCC). This condition causes mitochondrial rearrangements that sensitize these tumors to mitochondrial toxins, in particular to the mitochondrial complex II inhibitor alpha-tocopheryl succinate (αTOS). It has also been reported the GSNOR depletion impairs the selective degradation of mitochondria through mitophagy; however, if this contributes to GSNOR-deficient HCC cell sensitivity to αTOS and can be applied to anticancer therapies, is still not known. Here, we provide evidence that GSNOR-deficient HCC cells show defective mitophagy which contributes to αTOS toxicity. Mitophagy inhibition by Parkin (EC: 2.3.2.31) depletion enhances αTOS anticancer effects, thus suggesting that this drug could be effective in treating mitophagy-defective tumors.

Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites.

The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.

The Complex Role of Autophagy in Melanoma Evolution: New Perspectives From Mouse Models.

Despite tremendous efforts in the last decade to improve treatments, melanoma still represents a major therapeutic challenge and overall survival of patients remains poor. Therefore, identifying new targets to counteract melanoma is needed. In this scenario, autophagy, the "self-eating" process of the cell, has recently arisen as new potential candidate in melanoma. Alongside its role as a recycling mechanism for dysfunctional and damaged cell components, autophagy also clearly sits at a crossroad with metabolism, thereby orchestrating cell proliferation, bioenergetics and metabolic rewiring, all hallmarks of cancer cells. In this regard, autophagy, both in tumor and host, has been flagged as an essential player in melanomagenesis and progression. To pave the way to a better understanding of such a complex interplay, the use of genetically engineered mouse models (GEMMs), as well as syngeneic mouse models, has been undoubtedly crucial. Herein, we will explore the latest discoveries in the field, with particular focus on the potential of these models in unraveling the contribution of autophagy in melanoma, along with the therapeutic advantages that may arise.

S-nitrosylation drives cell senescence and aging in mammals by controlling mitochondrial dynamics and mitophagy.

S-nitrosylation, a prototypic redox-based posttranslational modification, is frequently dysregulated in disease. S-nitrosoglutathione reductase (GSNOR) regulates protein S-nitrosylation by functioning as a protein denitrosylase. Deficiency of GSNOR results in tumorigenesis and disrupts cellular homeostasis broadly, including metabolic, cardiovascular, and immune function. Here, we demonstrate that GSNOR expression decreases in primary cells undergoing senescence, as well as in mice and humans during their life span. In stark contrast, exceptionally long-lived individuals maintain GSNOR levels. We also show that GSNOR deficiency promotes mitochondrial nitrosative stress, including excessive S-nitrosylation of Drp1 and Parkin, thereby impairing mitochondrial dynamics and mitophagy. Our findings implicate GSNOR in mammalian longevity, suggest a molecular link between protein S-nitrosylation and mitochondria quality control in aging, and provide a redox-based perspective on aging with direct therapeutic implications.

Autophagy and the Cell Cycle: A Complex Landscape.

Autophagy is a self-degradation pathway, in which cytoplasmic material is sequestered in double-membrane vesicles and delivered to the lysosome for degradation. Under basal conditions, autophagy plays a homeostatic function. However, in response to various stresses, the pathway can be further induced to mediate cytoprotection. Defective autophagy has been linked to a number of human pathologies, including neoplastic transformation, even though autophagy can also sustain the growth of tumor cells in certain contexts. In recent years, a considerable correlation has emerged between autophagy induction and stress-related cell-cycle responses, as well as unexpected roles for autophagy factors and selective autophagic degradation in the process of cell division. These advances have obvious implications for our understanding of the intricate relationship between autophagy and cancer. In this review, we will discuss our current knowledge of the reciprocal regulation connecting the autophagy pathway and cell-cycle progression. Furthermore, key findings involving nonautophagic functions for autophagy-related factors in cell-cycle regulation will be addressed.

S-nitrosylation of the Mitochondrial Chaperone TRAP1 Sensitizes Hepatocellular Carcinoma Cells to Inhibitors of Succinate Dehydrogenase.

S-nitrosoglutathione reductase (GSNOR) represents the best-documented denitrosylase implicated in regulating the levels of proteins posttranslationally modified by nitric oxide on cysteine residues by S-nitrosylation. GSNOR controls a diverse array of physiologic functions, including cellular growth and differentiation, inflammation, and metabolism. Chromosomal deletion of GSNOR results in pathologic protein S-nitrosylation that is implicated in human hepatocellular carcinoma (HCC). Here we identify a metabolic hallmark of aberrant S-nitrosylation in HCC and exploit it for therapeutic gain. We find that hepatocyte GSNOR deficiency is characterized by mitochondrial alteration and by marked increases in succinate dehydrogenase (SDH) levels and activity. We find that this depends on the selective S-nitrosylation of Cys(501) in the mitochondrial chaperone TRAP1, which mediates its degradation. As a result, GSNOR-deficient cells and tumors are highly sensitive to SDH inhibition, namely to α-tocopheryl succinate, an SDH-targeting molecule that induced RIP1/PARP1-mediated necroptosis and inhibited tumor growth. Our work provides a specific molecular signature of aberrant S-nitrosylation in HCC, a novel molecular target in SDH, and a first-in-class therapy to treat the disease. Cancer Res; 76(14); 4170-82. ©2016 AACR.