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Kinases are enzymes that play a critical role in governing essential biological processes. Due to their pivotal involvement in cancer cell signaling, they have become key targets in the development of anti-cancer drugs. Among these drugs, those containing the 2,4-dihalophenyl moiety demonstrated significant potential. Here we show how this moiety, particularly the 2-fluoro-4-iodophenyl one, is crucial for the structural stability of the formed drug-enzyme complexes. Crystallographic analysis of reported kinase-inhibitor complex structures highlights the role of the halogen bonding that this moiety forms with specific residues of the kinase binding site. This interaction is not limited to FDA-approved MEK inhibitors, but it is also relevant for other kinase inhibitors, indicating its broad relevance in the design of this class of drugs., (© 2024 Wiley‐VCH GmbH.)

Caloric Restriction (CR) has established anti-cancer effects, but its clinical relevance and molecular mechanism remain largely undefined. Here, we investigate CR's impact on several mouse models of Acute Myeloid Leukemias, including Acute Promyelocytic Leukemia, a subtype strongly affected by obesity. After an initial marked anti-tumor effect, lethal disease invariably re-emerges. Initially, CR leads to cell-cycle restriction, apoptosis, and inhibition of TOR and insulin/IGF1 signaling. The relapse, instead, is associated with the non-genetic selection of Leukemia Initiating Cells and the downregulation of double-stranded RNA (dsRNA) sensing and Interferon (IFN) signaling genes. The CR-induced adaptive phenotype is highly sensitive to pharmacological or genetic ablation of LSD1, a lysine demethylase regulating both stem cells and dsRNA/ IFN signaling. CR + LSD1 inhibition leads to the re-activation of dsRNA/IFN signaling, massive RNASEL-dependent apoptosis, and complete leukemia eradication in ~90% of mice. Importantly, CR-LSD1 interaction can be modeled in vivo and in vitro by combining LSD1 ablation with pharmacological inhibitors of insulin/IGF1 or dual PI3K/MEK blockade. Mechanistically, insulin/IGF1 inhibition sensitizes blasts to LSD1-induced death by inhibiting the anti-apoptotic factor CFLAR. CR and LSD1 inhibition also synergize in patient-derived AML and triple-negative breast cancer xenografts. Our data provide a rationale for epi-metabolic pharmacologic combinations across multiple tumors., (© 2024. The Author(s).)

Background: The current therapeutic algorithm for Advanced Stage Melanoma comprises of alternating lines of Targeted and Immuno-therapy, mostly via Immune-Checkpoint blockade. While Comprehensive Genomic Profiling of solid tumours has been approved as a companion diagnostic, still no approved predictive biomarkers are available for Melanoma aside from BRAF mutations and the controversial Tumor Mutational Burden. This study presents the results of a Multi-Centre Observational Clinical Trial of Comprehensive Genomic Profiling on Target and Immuno-therapy treated advanced Melanoma., Methods: 82 samples, collected from 7 Italian Cancer Centres of FFPE-archived Metastatic Melanoma and matched blood were sequenced via a custom-made 184-gene amplicon-based NGS panel. Sequencing and bioinformatics analysis was performed at a central hub. Primary analysis was carried out via the Ion Reporter framework. Secondary analysis and Machine Learning modelling comprising of uni and multivariate, COX/Lasso combination, and Random Forest, was implemented via custom R/Python scripting., Results: The genomics landscape of the ACC-mela cohort is comparable at the somatic level for Single Nucleotide Variants and INDELs aside a few gene targets. All the clinically relevant targets such as BRAF and NRAS have a comparable distribution thus suggesting the value of larger scale sequencing in melanoma. No comparability is reached at the CNV level due to biotechnological biases and cohort numerosity. Tumour Mutational Burden is slightly higher in median for Complete Responders but fails to achieve statistical significance in Kaplan-Meier survival analysis via several thresholding strategies. Mutations on PDGFRB, NOTCH3 and RET were shown to have a positive effect on Immune-checkpoint treatment Overall and Disease-Free Survival, while variants in NOTCH4 were found to be detrimental for both endpoints., Conclusions: The results presented in this study show the value and the challenge of a genomics-driven network trial. The data can be also a valuable resource as a validation cohort for Immunotherapy and Target therapy genomic biomarker research., (© 2023. The Author(s).)

Despite the remarkable improvements achieved in the management of metastatic melanoma, there are still unmet clinical needs. A considerable fraction of patients does not respond to immune and/or targeted therapies owing to primary and acquired resistance, high-grade immune-related adverse events, and a lack of alternative treatment options. To design effective combination therapies, we set up a functional ex vivo preclinical assay on the basis of a drop-out genetic screen in metastatic melanoma patient-derived xenografts. We showed that this approach can be used to isolate actionable vulnerabilities predictive of drug efficacy. In particular, we highlighted that the dual targeting of AURKA and MAPK/extracellular signal-regulated kinase kinase employing the combination of alisertib and trametinib is highly effective in a cohort of metastatic melanoma patient-derived xenografts, both ex vivo and in vivo. Alisertib and trametinib combination therapy outperforms standard-of-care therapy in both BRAF-mutant patient-derived xenografts and targeted therapy-resistant models. Furthermore, alisertib and trametinib treatment modulates several critical cancer pathways, including an early metabolic reprogramming that leads to the transcriptional upregulation of the fatty acid oxidation pathway. This acquired trait unveiled an additional point of intervention for pharmacological targeting, and indeed, the triple combination of alisertib and trametinib with the fatty acid oxidation inhibitor etomoxir proved to be further beneficial, inducing tumor regression and remarkably prolonging the overall survival of the mice., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

Birt-Hogg-Dubé (BHD) syndrome is an inherited familial cancer syndrome characterized by the development of cutaneous lesions, pulmonary cysts, renal tumors and cysts and caused by loss-of-function pathogenic variants in the gene encoding the tumor-suppressor protein folliculin (FLCN). FLCN acts as a negative regulator of TFEB and TFE3 transcription factors, master controllers of lysosomal biogenesis and autophagy, by enabling their phosphorylation by the mechanistic Target Of Rapamycin Complex 1 (mTORC1). We have previously shown that deletion of Tfeb rescued the renal cystic phenotype of kidney-specific Flcn KO mice. Using Flcn/Tfeb/Tfe3 double and triple KO mice, we now show that both Tfeb and Tfe3 contribute, in a differential and cooperative manner, to kidney cystogenesis. Remarkably, the analysis of BHD patient-derived tumor samples revealed increased activation of TFEB/TFE3-mediated transcriptional program and silencing either of the two genes rescued tumorigenesis in human BHD renal tumor cell line-derived xenografts (CDXs). Our findings demonstrate in disease-relevant models that both TFEB and TFE3 are key drivers of renal tumorigenesis and suggest novel therapeutic strategies based on the inhibition of these transcription factors., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

The protocadherin proteins are cell adhesion molecules at the crossroad of signaling pathways playing a major role in neuronal development. It is now understood that their role as signaling hubs is not only important for the normal physiology of cells but also for the regulation of hallmarks of cancerogenesis. Importantly, protocadherins form a cluster of genes that are regulated by DNA methylation. We have identified for the first time that PCDHB15 gene is DNA-hypermethylated on its unique exon in the metastatic melanoma-derived cell lines and patients' metastases compared to primary tumors. This DNA hypermethylation silences the gene, and treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine reinduces its expression. We explored the role of PCDHB15 in melanoma aggressiveness and showed that overexpression impairs invasiveness and aggregation of metastatic melanoma cells in vitro and formation of lung metastasis in vivo. These findings highlight important modifications of the methylation of the PCDHβ genes in melanoma and support a functional role of PCDHB15 silencing in melanoma aggressiveness., (© 2022. The Author(s).)

Melanoma is a deadly form of cancer characterized by remarkable therapy resistance. Analyzing the transcriptome of MAPK inhibitor sensitive- and resistant-melanoma, we discovered that APAF-1 is negatively regulated by MITF in resistant tumors. This study identifies the MITF/APAF-1 axis as a molecular driver of MAPK inhibitor resistance. A drug-repositioning screen identified quinacrine and methylbenzethonium as potent activators of apoptosis in a context that mimics drug resistance mediated by APAF-1 inactivation. The compounds showed anti-tumor activity in in vitro and in vivo models, linked to suppression of MITF function. Both drugs profoundly sensitize melanoma cells to MAPK inhibitors, regulating key signaling networks in melanoma, including the MITF/APAF-1 axis. Significant activity of the two compounds in inhibiting specific epigenetic modulators of MITF/APAF-1 expression, such as histone deacetylases, was observed. In summary, we demonstrate that targeting the MITF/APAF-1 axis may overcome resistance and could be exploited as a potential therapeutic approach to treat resistant melanoma., Competing Interests: Declaration of interests P.A.A. reports grants and/or personal fees from BMS, Roche-Genentech and Array, MSD, Novartis, Merck Serono, Pierre Fabre, Incyte, Genmab, NewLink Genetics, Medimmune, AstraZeneca, Syndax, Sun Pharma, Sanofi, Idera, Ultimovacs, Sandoz, Immunocore, 4SC, Alkermes, and Nektar, Italfarmaco, outside the submitted work. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients' outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises. We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis. A total of 229 differentially hypermethylated genes was commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients. Five Cytosine-phosphate-Guanine (CpGs) identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations., Competing Interests: AC, CD, LP, LL, MB, JT, RH, CJ, LL, AD, GF, AD, FB, DH, JT, CE, JR, PA No competing interests declared, (© 2022, Carrier et al.)

Deubiquitinating enzymes are key regulators of the ubiquitin-proteasome system and cell cycle, and their dysfunction leads to tumorigenesis. Our in vivo drop-out screens in patient-derived xenograft models identify USP7 as a regulator of melanoma. We show that USP7 downregulation induces cellular senescence, arresting melanoma growth in vivo and proliferation in vitro in BRAF- and NRAS-mutant melanoma. We provide a comprehensive understanding of targets and networks affected by USP7 depletion by performing a global transcriptomic and proteomics analysis. We show that RRM2 is a USP7 target and is regulated by USP7 during S phase of the cell cycle. Ectopic expression of RRM2 in USP7-depleted cells rescues the senescent phenotype. Pharmacological inhibition of USP7 by P5091 phenocopies the shUSP7-induced senescent phenotype. We show that the bifunctional histone deacetylase (HDAC)/LSD1 inhibitor domatinostat has an additive antitumor effect, eliminating P5091-induced senescent cells, paving the way to a therapeutic combination for individuals with melanoma., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Long non-coding RNAs (lncRNAs) are key regulators of numerous intracellular processes leading to tumorigenesis. They are frequently deregulated in cancer, functioning as oncogenes or tumor suppressors. As they act through multiple mechanisms, it is not surprising that they may exert dual functions in the same tumor. In melanoma, a highly invasive and metastatic tumor with the propensity to rapidly develop drug resistance, lncRNAs play different roles in: (i) guiding the phenotype switch and leading to metastasis formation; (ii) predicting the response of melanoma patients to immunotherapy; (iii) triggering adaptive responses to therapy and acquisition of drug resistance phenotypes. In this review we summarize the most recent findings on the lncRNAs involved in melanoma growth and spreading to distant sites, focusing on their role as biomarkers for disease diagnosis and patient prognosis, or targets for novel therapeutic approaches.