MAPK inhibitors (MAPKi) have revolutionized the treatment of patients with advanced melanoma. However, primary and acquired resistance mechanisms limit their efficacy. Predicting MAPKi response from the tumor baseline features remains challenging due to the limited size of patient cohorts. Therefore, we collected data from nine different patient cohorts (total n = 417 patients with advanced melanoma treated with MAPKi) to identify clinical and molecular features. Our curated dataset, named MelanoDB, includes whole or partial exome sequencing data for 191 patients, copy number alteration information for 66 patients, and gene expression data for 132 patients. We provide a web application to explore the integrated dataset and data distribution across the collected studies, and we share this dataset with the scientific community according to the Findable, Accessible, Interoperable, Reusable (FAIR) principles.

Baseline genomic data have not demonstrated significant value for predicting the response duration to MAPK inhibitors (MAPKi) in patients with advanced BRAFV600-mutated melanoma. We used machine learning algorithms and pre-processed genomic data to test whether they could contain useful information to improve the progression-free survival (PFS) prediction. This exploratory analysis compared the predictive performance of a dataset that contained clinical features alone and supplemented with baseline genomic data. In the evaluation set (two cohorts, n = 111), the cross-validated model performance improved when pre-processed genomic data, such as mutation rates, were added to the clinical features. In the validation dataset (two cohorts, n = 73), the best model with genomic data outperformed the best model with clinical features alone. Finally, our best model outperformed with baseline genomic data, increasing the number of patients with a correctly predicted relapse by between +12% and +28%. In our models, baseline genomic data improved the prediction of response duration and could be incorporated into the development of predictive models of MAPKi treatment in melanoma.

Cutaneous melanoma is an invasive cancer with a worldwide annual death toll of 57,000 (Arnold et al., JAMA Dermatol 2022). In a metastatic state, surgical interventions are not curative and must be coupled with targeted therapy, or immunotherapy. However, resistance appears almost systematically and late-stage prognosis can remain poor. The complexity to eradicate melanoma stems from its plasticity; these cancer cells continually adapt to the tumor microenvironment, which leads to treatment resistance. Our primary assumption is that therapeutic resistance relies in part on a series of non-genetic transitions including changes in the metabolic states of these cancer cells. The 3D spatial distribution of blood vessels that are sources of nutrition and oxygen that drive this metabolic status is an important variable for understanding zoning aspects of this adaptation process. Using Whole Slide Images (WSI) of melanoma tumors from Patient-Derived Xenograft (PDX) mouse models, we build 3D vascular models to help predict and understand the metabolic states of cancer cells within the tumor. Our 3D reconstruction pipeline was based on PDX tumor samples sectioned over 2mm depth and stained with Hematoxylin and Eosin (H&E). The pipeline involves three primary steps, including 2D vessel segmentation using Deep Learning, intensity- and affine-based image registration, and 3D reconstruction using interpolation and 3D rendering (allowing for better interaction with biologists, pathologists, and clinicians). The originality of our computer-assisted pipeline is its capability to (a) deal with sparse data (i.e., not all tissue sections were readily available), and (b) adapt to a multitude of WSI-related challenges (e.g., epistemic uncertainty, extended processing times due to WSI scale, etc.). We posit both our 3D reconstruction pipeline, quantitative results of the major stages of the process, and a detailed illustration of the challenges faced, presenting resolutions to improve the pipeline's efficiency.

Purpose: Baseline genomic data have not demonstrated signifi cant value for predicting the response to MAPK inhibitors (MAPKi) in patients with BRAF V600 -mutated melanoma. We used machine learning algorithms and pre-processed genomic data to test whether they could contain useful information to improve the progression-free survival (PFS) prediction. Experimental design: This exploratory analysis compared the predictive performance of a dataset that contained clinical features alone and supplemented with baseline genomic data. Whole and partial exon sequencing data from four cohorts of patients with BRAF V600 -mutated melanoma treated with MAPKi were used: two cohorts as training/evaluation set (n = 111) and two as validation set (n = 73). Genomic data were pre-processed using three strategies to generate eight different genomic datasets. Several machine learning algorithms and one statistical algorithm were employed to predict PFS. The performance of these survival models was assessed using the concordance index, time-dependent receiver operating characteristic (ROC) curve and Brier score. Results: The cross-validated model performance improved when pre-processed genomic data, such as mutation rates, were added to the clinical features. In the validation dataset, the best model with genomic data outperformed the best model with clinical features alone. The trend towards improved prediction with baseline genomic data was maintained when data were censored according to the two clinical setting scenarios (duration of clinical benefi t and progression before 12 months). Conclusion: In our models, baseline genomic data improved the prediction of response duration and could be incorporated into the development of predictive models of response pattern to MAPKi in melanoma.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis work has been supported by the INCa-Canceropole GSO (Programme Emergence Intelligence Artificielle #2022/IA20 and Programme Emergence #2018/E1), the Fondation ARC pour la recherche sur le cancer (Projets ARC 2022 PJA2 #ARCPJA2022060005107), and the Ligue nationale contre le cancer (Comite du Gard JPB/GA/MV/39/2019). SD is supported by the Agence nationale pour la recherche (Intelligence Artificielle en Sante et Environnement / AXIAUM #ANR/20/THIA/0005/01) and the University of Montpellier/Ecole Doctorale CBS2.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethics Committee of the University of Montpellier (France): favorable advice UM 2022-009bisI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors.

The MAPK/ERK pathway is an essential intracellular signaling pathway. Its deregulation is involved in tumor transformation and progression. The discovery of activating mutations of BRAF in various cancers has opened new therapeutic avenues with BRAF protein kinase inhibitors. Depending on the type of cancers, these inhibitors have shown either insufficient efficacy due to primary resistance of tumor cells or transient efficacy due to the development of acquired resistance. In this review, we revisit the discoveries that led to the development of BRAF inhibitors and detail the molecular and cellular mechanisms of resistance in cancers treated with these inhibitors. Understanding these mechanisms is crucial for developing more efficient therapeutic strategies.

La voie de signalisation MAPK/ERK est une voie centrale de la signalisation intracellulaire. Sa dérégulation participe à la transformation et la progression tumorales. Dans plusieurs cancers, la découverte de mutations activatrices de BRAF, à l’origine de l’activation de cette voie, a ouvert de nouvelles perspectives thérapeutiques avec le développement d’inhibiteurs spécifiques de la protéine. Selon les cancers, ces inhibiteurs ont cependant montré soit une efficacité insuffisante, due à la résistance primaire des cellules tumorales, soit une efficacité transitoire, due à l’apparition d’une résistance acquise. Dans cette revue, nous revenons sur les découvertes qui ont conduit au développement de ces inhibiteurs de BRAF. Nous détaillons également les mécanismes moléculaires et cellulaires de la résistance à ces inhibiteurs observée dans différents types de cancers. Comprendre ces mécanismes est en effet primordial pour développer des stratégies thérapeutiques qui soient plus efficaces.

Spleen tyrosine kinase (SYK) can behave as an oncogene or a tumor suppressor, depending on the cell and tissue type. As pharmacological SYK inhibitors are currently evaluated in clinical trials, it is important to gain more information on the molecular mechanisms underpinning these opposite roles. To this aim, we reconstructed and compared its signaling networks using phosphoproteomic data from breast cancer and Burkitt lymphoma cell lines where SYK behaves as a tumor suppressor and promoter. Bioinformatic analyses allowed for unveiling the main differences in signaling pathways, network topology and signal propagation from SYK to its potential effectors. In breast cancer cells, the SYK target-enriched signaling pathways included intercellular adhesion and Hippo signaling components that are often linked to tumor suppression. In Burkitt lymphoma cells, the SYK target-enriched signaling pathways included molecules that could play a role in SYK pro-oncogenic function in B-cell lymphomas. Several protein interactions were profoundly rewired in the breast cancer network compared with the Burkitt lymphoma network. These data demonstrate that proteomic profiling combined with mathematical network modeling allows untangling complex pathway interplays and revealing difficult to discern interactions among the SYK pathways that positively and negatively affect tumor formation and progression.

Clinical data suggest that the protein tyrosine phosphatase PTPN13 exerts an anti-oncogenic effect. Its exact role in tumorigenesis remains, however, unclear due to its negative impact on FAS receptor-induced apoptosis. Methods: We crossed transgenic mice deleted for PTPN13 phosphatase activity with mice that overexpress human HER2 to assess the exact role of PTPN13 in tumor development and aggressiveness. To determine the molecular mechanism underlying the PTPN13 tumor suppressor activity we developed isogenic clones of the aggressive human breast cancer cell line MDA-MB-231 overexpressing either wild type or a catalytically-inactive mutant PTPN13 and subjected these to phosphoproteomic and gene ontology analyses. We investigated the PTPN13 consequences on cell aggressiveness using wound healing and Boyden chamber assays, on intercellular adhesion using videomicroscopy, cell aggregation assay and immunofluorescence. Results: The development, growth and invasiveness of breast tumors were strongly increased by deletion of the PTPN13 phosphatase activity in transgenic mice. We observed that PTPN13 phosphatase activity is required to inhibit cell motility and invasion in the MDA-MB-231 cell line overexpressing PTPN13. In vivo, the negative PTPN13 effect on tumor invasiveness was associated with a mesenchymal-to-epithelial transition phenotype in athymic mice xenografted with PTPN13-overexpressing MDA-MB-231 cells, as well as in HER2-overexpressing mice with wild type PTPN13, compared to HER2-overexpressing mice that lack PTPN13 phosphatase activity. Phosphoproteomic and gene ontology analyses indicated a role of PTPN13 in the regulation of intercellular junction-related proteins. Finally, protein localization studies in MDA-MB-231 cells and HER2-overexpressing mice tumors confirmed that PTPN13 stabilizes intercellular adhesion and promotes desmosome formation. Conclusions: These data provide the first evidence for the negative role of PTPN13 in breast tumor invasiveness and highlight its involvement in cell junction stabilization.

The malignant transformation of melanocytes causes several thousand deaths each year, making melanoma an important public health concern. Melanoma is the most aggressive skin cancer, which incidence has regularly increased over the past decades. We described here the preparation of new compounds based on the 1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxaline structure. Different positions of the quinoxaline moiety were screened to introduce novel substituents in order to study their influence on the biological activity. Several alkylamino or alkyloxy groups were also considered to replace the methylamine of our first generation of Imiqualines. Imidazo[1,2-a]pyrazine derivatives were also designed as potential minimal structure. The investigation on A375 melanoma cells displayed interesting in vitro low nanomolar cytotoxic activity. Among them, 9d (EAPB02303) is particularly remarkable since it is 20 times more potent than vemurafenib, the reference clinical therapy used on BRAF mutant melanoma. Contrary to the first generation, EAPB02303 does not inhibit tubulin polymerization, as confirmed by an in vitro assay and a molecular modelisation study. The mechanism of action for EAPB02303 highlighted by a transcriptomic analysis is clearly different from a panel of 12 well-known anticancer drugs. In vivoEAPB02303 treatment reduced tumor size and weight of the A375 human melanoma xenografts in a dose-dependent manner, correlated with a low mitotic index but not with necrosis.

Protein phosphorylation acts as an efficient switch controlling deregulated key signaling pathway in cancer. Computational biology aims to address the complexity of reconstructed networks but overrepresents well-known proteins and lacks information on less-studied proteins. A bioinformatic tool to reconstruct and select relatively small networks that connect signaling proteins to their targets in specific contexts is developed. It enables to propose and validate new signaling axes of the Syk kinase. To validate the potency of the tool, it is applied to two phosphoproteomic studies on oncogenic mutants of the well-known phosphatidyl-inositol 3-kinase (PIK3CA) and the unfamiliar Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites (SRMS) kinase. By combining network reconstruction and signal propagation, comprehensive signaling networks from large-scale experimental data are built and multiple molecular paths from these kinases to their targets are extracted. Specific paths from two distinct PIK3CA mutants are retrieved, and their differential impact on the HER3 receptor kinase is explained. In addition, to address the missing connectivities of the SRMS kinase to its targets in interaction pathway databases, phospho-tyrosine and phospho-serine/threonine proteomic data are integrated. The resulting SRMS-signaling network comprises casein kinase 2, thereby validating its currently suggested role downstream of SRMS. The computational pipeline is publicly available, and contains a user-friendly graphical interface (http://doi.org/10.5281/zenodo.3333687).

While first discovered in immunoreceptor signaling, the Syk protein kinase behaves as a tumor and metastasis suppressor in epithelial cells. Its reduced expression in breast and other carcinomas is correlated with decreased survival and increased metastasis risk, but its action mechanism remains largely unknown. Using phosphoproteomics we found that Syk phosphorylated E-cadherin and α-, β-, and p120-catenins on multiple tyrosine residues that concentrate at intercellular junctions. Increased Syk expression and activation enhanced E-cadherin/catenin phosphorylation, promoting their association and complex stability. In human breast cancer cells, Syk stimulated intercellular aggregation, E-cadherin recruitment and retention at adherens junctions, and promoted epithelial integrity, whereas it inhibited cell migration and invasion. Opposite effects were obtained with Syk knockdown or non-phosphorylatable mutant E-cadherin expression. Mechanistically, Syk stimulated the interaction of the E-cadherin/catenin complex with zonula occludens proteins and the actin cytoskeleton. Conditional Syk knockout in the lactating mouse mammary gland perturbed alveologenesis and disrupted E-cadherin localization at adherens junctions, corroborating the observations in cells. Hence, Syk is involved in the maintenance of the epithelial integrity of the mammary gland via the phosphorylation and stabilization of the E-cadherin/catenin adherens junction complex, thereby inhibiting cell migration and malignant tumor invasion.

The bidirectional regulation of epithelial-mesenchymal transitions (EMT) is key in tumorigenesis. Rho GTPases regulate this process via canonical pathways that impinge on the stability of cell-to-cell contacts, cytoskeletal dynamics, and cell invasiveness. Here, we report that the Rho GTPase activators Vav2 and Vav3 utilize a new Rac1-dependent and miR-200c-dependent mechanism that maintains the epithelial state by limiting the abundance of the Zeb2 transcriptional repressor in breast cancer cells. In parallel, Vav proteins engage a mir-200c-independent expression prometastatic program that maintains epithelial cell traits only under 3D culture conditions. Consistent with this, the depletion of endogenous Vav proteins triggers mesenchymal features in epithelioid breast cancer cells. Conversely, the ectopic expression of an active version of Vav2 promotes mesenchymal-epithelial transitions using E-cadherin-dependent and independent mechanisms depending on the mesenchymal breast cancer cell line used. In silico analyses suggest that the negative Vav anti-EMT pathway is operative in luminal breast tumors. Gene signatures from the Vav-associated proepithelial and prometastatic programs have prognostic value in breast cancer patients.

The ability to build in-depth cell signaling networks from vast experimental data is a key objective of computational biology. The spleen tyrosine kinase (Syk) protein, a well-characterized key player in immune cell signaling, was surprisingly first shown by our group to exhibit an onco-suppressive function in mammary epithelial cells and corroborated by many other studies, but the molecular mechanisms of this function remain largely unsolved. Based on existing proteomic data, we report here the generation of an interaction-based network of signaling pathways controlled by Syk in breast cancer cells. Pathway enrichment of the Syk targets previously identified by quantitative phospho-proteomics indicated that Syk is engaged in cell adhesion, motility, growth and death. Using the components and interactions of these pathways, we bootstrapped the reconstruction of a comprehensive network covering Syk signaling in breast cancer cells. To generate in silico hypotheses on Syk signaling propagation, we developed a method allowing to rank paths between Syk and its targets. We first annotated the network according to experimental datasets. We then combined shortest path computation with random walk processes to estimate the importance of individual interactions and selected biologically relevant pathways in the network. Molecular and cell biology experiments allowed to distinguish candidate mechanisms that underlie the impact of Syk on the regulation of cortactin and ezrin, both involved in actin-mediated cell adhesion and motility. The Syk network was further completed with the results of our biological validation experiments. The resulting Syk signaling sub-networks can be explored via an online visualization platform.

The spleen tyrosine kinase Syk was mainly studied in immunoreceptor-activated signaling in hematopoietic cells. We first demonstrated that Syk is also present in mammary epithelial cells and that its expression is lost in malignant breast cancer cells. Using mouse xenograft models injected with Syk-transfected cells we and others established that Syk acts as a tumor and metastasis suppressor. Moreover, clinical studies reveal a correlation between reduced Syk expression and a decreased survival and increased metastasis risk in breast cancer and other carcinomas. The main objective of our investigations is to unravel the mechanisms of the anti-oncogenic activity of Syk. For this, identification of its substrates and signaling pathways is crucial. A quantitative phospho-proteomic approach allowed to identify novel potential Syk substrates involved in intercellular adhesion and epithelial polarity, both characteristics of cell differentiation that are lost during tumor invasion and metastasis. Phosphorylation assays with recombinant proteins demonstrated that E-Cadherin (E-Cdh), α, β and p120-catenin (Ctn) are direct Syk substrates. Using mass spectrometry we identified the tyrosine residues phosphorylated by Syk and generated phospho-Tyr-peptide-specific (pY) antibodies. Using immuno-fluorescence Syk was shown to colocalize with E-Cdh at adherens junctions (AJ). Syk transfection increased E-Cdh, α- and β-Ctn phosphorylation at AJ in a kinase-dependent manner, whereas non-phosphorylatable mutant Syk exhibited a decreased AJ localization. This was biochemically confirmed by immunoprecipitation and Western blot experiments. Phosphorylated Ctns are still associated with E-Cdh and vice versa. In 2D culture, Syk transfection stimulated intercellular contact formation. Correspondingly, in Syk-knockdown cells the 2D and 3D cell-cell re-aggregation was decreased. Conversely, cell migration and invasion were inhibited by Syk transfection and stimulated by Syk knockdown. At the molecular level, Syk transfection increases the interaction between the E-Cdh/Ctn complex with zonula occludens proteins and the actin cytoskeleton. In conclusion, Syk seems to play a positive role in the formation and maintenance of AJ via the phosphorylation of the E-Cdh/Ctn complex. Loss of Syk expression or function might lead to the disruption of these complexes and consequently promote invasion and metastasis. (Supported by the Fondation ARC SL220110603480 and Plan Cancer/INCa ASC14021FSA) Citation Format: Toufic Kassouf, Philippe Montcourrier, Romain Larive, Anne Morel, Serge Urbach, Peter J. Coopman. The E-cadherin/catenin complex and the intercellular adhesion of breast cancer cells are positively regulated via phosphorylation by the Syk tyrosine kinase in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 917.

R-Ras2 is a transforming GTPase that shares downstream effectors with Ras subfamily proteins. However, little information exists about the function of this protein in tumorigenesis and its signalling overlap with classical Ras GTPases. Here we show, by combining loss- and gain-of-function studies in breast cancer cells, mammary epithelial cells and mouse models, that endogenous R-Ras2 has a role in both primary breast tumorigenesis and the late metastatic steps of cancer cells in the lung parenchyma. R-Ras2 drives tumorigenesis in a phosphatidylinostiol-3 kinase (PI3K)-dependent and signalling autonomous manner. By contrast, its prometastatic role requires other priming oncogenic signals and the engagement of several downstream elements. R-Ras2 function is required even in cancer cells exhibiting constitutive activation of classical Ras proteins, indicating that these GTPases are not functionally redundant. Our results also suggest that application of long-term R-Ras2 therapies will result in the development of compensatory mechanisms in breast tumours.

Abstract The non-receptor spleen tyrosine kinase Syk has mainly been studied in hematopoietic cells in which it plays a key role in the immune-response signalling. We previously demonstrated that Syk is also present in mammary epithelial cells and that its expression is lost during malignant progression of breast cancer cell lines. Clinical studies confirmed loss of Syk expression, not only in breast tumors but also in other carcinomas and melanoma. Xenotransplantation animal experiments evidenced that Syk acts as a tumor and metastasis suppressor in breast cancer and melanoma. The mechanisms by which Syk exerts its anti-oncogenic activity remain, however, unrevealed. Using a quantitative phospho-proteomic SILAC approach in breast cancer cells, we identified new potential Syk substrates involved in intercellular adhesion and epithelial cell polarity, both characters of cell differentiation that are lost during tumor progression. Using in vitro kinase assays, we demonstrated that E-cadherin and various catenins are direct Syk substrates. The tyrosine residues phosphorylated by Syk were identified by mass spectrometry and phospho-peptide-specific antibodies were generated. By immunofluorescence, we observed that endogenous Syk and E-cadherin colocalize at adherens junctions and that transient DsRed-Syk transfection induces phospho-E-cadherin- and phospho-catenin epitopes at cell-cell contacts. Immunoprecipitation experiments indicate that phosphorylated E-cadherin and catenins are associated in a complex. Syk-mediated phosphorylation of E-cadherin seems to be important for the proper localization of p120-catenin at adherens junctions. Syk knockdown in breast cancer cells partially inhibits the re-aggregation of cells and increases invasion in Matrigel. Using Syk knockdown and overexpression we explored its potential role in the maintenance of an epithelial phenotype, preventing EMT. In conclusion, Syk might play a role in the establishment and maintenance of intercellular junctions via the phosphorylation of the E-cadherin/catenin complex. Loss of Syk expression or function might lead to the destabilization of these complexes and promote invasion (supported by Fondation ARC SL220110603480) Citation Format: Toufic Kassouf, Philippe Montcourrier, Romain Larive, Nadir Bettache, Anne Morel, Fabrice Merezegue, Serge Urbach, Peter J. Coopman. The tyrosine kinase Syk plays a role in the maintenance of intercellular adhesive junctions in human breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1978. doi:10.1158/1538-7445.AM2014-1978

The adaptor SLAP is a negative regulator of receptor signalling in immune cells but its role in human cancer is ill defined. Here we report that SLAP is abundantly expressed in healthy epithelial intestine but strongly downregulated in 50% of colorectal cancer. SLAP overexpression suppresses cell tumorigenicity and invasiveness while SLAP silencing enhances these transforming properties. Mechanistically, SLAP controls SRC/EPHA2/AKT signalling via destabilization of the SRC substrate and receptor tyrosine kinase EPHA2. This activity is independent from CBL but requires SLAP SH3 interaction with the ubiquitination factor UBE4A and SLAP SH2 interaction with pTyr594-EPHA2. SRC phosphorylates EPHA2 on Tyr594, thus creating a feedback loop that promotes EPHA2 destruction and thereby self-regulates its transforming potential. SLAP silencing enhances SRC oncogenicity and sensitizes colorectal tumour cells to SRC inhibitors. Collectively, these data establish a tumour-suppressive role for SLAP in colorectal cancer and a mechanism of SRC oncogenic induction through stabilization of its cognate substrates.