A multi-omics integrative approach unravels novel genes and pathways associated with senescence escape after targeted therapy in NRAS mutant melanoma

Therapy Induced Senescence (TIS) leads to sustained growth arrest of cancer cells. The associated cytostasis has been shown to be reversible and cells escaping senescence further enhance the aggressiveness of cancers. Chemicals specifically targeting senescent cells, so-called senolytics, constitute a promising avenue for improved cancer treatment in combination with targeted therapies. Understanding how cancer cells evade senescence is needed to optimise the clinical benefits of this therapeutic approach. Here we characterised the response of three different NRAS mutant melanoma cell lines to a combination of CDK4/6 and MEK inhibitors over 33 days. Transcriptomic data show that all cell lines trigger a senescence programme coupled with strong induction of interferons. Kinome profiling revealed the activation of Receptor Tyrosine Kinases (RTKs) and enriched downstream signaling of neurotrophin, ErbB and insulin pathways. Characterisation of the miRNA interactome associates miR-211-5p with resistant phenotypes. Finally, iCell-based integration of bulk and single-cell RNA-seq data identifies biological processes perturbed during senescence and predicts 90 new genes involved in its escape. Overall, our data associate insulin signaling with persistence of a senescent phenotype and suggest a new role for interferon gamma in senescence escape through the induction of EMT and the activation of ERK5 signaling.VG is supported by the Luxembourg National Research Fond (FNR) PRIDE DTU CanBIO [grant reference: 21/16763386]. TR is supported by the FNR PRIDE DTU CriTiCS [grant reference: 10907093]. Project-related work performed by VG, HH, CM, DP, MTN, MB, AG, FT and SK were also supported by the University of Luxembourg and the Fondation Cancer, Luxembourg (grant “SecMelPro”). KM and NP are supported by funding from the European Union’s EU Framework Programme for Research and Innovation Horizon 2020, Innovative Training Networks (MSCA-ITN-2019), funded under EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions, Grant Agreement No 860895. KM, NMD, GC and NP are supported by funding from the European Research Council (ERC) Consolidator Grant 770827. NP is also supported by funding from the Spanish State Research Agency AEI 10.13039/501100011033 grant number PID2019-105500GB-I00.Peer Reviewed"Article signat per 22 autors/es: Vincent Gureghian, Hailee Herbst, Ines Kozar, Katarina Mihajlovic, Noël Malod-Dognin, Gaia Ceddia, Cristian Angeli, Christiane Margue, Tijana Randic, Demetra Philippidou, Milène Tetsi Nomigni, Ahmed Hemedan, Leon-Charles Tranchevent, Joseph Longworth, Mark Bauer, Apurva Badkas, Anthoula Gaigneaux, Arnaud Muller, Marek Ostaszewski, Fabrice Tolle, Nataša Pržulj & Stephanie Kreis"Postprint (published version

A multi-omics integrative approach unravels novel genes and pathways associated with senescence escape after targeted therapy in NRAS mutant melanoma

Therapy Induced Senescence (TIS) leads to sustained growth arrest of cancer cells. The associated cytostasis has been shown to be reversible and cells escaping senescence further enhance the aggressiveness of cancers. Chemicals specifically targeting senescent cells, so-called senolytics, constitute a promising avenue for improved cancer treatment in combination with targeted therapies. Understanding how cancer cells evade senescence is needed to optimise the clinical benefits of this therapeutic approach. Here we characterised the response of three different NRAS mutant melanoma cell lines to a combination of CDK4/6 and MEK inhibitors over 33 days. Transcriptomic data show that all cell lines trigger a senescence programme coupled with strong induction of interferons. Kinome profiling revealed the activation of Receptor Tyrosine Kinases (RTKs) and enriched downstream signaling of neurotrophin, ErbB and insulin pathways. Characterisation of the miRNA interactome associates miR-211-5p with resistant phenotypes. Finally, iCell-based integration of bulk and single-cell RNA-seq data identifies biological processes perturbed during senescence and predicts 90 new genes involved in its escape. Overall, our data associate insulin signaling with persistence of a senescent phenotype and suggest a new role for interferon gamma in senescence escape through the induction of EMT and the activation of ERK5 signaling.VG is supported by the Luxembourg National Research Fond (FNR) PRIDE DTU CanBIO [grant reference: 21/16763386]. TR is supported by the FNR PRIDE DTU CriTiCS [grant reference: 10907093]. Project-related work performed by VG, HH, CM, DP, MTN, MB, AG, FT and SK were also supported by the University of Luxembourg and the Fondation Cancer, Luxembourg (grant “SecMelPro”). KM and NP are supported by funding from the European Union’s EU Framework Programme for Research and Innovation Horizon 2020, Innovative Training Networks (MSCA-ITN-2019), funded under EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions, Grant Agreement No 860895. KM, NMD, GC and NP are supported by funding from the European Research Council (ERC) Consolidator Grant 770827. NP is also supported by funding from the Spanish State Research Agency AEI 10.13039/501100011033 grant number PID2019-105500GB-I00.Peer ReviewedArticle signat per 22 autors/es: Vincent Gureghian 1, Hailee Herbst 1, Ines Kozar 2, Katarina Mihajlovic 3, Noël Malod-Dognin 3, Gaia Ceddia 3, Cristian Angeli 1, Christiane Margue 1, Tijana Randic 1, Demetra Philippidou 1, Milène Tetsi Nomigni 1, Ahmed Hemedan 4, Leon-Charles Tranchevent 4, Joseph Longworth 5, Mark Bauer 1, Apurva Badkas 1, Anthoula Gaigneaux 1, Arnaud Muller 6, Marek Ostaszewski 4, Fabrice Tolle 1, Nataša Pržulj 3, 7, 8 and Stephanie Kreis 1 // 1 Department of Life Sciences and Medicine, University of Luxembourg, 6, Avenue du Swing, L-4367 Belvaux, Luxembourg; 2 Laboratoire National de Santé, Dudelange, Luxembourg; 3 Barcelona Supercomputing Center, 08034 Barcelona, Spain; 4 Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg; 5 Experimental and Molecular Immunology, Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg; 6 LuxGen, TMOH and Bioinformatics platform, Data Integration and Analysis unit, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg; 7 Department of Computer Science, University College London, London WC1E 6BT, UK; 8 ICREA, Pg. Lluís Companys 23, 08010 Barcelona, SpainPostprint (published version

A multi-omics integrative approach unravels novel genes and pathways associated with senescence escape after targeted therapy in NRAS mutant melanoma.

peer reviewedTherapy Induced Senescence (TIS) leads to sustained growth arrest of cancer cells. The associated cytostasis has been shown to be reversible and cells escaping senescence further enhance the aggressiveness of cancers. Chemicals specifically targeting senescent cells, so-called senolytics, constitute a promising avenue for improved cancer treatment in combination with targeted therapies. Understanding how cancer cells evade senescence is needed to optimise the clinical benefits of this therapeutic approach. Here we characterised the response of three different NRAS mutant melanoma cell lines to a combination of CDK4/6 and MEK inhibitors over 33 days. Transcriptomic data show that all cell lines trigger a senescence programme coupled with strong induction of interferons. Kinome profiling revealed the activation of Receptor Tyrosine Kinases (RTKs) and enriched downstream signaling of neurotrophin, ErbB and insulin pathways. Characterisation of the miRNA interactome associates miR-211-5p with resistant phenotypes. Finally, iCell-based integration of bulk and single-cell RNA-seq data identifies biological processes perturbed during senescence and predicts 90 new genes involved in its escape. Overall, our data associate insulin signaling with persistence of a senescent phenotype and suggest a new role for interferon gamma in senescence escape through the induction of EMT and the activation of ERK5 signaling

Contribution of transposable elements to drug resistance in melanoma

The main objective of this thesis was to investigate the involvement of the non- coding genome in drug resistance mechanisms of melanoma cells. Drug resistance is a major clinical problem as it renders currently available targeted therapies inefficient and is rapidly evolving in melanoma patients who are treated with combinations of Mitogen-activated protein kinase (MAPK) pathway and other specific inhibitors. For melanoma patients who carry a mutation in the NRAS gene (25% of patients), no clinically approved targeted therapy combinations are available. Combined cell cycle inhibition (targeting CDK4/6) and MAPK pathway inhibition (targeting MEK) are currently undergoing clinical trials. Here, we used this drug combination to evoke resistance in NRAS mutated cell lines and acquired different omics data sets along the adaptation processes to the treatment (Figure 1). First, we characterized the transcriptomic changes and performed Gene Set Enrichment Analysis to reveal deregulated biological processes. Our data show that all cell lines trigger an interferon and a senescence program. The interferon responses result from the expression of non-coding transcripts which trigger an innate immune response, a phenomenon called viral mimicry. Our data suggests an unforeseen role of interferon gamma in senescence escape. Then, we profiled the kinome of those cell lines and performed network- based enrichment to identify signaling pathways which could support adaptation to the treatment. Overlaying kinase activities on xml models of KEGG pathways allowed us to identify key molecular determinants. Our results associate insulin signaling and decreased activity of ERK5 with persistence of senescence. Moreover, we investigated the contribution of miRNAs to resistance. We acquired both small RNA-seq and miRNA interactions data and assessed their informativeness. We further constructed a miRNA-mRNA network and validated selected interactions by qPCR. Most miRNAs did not seem to exert a strong effect on their targets, as assessed by inverse expression patterns. Extending the analysis to 2 BRAF mutant cell lines, we found miR-211-5p to be upregulated in the proliferative cell lines. Finally, we integrated bulk and matching single-cell data to reveal perturbed processes and predicted novel genes associated with senescence escape. We applied a matrix factorization method to the adjacency matrices of a protein-protein interaction and a co-expression network together with the single- cell expression data and reconstructed condition-specific molecular network. Using graphlet-based topological analysis, we unrevealed perturbed biological processes and predicted a set of novel genes involved in senescence escape. Altogether, our results provide insights on how NRAS mutated melanoma cells adapt to the combination of CDK4/6 and MEK inhibitors. Our data are reminiscent of the Neural Crest Stem Cell (NCSC)-like state, which was further characterized as one of the Drug-Tolerant Persisters (DTPs) states in melanoma. DTPs are now considered important drivers of drug resistance in multiple cancer types. Our data reveal an unforeseen interplay between the expression of non-coding portions of the genomes, the activation of viral mimicry and senescence as processes underlying the DTP state