Linking metabolic reprogramming to chemo-immuno-resistance: identifying new predictive biomarkers and druggable targets moving from OMIC techniques to single cell analysis

Non-Small Cell Lung Cancer (NSCLC) patients develop resistance to platinum-based drugs. The transcription factor CCAAT/Enhancer Binding Protein-β (C/EBP-β) affects chemoresistance by increasing the ratio between its two splicing isoforms LAP and LIP. LAP/LIP splicing reprograms metabolism of murine embryonic fibroblasts via non-coding RNAs (ncRNAs). In this thesis, we ex-plored the involvement of C/EBP-β splicing in driving cisplatin (Pt) resistance in NSCLC by promot-ing metabolic rewiring, and the molecular mechanism beyond such splicing. Methods. Chemosensitivity assays, DNA damage, cell cycle distribution and ATP binding cassette (ABC) transporters levels were assessed in NSCLC cell lines overexpressing LAP/LIP or modulating the ncRNAs MALAT1 and hsa-miR-155-5p. Metabolic/lipidomic profiling and functional metabolic assays were performed to identify potential chemo-sensitizing agents, validated in Hu-CD34+NSG xenografts by tumor growth monitoring, mass spectrometry imaging and scRNA-seq, and in ex vivo co-cultures between cancer cells and natural killer (NK) cells. Results. LAPhigh cells had higher levels of fatty acyl-carnitine conjugates, higher fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) than LIPhigh cells. Consequently, they produced more mitochondrial ATP (mtATP) that boosted the Pt efflux through ABC transporters. This pheno-type was driven by the transcriptional up-regulation of carnitine palmitoyl transferase 1A (CPT1A) gene, the rate-limiting enzyme of FAO, induced by LAP. The FAO inhibitor etomoxir restored Pt sensitivity, causing a metabolic crash and a mitochondria-dependent apoptosis, redistributing fatty acids in tumor immune microenvironment (TIME) and boosting the anti-tumor activity of NK cells. LAP splicing was controlled by the ncRNAs MALAT1 and miR-155-5p competing endogenous RNAs (ceRNAs): MALAT1 increased and miR-155-5p reduced LAP splicing. LAPhighMA-LAT1highmiR-155lowcells treated with MALAT1 ASOs or miR-155-5p mimics reduced LAP/LIP ratio, OXPHOS flux and mtATP production. They shifted from chemoresistance to chemosensitivity, re-duced glucose-related metabolites and had a different pattern of saturated, mono-unsaturated or poly-unsaturated fatty acids. LAPlowMALAT1lowmiR-155highcells overexpressing MALAT1 or transfected with anti-miR-155-5p exhibited the opposite phenotype, shifting from chemosensitivity to chemo-resistance. A screening of glycolysis-targeting agents, saturated, mono- and poly-unsaturated fatty acids identified docosahexaenoic acid (DHA) as the most potent tool that rewired energetic metabo-lism reducing OXPHOS and mtATP, and re-sensitized resistant cells to Pt when combined with ASO MALAT1. 4 Conclusion. We unveiled a novel circuit controlling chemoresistance in NSCLC, based on MA-LAT1/miR-155-5p ceRNAs that control C/EBP-β splicing toward LAP. The latter caused chemo-resistance by increasing FAO/OXPHOS/mtATP in cancer cells, favoring the drug efflux through ABC transporters, and altering immune metabolism in NK cells, dampening their anti-tumor activity. Com-bined treatment of FAO inhibitors+Pt or ncRNA-modulating agents (as ASO MALAT1)+FA metab-olism targeting agents+Pt are promising chemo-sensitizing approaches in refractory NSCLC