TARGETING CANCER METABOLISM WITH FASTING-MIMICKING DIET¿BASED LOW-TOXIC COMBINATION THERAPIES: OVERCOMING RESISTANCE THROUGH METABOLIC SYNERGY

Aging represents the highest risk factor for cancer, defined by molecular hallmarks such as genomic instability, telomere attrition, deregulated nutrient sensing, and chronic inflammation promoting malignant transformation. Among age-associated cancers, non-small cell lung cancer (NSCLC) is the most prevalent and lethal, with KRAS mutations occurring in up to 25–30% of lung adenocarcinomas. Tumors harbouring this mutated oncogene are particularly resistant to targeted therapies, highlighting the need for novel targeted low-toxicity strategies. Nutrient restriction interventions, such as fasting and fasting-mimicking diets (FMD), modulate conserved growth pathways (IGF-1/AKT/mTOR, AMPK) and enhance cellular stress resistance, offering a promising approach to cancer therapy. FMD, if combined with treatments like pharmacological doses of ascorbate could effectively target NSCLC rewired metabolism that sustains NSCLC progression. In this thesis, I investigated the combinatorial effects of FMD and pharmacological doses of vitamin C in KRAS-mutant NSCLC. In vitro, short-term starvation (STS) synergized with vitamin C to induce dramatic cell death in KRAS-mutant and wild- type NSCLC cell lines, while protecting normal cells. In vivo, cycles of FMD combined with high-dose vitamin C significantly delayed tumor growth in both immunodeficient (NSG) and immunocompetent (C57BL/6) mouse models, without inducing major toxicity. However, as previously demonstrated in our studies, cancer cells resist FMD through upregulation of starvation escape pathways. Here, mechanistically, RNA sequencing (RNA-seq) analysis revealed that resistant subpopulations upregulated MAPK signaling as an acute adaptive response, while chronic exposure drove additional activation of PI3K-AKT pathways to resist FMD and vitamin C treatment. Pharmacological blockade of EGFR/ERK (Afatinib, Ulixertinib) or PI3K (Pictilisib), particularly in dual ERK/PI3K inhibition, synergized with FMD and vitamin C to overcome resistance, delay tumor progression, and induce tumor regression in vivo. Collectively, these findings identify the FMD–vitamin C axis as a safe and effective metabolic therapy for NSCLC and highlight MAPK and PI3K signaling as key resistance drivers. Rational combination of metabolic interventions with targeted inhibitors provides a promising framework for personalized strategies in KRAS- driven lung cancer.