In malignant transformation, cellular stress‐response pathways are dynamically mobilized to counterbalance oncogenic activity, keeping cancer cells viable. Therapeutic disruption of this vulnerable homeostasis might change the outcome of many human cancers, particularly those for which no effective therapy is available. Here, we report the use of fibroblast growth factor 2 (FGF2) to demonstrate that further mitogenic activation disrupts cellular homeostasis and strongly sensitizes cancer cells to stress‐targeted therapeutic inhibitors. We show that FGF2 enhanced replication and proteotoxic stresses in a K‐Ras‐driven murine cancer cell model, and combinations of FGF2 and proteasome or DNA damage response‐checkpoint inhibitors triggered cell death. CRISPR/Cas9‐mediated K‐Ras depletion suppressed the malignant phenotype and prevented these synergic toxicities in these murine cells. Moreover, in a panel of human Ewing's sarcoma family tumor cells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE‐821 (ATR inhibitor) induced cell death when combined with FGF2. Sustained MAPK‐ERK1/2 overactivation induced by FGF2 appears to underlie these synthetic lethalities, as late pharmacological inhibition of this pathway restored cell homeostasis and prevented these described synergies. Our results highlight how mitotic signaling pathways which are frequently overridden in malignant transformation might be exploited to disrupt the robustness of cancer cells, ultimately sensitizing them to stress‐targeted therapies. This approach provides a new therapeutic rationale for human cancers, with important implications for tumors still lacking effective treatment, and for those that frequently relapse after treatment with available therapies.

LKB1 and KRAS are the third most frequent co-mutations detected in non-small cell lung cancer (NSCLC) and cause aggressive tumor growth. Unfortunately, treatment with RAS-RAF-MEK-ERK pathway inhibitors has minimal therapeutic efficacy in LKB1-mutant KRAS-driven NSCLC. Autophagy, an intracellular nutrient scavenging pathway, compensates for Lkb1 loss to support Kras-driven lung tumor growth. Here we preclinically evaluate the possibility of autophagy inhibition together with MEK inhibition as a treatment for Kras-driven lung tumors. We found that the combination of the autophagy inhibitor hydroxychloroquine (HCQ) and the MEK inhibitor Trametinib displays synergistic anti-proliferative activity in KrasG12D/+;Lkb1-/- (KL) lung cancer cells, but not in KrasG12D/+;p53-/- (KP) lung cancer cells. In vivo studies using tumor allografts, genetically engineered mouse models (GEMMs) and patient-derived xenografts (PDXs) showed anti-tumor activity of the combination of HCQ and Trametinib on KL but not KP tumors. We further found that the combination treatment significantly reduced mitochondrial membrane potential, basal respiration, and ATP production, while also increasing lipid peroxidation, indicative of ferroptosis, in KL tumor-derived cell lines (TDCLs) and KL tumors compared to treatment with single agents. Moreover, the reduced tumor growth by the combination treatment was rescued by ferroptosis inhibitor. Taken together, we demonstrate that autophagy upregulation in KL tumors causes resistance to Trametinib by inhibiting ferroptosis. Therefore, a combination of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat NSCLC bearing co-mutations of LKB1 and KRAS.

Mitochondrial oxidation of nutrients is tightly regulated in response to the cellular environment and changes in energy demands. studies evaluating the mitochondrial capacity of oxidizing different substrates are important for understanding metabolic shifts in physiological adaptations and pathological conditions, but may be influenced by the nutrients present in the culture medium or by the utilization of endogenous stores. One such influence is exemplified by the Crabtree effect (the glucose-mediated inhibition of mitochondrial respiration) as most experiments are performed in glucose-containing media. Here, using high-resolution respirometry, we evaluated the oxidation of endogenous or exogenous substrates by cell lines harboring different metabolic profiles. We found that a 1-h deprivation of the main energetic nutrients is an appropriate strategy to abolish interference of endogenous or undesirable exogenous substrates with the cellular capacity of oxidizing specific substrates, namely glutamine, pyruvate, glucose, or palmitate, in mitochondria. This approach primed mitochondria to immediately increase their oxygen consumption after the addition of the exogenous nutrients. All starved cells could oxidize exogenous glutamine, whereas the capacity for oxidizing palmitate was limited to human hepatocarcinoma Huh7 cells and to C2C12 mouse myoblasts that differentiated into myotubes. In the presence of exogenous glucose, starvation decreased the Crabtree effect in Huh7 and C2C12 cells and abrogated it in mouse neuroblastoma N2A cells. Interestingly, the fact that the Crabtree effect was observed only for mitochondrial basal respiration but not for the maximum respiratory capacity suggests it is not caused by a direct effect on the electron transport system.