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- Harmine and Piperlongumine revert TRIB2-mediated drug resistancePublication . Machado, Susana; Silva, Andreia; De Sousa-Coelho, Ana Luísa; Duarte, Isabel; Grenho, Inês; Santos, Bruno F; Mayoral-Varo, Victor; Megias, Diego; Sánchez-Cabo, Fátima; Dopazo, Ana; Ferreira, Bibiana I.; Link, WolfgangTherapy resistance is responsible for most relapses in patients with cancer and is the major challenge to improving the clinical outcome. The pseudokinase Tribbles homologue 2 (TRIB2) has been characterized as an important driver of resistance to several anti-cancer drugs, including the dual ATP-competitive PI3K and mTOR inhibitor dactolisib (BEZ235). TRIB2 promotes AKT activity, leading to the inactivation of FOXO transcription factors, which are known to mediate the cell response to antitumor drugs. To characterize the downstream events of TRIB2 activity, we analyzed the gene expression profiles of isogenic cell lines with different TRIB2 statuses by RNA sequencing. Using a connectivity map-based computational approach, we identified drug-induced gene-expression profiles that invert the TRIB2-associated expression profile. In particular, the natural alkaloids harmine and piperlongumine not only produced inverse gene expression profiles but also synergistically increased BEZ235-induced cell toxicity. Importantly, both agents promote FOXO nuclear translocation without interfering with the nuclear export machinery and induce the transcription of FOXO target genes. Our results highlight the great potential of this approach for drug repurposing and suggest that harmine and piperlongumine or similar compounds might be useful in the clinic to overcome TRIB2-mediated therapy resistance in cancer patients.
- mTORC2 Is the major second layer kinase negatively regulating FOXO3 activityPublication . Jimenez, Lucia; Amenabar, Carlos; Mayoral-Varo, Victor; Mackenzie, Thomas A.; Ramos, Maria C.; Silva, Andreia; Calissi, Giampaolo; Grenho, Inês; Blanco-Aparicio, Carmen; Pastor, Joaquin; Megías, Diego; Ferreira, Bibiana; Link, WolfgangForkhead box O (FOXO) proteins are transcription factors involved in cancer and aging and their pharmacological manipulation could be beneficial for the treatment of cancer and healthy aging. FOXO proteins are mainly regulated by post-translational modifications including phosphorylation, acetylation and ubiquitination. As these modifications are reversible, activation and inactivation of FOXO factors is attainable through pharmacological treatment. One major regulatory input of FOXO signaling is mediated by protein kinases. Here, we use specific inhibitors against different kinases including PI3K, mTOR, MEK and ALK, and other receptor tyrosine kinases (RTKs) to determine their effect on FOXO3 activity. While we show that inhibition of PI3K efficiently drives FOXO3 into the cell nucleus, the dual PI3K/mTOR inhibitors dactolisib and PI-103 induce nuclear FOXO translocation more potently than the PI3Kδ inhibitor idelalisib. Furthermore, specific inhibition of mTOR kinase activity affecting both mTORC1 and mTORC2 potently induced nuclear translocation of FOXO3, while rapamycin, which specifically inhibits the mTORC1, failed to affect FOXO3. Interestingly, inhibition of the MAPK pathway had no effect on the localization of FOXO3 and upstream RTK inhibition only weakly induced nuclear FOXO3. We also measured the effect of the test compounds on the phosphorylation status of AKT, FOXO3 and ERK, on FOXO-dependent transcriptional activity and on the subcellular localization of other FOXO isoforms. We conclude that mTORC2 is the most important second layer kinase negatively regulating FOXO activity.