Autophagy requires poly(adp-ribosyl)ation-dependent AMPK nuclear export.

AMPK is a central energy sensor linking extracellular milieu fluctuations with the autophagic machinery. In the current study we uncover that Poly(ADP-ribosyl)ation (PARylation), a post-translational modification (PTM) of proteins, accounts for the spatial and temporal regulation of autophagy by modulating AMPK subcellular localisation and activation. More particularly, we show that the minority AMPK pool needs to be exported to the cytosol in a PARylation-dependent manner for optimal induction of autophagy, including ULK1 phosphorylation and mTORC1 inactivation. PARP-1 forms a molecular complex with AMPK in the nucleus in non-starved cells. In response to nutrient deprivation, PARP-1 catalysed PARylation, induced the dissociation of the PARP-1/AMPK complex and the export of free PARylated nuclear AMPK to the cytoplasm to activate autophagy. PARP inhibition, its silencing or the expression of PARylation-deficient AMPK mutants prevented not only the AMPK nuclear-cytosolic export but also affected the activation of the cytosolic AMPK pool and autophagosome formation. These results demonstrate that PARylation of AMPK is a key early signal to efficiently convey extracellular nutrient perturbations with downstream events needed for the cell to optimize autophagic commitment before autophagosome formation.

PARP targeting counteracts gliomagenesis through induction of mitotic catastrophe and aggravation of deficiency in homologous recombination in PTEN-mutant glioma.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults and one of the most aggressive cancers. PARP-1 is a nuclear protein involved in multiple facets of DNA repair and transcriptional regulation. In this study we dissected the action of PARP inhibition in different GBM cell lines with either functional or mutated PTEN that confers resistance to diverse therapies. In PTEN mutant cells, PARP inhibition induced a severe genomic instability, exacerbated homologous recombination repair (HR) deficiency and down-regulated the Spindle Assembly Checkpoint (SAC) factor BUBR1, leading to mitotic catastrophe (MC). EGFR gene amplification also represents a signature of genetic abnormality in GBM. To more effectively target GBM cells, co-treatment with a PARP inhibitor and an EGFR blocker, erlotinib, resulted in a strong suppression of ERK1/2 activation and in vivo the combined effect elicited a robust reduction in tumour development. In conclusion, PARP inhibition targets PTEN-deficient GBM cells through accentuation of SAC repression and aggravation of HR deficiency, leading to the induction of genomic instability and eventually deriving to mitotic catastrophe (MC); the inhibition of PARP and co-treatment with an inhibitor of pro-survival pathways strongly retarded in vivo gliomagenesis.

The DNA methyltransferase inhibitors zebularine and decitabine induce mitochondria-mediated apoptosis and DNA damage in p53 mutant leukemic T cells.

DNA methyltransferase (DNMT)-inhibiting nucleoside analogs reactivate the expression of tumor suppressor genes and apoptosis-related genes silenced by methylation, thus favoring the induction of apoptosis in tumor cells. Moreover, induction of DNA damage seems to contribute to their antitumor effect. However, the apoptotic signaling pathway induced by these demethylating drugs is not well understood. Here, we have investigated the induction of apoptosis by two nucleoside DNMT inhibitors, decitabine and zebularine, in leukemic T cells. Both inhibitors induced caspase-dependent apoptosis in Jurkat, CEM-6 and MOLT-4 leukemia T cell lines, all with mutant p53, whereas resting and activated normal T lymphocytes were highly resistant to these demethylating agents. Although decitabine and zebularine showed different ability to induce apoptosis and cell cycle arrest among the three cell lines, they similarly activated the intrinsic apoptotic pathway by inducing mitochondrial alterations such as Bak activation, loss of transmembrane potential and generation of reactive oxygen species (ROS). Accordingly, Bcl-2- and Bcl-x(L) -overexpressing Jurkat cells, as well as caspase-9-deficient Jurkat cells, were resistant to apoptosis induced by decitabine and zebularine. Interestingly, ROS production seemed to be necessary for the induction of apoptosis. Apoptotic events, such as Bak and caspase activation, started as soon as 20 hr after treatment with either decitabine or zebularine. In addition, progression of apoptosis triggered by both DNMT inhibitors was paralleled by the induction of DNA damage. Our results suggest that the mitochondrial apoptotic pathway activated by decitabine and zebularine in p53 mutant leukemic T cells depends mainly on the induction of DNA damage.