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SUMMARYNeurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here, we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion. This provides cells with resistance to pro-oxidants targeting complex I and decreases both respiration and intracellular NAD+. Expression of the alternative NADH dehydrogenase NDI1 raises NAD+/NADH ratio, enhances the activity of the mitochondrial NAD+-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. This anti-neoplastic effect is mimicked both in vitro and in vivo by administration of NAD+ precursors or by rising expression of the NAD+ deacetylase SIRT3, and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes of these cells. These findings shed light on chemotherapeutic resistance and on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD+/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.

CK2 is a Ser/Thr protein kinase overexpressed in many cancers. It is usually present in cells as a tetrameric enzyme, composed of two catalytic (&alpha, or &alpha, &rsquo, ) and two regulatory (&beta, ) subunits, but it is active also in its monomeric form, and the specific role of the different isoforms is largely unknown. CK2 phosphorylates several substrates related to the uncontrolled proliferation, motility, and survival of cancer cells. As a consequence, tumor cells are addicted to CK2, relying on its activity more than healthy cells for their life, and exploiting it for developing multiple oncological hallmarks. However, little is known about CK2 contribution to the metabolic rewiring of cancer cells. With this study we aimed at shedding some light on it, especially focusing on the CK2 role in the glycolytic onco-phenotype. By analyzing neuroblastoma and osteosarcoma cell lines depleted of either one (&alpha, ) or the other (&alpha, ) CK2 catalytic subunit, we also aimed at disclosing possible pro-tumor functions which are specific of a CK2 isoform. Our results suggest that both CK2 &alpha, and &alpha, contribute to cell proliferation, survival and tumorigenicity. The analyzed metabolic features disclosed a role of CK2 in tumor metabolism, and suggest prominent functions for CK2 &alpha, isoform. Results were also confirmed by CK2 pharmacological inhibition. Overall, our study provides new information on the mechanism of cancer cells addiction to CK2 and on its isoform-specific functions, with fundamental implications for improving future therapeutic strategies based on CK2 targeting.

Aims: TNF receptor-associated protein 1 (TRAP1), the mitochondrial paralog of the heat shock protein 90 (Hsp90) family of molecular chaperones, is required for neoplastic growth in several tumor cell models, where it inhibits succinate dehydrogenase (SDH) activity, thus favoring bioenergetic rewiring, maintenance of redox homeostasis, and orchestration of a hypoxia-inducible factor 1-alpha (HIF1α)-mediated pseudohypoxic program. Development of selective TRAP1 inhibitors is instrumental for targeted development of antineoplastic drugs, but it has been hampered up to now by the high degree of homology among catalytic pockets of Hsp90 family members. The vegetal derivative honokiol and its lipophilic bis-dichloroacetate ester, honokiol DCA (HDCA), are small-molecule compounds with antineoplastic activity. HDCA leads to oxidative stress and apoptosis in in vivo tumor models and displays an action that is functionally opposed to that of TRAP1, as it induces both SDH and the mitochondrial deacetylase sirtuin-3 (SIRT3), which further enhances SDH activity. We investigated whether HDCA could interact with TRAP1, inhibiting its chaperone function, and the effects of HDCA on tumor cells harboring TRAP1. Results: An allosteric binding site in TRAP1 is able to host HDCA, which inhibits TRAP1 but not Hsp90 ATPase activity. In neoplastic cells, HDCA reverts TRAP1-dependent downregulation of SDH, decreases proliferation rate, increases mitochondrial superoxide levels, and abolishes tumorigenic growth. Innovation: HDCA is a potential lead compound for the generation of antineoplastic approaches based on the allosteric inhibition of TRAP1 chaperone activity. Conclusions: We have identified a selective TRAP1 inhibitor that can be used to better dissect TRAP1 biochemical functions and to tailor novel tumor-targeting strategies.

Summary: TRAP1 is the mitochondrial paralog of the heat shock protein 90 (HSP90) chaperone family. Its activity as an energy metabolism regulator has important implications in cancer, neurodegeneration, and ischemia. Selective inhibitors of TRAP1 could inform on its mechanisms of action and set the stage for targeted drug development, but their identification was hampered by the similarity among active sites in HSP90 homologs. We use a dynamics-based approach to identify a TRAP1 allosteric pocket distal to its active site that can host drug-like molecules, and we select small molecules with optimal stereochemical features to target the pocket. These leads inhibit TRAP1, but not HSP90, ATPase activity and revert TRAP1-dependent downregulation of succinate dehydrogenase activity in cancer cells and in zebrafish larvae. TRAP1 inhibitors are not toxic per se, but they abolish tumorigenic growth of neoplastic cells. Our results indicate that exploiting conformational dynamics can expand the chemical space of chaperone antagonists to TRAP1-specific inhibitors with wide therapeutic opportunities. : The molecular chaperone TRAP1 regulates energy metabolism, and its activity is relevant in cancer and degenerative diseases. Here, Sanchez-Martin et al. identify highly selective allosteric inhibitors of TRAP1. These compounds revert biochemical and pro-neoplastic effects of TRAP1 and could both enlighten its mode of action and disclose novel therapeutic strategies. Keywords: chaperone inhibitors, anticancer compound, molecular dynamics, allosteric ligands, TRAP1, HSP90, mitochondria, mitochondrial biology, zebrafish, cancer cells, neurofibroma

Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+ -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.

The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In the yeast F-ATP synthase, subunits e and g of the F(O) sector constitute a lateral domain, which is required for dimer stability and cristae formation. Here, by using site-directed mutagenesis, we identified Arg-8 of subunit e as a critical residue in mediating interactions between subunits e and g, most likely through an interaction with Glu-83 of subunit g. Consistent with this hypothesis, (i) the substitution of Arg-8 in subunit e (eArg-8) with Ala or Glu or of Glu-83 in subunit g (gGlu-83) with Ala or Lys destabilized the digitonin-extracted F-ATP synthase, resulting in decreased dimer formation as revealed by blue-native electrophoresis; and (ii) simultaneous substitution of eArg-8 with Glu and of gGlu-83 with Lys rescued digitonin-stable F-ATP synthase dimers. When tested in lipid bilayers for generation of Ca(2+)-dependent channels, WT dimers displayed the high-conductance channel activity expected for the mitochondrial megachannel/permeability transition pore, whereas dimers obtained at low digitonin concentrations from the Arg-8 variants displayed currents of strikingly small conductance. Remarkably, double replacement of eArg-8 with Glu and of gGlu-83 with Lys restored high-conductance channels indistinguishable from those seen in WT enzymes. These findings suggest that the interaction of subunit e with subunit g is important for generation of the full-conductance megachannel from F-ATP synthase.

Introduction Hexokinase 2 (HK2) phosphorylates glucose for starting its utilisation in glycolysis and pentose phosphate pathway. In many cancer cell types these processes are enhanced and HK2 expression is strongly induced and mainly localised to the outer mitochondrial membrane, where it also exerts an anti-apoptotic activity. Genetic ablation in mouse highlights HK2 importance in tumour formation. Therefore, HK2 is a good target for antineoplastic strategies, but HK2 inhibitors can have important side effects as they affect glucose metabolism. Here we have developed an antineoplastic strategy based on HK2 detachment from mitochondria in order to induce tumour cell death without inhibiting hexokinase enzymatic activity. Material and methods Peptide design and synthesis; hexokinase enzymatic activity assays. Measurements of mitochondrial membrane potential, intracellular Ca2+ levels, cell death and in vitro and in vivo tumorigenic assays on human and mouse cancer cell models (CT26 colon cancer cells, 4 T1 breast cancer cells, HeLa cervix carcinoma cells and primary human B-CLL cells). Results and discussions We have observed that in cancer cells HK2 locates at contact sites between mitochondria and endoplasmic reticulum called MAMs (mitochondria-associated membranes). We could selectively detach HK2 from MAMs by using a peptide that does not perturb hexokinase enzymatic activity. This treatment rapidly induces opening of the Inositol-3-Phospate-Receptor and the ensuing Ca2+ transfer from endoplasmic reticulum to mitochondria. As a consequence, a Ca2+ overload occurs in mitochondria, leading to permeability transition pore opening, mitochondrial membrane depolarization and apoptosis in a caspase-independent way. Peptide administration reduces allografic growth of breast and colon cancer cells without any noxious effect on healthy tissues, and elicits death of B-cell chronic lymphocytic leukaemia (B-CLL) cells freshly obtained by patients and in vivo. Conclusion We have reported that HK2 locates in MAMs of cancer cells, where it acts as an important player in the control of their survival. Targeting HK2 with a peptide-based strategy constitutes a novel and promising anti-neoplastic approach.

p21Waf1/Cip1/Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs) and one of the best characterized p53 direct target genes. Moreover, it is one of the principal inducers of senescence, a permanent arrest phenotype related to ageing and the prevention of cell transformation. It was initially thought that p21 had tumour suppressor properties, due to its ability to stop cell cycle at different stages, either transitorily or permanently. However, recent evidence points to a much more complex picture. It is now well established that p21 itself can trigger apoptosis in certain situations, even independently of p53. On the other hand, p21-mediated cell arrest can actually limit the sensitivity to apoptotic stimuli. To complicate matters even further, p21 has been shown to have other direct pro-survival functions and could even be contributing to tumourigenesis through the secretion of growth factors by arrested cells. At the core of these antagonistic functions of p21 is the generation of Reactive Oxygen Species (ROS), which have been shown to be important both in the induction of apoptosis and the establishment of senescence, and could also participate in the negative effects on tissue homeostasis of the senescent cell secretome. p21 has the ability to stop cancer cell growth and is not mutated in cancer. However, without a better understanding of its pleiotropic functions we will not be able to harness its clinical potential. It is therefore important to investigate the mechanisms by which p21 affects cell physiology and its use of ROS as messengers and effectors.

p21(Waf1/Cip1/Sdi1) is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest. Prolonged p21 up-regulation induces a senescent phenotype in normal and cancer cells, accompanied by an increase in intracellular reactive oxygen species (ROS). However, it has been shown recently that p21 expression can also lead to cell death in certain models. The mechanisms involved in this process are not fully understood. Here, we describe an induction of apoptosis by p21 in sarcoma cell lines that is p53-independent and can be ameliorated with antioxidants. Similar levels of p21 and ROS caused senescence in the absence of significant death in other cancer cell lines, suggesting a cell-specific response. We also found that cells undergoing p21-dependent cell death had higher sensitivity to oxidants and a specific pattern of mitochondrial polarization changes. Consistent with this, apoptosis could be blocked with targeted expression of catalase in the mitochondria of these cells. We propose that the balance between cancer cell death and arrest after p21 up-regulation depends on the specific effects of p21-induced ROS on the mitochondria. This suggests that selective up-regulation of p21 in cancer cells could be a successful therapeutic intervention for sarcomas and tumors with lower resistance to mitochondrial oxidative damage, regardless of p53 status.