Krishnan Thirumoorthy

Methionine and Kynurenine Activate Oncogenic Kinases in Glioblastoma, and Methionine Deprivation Compromises Proliferation.

Purpose: We employed a metabolomics-based approach with the goal to better understand the molecular signatures of glioblastoma cells and tissues, with an aim toward identifying potential targetable biomarkers for developing more effective and novel therapies. Experimental Design: We used liquid chromatography coupled with mass spectrometry (LC-MS/Q-TOF and LC-MS/QQQ) for the discovery and validation of metabolites from primary and established glioblastoma cells, glioblastoma tissues, and normal human astrocytes. Results: We identified tryptophan, methionine, kynurenine, and 5-methylthioadenosine as differentially regulated metabolites (DRM) in glioblastoma cells compared with normal human astrocytes (NHAs). Unlike NHAs, glioblastoma cells depend on dietary methionine for proliferation, colony formation, survival, and to maintain a deregulated methylome (SAM:SAH ratio). In methylthioadenosine phosphorylase (MTAP)-deficient glioblastoma cells, expression of MTAP transgene did not alter methionine dependency, but compromised tumor growth in vivo. We discovered that a lack of the kynurenine-metabolizing enzymes kynurenine monooxygenase and/or kynureninase promotes the accumulation of kynurenine, which triggers immune evasion in glioblastoma cells. In silico analysis of the identified DRMs mapped the activation of key oncogenic kinases that promotes tumorigenesis in glioblastoma. We validated this result by demonstrating that the exogenous addition of DRMs to glioblastoma cells in vitro results in oncogene activation as well as the simultaneous downregulation of Ser/Thr phosphatase PP2A. Conclusions: We have connected a four-metabolite signature, implicated in the methionine and kynurenine pathways, to the promotion and maintenance of glioblastoma. Together, our data suggest that these metabolites and their respective metabolic pathways serve as potential therapeutic targets for glioblastoma. Clin Cancer Res; 22(14); 3513–23. ©2016 AACR.

Caveolae-Mediated Endocytosis Is Critical for Albumin Cellular Uptake and Response to Albumin-Bound Chemotherapy

Nab-paclitaxel, a nanoparticle conjugate of paclitaxel to human albumin, exhibits efficacy in pancreatic cancer, non-small cell lung cancer and breast cancer. However, there is a lack of predictive biomarkers to identify patients who might benefit most from its administration. This study addresses this gap in knowledge by identifying that caveolin-1 (Cav-1) is a candidate mechanism-based biomarker. Caveolae are small membrane invaginations important for transendothelial albumin uptake. Cav-1, the principal structural component of caveolae, is overexpressed in the cancers noted above that respond to nab-paclitaxel. Thus, we hypothesized that Cav-1 may be critical for albumin uptake in tumors and perhaps determine their response to this drug. Cav-1 protein levels correlated positively with nab-paclitaxel sensitivity. RNAi-mediated attenuation of Cav-1 expression reduced uptake of albumin and nab-paclitaxel in cancer cells and rendered them resistant to nab-paclitaxel-induced apoptosis. Conversely, Cav-1 overexpression enhanced sensitivity to nab-paclitaxel. Selection for cellular resistance to nab-paclitaxel in cell culture correlated with a loss of Cav-1 expression. In mouse xenograft models, cancer cells, where Cav-1 was attenuated, exhibited resistance to the antitumor effects of nab-paclitaxel therapy. Overall, our findings suggest Cav-1 as a predictive biomarker for the response to nab-paclitaxel and other albumin-based cancer therapeutic drugs. Cancer Res; 77(21); 5925-37. ©2017 AACR.

NNMT silencing activates tumor suppressor PP2A, inactivates oncogenic STKs and inhibits tumor forming ability.

Purpose: To identify potential molecular hubs that regulate oncogenic kinases and target them to improve treatment outcomes for glioblastoma patients. Experimental Design: Data mining of The Cancer Genome Atlas datasets identified nicotinamide-N-methyl transferase (NNMT) as a prognostic marker for glioblastoma, an enzyme linked to the reorganization of the methylome. We tested our hypothesis that NNMT plays a crucial role by modulating protein methylation, leading to inactivation of tumor suppressors and activation of oncogenes. Further experiments were performed to understand the underlying biochemical mechanisms using glioblastoma patient samples, established, primary, and isogenic cells. Results: We demonstrate that NNMT outcompetes leucine carboxyl methyl transferase 1 (LCMT1) for methyl transfer from principal methyl donor SAM in biological systems. Inhibiting NNMT increased the availability of methyl groups for LCMT1 to methylate PP2A, resulting in the inhibition of oncogenic serine/threonine kinases (STK). Further, NNMT inhibition retained the radiosensitizer nicotinamide and enhanced radiation sensitivity. We have provided the biochemical rationale of how NNMT plays a vital role in inhibiting tumor suppressor PP2A while concomitantly activating STKs. Conclusions: We report the intricate novel mechanism in which NNMT inhibits tumor suppressor PP2A by reorganizing the methylome both at epigenome and proteome levels and concomitantly activating prosurvival STKs. In glioblastoma tumors with NNMT expression, activation of PP2A can be accomplished by FDA approved perphenazine (PPZ), which is currently used to treat mood disorders such as schizophrenia, bipolar disorder, etc. This study forms a foundation for further glioblastoma clinical trials using PPZ with standard of care treatment. Clin Cancer Res; 23(9); 2325–34. ©2016 AACR.

Eight membered cyclic-borasiloxanes: synthesis, structural, photophysical, steric strain and DFT calculations

Eight-membered cyclic borasiloxanes, Ph2Si[OBArO]2SiPh2 [Ar = 4-EtC6H4 (1), 4-tBuC6H4 (2), 2-PhC6H4 (3), 4-PhC6H4 (4) and β-C10H7 (Nap) (5); Ph = phenyl], were synthesized via the reaction of diphenylsilanediol with aryl boronic acid through a condensation reaction. The compounds were characterized using elemental analysis, FT-IR and NMR (1H, 13C, 29Si and 11B). The compounds 1, 3 and 5 were further confirmed using single crystal X-ray diffraction studies. This showed the eight-membered ring (B2O4Si2) configuration and that organic substituents occupied the axial and equatorial positions. Furthermore, non-covalent C–H⋯π and π⋯π interactions were observed in the crystal packing pattern. These borasiloxanes exhibited strong solid state fluorescence. The thermal behavior of the compounds 1–5 has been investigated using thermogravimetric analysis (TGA), which shows that the borasiloxanes 1 and 2 are thermally stable up to 220 °C and 180 °C respectively, whereas 3 and 4 are stable up to ∼120 °C and 5 is stable up to 230 °C. The band gap was calculated using the diffuse reflectance spectroscopic method. Compound 5 exhibits a low band gap (3.28 eV) which indicates that the naphthyl group shows more π-bonding delocalization within the molecule (strong intra-molecular charge transfer). The band gap decreases in the order of the compounds, 1 > 2 > 3 > 4 > 5. The theoretically computed band gap values were in good agreement with the experimentally observed trend. HOMO–LUMO analysis, TD-DFT, and the electrophilicity index, dipole moment and hyperpolarizability were computed using the B3LYP/6-31+G** method. The steric strain energies of the borasiloxanes and their degree of puckering conformation (O–Si–O, O–B–O and B–O–Si) were also analysed using DFT. This confirms that compound 3 has more strain, which is due to having a phenyl group in a sterically hindered ortho-position.

Abstract 2514: Radiation-induced HIF-1α prevents apoptosis through reduction of ROS productions after irradiation in glioblastoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose/Objective(s): Glioblastoma is one of the most radioresistant tumors, and hypoxic regions are commonly observed. Molecular markers of hypoxia, Hypoxia inducible factor-1α (HIF-1α), a transcriptional factor, is strongly expressed in glioblastoma and associated with poor survival. Recent studies have shown that HIF-1α is induced by irradiation even if under normoxic conditions, although the mechanism still remains unclear. In this study, we firstly investigated the association radiation-induced HIF-1α with radioresistance in glioblastoma cells. Next, to investigate the function of HIF-1α, we focused Pyruvate dehydrogenase kinase (PDK1), one of the target genes of HIF-1α, which can decrease reactive oxygen species (ROS) productions in hypoxic conditions. We analyzed whether radiation-induced HIF-1α decreases ROS productions through activation of PDK1 after irradiation under normoxic conditions. Materials/Methods: Clonogenic survival assay was performed to investigate the effect of radiation on colony-forming ability in established cell lines (U87, LN18, and LN229) and primary cell lines (VC3 and MGH8). In these cell lines, HIF-1α and PDK1 expressions were analyzed by using Western blotting after irradiation. To investigate whether HIF-1α is associated with radiation resistance, we used two HIF-1α blockade strategies: stable knockdown cell lines of HIF-1α using lentivirus-based sh-RNA and YC-1 (A.G. Scientific, Inc.), a novel HIF-1α inhibitor. These blockade treatments were evaluated by clonogenic survival assay, MTS assay (Promega), and apoptotic proteins, such as cleaved caspase-3 and cleaved PARP. To measure ROS levels, CM-H2DCFDA (Invitrogen) was used at a dose of 10 µM. Results: Clonogenic survival assay showed that U87 and LN18 were more radioresistant than VC3, MGH8, and LN229. In this study, U87 and LN18 were defined as radioresistant cells and the others were radiosensitive cells. Under normoxic conditions radiation-induced HIF-1α was detected in radioresistant cells, but not in radiosensitive cells. Knockdown of HIF-1α decreased cell proliferation in clonogenic survival assay. YC-1 decreased the radiation-induced HIF-1α and decreased cell viability in MTS assay. These HIF-1α blockades treatments increased cleaved caspase-3 and cleaved PARP expression after irradiation. To evaluate the function of HIF-1α, PDK1 expression and ROS levels were analyzed. PDK1 was increased only in radioresistant cells that have radiation-induced HIF-1α. Knockdown of HIF-1α decreased PDK1 expressions and increased ROS levels after irradiation. Conclusions: We demonstrated that radiation-induced HIF-1α induces PDK1 expression and decreases ROS productions, resulting in prevention of apoptosis after irradiation. Additional studies are ongoing to determine the in vivo efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2514. doi:10.1158/1538-7445.AM2011-2514