Nicolaus Gordon

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.

Lack of Constitutively Active DNA Repair Sensitizes Glioblastomas to Akt Inhibition and Induces Synthetic Lethality with Radiation Treatment in a p53-Dependent Manner

Treatment refractory glioblastoma (GBM) remains a major clinical problem globally, and targeted therapies in GBM have not been promising to date. The Cancer Genome Atlas integrative analysis of GBM reported the striking finding of genetic alterations in the p53 and PI3K pathways in more than 80% of GBMs. Given the role of these pathways in making cell-fate decisions and responding to genotoxic stress, we investigated the reliance of these two pathways in mediating radiation resistance. We selected a panel of GBM cell lines and glioma stem cells (GSC) with wild-type TP53 (p53-wt) and mutant TP53, mutations known to interfere with p53 functionality (p53-mt). Cell lines were treated with a brain permeable inhibitor of P-Akt (ser473), phosphatidylinositol ether lipid analogue (PIA), with and without radiation treatment. Sensitivity to treatment was measured using Annexin-V/PI flow cytometry and Western blot analysis for the markers of apoptotic signaling, alkaline COMET assay. All results were verified in p53 isogenic cell lines. p53-mt cell lines were selectively radiosensitized by PIA. This radiosensitization effect corresponded with an increase in DNA damage and a decrease in DNA-PKcs levels. TP53 silencing in p53-wt cells showed a similar response as the p53-mt cells. In addition, the radiosensitization effects of Akt inhibition were not observed in normal human astrocytes, suggesting that this treatment strategy could have limited off-target effects. We demonstrate that the inhibition of the PI3K/Akt pathway by PIA radiosensitizes p53-mt cells by antagonizing DNA repair. In principle, this strategy could provide a large therapeutic window for the treatment of TP53-mutant tumors. Mol Cancer Ther; 17(2); 336–46. ©2017 AACR. See all articles in this MCT Focus section, “Developmental Therapeutics in Radiation Oncology.”

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.

Abstract LB-6: Exploring nicotinamide-N-methyltransferase kinetics through targeted metabolomic profiling for its prognostic value in glioblastoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose: Glioblastoma (GBM) is the most aggressive form of glioma. With average patient survival of about 14 months, largely due to its resistance to conventional therapies, there is opportunity for significant improvements to care. Nicotinamide-N-methyltransferase (NNMT) is an enzyme responsible for the methylation of Nicotinamide and other xenobiotic compounds. Although NNMTs exact role in the malignant phenotype remains poorly understood, it has been investigated for its potential use as a prognostic marker or therapeutic target in other cancers. It is detected at very low levels in normal brain tissue but at significantly higher levels in GBM. Additionally, Kaplan-Meier plots from publically available data sets suggest that higher NNMT expression is correlated with adverse patient outcome. In addition to gene interference and various functional assays, we employed a targeted metabolomic approach, using LC-MS Quadropole Time of Flight (Q-TOF) and LC-MS Triple Quad (QQQ) instruments to study intracellular levels of Nicotinamide and N-methylnicotinamide to gain a better understanding of NNMTs role in GBM. Methods: We determined the expression profile of NNMT in established (ATCC) and in our panel of patient derived, primary GBM cell lines. By generating an NNMT isogenic model in U87 cells, we were able to study the functional consequences of differential NNMT expression using in vitro assays such as MTS Assay, Clonogenic Survival Assay, Annexin V, ATP Assay, Comet Assay, and Western Blotting. We intracranially injected NOD-SCID mice with U87 NNMT cells to assess differences in tumorgenicity in vivo. We have also used LC-MS Quadropole Time of Flight (Q-TOF) and LC-MS Triple Quad (QQQ) instruments to measure intracellular levels of the metabolites relevant to NNMT enzymatic function. Results and Conclusions: Results indicate that NNMT protein is highly expressed in primary cell lines. Encouragingly, U87 NNMT Knockdown cells are less proliferative, more sensitive to radiation in vitro, and are less tumorgenic in vivo. Recapitulating the clinical observations, these results could justify why clinically patients with lower expression of NNMT enjoyed increased overall survival. These results also suggest that finding ways to decrease NNMT in patients tumors may improve response to radiation. Additional preliminary results indicate that intracellular N-methylnicotinamide levels correlate directly with NNMT protein expression and could be used a surrogate biomarker for intracellular NNMT levels, potentially predicting not only overall survival but perhaps response to radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-6. doi:1538-7445.AM2012-LB-6

Abstract 2478: Silencing Aurora Kinase A radio-sensitizes glioma cells

Purpose: It has been reported that Aurora Kinase A (AurKA) is overexpressed in many cancer types, including breast, ovarian, pancreatic, head and neck, esophageal, renal, and lung. Upregulation of AurKA can lead to oncogenic transformation and aneuploidy. AurKA is a member of the serine/threonine family of protein kinases that is localized to the mitotic spindle during cell division. AurKA is involved in spindle assembly and regulated by phosphorylation-dependent proteosomal degradation. Cells that overexpress AurKA surpass the G2/M checkpoint prematurely, resulting in chromosomal abnormalities. Silencing AurKA will prevent premature progression through the G2/M checkpoint, allowing for cells to be targeted in the radiosensitive G2/M phase. Materials and Methods: We first looked at the expression of AurKA in established (ATCC) and primary glioblastoma (GBM) cell lines. Stable silencing of AurKA was done via a lentiviral transfection with shRNA in U87, LN18, and LN229 cell lines. We performed clonogenic survival and MTS assays to measure the cell death after radiation of these AurKA knockdown (AurKA KD) cells versus cells transfected with non-targeting (NT) shRNA. Western blot was performed using these same cells post-radiation treatment to investigate the expression of proteins in both apoptotic and pro-survival pathways. To investigate the effect of AurKA KD on the formation of polyploidy cells and cell cycle regulation. Cell cycle analysis on AurKA KD and NT cells was conducted using flow cytometry. AurKA is also known to phosphorylate tumor suppressor protein p53 at Ser315, which leads to degradation of p53 via ubiquitination by Mdm2. We carried out experiments where AurKA KD cells, with either intact or mutant p53, were treated with a p53 inhibitor Pifithrin α and subsequently measured apoptosis using Annexin V and Western blot. Initial sequencing results of AurKA in our GBM cell lines did not lead to any conclusive results. Furthermore, we assessed the copy number variation of AurKA to correlate with radiosensitivity of our GBM cell lines. Results and Conclusions: Radiation treatment increases AurKA expression in our established and primary GBM cell lines. Next we silenced AurKA using lentiviral mediated shRNA transduction. AurKA KD cells exhibit greater radiosensitivity and reduced proliferation when compared to control cells, as shown by the clonogenic survival and MTS assays, respectively. Increased expression of apoptotic proteins, such as cleaved PARP and cleaved caspase-3, is seen in AurKA KD cells after treatment with radiation when compared to controls. Moreover, silencing AurKA prevented the accumulation of polyploid cells after radiation treatment. Our data suggest that AurKA could be a promising therapeutic target for radiosensitizing of GBM. 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 2478. doi:10.1158/1538-7445.AM2011-2478

Abstract 2504: Genetic and molecular profiles altering radiosensitivity in melanoma

Purpose/Objectives: Historically, melanoma has been considered a relatively radioresistant tumor. Newer data have challenged this viewpoint, and radiation therapy (RT) is now considered to be useful for treating patients with malignant melanoma. Most importantly, it can provide effective palliation for the 40 to 50 percent of patients who develop unresectable locally recurrent or metastatic disease producing bone pain, epidural spinal cord compression, central nervous system dysfunction due to brain involvement, and/or tumor hemorrhage. RT has also been applied after complete excision of a primary melanoma or after therapeutic lymphadenectomy for regional nodal disease as adjuvant therapy to reduce the rate of local recurrence for certain types of melanoma. Melanoma is the most lethal form of skin cancer and incidences in the United States have steadily increased over the past 30 years. According to the American Cancer Society, melanoma accounts for only 3 percent of all skin cancers, but is responsible for nearly 74 percent of the deaths resulting from skin cancer. Prognosis is particularly poor for patients with metastasis as only 15 percent of this patient population survives for more than 5 years. Melanoma has been regarded as resistant to most treatments including radiation therapy. Melanoma has been poorly studied from the perspective of radiation biology and it is our intentions to elucidate key molecular mechanisms that can improve the benefit of radiotherapy in melanoma. Materials/Methods: In this study we set out to study genes that are commonly mutated in melanoma: BRAF, N-RAS, CKIT, TP53, PTEN, etc. These genes were sequenced in our panel of melanoma cell lines for determination of mutational status. We then stratified the cell lines based on their response to radiation as measured in a clonogenic survival and other functional assays. Established (ATCC) and primary (OSUMC patients) cell lines were used in this study. From the radioresistant melanoma cell lines we were able to isolate a small subpopulation of cells which exhibited the properties of stem cells and currently we are conducting further studies to completely characterize this sub population. Results/Conclusion: Melanoma has been poorly studied from the perspective of radiation biology and it is our intentions to elucidate key molecular mechanisms that can improve the benefit of radiotherapy in melanoma. It appears that BRAF intact cell lines are radiosensitive, whereas a mutation in at least one of the BRAF alleles at codon 600 leads to a radioresistant phenotype. We have adapted a pathway based targeting approach rather than the conventional gene based methods. We will present our preclinical studies on how genetic and molecular signatures in melanoma play a pivotal role in radiosensitizing melanoma and which subset of melanoma patients would derive the benefit from RT. 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 2504. doi:10.1158/1538-7445.AM2011-2504

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

Abstract LB-165: Novel metabolic metyltransferase mediates radiation resistance in glioblastomas

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: Nicotinamide- N -methyltransferase(NNMT) is an enzyme involved in the metabolism of drugs and xenobiotic compounds. It is known to be highly expressed in a variety of cancers and has been investigated for its potential use as a diagnostic or prognostic marker. Furthermore, clinical data has shown that high NNMT expression is correlated with adverse patient outcome. In our glioblastoma (GBM) patient data set and other publically available data set NNMT is one among the top 25 genes which is differentially regulated when compared with normal adjacent tissue and also with various grades of brain tumor. NNMT is highly expressed in grade IV compared to low grade astrocytomas. Kaplan-Meier survival plots for NNMT gene shows that higher expression is associated with poor survival benefit and the median survival was found to be more than doubled in patient group with low expression of NNMT. Despite all this, the mechanistic behavior of NNMT in the malignant phenotype remains uncharacterized. We plan to gain an understanding of the signaling pathways with which NNMT interacts and elucidate its role in radioresistance. Experimental Design: The in vitro radiosensitivity assays were evaluated using wild type, NNMT-knockdown and NNMT-overexpression GBM cell lines. The assay conducted includes MTS, DNA damage and repair, Clonogenic survival assay, comet assay, Annexin V apoptosis assay, matrigel invasion assays, ATP assay, etc. The cell line after treatment was lysed and probed for pro-and anti-survival signaling pathways. Results and Discussion: We explored the expression profile of NNMT in established (ATCC) and in our panel of primary GBM cell lines. The expression levels correlates with relative radioresistance exhibited by the cell line. It appears NNMT knockdown cell lines exhibited an enhanced radiosensitivity. Currently we are probing signaling cascades which are differentially regulated in the NNMT knockdown and overexpression cell lines and also the differential response to radiation treatment based on differing levels of NNMT expression. The results will be presented at the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-165.

Abstract LB-164: Aurora Kinase A as a target for sensitizing glioma cells to radiation treatment

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: Eukaryotic cells have a complex cell cycle process that is regulated by many factors. Tumors arise when cell cycle progression is deregulated. Mitosis is a well coordinated event and occurs only when proper signals allow the cell cycle machinery to proceed beyond the G2/M checkpoint. Aurora kinase A (AurKA) is a mitotic regulator involved in bipolar-spindle assembly and chromosome segregation, and also recruits other proteins and cofactors that are important for mitotic events. AurKA is upregulated in many cancers and has been shown to cause aneuploidy and induce oncogenic transformation. AurKA overexpression allows cells to surpass the G2/M checkpoint and enter mitosis prematurely, which leads to chromosomal abnormalities and genomic instability. Experimental Design: Here we are investigating AurKA in established (ATCC) and primary glioblastoma (GBM) cell lines. Assays measuring radiosensitivity were conducted using wild-type and AurKA knockdown GBM cells. These include the clonogenic survival assay, MTS proliferation assay, cell-cycle analysis, and Annexin V apoptosis assay. Protein profiles were used to investigate pro-apoptotic, anti-apoptotic, and other downstream and upstream signaling pathways after radiation treatment. Results and Discussion: Irradiation induces AurKA expression in both our established and primary GBM cell lines. Next we silenced AurKA using lentiviral mediated shRNA transduction. AurKA knockdown cells exhibit greater radiosensitivity when compared to cells transduced with a mock vector. AurKA and p53 interact at multiple levels. Preclinical data with AurKA inhibitors suggest p53-negative tumors may be more sensitive to AurKA inhibitors than p53-positive tumors. To investigate which subset of GBMs show greater response to AurKA silencing, we will conduct experiments using our panel of isogenic GBM cells lines. Our hypothesis is that AurKA knockdown cells will show reduced proliferation and increased apoptosis, thereby leading to cell death. The outcome of this preclinical study would be to evaluate AurKA as a potential therapeutic target for the treatment of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-164.