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Supplementary Figure 1: Inhibition of MPS1 activity compromises the viability of GBM tumor cells.

Supplementary Figure 6. Kaplan-Meier curve analysis examining MPS1 expression (red- high expression, black- low expression) and overall survival association in 1115 breast (A) and 1405 lung (B) cancer patients using univariate analysis using KM-Plotter (www.kmplot.com).

Supplementary Figure 3. Western blot analysis of pDNAPK and DNAPK from 2.5 uM NMS-P715 treated and irradiated (4 Gy) U251 cells.

Supplementary Figure 2. Molecular profiling reveals changes in genes associated with DNA replication, recombination and repair.

Supplementary Figure 5. To assay nonhomologous end joining (NHEJ), U251 DR-GFP cells were transfected with pCBAScel vector and harvested at 48 hours after indicated treatments. Genomic DNA was harvested for PCR amplification using DR-GFP primers, followed by I-Scel+Bcgl enzyme digestion and PCR products were subjected to gel electrophoresis.

Supplementary Figure 4. To assay homologous recombination (HR), U251 DR-GFP cells were transfected with pCBAScel vector and harvested at 48 hours after indicated treatments. GFP+ve cells representing HR repair events were determined by flow cytometry and PCR based assays.

Supplementary methods

PDF - 89K, Functions associated with the top networks for the genes no longer decreased by radiation after INK128 treatment.

PDF - 93K, Genes comprising the DNA replication, recombination and repair biological function whose radiation-induced up-regulation was inhibited by INK128.

Purpose: Radiotherapy remains a primary treatment modality for pancreatic carcinoma, a tumor characterized by aberrant mTOR activity. Given the regulatory role of mTOR in gene translation, in this study, we defined the effects of the clinically relevant, ATP-competitive mTOR inhibitor, INK128 on the radiosensitivity of pancreatic carcinoma cell lines.Experimental Design: Clonogenic survival was used to determine the effects of INK128 on in vitro radiosensitivity of three pancreatic carcinoma cell lines and a normal fibroblast cell line with mTOR activity defined using immunoblots. DNA double-strand breaks were evaluated according to γH2AX foci. The influence of INK128 on radiation-induced gene translation was determined by microarray analysis of polysome-bound mRNA. Leg tumor xenografts grown from pancreatic carcinoma cells were evaluated for mTOR activity, eIF4F cap complex formation, and tumor growth delay.Results: INK128, while inhibiting mTOR activity in each of the cell lines, enhanced the in vitro radiosensitivity of the pancreatic carcinoma cells but had no effect on normal fibroblasts. The dispersal of radiation-induced γH2AX foci was inhibited in pancreatic carcinoma cells by INK128 as were radiation-induced changes in gene translation. Treatment of mice with INK128 resulted in an inhibition of mTOR activity as well as cap complex formation in tumor xenografts. Whereas INK128 alone had no effect of tumor growth rate, it enhanced the tumor growth delay induced by single and fractionated doses of radiation.Conclusion: These results indicate that mTOR inhibition induced by INK128 enhances the radiosensitivity of pancreatic carcinoma cells and suggest that this effect involves the inhibition of DNA repair. Clin Cancer Res; 20(1); 110–9. ©2013 AACR.

PDF - 99K, Functions associated with the top networks corresponding to the genes whose radiation-induced upregulation was inhibited by INK128

Nutritional intervention is becoming more prevalent as adjuvant therapy for many cancers in view of the tumor dependence on external sources for some nutrients. However, little is known about the mechanisms that render cancer cells dependent on certain nutrients from the microenvironment. Herein, we report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. Using several 13 C-tracers and analysis of cystathionine synthase and cystathioninase levels, we revealed that glioma cells were not able to support GSH synthesis through the transsulfuration pathway, which allows methionine to be converted to cysteine in cysteine/cystine deprived conditions. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine-free diet survived longer, although this increase did not attain statistical significance, with concomitant reductions in plasma glutathione and cysteine levels. At the end point, however, tumors displayed the ability to synthesize glutathione, although higher levels of oxidative stress were detected. We observed a compensation from the nutritional intervention revealed as the recovery of cysteine-related metabolites levels in plasma. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.

Dynamic nuclear polarization (DNP) of 13 C-labeled substrates enables the use of magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors and offers an opportunity to investigate these differences. In this study, DNP-MRI chemical shift imaging with hyperpolarized [1-13 C] pyruvate was conducted to evaluate the metabolic change in glycolytic profiles after radiation of two glioma stem-like cell-derived gliomas (GBMJ1 and NSC11) and an adherent human glioblastoma cell line (U251) in an orthotopic xenograft mouse model. The DNP-MRI showed an increase in Lac/Pyr at 6 and 16 h after irradiation (18% ± 4% and 14% ± 3%, respectively; mean ± SEM) compared with unirradiated controls in GBMJ1 tumors, whereas no significant change was observed in U251 and NSC11 tumors. Metabolomic analysis likewise showed a significant increase in lactate in GBMJ1 tumors at 16 h. An immunoblot assay showed upregulation of lactate dehydrogenase-A expression in GBMJ1 following radiation exposure, consistent with DNP-MRI and metabolomic analysis. In conclusion, our preclinical study demonstrates that the DNP-MRI technique has the potential to be a powerful diagnostic method with which to evaluate GBM tumor metabolism before and after radiation in the clinical setting.

Dynamic nuclear polarization (DNP) of 13C‐labeled substrates enables the use of magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors and offers an opportunity to investigate these differences. In this study, DNP‐MRI chemical shift imaging with hyperpolarized [1‐13C] pyruvate was conducted to evaluate the metabolic change in glycolytic profiles after radiation of two glioma stem‐like cell‐derived gliomas (GBMJ1 and NSC11) and an adherent human glioblastoma cell line (U251) in an orthotopic xenograft mouse model. The DNP‐MRI showed an increase in Lac/Pyr at 6 and 16 h after irradiation (18% ± 4% and 14% ± 3%, respectively; mean ± SEM) compared with unirradiated controls in GBMJ1 tumors, whereas no significant change was observed in U251 and NSC11 tumors. Metabolomic analysis likewise showed a significant increase in lactate in GBMJ1 tumors at 16 h. An immunoblot assay showed upregulation of lactate dehydrogenase‐A expression in GBMJ1 following radiation exposure, consistent with DNP‐MRI and metabolomic analysis. In conclusion, our preclinical study demonstrates that the DNP‐MRI technique has the potential to be a powerful diagnostic method with which to evaluate GBM tumor metabolism before and after radiation in the clinical setting., Orthotopic brain tumor mice were subjected to 13C‐MRI using hyperpolarized [1‐13C] pyruvate to investigate the effect of radiation exposure on glucose metabolism by measuring lactate‐to‐pyruvate ratio in the tumor. Tissue sampling was conducted independently to quantify the lactate concentration and LDHA expression by metabolomic and immunoblot analyses, respectively. These indicated an acceleration of lactate‐to‐pyruvate conversion in one of the stem‐like cell‐derived gliomas after an irradiation during a 24‐h period of time.

Nutritional intervention is becoming more prevalent as adjuvant therapy for many cancers in view of the tumor dependence on external sources for some nutrients. We report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. 13C-tracing and the analysis of cystathionine synthase and cystathioninase levels revealed the metabolic landscape attributable to cysteine deprivation, and the disconnection between the methionine cycle and the transsulfuration pathway. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine-free diet survived longer, with concomitant reductions in glutathione and cysteine plasma levels. At the end point, however, tumors displayed the ability to synthesize glutathione, although higher levels of oxidative stress were detected. We observed a compensation from the nutritional intervention revealed as the recovery of cysteine-related metabolites in plasma. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.

AZD0530, a potent small-molecule inhibitor of the Src kinase family, is an anticancer drug used in the treatment of various cancers. In the case of glioblastoma (GBM), where resistance to radiotherapy frequently occurs, Src kinase is known as one of the molecules responsible for imparting radioresistance to GBM. Thus, we evaluated the effect of AZD0530 on the radiosensitivity of human GBM cells and human glioblastoma stem-like cells (GSCs). We show that Src activity of GBM and GSC is increased by radiation and inhibited by AZD0530, and using clonogenic assays, AZD0530 enhances the radiosensitivity of GBM and GSCs. Also, AZD0530 induced a prolongation of radiation-induced γH2AX without specific cell cycle and mitotic index changes, suggesting that AZD0530-induced radiosensitization in GBM cells and GSCs results from the inhibition of DNA repair. In addition, AZD0530 was shown to inhibit the radiation-induced EGFR/PI3K/AKT pathway, which is known to promote and regulate radioresistance and survival of GBM cells by radiation. Finally, mice bearing orthotopic xenografts initiated from GBM cells were then used to evaluate the in vivo response to AZD0530 and radiation. The combination of AZD0530 and radiation showed the longest median survival compared with any single modality. Thus, these results show that AZD0530 enhances the radiosensitivity of GBM cells and GSCs and suggest the possibility of AZD0530 as a clinical radiosensitizer for treatment of GBM.

BACKGROUND Little is known about the mechanisms that render cancer cells dependent on certain nutrients from the microenvironment. Cysteine is a non-essential amino acid, since it can be synthetized from methionine through the transsulfuration pathway; moreover, cysteine is also uptake from the diet as cystine. We have investigated the metabolism of cysteine in glioma cell lines, and how cysteine/cystine-deprivation alters their antioxidant response in addition to the effect of this nutrient restriction to viability and proliferation in vitro and in vivo. METHODS Cysteine metabolism was investigated through LCMS-based 13C-tracing experiments and the expression levels of key enzymes in the transsulfuration pathway were also explored. Finally, a mouse model of IDH1 mutant glioma was subjected to a cysteine/cystine-free diet and tumor metabolism was analyzed by LCMS. RESULTS Herein, we report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. Using several 13C-tracers and analysis of cystathionine synthase and cystathioninase levels, we revealed that glioma cells were not able to upregulate the transulfuration pathway cysteine, which allows methionine to be converted to cysteine in cysteine/cystine deprived conditions. We demonstrated that exogenous cysteine/cystine are crucial for glutathione synthesis, and impact growth and viability. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine-free diet survived longer, with concomitant reductions in glutathione and cysteine plasma levels. At the endpoint higher levels of oxidative stress were detected despite the systemic recovery of cysteine-related metabolites in the plasma. CONCLUSION The results presented herein reveal an alternative therapeutic approach combining cysteine/cysteine-deprivation diets and treatments involving ROS production by limiting the ability of glioma cells to quench oxidative stress through dietary interventions. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.

Background Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Methods Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. Results PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Conclusions Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma.

Cholesterol is a critical component of membranes and a precursor for hormones and other signaling molecules. Previously, we showed that unlike astrocytes, glioblastoma cells do not downregulate cholesterol synthesis when plated at high density. In this report, we show that high cell density induces ABCA1 expression in glioblastoma cells, enabling them to get rid of excess cholesterol generated by an activated cholesterol biosynthesis pathway. Because oxysterols are agonists for Liver X Receptors (LXRs), we investigated whether increased cholesterol activates LXRs to maintain cholesterol homeostasis in highly-dense glioblastoma cells. We observed that dense cells had increased oxysterols, which activated LXRβ to upregulate ABCA1. Cells with CRISPR-mediated knockdown of LXRβ, but not ABCA1, had decreased cell cycle progression and cell survival, and decreased feedback repression of the mevalonate pathway in densely-plated glioma cells. LXRβ gene expression poorly correlates with ABCA1 in glioblastoma patients, and expression of each gene correlates with poor patient prognosis in different prognostic subtypes. Finally, gene expression and lipidomics analyses cells revealed that LXRβ regulates the expression of immune response gene sets and lipids known to be involved in immune modulation. Thus, therapeutic targeting of LXRβ in glioblastoma might be effective through diverse mechanisms.

BACKGROUND Cysteine is a non-essential amino acid, since it can be synthetized from methionine through the transsulfuration pathway; moreover, cysteine is also uptake from the diet as cystine. We have investigated the metabolism of cysteine in glioma cell lines, and how cysteine/cystine-deprivation alters their antioxidant response in addition to the effect of this nutrient restriction to viability and proliferation in vitro and in vivo. METHODS Cysteine metabolism was investigated through LCMS-based 13C-tracing experiments involving different probes such as 13C-methyl-Methionine, 13C-C3-Cysteine, 13C-C3,3’-Cystine, 13C-C3-Serine and 13C-U-Glutamine and the expression levels of key enzymes in the transsulfuration pathway were also explored. Finally, a mouse model of IDH1 mutant glioma was subjected to a cysteine/cystine-free diet and tumor metabolism was analyzed by LCMS. RESULTS We demonstrated that exogenous cysteine/cystine are crucial for glutathione synthesis, and impact growth and viability. We also found that methionine cycle is disconnected from the transsulfuration pathway based on 13C-tracing data and protein expression levels of cystathionine synthase and cystathioninase. Accordingly, cysteine-related metabolites such as GSH, involved in REDOX hemostasis, are downregulated, revealing a hypersensitive phenotype to ROS. Animal models upon a cysteine/cystine-free diet experienced an increase in survival and elevated levels of oxidative stress in tumor tissue. CONCLUSION This results presented herein reveal an alternative therapeutic approach combining cysteine/cysteine-deprivation diets and treatments involving ROS production by limiting the ability of glioma cells to quench oxidative stress through dietary interventions.

// Uday B. Maachani 1 , Uma Shankavaram 1 , Tamalee Kramp 1 , Philip J. Tofilon 1 , Kevin Camphausen 1 , Anita T. Tandle 1 1 Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA Correspondence to: Anita T. Tandle, email: tandlea@mail.nih.gov Keywords: glioblastoma multiforme, FOXM1, STAT3, glioma stem cells, radio resistance Received: June 30, 2016 Accepted: September 28, 2016 Published: October 14, 2016 ABSTRACT Glioblastoma multiforme (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Adjuvant chemo-radiotherapy remains the standard of care following surgical resection. In this study, using reverse phase protein arrays (RPPAs), we assessed the biological effects of radiation on signaling pathways to identify potential radiosensitizing molecular targets. We identified subsets of proteins with clearly concordant/discordant behavior between irradiated and non-irradiated GBM cells in vitro and in vivo . Moreover, we observed high expression of Forkhead box protein M1 (FOXM1) in irradiated GBM cells both in vitro and in vivo . Recent evidence of FOXM1 as a master regulator of metastasis and its important role in maintaining neural, progenitor, and GBM stem cells, intrigued us to validate it as a radiosensitizing target. Here we show that FOXM1 inhibition radiosensitizes GBM cells by abrogating genes associated with cell cycle progression and DNA repair, suggesting its role in cellular response to radiation. Further, we demonstrate that radiation induced stimulation of FOXM1 expression is dependent on STAT3 activation. Co-immunoprecipitation and co-localization assays revealed physical interaction of FOXM1 with phosphorylated STAT3 under radiation treatment. In conclusion, we hypothesize that FOXM1 regulates radioresistance via STAT3 in GBM cells. We also, show GBM patients with high FOXM1 expression have poor prognosis. Collectively our observations might open novel opportunities for targeting FOXM1 for effective GBM therapy.

Most cancer patients with brain metastases are treated with radiation therapy, yet this modality has not yet been meaningfully incorporated into preclinical experimental brain metastasis models. We applied two forms of whole brain radiation therapy (WBRT) to the brain-tropic 231-BR experimental brain metastasis model of triple-negative breast cancer. When compared to sham controls, WBRT as 3 Gy × 10 fractions (3 × 10) reduced the number of micrometastases and large metastases by 87.7 and 54.5 %, respectively (both p < 0.01); whereas a single radiation dose of 15 Gy × 1 (15 × 1) was less effective, reducing metastases by 58.4 % (p < 0.01) and 47.1 % (p = 0.41), respectively. Neuroinflammation in the adjacent brain parenchyma was due solely to a reaction from metastases, and not radiotherapy, while adult neurogenesis in brains was adversely affected following both radiation regimens. The nature of radiation resistance was investigated by ex vivo culture of tumor cells that survived initial WBRT (“Surviving” cultures). The Surviving cultures surprisingly demonstrated increased radiosensitivity ex vivo. In contrast, re-injection of Surviving cultures and re-treatment with a 3 × 10 WBRT regimen significantly reduced the number of large and micrometastases that developed in vivo, suggesting a role for the microenvironment. Micrometastases derived from tumor cells surviving initial 3 × 10 WBRT demonstrated a trend toward radioresistance upon repeat treatment (p = 0.09). The data confirm the potency of a fractionated 3 × 10 WBRT regimen and identify the brain microenvironment as a potential determinant of radiation efficacy. The data also nominate the Surviving cultures as a potential new translational model for radiotherapy.

Monopolar spindle 1 (MPS1) is an essential spindle assembly checkpoint (SAC) kinase involved in determining spindle integrity. Beyond its mitotic functions, it has been implicated in several other signaling pathways. Our earlier studies have elaborated on role of MPS1 in glioblastoma (GBM) radiosensitization. In this study using reverse phase protein arrays (RPPAs), we assessed MPS1 mediated cell signaling pathways and demonstrated that inhibiting MPS1 could upregulate the expression of the tumor suppressor PDCD4 and MSH2 genes, by down regulating micro RNA-21 (miR-21). In GBMs miR-21 expression is significantly elevated and is associated with chemo and radioresistance. Both MPS1 and miR-21 depletion suppressed GBM cell proliferation, whereas, ectopic expression of miR-21 rescued GBM cell growth from MPS1 inhibition. Further, we demonstrate that MPS1 mediates phosphorylation of SMAD3 but not SMAD2 in GBM cells; A possible mechanism behind miR-21 modulation by MPS1. Collectively, our results shed light onto an important role of MPS1 in TGF-β/SMAD signaling via miR-21 regulation. We also, show the prognostic effect of miR-21, PDCD4 and MSH2 levels to patient survival across different GBM molecular subtypes. This scenario in which miR-21 is modulated by MPS1 inhibition may be exploited as a potential target for effective GBM therapy.

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell–specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells were analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor NMS-P715 on radiosensitivity in multiple model systems, including GBM cell lines, a normal astrocyte, and a normal fibroblast cell line. DNA double-strand breaks (DSB) were evaluated using γH2AX foci, and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Furthermore, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of postradiation mitotic catastrophe. NMS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1-silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair, and replication, including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. Implications: Inhibition of MPS1 kinase in combination with radiation represents a promising new approach for glioblastoma and for other cancer therapies. Mol Cancer Res; 13(5); 852–62. ©2015 AACR.

Atypical and anaplastic meningiomas (AAM) represent 20% of all meningiomas. They are associated with poor outcomes due to their tendency to recur. While surgery and radiation (RT) are first line therapy, no effective systemic medical treatment has been identified. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in cell cycle regulation and DNA repair. Here, we examined radiosensitizing effects of LB-100, a novel inhibitor of PP2A against AAM as a novel treatment strategy. Three human-derived immortalized meningioma cell lines, IOMM-LEE, GAR, and CH-157, were used to investigate the radio-sensitizing potential of LB-100 in AAM. Survival fraction by clonogenic assay, immunofluorescence, cell cycle analysis and protein expression were evaluated in vitro. The antitumor effects of combining LB-100 with RT were verified in vivo by using intracranial orthotopic xenograft mouse model. Pharmacologic PP2A inhibition with LB-100 prior to RT enhanced the radiosensitivity of meningioma cells and reduced survival fraction in clonogenic assays. LB-100 increased DNA double-strand breakage (measured by γ-H2AX), mitotic catastrophe cell death, and G2/M cell cycle arrest in irradiated meningioma cells. Also, LB-100 decreased activation of STAT3 and expression of its downstream proteins. In vivo, LB-100 and RT combined treatment prolonged the survival of mice with xenografts compared to RT alone. Taken together, these results provide convincing preclinical data to support the use of LB-100 as a radiosensitizing agent for treatment of malignant meningioma. Its potential for clinical application deserves further investigation.

OBJECTIVES: Since the cancer stem-like cells (CSCs) play an important role in the resistance against radiation therapy due to its different metabolic profile, investigation of the specific metabolic characteristics of CSCs is valuable. Dynamic nuclear polarization (DNP) technique using 13C-labeled substrates enables magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors. In this study, DNP-MRI along with hyperpolarized [1-13C] pyruvate was conducted to evaluate the metabolic change in glycolytic profiles after irradiation between a glioblastoma cell line and a CSC-derived glioma using orthotopic xenograft mouse model. A microarray analysis was also performed to compare to the imaging study. METHODS: DNP-MRI A 3T MRI scanner along with a custom-made head coil was used. To obtain hyperpolarized [1-13C] pyruvate, 30 μL of [1-13C] pyruvic acid containing 15 mM of OX063 (trityl radical compound) and 2.5 mM of the gadolinium chelate were polarized at 3.35 T and 1.4 K in the Hypersense DNP polarizer. The hyperpolarized sample dissolved in a buffered medium was injected via the tail vein followed by an acquisition of 13C-two-dimensional MR-spectroscopy to measure the pyruvate-to-lactate ratios (Lac/Pyr) in the tumor region and the contralateral normal brain separately. Cell lines A human glioblastoma cell line, U251 and a glioma CSC line, NSC11 were cultured for stereotactic intracranial injection. Animal model Orthotopic brain tumor model was developed in athymic nude mice by intracranial implantation. When the tumor volume reached 50 +/- 10 mm3 on T2-weighted MRI, a sequence of DNP-MRI scans was performed 24 hours before and 6, 16 and 24 after 6-Gy whole-brain irradiation (N = 4 each with a non-irradiated control group). Independently, a microarray analysis using the tumor tissue samples excised 6 and 24 hours after irradiation was performed on Affymetrix Human Genome U133 Plus 2.0 array. RESULTS: DNP-MRI showed a decrease in Lac/Pyr 6 and 16 hours after irradiation compared to the control group in U251 whereas no significant change was observed in the NSC11 tumor. The microarray analysis showed a significant down-regulation of lactate dehydrogenase A (LDH-A) expression in U251 6 and 24 hours after irradiation, consistent with the finding of DNP-MRI. In the microarray analysis, SIRT2 and KLF4 expression also changed in the U251 tumor, suggested to regulate the LDH-A activity/expression. CONCLUSION: DNP-MRI demonstrated the difference in radiation response in glycolytic metabolism between the glioblastoma cell line and the CSC-derived glioma. This technique might be a feasible diagnostic method evaluating metabolic profiles of the brain tumors in the clinical settings of pretreatment and post-treatment. Citation Format: Tatsuya Kawai, Murali C. Krishna, Tamalee Kramp, Jeffery R. Brender, Philip Tofilon, Kevin A. Camphausen. Detection of metabolic change in glioblastoma after radiotherapy using hyperpolarized 13C-MRI: Glycolytic metabolism in cancer stem cell-like cell-derived tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1944.

Purpose: Radiotherapy remains a primary treatment modality for pancreatic carcinoma, a tumor characterized by aberrant mTOR activity. Given the regulatory role of mTOR in gene translation, in this study, we defined the effects of the clinically relevant, ATP-competitive mTOR inhibitor, INK128 on the radiosensitivity of pancreatic carcinoma cell lines. Experimental Design: Clonogenic survival was used to determine the effects of INK128 on in vitro radiosensitivity of three pancreatic carcinoma cell lines and a normal fibroblast cell line with mTOR activity defined using immunoblots. DNA double-strand breaks were evaluated according to γH2AX foci. The influence of INK128 on radiation-induced gene translation was determined by microarray analysis of polysome-bound mRNA. Leg tumor xenografts grown from pancreatic carcinoma cells were evaluated for mTOR activity, eIF4F cap complex formation, and tumor growth delay. Results: INK128, while inhibiting mTOR activity in each of the cell lines, enhanced the in vitro radiosensitivity of the pancreatic carcinoma cells but had no effect on normal fibroblasts. The dispersal of radiation-induced γH2AX foci was inhibited in pancreatic carcinoma cells by INK128 as were radiation-induced changes in gene translation. Treatment of mice with INK128 resulted in an inhibition of mTOR activity as well as cap complex formation in tumor xenografts. Whereas INK128 alone had no effect of tumor growth rate, it enhanced the tumor growth delay induced by single and fractionated doses of radiation. Conclusion: These results indicate that mTOR inhibition induced by INK128 enhances the radiosensitivity of pancreatic carcinoma cells and suggest that this effect involves the inhibition of DNA repair. Clin Cancer Res; 20(1); 110–9. ©2013 AACR.

The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of gene translation and has been suggested as a potential target for radiosensitization. The goal of this study was to compare the radiosensitizing activities of the allosteric mTOR inhibitor rapamycin with that of the competitive mTOR inhibitor PP242. On the basis of immunoblot analyses, whereas rapamycin only partially inhibited mTOR complex 1 (mTORC1) activity and had no effect on mTOR complex 2 (mTORC2), PP242 inhibited the activity of both mTOR-containing complexes. Irradiation alone had no effect on mTORC1 or mTORC2 activity. Clonogenic survival was used to define the effects of the mTOR inhibitors on in vitro radiosensitivity. In the two tumor cell lines evaluated, PP242 treatment 1 hour before irradiation increased radiosensitivity, whereas rapamycin had no effect. Addition of PP242 after irradiation also enhanced the radiosensitivity of both tumor lines. To investigate the mechanism of radiosensitization, the induction and repair of DNA double-strand breaks were evaluated according yH2AX foci. PP242 exposure did not influence the initial level of yH2AX foci after irradiation but did significantly delay the dispersal of radiationinduced yH2AX foci. In contrast to the tumor cell lines, the radiosensitivity of a normal human fibroblast cell line was not influenced by PP242. Finally, PP242 administration to mice bearing U251 xenografts enhanced radiationinduced tumor growth delay. These results indicate that in a preclinical tumor model PP242 enhances tumor cell radiosensitivity both in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair.

OBJECTIVES: Since the cancer stem-like cells (CSCs) play an important role in the resistance against radiation and/or chemotherapy due to its different metabolic profile, to investigate the specific metabolic characteristics of CSCs is valuable. Dynamic nuclear polarization (DNP) technique using 13C-labeled substrates enables magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors. In this study, DNP-MRI along with hyperpolarized [1-13C] pyruvate was conducted to evaluate the difference in glycolytic profile between a glioblastoma cell line and a CSC-derived glioma using orthotopic xenograft mouse model. The imaging findings were compared to those in immunohistology and cell cultures. METHODS: A 3T MRI scanner along with a custom-made head coil was used for imaging. To obtain hyperpolarized [1-13C] pyruvate, 30 μL of [1-13C] pyruvic acid containing 15 mM of OX063 (trityl radical compound) and 2.5 mM of the gadolinium chelate were polarized at 3.35 T and 1.4 K in the Hypersense DNP polarizer. The hyperpolarized sample dissolved in a buffered medium was injected via the tail vein followed by a 13C-two-dimensional spectroscopic imaging. During the acquisition, the body temperature was kept in the range of 35.0 to 36.9 Celsius degrees. Orthotopic brain tumor model was developed in nude mice by intracranial implantation of glioblastoma cell lines, U251 and glioma stem-like cells, NSC11. DNP-MRI was performed when the tumor size ranged 50 + 10 mm3 on T2-weighted MRI. Independently, immunofluorescent analyses of monocarboxylate transporter 1(MCT1) and MCT4 was performed using paraffin-embedded tumor tissue. Expressed protein level of lactate dehydrogenase A (LDHA), LDHB, MCT1 and MCT4 under the normal cell culture conditions were evaluated by Western blotting. RESULTS: 13C-MRI showed an increase in lactate-to-pyruvate ratio (Lac/Pyr) both in U251 and NSC11 compared to the contralateral normal brain tissue, suggested elevated anaerobic glycolysis (P CONCLUSION: The quality of 13C-DNP-MRI using [1-13C] pyruvate was validated and considered to be feasible to apply to the brain tumors. The result was reasonable in that malignant tumors enhance anaerobic glycolytic pathway even in the aerobic environment. 13C-DNP-MRI also demonstrated a difference in the extent of Lac/Pyr in relation to the normal brain between the two tumor models probably related to expression levels of glycolysis-related proteins. Citation Format: Tatsuya Kawai, Murali C. Krishna, Jeffery R. Brender, Jennifer Lee, Tamalee Kramp, Kazu Yamamoto, Shun Kishimoto, Tomohiro Seki, Kevin A. Camphausen. Metabolic imaging of glioblastoma using hyperpolarized 13C-MRI: Glycolytic metabolism in cancer stem cell-like cell-derived tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4110.

The mammalian target of rapamycin (mTOR) has been suggested as a target for radiosensitization. Given that radiotherapy is a primary treatment modality for glioblastoma (GBM) and that mTOR is often dysregulated in GBM, the goal of this study was to determine the effects of AZD2014, a dual mTORC1/2 inhibitor, on the radiosensitivity of GBM stem-like cells (GSCs).mTORC1 and mTORC2 activities were defined by immunoblot analysis. The effects of this mTOR inhibitor on the in vitro radiosensitivity of GSCs were determined using a clonogenic assay. DNA double strand breaks were evaluated according to γH2AX foci. Orthotopic xenografts initiated from GSCs were used to define the in vivo response to AZD2014 and radiation.Exposure of GSCs to AZD2014 resulted in the inhibition of mTORC1 and 2 activities. Based on clonogenic survival analysis, addition of AZD2014 to culture media 1 hour before irradiation enhanced the radiosensitivity of CD133+ and CD15+ GSC cell lines. Whereas AZD2014 treatment had no effect on the initial level of γH2AX foci, the dispersal of radiation-induced γH2AX foci was significantly delayed. Finally, the combination of AZD2014 and radiation delivered to mice bearing GSC-initiated orthotopic xenografts significantly prolonged survival as compared with the individual treatments.These data indicate that AZD2014 enhances the radiosensitivity of GSCs both in vitro and under orthotopic in vivo conditions and suggest that this effect involves an inhibition of DNA repair. Moreover, these results suggest that this dual mTORC1/2 inhibitor may be a radiosensitizer applicable to GBM therapy.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in the United States of America (USA) with a median survival of approximately 14 months. Low survival rates are attributable to the aggressiveness of GBM and a lack of understanding of the molecular mechanisms underlying GBM. The disruption of signalling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. To identify protein kinases required for the survival of GBM we performed a siRNA-based RNAi screen focused on the human kinome in GBM. Inhibition of the polo-like kinase 1 (PLK1) induced a reduction in the viability in two different GBM cell lines. To assess the potential of inhibiting PLK1 as a treatment strategy for GBM we examined the effects of a small molecule inhibitor of PLK1, GSK461364A, on the growth of GBM cells. PLK1 inhibition arrested cells in the mitotic phase of the cell cycle and induced cell kill by mitotic catastrophe. GBM engrafts treated with GSK461364A showed statistically significant inhibition of tumour growth. Further, exposure of different GBM cells to RNAi or GSK461364A prior to radiation resulted in an increase in their radiosensitivity with dose enhancement factor ranging from 1.40 to 1.53 with no effect on normal cells. As a measure of DNA double strand breaks, γH2AX levels were significantly higher in the combined modality as compared to the individual treatments. This study suggests that PLK1 is an important therapeutic target for GBM and can enhance radiosensitivity in GBM.

In the event of a radiological or nuclear attack, advanced clinical countermeasures are needed for screening and medical management of the exposed population. In such a scenario, minimally invasive biomarkers that can accurately quantify radiation exposure would be useful for triage management by first responders. In this murine study, we evaluated the efficacy of a novel combination of radiation responsive proteins, Flt3 ligand (FL), serum amyloid A (SAA), matrix metalloproteinase 9 (MMP9), fibrinogen beta (FGB) and pentraxin 3 (PTX3) to predict the received dose after whole- or partial-body irradiation. Ten-week-old female C57BL6 mice received a single whole-body or partial-body dose of 18 Gy from a Pantak X-ray source at a dose rate of 2.28 Gy/min. Plasma was collected by cardiac puncture at 24, 48, 72 h and 1 week postirradiation. Plasma protein levels were determined via commercially available ELISA assay. A multivariate discriminant analysis was utilized to generate best-fit dose prediction models for whole-body exposures using the selected biomarker panel and its potential application to partial-body exposures was examined. The combination of values from FL, SAA, MMP9, FGB and PTX3 between 24 h and 1 week postirradiation yielded novel dose-response relationships. For day 1 postirradiation, the best-fit model yielded a predictive accuracy of 81% utilizing FL alone. The use of additional proteins did not enhance the model accuracy whereas, at day 2 postirradiation, the addition of PTX3 and FGB to FL increased the accuracy to 100%. At day 3 the use of FL and PTX3 yielded a predictive accuracy of 93% and at day 7 use of FL and SAA had an accuracy of 90%. Dose prediction of partial-body exposures based on the TBI model had a higher predictive accuracy when the percentage of the body exposed to radiation increased. Our findings indicate that this novel combination of radiation responsive biomarker proteins are an efficient method for predicting radiation exposure and are more accurate when used in concert compared to using any single biomarker protein alone.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 μl every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

Purpose Vosaroxin is a first in class naphthyridine analog structurally related to quinolone antibacterials, that intercalates DNA and inhibits topoisomerase II. Vosaroxin is not a P-glycoprotein receptor substrate and its activity is independent of p53, thus evading common drug resistance mechanisms. To evaluate vosaroxin as a clinically applicable radiation sensitizer, we investigated its effects on tumor cell radiosensitivity in vitro and in vivo. Methods Vosaroxin's effect on post-irradiation sensitivity of U251, DU145, and MiaPaca-2 cells was assessed by clonogenic assay. Subsequent mechanistic and in vivo studies were performed with U251 cells. Cell cycle distribution and G2 checkpoint integrity was analyzed by flow cytometry. DNA damage and repair was evaluated by a high throughput gamma-H2AX assay. Apoptosis was assessed by flow cytometry. Mitotic catastrophe was assessed by microscopic evidence of fragmented nuclei by immunofluorescence. In vivo radiosensitization was measured by subcutaneous tumor growth delay. Results 50-100 nmol/L treatment with vosaroxin resulted in radiosensitization of all 3 cell lines tested with a dose enhancement factor of 1.20 to 1.51 measured at a surviving fraction of 0.1. The maximal dose enhancement was seen in U251 cells treated with 75 nmol/L vosaroxin (DEF 1.51). Vosaroxin exposure did not change cell cycle distribution prior to irradiation nor alter G2 checkpoint integrity after irradiation. No difference was seen in the apoptotic fraction regardless of drug or radiation treatment. The number of cells in mitotic catastrophe was significantly greater in irradiated cells treated with vosaroxin than cells receiving radiation only at 72 hr (p = 0.009). Vosaroxin alone did not significantly increase mitotic catastrophe over control (p = 0.53). Cells treated with vosaroxin and radiation maintained significantly higher gamma-H2AX levels than cells treated with vehicle control (p = 0.014), vosaroxin (p = 0.042), or radiation alone (p = 0.039) after 24 hr. In vivo tumor growth delay was 1.5 days for vosaroxin alone (IV 10 mg/kg), 1.0 days for radiation (3 Gy) alone, and 8.6 days for the group treated with vosaroxin 4 hours prior to radiation. Conclusions Vosaroxin enhanced tumor cell radiosensitivity in vitro and in vivo. The mechanism appears to be related to inhibition of DNA repair and increased mitotic catastrophe.

Aerobic glycolysis (the Warburg effect), first recognized almost a century ago, by Otto Warburg is a core hallmark of cancer. The Warburg effect describes a switch in glucose metabolism from oxidative phosphorylation to glycolysis. Recently links have been established between the oncogenic pathways that drive tumorigenesis and the mechanistic basis of tumor cell metabolism. The kinase mTOR is a major driver of tumor metabolism and proliferation of cancer cells, acts downstream of numerous oncogenic pathways. Several drugs targeting the mTOR pathway are being developed, however the most common drug rapamycin does not inhibit mTOR complex-2. Therefore in the current study, we examined the potential benefit of MLN0128, a novel potent mTOR ATP competitive inhibitor, as a therapeutic strategy for glioblastoma (GBM), one of the deadliest brain tumors. We studied the action of MLN0128 in human GBM tumor cells and patient derived stem cells. We show the inhibitory effect of MLN0128 in nanomolar concentrations on tumor proliferation in multiple GBM cell lines via the blockage of phospho-AKT, -ribosomal protein, -4EBP1 inhibiting both mTORC1 and mTORC2 signaling in GBM. Reduction of invasive potential and disruption of paxillin localization to focal adhesions was observed in vitro. MLN0128 treatment decreased glucose consumption and lactate production. It inhibited rate of glycolysis, glycolytic capacity and glycolytic reserve. It also inhibited ATP production, mitochondrial respiration and spare respiratory capacity. MLN0128 was superior in inhibiting metabolism compared to rapamycin. On clonogenic survival assay MLN0128 decreased GBM cell survival and increased radiosensitivity. In vivo treatment of mouse xenografts with MLN0128 downregulated mTOR targets and significantly inhibited tumor volume compared to drug and irradiation alone. Combination treatment of GBM cells with MLN0128 and other chemotherapeutic agents like carboplatin, irinotecan and TMZ showed synergistic effects when combination index was calculated in GBM tumor cells and stem cells. However, the therapeutic efficacy of TMZ combination was greater than MLN0128 combination with either carboplatin or irinotecan. Moreover, similar TMZ chemosensitivity was observed in MGMT non-methylated, TMZ in-sensitive GBM stem cells and MGMT methylated GBM cells. These findings support the rationale for clinical testing of MLN0128 in patients with GBM to provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors with standard care. Citation Format: Anita Tandle, Tamalee Kramp, Philip Tofilon, Kevin Camphausen. Targeting mTOR metabolism for glioblastoma cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A51.

Glioblastoma Multiforme (GBM) is a highly aggressive, therapeutically resistant brain tumor that arises from cells of astrocytic lineage. The five year survival rate is just below 10% and this statistic has changed very little in the past decade. Amplification and activating mutations of receptor tyrosine kinases (RTKs) are common in GBM; however despite their oncogenic importance, RTK inhibition with tyrosine kinase inhibitors (TKIs) has been clinically ineffective for GBM. The gap between promising laboratory results and clinical success is a common obstacle in cancer research in part because cell culture models do not accurately mimic complex in vivo conditions and cell behavior, such as therapeutic resistance. Many cell culture studies and high throughput screens of pharmacological agents are performed in cells plated at sub-confluence, or low density. We have found that GBM tumor initiating cells (GBM-TICs) are sensitive to TKIs at low density; however as culture density increases they become resistant, despite equal inhibition of the target RTK and downstream signals. Thus, culturing cells at low and high density provides a novel, isogenic system for studying the cellular mechanisms of therapeutic response. While density does not affect the cell cycle profile of GBM-TICs, we have found that high density cells have lower levels of reactive oxygen species and ATP than low density cells and reduced mitochondrial membrane potential. This suggests a switch to aerobic glycolysis (the Warburg Effect); however lactate levels, which should increase under conditions of aerobic glycolysis, are unaffected by cell density. Additionally, assessment of extracellular metabolic flux does not support an increase in aerobic glycolysis in dense GBM-TICs. Extracellular acidification rate (ECAR), which should increase with aerobic glycolysis, does not show a tumor-specific effect. Oxygen consumption rate (OCR), which should decrease during aerobic glycolysis, increases significantly with density in GBM-TICs but shows no density-dependent change in healthy astrocytes. These data indicate a tumor-specific, non-Warburg metabolic shift in dense GBM-TICs that is not present in sparse GBM-TICs or normal human astrocytes at any density. Further analysis has implicated changes in cholesterol metabolism and transport in density-dependent metabolic change. Specifically, densely cultured GBM-TICs have higher levels of cholesterol and elevated expression of cholesterol transport proteins such as ABCA1 and ABCG1 than cells cultured sparsely. Pharmacologic inhibition of cholesterol biosynthesis results in reduced OCR and increased ATP/cell in dense but not sparse GBM-TICs, which corroborates the importance of cholesterol synthesis and metabolism in dense GBM-TICs. We are currently investigating the connection between cholesterol metabolism and therapeutic resistance in GBM in the hope of identifying novel therapeutic targets to improve clinical outcomes for GBM patients. Citation Format: Diane M. Kambach, Tamalee Kramp, Jie Liu, Michael L. Oshinsky, Federico Bernal, Jayne M. Stommel. Changes in cholesterol metabolism support high-density cell growth and therapeutic resistance in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A18.

Monopolar spindle 1 (MPS1) is an essential spindle assembly checkpoint (SAC) kinase involved in determining spindle integrity. Beyond its mitotic functions, it has been implicated in several other signaling pathways. Its overexpression in cancer cells is known to contribute to genomic instability and tumorigenesis. Our earlier studies have elaborated on role of MPS1 in glioblastoma (GBM) radiosensitization. In this study using reverse phase protein arrays (RPPA), we assessed MPS1 mediated cell signaling pathways and demonstrated that inhibiting MPS1 could upregulate the expression of the tumor suppressor genes PDCD4 and MSH2 by downregulating micro RNA-21 (MiR-21). In GBMs MiR-21 expression is significantly elevated and is associated with chemo and radioresistance. Here, we show that MPS1 inhibition significantly repressed MiR-21 levels and elevated PDCD4 and MSH2 expression in GBM cells both in vitro and in vivo. Both MPS1 and MiR-21 depletion suppressed GBM cell proliferation, whereas, ectopic expression of MiR-21 rescued GBM cell growth from MPS1 inhibition. Further, we demonstrate that MPS1 mediates phosphorylation of SMAD3 but not SMAD2 in GBM cells; A possible mechanism behind MiR-21 modulation by MPS1. Collectively, our results shed light onto an important role of MPS1 in TGF-β/SMAD signaling via MiR-21 regulation. We also, evaluated the prognostic effect of MiR-21, PDCD4 and MSH2 expression on survival in GBM patients of different molecular subtypes from The Cancer Genome Atlas (TCGA) database, showing tumors with high MiR-21 expression and low PDCD4 and MSH2 expression were associated with significantly shorter survival times. To our knowledge this is the first report to demonstrate the role of MPS1 in MiR-21 modulation. This scenario in which MiR-21 is modulated by MPS1 inhibition may be exploited as a potential target for effective GBM therapy. Citation Format: Uday Bhanu Maachani, Anita Tandle, Uma Shankavaram, Tamalee Kramp, Kevin A. Camphausen. Modulation of MiR-21 signaling by MPS1 in human glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 190. doi:10.1158/1538-7445.AM2015-190

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults and, even with aggressive treatment that includes surgical resection, radiation, and temozolomide administration, prognosis is poor due to tumor recurrence. There is evidence that within GBMs a small number of glioma stem cells (GSCs) exist, which are thought to be radiation resistant and may be capable of repopulating a tumor after treatment. Like most cancers, GBMs largely employ aerobic glycolysis to create ATP, a phenomenon known as the Warburg effect. Experiments have shown that the cellular metabolism of GSCs differs from that of differentiated cells, making them an attractive target for novel therapeutic approaches. Much work has been done to analyze the metabolic profiles of GSCs with the goal of identifying potential therapeutic targets, but little data exists linking metabolic changes to radiation resistance. The purpose of this study is to determine the impact of radiation on the cellular metabolism of GSCs and to identify changes that can be exploited for treatment in conjunction with radiation therapy. To this end, we analyzed three human GSC lines harboring unique sets of genetic mutations that were hypothesized to differentially impact cellular metabolism. Experimental approaches include in vitro examination of metabolic rates and mitochondrial stress of the GSC lines via Seahorse extracellular flux analysis with and without radiation administration as well as analysis of tumor xenografts in mice via 13C MR spectroscopic imaging using hyperpolarized [1-13C] pyruvate as a tracer. Additionally, we are employing genomic and proteomic profiling of the GSC lines both in culture and in vivo to categorize metabolic changes following radiation treatment. We show that the basal metabolic rates of GSC lines with distinctive genetic mutations can be differentiated using Seahorse analysis. This is confirmed in vivo by 13C MR spectroscopic imaging, thus revealing a potential non-invasive diagnostic modality. The underlying mechanism behind these metabolic differences and how this relates to each GSC's response to radiation is currently being examined. We expect that this data will result in the identification of molecular targets within the metabolic pathways of cells that can be exploited with drugs in conjunction with radiation therapy to improve the prognosis and survival of patients presenting with GBMs in the clinic. Citation Format: Elizabeth I. Spehalski, Keita Saito, Tamalee Kramp, Anita Tandle, Barbara Rath, Murali Cherukuri, Kevin Camphausen. Exploiting glioma stem cell metabolism to abrogate radioresistance in glioblastomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1174. doi:10.1158/1538-7445.AM2015-1174

There is a need for minimally invasive biomarkers that can accurately and quickly quantify radiation exposure. Radiation-responsive proteins have applications in clinical medicine and for mass population screenings after a nuclear or radiological incident where the level of radiation exposure and exposure pattern complicate medical triage for first responders. In this study, we evaluated the efficacy of the acute phase protein serum amyloid A (SAA) as a biomarker for radiation exposure using plasma from irradiated mice. Ten-week-old female C57BL6 mice received a 1-8 Gy single whole-body or partial-body dose from a Pantak X-ray source at a dose rate of 2.28 Gy/min. Plasma was collected by mandibular or cardiac puncture at 6, 24, 48 and 72 h or 1-3 weeks postirradiation. SAA levels were determined using a commercially available ELISA assay. Data was pooled to generate SAA μg/ml threshold values correlating plasma SAA levels with radiation dose. SAA levels were statistically significant over control at all exposures between 2 and 8 Gy at 24 h postirradiation but not at 6, 48 and 72 h or 1-3 weeks postirradiation. SAA levels at 1 Gy were not significantly elevated over control at all time points. Total-body-irradiated (TBI) SAA levels at 24 h were used to generate a dose prediction model that successfully differentiated TBI mice into dose received cohorts of control/1 Gy and ≥ 2 Gy groups with a high degree of accuracy in a blind study. Dose prediction of partial-body exposures based on the TBI model correlated increasing predictive accuracy with percentage of body exposure to radiation. Our findings indicate that plasma SAA levels might be a useful biomarker for radiation exposure in a variety of total- and partial-body irradiation settings.

Radiation-induced translational control of gene expression has been shown to be a component of the cellular radioresponse. Furthermore, recent data suggest strategies targeting eIF4E may be effective for tumor cell radiosensitization. The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of eIF4E and mRNA translation. The newly developed ATP-competitive mTOR inhibitor, INK128, inhibits both mTORC1 and mTORC2 activity, and is in currently undergoing evaluation in Phase 1 clinical trials. In the study described here we evaluated the effects of INK128 treatment on tumor cell radiosensitivity in vitro using a clonogenic survival assay and in vivo according to tumor growth delay. For tumor cell lines initiated from different histologic origins in vitro exposure to INK128 resulted in the complete inhibition of both mTORC1 and 2. Treatment with the same concentration of INK128 immediately after irradiation increased tumor cell radiosensitivity with DEFs greater than 1.3. To determine whether the INK128 induced radiosensitization could be extended to an in vivo model we used subcutaneous xenografts initiated from U251 or PSN1 tumor cells. INK128 treatment (oral gavage) inhibited mTOR activity in tumors established from both cell lines as defined by loss of 4E-BP1 phosphorylation. Furthermore, m7-GTP batch chromatography showed that INK128 treatment decreased cap-binding activity in xenografts. To evaluate the effects of INK128 on in vivo tumor radiosensitivity a tumor growth delay assay was performed using mice bearing U251 tumor xenografts. When tumors reached approximately 210mm3 mice were randomized into 4 groups: vehicle, radiation, INK128, and INK128 plus radiation. INK128 was delivered once a day (3 mg/kg, oral gavage) for four days with the tumor locally irradiated (2 Gy) 2h after each of the four drug treatments. Absolute growth delays were calculated as the time in days for tumors in treated mice to grow from 210 to 1000mm3 minus the time in days for tumors to reach the same size in vehicle treated mice. The absolute growth delays for INK128 alone and radiation alone groups were 1.7 and 12.7 days respectively. The growth delay in mice treated with the combination of INK128 and radiation was 22.2 days, which is greater than the sum of the growth delays caused by INK128 and radiation alone. The DEF, obtained by dividing the normalized tumor growth delay in mice treated with the radiation/PP242 combination (20.5) by the absolute growth delay in mice treated with radiation only (12.7), was 1.6. These results suggest that mTOR inhibition by the clinically relevant ATP-competitive mTOR inhibitor, INK128, enhances tumor cell radiosensitivity both in vitro and in vivo. Citation Format: Thomas J. Hayman, Tamalee Kramp, Kevin Camphausen, Philip J. Tofilon. In vitro and in vivo tumor cell radiosensitization by the competitive mTOR inhibitor INK128. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 69. doi:10.1158/1538-7445.AM2013-69