Murugesan K. Gounder

Targeting tumor metabolism with 2-deoxyglucose in patients with castrate-resistant prostate cancer and advanced malignancies.

A profound difference between cancer and normal tissues is the preferential utilization of glycolysis by cancer cells. To translate this paradigm in the clinic, we completed a phase I study of 2‐deoxyglucose (2DG), and assessed 2DG uptake with fluorodeoxyglucose (FDG) positron emission tomography (PET) and the autophagy substrate p62 as a marker of 2DG resistance.

Contribution of serine, folate and glycine metabolism to the ATP, NADPH and purine requirements of cancer cells

Recent observations on cancer cell metabolism indicate increased serine synthesis from glucose as a marker of poor prognosis. We have predicted that a fraction of the synthesized serine is routed to a pathway for ATP production. The pathway is composed by reactions from serine synthesis, one-carbon (folate) metabolism and the glycine cleavage system (SOG pathway). Here we show that the SOG pathway is upregulated at the level of gene expression in a subset of human tumors and that its level of expression correlates with gene signatures of cell proliferation and Myc target activation. We have also estimated the SOG pathway metabolic flux in the NCI60 tumor-derived cell lines, using previously reported exchange fluxes and a personalized model of cell metabolism. We find that the estimated rates of reactions in the SOG pathway are highly correlated with the proliferation rates of these cell lines. We also observe that the SOG pathway contributes significantly to the energy requirements of biosynthesis, to the NADPH requirement for fatty acid synthesis and to the synthesis of purines. Finally, when the PC-3 prostate cancer cell line is treated with the antifolate methotrexate, we observe a decrease in the ATP levels, AMP kinase activation and a decrease in ribonucleotides and fatty acids synthesized from [1,2-13C2]-D-glucose as the single tracer. Taken together our results indicate that the SOG pathway activity increases with the rate of cell proliferation and it contributes to the biosynthetic requirements of purines, ATP and NADPH of cancer cells.

Effects of drug efflux proteins and topoisomerase I mutations on the camptothecin analogue gimatecan

SummaryClinically relevant resistance to the currently approved camptothecins, irinotecan and topotecan, is poorly understood but may involve increased expression of ATP-dependent drug transporters such as ABCG2 (breast cancer resistant protein, BCRP). Gimatecan (ST1481) is a lipophilic 7-substituted camptothecin derivative that exhibits potent anti-tumor activity in a variety of preclinical cancer models and is under investigation in the clinic. Previous studies reported that gimatecan cytotoxicity was not affected by expression of ABCG2. To confirm and extend this finding, we assessed the cytotoxicity of gimatecan in pairs of isogenic cell lines consisting of transfectants expressing either ABCG2 (including wild-type, R482T, or R482G mutants), ABCB1 (P-glycoprotein), ABCC1 (MRP1), ABCC2 (MRP2), or ABCC4 (MRP4). Expression of wild-type or mutant ABCG2 in human cell lines conferred resistance to topotecan but not to gimatecan. Similarly, intracellular accumulation of gimatecan was unaffected by expression of wild-type ABCG2. Furthermore, expression of P-glycoprotein or MRP2 did not alter gimatecan cytotoxicity. Whereas expression of MRP1 had a minor effect on gimatecan cytotoxicity, expression of ABCC4 was found to significantly reduce the anti-proliferative effects of this drug. Cells containing resistance-conferring mutations in topoisomerase I were also resistant to gimatecan. These results suggest that gimatecan may be more effective than irinotecan or topotecan in cancers that express ABCG2, but not in cancers that express high levels of ABCC4 or contain certain topoisomerase I (TOP1) mutations.

Deficiency of the dual ubiquitin/SUMO ligase Topors results in genetic instability and an increased rate of malignancy in mice

BackgroundTopors is a nuclear protein that co-localizes with promyelocytic leukemia bodies and has both ubiquitin and SUMO E3 ligase activity. Expression studies implicated Topors as a tumor suppressor in various malignancies. To gain insight into the function of Topors, we generated a Topors-deficient mouse strain.ResultsMice homozygous for a mutant Topors allele exhibited a high rate of perinatal mortality and decreased lifespan. In addition, heterozygotes were found to have an increased incidence of malignancy, involving a variety of tissues. Consistent with this finding, primary embryonic fibroblasts lacking Topors exhibited an increased rate of malignant transformation, associated with aneuploidy and defective chromosomal segregation. While loss of Topors did not alter sensitivity to DNA-damaging or microtubule-targeting agents, cells lacking Topors exhibited altered pericentric heterochromatin, manifested by mislocalization of HP1α and an increase in transcription from pericentric major satellite DNA. Topors-deficient cells exhibited a transcriptional profile similar to that of cells treated with histone deacetylase inhibitors, and were resistant to the anti-proliferative effects of the histone deacetylase inhibitor trichostatin A.ConclusionThese results indicate a unique role for Topors in the maintenance of genomic stability and pericentric heterochromatin, as well as in cellular sensitivity to histone deacetylase inhibitors.

A validated HPLC assay for the determination of R-(-)-gossypol in human plasma and its application in clinical pharmacokinetic studies

R-(-)-gossypol acetic acid (AT-101), a natural BH3 mimetic, is investigated in a Phase I/II clinical trial for the treatment of advanced solid tumor malignancies. Gossypol undergoes rapid degradation in solution phase, which causes major technical difficulty for its quantitation in plasma. We developed and validated a sensitive HPLC assay for pharmacokinetic evaluation of gossypol. Acetonitrile deproteinization method was chosen for sample preparation and Schiffs base derivative, R-(-)-gossypol-diamino-propanol (GDP), was used as internal standard. Chromatographic separation of gossypol in plasma was performed using a Zorbax Eclipse XDB column C(18) at 30 °C. The mobile phase consists of 10 mmol/L KH(2)PO(4) (pH 3.0) and acetonitrile (20:80) at 1.0 mL/min flow rate. Linearity ranged over 56-3585 ng/mL (R(2)=0.9997±0.0003, n=4), and the limit of detection was 28 ng/mL. The intra- and inter-assay precision was less than 13.7% and the bias ranged from -7.4 to 7.0%. The method was successfully applied to characterize the pharmacokinetics of AT-101 in a Phase I clinical trial. The validated assay is accurate, and sensitive with minimum loss and rapid analysis time and suitable for quantification of gossypol for pharmacokinetics evaluation.

A validated bioanalytical HPLC method for pharmacokinetic evaluation of 2-deoxyglucose in human plasma.

2-Deoxyglucose (2-DG), an analog of glucose, is widely used to interfere with glycolysis in tumor cells and studied as a therapeutic approach in clinical trials. To evaluate the pharmacokinetics of 2-DG, we describe the development and validation of a sensitive HPLC fluorescent method for the quantitation of 2-DG in plasma. Plasma samples were deproteinized with methanol and the supernatant was dried at 45°C. The residues were dissolved in methanolic sodium acetate-boric acid solution. 2-DG and other monosaccharides were derivatized to 2-aminobenzoic acid derivatives in a single step in the presence of sodium cyanoborohydride at 80°C for 45 min. The analytes were separated on a YMC ODS C₁₈ reversed-phase column using gradient elution. The excitation and emission wavelengths were set at 360 and 425 nm. The 2-DG calibration curves were linear over the range of 0.63-300 µg/mL with a limit of detection of 0.5 µg/mL. The assay provided satisfactory intra-day and inter-day precision with RSD less than 9.8%, and the accuracy ranged from 86.8 to 110.0%. The HPLC method is reproducible and suitable for the quantitation of 2-DG in plasma. The method was successfully applied to characterize the pharmacokinetics profile of 2-DG in patients with advanced solid tumors.

Abstract 3513: Hydroxychoroquine (HCQ) modulates autophagy in melanoma: preliminary results of a phase 0 trial in patients with resectable melanoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Autophagy enables tumors to survive metabolic stress by recycling intracellular components to sustain metabolism. This suggests that inhibiting autophagy with drugs such as HCQ, a lysosomotropic agent that blocks the final step of autophagy when autophagosomes fuse with lysosomes, may promote tumor cell death. In melanoma cell lines and animal models, HCQ administration resulted in inhibition of flux through the autophagy pathway as measured by visualization of increased numbers of autophagic vesicles (AVs) and processing of the autophagosome marker LC3 from LC3-I to LC3-II. We hypothesized that administration of HCQ can modulate autophagy in human melanoma tumors that can be detected by increased autophagic vesicle (AV) formation and by increased LC3-II/I ratio. Methods: To test this hypothesis, we conducted a phase 0 trial in patients with resectable stage III or IV melanoma. Patients who consented underwent pretreatment tumor biopsies, were treated with either 200 or 400 mg bid of HCQ for 14 days, and then underwent surgery. Paired tumor samples were processed for electron microscopy (EM) and scored by counting AVs per high powered field in multiple fields. Changes in the LC3II/I ratio were examined by Western blot. Quantitation of HCQ in blood was performed using HPLC-fluorescence method in whole blood, plasma and ultrafiltrates. Changes in AV formation on EM, LC3 II/I ratio, and pharmacokinetic (PK) data from the paired observations were to be correlated. Otherwise, the statistics utilized were descriptive. Results: 12 of 14 patients enrolled had tumor sufficient for evaluation. 8 patients were enrolled at 200 mg bid and 4 at 400 mg bid. No significant toxicities were observed. Autophagy modulation was observed in 4 of the 12 tumors as evidenced by increased LC3II/I ratio on Western blot. Observed changes in AV formation correlated with pre-post changes in LC3II/I ratio (correlation r = 0.42, p = 0.20) in this small sample size. HCQ concentrations (ssmin) showed dose proportional increases in blood, plasma and ultrafiltrate. No correlations were evident between changes on EM and HCQ concentration in plasma, whole blood, or plasma ultrafiltrates. Analyses for common driver mutations in melanoma are ongoing. Conclusions: Treatment with HCQ for 14 days resulted in increased LC3II/I ratio indicative of autophagy modulation in 1/3 of tumors from patients with resectable stage III or IV melanoma. The correlation between pre-post changes in AV formation and LC3II/I ratio suggests that LC3II/I ratio may be a useful measurement of autophagy in human tumors, as in the laboratory. The degree of modulation after 14 days of therapy did not appear to be dose or exposure dependent, although HCQ levels had likely not reached steady state at 14 days. Enrollment continues at 600 mg bid to assess if higher doses produce saturable PK, increased AV formation, and stronger correlations among them. Citation Format: Janice M. Mehnert, Xiaoqi Xie, Megha Rajpal, Liesel Dudek, Monal Mehta, Vassiliki Karantza, Murugesan Gounder, Joseph Aisner, Weichung Shih, Rajesh Patel, Chunxia Chen, James Goydos, Ravi K. Amaravadi, Eileen P. White. Hydroxychoroquine (HCQ) modulates autophagy in melanoma: preliminary results of a phase 0 trial in patients with resectable melanoma. [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 3513. doi:10.1158/1538-7445.AM2013-3513

Abstract 2759: Validated liquid chromatography-mass spectrometry assay for determination of busulfan: Application to clinical pharmacokinetics studies

Busulfan (BU), an alkylating agent, also known to have anti-leukemic efficacy, is commonly used in preparative regimens for allogenic and autologous stem cell marrow transplantation. The efficacy of BU is related to systemic levels and the area under concentration-time curve (AUC), and hence, a timely pharmacokinetic (PK) report is critical for subsequent dose adjustment. We developed two highly sensitive methods: a rapid, accurate, and sensitive liquid chromatography-mass spectrometry (LC-MS) method using direct inject tandem mass spectrometry with SIM mode, and an alternate equally sensitive high performance liquid chromatographic fluorometric (HPLC-FL) method for the quantitation of BU in human plasma. Method: BU concentrations were quantified in 50 μL of plasma spiked with [ 2 H 8 ]-busulfan, followed by liquid-liquid extraction with ethyl acetate. The solvent extract was dried under nitrogen and the residue was dissolved in mobile phase and analyzed using LC-MS. The mobile was 10mmol/L ammonium acetate and 10mL/L acetic acid in water and acetonitrile, (70:30) and flow rate was 0.5ml/min. Separation of BU was performed on a Gemini C 18 column (4.6 × 150 mm, 3μm) at 30°C. BU and IS were detected as ammonium adducts in selected-ion monitoring mode at m/z 264.2 and 272.2 at the retention time of 5.8 min. When plasma samples were analyzed by HPLC-FL method, 200μL plasma spiked with 1,6 (methanesulfonyloxy) octane was extracted in ethyl acetate and dried under air at 45°C. The residue was dissolved in 200μL ethanol and derivatized with 2-naphthelenethiol according to Nara S., et al (Analytical Sci. 2000,16, 287). The samples were analyzed on a HPLC system with fluorescence detector set at excitation wavelength 255 nm and emission wavelength 370 nm. The mobile was a mixture of methanol: acetonitrile: sodium acetate buffer (0.1M, pH 7.0) using gradient elution, and flow rate was 1.0 mL/min. Results: Both assay methods are comparable in precision, accuracy, linearity, and sensitivity. The calibration curve for LC-MS assay was linear at 31.25-1000ng/ml. The LOD of assay was 7.8ng/ml and the LOQ was 31.25ng/ml. The correlation coefficient for calibration curve was 0.997±0.003 (n =4). The intra and inter-assay precision was less than 3.0% and the accuracy ranged from 95.4% to 101.7%. Busulfan AUC last calculation comparison with HPLC-FL derivatization method showed an average difference between the assays of 7.7%. Conclusion: The method LC-MS is highly accurate, reproducible, and requires less specimen, sample preparation, and analysis time over the HPLC-FL derivitization method. The new LC-MS assay is rapid and sensitive and provides an appropriate method for quantification of BU in human plasma, making therapeutic drug monitoring of BU faster and easier in clinical practice. Acknowledgement: The study is supported by funds from Century for the Cure and U01 grant from NCI. 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 2759.