Eduard H. Panosyan

Asparaginase antibody and asparaginase activity in children with higher-risk acute lymphoblastic leukemia: Children's Cancer Group Study CCG-1961.

We investigated the anti-asparaginase antibody (Ab) and asparaginase enzymatic activity in the sera of 1,001 patients (CCG-1961) with high-risk acute lymphoblastic leukemia (HR-ALL). Patients received nine doses of native Escherichia coli asparaginase during induction. Half of rapid early responders (RER) were randomly assigned to standard intensity arms and continued to receive native asparaginase. The other RER patients and all slow early responders received 6 or 10 doses of PEG-asparaginase. Serum samples (n = 3,193) were assayed for determination of asparaginase Ab titers and enzymatic activity. Three hundred ninety of 1,001 patients (39%) had no elevation of Ab among multiple evaluations—that is, were Abnegative (<1.1 over negative control)—and 611 patients (61%) had an elevated Ab titer (>1.1). Among these 611 patients, 447 had no measurable asparaginase activity during therapy. Patients who were Ab-positive but had no clinical allergies continued to receive E. coli asparaginase, the activity of which declined precipitately. No detectable asparaginase activity was found in 81 of 88 Ab-positive patients shortly after asparaginase injections (94% neutralizing Ab). The Ab-positive patients with clinical allergies subsequently were given Erwinase and achieved substantial activity (0.1–0.4 IU/ml). An interim analysis of 280 patients who were followed for 30 months from induction demonstrated that the Ab-positive titers during interim maintenance-1 and in delayed intensification-1 were associated with an increased rate of events. The CCG-1961 treatment schedule was very immunogenic, plausibly due to initially administrated native asparaginase. Anti-asparaginase Ab was associated with undetectable asparaginase activity and may be correlated with adverse outcomes in HR ALL.

A Molecular Screening Approach to Identify and Characterize Inhibitors of Glioblastoma Stem Cells

Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types. Mol Cancer Ther; 10(10); 1818–28. ©2011 AACR.

Clinical outcome in pediatric glial and embryonal brain tumors correlates with in vitro multi‐passageable neurosphere formation

Cultured brain tumors can form neurospheres harboring tumorigenic cells with self renewal and differentiation capacities. Renewable neurosphere formation has clinical predictive value in adult malignant gliomas, yet its prognostic role for pediatric brain tumors is unknown.

Asparagine Depletion Potentiates the Cytotoxic Effect of Chemotherapy against Brain Tumors

Targeting amino acid metabolism has therapeutic implications for aggressive brain tumors. Asparagine is an amino acid that is synthesized by normal cells. However, some cancer cells lack asparagine synthetase (ASNS), the key enzyme for asparagine synthesis. Asparaginase (ASNase) contributes to eradication of acute leukemia by decreasing asparagine levels in serum and cerebrospinal fluid. However, leukemic cells may become ASNase-resistant by upregulating ASNS. High expression of ASNS has also been associated with biologic aggressiveness of other cancers, including gliomas. Here, the impact of enzymatic depletion of asparagine on proliferation of brain tumor cells was determined. ASNase was used as monotherapy or in combination with conventional chemotherapeutic agents. Viability assays for ASNase-treated cells demonstrated significant growth reduction in multiple cell lines. This effect was reversed by glutamine in a dose-dependent manner—as expected, because glutamine is the main amino group donor for asparagine synthesis. ASNase treatment also reduced sphere formation by medulloblastoma and primary glioblastoma cells. ASNase-resistant glioblastoma cells exhibited elevated levels of ASNS mRNA. ASNase cotreatment significantly enhanced gemcitabine or etoposide cytotoxicity against glioblastoma cells. Xenograft tumors in vivo showed no significant response to ASNase monotherapy and little response to temozolomide alone. However, combinatorial therapy with ASNase and temozolomide resulted in significant growth suppression for an extended duration of time. Taken together, these findings indicate that amino acid depletion warrants further investigation as adjunctive therapy for brain tumors. Implications: Findings have potential impact for providing adjuvant means to enhance brain tumor chemotherapy. Mol Cancer Res; 12(5); 694–702. ©2014 AACR.

In search of druggable targets for GBM amino acid metabolism

BackgroundAmino acid (AA) pathways may contain druggable targets for glioblastoma (GBM). Literature reviews and GBM database (http://r2.amc.nl) analyses were carried out to screen for such targets among 95 AA related enzymes.MethodsFirst, we identified the genes that were differentially expressed in GBMs (3 datasets) compared to non-GBM brain tissues (5 datasets), or were associated with survival differences. Further, protein expression for these enzymes was also analyzed in high grade gliomas (HGGs) (proteinatlas.org). Finally, AA enzyme and gene expression were compared among the 4 TCGA (The Cancer Genome Atlas) subtypes of GBMs.ResultsWe detected differences in enzymes involved in glutamate and urea cycle metabolism in GBM. For example, expression levels of BCAT1 (branched chain amino acid transferase 1) and ASL (argininosuccinate lyase) were high, but ASS1 (argininosuccinate synthase 1) was low in GBM. Proneural and neural TCGA subtypes had low expression of all three. High expression of all three correlated with worse outcome. ASL and ASS1 protein levels were mostly undetected in high grade gliomas, whereas BCAT1 was high. GSS (glutathione synthetase) was not differentially expressed, but higher levels were linked to poor progression free survival. ASPA (aspartoacylase) and GOT1 (glutamic-oxaloacetic transaminase 1) had lower expression in GBM (associated with poor outcomes). All three GABA related genes -- glutamate decarboxylase 1 (GAD1) and 2 (GAD2) and 4-aminobutyrate aminotransferase (ABAT) -- were lower in mesenchymal tumors, which in contrast showed higher IDO1 (indoleamine 2, 3-dioxygenase 1) and TDO2 (tryptophan 2, 3-diaxygenase). Expression of PRODH (proline dehydrogenase), a putative tumor suppressor, was lower in GBM. Higher levels predicted poor survival.ConclusionsSeveral AA-metabolizing enzymes that are higher in GBM, are also linked to poor outcome (such as BCAT1), which makes them potential targets for therapeutic inhibition. Moreover, existing drugs that deplete asparagine and arginine may be effective against brain tumors, and should be studied in conjunction with chemotherapy. Last, AA metabolism is heterogeneous in TCGA subtypes of GBM (as well as medulloblastomas and other pediatric tumors), which may translate to variable responses to AA targeted therapies.

Allergic reactions and antiasparaginase antibodies in children with high-risk acute lymphoblastic leukemia: A children's oncology group report

The objectives of this study were to assess the incidence of clinical allergy and end‐induction antiasparaginase (anti‐ASNase) antibodies in children with high‐risk acute lymphoblastic leukemia treated with pegylated (PEG) Escherichia coli ASNase and to determine whether they carry any prognostic significance.

A phase 3 trial of l-glutamine in sickle cell disease

BACKGROUND Oxidative stress contributes to the complex pathophysiology of sickle cell disease. Oral therapy with pharmaceutical‐grade l‐glutamine (USAN, glutamine) has been shown to increase the proportion of the reduced form of nicotinamide adenine dinucleotides in sickle cell erythrocytes, which probably reduces oxidative stress and could result in fewer episodes of sickle cell–related pain. METHODS In a multicenter, randomized, placebo‐controlled, double‐blind, phase 3 trial, we tested the efficacy of pharmaceutical‐grade l‐glutamine (0.3 g per kilogram of body weight per dose) administered twice daily by mouth, as compared with placebo, in reducing the incidence of pain crises among patients with sickle cell anemia or sickle β0‐thalassemia and a history of two or more pain crises during the previous year. Patients who were receiving hydroxyurea at a dose that had been stable for at least 3 months before screening continued that therapy through the 48‐week treatment period. RESULTS A total of 230 patients (age range, 5 to 58 years; 53.9% female) were randomly assigned, in a 2:1 ratio, to receive l‐glutamine (152 patients) or placebo (78 patients). The patients in the l‐glutamine group had significantly fewer pain crises than those in the placebo group (P=0.005), with a median of 3.0 in the l‐glutamine group and 4.0 in the placebo group. Fewer hospitalizations occurred in the l‐glutamine group than in the placebo group (P=0.005), with a median of 2.0 in the l‐glutamine group and 3.0 in the placebo group. Two thirds of the patients in both trial groups received concomitant hydroxyurea. Low‐grade nausea, noncardiac chest pain, fatigue, and musculoskeletal pain occurred more frequently in the l‐glutamine group than in the placebo group. CONCLUSIONS Among children and adults with sickle cell anemia, the median number of pain crises over 48 weeks was lower among those who received oral therapy with l‐glutamine, administered alone or with hydroxyurea, than among those who received placebo, with or without hydroxyurea. (Funded by Emmaus Medical; ClinicalTrials.gov number, NCT01179217.)

Can we target amino acid metabolism to affect brain tumor growth

I is estimated that more than 500000 tons of rice bran is produced in Egypt every year. Though its nutritional values and potential health benefits, it is used, due to its instability, as animal feed rather high value functional food and/or high value added nutraceutical. A number of papers were published showing, the stabilized rice bran extract, potential health benefits in some diseases like Alzheimer’s disease. The product is registered (Oryza), another one under registration at the Egyptian Ministry of Health (Riciplex). A functional food against Alzheimer is currently developed in Germany, based on the stabilized Egyptian rice bran supplied. In the present study the effects of RBE were examined in comparison to a well-known PPARγ agonist pioglitazone. RBE administration significantly improved the spatial working and reference memory in addition to non-spatial recognition memory in the LPS mouse model as shown by object recognition test, y-maze and water maze test. Pioglitazone improved memory, in the Y-maze and object recognition test with no effect in the water maze test. Interestingly, the effect of RBE on memory was abolished in the group injected with PPARγantagonist before RBE treatment, indicating the important role of PPARγ in the mechanism of action of RBE. Furthermore, the RBE -PPARγ DNA binding activity was measured in the brain extract samples of the mouse treated groups using transcription factor assay kit. Results showed a significant increase in PPARγ binding to PPRE with RBE treatment and this effect was reversed upon PPARγ antagonist injection before RBE treatment. These findings demonstrate that the involvement of RBE in the beneficial effects on cognitive performance is correlated with its action on PPARγ modulation, providing novel insight into its neuroprotective role in AD.