Asparagine Depletion Research Articles

Elucidation of the Specific Function of the Conserved Threonine Triad Responsible for Human l-Asparaginase Autocleavage and Substrate Hydrolysis

Our long-term goal is the design of a human l-asparaginase (hASNase3) variant, suitable for use in cancer therapy without the immunogenicity problems associated with the currently used bacterial enzymes. Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. The key property allowing for the depletion of blood asparagine by bacterial asparaginases is their low micromolar KM value. In contrast, human enzymes have a millimolar KM for asparagine. Toward the goal of engineering an hASNase3 variant with micromolar KM, we conducted a structure/function analysis of the conserved catalytic threonine triad of this human enzyme. As a member of the N-terminal nucleophile family, to become enzymatically active, hASNase3 must undergo autocleavage between residues Gly167 and Thr168. To determine the individual contribution of each of the three conserved active-site threonines (threonine triad Thr168, Thr186, Thr219) for the enzyme-activating autocleavage and asparaginase reactions, we prepared the T168S, T186V and T219A/V mutants. These mutants were tested for their ability to cleave and to catalyze asparagine hydrolysis, in addition to being examined structurally. We also elucidated the first N-terminal nucleophile plant-type asparaginase structure in the covalent intermediate state. Our studies indicate that, while not all triad threonines are required for the cleavage reaction, all are essential for the asparaginase activity. The increased understanding of hASNase3 function resulting from these studies reveals the key regions that govern cleavage and the asparaginase reaction, which may inform the design of variants that attain a low KM for asparagine.

Cerebrospinal fluid asparagine depletion during pegylated asparaginase therapy in children with acute lymphoblastic leukaemia

L-asparaginase is an important drug in the treatment of childhood acute lymphoblastic leukaemia (ALL). Cerebrospinal fluid (CSF) asparagine depletion is considered a marker of asparaginase effect in the central nervous system (CNS) and may play a role in CNS-directed anti-leukaemia therapy. The objective of this study was to describe CSF asparagine depletion during 30weeks of pegylated asparaginase therapy, 1000iu/m(2) i.m. every second week, and to correlate CSF asparagine concentration with serum L-asparaginase enzyme activity. Danish children (1-17years) with ALL, treated according to the Nordic Society of Paediatric Haematology and Oncology ALL2008 protocol, standard and intermediate risk, were included. CSF samples were obtained throughout L-asparaginase treatment at every scheduled lumbar puncture. A total of 128 samples from 31 patients were available for analysis. Median CSF asparagine concentration decreased from a pre-treatment level of 5·3μmol/l to median levels ≤1·5μmol/l. However, only 4/31 patients (five samples) had CSF asparagine concentrations below the limit of detection (0·1μmol/l). In 11 patients, 24 paired same day serum and CSF samples were obtained. A decrease in CSF asparagine corresponded to serum enzyme activities above 50iu/l. Higher serum enzyme activities were not followed by more extensive depletion. In conclusion, pegylated asparaginase 1000iu/m(2) i.m. every second week effectively reduced CSF asparagine levels.

Asparaginase in the treatment of non-ALL hematologic malignancies

Asparaginases are among the most effective agents against acute lymphoblastic leukemia (ALL) and are Food and Drug Administration-approved for the treatment of pediatric and adult ALL. However, the efficacy of these drugs for the treatment of other hematologic malignancies particularly acute myeloid leukemia is not well established. The mechanism of action of asparaginases has thought to be related to a swift and sustained reduction in serum L-asparagine, which is required for rapid proliferation of metabolically demanding leukemic cells. However, asparagine depletion alone appears not to be sufficient for effective cytotoxic activity of asparaginase against leukemia cells, because glutamine can rescue asparagine-deprived cells by regeneration of asparagine via a transamidation chemical reaction. For this reason, glutamine reduction is also necessary for full anti-leukemic activity of asparaginase. Indeed, both Escherichia coli and Erwinia chrysanthemi asparaginases possess glutaminase enzymatic activity, and their administrations have shown to reduce serum glutamine level by deamidating glutamine to glutamate and ammonia. Emerging data have provided evidence that several types of neoplastic cells require glutamine for the synthesis of proteins, nucleic acids, and lipids. This fundamental role of glutamine and its metabolic pathways for growth and proliferation of individual malignant cells may identify a special group of patients whose solid or hematologic neoplasms may benefit significantly from interruption of glutamine metabolism. To this end, asparaginase products deserve a second look particularly in non-ALL malignant blood disorders. Here, we review mechanisms of anti-tumor activity of asparaginase focusing on importance of glutamine reduction, pharmacology of asparaginase products, in vitro activities as well as clinical experience of incorporating asparaginase in therapeutic regimens for non-ALL hematologic malignancies.

Targeted metabolomic analysis of amino acid response to L-asparaginase in adherent cells.

L-asparaginase (L-ASP) is a therapeutic enzyme used clinically for the treatment of childhood acute lymphoblastic leukemia. L-ASP’s anticancer activity is believed to be associated primarily with depletion of asparagine, but secondary glutaminase activity has also been implicated in its anticancer mechanism of action. To investigate the effects of L-ASP on amino acid metabolism, we have developed an LC–MS/MS metabolomics platform for high-throughput quantitation of 29 metabolites, including all 20 proteinogenic amino acids, 6 metabolically related amino acid derivatives (ornithine, citrulline, sarcosine, taurine, hypotaurine, and cystine), and 3 polyamines (putrescince, spermidine, and spermine) in adherent cultured cells. When we examined the response of OVCAR-8 ovarian cancer cells in culture to L-ASP, asparagine was depleted from the medium within seconds. Interestingly, intracellular asparagine was also depleted rapidly, and the mechanism was suggested to involve rapid export of intracellular asparagine followed by rapid conversion to aspartic acid by L-ASP. We also found that L-ASP-induced cell death was more closely associated with glutamine concentration than with asparagine concentration. Time-course analysis revealed the dynamics of amino acid metabolism after feeding cells with fresh medium. Overall, this study provides new insight into L-ASP’s mechanism of action, and the optimized analytical method can be extended, with only slight modification, to other metabolically active amino acids, related compounds, and a range of cultured cell types.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-014-0634-1) contains supplementary material, which is available to authorized users.

Change In Albumin Levels During Induction Predicts Survival Outcomes In Adult Acute Lymphoblastic Leukemia

BackgroundAsparaginase is an important component of induction and consolidation chemotherapy for acute lymphoblastic leukemia (ALL). Effective asparagine depletion in adult patients with ALL results in a longer duration of overall survival and disease free survival. Variation in asparaginase activity is in part due to the formation of anti-asparaginase antibodies that inactivate asparaginase and result in inadequate asparagine depletion. In addition, the presence of anti-asparaginase antibodies influences dexamethasone pharmacokinetics by increasing dexamethasone clearance, which has been shown to correlate with a higher risk of relapse. Hypoalbuminemia is a recognized side effect of asparaginase, and has been studied as a measure of asparaginase inhibition of liver protein synthesis. The purpose of this retrospective study was to evaluate the effect of asparaginase activity during induction, using serum albumin as a surrogate marker, on overall outcomes. We hypothesized that patients with lower albumin levels, and thus increased asparaginase activity, would have improved survival. MethodsA retrospective electronic chart review was performed on 108 adult patients with newly diagnosed ALL who underwent induction chemotherapy treatment with Cancer and Leukemia Group B (CALGB) 9511 protocol at Stanford Hospital and Clinics between 2004 and 2012. PEG-asparaginase (2000 units per m2, capped at 3750 units) administration on day 5 of induction was confirmed on the electronic medical administration record. Patients also received therapy per protocol including prednisone (60mg per m2 per day) from days 1 through 21, with the exception of patients >60 years old who received prednisone from days 1 through 7. The primary outcomes measured were median overall survival and disease free survival. Patients were divided based on percent change in albumin level at day 14 of induction, using 20% decrease from pre-treatment baseline as a cut-off. The log rank test was used to calculate differences in survival and the Cox proportional hazards model was used to calculate hazard ratios. Baseline characteristics between the two groups were compared using chi-square or t-test analysis. ResultsA total of 104 patients with newly diagnosed ALL were included in the final analysis (1 patient did not receive PEG-asparaginase and 3 were lost early to follow-up). Of these, 52% were male. The median age was 49 years, and 20% of patients were 60 or older. The majority had B cell ALL (88%). Cytogenetics were normal in 28% of patients; t(9;22) was observed in 28% and t(4;11) in 4%. The induction mortality was 9% and 88% achieved complete remission (CR). In the entire patient population, the median overall survival was 27.4 months, and the median disease free survival was 25.0 months. For the patients who achieved at least a 20% decrease in albumin at day 14 of induction (57 patients), there was a statistically significant difference in median overall survival compared to those who had less than a 20% decrease in albumin, with an overall survival duration of 47.4 months and 15.8 months, respectively (HR = 2.23, P = 0.007). The median duration of disease free survival in those who achieved at least a 20% decrease in albumin at day 14 was 39 months compared to 13 months in those with less than a 20% decrease (HR = 1.93, P = 0.039). There was no statistically significant difference in the rate of CR between the two groups (P = 0.503). There was also no statistically significant difference in the baseline characteristics (age, WBC at diagnosis, presence of Philadelphia chromosome, and proportion of patients who eventually underwent BMT) between the two groups. ConclusionThis study found a correlation between a decrease in albumin levels during induction, which was used as a surrogate measure of asparaginase activity, and duration of overall survival and disease free survival. This suggests that lower albumin levels associated with higher asparaginase activity and adequate asparagine depletion are important predictors of outcomes. Further studies assessing the effect of optimal individualized dosing of asparaginase based on albumin levels and/or asparagine depletion might be helpful to improve outcomes of adult patients with ALL. Disclosures:No relevant conflicts of interest to declare.

Nanoparticle Encapsulated L-Asparaginase

Amino acid depletion with catabolizing enzymes is a potential therapeutic modality for many cancer types. L-asparaginase has been used to treat Acute Lymphoblastic Leukemia (ALL) for many decades and is part of treatment regimen that has a roughly 80% cure rate. The current frontline formulation is L-asparaginase from E.coli that has been conjugated to polyethylene glycol (PEG) for extended circulation half life and protection from immune responses. However, neutralizing antibodies still occur in a subset of patients, necessitating a switch to a different L-asparaginase, such as the less active enzyme from Erwinia chrysanthemi. This complicates therapy, risks severe allergic reactions, and if antibodies develop to the second enzyme the options become limited. The threat of immune reactions, as well as other side effects, has largely restricted L-asparaginase therapy to ALL, despite in vitro evidence that many other cancers might be sensitive to asparagine depletion.Our group has developed a novel nanoparticle enzyme encapsulation technology that overcomes the limitation of immune responses to enzymes or PEG. These particles, termed Synthetic Hollow Enzyme Loaded Porous nanoShells (SHELS), are produced in a two-step process where the first creates silica shells with large pores through which the enzyme can be loaded, and a second sealing step that encapsulates the mesoporous shell and the loaded enzyme cargo with a nanoporous silica layer. The work below was done with 200nm particles that have a total shell wall thickness of approximately 10nm.SHELS were loaded with L-asparaginase (Elspar). No loss of activity was seen when enzyme activity was measured in vitro. Groups of mice were injected IM with 5 IU of either free or SHELS encapsulated L-asparaginase. In each case, half of the mice were passively immunized with rabbit polyclonal anti-asparaginase antibodies to a blood concentration of ∼0.5 mg/ml. Serum asparaginase levels pre and post injection were measured by HPLC at 2, 5 and 8 days. Both free and encapsulated enzyme produced complete depletion of asparagine at day 2, but by day 5 asparagine levels had returned to baseline in the animals given free enzyme, whereas those given encapsulated enzyme maintained depletion at day 5 with restoration to baseline at day 8. There was no asparagine depletion in immunized animals with the free L-asparaginase, but SHELS encapsulated asparaginase produced an identical depletion both in the presence or absence of the neutralizing antibodies, indicating that the SHELS effectively shield the enzyme from the antibodies. Experiments in tumor models are ongoing as well as dose response and repeated administration studies. Disclosures:No relevant conflicts of interest to declare.

The Number Of Peg-Asparaginase Doses Administered Is a Determinant Of Relapse Risk In Adult ALL Treated With a Pediatric-Like Regimen

IntroductionAsparaginase (ASN) is an essential drug in treating acute lymphoblastic leukemia (ALL). Pediatric studies have highlighted the significance of adequate and prolonged depletion of asparagine in preventing leukemia relapse. However, similar studies are lacking in adults as ASN is used less frequently mainly due to increased toxicities associated with age. The objective of this study was to evaluate the association between the number of pegaspargase (PEG-ASN) doses administered in a pediatric-inspired regimen and the risk of late leukemia relapse in adult ALL. MethodsWe reviewed patients with adult ALL who were treated at our institution with a modified BFM regimen (Douer D et al. Blood. 2012;120:1495) between 2004 and 2013. This regimen plan was to administer a total of 6 doses of PEG-ASN (2000 units/m2 per dose) administered through 2 cycles of induction, 4 cycles of consolidation and 2 cycles of delayed re-induction. The (PEG-ASN) dose was based on pharmacokinetic and asparagine depletion data previously established for adults (Douer D et al. Blood. 2007;109:2744-50). The study included patients aged 18 to 59 years who received all 8 cycles of the planned treatment courses and were started on POMP maintenance therapy. We excluded patients who relapsed, died, underwent HSCT or were lost follow up before starting maintenance therapy. Relapse was defined as recurrence of disease at any site (bone marrow, CNS, or extramedullary sites). The endpoint considered in the analyses was leukemia free survival (LFS). LFS was defined as the duration of time between the date when the maintenance therapy started and date of first relapse or date of death. ResultsWe identified 60 patients who met the eligibility criteria. Median age was 32 years (range 18- 59) and 42 patients were males. Median presenting WBC at diagnosis was 7000/dL (range 1-350). Six patients had T-cell ALL and 37 patients were classified as high-risk (age > 35 years, initial WBC> 30,000/dL in B-cell ALL and > 100,000/dL in T-cell ALL, and/or unfavorable cytogenetics). The average number of PEG-ASN doses administered was 5 (range 0-6). Among all included patients, 43 had received all 6 doses, 2 received 5 doses, 4 received 4 doses, 5 received 3 doses, 3 received 2 doses, 2 received 1 dose and 1 received no PEG-ASN. Reasons for not completing all doses of PEG-ASN included anaphylaxis, pancreatitis, life-threatening thrombosis, and prolonged delay in treatment due to hyperbilirubinemia. The two- and five-year LFS from the time of starting maintenance therapy were 62±7% and 52±8%, respectively, for all patients. Patients risk category (standard risk vs. high risk) was not significantly associated with LFS. However, a significant association was found between the number of doses of PEG-ASN that patients received during the induction/consolidation cycles and LFS (Figure 1). Patients who received a higher number of doses of PEG-asp had significantly better LFS compared to those who received a lower number of doses (trend test: p=0.009). [Display omitted] ConclusionIn adult patients with ALL who had remained in first remission throughout the entire induction and consolidations period of a pediatric-like regimen, less prior exposure to PEG-ASN (<3 doses) was associated with increased late relapse risk after starting maintenance therapy. These results support the importance of ASN treatment in adult ALL. Disclosures:Douer:Sigma Tau: Membership on an entity's Board of Directors or advisory committees.

Thromboembolic and Hemorrhagic Complications In Adult Patients With Acute Lymphoblastic Leukemia (ALL) Treated With Asparaginase-Containing Combination Chemotherapy: A Single Center Experience

BackgroundL-asparaginase (LASP) depletion of asparagine reduces antithrombin (AT) and fibrinogen and is associated with thromboembolic (TE) and hemorrhagic events (HE). AT replacement in pediatric ALL patients demonstrated safety and potential benefit as thromboprophylaxis in a prospective, randomized trial.(Mitchell et al, 2003) Current consensus supports AT replacement when AT activity is below 60%.(Stock et al, 2011) The incidence of TE and HE in adults receiving AT repletion during LASP or pegylated asparaginase (PEGASP) requires further investigation. MethodsThis single center, retrospective, observational study was approved by the Mayo Clinic Institutional Review Board. Consecutive adult patients with a confirmed diagnosis of ALL receiving LASP or PEGASP containing chemotherapy regimens between January 2000 and October 2012 were evaluated. Patients with documented thrombophilia and pre-existing TE or HE were excluded. Patients receiving AT concentrate (ATC) compared to those that did not and were evaluated up to 60 days after the last LASP or PEGASP dose for objectively confirmed TE and HE. ResultsOf the 74 patients screened, 54 (73%) met inclusion criteria. Forty-seven (87%) received ATC and were evaluable. The remaining 7 (13%) did not meet follow up duration and were excluded from further analysis. Twenty-three (49%) patients received LASP, 20 (42%) received PEGASP and 4 (9%) received both. Distribution per treatment protocols was; CALGB 9111 (n=16 (34%), Larson et al, 1998), E2993 (n=12 (25%), Goldstone et al, 2008), C10403 (n=8 (17%), NLM: NCT00558519), CCG 1941 (n=6 (13%), Gaynon et al, 2006) and Augmented HyperCVAD (n=5 (11%), Faderl et al, 2011). A total of 1465 AT activity levels were analyzed, resulting in 378 doses of ATC. Ten patients (21%) experienced a clinical event, with all events occurring during cycle 1 of chemotherapy (table 1). Median time from last LASP or PEGASP dose to TE was 13 days (range 3-56) with a median of 4 days (range 3-13) from last ATC dose to TE. Median AT activity level at time of TE was 56% (range 47-72). Thrombotic events included; line-related deep vein thrombosis (LRDVT, n=7), cerebral venous sinus thrombosis (CVST, n=1) and pulmonary embolism (PE, n=1). One patient with cerebral hemorrhage constituted the only HE. All LRDVT occurred in central venous catheters and management included; line removal (n=3), anticoagulation (n=1), line removal and anticoagulation (n=2) and no intervention (n=1). The single case of PE was managed with anticoagulation. Two patients had a recurrent LRDVT in different locations during subsequent chemotherapy in spite of ongoing ATC supplementation. ConclusionsThe incidence of TE in adult ALL patients receiving LASP or PEGASP during induction chemotherapy with AT thromboprophylaxis was 19%. LRDVT was the most common TE with a comparable incidence to adult ALL patients not receiving ATC (15%) (Grace et al 2011). There was a small incidence of non-LRDVT (4%) and HE (2%). As induction-phase chemotherapy and placement of central lines are independent risk factors for thrombosis, the cost-effectiveness of AT replacement for prevention of TE during adult ALL treatment requires prospective evaluation. Disclosures:No relevant conflicts of interest to declare.

Feasibility Of Pegylated-Asparaginase (PEG-ASP) During Induction In Adults With Acute Lymphoblastic Leukaemia (ALL): Results From The UK Phase 3 Multicentre Trial UKALL 14

▪L- asparaginase treatment improves clinical outcomes in childhood ALL and Pegylated asparaginase (PEG-ASP) ) has become the standard of care for this patient group. By contrast, safety and efficacy data on PEG-ASP in adult ALL is limited. A key question is the impact of asparaginase associated toxicity on the delivery of multiagent chemotherapy in adult treatment regimes. UKALL14 prospectively evaluated the tolerability of PEG-ASP as part of a 5 drug induction regimen in adults with ALL between 25-65 yrs. The primary-end point of the PEG-ASP evaluation was toxicity. Here, we report on the toxicity of PEG-ASP in the first 91 subjects, after which the trial protocol was amended due to safety concerns.UKALL 14 Induction Phase 1 consisted of intravenous PEG-ASP (1000 IU/m2) on days (d) 4 and 18, Daunorubicin 60mg/m2 and Vincristine 1.4mg/m2 (2mg max) on d1,8,15 and 22, Dexamethasone 10mg/m2 d1,-4, 8-11,15-18 and a single intrathecal (IT) Methotrexate 12.5mg on d14. Patients with pre-B lineage ALL were randomized to receive Rituximab or not. Patients with Philadelphia chromosome (Ph) positive disease received continuous Imatinib 400mg escalating to 600mg daily throughout induction. Phase 2 induction comprised of cyclophosphamide 1000mg/m2 d1+ 15, Ara-C 75mg/m2 d2-5, 9-12, 16-19 + 23-26, mercaptopurine 60mg/m2 throughout and IT methotrexate d1,8,15,22. ResultsThe median age was 47 years (25-65yrs). Overall, 80.2% (73/91) patients completed phase 1 induction. Of these 60 achieved CR, 9 did not, and 4 had missing CR data. Thirteen of the 18 patients who did not complete phase 1 induction suffered early death. The overall induction mortality was 19.8% (18/91). Sixteen deaths occurred pre phase 2 induction and 2 deaths post phase 2. The most common cause of induction death was sepsis in combination with hepatotoxicity (55.6%,10/18) followed by sepsis alone (16.7%, n= 3). Additional causes of death were bowel ischaemia in combination with hepatotoxicity (n=2), acute coronary syndrome (n=1), haemorrhage (n=1) and pancreatitis + bowel perforation (n=1). Ten of the 12 hepatotoxicity associated induction deaths had NCI Grades 3-4 hyperbilirubinaemia. The median time from last dose of PEG-ASP to induction death was 14.5 days (6-71). Taking into account the multiple risk factors contributing to hepatoxicity associated morbidity (concomitant administration of hepatotoxic drugs and sepsis itself), a PEG-ASP related cause of death was “definitely or probably” implicated in 11/18 induction deaths. Logistic regression showed that age and Ph status were independent variables predicting induction death<40yrs vs ≥40yrs, odds ratio (OR) 5.27 (95% CI 1.13-24.7), p =0.035; age <55yrs vs ≥55yrs OR 4.47 (1.51-13.18), p =0.007 and Ph pos vs Ph neg disease, OR 6.08 (2.01 – 18.34), p =0.001. The trial steering committee confirmed that there was no relationship to Rituximab randomization.The incidence of non-fatal PEG-ASP toxicity during induction was 36.7% (n=33).The most common toxicity was abnormal liver function test (24.5%,n=22) followed by coagulopathy (7.7%,n=7) and thrombosis (5.6%,n=5). Grade 3-4 PEG-ASP related liver dysfunction was significantly associated with older age (<40 vs >40, p= 0.031).Enzyme activity, assessed by MAAT, after the first PEG-ASP dose in phase 1 induction was available for a subset of patients (n=27). Nearly all patients (88.9%) achieved a therapeutic level of enzyme activity of >100IU/ml 14 days after the first PEG-ASP. ConclusionSignificant levels of treatment associated mortality occurred in adults treated with a PEG-ASP containing induction regimen on UKALL 14 which was strongly associated with older age and Ph pos disease. As a consequence of the toxicity the protocol was amended to omit d4 PEG-ASP for those over 40yrs. PEG-ASP was completely removed from induction treatment for Ph positive patients. Furthermore, due to the high level of sepsis related to profound myelosuppression, the daunorubicin dose was halved to 30mg /m2 on d1,8,15 and 22. One year following the amendment 123 further patients have been recruited and 8 (6.5%) induction deaths recorded. Our data indicate that whilst PEG-ASP achieves effective asparagine depletion in adult patients it is difficult to deliver safely to those over the age of 40yrs with particularly pronounced toxicity in the over 55s, and should not be started early during induction. Caution should be exercised in co-administration with Imatinib. Disclosures:Fielding:Medac: Research Funding.

Preliminary Results Of a Pharmacokinetic Study Of Intravenous Asparaginase Erwinia Chrysanthemi Following Allergy To E Coli-Derived Asparaginase In Children, Adolescents, and Young Adults With Acute Lymphoblastic Leukemia Or Lymphoblastic Lymphoma

BackgroundAsparaginase is an important component of therapy for acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL). However, up to 30% of patients (pts) develop an allergy to Escherichia coli (E coli)–derived asparaginase. In those pts, Erwinia asparaginase (Erwinia), an antigenically distinct preparation derived from Erwinia chrysanthemi, can be used. Although Erwinia is typically administered intramuscularly (IM), intravenous (IV) asparaginase would offer a more convenient, less painful administration route. In an open-label, single-arm, multicenter, pharmacokinetic (PK) study, we evaluated IV administration of Erwinia in children and adolescents with ALL who developed hypersensitivity to E coli–derived asparaginase. MethodsPts (aged ≥1 to ≤30 years) with ALL or LBL who developed hypersensitivity (grade ≥2) to E coli–derived asparaginase were eligible for enrollment. Pts received Erwinia IV, 25,000 IU/m2/dose, on a Monday/Wednesday/Friday (M/W/F) schedule for 2 consecutive weeks (6 doses=1 cycle) for each dose of pegaspargase remaining in the original treatment plan. All other chemotherapy was continued per the original treatment plan. Blood samples were collected during the 1st treatment cycle, including predose, 5 min, 48 hrs and 72 hrs postdose. The primary study objective was to determine the proportion of pts who achieved nadir serum asparaginase activity (NSAA) ≥0.1 IU/mL, which has been associated with complete asparagine depletion, at 48 hrs after dose 5 in cycle 1. Secondary objectives included determining the proportion of pts who achieved NSAA ≥0.1 IU/mL at 72 hrs after dose 6 in cycle 1 and the incidence of asparaginase-related toxicities. Only pts who completed 1 cycle of Erwinia were considered evaluable for NSAA assessment. ResultsBetween November 2012 and June 2013, 30 pts were enrolled from 10 centers; 26 pts completed cycle 1. Twenty-three pts were evaluable for the primary NSAA endpoint (3 pts were not evaluable; 2 for PK data collected outside the protocol-specified window and 1 for nonadherence to the dosing schedule). Median age was 6.5 years (1-17 years), 63% were male, and 83% Caucasian. Predose NSAA levels were below the limit of quantification (defined as 0.0129 IU/mL) for nearly all (91%) pts. Of the 23 evaluable pts who completed cycle 1, 19 (83%) achieved NSAA levels ≥0.1 IU/mL at 48 hrs postdose 5, with mean ±SD NSAA 0.32 ±0.23 (Table 1). At 72 hrs postdose 6, 9 pts (45%) achieved NSAA levels ≥0.1 IU/mL (mean ±SD 0.088 ±0.076).Four pts discontinued Erwinia treatment before completing cycle 1; 3 for hypersensitivity and 1 for pancreatitis. The toxicity analysis included all 30 enrolled pts. The most common asparaginase-related toxicities reported during cycle 1 were: hypersensitivity (23% of pts), vomiting (20%), nausea (20%), and hyperglycemia (13%). Pancreatitis occurred in 3% and thrombosis in 3% of pts. One pt experienced a transient ischemic attack. The most common grade 3 or 4 adverse event was febrile neutropenia (7%). There were no deaths. ConclusionsThe majority (83%) of children and adolescents with ALL or LBL receiving Erwinia IV at 25,000 IU/m2/dose on a M/W/F schedule, achieved NSAA ≥0.1 IU/mL at 48 hrs postdose 5 (considered the therapeutic goal); however the majority did not achieve this goal at 72 hrs post Erwinia dose 6. With IV administration, asparaginase-related toxicities appeared to be consistent with previous reports in children treated with IM Erwinia. Based on these results, we are now evaluating IV Erwinia at 25,000 IU/m2/dose administered every 48 hrs rather than on a M/W/F schedule.This study was funded by Jazz Pharmaceuticals plc. Disclosures:Off Label Use: ERWINAZE (asparaginase Erwinia chrysanthemi) is indicated as a component of a multi-agent chemotherapeutic regimen for the treatment of patients with acute lymphoblastic leukemia (ALL)who have developed hypersensitivity to E. coli-derived asparaginase. The recommended dose is 25,000 International Units/m2 administered intramuscularly three times a week (Monday/Wednesday/Friday) for six doses for each planned dose of pegaspargase. Hijiya:Jazz Pharmaceuticals: Consultancy. Brown:Jazz Pharmaceuticals: advisory board Other. Drachtman:Jazz Pharmaceuticals: Honoraria. Messinger:Jazz Pharmaceuticals: Advisory Board Other. Plourde:Jazz Pharmaqceuticals: Employee of Jazz Pharmaceuticals, Inc, who in the course of this emplyment has received stock options exercisable for, and other stock awards of, ordinary shares of Jazz Pharmaceuticals plc Other, Employment. Silverman:Enzon Pharmaceuticals: Consultancy; Sigma Tau Pharmaceuticals: Advisory Board, Advisory Board Other; EUSA Pharma: Advisory Board Other.

Deficiency of Asparagine Synthetase Causes Congenital Microcephaly and a Progressive Form of Encephalopathy

We analyzed four families that presented with a similar condition characterized by congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. We show that recessive mutations in the ASNS gene are responsible for this syndrome. Two of the identified missense mutations dramatically reduce ASNS protein abundance, suggesting that the mutations cause loss of function. Hypomorphic Asns mutant mice have structural brain abnormalities, including enlarged ventricles and reduced cortical thickness, and show deficits in learning and memory mimicking aspects of the patient phenotype. ASNS encodes asparagine synthetase, which catalyzes the synthesis of asparagine from glutamine and aspartate. The neurological impairment resulting from ASNS deficiency may be explained by asparagine depletion in the brain or by accumulation of aspartate/glutamate leading to enhanced excitability and neuronal damage. Our study thus indicates that asparagine synthesis is essential for the development and function of the brain but not for that of other organs.

Pediatric Acute Lymphoblastic Leukemia: Efficacy and safety of recombinant E. coli-asparaginase in infants (less than one year of age) with acute lymphoblastic leukemia

The pharmacokinetics, pharmacodynamics, efficacy and safety of a new recombinant E. coli-asparaginase preparation were evaluated in infants (<1 year of age) with de novo acute lymphoblastic leukemia. Twelve patients were treated according to the INTERFANT-06 protocol and received up to 10,000 U/m(2) recombinant asparaginase as intravenous infusions on days 15, 18, 22, 25, 29 and 33 of remission induction treatment. The asparaginase dose was individually adjusted by protocol to 67% of the calculated dose for infants <6 months, and to 75% of the calculated dose for infants aged 6-12 months. The trough serum asparaginase activities observed were above 20, 50, and 100 U/L in 86%, 71%, and 51% of measured samples, respectively. Looking only at the data assessed 3 days after asparaginase infusion these percentages were 91%, 84%, and 74%, respectively. Asparagine was completely depleted in serum in all but one patient who was the youngest in the study. No anti-asparaginase antibodies were detected during this treatment phase. Observed adverse reactions are known to be possible and are labeled side effects of asparaginase treatment and chemotherapy. We conclude that the asparaginase dose regimen used in infants is safe and provides complete asparagine depletion for the desired time period in nearly all patients. Measured asparaginase trough serum levels justify the higher doses used in infants compared to in older children and show that 3-day intervals are preferred over 4-day intervals. (This trial was registered at www.clinicaltrialsregister.eu as EudraCT number 2008-006300-27).

Optimizing asparaginase therapy for acute lymphoblastic leukemia

Asparaginases are important agents used in the treatment of children with acute lymphoblastic leukemia (ALL). Three types of asparaginase are currently available: two are derived from Escherichia coli [native asparaginase and pegylated asparaginase (PEG-asparaginase)] and one from Erwinia chrysanthemi (crisantaspase). All three products share the same mechanism of action but have different pharmacokinetic properties, which do not make them easily interchangeable. Among the known toxicities and side-effects, allergic reactions and silent inactivation represent the most important limitations to the prolonged use of any asparaginase product, with associated reduced therapeutic effects and poorer outcomes. Routine real time monitoring can help to identify patients with silent inactivation and facilitate a switch to a different product to ensure continued depletion of asparagine, completion of the treatment schedule and maintenance of outcomes. However, the most appropriate second-line treatment is still a matter of debate. PEG-asparaginase has lower immunogenicity and a longer half-life than native Escherichia coli (E. coli) asparaginase, which makes it useful for both first-line and second-line use with a reduced number of doses. However, PEG-asparaginase displays cross-reactivity with native E. coli asparaginase that may harm its therapeutic effects. Crisantaspase does not display cross-reactivity to either of the E. coli-derived products, which has made crisantaspase the second-line treatment option in a number of recent protocols. As crisantaspase has a much shorter biological half-life than the E. coli-derived products, the appropriate dosage and administration schedule are of paramount importance in delivering treatment with this product. In the ongoing trial AIEOP-BFM ALL 2009 (Associazione Italiana Ematologia Oncologia Pediatrica - Berlin-Franklin-Munster), in which PEG-asparaginase is used first-line, one dose of PEG-asparaginase is substituted by seven doses of crisantaspase given intravenously at 20,000 IU/m2 on alternate days when clinical allergy or silent inactivation is present. Based on the indications of different protocols, lack of cross-reactivity to the E. coli-derived products and taking into consideration regulatory factors and availability, crisantaspase may be considered a viable second-line therapy.

Risk of Thrombosis with Different Approaches to Central Venous Access During Acquired Asparaginase Related Antithrombin Deficiency in Children with Leukemia.

Abstract 2241 BackgroundChildren with acute lymphoblastic leukemia (ALL) have several risk factors for thrombosis including asparaginase related acquired antithrombin (AT) deficiency and central venous catheters (CVCs). Both external tunneled and internal CVCs (tunneled, fully implantable devices known as ports) are used to provide reliable venous access. After introduction of a new ALL protocol with prolonged continuous asparaginase treatment aiming at approximately 30 weeks depletion of asparagine more symptomatic thromboses than expected were observed at the Children's Hospital, Helsinki University Central Hospital, Finland. To ensure asparaginase treatment and prevent thromboses an on-demand AT substitution regimen was adopted. Previous studies have described AT substitution during ALL induction; this is the first description and evaluation of on-demand AT substitution over a longer period of time in children. AimThe aim of this study is to describe the prolonged on-demand AT substitution practice in Helsinki during depletion of asparagine from the blood with PEG-ASP and to compare experiences from two Nordic pediatric oncology centers (Children's Hospital in Helsinki and the Astrid Lindgren Children's Hospital Karolinska University Hospital, in Stockholm, Sweden) with the same leukemia protocol but different approaches to central venous access. ProcedureAll Finnish children with ALL and external tunneled CVCs diagnosed at Children's Hospital in Helsinki between May 2008 and November 2011 (n=38) were compared with Swedish children with ALL and internal CVCs treated concurrently with the same ALL protocol (n=39). Children treated in Helsinki according to the standard or intermediate risk NOPHO-ALL 2008 protocol after initiation of the AT substitution regiment received AT concentrate when their AT level decreased below 55% (the intervention group, n=25). The thrombosis rate and laboratory parameters were compared with children treated earlier in the same unit (the control group, n=10). ResultsIn Helsinki, 2/10 (20%) children in the control group had 3 symptomatic thromboses and 2/25 (8%) children in the intervention group with on-demand AT substitution had one symptomatic thrombotic event each. Both thromboses in the intervention group were associated with concomitant steroid treatment. In the intervention group 14/25 (56%) received AT concentrate (median number of infusions 3, range 1–17).Altogether, 4/38 Finnish patients with external CVCs and 0/39 Swedish patients with internal CVCs had thromboses (Mann Whitney U test, p=0.039) in spite of similar exposure to asparaginase and low antithrombin. A significant difference was also observed in the use of tissue plasminogen activator (tPA): 28/38 Swedish patients had received tPA at least once to restore dysfunctional CVCs (usually loss of the ability to withdraw blood) compared to 2/38 Finnish patients (Mann Whitney U test, p≤0.001). ConclusionsMost children are exposed to low AT levels during ASP treatment predisposing to thrombosis especially with concomitant steroids. Larger studies are needed to evaluate the benefit of prophylactic antithrombin treatment. Internal CVCs and tPA may be beneficial in preventing symptomatic thrombosis during leukemia treatment. Disclosures:No relevant conflicts of interest to declare.

Two Years Follow-up Results of Graspall/Graall-SA2–2008 Study: L-Asparaginase-Loaded Red Blood Cell Combined with Standard EWALL Chemotherapy in Older Patients with Newly Diagnosed Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia (Ph-ALL)

AbstractAbstract 1473 Introduction:L-asparaginase-loaded red blood cells (GRASPA®) has been shown to be a new available option for combining L-asparaginase with standard chemotherapy in different ALL population, including older patients with the disease. Tolerability and preliminary results of efficacy of GRASPA® in GRASPALL/GRAALL-SA2–2008 study have already been presented. We report below the 2-year follow-up efficacy results of this study. Methods:GRASPALL/GRAALL-SA2–2008 study aimed at determining optimal dose of GRASPA® that could be combined with standard EWALL chemotherapy backbone in patients aged >55y with newly diagnosed Ph-ALL. It was an open label Phase II dose escalation study. Primary endpoint combined tolerance and efficacy (asparagine depletion ≥7 days). Three doses of L-Asparaginase-Loaded Red Blood Cells (50, 100 and 150 IU/kg), infused twice during induction cycles, were investigated. EWALL backbone consisted of dexamethasone prephase followed by induction-1 (dexamethasone d1–2/8–11, vincristine d1,8, and idarubicine d1–2/8–9) and induction-2 (cyclophosphamide d15–17 and cytarabine d16–19/23–26). Consolidation consisted of 6 monthly alternating cycles with methotrexate (d1) / E.coli asparaginase (d2) and high-dose cytarabine (d1, 3, 5). Maintenance included mercaptopurine, methotrexate and vincristine/dexamethasone pulses for 2 years. Hematological and molecular Ig/TCR minimal residual disease (MRD) response rates, survival were secondary endpoints. Results:Between March 2009 and October 2010, 30 patients were recruited in 20 centres in France by the GRAALL network. The 50, 100 and 150 dose levels included 3, 13 and 14 patients, respectively. Median age was 67 years (range 59–77). No differences in baseline characteristics were observed across the 3 dose level groups.The tolerability with L-Asparaginase-related side effects is reported below:GRASPA Dose (IU/kg)50100150Number patients/group31314Allergic reaction000Clinical thrombosis1 (33%)1 (8%)2 (14%)Antithrombin III decrease <60%2 (67%)7 (54%)12 (86%)Other Coagulation disorder2 (67%)3 (23%)11 (79%)Clinical pancreatitis000Pancreatic enzymes increased >2N1 (33%)5 (38%)5 (36%)Protein synthesis dicrease (hypoalbuminemia <30g/l)1 (33%)1 (8%)6 (43%)Transaminases increase >5N2 (67%)4 (31%)6 (43%)Overall L-asp expected adverse events tended to be lower in the 100 UI/kg group.Regarding the efficacy and benefit/risk, asparagines depletion, remission rate, EFS and OS are reported below:Dose levelNPts with at least 7 days depletion N (%)Patients with DLT N (%)Pts with at least 7 days depletion and no DLT N (%)Remission After Induction CREFSOSMedian1 year2 yearsMedian1 year2 years100 IU/Kg1311 (85%)2 (15%)9 (69%)10 (77%)11.8 mo46%23%15.6 mo62%23%150 IU/Kg1410 (71%)5 (36%)7 (50%)9 (64%)4.0 mo33%–9.5 mo43%–Three patients received the dose of 50 UI/kg but this dose was insufficient to reach a 7-day asparagine depletion.Survival analysis showed that the dose of 100IU/kg was associated with median OS of 15.6 months an absolute value that compared favorably with historical controls: 8,8 mo (Rousselot et al. Drugs Aging,2011) and 10,5 mo (Hunault et al. Haematologica,2011) Conclusion:GRASPA® at a dose of 100 UI/kg appears to be a safe and active manner to introduce L-asparaginase during initial induction chemotherapy of older patients with Ph- ALL with a sustained asparagine depletion and a good efficacy/safety profile. Disclosures:Godfrin:ERYTECH Pharma: COO Other.

L-Asparaginase Strongly Affects Bioenergetics in Leukemic Cells

AbstractAbstract 779L-asparaginase (L-asp) is an important component of childhood acute lymphoblastic leukemia (ALL) therapy. Its cytotoxic effect is based on the depletion of extracellular asparagine and glutamine. Leukemic cells are sensitive to this depletion due to the lower activity of asparagine synthetase compared to healthy cells. However, the mechanism of resistance development remains unclear. The aim of this study was to obtain further insights into the mechanisms underlying the cytotoxic effect of L-asp.We used two models: resistant preB ALL leukemic cells derived from the REH (TEL/AML1[+]; very sensitive) and NALM6 (TEL/PDGFRB[+]; medium sensitive) cell lines by long-term incubation with L-asp. As a second model we used REH and NALM6 incubated with L-asp for 24 hrs, mimicking acute phase of treatment.We performed GEP of REH, res-REH, NALM6 and res-NALM6. There was an overlap of 30 genes changed in both cases. Applying the pathway analysis we found that L-asp influences the regulation of lipid metabolism and apoptosis regulated by mitochondrial proteins. Next we merged our data with GEP data published by Holleman et al. (NEJM, 2004) of ALL patients′ samples sensitive/resistant to L-Asp. Two pathways, one regulating protein translation, the other metabolism, scored very highly. Next to glucose, glutamine is the other major cellular energy source, it is also important for activation of PI3K/Akt/mTOR pathway - the key regulator of translation. Since L-asp also depletes glutamine, we focused on the effect of L-asp treatment on bioenergetics and translation in leukemic cells. To confirm the effect of L-asp on PI3K/Akt/mTOR pathway we measured the amount of Akt protein and P70S6K/p-P70S6K (downstream target of mTOR) in REH and NALM6 treated for 24 hours with L-asp. Akt and p-P70S6K expression fell in both cell lines. Next we were interested if PI3K/mTOR inhibition affects the sensitivity of resistant cell lines. We measured the cytostatic effect of co-treatment: L-asp with mTORC1 inhibitor (rapamycin); with dual inhibitor of mTORC1 and PI3K (LY294002); and with specific inhibitor of PI3K (PX866) on REH, res-REH, NALM6 and res-NALM6. The MTS assay proved the L-asp/rapamycin combination the most effective. It strongly diminished viability of all cell lines, even in those resistant to L-asp.c-Myc as an activator of glutamine catabolism showed a significant decrease in REH and NALM6 after incubation with L-asp. In addition, c-Myc was also decreased in res-REH compared with REH. Importantly, c-Myc activates glycolysis which is a main energy generator in cancer cells rather than oxidative phosphorylation (OXPHOS). We showed diminishement of Glucose transporter type 1 protein expression and 2.5-times reduced level of lactate, the product of cancer cell glycolysis, in media after L-asp exposure. Next we measured cell respiration that mirrors oxidative fosforylation (OXPHOS). REH and NALM6 showed increased capacity of respiratory chain after L-asp exposure, which was observed as an increase of endogenous respiration in uncoupled state. REH boosted the capacity of the respiratory chain from 163 to 236 pmol O2/s/mg (p<0.01). NALM6 have increased the capacity of the respiratory chain from 78 to 155 pmol O2/s/mg (p<0.01). Interestingly, REH has a significantly higher capacity of respiratory chain compared with NALM6 and a lower ability to regulate it. Moreover, res-REH had elevated the level of the endogenous oxygen consumption whereas res-NALM6 levels did not change. Our results indicate that leukemic cells, which normally depend on glycolysis for energy generation rather than OXPHOS, are able to swap these two mechanisms of energy generation under amino acid deprivation stress.The data show that L-asp inhibits mTORC1 and strongly affects bioenergetics pathways of leukemic cells. It decreased c-Myc-regulated glycolysis and increased OXPHOS. We believe that these metabolic changes are mainly caused by the effort of the cells to mobilize another mitochondrial pathway as Krebs cycle and β-oxidation, with the aim to supply depleted amino acids. We conclude from these data that resistance of the cells is caused by better biochemical adaptability to the nutrient deprived environment.Support: GAUK 92710, IGA NT1249, UNCE204012 Disclosures:No relevant conflicts of interest to declare.

Pancreatic Tumor Sensitivity to Plasma L-Asparagine Starvation

In this study, our aim was to test whether asparagine synthetase (ASNS) deficiency in pancreatic malignant cells can lead to sensitivity to asparagine starvation. We also investigated, in tumor-bearing mice, the efficacy of L-asparaginase entrapped in red blood cells (RBCs), a safe formulation, to induce asparagine depletion. First, ASNS expression was evaluated by immunohistochemistry in sporadic pancreatic ductal adenocarcinoma. Then, 4 pancreatic carcinoma cell lines were examined by Western blot, immunocytochemistry, and cytotoxicity assay to L-asparaginase and in asparagine-free or reduced-asparagine media. Finally, mice bearing the most in vitro sensitive cell line received RBC-entrapped L-asparaginase to investigate the anticancer efficacy of serum asparagine depletion in vivo. Approximately 52% of pancreatic adenocarcinomas expressed no or low ASNS. The highest in vitro cytotoxicity to L-asparaginase or to reduced asparagine medium was observed with SW1990 line when ASNS expression was the lowest. In vivo sensitivity was confirmed for this cell line. Plasma asparagine depletion by RBC-entrapped L-asparaginase in selected patients having no low or no ASNS may be a promising therapeutic approach for pancreatic cancer.

L-asparaginase loaded red blood cells (RBC) in pancreatic cancer: A phase I clinical study.

e14650 Background: L-asparaginase (L-asp) efficacy is based on sensitivity of tumoral cells to asparagine deprivation due to a lack of asparagine synthethase (ASNS). However its use is hampered by significant toxicities of which allergic reaction, coagulation disorders and pancreatic impairment take a prominent place. Deficiency in ASNS and therefore potential sensitivity to L-asp treatment has been reported in multiple tumor cell lines including pancreatic cancer, however tentative to use L-asp in solid tumor, and especially in pancreatic cancer is reported in the literature but all attempts so far have failed due to toxicity. Recently, a new formulation of L-asp loaded inside RBC (GRASPA) has shown a very promising safety profile in leukemic patients. Methods: GRASPANC is the first phase I clinical study with GRASPA in patients with pancreatic carcinoma. Primary objective was to find the Dose Limiting Toxicity (DLT) of this cell-based formulation in this setting. Secondary objectives were to assess overall safety profile and activity (defined as serum asparagine depletion) of GRASPA. Patients with non resectable, relapsed pancreatic cancer were included by cohort of 3 and treated with one injection of increasing dose (25, 50, 100 or 150 IU/kg) of GRASPA. An independent Safety Monitoring Board assessed toxicities at the end of follow-up of each cohort to decide for next dose. Results: Twelve patients were included between Dec 2009 and Feb 2011 by 3 centres in France. Mean age was 60.4 years (min: 42.6; max: 71.9) with 9 Male and 3 female. No limiting toxicity has been found up to the GRASPA highest dose (150 IU/kg) planned in the study and the DLT was not reached. No specific other unexpected non limiting toxicity was evidenced. In addition, at all doses GRASPA was shown to be effective at depleting serum asparagine with a trend toward longer activity with increasing dose. Conclusions: Preliminary conclusion is that the dose of 150 IU/kg of GRASPA is safe and appears suitable for further clinical development in patients with pancreatic carcinoma. In order to maximise potential efficacy of this new therapy, selection of patients should be made according to their screening for a none or low expression of ASNS in their tumour cells.

Asparagine and aspartic acid concentrations in bone marrow versus peripheral blood during Berlin–Frankfurt–Münster-based induction therapy for childhood acute lymphoblastic leukemia

A recent study suggested that mesenchymal cells in bone marrow (BM) may counteract l-asparaginase (L-Asp)-containing acute lymphoblastic leukemia (ALL) therapy by secreting asparagine. Herein, we compared asparagine and aspartic acid concentrations in the BM and peripheral blood (PB), in order to determine whether this in vitro observation could be translated into in vivo differences of amino acid levels between both compartments. Asparagine and aspartic acid concentrations in BM (days 15 and 33) and PB (days 12, 15 and 33) were measured during L-Asp-containing Berlin–Frankfurt–Münster (BFM)-based 5-week multi-agent remission induction therapy in 11 children diagnosed with ALL at the St. Anna Children's Hospital in Vienna, Austria. The level of asparagine depletion did not differ significantly between both compartments at any time point measured, but aspartic acid concentrations were significantly higher in BM than PB at days 15 and 33 (p < 0.05). In the context of the reported mesenchymal asparagine production in BM, an increased asparagine production may indeed take place in BM. However, it may be overcome by continuous action of L-Asp, which is mirrored by increased aspartic acid levels but unchanged low asparagine levels in BM, suggesting a higher BM turnover of asparagine generated by L-Asp during induction therapy.

Abstract 5245: Adipocyte production of glutamine protects leukemia cells from L-asparaginase

Abstract Obesity is associated with increased cancer incidence and mortality. Adipose tissue has been demonstrated to play a role in several types of cancer, including acute lymphoblastic leukemia (ALL). We have shown that obesity impairs the survival of mice transplanted with ALL and treated with the front-line chemotherapy, L-Asparaginase (ASNase), which acts through the deamination of asparagine (ASN) and glutamine (GLN). We have also shown that adipocytes protect ALL cells from ASNase in vitro. We hypothesized that adipocytes protect ALL cells from ASNase via production of GLN. Fat pads were excised from control and diet-induced obese mice. Glutamine synthetase, the rate-limiting enzyme for GLN synthesis, was analyzed by western blot. Amino acids secreted by fat pads were measured using HPLC. ALL cell sensitivity to ASN and/or GLN depletion, and ASNase, was determined by trypan blue exclusion in culture alone and in transwells over 3T3-L1 adipocytes. Mouse fat pads expressed high levels of glutamine synthetase protein, and this was not affected by obesity or 5 days of L-asparaginase treatment (3,000 IU/kg/day). Fat pad explants secreted GLN (251±50 nmoles/100 mg tissue), but not ASN, over 72 hours. Confirming that GLN secretion could protect ALL from ASNase, twice daily addition of 500 nmoles of GLN to murine ALL cells completely reversed ASNase cytotoxicity (64.0x10^4 vs. 7.9x10^4 viable cells after 72 hours of 1 IU/mL ASNase, p&amp;lt;0.01). Testing a panel of human ALL cell lines, we found that proliferation of 3 of 9 cell lines were decreased when ASN was removed from the media (RS4;11, SupB15, Molt4), while 8 of 9 cell lines were decreased when GLN was removed from the media (RS4;11, SupB15, Molt4, SEM, RCH-ACV, BV-173, SD-1, HL60). To test whether blocking adipocyte production of GLN would reverse their ability to protect ALL cells, we pretreated 3T3-L1 adipocytes with methionine sulfoximine (MSO), an irreversible inhibitor of glutamine synthetase. MSO pretreatment blocked GLN secretion by adipocytes and completely reversed adipocyte protection of ALL cells (with no adipocytes: 7.5x10^4; with untreated adipocytes: 14.8x10^4; with MSO treated adipocytes: 6.2x10^4, p=0.03 vs. untreated adipocytes). Finally, we tested whether an alternative preparation of ASNase with a higher glutaminase activity, ERWinase, could overcome adipocyte protection of three GLN dependent cell lines: SD-1, HL60, and Molt-4. Compared to E. coli ASNase, ERWinase treatment resulted in significantly decreased survival of all 3 cell lines in the presence of adipocytes (SD-1 4.2x10^4 vs. 6.7x10^4, p&amp;lt;0.05; HL60 4.1x10^4 vs. 8.8x10^4, p&amp;lt;0.05; Molt-4 16.9x10^4 vs. 26.4x10^4, p&amp;lt;0.05). These studies uncover a novel mechanism of adipocyte protection of leukemia cells from chemotherapy: adipocytes protect ALL cells from ASNase treatment via the production of GLN. Adipocyte production of GLN is both necessary and sufficient to protect ALL cells from ASNase treatment. 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 5245. doi:1538-7445.AM2012-5245