ALBERT

Description: Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children, and cure rates for relapsed/refractory ALL in children and adults remain dismal, highlighting the need for novel targeted therapies capable of overcoming resistance in relapsed/refractory disease. We previously uncovered that ALL cells are vulnerable to metabolic/energy stress and endoplasmic reticulum (ER)-stress via AMP-activated protein kinase (AMPK) activation leading to unfolded protein response (UPR)-mediated apoptosis. In order to identify genome-wide metabolic-stress and AMPK-transcriptionally regulated genes in ALL cells undergoing metabolic/energy stress, we used RNA-Seq and compared mRNA transcript profiles in ALL cells treated with acadesine (adenosine analog 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside or AICAR), known to activate AMPK. RNA-Seq data indicated that acadesine treatment (15 mM/45 min) induced a robust and rapid alteration in gene expression in ALL cells. The most significant acadesine-induced gene signature represented a cluster of genes known as immediate early genes (IEGs), which are fundamental in critical biological pathways for cell survival/proliferation/adaptation. We interpreted these changes as a compensatory pro-survival mechanism in ALL cells undergoing energy/metabolic stress. Among the acadesine-induced downregulated IEGs, we selected DUSP1, JUNB and NFKBIA for further characterization. Downregulation of these IEGs was confirmed using RT-qPCR. We found that the effect of acadesine-induced downregulation on IEGs expression was dose- and time-dependent, and these effects were observed in other cell types (HeLa, HEK293T, mouse embryonic fibroblasts(MEF)), indicating this mechanism of acadesine-induced downregulation of IEGs expression is conserved in mammalian cells. Interestingly, when we used lower doses of acadesine (the half-maximal inhibition concentration for IEGs (IC50)), the IEGs mRNA levels returned to baseline after 3 hours of exposure, suggesting the effect of acadesine on these IEGs was transient at IC50 dose. Using NALM6 AMPKα1 knockdown and MEF AMPKα1/α2 knockout cell lines, we uncovered that high-dose/short-time exposure to acadesine led to changes in IEGs expression that were independent of AMPK. Consistent with these findings, ALL cells co-treated with acadesine plus adenosine kinase inhibitors (ABT702 or 5-Iodotubercidin), which prevent its conversion to ZMP, exhibited the same gene expression signature. Characterization of acadesine's mechanism of action identified protein kinase D1 (PKD1) as responsible for acadesine-induced downregulation of IEGs. PKD1 is a serine/threonine protein kinase involved in many cellular processes important to cancer development and progression, including proliferation, survival, apoptosis, motility, cell adhesion and angiogenesis. Acadesine induced strong inhibition of PKD1 activity which resulted in PKD1 accumulation in the cytoplasm and prevented its nuclear translocation. When ALL cells were treated with protein kinase D (PKD) inhibitors (CRT0066101, GF109203X), we observed a similar rapid, robust and transient downregulation of IEGs, suggesting acadesine interacts with the PKD1 pathway. Conversely, the effect of acadesine on IEGs expression was abrogated by phorbol 12-myristate 13-acetate (PMA), a direct activator of PKD. Further, we determined that acadesine suppresses PKD1-regulated class II Histone deacetylase (HDAC4/5) phosphorylation and nuclear export, which led to decreased histone H3 acetylation levels at the IEG's promoter region. Finally, ChIP-qPCR experiments uncovered that the acadesine/PKD1 axis regulates the recruitment of nuclear factor-κB (NF-κB) to the promoter region of selected IEGs. Consequently, we have identified a novel, AMPK-independent transcription regulation mechanism of acadesine thorugh PKD1 in ALL cells, and co-targeting PDK1 and other pro-survival stress response pathways in ALL cells vulnerable to energy/metabolic stress offers potential novel strategies to overcome therapeutic resistance. Disclosures No relevant conflicts of interest to declare.

Description: Methotrexate (MTX) is an antifolate widely used to treat childhood acute lymphoblastic leukemia (ALL). MTX is retained within cells as long-chain polyglutamates (MTX-PGs), after metabolism by the enzyme folylpoly-γ-glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR), and the additional inhibition of thymidylate synthetase (TS). The FPGS gene was shown to be regulated by the transcription factors Sp1 and NFY. We performed DNaseI hypersensitive assays and identified a hypersensitive site mapping closely upstream of exon 1 suggesting that chromatin remodeling may contribute to FPGS gene regulation. Using co-immunoprecipitation and Western blotting we investigated the role of histone modifications and chromatin remodeling on the expression of FPGS and uncovered interactions between NFY, Sp1 and HDAC1. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assay (DAPA) revealed that the HDAC1-NFY and HDAC1-Sp1 complex binds to the NFY and Sp1 binding sites in the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by acetylation of NFY and Sp1 factors and interaction with HDAC1, and/or chromatin remodeling. We then examined the effect of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaBu) on the expression of FPGS and other folate-related genes. The level of FPGS, ATP-binding cassette subfamily C (ABCC1), ATP-binding cassette subfamily G (ABCG2), DHFR, γ-glutamyl hydrolase (GGH), solute carrier family 19/folate transporter (SLC19A1), and TS mRNA gene expression was determined by qRT-PCR in NALM6 (Bp-ALL), REH (Bp-ALL, t(12,21)/TEL-AML1), SupB15 (Bp-ALL, t(9,22)/BCR-ABL), and CCRF-CEM (T-ALL) cells treated with NaBu [2mM-5mM]. In all cell lines examined, treatment with NaBu induced 2- to 5-fold the level of FPGS and ABCC1 mRNA expression whereas the level of DHFR, SLC19A1, and TS mRNA expression was decreased. Expression of GGH and ABCG2 mRNAs was increased 2-fold in CCRF-CEM but remained unaltered in Bp-ALL NaBu treated cells. Promoters of butyrate-responsive genes have been shown to contain genetic elements such as Sp1/Sp3 binding sites which interact with HDAC1 to mediate the action of NaBu. On this basis, we hypothesized that pre-treatment of ALL cells with NaBu should lead to induction of FPGS expression and subsequently, higher synthesis of MTX-PG and enhanced MTX cytotoxicity in ALL cells. To test this hypothesis, CCRF-CEM, NALM6, REH, and SupB15 cells were treated sequentially with NaBu (24h) and MTX (4h), and assessed for cell viability. Treatment of NaBu and MTX increased cell death by ∼40% in NALM6, REH, and SupB15 Bp-cells, and ∼60% in CCRF-CCEM cells when compared to treatment with each drug alone. These data suggest that combination of HDACi and MTX may represent a novel therapeutic strategy for treatment of ALL. This strategy may be particularly useful to overcome MTX resistance in patients diagnosed with phenotypes that accumulate low levels of MTX-PGs and for patients after relapse.

Description: Methotrexate (MTX) is a universal component of chidlhood ALL therapies and its conversion to long chain polyglutamates (PG) by folylpoly-γ-glutamate synthetase (FPGS) is essential for its antileukemic acitivity. Expression of FPGS appears to be controlled by tissue/lineage specific and proliferation-dependent mechanisms. Levels of FPGS mRNA, protein, and enzyme activity are 2-3 fold higher in B-precursor (Bp) ALL cells when compared to T-lineage ALL, and these differences correlate with intracellular accumulation of long chain MTX-PG and lymphoblast sensitivity to MTX. To characterize these lineage differences in FPGS expression between B and T lymphoblasts we examined its mRNA expression in Nalm6 (Bp-ALL) and CCRF-CEM (T-ALL) cells during cellular growth and cell cycle checkpoints. During early exponential growth (24 hrs), FPGS expression was 6-fold higher in Nalm6 when compared to CCRF-CEM but decreased significantly after 72 hrs while it was unchanged in CCRF-CEM cells. During G1/G0 phase we found that FPGS expression was 15-fold higher in Nalm6 when compared to CCRF-CEM cells. Taken together, these data suggest that during proliferation and cell cycle progression FPGS gene expression is regulated differently in Bp-ALL and T-ALL cells. To determine whether this lineage-specific regulation occurs at the transcriptional level we performed nuclear run-on assays. We found that FPGS mRNA transcription initiation rate was 1.7-fold higher in Nalm6 when compared to CCRF-CEM cells, indicating that differences in promoter regulation lead to the observed lineage differences in FPGS expression in Bp- vs. T-ALL. We then used a methylation specific PCR assay to investigate the methylation status of the FPGS proximal promoter region from both Nalm6 and CCRF-CEM cells. Our data indicate that the FPGS proximal promoter region is unmethylated in both cell lines and therefore can not explain the observed lineage-specific differences. 5′-RACE experiments demonstrated that Nalm6 and CCRF-CEM have the same FPGS transcriptional start sites, but a reporter gene assay indicated that the minimal promoter region that directs FPGS transcription in CCRF-CEM cells is insufficient to drive FPGS mRNA transcription in NALM6 cells. In order to identify potential regulatory regions directing FPGS transcription in Bp-lymphoblasts, we used DNaseI hypersensitivity assays. We identified a hypersensitive region located 8.5 kbp upstream to exon 1 in Nalm6 cells suggesting that tissue-specific regulatory elements responsible for lineage-specific FPGS expression in Bp-ALL cells may be localized within this region. Finally, we detected reduced levels of FPGS mRNA expression in RCH-ACV (Bp-ALL, t(1:19)/E2A-PBX1) and REH (Bp-ALL, t(12:21)/TEL-AML1) cells expressing chromosomal translocated fusions when compared to control (Nalm6). To characterize the molecular basis of the E2A-PBX1 and TEL-AML1 interactions with FPGS mRNA expression in Bp lymphoblasts we used antisense and RNAi technology to downregulate these two genetic fusions. Our data lead us to hypothesize that in addition to lineage-specific regulatory differences in FPGS expression, molecular mechanisms associated with non-random translocations may alter FPGS mRNA expression and influence MTX sensitivity in Bp-ALL lymphoblasts.

Description: Between 1995 and 2004, two NIH-sponsored studies (STOP/STOP II) showed that children with sickle cell disease (SCD) and abnormal transcranial Doppler blood flow measurements (high stroke risk) are protected from stroke with regular blood transfusions. Iron overload, which may lead to complications and requires iron removal therapy, was monitored by serum ferritin (SF). Liver iron concentration (LIC) measurement was not mandated by protocol and was performed at investigator discretion. Biopsy dates and lab values were captured during STOP/STOP II, providing an opportunity to validate SF against LIC. 75 LICs on 36 patients (19 female, 17 male) at 8 centers were obtained. No liver biopsy complications were reported. LICs were correlated with STOP/STOP II core laboratory SF and alanine aminotransferase (ALT) obtained within 180 days of LICs. Median age at first biopsy was 11.1 years (range, 4.5–17.8), median time from start of transfusion was 36 months (range, 2–100). Iron removal treatment was initiated a median 23 months (range, 4–108) from start of transfusion, with deferoxamine (n=27), and/or exchange transfusion (n=9). 21 pts (58%) had multiple LIC measures: 2 (n=9), 3 (n=8), 4 (n=2), 5 (n=2). Last LICs on iron removal therapy were obtained a median 72 months (range, 35–124) from start of transfusion. Correlation between SFs and LICs were r=-0.06 (n=18) for first LICs obtained prior to iron removal therapy, r=0.50 (n=17) for last LICs obtained on iron removal therapy, and r=0.51 for all LICs (n=60). Pts with single/last LIC 〉=15 mg/gram dry liver were significantly more likely to have ALTs 〉=45 IU/L compared to those with LICs =15 mg/gram and ALT 〉=45 IU/L tended to have higher SFs then those with normal ALT (mean SF 4927 ng/ml, 95% CI 1739–8115 vs. mean SF 2255 ng/ml, 95% CI 1599–2912). 37% (7/19) of pts with LIC 〉=15 mg/gram had SFs

Description: Background: Approximately 3540 children are diagnosed with leukemia in the United States yearly (Bhatia and Robison, Oncology of Infancy and Childhood, 2008). Cooperative group trials have increased survival, particularly for acute lymphoblastic leukemia (ALL), but successful treatment of recurrent leukemia remains an unmet medical need. Resistance pathways and epigenetic alterations suggest a role for histone deacetylase (HDAC) inhibitors in children with leukemia (Burke and Bhatla, Frontiers in Pediatrics, 2014). Panobinostat is an orally administered pan-deacetylase inhibitor with activity against HDACs at concentrations in the nanomolar range (Atadja, Cancer Letters, 2009), and for which there is pre-clinical evidence of activity in pediatric leukemia (Stubbs, et al., ASH 2010). Panobinostat shows promise in a variety of adult hematologic malignancies (Khot et al., Expert Opinion on Investigational Drugs, 2013). We undertook a phase I trial of panobinostat in children with recurrent hematologic malignancies, and herein report the safety and pharmacokinetics (PK) from enrolled children with leukemia. Methods: T2009-012 is a first-in-child study coordinated by Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL). Children with relapsed or refractory leukemia between the ages of 1 and 21 years were enrolled to a multi-center, single agent trial of panobinostat dosed once per day three days per week for four successive weeks. Dose escalation was a standard 3+3 design with three dose levels planned. Subjects underwent lumbar puncture with prophylactic chemotherapy at treatment start and after a 28 day course. Blood was sampled pre-dose, at 0.5, 1, 6, 24 and 28-48 hours following the first dose. PK was obtained from blood on patients concurrently with optional specimens obtained from cerebrospinal fluid (CSF) on Day 29. Subjects who received fewer than 11 of the 12 planned doses and did not experience a dose limiting toxicity (DLT) were considered not evaluable for DLT, but were included in the summary of toxicities. Serial ECGs were monitored. Results: Seventeen subjects were enrolled with a diagnosis of acute leukemia, 10 with ALL and 7 with acute myelogenous leukemia (AML). Five were enrolled at dose level 1, 24 mg/m2/dose, 6 at dose level 2, 30 mg/m2/dose, and 6 at dose level 3, 34 mg/m2/dose. There have been no DLTs. Nine subjects are evaluable for DLT and 4 subjects were taken off study early due to increasing blast count. No subjects required removal from protocol therapy for QTc prolongation. One subject with infant ALL was removed early for progressive Aspergillus infection, 1 subject only received 10 doses owing to electrolyte abnormalities, and 2 subjects had nausea and vomiting after administration of 4 doses and did not continue. Grade 3/4 adverse events occurring in more than 20% of subjects included anemia in 82%, diarrhea in 24%, febrile neutropenia in 65%, hypokalemia in 41%, and hypophosphatemia in 24%. Concentration-time profiles were obtained from 9 subjects ages 16 months to 14 years in the 3 dose levels. Mean ± SE of PK for all subjects were Cmax 28.8 ± 6.1 ng/mL, Tmax 2.0 ± 0.8 hours, and T1/2 12.8 ± 3.0 hours. Two toddlers had the highest dose-normalized AUC0-inf and lowest oral clearance. Apparent oral clearance proportionally increased with increase in BSA. To date, 4 CSF specimens have been evaluated and found to have panobinostat below the lower limit of the quantification of 0.1 ng/mL, despite appreciable levels in the plasma. Two subjects on dose level two began a second cycle of therapy; one completed a second cycle for MLL rearranged leukemia and one discontinued study participation in the second cycle to undergo hematopoietic stem cell transplant for secondary AML after achieving a CRp in the first cycle. Conclusions: Panobinostat was tolerated these heavily pre-treated patients without unanticipated toxicities. PK in larger children and adults appears similar but PK in smaller children needs to be further explored. Penetration of panobinostat into the CSF was negligible. Two of 17 patients were able to receive a second cycle of therapy, but 4 had to be withdrawn early because of rapid increase in blast counts. Future trials will explore combination therapy in children with refractory hematologic malignancies, particularly those known to be driven by epigenetic mechanisms, in order to better control risk of rapid progression and improve efficacy through synergy. Disclosures Off Label Use: panobinostat for leukemia. Manley:Seattle Genetics, Inc.: Employment, Equity Ownership. Thomson:Epizyme, Inc: Employment.

Description: Chronic blood transfusion is increasingly indicated in patients with sickle cell disease. Measuring resulting iron overload remains a challenge. Children without viral hepatitis enrolled in 2 trials for stroke prevention were examined for iron overload (STOP and STOP2; n = 271). Most received desferrioxamine chelation. Serum ferritin (SF) changes appeared nonlinear compared with prechelation estimated transfusion iron load (TIL) or with liver iron concentrations (LICs). Averaged correlation coefficient between SF and TIL (patients/observations, 26 of 164) was r = 0.70; between SF and LIC (patients/observations, 33 of 47) was r = 0.55. In mixed models, SF was associated with LIC (P = .006), alanine transaminase (P = .025), and weight (P = .026). Most patients with SF between 750 and 1500 ng/mL had a TIL between 25 and 100 mg/kg (72.8% ± 5.9%; patients/observations, 24 of 50) or an LIC between 2.5 and 10 mg/g dry liver weight (75% ± 0%; patients/observations, 8 of 9). Most patients with SF of 3000 ng/mL or greater had a TIL of 100 mg/kg or greater (95.3% ± 6.7%; patients/observations, 7 of 16) or an LIC of 10 mg/g dry liver weight or greater (87.7% ± 4.3%; patients/observations, 11 of 18). Although SF changes are nonlinear, levels less than 1500 ng/mL indicated mostly acceptable iron overload; levels of 3000 ng/mL or greater were specific for significant iron overload and were associated with liver injury. However, to determine accurately iron overload in patients with intermediately elevated SF levels, other methods are required. These trials are registered at www.clinicaltrials.gov as #NCT00000592 and #NCT00006182.

Description: Folate cofactors are essential components of one carbon metabolism and are required for the biosynthesis of purines, pyrimidines, serine and methionine. The classical folate antagonist methotrexate (MTX) continues to be a universal component of most ALL treatment regimens. MTX is retained within cells as long-chain polyglutamates (MTX-PGs) after metabolism by the enzyme folylpoly-γ -glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR) and thymidylate synthetase (TS). The FPGS gene is regulated by the transcription factors NFY and Sp1. Using DNaseI assays we identified a hypersensitive site mapping closely upstream of exon 1, suggesting that chromatin remodeling may contribute to FPGS gene regulation. To investigate the role of histone modifications and chromatin remodeling on FPGS expression and uncover interactions between NFY, Sp1 and HDAC1, we performed co-immunoprecipitation and Western blotting. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assays (DAPA) revealed that HDAC1 is recruited by NFY and Sp1 to the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by NFY and Sp1 factors interacting with HDAC1, and leading to chromatin remodeling. We then examined the effect of the histone deacetylase inhibitors (HDACIs) sodium butyrate (NaBu) and suberoylanilide hydroxamic acid (SAHA) on the expression of FPGS and other folate-related genes in NALM6 (Bp-ALL), REH (TEL/AML1+, Bp-ALL), SupB15 (BCR/ABL+, Bp-ALL), and CCRF-CEM (T-ALL) cells using qRT-PCR. In all cell lines examined, treatment with HDACIs increased FPGS mRNA expression by 2- to 5-fold, whereas the level of DHFR and TS mRNA expression were decreased. On this basis, we hypothesized that induction of FPGS expression by HDACIs, results in higher accumulation of MTX-PG and enhanced MTX cytotoxicity in ALL cells. Further, the concomitant decrease in the expression of the MTX-PG target enzymes DHFR and TS, would enhance the cytotoxicity of the combination of HDACIs plus MTX in ALL cells. To test this hypothesis, NALM6, REH, and SupB15 cells were treated with MTX (4h) + SAHA (24h), and cell viability assessed. We determined that SAHA increased the intracellular accumulation of long chain MTX-PGs (n ≥3 Glu) in ALL cells, correlating with the upregulation of FPGS expression in SAHA-treated cells. Treatment with MTX + SAHA increased cytotoxicity by ~30% with a calculated combination index of ≤ 0.8 indicating synergy. Analysis of apoptosis using AnnexinV/PI staining revealed a 2 to 3-fold increase in apoptotic cell death in all cell lines treated with this combination. Our data suggest HDACIs enhance MTX cytotoxicity by upregulation of FPGS expression, increased accumulation of MTXPG and downregulation of DHFR and TS. The synergism exhibited by the combination of MTX and SAHA suggests it should be tested in ALL patients, in particular those who exhibit phenotypes with de novo or acquired resistance to MTX.

Description: Abstract 2439 Tumor metabolism has emerged as a hallmark of cancer by which the oncogenic profile of cancer cells pairs energy availability with cell growth and survival. BCR-ABL positive acute lymphoblastic leukemia (BCR-ABL+ ALL) is a highly resistant phenotype, and the BCR-ABL fusion protein has been correlated with alterations in glucose metabolism. The glucose analogue 2-deoxy-D-glucose (2DG) has been found to be an effective antitumor agent in both animal models and human clinical trials for solid tumors based on the dependency of hypoxic tumor cells on anaerobic glycolysis to generate ATP. 2DG inhibits two key glycolytic enzymes, hexokinase (HK) and phosphoglucose isomerase (PGI), and interfers with N-linked glycosylation by its incorporation in place of mannose into lipid-linked oligosaccharide (LLO) chains which leads to premature termination of LLO synthesis. Unlike hypoxic solid tumors, we recently demonstrated that 2DG induces cell death in ALL under normoxia preferentially by inhibiting N-linked glycosylation resulting in ER stress/UPR-mediated apoptosis, although inhibition of glycolysis also contributed to cell death in a phenotype specific-manner (Mol Cancer Res 10:969, 2012). Among ALL subtypes, BCR-ABL+ ALL t(9;22) cell lines SupB15 and TOM1 exhibited the highest sensitivity to 2-DG suggesting that BCR-ABL expression may be linked to sensitivity to 2DG. To investigate the role of BCR-ABL expression in this process, we constructed NALM6 (Bp-ALL) stable cell lines expressing BCR-ABL p190 fusion, and assessed their level of sensitivity to 2DG under normoxia. Expression of the BCR-ABL p190 fusion in NALM6 cells significantly increased 2DG-induced apoptosis compared to mock transfected NALM6 cells, indicating that BCR-ABL expression results in higher sensitivity to 2DG in this experimental system. Western blot analysis of BCR-ABL+ ALL (SUPB15 and TOM1) and Bp-ALL (NALM6) cell lines demonstrated that BCR-ABL expression induced alterations in the PI3K/Akt/p70S6K, GSK-3β/Mcl-1, p-STAT3 (Ser727), p-PKCδ (Thr505), and GRP78/p-eIF2α (Ser51) signaling pathways. Treatment with 2DG resulted in upregulation of p-AMPK (Thr172) and p-GSK-3β (Ser9), and downregulation of p-mTOR (Ser2448), p-p70S6K (Thr389), p-STAT3 (Ser727) and Mcl-1 in both BCR-ABL+ ALL (SUPB15 and TOM1) and Bp-ALL cells (NALM6). As we have previously described, 2DG induces ER stress and the expression of the unfolded protein response (UPR) markers (Mol Cancer Res 10:969, 2012). In BCR-ABL+ ALL cells, 2DG led to upregulation of IRE1α, GRP78 and CHOP (a marker of UPR induced cell death) and resulted in apoptotic cell death (〉60%), which correlated with lower level of Mcl-1 expression. To further investigate the role of Mcl-1 on 2DG's sensitivity we used pharmacological inhibitors and shRNA. We found that Mcl-1 down-regulation sensitized Bp-ALL cells to 2DG (36% ± 4% cell death; p

Description: Abstract 3254 Cancer cells exhibit an increased dependency on the glycolytic pathway as the predominant energy source over mitochondrial oxidative phosphorylation (OXPHOS). This unique alteration in glucose metabolism gives malignant cells significant proliferative advantages by enabling cancer cells to better adapt to the hypoxic microenvironment. The glucose analogue 2-Deoxy-D-Glucose (2-DG) has been found to be an effective antitumor agent in both animal models and human clinical trials. In childhood acute lymphoblastic leukemia (ALL), elevated glycolytic rates have been identified and increased glucose consumption has been postulated to be responsible for chemotherapy resistance. We have previously reported that B-precursor ALL (Bp-ALL) cells exhibit significant sensitivity to 2-DG under normoxia, and that PTEN mutant T-ALL CCRF-CEM cells are less sensitive to 2-DG compared to Bp-ALL cells. In this study, we further investigated signaling alterations in critical metabolic pathways linked to cell survival and proliferation and the mechanisms of apoptotic cell death following inhibition of glycolysis in ALL. Warburg suggested that the deficiency of mitochondrial respiration plays an important role in the metabolic shift seen in cancer cells. We assessed mitochondrial function in selective Bp- and T-ALL cell models including CCRF-CEM, NALM6, REH (RUNX/ETV1 + Bp-ALL), SupB15 and TOM1 (both BCR/ABL + Bp-ALL). The K562 cell line (CML) and an EBV-immortalized non-malignant lymphocyte cell line HCC1187BL were tested as controls. Our data indicate that oxygen consumption rates, and steady-state levels of representative protein markers of OXPHOS were decreased in the ALL cell lines, which correlated with a decrease in mtDNA levels when compared to CML and EBV immortalized lymphocyte controls. We previously demonstrated that 2-DG also interferes with protein synthesis and processing in ALL cells by inhibiting N-linked glycosylation. On this basis, we investigated the induction of prolonged ER stress leading to an unfolded protein response (UPR) in ALL cells following treatment with 2-DG. The expression of UPR specific markers Grp78/BiP and Grp94 were probed in CCRF-CEM, NALM6, and SupB15 cells using specific antibodies by Western immunoblotting. In the presence of 4mM 2-DG, significant increase in the expression of both Grp78/BiP and Grp94 was detected in all ALL cell lines tested. Western immunoblotting also identified increased expression of CHOP/GADD153 and cleavage of Poly (ADP-ribose) polymerase (PARP) following 2-DG treatment, indicating that 2-DG leads to UPR-induced apoptotic cell death in ALL cells. Among ALL cells studied, the PTEN mutant CCRF-CEM cells exhibit constitutive activation of Akt and resistance to 2-DG. We have reported that simultaneous inhibition of glycolysis and Akt signaling results in greater induction of cell death. In this study we further investigated signaling changes within these pathways to determine the mechanism of synergistic cell death following combination treatment. Western immunoblotting demonstrated that the combination of 2-DG and the Akt inhibitor × (AIX) led to almost complete abrogation P-Akt expression at both Ser473 and Thr308, and significant down-regulation of P-mTOR signaling as compared to treatment with each drug alone. Most important, the combination treatment led to a significant decrease in the expression of Hexokinase and GLUT1 in CCRF-CEM cells, and “sensitized” these cells to apoptotic death by 2-DG as demonstrated by significant cleavage of PARP.These data demonstrate for the first time that ALL cells are unable to effectively utilize mitochondrial OXPHOS for ATP generation, providing an explanation for their sensitivity to 2-DG under normoxic conditions. We also demonstrate that the cytotoxicity of 2-DG in ALL cells is due to concomitant inhibition of N-linked glycosylation leading to ER stress and UPR-induced apoptosis. Finally, our data indicate that hyper-activation of Akt signaling is responsible for the relative resistance of PTEN mutant CCRF-CEM cells to inhibition of glycolysis, and that simultaneous inhibition of Akt signaling is capable of overcoming this relative resistance to 2-DG. We propose that glycolytic inhibitors alone or in combination with selected targeted agents aimed at key metabolic and oncogenic pathways show promise as novel strategies for ALL therapy. Disclosures: No relevant conflicts of interest to declare.