Search

Table 4 Excel Document

Supplemental Figures 1-8, Supplemental Tables 1-3

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)–grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

Legends for Supplementary Figures 1-8 and Supplementary Tables 1-5

Table 5 Excel Document

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-d-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with d-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG–treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG–treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival. Mol Cancer Res; 10(7); 969–78. ©2012 AACR.

PDF file - 114K

PDF file - 707K, Salubrinal does not protect ALL from apoptosis induced by the inhibitors of N-linked glycosylation

PDF file - 240K, Effects of D-mannose on ALL cytotoxicity induced by equimolar concentrations of 2-DG and 2-FDG

PDF file - 1.3MB, Dose-response of 2-DG and AIX in CCRF-CEM

PDF file - 415K, Inhibition of mTOR with CCI-779 does not protect ALL cells from 2-DG-cytotoxicity

PDF file - 385K, The AMP analogue AICAr does not significantly affect 2-DG-induced cytotoxicity in ALL cell lines

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)–grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

Context Simulated clinical immersion (SCI), in which clinical situations are simulated in a realistic environment, safely and gradually exposes novices to complex problems. Given their limited experience, undergraduate students can potentially be quite overwhelmed by SCI learning tasks, which may result in misleading learning outcomes. Although task complexity should be adapted to the learner's level of expertise, many factors, both intrinsic and extraneous to the learning task, can influence perceived task complexity and its impact on cognitive processes. Objectives The purpose of this mixed-methods study was to understand the effects of task complexity on undergraduate pharmacy students' cognitive load, task performance and perception of learning in SCI. Methods A total of 167 second-year pharmacy students were randomly assigned to undertake one simple and one complex learning task in SCI consecutively. Participants' cognitive load was measured after each task and debriefing. Task performance and time on task were also assessed. As part of a sequential explanatory design, semi-structured interviews were conducted with students showing maximal variations in intrinsic cognitive load to elucidate their perceptions of learning when dealing with complexity. Results Although the complex task generated significantly higher cognitive load and time on task than the simple task, performance was high for both tasks. Qualitative results revealed that a lack of clinical experience, an unfamiliar resource in the environment and the constraints inherent to SCI, such as time limitations, hindered the clinical reasoning process and led to poorer self-evaluation of performance. Simple tasks helped students gain more self-confidence, whereas complex tasks further encouraged reflective practice during debriefings. Conclusions Although complex tasks in SCI were more cognitively demanding and took longer to execute, students indicated that they learned more from them than they did from simple tasks. Complex tasks constitute an additional challenge in terms of clinical reasoning and thus provide a more valuable learning experience from the student's perspective.

De novo and acquired drug resistance and subsequent relapse remain major challenges in acute lymphoblastic leukemia (ALL). We previously identified that pevonedistat (TAK-924, MLN4924), a first-in-class inhibitor of NEDD8 activating enzyme (NAE), elicits ER stress and has potent in vitro and in vivo efficacy against ALL. However, in pevonedistat-treated ALL cell lines, we found consistent activation of the pro-survival MEK/ERK pathway, which has been associated with relapse and poor outcome in ALL. We uncovered that inhibition of the MEK/ERK pathway in vitro and in vivo sensitized ALL cells to pevonedistat. The observed synergistic apoptotic effect appears to be mediated by inhibition of the MEK/ERK pro-survival cascade leading to de-repression of the pro-apoptotic BIM protein. Mechanistically, Ca2+ influx via the Ca2+-release-activated Ca2+ (CRAC) channel induced protein kinase C β2 (PKC-β2) was responsible for activation of the MEK/ERK pathway in pevonedistat-treated ALL cells. Sequestration of Ca2+ using BAPTA-AM or blockage of store-operated Ca2+ entry (SOCE) using BTP-2 both attenuated the compensatory activation of MEK/ERK signaling in pevonedistat-treated ALL cells. Pevonedistat significantly altered the expression of Orai1 and stromal interaction molecule 1 (STIM1), resulting in significantly decreased STIM1 protein levels relative to Orai1. Further, we identified eIF2α as an important post-transcriptional regulator of STIM1, suggesting that pevonedistat-induced eIF2α de-phosphorylation selectively down-regulates translation of STIM1 mRNA. Consequently, our data suggest that pevonedistat potentially activates SOCE and promotes Ca2+ influx leading to activation of the MEK/ERK pathway by altering the stoichiometric Orai1:STIM1 ratio and inducing ER stress in ALL cells.

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.

Recent evidence using GT1-7 cells indicates that GnRH pulsatility depends on exocytotic-release and gene transcription events. To determine whether calcium or DREAM may play a role in linking these processes, we used an L-type Ca(2+)-blocker (nimodipine) and found that not only GnRH gene expression (GnRH-GE) pulse activity was abolished but also that binding of proteins to OCT1BS-a (essential site for GnRH-GE pulses) was reduced. We further found that only EF-hand forms of DREAM were expressed in GT1-7 and that DREAM was part of the complex binding to OCT1BS-a. Finally, microinjection of DREAM antibody into cells abolished GnRH-GE pulses demonstrating its importance in pulsatility. These results reveal that calcium and DREAM may bridge cytoplasmic and nuclear events enabling temporal coordination of intermittent activity. Expression of DREAM in various cell types coupled with the universal role of calcium raise the possibility that these factors may play similar role in other secretory cells.

BCR-ABL positive (+) acute lymphoblastic leukemia (ALL) accounts for ∼30% of cases of ALL. We recently demonstrated that 2-deoxy-d-glucose (2-DG), a dual energy (glycolysis inhibition) and ER-stress (N-linked-glycosylation inhibition) inducer, leads to cell death in ALL via ER-stress/UPR-mediated apoptosis. Among ALL subtypes, BCR-ABL+ ALL cells exhibited the highest sensitivity to 2-DG suggesting BCR-ABL expression may be linked to this increased vulnerability. To confirm the role of BCR-ABL, we constructed a NALM6/BCR-ABL stable cell line and found significant increase in 2-DG-induced apoptosis compared to control. We found that Mcl-1 was downregulated by agents inducing ER-stress and Mcl-1 levels correlated with ALL sensitivity. In addition, we showed that Mcl-1 expression is positively regulated by the MEK/ERK pathway, dependent on BCR-ABL, and further downregulated by combining ER-stressors with TKIs. We determined that energy/ER stressors led to translational repression of Mcl-1 via the AMPK/mTOR and UPR/PERK/eIF2α pathways. Taken together, our data indicate that BCR-ABL+ ALL exhibits heightened sensitivity to induction of energy and ER-stress through inhibition of the MEK/ERK pathway, and translational repression of Mcl-1 expression via AMPK/mTOR and UPR/PERK/eIF2α pathways. This study supports further consideration of strategies combining energy/ER-stress inducers with BCR-ABL TKIs for future clinical translation in BCR-ABL+ ALL patients.

Recent reports demonstrate that the rat GnRH promoter is activated in an episodic fashion in immortalized GnRH neurons, but little information is available on molecular processes that contribute to this phenomenon. In this study, we dissected the regions of the rat GnRH promoter that mediate these effects by testing a series of 5' deletion luciferase reporter constructs on the pattern of photonic emissions from single, living GT1-7 GnRH neuronal cells. Deletion analysis revealed that the region -2012/-1597 that contains the neuron-specific enhancer (NSE) was required for the elaboration of pulses of GnRH promoter activity. The importance of this region was supported by observations that episodic reporter activity could be transferred to a neutral nonpulsatile promoter (Rous sarcoma virus, RSV(180)). Immunoneutralization of Oct-1 as well as mutation of an octamer binding site located at -1787/-1783 (AT-a site) blocked the pulsatile GnRH promoter activity in GT1-7 neuronal cells. Taken together, our findings indicate that episodic GnRH gene expression is a promoter-dependent phenomenon, which is mediated by Oct-1 interaction with regulatory elements in the NSE region.

A comparative study was made on the physicochemical characteristics of two isozymes of methylcobamide:- coenzyme M methyltransferase (MT2). Both isozymes catalyzed S-methylation of 2-thioethanesulfonate (coenzyme M) and exhibited similar apparent Km values for coenzyme M of 35 microM (MT2-A) and 20 microM (MT2-M). Weak binding to methylcobalamin was indicated by the apparent Km of 14 mM for both isozymes. Cob(I)alamin was established as the major product of the reaction, demonstrating heterolytic cleavage of the methylcobamide carbon-cobalt bond. The isozymes were shown to be zinc-containing metalloproteins. Metal ion chelators strongly inhibited both isozymes. A variety of coenzyme M analogs were tested for activity and/or inhibition. One alternative substrate 3-mercaptopropionate was discovered, with apparent Km 9 mM (MT2-A) and 10 mM (MT2-M). The results suggested an active site geometry in which coenzyme M is bound both by S-coordination to zinc, and electrostatic interaction of the sulfonate with a cationic group on the enzyme. Methanosarcina barkeri genes cmtA and cmtM encoding both isozymes were cloned and sequenced. Both genes encoded proteins with 339 amino acids and predicted molecular masses of 36-37 kDa. Active forms of both isozymes were expressed in Escherichia coli. A conserved segment with the potential for metal binding was found. The possibility of zinc involvement in catalysis of coenzyme M methylation is considered.

— Meiotic gynogens were produced using hybrid striped bass (♀ white bass, Morone chrysops, ×♂ striped bass, M. saxatilis) eggs and white perch M. americana UV-irradiated sperm. Diploidy of the fertilized eggs was restored by application of hydrostatic pressure, which induced retention of the second polar body. Use of white perch sperm provided an unmistakable marker for detection of a paternal genetic contribution. Two assays were developed using the polymerase chain reaction (PCR) to amplify specific regions of the Morone genome. Primers for gene amplification were developed based on the DNA sequence of the striped bass growth hormone gene (SB-GH) or an anonymous striped bass locus (SBI-10). Control experiments using DNA from the three Morone species demonstrated that gene amplification yielded species-specific patterns of DNA fragments for both of these loci. Therefore, any progeny with a paternal contribution of a set of white perch chromosomes could be identified. Using these assays, we demonstrated that greater than 75% of the progeny obtained from successful experiments were true gynogens. These striped bass hybrid gynogens will provide the basis for future efforts to calculate gene-centromere distances and to identify markers linked to specific traits of interest to aquaculture.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children and the relapse rate in adult ALL patients is about 50%, highlighting the need for new therapeutic strategies. Data from our laboratory and others showed that ALL cells are sensitive to drugs that induce endoplasmic reticulum (ER) stress/unfolded protein response (UPR). In search for novel strategies to target the ER stress/UPR in ALL, we tested the efficacy of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924, pevo) in ALL. We found ALL cells exhibited significant in vitro and in vivo sensitivity to pevo-induced ER stress/UPR. Specifically, proteotoxic/ER stress was observed in pevo-treated ALL cells secondary to their inability to halt protein translation following pevo-induced activation of the mTOR pathway and concomitant de-phosphorylation of p-eIF2α (S51). In addition, aberrant activation of MEK/ERK has been correlated with resistance/relapse in pediatric ALL (Blood 2014; 124: 3420-3430). In our Bp- and T-ALL cell line models, we found consistent induction of p-ERK1/2 (T202/Y204) following pevo treatment, suggesting phosphorylation of ERK1/2 as a compensatory survival mechanism in response to pevo's cytotoxicity. Supporting this hypothesis, we observed significant in vitro synergy between the MEK inhibitor selumetinib (SEL) and pevo (CI = 0.017). On this basis, we tested the in vivo efficacy of pevo + SEL in NSG mice injected with NALM6 cells expressing the luciferase gene (NALM6/LUC). Engrafted NSG mice were treated with pevo (s.c., 66 mg/kg) and SEL (p.o., 50 mg/kg) twice daily on weekdays and once per day on weekends. Bioluminescence analysis of animals 21 days post ALL injection revealed significant reduction of tumor burden in mice treated with pevo alone or pevo + SEL (p Citation Format: Shuhua Zheng, Gilles M. Leclerc, Guy J. Leclerc, Joanna DeSalvo, Ronan T. Swords, Julio C. Barredo. Rebalancing of Bcl-2 family proteins mediate the vulnerability of pevonedistat-treated acute lymphoblastic leukemia cells towards MEK/ERK pathway inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4547.

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-d-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with d-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG–treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG–treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival. Mol Cancer Res; 10(7); 969–78. ©2012 AACR.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children. Most adults will die from the disease. Hence, current therapy in ALL is inadequate. We previously showed that ALL cells were differentially sensitive to agents inducing endoplasmic reticulum (ER) stress (Mol Cancer Res 2012; 10:969-978; PLoS One 2013; 8:e74420). In this regard, we investigated the anti-leukemic activity of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924). Pevonedistat prevents neddylation of cullin-RING E3 ligases, leading to accumulation of cullin dependent substrates with anti-proliferative effects. We demonstrated that pevonedistat induced dose-dependent cell growth inhibition (IC50 between 159-300 nM) and cell death (EC50 between 317-463 nM) in both T- and B-ALL cells (cell lines and primary patient material). We found that neddylated cullin levels were significantly decreased confirming inhibition of the NEDD8 conjugation pathway by pevonedistat. Pevonedistat induced ER stress and unfolded protein response (UPR) mediated cell death in ALL cell lines and primary cells as evidenced by increased expression of UPR markers (GRP78 and CHOP), and cleaved-PARP. Mechanistically, pevonedistat caused proteotoxic/ER stress from failure to halt protein translation mediated by p-eIF2α (Ser51) de-phosphorylation via up-regulation of the PERK inhibitor p58IPK. We also observed up-regulation of mTOR/p70S6K, further increasing protein synthesis and augmenting proteotoxic/ER stress. Indeed, we demonstrated that pevonedistat induced nascent protein synthesis, and that co-treatment with protein synthesis inhibitors (rapamycin, cycloheximide) rescued ALL cells from pevonedistat induced cytotoxicity (p < 0.01 for combination treatment vs. pevonedistat alone). The observed rescue correlated with decreased UPR markers and P-p70S6K (Thr389) expression, suggesting that proteotoxic stress is a central determinant in pevonedistat-induced ALL cell death. Additional studies into the mechanism of pevonedistat-induced apoptosis revealed that homeostasis of pro- and anti-apoptotic proteins was rebalanced in favor of cell death through decreased Mcl-1 pro-survival activity, via sequestration by NOXA and BIM. Co-immunoprecipitation experiments showed that pevonedistat increased the interaction between NOXA and Mcl-1, and BIM and Mcl-1 proteins, resulting in the reduction of Mcl-1 pro-survival activity. siRNA-mediated down-regulation of NOXA and BIM expression decreased the sensitivity of ALL cells to pevonedistat, supporting the sequestration by NOXA and BIM of Mcl-1's pro-survival activity. Activation of the MEK/ERK/Mcl-1 pathway following pevonedistat-induced cell death was also noted, possibly as a compensatory mechanism. We found that pevonedistat plus a MEK inhibitor (PD98059, selumetinib) induced significant cell death compared to each drug alone (p < 0.0001), which was associated with decreased Mcl-1 expression and increased cleaved-PARP cleavage, supporting a critical role of Mcl-1 in ALL cell survival. In addition, we demonstrated synergy between pevonedistat and effective anti-leukemic agents such as dexamethasone, doxorubicin and cytarabine in ALL cell line models, with CI values of 0.17, 0.46, and 0.23, respectively. More importantly, we showed that NSG mice engrafted with human ALL cells had statistically significant increased survival when treated with pevonedistat (pevo) plus dexamethasone (dex) compared to single agent therapy (p = 0.0076 for pevo+dex vs. dex alone; p = 0.0182 pevo+dex vs. pevo alone), lending support for the use of pevonedistat as part of a multi-agent approach. Taken together, our data demonstrate that the NAE inhibitor pevonedistat alters cellular translational machinery leading to ER stress/UPR-mediated cell death, and suggest that pevonedistat may have a "priming" effect on ALL cells by altering the apoptotic threshold through modulation of Mcl-1's pro-survival activity. Disclosures No relevant conflicts of interest to declare.

AICAr is a cell-permeable nucleotide that has been used in vivo and in vitro to activate AMPK. Our previous findings have shown that AICAr as a single agent induces dose- and time-dependent growth inhibition in acute lymphoblastic leukemia (ALL) cell lines. In addition, the combination of AICAr with antifolates [methotrexate (MTX) or pemetrexed] has been shown to further potentiate AMPK activation and to lead to greater cytotoxicity and growth inhibition in leukemia and other malignant cell types. Our data presented herein show that sustained endoplasmic reticulum (ER) stress is the predominant mechanism behind the synergistic induction of cell death by the combination of AICAr plus the inhibitor of one-carbon metabolism, MTX, in Bp- and T-ALL, as evidenced by induction of several unfolded protein response markers leading to apoptosis. We also show for the first time that AICAr in combination with MTX significantly induces Akt phosphorylation in ALL. Under these conditions, the concomitant inhibition of Akt, a cellular antagonist of AMPK, leads to further upregulation of AMPK activity and alleviates AICAr plus MTX-induced ER stress and apoptosis. Therefore, we also show that the concomitant activation of AMPK actually rescues the cells from AICAr plus MTX-induced ER stress and apoptosis. Our data suggest that the effects of AMPK activation on cell death or survival differ contextually depending on its signaling alterations with related oncogenic pathways and provide insight into the reported paradoxical proapoptotic versus prosurvival effects of AMPK activation. Mol Cancer Ther; 10(3); 437–47. ©2011 AACR.

Background: Children with Acute Lymphoblastic Leukemia (ALL) diagnosed with resistant phenotypes and those who relapse have a dismal prognosis for cure. In search for novel treatment strategies, we identified the AMP activated protein kinase (AMPK) as a potential drug target based on its effects on cell growth and survival. We have shown previously that AICAR-induced AMPK activation also induced a compensatory survival mechanism via PI3K/Akt signaling.Results: In the present study, we further investigated the downstream signaling induced by AMPK activation in ALL cells. We found that AICAR-induced AMPK activation resulted in up-regulation of P-Akt (Ser473 and Thr308) and decrease of P-mTOR (Ser2448) expression and downstream signaling. We determined that activation of P-Akt (Thr308) was mediated by AMPK-induced IGF-1R activation via phosphorylation of the insulin receptor substrate-1 (IRS-1) at Ser794. Inhibition of IGF-1R signaling using the tyrosine kinase inhibitor HNMPA(AM)3 resulted in significant decrease in P-IRS-1 (Ser794) and P-Akt (Thr308). Co-treatment of AICAR plus HNMPA(AM)3 prevented AMPK-induced up-regulation of P-Akt (Thr308) but did not alter the activation of P-Akt (Ser473). Inhibition of AMPK using compound-C resulted in decreased P-Akt expression at both residues, suggesting a central role for AMPK in Akt activation. In addition, inhibition of IGF-1R signaling in ALL cells resulted in cell growth arrest and apoptosis. Additional Western blots revealed that P-IGF-1R (Tyr1131) and P-IRS-1 (Ser794) levels were higher in NALM6 (Bp- ALL) than CEM (T-ALL), and found differences in IGF-1R signaling within Bp-ALL cell line models NALM6, REH (TELAML1, [t(12;21)]), and SupB15 (BCR-ABL, [t(9;22)]). In these models, higher sensitivity to IGF-1R inhibitors correlated with increased levels of IGF-1R expression. Combined therapy simultaneously targeting IGF-1R, AMPK, Akt, and mTOR pathways resulted in synergistic growth inhibition and cell death.Conclusions: Our study demonstrates that AMPK activates Akt through IGF-1R dependent and independent mechanisms. Co-targeting IGF-1R and related downstream metabolic and oncogenic signaling pathways represent a potential strategy for future translation into novel ALL therapies.

GnRH is released from hypothalamic neurons in coordinated pulses, but the cellular basis for this process is poorly understood. Previously, we found that secretory pulses from GT1-7 cells became synchronized with time in culture. Using this culture model, we investigated whether the gap junction proteins connexin43 (Cx43) and/or connexin26 (Cx26) are involved in this synchronization. Our results reveal that cytoplasmic densities immunoreactive for Cx43, and mRNA or protein for Cx43 increase with time in culture. Also, microinjection of day-3 cultures with siRNA for Cx43 abolished synchronized activity at day 7. Interestingly, cytoplasmic plaques, mRNA, or protein for Cx26 remained stable with culture time and Cx26 siRNA administration did not alter secretory activity. Our findings demonstrate that Cx43, but not Cx26 is necessary for synchronized secretory activity in these GT1-7 cultures and raise the possibility that Cx43-related gap junctions may be important in GnRH neuronal coordination in the hypothalamus.

Tumor metabolism has emerged as a hallmark of cancer by which the oncogenic profile of cancer cells pairs energy availability with growth and survival. BCR-ABL+ acute lymphoblastic leukemia (BCR-ABL+ ALL) accounts for 25% of ALL and the BCR-ABL fusion protein has been correlated with alterations in glucose metabolism. The glucose analogue 2-deoxy-D-glucose (2DG), induces simultaneous energy and ER-stress by inhibiting glycolysis (energy stress) via blocking HK and PGI enzymes, and interfering with N-linked glycosylation through its incorporation in place of mannose into the lipid-linked oligosaccharide (LLO) chain leading to premature termination of LLO synthesis (ER stress). We recently demonstrated that inhibition of both aerobic glycolysis and N-linked glycosylation by 2DG led to apoptosis in ALL (Mol Cancer Res 10:969, 2012). Among ALL subtypes, BCR-ABL+ ALL cell lines SupB15 and TOM1 exhibited the highest sensitivity to 2DG suggesting that in ALL cells BCR-ABL expression may be linked to heightened sensitivity to energy and/or ER stress. To test this premise, we constructed NALM6 (Bp-ALL) stable cell lines expressing the BCR-ABL p190 fusion and found that expression of the BCR-ABL p190 fusion in NALM6 cells significantly increased 2DG-induced apoptosis compared to mock transfected controls. To investigate the contribution of each form of stress (energy and ER stress) in the increased sensitivity of BCR-ABL+ cells to 2DG, we examined the effects of agents known to preferentially induce energy stress (lowering ATP) such as 2-fluoro-2-deoxy-D-glucose and oxamate, and ER stress such as tunicamycin. We found no differences in cell death or ATP levels between BCR-ABL+ and control ALL cells treated with agents solely inducing energy or ER stress, indicating the increased susceptibility of BCR-ABL+ ALL cells to 2DG results from the simultaneous induction of both types of stress. Consistent with these data, Fluorophore-Assisted Carbohydrate Electrophoresis and immunoblotting demonstrated that 2DG induced equivalent inhibition of N-linked glycosylation and upregulation of the UPR signaling in BCR-ABL+ and control ALL cells. Similar findings were seen in primary ALL patient samples. Consequently, simultaneous energy and ER stress are required for the heightened vulnerability to these forms of stress conferred by BCR-ABL in ALL. Based on the known function of Mcl-1 promoting survival in BCR-ABL+ ALL, we investigated its role in the mechanism of cell death induced by energy and/or ER stress agents. We uncovered that BCR-ABL+ ALL cell lines exhibited the lowest levels of Mcl-1 expression compared to control BCR-ABL- ALL and CML cells, and this basal level of Mcl-1 expression directly correlated with their resistance to energy and/or ER stressing agents. Further, we found that Mcl-1 knock-down by shRNA sensitized ALL and CML cells to the dual induction of energy and ER stress by 2DG. We also found that Mcl-1 expression was further downregulated in BCR-ABL+ and - ALL cells treated with energy and/or ER stressors which correlated with the level of apoptosis. Using qRT-PCR or co-treatment with the pancaspase inhibitor qVD-OPH or the proteasome inhibitor bortezomib, we showed that Mcl-1 downregulation by energy and/or ER stressors is neither due to transcriptional, caspase dependent cleavage, or post-translational regulation mechanisms but likely due to translational control of the Mcl-1 protein via AMPK/mTOR and the UPR PERK/p-eIF2α. Finally, because TKIs lead to decrease ATP (energy stress) in BCR-ABL+ cells, we co-treated BCR-ABL+ ALL cells with TKIs plus ER stressors and found that the simultaneous induction of these forms of stress by these combinations significantly increased cell death compared to single drug. Further, we found that TKIs + ER stressors further downregulated Mcl-1 and GRP78 expression compared to single agent treatment. Taken together, our data demonstrate that simultaneous induction of energy and ER stress leads to significant induction of apoptosis in BCR-ABL+ ALL cells, and these heightened vulnerability to these forms of stress is likely mediated by downregulation of Mcl-1 expression via AMPK activation/mTOR inhibition and PERK/p-eIF2α. Our study demonstrates that strategies with agents that induce energy and/or ER stress plus BCR-ABL TKIs warrant further consideration as a potential approach for future clinical translation into trials for BCR-ABL+ ALL patients. Disclosures No relevant conflicts of interest to declare.

Recent findings from our laboratory and those of others demonstrated that prolactin gene expression (PRL-GE) oscillates in single living mammotropes, but little information is available on the molecular processes that contribute to this phenomenon. To elucidate the source of this activity, we generated a series of constructs containing decreasing lengths of the PRL promoter fused to a luciferase reporter gene. These constructs were injected into single cells and assayed for photonic activity. We found pulse activity with all plasmids tested, even with the smallest promoter fragment of 331 bp. Sequence analysis of this fragment identified two potential E-boxes (elements known to bind CLOCK and BMAL1 circadian proteins). Furthermore, RT-PCR of PRL cells (pituitary, MMQ, and GH3) revealed expression of clock and bmal1 as well as five other clock genes (per1, per2, cry1, cry2, and tim), suggesting that the circadian system may function in PRL cells. Next, we mutated the core sequences of both E-boxes within the 2.5-kb PRL promoter and found that only mutation of the E-box133 completely abolished PRL-GE pulses. EMSAs revealed that CLOCK and BMAL1 were able to bind to the E-box133 site in vitro. Our results demonstrate that PRL-GE pulses are dependent on a specific E-box binding site in the PRL promoter. Moreover, the indication that CLOCK/BMAL1 can bind to this site suggests that these circadian proteins, either alone or in conjunction with other factors, may regulate intermittent PRL promoter activity in mammotropes, perhaps by acting as a temporal switch for the on/off expression of PRL.

PRL gene expression in the anterior pituitary has been the focus of intensive investigation for many years, but very little information is available on the actual dynamics by which this process occurs in individual mammotrope cells. Here, we used single cell bioluminescent imaging microscopy and a recently refined reporter gene strategy to measure PRL promoterdriven gene expression (PRL-GE) in individual living primary mammotropes. Using this approach we report a new phenomenon involving repetitive on/off gene expression bursts that occurred in a distinctly noncircadian oscillatory pattern. Furthermore, we demonstrate a functional basis for these gene expression oscillations, inasmuch as PRL-GE pulses were sensitive to calcium-dependent modulation, which we show arose exclusively as changes in the shape of individual pulse episodes. Our results provide the first clear evidence that PRLGE, in its homologous cell environment, displays oscillatory bursts of activity. Moreover, they strongly support the idea that these discrete on/off bursts of activity serve as an important determinant of the timing and level of PRL-GE under both basal and stimulated conditions. (Endocrinology 143: 1126 –1133, 2002)

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, and cure rates for patients with resistant/relapsed ALL remain poor. In addition, many childhood cancer survivors experience significant long-term therapy related toxicity, highlighting the need for novel targeted anti-leukemic agents. We recently uncovered that ALL cells are vulnerable to undergo apoptosis via endoplasmic reticulum (ER) stress/unfolded protein response (UPR)-mediated mechanisms (Mol Cancer Res 2012;10:969-978). We reported that the AMP activated protein kinase (AMPK) is activated in response to ER stressors (metformin, 2-DG, tunicamycin), and its activation suppressed the UPR preventing ALL cells from processing misfolded/unfolded proteins in the ER lumen, leading to apoptosis. In search for novel agents targeting AMPK, we investigated the anti-leukemic activity of a new AMP analogue, MLN4924, in Bp- and T-ALL cell line models. This agent targets the ubiquitin-like protein NEDD8 conjugation pathway leading to inhibition of cullin-based E3 ligase dependent proteasomal degradation of proteins essential for cancer cell growth and survival. We demonstrated that MLN4924 induced dose-dependent cell growth inhibition and apoptosis in both Bp-ALL (EC50 = 372 nM, p Disclosures: No relevant conflicts of interest to declare.

The outcome of patients with resistant phenotypes of acute lymphoblastic leukemia (ALL) or those who relapse remains poor. We investigated the mechanism of cell death induced by metformin in Bp- and T-ALL cell models and primary cells, and show that metformin effectively induces apoptosis in ALL cells. Metformin activated AMPK, down-regulated the unfolded protein response (UPR) demonstrated by significant decrease in the main UPR regulator GRP78, and led to UPR-mediated cell death via up-regulation of the ER stress/UPR cell death mediators IRE1α and CHOP. Using shRNA, we demonstrate that metformin-induced apoptosis is AMPK-dependent since AMPK knock-down rescued ALL cells, which correlated with down-regulation of IRE1α and CHOP and restoration of the UPR/GRP78 function. Additionally rapamycin, a known inhibitor of mTOR-dependent protein synthesis, rescued cells from metformin-induced apoptosis and down-regulated CHOP expression. Finally, metformin induced PIM-2 kinase activity and co-treatment of ALL cells with a PIM-1/2 kinase inhibitor plus metformin synergistically increased cell death, suggesting a buffering role for PIM-2 in metformin's cytotoxicity. Similar synergism was seen with agents targeting Akt in combination with metformin, supporting our original postulate that AMPK and Akt exert opposite regulatory roles on UPR activity in ALL. Taken together, our data indicate that metformin induces ALL cell death by triggering ER and proteotoxic stress and simultaneously down-regulating the physiologic UPR response responsible for effectively buffering proteotoxic stress. Our findings provide evidence for a role of metformin in ALL therapy and support strategies targeting synthetic lethal interactions with Akt and PIM kinases as suitable for future consideration for clinical translation in ALL.

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 Disclosures: No relevant conflicts of interest to declare.

In Canada, the Faculty of Pharmacy of the Universite de Montreal was the first to develop and implement an entry-level doctor of pharmacy (PharmD) degree in 2007. With this initiative, the curriculum was entirely redesigned and various pedagogical approaches were implemented.1,2 A custom learning management system based on the concept of the learning object (http://www.UGOpharm.umontreal.ca) was developed and implemented and most of the academic program was posted on a secure-access Web site. Incoming first-year pharmacy students were expected to purchase a preconfigured laptop for personal and academic use. In this letter, we report on the successes and pitfalls we experienced using 3 different technology initiatives in the 2011 semester of a pharmacy law course.3 We have been using WebCT (Blackboard, Inc) since 19994,5 as the platform offered additional tools for interaction with students including educational and certification tests. In 2011, the Universite de Montreal decided to replace WebCT with Moodle (Moodle Pty Ltd, Perth, Australia; called locally StudiUM6) as its new open source Web platform for online courses. Moodle is used on more than 56,400 Web sites in at least 214 countries for more than 4,784,691 courses taken by more than 45 million students and more than 1.1 million teachers, but there is relatively little data about its use in pharmacy curricula. A search on moodle.org with “pharmacy” and “pharmacie” terms results in only 123 hits.7 Moodle was used to host the law course’s pedagogical material using a 9-step approach: (1) learning Moodle to define its benefits, disadvantages, and host of functions; (2) reviewing the LSS print seventh edition; (3) developing a framework to write multiple-choice and short development educational and summary questions, including the random generation of questions from a bank of questions; (4) developing standard content for the Web platform; (5) developing new pedagogical tools; (6) drafting questions and initiating the platform; (7) conducting platform tests; (8) logging management problems and debugging in conjunction with the Moodle support team; and (9) drafting policies and procedures. The implementation phase took place from May through August 2011, and the platform went online September 1, 2011. Moodle is a tool-rich user-friendly platform (eg, newswire for news, glossaries, a chatting tool, wiki, databases, blogs, RSS flux, calendars, etc). Moodle integrates more easily into Web sites and offers management tools such as detailed student usage reports. The use of StudiUM has helped us to implement a meta-course that allows us to link one or more courses to the same core material so that content, questionnaire, and tools can be selectively shared). A meta-course avoids needless duplication of content and makes it easier to update and correct content in case of error.8 In this way, for each chapter, the student may consult a history of commented pertinent news, a list of laws, regulations, and other normative documents dealt with, including hyperlinks to source documents in the form of summary tables and databases, a history of the development of the legislative framework, suggested hyperlinks, pivotal documents deposited in the platform to facilitate their consultation within the pedagogical framework, commented decisions from various courts, educational questions, and summary questions. For example, the database function allows for structured development, setting how various types of knowledge are displayed, long-term content entry, and more efficient searches by the students. Finally, the platform can offer access to various student cohorts throughout their university studies and promote longitudinal use of content for integration in various courses, workshops, laboratories, and training sessions. As of September 1, 2011, StudiUM hosted 950 courses at the Universite de Montreal. The ExamSoft suite (ExamSoft Worldwide, Inc) was chosen for the administration of formal examinations (mid-terms and finals) in class, starting in the fall of 2011. ExamSoft provides a solution for secure examination preparation, administration, and grading. The Web site reports that it is used by 8 faculties of pharmacy.9 The SofTeach application makes it possible for the professor to create items and manage a bank of items (ie, multiple-choice questions, short-answer questions, etc). The centralized depository allows the collaborative management of item banks. Items can be added to an examination using one or more banks of questions. In the case of interrelated questions that require chronological presentation for comprehension and response purposes, the order of display may be forced. When required, the order of display is random, reducing the risk of plagiarism among students. An encrypted and password-protected examination file is then produced. The examination file is published so that students can download it before the scheduled examination period. Only students who have downloaded the file will be authorized to enter the examination room. Student identification is required and students are assigned a seat according to a specific room plan. When all the students are present and the instructions are given, the password is given. A technician is present in the room 30 minutes before the examination and stays for 45 minutes after the examination starts to answer the student’s technical questions. To this date, no major technical problem has been encountered. On 3 occasions, students had to reboot their computers, but did so without any data loss. A team of 5 invigilators (proctors) makes sure that the examination runs smoothly and confirms that all the students end their examination at the end of the examination period by checking each student screen by screen. Given the time allowed for the examination and its level of difficulty, this procedure has not been able to handle a large number of students all leaving at the same time after the statutory examination time is up. The development questions raised a number of issues with the students. First, lengthy reading on the screen led to eye strain in the case of 1 student and some students also questioned the speed of typing on a keyboard versus their handwriting speed. Some students had problems with the display of French accent characters on the screen when reading or writing and some students experienced problems with closing or uploading the examination (eg, error messages that the test had not closed correctly). The presence of a support person allowed us to solve these problems. ExamSoft greatly facilitated the process of correcting the examinations. In the past, a similar examination took on average 40 hours to correct by hand. With ExamSoft, the amount of time required to correct examinations was reduced to approximately 25 hours. A number of reasons can explain the time saved. First, automatic correction of multiple-choice questions included item analysis and facilitated the review of questions (cancelling, identifying answer keys, for example). Next, having the students write directly on a computer facilitated reading their answers. It was even possible to use the search function in the text to look for and validate certain elements in the answer in the short/long development questions. In addition, it was possible to correct 1 question for all the students, thereby ensuring a uniform correction of a question versus correcting each student’s examination individually, one after another. The presence of statistical data made it possible to see the development of the correction and also the average score for each question. A single corrector performed all the examination corrections. ExamSoft made it possible to provide the corrector with targeted access, for example, to hone in on development questions alone. Furthermore, it enabled a collaborative approach between the person in charge of the course and the corrector so that 2 people could connect to ExamSoft at the same time from a distance. When ExamSoft was first used to correct an examination, some problems were encountered. First, the list of questions being corrected was not displayed appropriately and the corrector had to disconnect from ExamSoft and return in order to download the updated list of students again. Another problem encountered was that some comments shown for 1 student were automatically copied into the comment file of the next student. Furthermore, once a comment had been added by the corrector or a mark had been given to a student, it was impossible to remove the comment and leave the space blank. Finally, we tried to use Twitter (Twitter Inc) within the context of a summary course. All the students were invited on a voluntary basis to open an account in order to test student-professor interaction with this social medium. The tool TweetDeck was installed on the teachers’ laptop. During the course, the students were asked to interact and ask questions by raising their hands or using Twitter. Although 99 of the 200 students had a Twitter account (only a few had an account before this initiative), only 8 students wrote a tweet. At the end of the course, the students were asked to answer 4 multiple-choice questions using StudiUM to measure their level of agreement with this initiative. While the trial was not a success (only 8 questions were asked in class using a Twitter account during a 3-hour course and about 30 questions were asked by students raising their hand), 41% of the students stressed that using Twitter in class could allow individuals to express themselves when they would not otherwise do so. Teaching pharmacy legislation can benefit from novel technologies. Migrating to Moodle and using ExamSoft were both a success in 2011 at the Faculty of Pharmacy of the Universite de Montreal. We do not plan to use Twitter as a complementary communications tool in class at this time.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, and despite significant overall improvements in cure rates, outcome for patients diagnosed with resistant phenotypes or those who relapse is dismal. We investigated the mechanism of cell death induced by metformin (MET) in ALL cell models. We showed that MET induced significant growth inhibition and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cells. Western blots revealed that MET activated p-AMPK, p-ACC, p-Akt, and p-4EBP1. The latter suggested that regulation of protein translation may be an important determinant in MET-induced cell death. Indeed, inhibition of mTOR/protein translation with rapamycin (RAPA) rescued MET-induced cell death in ALL cells. In addition, knockdown of AMPK expression in ALL cells using shRNA (shAMPK) abrogated MET-induced apoptosis as compared to control cells expressing scramble shRNA (shCTRL), indicating that AMPK mediated MET's cytotoxicity. Western blots demonstrated that shAMPK cells expressed lower level of total AMPK, p-p38MAPK, p-mTOR, and p-4EBP1 compared to shCTRL cells, indicating that AMPK and protein translation are critical in MET sensitivity. Indeed, pulse labeled 35S-methionine experiments demonstrated increase incorporation confirming the importance of protein synthesis is MET-induced cytotoxicity. More important, we uncovered that MET-induced apoptosis correlated with induction of ER stress as evidenced by up-regulation of IRE1α and CHOP although GRP78 decreased significantly. We further demonstrated that RAPA rescued MET-treated cells by relieving ER stress/UPR mediated cell death. We previously showed that the unfolded protein response (UPR) in ALL cells is regulated by the contextual crosstalk between AMPK and Akt (Mol Cancer Ther 10:437, 2011). Therefore, we evaluated the effects of co-targeting Akt and AMPK using the Akt inhibitor X/perifosine + MET and found these combinations to be synergistic. To further investigate the relationship between protein translation and ER stress/UPR in MET-induced cell death, we examined the role of PIM1/2 kinases. We found that expression of PIM2 was increased in MET-treated ALL with concomitant decreased in the expression of IRE1α, ATF6, and CHOP, suggesting that PIM2 maybe up-regulated as a compensatory survival mechanism aimed at relieving MET-induced ER stress/UPR mediated cell death. To test this hypothesis, we co-treated ALL cells with a PIM1/2 kinase inhibitor + MET and found that PIM2 inhibition synergistically sensitized ALL cells to MET (CI=0.28). Taken together, our data indicate that PIM2 plays a role in buffering cell death in MET-treated cells, and that regulation of protein translation modulates ER stress/UPR induced apoptosis in ALL cells. Consequently, our data support strategies targeting these synthetic lethal interactions as suitable for clinical translation in patients with ALL. 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 2736. doi:1538-7445.AM2012-2736

Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy and the main cause of cancer-related death in children. In search for novel treatment strategies, we investigated the mechanism of cell death induced by the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) in ALL cell models. We found that 2-DG induced significant cell death under normoxia in Bp-ALL and T-ALL cells. Similar treatments with 2-FDG, a more selective glycolytic inhibitor, failed to induce equivalent degrees of cell death in ALL, suggesting that 2-DG induces apoptosis through pathways independent of glycolysis, such as inhibition of N-linked glycosylation (N-LG). Co-treatment with mannose, a sugar essential for N-LG, rescued 2-DG induced cell death in ALL cells (p < 0.01), indicating that inhibition of N-LG leads to apoptosis in ALL. Western blot analysis of 2-DG treated cells showed sustained activation of P-AMPK (T172) and transient up-regulation of P-Akt (S473). More important, treatment with 2-DG resulted in up-regulation of the UPR markers GRP78, GRP94, IRE1α, P-eIF2α, and CHOP, and correlated with increased cleavage of PARP and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL). Therefore, inhibition of the N-LG by 2-DG leads to ER stress/UPR mediated cell death in ALL. We then evaluated the role of Akt using the Akt inhibitor X (AIX) and shRNA against Akt (shAkt). Inhibition of Akt synergistically sensitized CCRF-CEM and NALM6 cells to 2-DG (CI = 0.15; p < 0.05 for AIX vs. each drug alone; p < 0.01 for shAkt vs. scramble shRNA). Western blots showed that P-AMPK was increased in shAkt vs. scramble shRNA expressing cells, whereas P-mTOR, IRE1α, GRP78, P-eIF2α, and CHOP were down-regulated. Again, mannose completely reversed 2-DG+AIX induced cytotoxicity (p < 0.0001, 7% apoptosis for 2DG+AIX+MAN vs. 85% for 2DG+AIX), and correlated with down-regulation of UPR markers, confirming that inhibition of N-LG is the main mechanism by which 2-DG induces apoptosis in ALL. Next, we examined the role of AMPK using shRNA and found that inhibition of AMPK rescued 2-DG+AIX induced cell death and growth inhibition (p < 0.001), and led to up-regulation of P-Akt, P-mTOR, and UPR markers, suggesting that functional UPR activity is critical for ALL cell survival following treatment with 2-DG. Indeed, inhibition of UPR via down-regulation of GRP78 increased cell death in both CCRF-CEM and NALM6 cells treated with 2-DG (p < 0.01). Taken together, we demonstrate that 2-DG induces ER-stress mediated cell death in ALL cells by inhibiting N-LG, and that the ability of ALL cells to effectively engage UPR determines ALL cell fate (survival vs. death). Our data also demonstrate that Akt and AMPK differentially modulate UPR activity in response to 2-DG in ALL. These findings provide a rationale to develop novel strategies for ALL therapy using glycolytic inhibitors alone or in combination with agents targeting these metabolic and oncogenic pathways. 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 4991. doi:1538-7445.AM2012-4991

Abstract 3496 Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children and adolescents. Despite significant overall improvements in cure rates, outcome remains dismal for patients with resistant phenotypes or after relapse. Therefore, novel treatment strategies are warranted. Recently, we identified the AMP activated protein kinase (AMPK), a regulator of energy homeostasis in eukaryotic cells, as a potential target for ALL therapy due to its effects on cell growth, proliferation, and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We showed that activation of AMPK using metformin (1-5 mM) induced significant cell growth inhibition and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cell line models. Western blot analysis revealed that metformin led to activation of p-AMPK (Thr172) and its downstream target p-ACC (Ser79), the cell proliferation regulator p-Akt (Ser473), and the protein translation regulator p-4EBP1 (Thr70), suggesting that protein translation may be an important determinant in the mechanism of metformin-induced cell death. Indeed, we demonstrated that blocking protein translation with the mTOR inhibitor rapamycin (1 μg/ml) rescued ALL cells from metformin-induced cell death (p < 0.01). In addition, knockdown of AMPK1α expression using shRNAs (shAMPK) abrogated metformin-induced growth inhibition and apoptosis in ALL cells as compared to control cells expressing scramble shRNAs (shCTRL), indicating that AMPK mediates metformin's cytotoxicity in our models. Western blots demonstrated that ALL cells expressing shAMPK exhibit decreased expression of total AMPK, p-p38MAPK (Thr180), p-mTOR (Ser2448), and p-4EBP1 (Thr70) compared to shCTRL cells, implicating regulation of protein translation in the mechanism of cell death induced by metformin. In addition, metformin-induced p-Akt (Ser473) activation observed in shCTRL cells is blocked in shAMPK expressing cells, suggesting that the contextual crosstalk between AMPK and Akt is relevant for metformin's cytotoxicity. Indeed, experiments co-targeting Akt and AMPK using perifosine (6 μM) or the Akt inhibitor X (AIX, 5 μM) plus metformin (5 mM) for 72 h induced synergistic cell death in NALM6 cells (Combination Index (CI) values of 0.21 for perifosine + metformin, and 0.19 for AIX + metformin). Our studies uncovered that apoptotic death in NALM6 and CCRF-CEM cells treated with metformin correlated with metformin's induction of ER stress/UPR in ALL cells, as demonstrated by increased expression of the UPR markers IRE1α and CHOP. More important, rapamycin rescued metformin-treated ALL cells by relieving ER stress/UPR as demonstrated by decreased IRE1α and CHOP. These observations support our previous findings that ER stress/UPR mediates cell death in ALL cells under metabolic stress, and is tightly coupled to regulation of protein translation (Mol Cancer Ther 10:437, 2011). To further investigate the relationship between protein translation and ER stress/UPR, we examined the role of PIM1/2 kinases, particularly PIM2 known to regulate CAP protein translation, in metformin-induced ALL cell death. Our results indicate that expression of PIM2 is significantly increased in NALM6 cells treated with metformin (5–10 mM) for 72 h. We also observed concomitant decrease in the expression of the UPR markers IRE1α, ATF6, and CHOP, raising the possibility that PIM2 upregulation may be a compensatory survival mechanism to regulate protein translation and suppress metformin-induced ER stress/UPR. To test this hypothesis, we co-treated NALM6 cells with the small molecule PIM1/2 kinase inhibitor V (80 μM) and metformin (5 mM) and found that inhibition of PIM2 in metformin-treated NALM6 cells induced synergistic cell death (CI = 0.28). Taken together, our data indicate that PIM2 plays a role in buffering cell death in metformin treated cells, and that regulation of protein translation modulates ER stress/UPR induced apoptosis in ALL cells. Consequently, our data support strategies that exploit synthetic lethality by combining activators of AMPK such as metformin and compounds that target regulation of protein translation or protein degradation as suitable for clinical translation in patients with ALL. Disclosures: No relevant conflicts of interest to declare.

Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy and the main cause of cancer-related death in children. In search for novel treatment strategies, we identified AMP activated protein kinase (AMPK) as a potential target for ALL therapy due to its effects on cell proliferation and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We demonstrated that treatment of NALM6 and CCRF-CEM cells with the AMPK activators AICAR and metformin induced significant cell growth inhibition and apoptosis. Rescue experiments using the AMPK inhibitor compound-C (CC) failed to completely abrogate the cytotoxic effects induced by AICAR, attenuating AICAR's apoptosis by 42% in CCRF-CEM and 45% in NALM6. Whereas when used alone, both CC and Ara-A induced significant apoptosis in ALL cells. Using the caspase inhibitors Z-VAD and Z-IETD, we demonstrated that activation of AMPK by AICAR induced cell death via both caspase-dependent and independent mechanisms, whereas inhibition of AMPK by CC induced apoptosis mainly via the intrinsic caspase-dependent pathway. To examine the effects of AMPK activation vs. inhibition on downstream signaling, we used Western blot analysis of key signaling factors associated with the PI3K/Akt/mTOR and RAS/RAF/ERK pathways in ALL cells. We found that AICAR and CC exerted opposite effects on PI3K/Akt and RAS pathway signaling. CC decreased P-Akt (Ser473) and activated the RAS pathway, whereas AICAR increased P-Akt, suggesting that AMPK may play a role as regulator of Akt and RAS activity. We showed that activation of AMPK and Akt by AICAR down-regulated the RAS pathway via phosphorylation of cRAF at Ser621 and Ser259, respectively. Inhibition of the RAS pathway with farnesylthiosalicylic acid (FTS) and U-0126 induced significant cell growth arrest and apoptosis in ALL cells (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 605. doi:10.1158/1538-7445.AM2011-605

Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy and the main cause of cancer-related deaths in children. Therefore, search for novel treatment strategies is warranted. We identified AMP activated protein kinase (AMPK), a master regulator of bioenergetics, as a potential target for ALL therapy due to its effects on cell proliferation and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We demonstrated that treatment of NALM6 (Bp-ALL) and CEM (T-ALL) cells with AICAR, an AMPK activator, induced growth inhibition and apoptosis. Using metformin, another AMPK agonist, we found 40% growth inhibition, and up to five- and three-fold greater induction of apoptosis relative to controls in CEM and NALM6, respectively. Unexpectedly, rescue experiments with AMPK inhibitors Ara-A and compound-C (CC) failed to abrogate the cytotoxic effects induced by AICAR. When used alone, Ara-A induced 60% and 40% cell death in NALM6 and CEM cells, respectively, whereas CC induced 75- and 15-fold more apoptotic death relative to controls. To investigate the mechanism by which AMPK activation vs. inhibition induced apoptosis, we determined levels of P-AMPK (T172) and factors associated with the PI3K/Akt/mTOR and RAS/cRAF/Erk signaling pathways in NALM6 and CEM cells treated with either CC, AICAR, or in combination. Our data show that P-AMPK levels were decreased by CC and increased by AICAR. Additional Western blots demonstrated that these agents exerted opposite effects on Akt and RAS signaling. CC decreased P-Akt (S473) and activated the RAS pathway, while AICAR increased P-Akt. We showed that activation of Akt by AICAR down-regulated the RAS pathway via phosphorylation of cRAF (S259). P-mTOR (S2448) and P-4EBP1 (T70) exhibited a greater decrease in cells treated with CC + AICAR as compared to each agent alone. A significant decrease in P-Akt was also detected in cells treated with both agents vs. each drug alone. Together, our data indicate that AICAR and CC induce cell death in ALL cells by two different mechanisms mediated by AMPK: AICAR-activation of AMPK inhibited the RAS-dependent cell proliferation pathway, and CC-inhibition of AMPK by down-regulating the Akt cell survival pathway. These results suggest that alterations in AMPK signaling may regulate the cross-talk between the PI3K/Akt/mTOR and RAS/cRAF/Erk cascades and may dictate the fate of ALL cells by regulating apoptosis after exposure to agents targeting these pathways. Experiments co-targeting AMPK and Akt using AICAR and Akt-inhibitor X, respectively, induced synergistic growth inhibition in CEM (CI=0.90) and NALM6 (CI=0.85) cells compared to each drug alone. These findings provide a rationale for simultaneously targeting AMPK and key signaling factors associated with either PI3K/Akt/mTOR or RAS/cRAF/Erk pathways in ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5089.

Cells of Escherichia coli that harbor supH (an allele of the wild-type gene serU) are sensitive to UV irradiation and temperature and appear to have an impaired cell division control mechanism. We found that a gene located at the 2-min region, designated shl, inhibited the growth of supH-harboring cells when carried by a high-copy-number plasmid, whereas the same plasmid had no visible effect when present in parental cells. The amino acid sequence predicted from the nucleotide sequence of the shl gene indicated a similarity to the GalR and LacI repressor proteins, suggesting it is a transcription regulator. The sequence between the promoter and the structural genes revealed the presence of a short open reading frame of 28 amino acid residues followed by a segment of 81 base pairs. These structural features suggest that a transcription antitermination mechanism may be involved in the regulation of expression of the shl gene. The possibility that shl is a regulator of serU is discussed.

Background: We describe an alternative method to determine mRNA half-life (t1/2) based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results: Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One µg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp). To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h). Conclusions: We have developed a rapid, sensitive, and reliable method based on Real-Time RTPCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

The ftsM1 mutation is believed to be in a gene implicated in the regulation of cell division in Escherichia coli because it displayed the lon mutation phenotypes. In this study, we show that this mutation is located in serU, a gene which codes for tRNA(Ser)2, and has the phenotypes of the serU allele supH. Both ftsM1 and supH suppressed the leuB6 and ilvD145 missense mutations, and both conferred temperature and UV light irradiation sensitivity to the harboring cells. Cells which carried the ftsM1 mutation or the supH suppressor had very low colony-forming abilities on salt-free L agar, and this phenotype was almost completely abolished by the presence of plasmids bearing the ftsZ+ gene. Furthermore, sensitivity of the mutant cells to UV irradiation was also markedly diminished when they carried a ftsZ+-bearing plasmid. These results suggest that supH-containing cells have reduced FtsZ activities, in accordance with their displaying the phenotypes of the lon mutant cells. The possibility that ftsM1 (supH) is functionally involved in the biosynthesis of a specific protein which affects cell division is discussed.

BackgroundExpression of folylpoly-γ-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells.MethodsTo determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA.ResultsFPGS transcription initiation rate was 1.6-fold higher in NALM6vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage.ConclusionWe demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory elements, transcription initiation rates, and mRNA processing. Understanding the lineage-specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance or inducing apoptosis in leukemic cells.

Background Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells. Results We found that treatment with AICAR inhibited cell proliferation, induced cell cycle arrest in G1-phase, and apoptosis in CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL, TEL/AML1) and SupB15 (Bp-ALL, BCR/ABL) cells. These effects were abolished by treatment with the adenosine kinase inhibitor 5'-iodotubericidin prior to addition of AICAR indicating that AICAR's cytotoxicity is mediated through AMPK activation. Moreover, we determined that growth inhibition exerted by AICAR was associated with activation of p38-MAPK and increased expression of the cell cycle regulators p27 and p53. We also demonstrated that AICAR mediated apoptosis through the mitochondrial pathway as revealed by the release of cytochrome C and cleavage of caspase 9. Additionally, AICAR treatment resulted in phosphorylation of Akt suggesting that activation of the PI3K/Akt pathway may represent a compensatory survival mechanism in response to apoptosis and/or cell cycle arrest. Combined treatment with AICAR and the mTOR inhibitor rapamycin resulted in additive anti-proliferative activity ALL cells. Conclusion AICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear to be mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL. Therefore, activation of AMPK by AICAR represents a novel approach to targeted therapy, and suggests a role for AICAR in combination therapy with inhibitors of the PI3K/Akt/mTOR pathways for the treatment of childhood in ALL.