AMPK Associates with Chromatin and Phosphorylates the TAF-1 Subunit of the Transcription Initiation Complex to Regulate Histone Gene Expression in ALL Cells.

The survival rates for relapsed/refractory acute lymphoblastic leukemia (ALL) remain poor. We and others have reported that ALL cells are vulnerable to conditions inducing energy/ER-stress mediated by AMP-activated protein kinase (AMPK). To identify the target genes directly regulated by AMPKα2, we performed genome-wide RNA-seq and ChIP-seq in CCRF-CEM (T-ALL) cells expressing HA-AMPKα2 (CN2) under normal and energy/metabolic stress conditions. CN2 cells show significantly altered AMPKα2 genomic binding and transcriptomic profile under metabolic stress conditions, including reduced histone gene expression. Proteomic analysis and in vitro kinase assays identified the TATA-Box-Binding Protein-Associated Factor 1 (TAF1) as a novel AMPKα2 substrate that downregulates histone gene transcription in response to energy/metabolic stress. Knockdown and knockout studies demonstrated that both AMPKα2 and TAF1 are required for histone gene expression. Mechanistically, upon activation, AMPKα2 phosphorylates TAF1 at Ser-1353 which impairs TAF1 interaction with RNA polymerase II (Pol II), leading to a compromised state of p-AMPKα2/p-TAF1/Pol II chromatin association and suppression of transcription. This mechanism was also observed in primary ALL cells and in vivo in NSG mice. Consequently, we uncovered a non-canonical function of AMPK that phosphorylates TAF1, both members of a putative chromatin-associated transcription complex that regulate histone gene expression, among others, in response to energy/metabolic stress. IMPLICATIONS: Fully delineating the protein interactome by which AMPK regulates adaptive survival responses to energy/metabolic stress, either via epigenetic gene regulation or other mechanisms, will allow the rational development of strategies to overcome de novo or acquired resistance in ALL and other cancers.

Protein Kinase D-Dependent Downregulation of Immediate Early Genes through Class IIA Histone Deacetylases in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death in children and adolescents, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. To identify genome-wide metabolic-stress regulated genes, we used RNA-sequencing in ALL cells treated with AICAR, an AMPK activator. RNA-sequencing identified the immediate early genes (IEGs) as a subset of genes downregulated by AICAR. We show that AICAR-induced IEGs downregulation was blocked by an adenosine uptake inhibitor indicating AICAR was responsible for IEGs reprogramming. Using pharmacologic and genetic models we established this mechanism was AMPK-independent. Further investigations using kinase assays, PKD/PKC inhibitors and rescue experiments, demonstrated that AICAR directly inhibited PKD kinase activity and identified PKD as responsible for IEGs downregulation. Mechanistically, PKD inhibition suppressed phosphorylation and nuclear export of class IIa HDACs, which lowered histone H3 acetylation and decreased NFκB(p65) recruitment to IEGs promoters. Finally, PKD inhibition induced apoptosis via DUSP1/DUSP6 downregulation eliciting a DNA damage response. More importantly, ALL patient cells exhibited the same PKD-HDACs-IEGs-mediated mechanism. As proof of principle of the therapeutic potential of targeting PKD, we established the in vivo relevance of our findings using an NSG ALL mouse model. In conclusion, we identified a previously unreported PKD-dependent survival mechanism in response to AICAR-induced cellular stress in ALL through regulation of DUSPs and IEGs' expression. IMPLICATIONS: PKD mediates early transcriptional responses in ALL cells as an adaptive survival mechanism to overcome cellular stress.

The NEDD8-activating enzyme inhibitor pevonedistat activates the eIF2α and mTOR pathways inducing UPR-mediated cell death in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children, and cure rates for adults remain dismal. Further, effective treatment strategies for relapsed/refractory ALL remain elusive. We previously uncovered that ALL cells are prone to apoptosis via endoplasmic reticulum (ER) stress/unfolded protein response (UPR)-mediated mechanisms. We investigated the antineoplastic activity of pevonedistat®, a novel NEDD8-activating enzyme inhibitor that targets E3 cullin-RING ligases (CRLs) dependent proteasomal protein degradation, in ALL. Herein, we report that pevonedistat induces apoptosis in ALL cells by dysregulating the translational machinery leading to induction of proteotoxic/ER stress and UPR-mediated cell death. Mechanistically, pevonedistat led to P-eIF2a dephosphorylation causing atypical proteotoxic/ER stress from failure to halt protein translation via the UPR and upregulation of mTOR/p70S6K. Additional studies revealed that pevonedistat re-balanced the homeostasis of pro- and anti-apoptotic proteins to favor cell death through altered expression and/or activity of Mcl-1, NOXA, and BIM, suggesting that pevonedistat has a "priming" effect on ALL by altering the apoptotic threshold through modulation of Mcl-1 activity. Further, we demonstrated that pevonedistat synergizes with selected anti-leukemic agents in vitro, and prolongs survival of NSG mice engrafted with ALL cells, lending support for the use of pevonedistat as part of a multi-agent approach.

Mcl-1 downregulation leads to the heightened sensitivity exhibited by BCR-ABL positive ALL to induction of energy and ER-stress.

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.

Metformin induces apoptosis through AMPK-dependent inhibition of UPR signaling in ALL lymphoblasts.

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.

Inhibition of Akt potentiates 2-DG-induced apoptosis via downregulation of UPR in acute lymphoblastic leukemia.

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.

AMPK and Akt determine apoptotic cell death following perturbations of one-carbon metabolism by regulating ER stress in acute lymphoblastic leukemia.

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.

AMPK-induced activation of Akt by AICAR is mediated by IGF-1R dependent and independent mechanisms in acute lymphoblastic leukemia.

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 (TEL-AML1, [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.

Folylpolyglutamate synthetase gene transcription is regulated by a multiprotein complex that binds the TEL-AML1 fusion in acute lymphoblastic leukemia.

Acute Lymphoblastic Leukemia (ALL) non-random fusions influence clinical outcome and alter the accumulation of MTX-PGs in vivo. Analysis of primary ALL samples uncovered subtype-specific patterns of folate gene expression. Using an FPGS-luciferase reporter gene assay, we determined that E2A-PBX1 and TEL-AML1 expression decreased FPGS transcription. ChIP assays uncovered HDAC1, AML1, mSin3A, E2F, and Rb interactions with the FPGS promoter region. We demonstrate that FPGS expression is epigenetically regulated through binding of selected ALL fusions to a multiprotein complex, which also controls the cell cycle dependence of FPGS expression. This study provides insights into the pharmacogenomics of MTX in ALL subtypes.

Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy.

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.

Molecular basis for decreased folylpoly-gamma-glutamate synthetase expression in a methotrexate resistant CCRF-CEM mutant cell line.

A CCRF-CEM mutant, CEM-p, has been shown to exhibit resistance to methotrexate due to decreased methotrexate polyglutamate accumulation. To ascertain the mechanism(s) responsible for this phenotype, we analyzed FPGS and SLC19A1 mRNA expression, examined FPGS promoter activity, and determined nucleotide sequence of the FPGS promoter and full length cDNA from CCRF-CEM and CEM-p cells. We identified in FPGS from CEM-p cells three amino acid substitutions that altered the ATP binding P-loop, glutamate/folate binding, and a conserved domain located at the carboxyl-terminal. Our data demonstrated for the first time the importance of the highly conserved domain (VTGSLHLVGGV) located at the carboxyl-terminal for FPGS activity.

Analysis of folylpoly-gamma-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells.

BACKGROUND: Expression of folylpoly-gamma-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. METHODS: To 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. RESULTS: FPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. 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 NALM6 vs. 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. CONCLUSION: We 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.

Cloning and mRNA expression of the Ca2+-binding DREAM protein in the pituitary.

It is well recognized that the level of intracellular calcium governs several cellular processes such as gene expression and secretion in the pituitary. Recently, a novel gene has been identified in neuroendocrine cells that encodes DREAM, a calcium-binding protein that acts as a transcriptional repressor by binding specific downstream regulatory elements (DRE) on DNA. To explore the possibility that DREAM may be expressed in the rat pituitary and may function in endocrine activity, we analyzed its mRNA expression by RT-PCR. Using oligonucleotide primers derived from the mouse DREAM cDNA, we amplified, cloned, and characterized a 852-bp RT-PCR product from rat pituitary tissue. Two splice variants of the rat DREAM gene differing by four nucleotides (tetramer ACAG) were identified. The ACAG(+) variant (ORF1) consisted of 768bp encoding a protein of 256 residues with an estimated molecular weight of 29.5kDa. Amino acid sequence analysis of ORF1 indicated 92.6% and 98.1% identity to the DREAM gene product from human and mouse, respectively. The second variant, ACAG(-) (ORF2), was 567-bp long and was predicted to encode a peptide of 189 residues with a molecular mass of about 20.8kDa. To determine which endocrine pituitary cells were expressing DREAM, we evaluated several different clonal populations containing cells that expressed specific pituitary hormones. We found that both DREAM splice variants were expressed in each pituitary cell types examined, which included the mammotropes (MMQ cells), somatotropes (GC cells), mammosomatotropes (GH(3) cells), gonadotropes (LbetaT2 cells), thyrotropes (TalphaT1 cells), and corticotropes (AtT-20 cells). Interestingly, the levels of the two variants differed between the cell types tested with the ACAG(+) variant comprising about two-thirds of the DREAM expression for the mammotropes, somatotropes, mammosomatotropes, and corticotropes as compared to less than one-half for the thyrotropes and the gonadotropes. Our initial attempts to identify pituitary-specific genes regulated by DREAM revealed that prolactin gene expression was not influenced by DREAM suggesting that an action of DREAM may involve other pituitary hormones or be mediated by other cell processes. When taken together, our findings of DREAM expression in the pituitary in a manner specific to pituitary endocrine cell type raises the possibility that this protein may play a role in determining specific pituitary cell function.

Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines.

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 beta-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 RiboGree&ncircledR; fluorescent dye with the VersaFluor Fluorometer System. One &mgr;g of total RNA was reverse transcribed and used as template for the amplification of a region of the beta-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, beta-actin mRNAs were quantified by Real-Time RT-PCR using the SYB&RcircledR; Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the beta-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 RT-PCR for measuring mRNA half-life. Our results confirm that beta-actin mRNA half-life can be affected by the cellular growth rate.