Genome-Wide shRNA Screen Identifies WEE1 As a Critical Mediator of Cell Fate and Novel Therapeutic Target in AML,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3503-3503
Author(s):  
Christopher C. Porter ◽  
Jihye Kim ◽  
Susan Fosmire ◽  
Christy M. Gearheart ◽  
Annemie van Linden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Cell Cycle Checkpoint ◽  
Phase Ii Trials ◽  
Genome Wide ◽  
Cycle Checkpoint

Abstract Abstract 3503 Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge of the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to specific molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational strategies, we performed genome-wide shRNA screens to identify proteins which mediate AML cell fate in cytarabine in 2 AML cell lines (Molm13 and MV4-11). Over 55,000 shRNAs targeting over 11,000 genes were quantified by deep sequencing to identify shRNAs under-represented in the context of cytarabine as compared to no treatment. Complementary analyses identified 125 genes as mediators of AML cell fate in cytarabine. In addition we performed gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context and examined existing gene expression data from primary patient samples. The integration of these independent analyses strongly implicates cell cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell fate in cytarabine. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival in cytarabine. Pharmacologic inhibition of WEE1 in several, but not all, AML cell lines is synergistic with cytarabine, suggesting underlying molecular susceptibility to this combination of drugs. A WEE1 inhibitor is in Phase II trials in solid tumors, primarily as a means to abrogate the G2/M checkpoint in tumors with TP53 dysfunction. Further experiments demonstrate that inhibition of WEE1 prevents slowed S-phase progression induced by cytarabine in AML cells, broadening the functions of WEE1 that may be exploited therapeutically. Preliminary experiments indicate synergistic inhibition of AML cellular proliferation with daunorubicin in some AML cell lines. Experiments to determine whether WEE1 inhibition in combination with chemotherapy prolongs survival of mice with leukemia are underway. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as potential therapeutic target in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Gene expression signatures but not cell cycle checkpoint functions distinguish AT carriers from normal individuals

2013 ◽  
Vol 45 (19) ◽  
pp. 907-916
Author(s):  
Liwen Zhang ◽  
Dennis A. Simpson ◽  
Cynthia L. Innes ◽  
Jeff Chou ◽  
Pierre R. Bushel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Lines ◽  
Cell Cycle Checkpoint ◽  
G2 Checkpoint ◽  
P53 Signaling ◽  
Normal Individuals ◽  
Gene Expression Signatures ◽  
Cycle Checkpoint ◽  
Atm Cell

Ataxia telangiectasia (AT) is a rare autosomal recessive disease caused by mutations in the ataxia telangiectasia-mutated gene ( ATM). AT carriers with one mutant ATM allele are usually not severely affected although they carry an increased risk of developing cancer. There has not been an easy and reliable diagnostic method to identify AT carriers. Cell cycle checkpoint functions upon ionizing radiation (IR)-induced DNA damage and gene expression signatures were analyzed in the current study to test for differential responses in human lymphoblastoid cell lines with different ATM genotypes. While both dose- and time-dependent G1 and G2 checkpoint functions were highly attenuated in ATM−/− cell lines, these functions were preserved in ATM+/− cell lines equivalent to ATM+/+ cell lines. However, gene expression signatures at both baseline (consisting of 203 probes) and post-IR treatment (consisting of 126 probes) were able to distinguish ATM+/− cell lines from ATM+/+ and ATM−/− cell lines. Gene ontology (GO) and pathway analysis of the genes in the baseline signature indicate that ATM function-related categories, DNA metabolism, cell cycle, cell death control, and the p53 signaling pathway, were overrepresented. The same analyses of the genes in the IR-responsive signature revealed that biological categories including response to DNA damage stimulus, p53 signaling, and cell cycle pathways were overrepresented, which again confirmed involvement of ATM functions. The results indicate that AT carriers who have unaffected G1 and G2 checkpoint functions can be distinguished from normal individuals and AT patients by expression signatures of genes related to ATM functions.

HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 832-832 ◽  
Author(s):  
Michael A Chapman ◽  
Jean-Philippe Brunet ◽  
Jonathan J Keats ◽  
Angela Baker ◽  
Mazhar Adli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Histone Modification ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Specific Expression ◽  
Aberrant Expression ◽  
High Level

Abstract Abstract 832 We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM. Disclosures: No relevant conflicts of interest to declare.

Bortezomib Inhibits mTOR Pathway in Multiple Myeloma Cell Lines Via Induced Expression of REDD1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 242-242
Author(s):  
Olivier Decaux ◽  
Monique Clement ◽  
Florence Magrangeas ◽  
Laurence Lode ◽  
Catherine Charbonnel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Size ◽  
Cell Lines ◽  
Stress Responses ◽  
Cellular Proliferation ◽  
Expression Profiles ◽  
Mtor Pathway ◽  
Molecular Signature ◽  
Gene And Protein Expression

Abstract Pharmacogenomic profiles of genes involved in bortezomib - dexamethasone response may help to understand resistance and could provide new therapeutic targets as well as contributing to novel prognostic markers in multiple myeloma. We have used gene expression profiling to analyze the complex signaling pathways regulating the response to bortezomib - dexamethasone. Gene expression profiles were established in 9 cell lines, derived from 9 myeloma patients, incubated or not with a combination of bortezomib 10 nM and dexamethasone 1 μM. These concentrations correspond to the ones used for patients in the IFM 2005-01. Cells were collected after 6 hours of treatment. We focused our interest in early response genes, making the hypothesis that the comprehension of early effects would help to better understand the mechanisms of resistance that take place in at least two third of myeloma patients. Supervised analysis with permutations identified significantly up regulated genes involved in stress responses (heat shocks proteins, RTP801/dig2/REDD1/DDIT4), endoplasmic reticulum stress (HERP/HERPUD1, gadd145/CHOP/DDIT3), ubiquitin/proteasome pathway (proteasome 26S subunits PSMB7, PSMC4, PSMD3 and PSMD13), unfolded protein response (such as SQSTM1, ATF4) or redox equilibrium (PLRX, PRDX1). We assumed that these genes might represent a molecular signature of response to bortezomib and provide important insight into the complex mechanisms of action of these drugs. We focused on REDD1 a gene cloned in 2002 that is known to be rapidly induced by a wide variety of stress conditions (arsenic, hypoxia, dexamethasone, thapsigargin, tunimycin and heat shock) and DNA damages (ionizing radiation, ultraviolet radiation, DNA alkylant). We found that both REDD1 gene and protein expression were early and highly induced after bortezomib exposure alone or in combinaison with dexamethasone. This effect was dependent upon cell line: REDD1 was overexpressed within two hours in resistant cell lines in association with a cell size decrease while in sensitive cell lines, neither REDD1 induction nor morphological changes occured. REDD1 induction was associated with the dephosphorylation of S6K1, a key substrat of mTOR, a protein kinase which controls cell growth and cell size in response to various signals. SiRNA studies confirmed that bortezomib lead to a negative regulation of mRTor activity mediated by REDD1: disruption of REDD1 abrogates both S6K1 phosphorylation and early transitory cell size reduction. Our results are in accordance with data obtained in mouse showing an early regulation of mTOR pathway and cellular proliferation induced by REDD1 expression in response to stress. Our study suggests that mTOR regulation could be a resistance mechanism mediated by REDD1 expression. As we found that REDD1 was differentially induced in primary plasma cells from patients, this gene expression could help to predict response to bortezomib. Our objective is now to clarify the pathway that links bortezomib to REDD1 in multiple myeloma and to investigate REDD1 expression in patients enrolled in IFM 2005-01 clinical trial.

Gene Expression Profiling Reveals a Crucial Role for C/EBPbeta in Proliferation Pathways of ALK+ ALCL Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2818-2818
Author(s):  
Irina Bonzheim ◽  
Martin Irmler ◽  
Natasa Anastasov ◽  
Margit Klier ◽  
Johannes Beckers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Statistical Analysis ◽  
Gene Expression Profiling ◽  
Cell Lines ◽  
Expression Profiling ◽  
Leucine Zipper ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
Large Cell

Abstract Introduction: ALK+ anaplastic large cell lymphomas (ALCL) overexpress C/EBPβ, as a consequence of NPM-ALK kinase activity. C/EBPβ is a leucine zipper transcription factor, which plays a major role in cellular differentiation, inflammation, proliferation and metabolism control. To determine the role of C/EBPβ in ALK+ ALCL transformation, and to identify its downstream targets, a highly specific C/EBPβ-shRNA was used to knockdown C/EBPβ. The consequences of C/EBPβ gene-silencing were analyzed by gene expression profiling. Materials and Methods: Four ALK+ ALCL cell lines, SUDHL-1, Kijk, Karpas 299 and SUP-M2 were transfected with lentivirus containing the C/EBPβ shRNA or the vector without shRNA in triplicates. Western Blot analysis and qRT-PCR were performed to quantify the knockdown effect. At day three after infection, RNA was extracted and used for Gene Chip expression analysis (Affymetrix). Using Anova software for statistical analysis, we identified genes, which were regulated in all four cell lines. The effect of C/EBPβ knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis. Results: In all four ALK+ ALCL, efficient C/EBPβ knockdown resulted in profound growth retardation (up to 84%) compared to control cells after 6 days of infection, and a clear shift from the S phase to the G1 phase in the cell cycle was observed. To identify genes regulated by C/EBPβ in all four cell lines, we performed statistical analysis applying a false discovery rate of 20%, and accepted only genes with a >1,1 and <0,9 fold ratio. We identfied 435 genes regulated after C/EBPβ knockdown (117 upregulated, 318 downregulated). Classification of the differentially expressed genes into biological categories revealed overrepresentation of genes involved in the regulation of kinase activity, cell cycle and proliferation, lymphocyte differentiation, and metabolic processes. In particular, kinases involved in the regulation of JNK activity, which have been shown previously to be involved in proliferation of ALCL, were highly affected by C/EBPβ knockdown. Genomatix Bibliosphere Pathway Analysis revealed C/EBPβ to be connected to pathways involving cell cycle (RUNX3, CCNG1, CDKN2A), apoptosis (FAS, PTPRC, BCL2A1, BIRC3) and MAPK cascades (TRIB1 and several MAP3Ks). Several of the genes identified contain known C/EBPβ binding sites. Conclusions: C/EBPβ silencing induces growth arrest in ALK+ALCL, which correlates with differential expression of genes involved in cell cycle, apoptosis and differentiation. This study reveals C/EBPβ as a master transcription regulator of NPM-ALK induced cellular proliferation, and therefore, an ideal candidate for targeted therapeutic intervention.

A Genome-Wide Approach Identifies Variations in the Aspartate Metabolism Pathway That Are Associated with Asparaginase Sensitivity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 213-213
Author(s):  
Shih-Hsiang Chen ◽  
Wenjian Yang ◽  
Yiping Fang ◽  
Gabriele Stocco ◽  
Kristine R. Crews ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Interindividual Variation ◽  
European Ancestry ◽  
Mixed Effect ◽  
Metabolism Pathway ◽  
Genome Wide ◽  
Individual Snps ◽  
A Genome ◽  
Aspartate Pathway

Abstract Asparaginase is an important drug for acute lymphoblastic leukemia (ALL). The basis for interindividual differences in asparaginase sensitivity remains unclear. To comprehensively identify genetic variants important in asparaginase sensitivity, we employed a genome-wide association approach using the HapMap lymphoblastoid cell lines from 87 individuals of European ancestry (CEU) and diagnostic ALL blasts from 42 newly diagnosed, genomically-determined white patients. In vitro sensitivity was based on IC50 values measured following 48 hour exposures to native E. coli asparaginase (0.003–100 IU/ml) in CEU cell lines and 96 hour exposures (0.003–10 IU/ml) in patient samples using the methylthiazol tetrazolium assay. For CEU cell lines, single nucleotide polymorphism (SNP) genotypes were downloaded from the International HapMap database (www.hapmap.org) and gene expression data (Affymetrix GeneChip Human Exon 1.0 ST Array) were downloaded from http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE7761. For patients with ALL, we used the 500K SNP arrays to interrogate germline DNA and Affymetrix U133A GeneChip Array to assess gene expression in ALL blasts. We tested whether 2,390,203 SNP genotypes were associated with asparaginase IC50 using a linear mixed effect model in CEU cell lines, setting a p value threshold of p < 0.001 for individual SNPs and p < 0.05 at the gene level. This approach yielded 329 SNPs representing 94 genes. Combining these germline SNPs with those representing genes whose expression was also associated with IC50 at the p < 0.05 level (1,706 genes), there were 6 SNPs representing 5 genes, two of which (rs8135371 and rs17001863, both in the ADSL gene) contributed to asparaginase sensitivity (p = 6.9 × 10−4 and 9.1 × 10− 4, respectively) through their effects on ADSL gene expression. The top ranked KEGG pathway overrepresented by the 94 top-ranked genes (329 SNPs) was that of aspartate metabolism, which may be directly linked to the mechanism of action of asparaginase. The two most highly ranked genes (ADSL and DARS) in this pathway encompassed 7 SNPs (rs8135371, rs17001863, rs3768998, rs2278683, rs11893318, rs2322725, and rs7587285), all with p < .001. Using multiple linear regression analysis, 32% of the variability in asparaginase IC50 among the CEU cell lines could be accounted for by these 7 SNPs (p = 5.9 × 10−7). To examine the overall contribution of the aspartate metabolism pathway to asparaginase IC50, we compared all SNPs (935 in cell lines, 717 in patients) representing the aspartate pathway with those SNPs representing other pathways, using a random forest model. We found that the SNP genotypes in the aspartate pathway explained significantly more variation in asparaginase IC50 in cell lines (11.4%, p = 6.9 × 10−4) and in ALL patient samples (11.2%, p = 0.02) than other pathways. The expression of ADSL differed among ALL subtypes, with more sensitive subtypes (hyperdiploid and TEL-AML1 ALL) having lower ADSL expression than more resistant subtypes (T-ALL) (p = 1.1 × 10−5 and 2.9 × 10−9, respectively). Genome-wide interrogation of CEU cell lines and primary ALL blasts revealed that inherited and acquired genomic interindividual variation in a plausible candidate pathway contribute to asparaginase sensitivity.

RNAi Screen of the Druggable Genome Identifies Modulators of Proteasome Inhibitor Sensitivity in Myeloma Including CDK5.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 602-602
Author(s):  
Yuan Xiao Zhu ◽  
Rodger E. Tiedemann ◽  
Chang-Xin Shi ◽  
Jessica Schmidt ◽  
Laura Bruins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Shrna Expression ◽  
Primary Screening ◽  
A Genome ◽  
Proteasome Subunits ◽  
Druggable Genome ◽  
Sensitization Effect

Abstract Abstract 602 The molecular target(s) which co-operate with proteasome inhibition in inducing drug sensitivity or resistance in Multiple Myeloma (MM) remain unknown. We therefore conducted a genome scale small interfering RNA (siRNA) lethality study in KMS11 MM cells in the presence or absence of bortezomib without regard to pre-conceived mechanistic notions. Primary screening was performed in a single-siRNA-per-well format with the human druggable genome siRNA set V4 comprising 13,984 siRNA targeting 6,992 genes and comprising two RNAi per gene. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. Bortezomib was added (at the IC10, 25, 70 and 90) 24 hours post transfection. After 96 hours (72 hours after bortezomib), viability was measured by ATP-dependent luminescence. In primary screening 320 candidate bortezomib sensitizing genes were identified and rescreened with four siRNA oligos each gene. 57 of the top sensitizer hits were selected for which at least two distinct siRNA decreased the EC50 by 2 standard deviations from cells treated with control siRNAs. By cross referencing gene expression profile (GEP) data of KMS11, we furthered pared the list to 37 plausibly expressed targets (0.5% of those genes originally screened) as bortezomib sensitizers. After silencing, 50% of these 37 genes also sensitized the lung cancer cell line A549 to bortezomib. After further parsing of genes which also modulated the sensitivity of MM to Melphalan (non specific chemosensitizers), 34 genes remained: The strongest sensitizers to bortezomib were the proteasome subunits PSMA5, PSMB2, PSMB3, PSMB7 but included less obvious targets such as BAZ1B, CDK5, CDC42SE2, MDM4, NME7, TFE3, TNFAIP3, TNK1, TOP1, VAMP2 and YY1 were also identified. Of these, the most potent synergetic effects were observed with siRNAs against the proteasome subunits and against cyclin dependent kinase 5 (CDK5), which caused the greatest shift in EC50. CDK5 is of particular interest as a therapeutic target as it is expressed at high levels in MM and neural tissues but has low expression in other organs. Using viral shRNA expression, silencing CDK5 consistently increased the sensitivity of genetically variable MM cell lines (n=5) to all of the proteasome inhibitors tested: bortezomib, carfilzomib and PR047, and the effect could be at least partially rescued by overexpression of an RNAi resistant CDK5. To explore therapeutic relevance the small molecule CDK5 inhibitor, Roscovitin, was shown to be synergistic or additive with bortezomib in both MM cell lines and primary patient samples. Gene expression profiling was then performed to seek an explanation for the CDK5 sensitization effect and regulation of a proteasome subunit PSMB5 by CDK5 was identified as a probable route to sensitization. In summary inhibition of the existing proteasome either directly by suppression of proteasome subunits, or indirectly by suppression of modulators such as CDK5 appears to confer the greatest sensitization effect suggesting that combinations of bortezomib with other unique proteasome inhibitor drugs or combinations with inhibitors of CDK5 is a logical avenue for clinical exploration. Disclosures: No relevant conflicts of interest to declare.

PDK1 Overexpression In Acute Myeloid Leukemia; Clinical Significance and Potential as a Therapeutic Target

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2162-2162
Author(s):  
Joanna Zabkiewicz ◽  
Lorna Pearn ◽  
Robert Hills ◽  
Gareth Morgan ◽  
Alan Burnett ◽  
...  
Keyword(s):  
Overall Survival ◽  
Clinical Significance ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Cyclin D3 ◽  
Significant Heterogeneity ◽  
Selective Treatment ◽  
Cd34 Cells

Abstract Abstract 2162 PDK1 is a master kinase responsible for regulating at least six kinase groups including AKT, PKC and S6K. Many of these kinases have been shown to be constitutively active in tumour tissues including leukemia suggesting PDK1 is frequently dysregulated. Here we describe the frequency and significance of PDK1 overexpression in AML and determine the potential of PDK1 as therapeutic target. Analysis of 113 AML patients showed that overexpression of PDK1 compared with normal blast cells was frequently observed in 2 groups of patients defined by FAB M1/M0 (38% PDK1Hi) and M4/M5 (44% PDK1Hi). To establish whether overexpression was a property of the entire leukemic population (including putative leukemic stem cells) we carried out multiparameter flow cytometric intracellular staining. In 7/8 patients, we found PDK1 overexpression to be uniformly expressed in all leukemic sub-populations. Ectopic overexpression of PDK1 in normal CD34+ cells promoted their survival in unsupplemented medium (155% ±38, P<0.01, n=6). Similarly, we found that primary AML blasts overexpressing PDK1 displayed significantly higher survival in vitro when cultured in the absence of growth factors than those with normal PDK1 levels (82.4%±10.1 in PDK1Hi vs 64.4%±12.2 in PDK1Norm; P<0.0001, n=71). To determine whether PDK1Hi AML blasts were more dependent on PDK1 activity for their survival, we assessed their sensitivity to PDK1 inhibition compared with PDK1Norm blasts, using a selective inhibitor, BX-795. We found that PDK1Hi blasts showed significantly increased sensitivity to PDK1 inhibition (PDK1Hi 6.4μM +/−3.07 n=30; PDK1Norm 13.4μM +/−7.4 n=37, P=0.001) indicating that oncogene addiction is a feature of PDK1 overexpression in AML. In contrast, normal bone marrow CD34+ cells were resistant to BX-795 suggesting that PDK1 inhibition selectively targets the survival of AML blasts. Western blotting of BX-795-treated AMLs revealed dose dependent knock down of all detectable PDK1 targets substantiating the specificity of this inhibitor; furthermore BX-795 showed synergistic action when used in combination with AraC (CI= 0.7). We next investigated the clinical significance of PDK1 overexpression (using a statistical model to adjust for known prognostic factors) and discovered significant heterogeneity in overall survival between M1/M0 and M4/M5 AMLs (p=0.001 for interaction) with M1/M0 PDK1Hi patients showing significantly improved overall survival (HR 0.14 (0.003-0.61) p=0.004) whereas M4/M5 PDK1Hi patients showed poorer overall survival (although this did not reach significance: HR 2.08 (0.97-4.46) p=0.06). To examine the basis of this differential survival pattern, we surveyed the targets previously associated with PDK1 hyperactivation. We discovered that PDK1 overexpression in M4/M5 patients exclusively suppressed expression of cyclin D3 (R2 =0.675). Ectopic expression of PDK1 in AML cell lines confirmed that PDK1 suppressed D3 expression in monocytic AML cell lines whereas it induced cyclin D3 in AML lines derived from undifferentiated AML. These data indicate that suppression of cyclin D3 expression by PDK1 may reduce the rate of proliferation in M4/M5 AML, decreasing their sensitivity to standard chemotherapeutic treatments (which are most effective in targeting highly proliferative cells). Taken as a whole these data suggest that therapeutic targeting of PDK1 is an effective and selective treatment for AML, but is likely to be most beneficial for M4/M5 patients. Further, these data demonstrate that developmental context can be a significant factor when establishing the role and significance of abnormalities in AML. Disclosures: No relevant conflicts of interest to declare.

CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4174-4174
Author(s):  
Jiaying Tan ◽  
Jay L. Hess
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Polycomb Group ◽  
Human Leukemia ◽  
Interaction Sites

Abstract Abstract 4174 Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

The Alteration of SEPT5 Gene Expression in BCR-ABL Positive Cells and Its Possible Correlation with the Development and / or Progression of Chronic Myeloid Leukemia (CML)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4415-4415
Author(s):  
Cintia Do Couto Mascarenhas ◽  
Anderson Ferreira Cunha ◽  
Ana Flavia Brugnerotto ◽  
Sheley Gambero ◽  
Joao Machado-Neto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Blood Donors ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Blood Platelets ◽  
Gene Expression Pattern ◽  
P Value

Abstract Abstract 4415 The CML is a clonal disease of stem cells and its main feature is the unregulated production of a tyrosine kinase protein called BCR-ABL, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. Because of this, the use of tools for the study of gene expression could bring new insights in the understanding of these mechanisms in the CML. In a recent study using SSH libraries, we compared the gene expression pattern between granulocytes of health control and CML patients, and we identified the gene SEPT5 expressed only in CML patients. Although the studies in the literature, there is not a clear relationship between the expression of this gene and the development or progression of CML. SEPT5 is a member of nucleotide binding proteins called septins that were firstly described in yeast as cell division cycle regulatory proteins. This gene was reported in patients with AML translocated with MLL gene, in adult human brain and heart; it is also associated with alpha granules of human blood platelets. The aims of this study are to carry a functional analysis of SEPT5 in differents cells line and to study the relationship of this gene and the development and/or progression of CML. The gene expression evaluation was made in granulocytes, mononuclear cells and total leukocytes of CML patients and healthy blood donors in peripheral blood. It was also evaluated in bone marrow donors, in human cell lines (K562, HL60 and NB4) and in mice cell lines (BaF3/BCR-ABLp210 and BaF3T315I), performed by real-time PCR for the following genes: SEPT5, β-actin and GAPDH. Experiments were also performed to verify the difference between the chemotaxis of granulocytic cells from controls and patients by ELISA. Data were analysed statistically using the ANOVA followed by Dunnett’s test – P value of less than 0.05 was considered to be significant. The study was approved by the Research Ethic Committee of the Faculty of Medical Sciences of University of Campinas. The gene expression of SEPT5 was evaluated by real time PCR using the same samples used in the library construction to validate the results found in the SSH library. The data confirmed our previous results, showing that the SEPT5 expression is increased in all cells of patients compared to controls. The same results were observed when we studied the expression comparing individually patients and health blood donors, suggesting that this protein could be increased in all human cells that present the translocation BCR-ABL. The level of expression of this gene in HL60 and NB4 was significantly lower than in K562 cell line. The experiments with mice cell lines showed a higher expression of this gene in BaF3T315I when compared to BaF3BCR-ABLp210. We obtained a significant expression difference in all experiments (p <0.05). The spontaneous and stimulated with IL-8 chemotaxis assays used granulocytes and were assessed using chamber containing 96 wells. However, although the results suggest an increased chemotactic activity in patients, there were no significant differences (p<0.05) between controls and patients – regardless of whether the chemotaxis was spontaneous or stimulated with IL-8. In mammals the SEPT5 gene is associated with cellular processes such as exocytosis, apoptosis, leukemogenesis, carcinogenesis and neurodegeneration. Therefore, molecules capable of interacting with the septins, either at biochemical or molecular level, can bring information about their functions in cytokinesis. Studies indicate that the human septins can interact among themselves and with other components of the cytoskeleton – this may be a relevant observation regarding the function of this gene in cancer. The SEPT5 can be activated by different pathways – this may increase expression in translocated cells. Despite major advances in the treatment of CML, the treatments available are not capable of inactivating all the signaling pathways activated by BCR/ABL. Our results demonstrate that SEPT5 may be involved in the pathophysiology of CML. Also, it is clear the importance of the study of pathways that could culminate in its high expression or the triggering of other unknown pathways involved in the development of CML. The increased expression of this gene may be related to disease progression, and finally, the identification of several important genes may lead to a better understanding of CML and helping to identify new therapeutic targets. FAPESP/INCT. Disclosures: No relevant conflicts of interest to declare.

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