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1.
Oncotarget ; 8(10): 16712-16727, 2017 Mar 07.
Article in English | MEDLINE | ID: mdl-28187429

ABSTRACT

The molecular determinants governing escape of Acute Myeloid Leukemia (AML) cells from DNA damaging therapy remain poorly defined and account for therapy failures. To isolate genes responsible for leukemia cells regeneration following multiple challenges with irradiation we performed a genome-wide shRNA screen. Some of the isolated hits are known players in the DNA damage response (e.g. p53, CHK2), whereas other, e.g. SMYD2 lysine methyltransferase (KMT), remains uncharacterized in the AML context. Here we report that SMYD2 knockdown confers relative resistance to human AML cells against multiple classes of DNA damaging agents. Induction of the transient quiescence state upon SMYD2 downregulation correlated with the resistance. We revealed that diminished SMYD2 expression resulted in the upregulation of the related methyltransferase SET7/9, suggesting compensatory relationships. Indeed, pharmacological targeting of SET7/9 with (R)-PFI2 inhibitor preferentially inhibited the growth of cells expressing low levels of SMYD2.Finally, decreased expression of SMYD2 in AML patients correlated with the reduced sensitivity to therapy and lower probability to achieve complete remission. We propose that the interplay between SMYD2 and SET7/9 levels shifts leukemia cells from growth to quiescence state that is associated with the higher resistance to DNA damaging agents and rationalize SET7/9 pharmacological targeting in AML.


Subject(s)
Histone-Lysine N-Methyltransferase/genetics , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/genetics , Cell Growth Processes/physiology , DNA Damage/physiology , Down-Regulation , Drug Resistance, Neoplasm , Gene Knockdown Techniques , HEK293 Cells , Histone-Lysine N-Methyltransferase/metabolism , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , RNA, Small Interfering/genetics , Transfection
2.
ACS Synth Biol ; 6(4): 628-637, 2017 04 21.
Article in English | MEDLINE | ID: mdl-28033709

ABSTRACT

It is increasingly practical to co-opt many native cellular components into use as elements of synthetic biological systems. We present the design and experimental investigation of the first exogenous genetic construct to be successfully targeted by RNA activation, a phenomenon whereby small double-stranded RNAs increase gene expression from sequence-similar promoters by a mechanism thought to be related to that of RNA interference. Our selection of activating RNA candidates was informed by a custom-written computer program designed to choose target sites in the promoter of interest according to a set of empirical optimality criteria drawn from prior research. Activating RNA candidates were assessed for activity against two exogenously derived target promoters, with successful candidates being subjected to further rounds of validation as a precaution against potential off-target effects. A genetic platform was assembled that allowed activating RNA candidates to be simultaneously screened both for positive activity on the target reporter gene and for possible nonspecific effects on cell metabolism. Several candidate sequences were tested to appraise the utility of this platform, with the most successful achieving a moderate activation level with minimal off-target effects.


Subject(s)
Gene Targeting/methods , RNA, Double-Stranded/metabolism , Cell Line , Gene Expression , Genes, Reporter , Genetic Vectors/genetics , Genetic Vectors/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Promoter Regions, Genetic , RNA Interference , RNA, Double-Stranded/antagonists & inhibitors , RNA, Double-Stranded/genetics , Transfection , User-Computer Interface
3.
Mol Syst Biol ; 9: 696, 2013 Oct 08.
Article in English | MEDLINE | ID: mdl-24104479

ABSTRACT

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.


Subject(s)
Breast Neoplasms/genetics , Epistasis, Genetic , Genes, Essential , Neoplasm Proteins/genetics , Ovarian Neoplasms/genetics , PTEN Phosphohydrolase/genetics , Pancreatic Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Coculture Techniques , Female , Gene Regulatory Networks , Genome, Human , Humans , Mutation , Neoplasm Proteins/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , PTEN Phosphohydrolase/deficiency , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism
4.
Cancer Discov ; 2(2): 172-189, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22585861

ABSTRACT

UNLABELLED: Genomic analyses are yielding a host of new information on the multiple genetic abnormalities associated with specific types of cancer. A comprehensive description of cancer-associated genetic abnormalities can improve our ability to classify tumors into clinically relevant subgroups and, on occasion, identify mutant genes that drive the cancer phenotype ("drivers"). More often, though, the functional significance of cancer-associated mutations is difficult to discern. Genome-wide pooled short hairpin RNA (shRNA) screens enable global identification of the genes essential for cancer cell survival and proliferation, providing a "functional genomic" map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting ~16,000 genes and a newly developed, dynamic scoring approach, we identified essential gene profiles in 72 breast, pancreatic, and ovarian cancer cell lines. Integrating our results with current and future genomic data should facilitate the systematic identification of drivers, unanticipated synthetic lethal relationships, and functional vulnerabilities of these tumor types. SIGNIFICANCE: This study presents a resource of genome-scale, pooled shRNA screens for 72 breast, pancreatic, and ovarian cancer cell lines that will serve as a functional complement to genomics data, facilitate construction of essential gene profiles, help uncover synthetic lethal relationships, and identify uncharacterized genetic vulnerabilities in these tumor types. SIGNIFICANCE: This study presents a resource of genome-scale, pooled shRNA screens for 72 breast, pancreatic, and ovarian cancer cell lines that will serve as a functional complement to genomics data, facilitate construction of essential gene profiles, help uncover synthetic lethal relationships, and identify uncharacterized genetic vulnerabilities in these tumor types.


Subject(s)
Breast Neoplasms/genetics , Ovarian Neoplasms/genetics , Pancreatic Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Female , Gene Library , Humans , Male , Ovarian Neoplasms/metabolism , Pancreatic Neoplasms/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Transcriptome
5.
BMC Genomics ; 10: 642, 2009 Dec 30.
Article in English | MEDLINE | ID: mdl-20042094

ABSTRACT

BACKGROUND: Emergence of multi-drug resistant (MDR) serotype 19A Streptococcus pneumoniae (SPN) is well-documented but causal factors remain unclear. Canadian SPN isolates (1993-2008, n = 11,083) were serotyped and in vitro susceptibility tested. A subset of MDR 19A were multi-locus sequence typed (MLST) and representative isolates' whole genomes sequenced. RESULTS: MDR 19A increased in the post-PCV7 era while 19F, 6B, and 23F concurrently declined. MLST of MDR 19A (n = 97) revealed that sequence type (ST) 320 predominated. ST320 was unique amongst MDR 19A in that its minimum inhibitory concentration (MIC) values for penicillin, amoxicillin, ceftriaxone, and erythromycin were higher than for other ST present amongst post-PCV7 MDR 19A. DNA sequencing revealed that alleles at key drug resistance loci pbp2a, pbp2x, pbp2b, ermB, mefA/E, and tetM were conserved between pre-PCV7 ST 320 19F and post-PCV7 ST 320 19A most likely due to a capsule switch recombination event. A genome wide comparison of MDR 19A ST320 with MDR 19F ST320 identified 822 unique SNPs in 19A, 61 of which were present in antimicrobial resistance genes and 100 in virulence factors. CONCLUSIONS: Our results suggest a complex genetic picture where high-level drug resistance, vaccine selection pressure, and SPN mutational events have created a "perfect storm" for the emergence of MDR 19A.


Subject(s)
Comparative Genomic Hybridization , Drug Resistance, Multiple, Bacterial/genetics , Genome, Bacterial , Streptococcus pneumoniae/genetics , Anti-Bacterial Agents/pharmacology , Canada , Computational Biology , DNA, Bacterial/genetics , Heptavalent Pneumococcal Conjugate Vaccine , Microbial Sensitivity Tests , Pneumococcal Vaccines , Sequence Analysis, DNA , Serotyping , Streptococcus pneumoniae/classification , Streptococcus pneumoniae/drug effects
6.
PLoS Genet ; 4(4): e1000051, 2008 Apr 11.
Article in English | MEDLINE | ID: mdl-18404216

ABSTRACT

The Locus Control Region (LCR) requires intronic elements within beta-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional beta-globin intron 2 elements that rescue LCR activity directed by 5'HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igmu 3'MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igmu 3'MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5'HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5'HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors.


Subject(s)
Genetic Therapy/methods , Globins/genetics , Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/therapy , Animals , Base Sequence , Binding Sites/genetics , Cell Line , DNA Primers/genetics , Enhancer Elements, Genetic , Female , Gene Expression , Genetic Complementation Test , Genetic Vectors , Introns , Locus Control Region , Male , Mice , Mice, Transgenic , Octamer Transcription Factor-1/metabolism , Pregnancy , Recombinant Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction
7.
Nucleic Acids Res ; 33(14): 4412-24, 2005.
Article in English | MEDLINE | ID: mdl-16085752

ABSTRACT

The origin of DNA replication in the human beta-globin gene contains an initiation region (IR) and two flanking auxiliary elements. Two replicator modules are located within the upstream auxiliary sequence and the IR core, but the functional sequences in the downstream auxiliary element are unknown. Here, we use a combination of benzoylated-naphthoylated DEAE (BND) cellulose purification and nascent strand abundance assays to show that replication initiation occurs at the beta-globin 3' enhancer on human chromosome 11 in the Hu11 hybrid murine erythroleukemia (MEL) cell line. To examine replicator function, 3' enhancer fragments were inserted into an ectopic site in MEL cells via an optimized FRT/EGFP-FLP integration system. These experiments demonstrate that the 1.6 kb downstream auxiliary element is a third replicator module called bGRep-E in erythroid cells. The minimal 260 bp 3' enhancer is required but not sufficient to initiate efficient replication, suggesting cooperation with adjacent sequences. The minimal 3' enhancer also cooperates with elements in an expressing HS3beta/gamma-globin construct to initiate replication. These data indicate that the beta-globin replicator has multiple initiation sites in three closely spaced replicator modules. We conclude that a mammalian enhancer can cooperate with adjacent sequences to create an efficient replicator module.


Subject(s)
3' Flanking Region , DNA Replication , Enhancer Elements, Genetic , Globins/genetics , Replication Origin , Animals , Cell Line , Chromatography, Affinity , DNA, Single-Stranded/isolation & purification , Humans , Locus Control Region , Mice , Recombination, Genetic
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