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RNA interference (RNAi) Natasha Caplen Gene Silencing Section, Genetics Branch, CCR, NCI

RNA interference (RNAi) Natasha Caplen Gene Silencing Section, Genetics Branch, CCR, NCI. RNAi: Truly a story of translational research.

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RNA interference (RNAi) Natasha Caplen Gene Silencing Section, Genetics Branch, CCR, NCI

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  1. RNA interference(RNAi)Natasha CaplenGene Silencing Section, Genetics Branch, CCR, NCI

  2. RNAi: Truly a story of translational research ●RNAi: ● Truly a story of translational research ●Plants & Fungi●C. elegans●Mammals● Clinical trialsDescription of phenomena● Identification of dsRNA as mediator● Application of siRNAs Humanapplication● Early 1990s ● Late 1990s● Early 2000s● Mid 2000s Plants & Fungi C. elegans Mammals Clinical trials Description of phenomena Identification of dsRNA as mediator Application of siRNAs Humanapplication Early 1990s Late 1990s Early 2000s Mid 2000s

  3. Plants C. elegans PlantsNapoli et al., Plant Cell (1990) 2 289 Van der Krol et al., Plant Cell (1990) 2 291RNA interferenceDouble stranded RNAdsRNA processingSmall interfering RNAs (SiRNAs) RNA-induced silencing complex (RISC) Pasquienelli et al., Nature (2000) 408 37Lee & Ambros Science (2001) 294 862 C. elegansFire et al., Nature (1998) 391 806DrosophilaMisquitta & Paterson PNAS (1999) 96 1451Kennerdell & Carthew Cell (1998) 95 1017MammalsCaplen et al., PNAS (2001) 98 9742Elbashir et al., Nature (2001) 411 494 RNA interference Double stranded RNA Napoli et al., Plant Cell (1990) 2 289 Van der Krol et al., Plant Cell (1990) 2 291 Fire et al., Nature (1998) 391 806 dsRNA processing Fungi Drosophila Small interfering RNAs (SiRNAs) s Misquitta & Paterson PNAS (1999) 96 1451 Kennerdell & Carthew Cell (1998) 95 1017 RNA-induced silencing complex (RISC) Romano & Macino Mol. Microbiol (1992) 6 3343 Mammals S. pombe Caplen et al., PNAS (2001) 98 9742 Elbashir et al., Nature (2001) 411 494 Pasquienelli et al., Nature (2000) 408 37 Lee & Ambros Science (2001) 294 862 Volpe et al., Science (2003) 292 1833 Caplen, Genetics Branch, CCR, NCI

  4. RNAi gene silencing pathways RNAi gene silencing pathways Caplen, Genetics Branch, CCR, NCI

  5. Implications for the identification and application of RNAi Basic Science Places RNA as a central player in the regulation of gene expression Role of RNAi in cellular responses to external stimuli The role of RNA and RNAi in host-pathogen (foreign nucleic acid) responses Role of RNAi in disease initiation, progression and response to therapy Applications A biological tool that can be used to test hypotheses drawn from the large amount of genetic, expression and functional data now available - Gene: protein function discovery and analysis - Pathway/network analysis - Molecular target identification, validation and synergistic interactions - Drug Development: mechanism of action, drug/protein relationships Enables large scale gene-phenotype analysis Development of novel model systems Development of novel RNAi therapeutic approaches Caplen, Genetics Branch, CCR, NCI

  6. RNAi1. The use of RNAi-based technologies to discover and interrogate the function of cancer genes, including those that impact anti-cancer drug activity.2. Investigation of the role that RNAi plays in the dysregulated gene expression that is the hallmark of cancer.● Caplen, Genetics Branch, CCR, NCI RNAi The use of RNAi-based technologies to discover and interrogate the function of cancer genes, including those that impact anti-cancer drug activity. Investigation of the role that RNAi plays in the dysregulated gene expression that is the hallmark of cancer. Caplen, Genetics Branch, CCR, NCI

  7. RNAi1: The induction of gene-specific RNAi for the study of cancer biology.2: The application of RNAi analysis for the study of gene:drug interactions relevant to anti-cancer therapeutic approaches.3: The role of miRNA-mediated RNAi in the biology of cancer.●Caplen, Genetics Branch, CCR, NCI RNAi 1: The induction of gene-specific RNAi for the study of cancer biology. 2: The application of RNAi analysis for the study of gene:drug interactions relevant to anti-cancer therapeutic approaches. 3: The role of miRNA-mediated RNAi in the biology of cancer. Caplen, Genetics Branch, CCR, NCI

  8. RNAi: 1: The induction of gene-specific RNAi for the study of cancer biology.2: The application of RNAi analysis for the study of gene:drug interactions relevant to anti-cancer therapeutic approaches.

  9. The induction of gene-specific RNAi against cancer-associated genes ● Caplen, Genetics Branch, CCR, NCI The induction of gene-specific RNAi against cancer-associated genes Caplen, Genetics Branch, CCR, NCI

  10. The induction of gene-specific RNAi against cancer-associated genes●Caplen, Genetics Branch, CCR, NCI The induction of gene-specific RNAi against cancer-associated genes Delivery siRNA shRNA Optimization Model system Efficacy Specificity Assays mRNA Protein Function Caplen, Genetics Branch, CCR, NCI

  11. siRNA transfection protocol ●Caplen, Genetics Branch, CCR, NCI siRNA transfection protocol Caplen, Genetics Branch, CCR, NCI

  12. RNAi characterization: RNA assay ß-catenin ● RNAi characterization: RNA assay ß-catenin ●Normalized mRNA levels are measured by Branched DNA RNA assay (Panomics Inc.) Cyclophilin B used for normalization●Martin, et al., Nuc. Acids Res. (2007) 35(8) e57●Caplen, Genetics Branch, CCR, NCI Normalized mRNA levels are measured by Branched DNA RNA assay (Panomics Inc.) Cyclophilin B used for normalization Martin, et al., Nuc. Acids Res. (2007) 35(8) e57 Caplen, Genetics Branch, CCR, NCI

  13. RNAi characterization: Protein analysis Quantitative Western based assay ● RNAi characterization: Protein analysis Quantitative Western based assay●Martin, et al., Nuc. Acids Res. (2007) 35(8) e57 ●Caplen, Genetics Branch, CCR, NCI Caplen, Genetics Branch, CCR, NCI Martin, et al., Nuc. Acids Res. (2007) 35(8) e57

  14. ● The induction of gene-specific RNAi against cancer-associated genes●Martin et al., Nuc. Acids. Res. (2007) 35 e57●Caplen, Genetics Branch, CCR, NCI The induction of gene-specific RNAi against cancer-associated genes Martin et al., Nuc. Acids. Res. (2007) 35 e57 Caplen, Genetics Branch, CCR, NCI

  15. shRNA analysis●Gehlhaus & Caplen unpublished In collaboration with A. Glatfelter, R. Cornelison, P. Meltzer●Caplen, Genetics Branch, CCR, NCI shRNA analysis Gehlhaus & Caplen unpublished In collaboration with A. Glatfelter, R. Cornelison, P. Meltzer Caplen, Genetics Branch, CCR, NCI

  16. Ranking of the percent decrease in mRNA levels mediated by 258 siRNAs (129 human genes) ● Ranking of the percent decrease in mRNA levels mediated by 258 siRNAs (129 human genes) ●Martin, et al., Nuc. Acids Res. (2007) 35(8) e57 ● Caplen, Genetics Branch, CCR, NCI Martin, et al., Nuc. Acids Res. (2007) 35(8) e57 Caplen, Genetics Branch, CCR, NCI

  17. Correlation of silencing mediated by two different siRNAs corresponding to the same gene TP53 BAG1 Correlation of silencing mediated by two different siRNAs corresponding to the same gene ● Martin, et al., Nuc. Acids Res. (2007) 35(8) e57 Martin, et al., Nuc. Acids Res. (2007) 35(8) e57 Caplen, Genetics Branch, CCR, NCI

  18. Sequence variation and RNAi Sequence variation and RNAi ● Martin, et al., Nuc. Acids Res. (2007) 35(8) e57 Martin, et al., Nuc. Acids Res. (2007) 35(8) e57 Caplen, Genetics Branch, CCR, NCI

  19. Sequence variation and RNAi ● Sequence variation and RNAi ●Klootwijk et al., FASEB J. (2008) Jul 24 ● Caplen, Genetics Branch, CCR, NCI Klootwijk et al., FASEB J. (2008) Jul 24 Caplen, Genetics Branch, CCR, NCI

  20. Transcript variation and RNAi Transcript variation and RNAi ● Martin, et al., Nuc. Acids Res. (2007) 35(8) e57 ●Caplen, Genetics Branch, CCR, NCI Martin, et al., Nuc. Acids Res. (2007) 35(8) e57 Caplen, Genetics Branch, CCR, NCI

  21. Transcript variation and RNAi Transcript variation and RNAi ● Martin, et al., Nuc. Acids Res. (2007) 35(8) e57● Caplen, Genetics Branch, CCR, NCI Martin, et al., Nuc. Acids Res. (2007) 35(8) e57 Caplen, Genetics Branch, CCR, NCI

  22. Transcript variation and RNAi ● SpliceCenter: a suite of web-based bioinformatic applications for evaluating the impact of alternative splicing on RT-PCR, RNAi (siRNA-Check), microarray, and peptide based studies ●Ryan et al., BMC Bioinformatics. (2008) 9 313● Caplen, Genetics Branch, CCR, NCI Transcript variation and RNAiSpliceCenter: a suite of web-based bioinformatic applications for evaluating the impact of alternative splicing on RT-PCR, RNAi (siRNA-Check), microarray, and peptide based studies Ryan et al., BMC Bioinformatics. (2008) 9 313 Caplen, Genetics Branch, CCR, NCI

  23. ● Functional effect of silencing PLK1●P. Lorenzi & J. Llamas unpublished●Caplen, Genetics Branch, CCR, NCI Functional effect of silencing PLK1 P. Lorenzi & J. Llamas unpublished Caplen, Genetics Branch, CCR, NCI

  24. RNAi screens • C. elegans - whole organism analysis, • plasmid based feeding libraries • Drosophila - passive dsRNA into established and primary cell lines • Human - siRNA and shRNA based libraries up to to whole genome • C. elegans - whole organism analysis, plasmid based feeding libraries • Drosophila - passive dsRNA into established and primary cell lines • Human - siRNA and shRNA based libraries up to to whole genome●Caplen, Genetics Branch, CCR, NCI Caplen, Genetics Branch, CCR, NCI

  25. ARRAYED RNAi SCREENS ●ARRAYED RNAi SCREENS ●Adapted from Martin & Caplen Annu. Rev. Genomics Human. Genet. (2007)●Caplen, Genetics Branch, CCR, NCI Adapted from Martin & Caplen Annu. Rev. Genomics Human. Genet. (2007) Caplen, Genetics Branch, CCR, NCI

  26. Large scale RNAi analysis: siRNAs Sensitized RNAi screens of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance. MacKeigan et al. Nature Cell Biology (2005). ● Sensitized RNAi screens of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance. MacKeigan et al. Nature Cell Biology (2005). ●HeLa cells seeded in 96-well plates were transfected with siRNAs directed against all known and putative human kinases. Cells were incubated for 72 h to allow target knockdown, and apoptosis was measured by a DNA-fragmentation ELISA. The graph shows the relative apoptosis for all 650 kinase siRNA targets, and basal cell survival is set at 1. ●Caplen, Genetics Branch, CCR, NCI HeLa cells seeded in 96-well plates were transfected with siRNAs directed against all known and putative human kinases. Cells were incubated for 72 h to allow target knockdown, and apoptosis was measured by a DNA-fragmentation ELISA. The graph shows the relative apoptosis for all 650 kinase siRNA targets, and basal cell survival is set at 1. Caplen, Genetics Branch, CCR, NCI

  27. RNAi screens in human cells ●RNAi screens in human cells ●Adapted from Martin & Caplen Annu. Rev. Genomics Human. Genet. (2007) ●Caplen, Genetics Branch, CCR, NCI Adapted from Martin & Caplen Annu. Rev. Genomics Human. Genet. (2007) Caplen, Genetics Branch, CCR, NCI

  28. ● Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB Brummelkamp et al., (2003) Nature 424 797●CYLD Cylindromatosis (turban tumor syndrome) ●DUB De-ubiquitination enzyme focused library●Caplen, Genetics Branch, CCR, NCI Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB Brummelkamp et al., (2003) Nature 424 797 CYLD Cylindromatosis (turban tumor syndrome) DUB De-ubiquitination enzyme focused library Caplen, Genetics Branch, CCR, NCI

  29. An evaluation of the efficacy of topical application of salicylic acid for the treatment of familial cylindromatosisOosterkamp et al., British Journal of Dermatology (2006) 155 182 ● ● An evaluation of the efficacy of topical application of salicylic acid for the treatment of familial cylindromatosisOosterkamp et al., British Journal of Dermatology (2006) 155 182 ● Caplen, Genetics Branch, CCR, NCI Caplen, Genetics Branch, CCR, NCI

  30. ● Echeverri & Perrimon.Nature Reviews Genetics;published online 11 April 2006 | doi:10.1038/nri1836● Caplen, Genetics Branch, CCR, NCI Echeverri & Perrimon.Nature Reviews Genetics; published online 11 April 2006 | doi:10.1038/nri1836 Caplen, Genetics Branch, CCR, NCI

  31. Large scale RNAi analysis: shRNAs Extramural consortia (US) developing whole genome shRNA libraries Hannon (CSH)-Elledge (Harvard): distributed by Open Biosystems. Human library: : Retoviral backbone (pyro selection). : 2.8 shRNA/accession number : miRNA flanking sequence for improved processing. : “pMagic” cloning strategy & optimized bacterial background. : 60mer barcode adjacent to shRNA for genetic screen. The RNAi consortium (TRC) : distributed by Sigma and Open Biosystems The Broad Institute, Harvard, MIT, (Whitehead) Human library : Lentiviral backbone : 10 shRNA/ accession number : No miRNA sequences : No separate barcode Extramural consortia (US) developing whole genome shRNA libraries Hannon (CSH)-Elledge (Harvard): distributed by Open Biosystems. Human library: : Retoviral backbone (pyro selection). : 2.8 shRNA/accession number : miRNA flanking sequence for improved processing. : “pMagic” cloning strategy & optimized bacterial background. : 60mer barcode adjacent to shRNA for genetic screen. The RNAi consortium (TRC) : distributed by Sigma and Open Biosystems The Broad Institute, Harvard, MIT, (Whitehead) Human library : Lentiviral backbone : 10 shRNA/ accession number : No miRNA sequences : No separate barcode●Caplen, Genetics Branch, CCR, NCI Caplen, Genetics Branch, CCR, NCI

  32. Cancer Proliferation Gene Discovery Through Functional GenomicsSchlabach et al., Science (2008) 319 620 – 624 ● Cancer Proliferation Gene Discovery Through Functional GenomicsSchlabach et al., Science (2008) 319 620 – 624 ●Overview of the pool-based dropout screen with barcode microarrays. (A) Schematic of library construction and screening protocol. (B) Schematic of the HH barcode hybridization. (C) Comparison between HH amplicons (top) and full-hairpin PCR amplicons (bottom) on an HH probe microarray● Caplen, Genetics Branch, CCR, NCI Overview of the pool-based dropout screen with barcode microarrays. (A) Schematic of library construction and screening protocol. (B) Schematic of the HH barcode hybridization. (C) Comparison between HH amplicons (top) and full-hairpin PCR amplicons (bottom) on an HH probe microarray Caplen, Genetics Branch, CCR, NCI

  33. Cancer Proliferation Gene Discovery Through Functional GenomicsSchlabach et al., Science (2008) 319 620 – 624 ●Cancer Proliferation Gene Discovery Through Functional GenomicsSchlabach et al., Science (2008) 319 620 – 624 ●Caplen, Genetics Branch, CCR, NCI Genes commonly required for proliferation or survival of normal and cancer cells. (A) Representative candidate shRNAs that reduce viability of all four cell lines. (B) Examples of core cellular modules required in all four cell lines. Shown are the APC/C (top left), the COP9 signalosome (top right), the eIF3 translation initiation complex (bottom left), and E3 ubiquitin ligases (bottom right). Genes required for viability of one (yellow), two (orange), three (red), or all four (black) cell lines. (C) Validation of selected shRNAs from the screen that reduce viability of all four cell lines. Caplen, Genetics Branch, CCR, NCI

  34. Issues to remember 1 • RNAi is a knock-down (not a knock-out) • The degree of knockdown at an RNA and protein level can be highly variable even for RNAi effectors targeting the same transcript and • An RNAi phenotype will be dependent on a large number of parameters (most of which will not be linear). • RNAi efficacy • What knock down level is enough? Where is the “tipping point”? • Magic number usually reported is at a least a ~70% knockdown • Will be protein specific - some enzymes 95% knockdown may still be enough for activity, some structural proteins may only require 40 to 50% knockdown to induce a phenotype • The generation of hypomorphs may be of enormous use in assessing protein function • One gene may not be enough to generate phenotype • Functional redundancy • Multiple members of a complex • Multiple members of a pathway or network • Caplen, Genetics Branch, CCR, NCI Caplen, Genetics Branch, CCR, NCI

  35. Issues to remember: 2 • The timing of any analysis may be critical • The rate of RNA cleavage following RNAi is relatively predictable but changes in protein and down stream effects are not. • Further, synthetic siRNA mediated RNAi is transient (24-96 hrs), and longer term silencing through use of shRNA may select against the RNAi effect (loss of silencing effect, functional redundancy). • The specificity of RNAi is relative • Need to maximize the specific effect of targeting a transcript through perfect sequence alignment and RNA cleavage while minimizing off-target effects as a result of miRNA-like interactions - partial sequence. • Need to minimize mammalian cellular responses to dsRNA. • Need to minimize effects on the endogenous role of the RNAi pathway. • The timing of any analysis may be critical • The rate of RNA cleavage following RNAi is relatively predictable but changes in protein and down stream effects are not. • Further, synthetic siRNA mediated RNAi is transient (24-96 hrs), and longer term silencing through use of shRNA may select against the RNAi effect (loss of silencing effect, functional redundancy). • The specificity of RNAi is relative • Need to maximize the specific effect of targeting a transcript through perfect sequence alignment and RNA cleavage while minimizing off-target effects as a result of miRNA-like interactions - partial sequence. • Need to minimize mammalian cellular responses to dsRNA. • Need to minimize effects on the endogenous role of the RNAi pathway. ●Caplen, Genetics Branch, CCR, NCI Caplen, Genetics Branch, CCR, NCI

  36. RNAi and off target effects RNAi and off target effects ●Caplen, Genetics Branch, CCR, NCI Caplen, Genetics Branch, CCR, NCI

  37. In vivo RNAi in mice: Transient In vivo RNAi in mice: Transient ●Mediated by eithersiRNAs or short hairpin RNA (shRNA) transcripts ●McCaffrey et al., Gene expression: RNA interference in adult mice Nature (2002) 418 38●See also Lewis et al., (2002) Nature Genetics 32 107Caplen, Genetics Branch, CCR, NCI Mediated by eithersiRNAs or short hairpin RNA (shRNA) transcripts McCaffrey et al., Gene expression: RNA interference in adult mice Nature (2002) 418 38 See also Lewis et al., (2002) Nature Genetics 32 107 Caplen, Genetics Branch, CCR, NCI

  38. In vivo RNAi in mice: Transgenics In vivo RNAi in mice: Transgenics Acceleration of E-Myc–induced lymphomagenesis by TP53 shRNAs generating RNAi hypomorphs.●Hermann et al., (2003) ●Nature Genetics 33 396 Acceleration of E-Myc–induced lymphomagenesis by TP53 shRNAs generating RNAi hypomorphs. • Germ-line transmission of expressed shRNAs in ES cells ●Generation of GFP knockdown mice.●Tiscornia et al., (2003) • PNAS USA 100 1844 ●Caplen, Genetics Branch, CCR, NCI Hermann et al., (2003) Nature Genetics 33 396 Germ-line transmission of expressed shRNAs in ES cells Generation of GFP knockdown mice. Tiscornia et al., (2003) PNAS USA 100 1844 Caplen, Genetics Branch, CCR, NCI

  39. ‘Yes, it was disappointing. Best hope now apparently is RNA interference.’ ‘Yeah. Gene Silencing. One day perhaps. After I’m dead.’ “Saturday” Ian McEwan (2005) Therapeutic potential of RNAi ‘Yes, it was disappointing. Best hope now apparently is RNA interference.’ ‘Yeah. Gene Silencing. One day perhaps. After I’m dead.’ “Saturday” Ian McEwan (2005) ●Caplen, Genetics Branch, CCR, NCI Caplen, Genetics Branch, CCR, NCI

  40. Therapeutic potential of RNAi: Cancer Brummelkamp et al., Stable suppression of tumorigenicity by virus-mediated RNA interference Cancer Cell (2002) 2 243 ●Therapeutic potential of RNAi: Cancer ●Brummelkamp et al., Stable suppression of tumorigenicity by virus-mediated RNA interference Cancer Cell (2002) 2 243●RAS genes are frequently mutated in human cancers, e.g. 85% of pancreatic cancers, 40% or colon carcinomas harbor mutations. RAS genes are guanine nucleotide binding proteins and act as intracellular a signaling proteins and are required for regulation of cell proliferation, differentiation and survival.Therapeutic intervention requires specific elimination of the product from the mutant oncogenic allele ●Stable & selective loss of tumorigenicity by a retroviral vector that targets the K-RASV12 oncogene.●Caplen, Genetics Branch, CCR, NCI RAS genes are frequently mutated in human cancers, e.g. 85% of pancreatic cancers, 40% or colon carcinomas harbor mutations. RAS genes are guanine nucleotide binding proteins and act as intracellular a signaling proteins and are required for regulation of cell proliferation, differentiation and survival. Therapeutic intervention requires specific elimination of the product from the mutant oncogenic allele Stable & selective loss of tumorigenicity by a retroviral vector that targets the K-RASV12 oncogene. Caplen, Genetics Branch, CCR, NCI

  41. In vivo development of RNAi therapeutics ●In vivo development of RNAi therapeutics ●Martin & Caplen Annu. Rev. Genomics Human. Genet. (2007) ●Caplen, Genetics Branch, CCR, NCI Martin & Caplen Annu. Rev. Genomics Human. Genet. (2007) Caplen, Genetics Branch, CCR, NCI

  42. RNAi RNAi 1: The induction of gene-specific RNAi for the study of cancer biology. 2: The application of RNAi analysis for the study of gene:drug interactions relevant to anti-cancer therapeutic approaches. 1: The induction of gene-specific RNAi for the study of cancer biology. 2: The application of RNAi analysis for the study of gene:drug interactions relevant to anti-cancer therapeutic approaches. ●Caplen, Genetics Branch, CCR, NCI Caplen, Genetics Branch, CCR, NCI

  43. ●Application of RNAi analysis and screening to the drug development pipeline●Adapted from the Pharmacogenetics and Pharmacogenomics Knowledge Base●Caplen, Genetics Branch, CCR, NCI Application of RNAi analysis and screening to the drug development pipeline Adapted from the Pharmacogenetics and Pharmacogenomics Knowledge Base Caplen, Genetics Branch, CCR, NCI

  44. The induction of gene-specific RNAi against cancer-associated genes ●The induction of gene-specific RNAi against cancer-associated genes ●Caplen, Genetics Branch, CCR, NCI Caplen, Genetics Branch, CCR, NCI

  45. The functional validation of gene-drug interactions using RNAi analysisCaplen, Genetics Branch, CCR, NCI The functional validation of gene-drug interactions using RNAi analysis 1 2 3 Caplen, Genetics Branch, CCR, NCI

  46. Discovery of a correlation between L-ASP activity and asparagine synthetase (ASNS) expression Discovery of a correlation between L-ASP activity and asparagine synthetase (ASNS) expression ●Caplen, Genetics Branch, CCR, NCI Scherf et al., Nat. Genet (2000) 24 236 Bussey et al., Mol Cancer Ther. (2006) 5 853 Lorenzi et al, Mol. Cancer Ther. (2006) 5 2613 Question: Is the relationship causal? Caplen, Genetics Branch, CCR, NCI

  47. Rationale for Therapy with L-Asparaginase (L-ASP)●Caplen, Genetics Branch, CCR, NCI Rationale for Therapy with L-Asparaginase (L-ASP) L-ASP depletes blood levels of the amino acid asparagine effecting tumor growth L-asparaginase (L-ASP) has been FDA-approved for more than 30 yrs. for the treatment of acute lymphoblastic leukemia (ALL). Cancer Cell: Acute Lymphoblastic Leukemia Bacterial L-ASP Asparagine from Bloodstream Endogenous ASNS Cell Starves Asparagine Cell proliferation Individual response to L-ASP treatment is highly variable. Caplen, Genetics Branch, CCR, NCI

  48. ASNS mRNA expression after transfection of siRNAs corresponding to ASNS●Lorenzi et al, Mol. Cancer Therapeutics (2006) 5 2613 ●Caplen, Genetics Branch, CCR, NCI ASNS mRNA expression after transfection of siRNAs corresponding to ASNS 2 Asparagine synthetase expression (log2) OVCAR-3 OVCAR-4 0 -2 OVCAR-8 -1 0 1 L-asparaginase activity [-log(GI50)] Lorenzi et al, Mol. Cancer Therapeutics (2006) 5 2613 Caplen, Genetics Branch, CCR, NCI ASNS mRNA expression after transfection of siRNAs corresponding to ASNS

  49. ASNS Protein Expression after transfection of ASNS siRNAs● Lorenzi et al, Mol. Cancer Therapeutics (2006) 5 2613●Caplen, Genetics Branch, CCR, NCI ASNS Protein Expression after transfection of ASNS siRNAs MTS Assay 2 L-ASP Asparagine synthetase expression (log2) OVCAR-4 OVCAR-3 MTS Assay L-ASP MTS Assay L-ASP -2 OVCAR-8 -1 0 1 L-asparaginase activity [-log(GI50)] 0 Caplen, Genetics Branch, CCR, NCI

  50. L-ASP Cytotoxicity in ovarian cell lines is potentiated by ASNS siRNAs●Lorenzi et al, Mol. Cancer Therapeutics (2006) 5 2613 ●Caplen, Genetics Branch, CCR, NCI ~5-fold potentiation ~4-fold potentiation >500-fold potentiation L-ASP Cytotoxicity in ovarian cell lines is potentiated by ASNS siRNAs 2 Asparagine synthetase expression (log2) OVCAR-4 OVCAR-3 0 -2 OVCAR-8 -1 0 1 L-asparaginase activity [-log(GI50)] Lorenzi et al, Mol. Cancer Therapeutics (2006) 5 2613 Caplen, Genetics Branch, CCR, NCI

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