DOCSLIB.ORG

Sarcoma Genome Project (Phase I) Genome-Wide Molecular Genetic Analysis of 7 Sarcoma Types

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Sarcoma Genome Project (Phase I) Genome-Wide Molecular Genetic Analysis of 7 Sarcoma Types Liposarcoma Genomic Alterations Define New Targets for Therapy Samuel Singer, MD Memorial Sloan-Kettering Cancer Center Sarcoma Disease Management Program Chief Gastric and Mixed Tumor Service Liposarcoma • ~20% of 12,000 new STS cases in US each year, the majority are sporadic • Distinct cytogenetic subgroups comprising 5 subtypes • Simple (translocation-associated) − Myxoid and round cell liposarcoma: >90% t(12;16) • Complex rearrangements (alterations in cell cycle genes and checkpoint defects) – Well-differentiated and Dedifferentiated liposarcoma: ~90% 12q amplification – Pleomorphic liposarcoma • Genetic events associated with liposarcomagenesis remain undiscovered Therapeutic Challenges • More than 60% of patients with newly diagnosed liposarcoma eventually die of disease • Diverse histopathology and biological behavior • WDLS and DDLS are not very responsive to chemotherapy • Pressing need to develop subtype specific molecularly targeted therapeutics for patients with advanced/recurrent disease Histologic distribution of Liposarcoma subtype MSKCC Clinical Sarcoma Database 1772 liposarcoma patients over 29 years (1982–2011) Site‐specific histologic subtype distribution WD Sarcoma Genome Project (Phase I) Genome-wide molecular genetic analysis of 7 sarcoma types (Phase I) DNA copy number + LOH 207 sarcomas and Sequencing 225 matched normals genes and 451 Clinical microRNAs annotation database Transcriptional changes (MSKCC) (protein-coding genes) Functional genetic screen of amplified genes in DDLPS Sarcoma Genome Project (Phase I) Analysis of DNA sequence in 6 sarcoma types • Kit in GIST was the most frequently mutated gene • Next most frequently mutated genes in more than 5% of samples within a subtype were: genes sarcoma type % of samples drug able mutated mutated target PIK3CA myxoid/round cell 18 % PI3K inhibitors liposarcoma TP53 pleomorphic 17 % Small molecules liposarcoma that reactivate mutant p53 (PRIMA-1) NF1 myxofibrosarcoma 10.5 % MEK or mTOR inhibitors pleomorphic 8 % liposarcoma PIK3CA mutations in myxoid/round cell liposarcoma are associated with shorter disease- specific survival compared with wildtype (p-value = 0.036) E545K helical-domain mutations were associated with increased Akt phosphorylation relative to wildtype in myxoid/round cell liposarcoma TORC2 phosphorylation site PDK1 phosphorylation site Nucleotide and Copy-number alterations in Dedifferentiated Liposarcoma (n=50) and myxoid/round cell liposarcoma (n=21) • Consensus plot of statistically significant genome-wide copy-number alterations assessed by RAE Knockdown of the Dedifferentiated Liposarcoma “Amplicome” as a Functional Screen for Potential Driver Genes • Which amplified genes in DDLS are necessary for cancer cell proliferation and survival? – Genomics-driven RNAi screen in 3 genotype-matched DDLS cell lines – Systematic knockdown with shRNAs on 385 significantly amplified genes – 99 genes whose knockdown decreased cell growth, 27 of 99 were amplified in at least one cell line Functional Validation of CDK4 as a Therapeutic Target in DDLS Effect of three validated shRNAs targeting 1m selective CDK4/CDK6 CDK4 on the proliferation of two DDLS cell inhibitor PD0332991 induces a lines G1 growth arrest and senescence Therapeutics for DDLS • Potential targets: MDM2 antagonists Apoptosis Following 48hr Nutlin Treatment No Drug 60 • Nutlin-3 5uM Nutlin – MDM2 50 40 • Roche (R7112) – 30 20 10 prevents MDM2-p53 Annexin Annexin / / % % Apoptosis Apoptosis 0 interaction NADIP DDLS LS141 – CDK4 CDK4/CDK6 inhibitor senescence associated heterochromatic foci • PD0332991 – G1 arrest and senescence – AURKA AURKA inhibitor DDLS ND DDLS AK (5 µM) • MLN8237 – oral, ATP- competitive, disrupts assembly of mitotic spindle – mitotic delay – …other kinases (IRAK3, PCTK2)? Multi-center phase I trial of R7112 in patients with advanced malignancies • R7112 (Roche) is a small molecule antagonist of MDM2 – binds to the p53 site on the surface of MDM2 and blocks protein-protein interaction between MDM2 and p53 wt protein • Oral administration – MTD: 2500 mg/day, BID x 10 days with 18 days of rest associated with significant thrombocytopenia • Dosing schedule revised to 2500 mg/day, QD dosing x 5 days with 23 days rest • 20 patients with WDLS and DDLS treated to date on BID dosing schedule – 8 with stable disease at 8 weeks • Molecular and pharmacokinetic analysis – p53 mutation, MDM2 levels Phase II clinical trial of PD0332991 in patients with WDLS/DDLS that have CDK4 amplification by FISH and express the Rb protein by IHC • Patients with locally advanced, recurrent or metastatic WDLS or DDLS and disease progression on one prior systemic therapy • PD0332991 dose of 200 mg PO QD for 14 days, followed by 7 days of rest. – safe and tolerable in a phase I study – In phase 1 study two patients with advanced WDLS had stable disease for 3.1 and 2.3 years • Primary endpoint: patients who are progression-free at 12 weeks Phase II clinical trial of MLN8237 in patients with WDLS/DDLS and pleomorphic liposarcoma • Test a second-generation selective AURKA inhibitor in patients with advanced / metastatic liposarcoma • Primary objective: response rate (CR + PR) assessed at 12 weeks • Based on a completed phase I study, patients will be treated with MLN8237 50mg PO BID for 7 days, followed by 14 days of rest. Treatment is repeated every 3 weeks (one cycle) Sarcoma Genome Project (Phase II) • Perform a genome-wide genetic and functional analysis of well-differentiated (WDLS) and dedifferentiated (DDLS) Liposarcoma to identify: • distinct genomic subtypes • molecular markers that associate with outcome and pathologic features • genetic alterations associated with sarcoma progression • subtype specific therapeutic targets MSKCC Rockefeller BROAD Samuel Singer Thomas Tuschl Matthew Meyerson Marc Ladanyi Markus Hafner Jordi Barretina Chris Sander Alex Ramos Barry Taylor Shantanu Banerji Sample procurement and microRNA profiling Systematic shRNA screens selection / RNA and DNA extraction microRNA cloning and sequencing Solexa single molecule cDNA sequencing U133A Affy transcript arrays microRNA ISH 244K Agilent CGH array, Validation of candidate fusion array mutations Agilent microRNA array Computational analysis for protein-coding genes, microRNAs, amplified genes, gene rearrangements, activating mutations and activated pathways microRNA functional analysis Validate genetic, genomic and functional alterations Well-differentiated and Dedifferentiated Liposarcoma Disease-specific survival for primary Local recurrence-free survival for primary retroperitoneal liposarcoma: WDLS vs. DDLS retroperitoneal liposarcoma: WDLS vs. DDLS Data from MSKCC Sarcoma Database (n=345, 7/1/82 to 6/30/2010) • Resistant to chemotherapy • Can we exploit differentially expressed microRNAs as therapeutic targets for this deadly disease? Role of miRNAs in liposarcomagenesis • miRNAs regulate cell proliferation, apoptosis, and differentiation • miRNAs are abnormally regulated in cancer – may serve as oncogenes but generally down-regulated in tumors compared to normal tissue – inhibiting miRNA processing enhances tumorigenesis suggest miRNAs act mainly as tumorsuppressors • Profile miRNAs in normal fat (NF), WDLS and DDLS tissue samples to identify miRNAs that associate tissue type and tumor progression – Agilent microarrays (17 NF, 32 WDLS, 30 DDLS) – Deep sequencing of small RNA cDNA libraries (11 NF, 22 WDLS, 22 DDLS) Small RNA cDNA library preparation for sequencing • Developed a set of bar-coded sequencing adapters • parallel sequencing of up to 20 samples in one Solexa sequencing run, from < 2 µg total RNA / sample • > 200,000 sequence reads / sample at a lower cost than miRNA microarrays • map small RNAs to the genome; annotate by functional type • convert cloning frequencies to miRNA expression levels Hafner, M., Tuschl, T. Methods 2008 44:3-12 Unsupervised clustering of miRNAs in normal fat, WDLS and DDLS samples analyzed by deep sequencing DDLPS Normal fat tissue WDLPS Deep sequencing compared to Agilent microarray (DDLS / NF) miR-21 and and miR-26a are significantly over- expressed in DDLS compared to normal fat miR-21 miR-26a PTEN expression Normalized Counts Normalized NF WD DD NF WD DD NF WD DD miR-26a-2 encoded in the intron of CTDSP2, adjacent to CDK4 on chr12, is amplified in 88% of DDLS miR-143 and miR-145 are down-regulated in WDLS and DDLS compared to NF • miR-143 - most strongly expressed miRNA in normal fat (8% of total) • miR-143 and miR-145 located within 1.8 kb of each other on 5q miR‐143 miR‐145 Normalized Counts Normalized N WD DD N WD DD miR‐143F Fold FDR miRF‐145 Fold FDR Change Change WD/NF ‐3.3 1.5e‐5 WD/NF ‐2.8 > 0.05 DD/NF ‐7.9 4.0e‐12 DD/NF ‐6.6 3.2e‐7 FDR= false discovery rate miR-143 and miR-145 are down-regulated in WDLS and DDLS cell lines compared to ASCs by deep sequencing and quantitative PCR In situ hybridization for miR-143 DDLS cell lines WDLS cell lines DDLS cell lines WDLS cell lines A lentiviral system was used for stable re-expression of miR-143 and miR-145 in liposarcoma cell lines miRNA precursor in Tranform E. coli Culture and extract Transfect HEK293T lentiviral vector cells plasmid DNA Cells miR‐143; miR‐145 re‐ expression Harvest viral Functional supernatant Studies Select with Puromycin Infect cells of interest (Day 1) (Day 0) Re-expression of miR-143 inhibits proliferation in two DDLS cell lines DNA content (relative to day 2) (relative to day Time (Days) Time (Days) Re-expression of miR-143, but not miR-145, induces apoptosis in DDLS cells DDLS14 DDLS8817
Load more

Recommended publications
  • Profiling Data
    Compound Name DiscoveRx Gene Symbol Entrez Gene Percent Compound Symbol Control Concentration (nM) JNK-IN-8 AAK1 AAK1 69 1000 JNK-IN-8 ABL1(E255K)-phosphorylated ABL1 100 1000 JNK-IN-8 ABL1(F317I)-nonphosphorylated ABL1 87 1000 JNK-IN-8 ABL1(F317I)-phosphorylated ABL1 100 1000 JNK-IN-8 ABL1(F317L)-nonphosphorylated ABL1 65 1000 JNK-IN-8 ABL1(F317L)-phosphorylated ABL1 61 1000 JNK-IN-8 ABL1(H396P)-nonphosphorylated ABL1 42 1000 JNK-IN-8 ABL1(H396P)-phosphorylated ABL1 60 1000 JNK-IN-8 ABL1(M351T)-phosphorylated ABL1 81 1000 JNK-IN-8 ABL1(Q252H)-nonphosphorylated ABL1 100 1000 JNK-IN-8 ABL1(Q252H)-phosphorylated ABL1 56 1000 JNK-IN-8 ABL1(T315I)-nonphosphorylated ABL1 100 1000 JNK-IN-8 ABL1(T315I)-phosphorylated ABL1 92 1000 JNK-IN-8 ABL1(Y253F)-phosphorylated ABL1 71 1000 JNK-IN-8 ABL1-nonphosphorylated ABL1 97 1000 JNK-IN-8 ABL1-phosphorylated ABL1 100 1000 JNK-IN-8 ABL2 ABL2 97 1000 JNK-IN-8 ACVR1 ACVR1 100 1000 JNK-IN-8 ACVR1B ACVR1B 88 1000 JNK-IN-8 ACVR2A ACVR2A 100 1000 JNK-IN-8 ACVR2B ACVR2B 100 1000 JNK-IN-8 ACVRL1 ACVRL1 96 1000 JNK-IN-8 ADCK3 CABC1 100 1000 JNK-IN-8 ADCK4 ADCK4 93 1000 JNK-IN-8 AKT1 AKT1 100 1000 JNK-IN-8 AKT2 AKT2 100 1000 JNK-IN-8 AKT3 AKT3 100 1000 JNK-IN-8 ALK ALK 85 1000 JNK-IN-8 AMPK-alpha1 PRKAA1 100 1000 JNK-IN-8 AMPK-alpha2 PRKAA2 84 1000 JNK-IN-8 ANKK1 ANKK1 75 1000 JNK-IN-8 ARK5 NUAK1 100 1000 JNK-IN-8 ASK1 MAP3K5 100 1000 JNK-IN-8 ASK2 MAP3K6 93 1000 JNK-IN-8 AURKA AURKA 100 1000 JNK-IN-8 AURKA AURKA 84 1000 JNK-IN-8 AURKB AURKB 83 1000 JNK-IN-8 AURKB AURKB 96 1000 JNK-IN-8 AURKC AURKC 95 1000 JNK-IN-8
    [Show full text]
  • PCTK2 Protein Full-Length Recombinant Human Protein Expressed in Sf9 Cells
    Catalog # Aliquot Size P10-34G-20 20 µg P10-34G-50 50 µg PCTK2 Protein Full-length recombinant human protein expressed in Sf9 cells Catalog # P10-34G Lot # Z1204 -3 Product Description Purity Recombinant full-length human PCTK2 (CDK17) was expressed by baculovirus in Sf9 insect cells using an N- terminal GST tag. This gene accession number is NM_002595 . The purity of PCTK2 protein was determined to be >75% by Gene Aliases densitometry. Approx. MW 88 kDa . PCTAIRE2; PCTK2 Formulation Recombinant protein stored in 50mM Tris-HCl, pH 7.5, 50mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25mM DTT, 0.1mM PMSF, 25% glycerol. Storage and Stability Store product at –70 oC. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles. Scientific Background PCTK2 (CDK17) or cyclin-dependent kinase 17 belongs to the cdc2/cdkx subfamily of the ser/thr family of protein kinases. PCTK2 has similarity to a rat protein that is thought to play a role in terminally differentiated neurons (1). The identification of a large family of cdc2-related kinases PCTK2 Protein opens the possibility of combinatorial regulation of the Full-length recombinant human protein expressed in Sf9 cells cell cycle together with the emerging large family of cyclins (2). Catalog # P10-34G Lot # Z1204-3 References Purity >75% Concentration 0.1 µ g/ µl 1. Hirose T. et.al: PCTAIRE 2, a Cdc2-related serine/threonine Stability 1yr At –70 oC from date of shipment kinase, is predominantly expressed in terminally Storage & Shipping Store product at –70 oC.
    [Show full text]
  • Targeting Cyclin-Dependent Kinase 9 and Myeloid Cell Leukaemia 1 in MYC-Driven B-Cell Lymphoma
    Targeting cyclin-dependent kinase 9 and myeloid cell leukaemia 1 in MYC-driven B-cell lymphoma Gareth Peter Gregory ORCID ID: 0000-0002-4170-0682 Thesis for Doctor of Philosophy September 2016 Sir Peter MacCallum Department of Oncology The University of Melbourne Doctor of Philosophy Submitted in total fulfilment of the degree of Abstract Aggressive B-cell lymphomas include diffuse large B-cell lymphoma, Burkitt lymphoma and intermediate forms. Despite high response rates to conventional immuno-chemotherapeutic approaches, an unmet need for novel therapeutic by resistance to chemotherapy and radiotherapy. The proto-oncogene MYC is strategies is required in the setting of relapsed and refractory disease, typified frequently dysregulated in the aggressive B-cell lymphomas, however, it has proven an elusive direct therapeutic target. MYC-dysregulated disease maintains a ‘transcriptionally-addicted’ state, whereby perturbation of A significant body of evidence is accumulating to suggest that RNA polymerase II activity may indirectly antagonise MYC activity. Furthermore, very recent studies implicate anti-apoptotic myeloid cell leukaemia 1 (MCL-1) as a critical survival determinant of MYC-driven lymphoma. This thesis utilises pharmacologic and genetic techniques in MYC-driven models of aggressive B-cell lymphoma to demonstrate that cyclin-dependent kinase 9 (CDK9) and MCL-1 are oncogenic dependencies of this subset of disease. The cyclin-dependent kinase inhibitor, dinaciclib, and more selective CDK9 inhibitors downregulation of MCL1 are used
    [Show full text]
  • PCTK2 (CDK17)/Cycliny, Active Recombinant Full-Length Human Proteins Expressed in Sf9 Cells
    Catalog # Aliquot Size P10-10G -05 5 µg P10-10G -10 10 µg PCTK2 (CDK17)/CyclinY, Active Recombinant full-length human proteins expressed in Sf9 cells Catalog # P10-10G Lot # Z1168-3 Product Description Specific Activity Recombinant full-length human PCTK2 (CDK17) and Cyclin Y (2-end) were co-expressed by baculovirus in Sf9 140,000 insect cells using N-terminal GST tags. The PCTK2 (CDK17) gene accession number is NM_002595 and Cyclin Y is 105,000 NM_145012. 70,000 Gene Aliases 35,000 Activity (cpm) Activity PCTK2 (CDK17): PCTAIRE2; PCTK2 CyclinY: CCNY; C10orf9; CBCP1; CCNX; CFP1 0 0 40 80 120 160 200 Formulation Protein (ng) Recombinant protein stored in 50mM Tris-HCl, pH 7.5, The specific activity of PCTK2 (CDK17)/Cyclin Y was 150mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25mM determined to be 24 nmol/min/mg as per activity assay DTT, 0.1mM PMSF, 25% glycerol. protocol. Storage and Stability Purity Store product at –70oC. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable The purity of PCTK2 (CDK17) performance, avoid repeated handling and multiple /Cyclin Y was determined to be freeze/thaw cycles. >70% by densitometry, PCTK2 (CDK17) approx. MW 88kDa and Scientific Background Cyclin Y Approx. MW 64kDa. PCTK2 (CDK17) or cyclin-dependent kinase 17 belongs to the cdc2/cdkx subfamily of the ser/thr family of protein kinases. PCTK2 has similarity to a rat protein that is thought to play a role in terminally differentiated neurons (1). The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell PCTK2 (CDK17)/CyclinY, Active cycle together with the emerging large family of cyclins Recombinant full-length human proteins expressed in Sf9 cells (2).
    [Show full text]
  • PCTK2 (CDK17)/Cycliny, Active Recombinant Full-Length Human Proteins Expressed in Sf9 Cells
    Catalog # Aliquot Size P10-10G -05 5 µg P10-10G -10 10 µg PCTK2 (CDK17)/CyclinY, Active Recombinant full-length human proteins expressed in Sf9 cells Catalog # P10-10G Lot # Z1335 -1 Product Description Specific Activity Recombinant full-length human PCTK2 (CDK17) and Cyclin Y (2-end) were co-expressed by baculovirus in Sf9 160,000 insect cells using N-terminal GST tags. The PCTK2 (CDK17) gene accession number is NM_002595 and Cyclin Y is 120,000 NM_145012 . 80,000 Gene Aliases 40,000 (cpm) Activity PCTK2 (CDK17): PCTAIRE2; PCTK2 0 CyclinY: CCNY; C10orf9; CBCP1; CCNX; CFP1 0 120 240 360 480 Protein (ng) Formulation The specific activity of PCTK2 (CDK17)/Cyclin Y was determined Recombinant protein stored in 50mM Tris-HCl, pH 7.5, to be 21 nmol /min/mg as per activity assay protocol. 150mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25mM Purity DTT, 0.1mM PMSF, 25% glycerol. Storage and Stability Store product at –70 oC. For optimal storage, aliquot The purity of PCTK2 (CDK17) /Cyclin target into smaller quantities after centrifugation and Y was determined to be >75% by store at recommended temperature. For most favorable densitometry, PCTK2 (CDK17) performance, avoid repeated handling and multiple approx. MW 88kDa and Cyclin freeze/thaw cycles. Y Approx. MW 64kDa . Scientific Background PCTK2 (CDK17) or cyclin-dependent kinase 17 belongs to the cdc2/cdkx subfamily of the ser/thr family of protein kinases. PCTK2 has similarity to a rat protein that is thought to play a role in terminally differentiated neurons (1). The PCTK2 (CDK17)/CyclinY, Active identification of a large family of cdc2-related kinases Recombinant full-length human proteins expressed in Sf9 cells opens the possibility of combinatorial regulation of the Catalog # cell cycle together with the emerging large family of P10-10G Specific Activity 21 nmol/min/mg cyclins (2).
    [Show full text]
  • Human Kinases Info Page
    Human Kinase Open Reading Frame Collecon Description: The Center for Cancer Systems Biology (Dana Farber Cancer Institute)- Broad Institute of Harvard and MIT Human Kinase ORF collection from Addgene consists of 559 distinct human kinases and kinase-related protein ORFs in pDONR-223 Gateway® Entry vectors. All clones are clonal isolates and have been end-read sequenced to confirm identity. Kinase ORFs were assembled from a number of sources; 56% were isolated as single cloned isolates from the ORFeome 5.1 collection (horfdb.dfci.harvard.edu); 31% were cloned from normal human tissue RNA (Ambion) by reverse transcription and subsequent PCR amplification adding Gateway® sequences; 11% were cloned into Entry vectors from templates provided by the Harvard Institute of Proteomics (HIP); 2% additional kinases were cloned into Entry vectors from templates obtained from collaborating laboratories. All ORFs are open (stop codons removed) except for 5 (MST1R, PTK7, JAK3, AXL, TIE1) which are closed (have stop codons). Detailed information can be found at: www.addgene.org/human_kinases Handling and Storage: Store glycerol stocks at -80oC and minimize freeze-thaw cycles. To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip, puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate. To patch the hole, use sterile tape or a portion of a fresh aluminum seal. Note: These plasmid constructs are being distributed to non-profit institutions for the purpose of basic
    [Show full text]
  • Targeted MS Quantitation Assays for Signal Transduction Protein Pathways
    Targeted MS quantitation assays for signal transduction protein pathways Paul Haney R&D Platform Manager Thermo Scientific Protein Research Products Rockford, IL Why Targeted Quantitative Proteomics via Mass Spec? . Measure protein abundance and protein isoforms (e.g. splice variants, PTMs) without the need for antibodies . Avoid immuno-based cross- reactivity during multiplexing. Validate relative quantitation data from discovery proteomic experiments 2 How Do You Select Peptides for Targeted MS Assays? List of Proteins Spectral library Discovery data repositories In silico prediction Pinpoint 1.2 Hypothesis Experimental List of target peptides and transitions 3 How Do You Select Peptides for Targeted MS Assays? List of Proteins Spectral library Discovery data repositories In silico prediction Pinpoint 1.2 Hypothesis Experimental Validation Assay results List of target peptides and transitions 4 Tools for Target Peptide Identification and Scheduling . Active-site probes for enzyme subclass enrichment . Rapid recombinant heavy protein expression using human cell-free extracts . Peptide retention time calibration mixture for chromatography QC and targeted method 100 80 60 acquisition scheduling Intensity 40 20 0 5 10 15 20 25 30 5 Tools for Target Peptide Identification and Scheduling . Active-site probes for enzyme subclass enrichment . Rapid recombinant heavy protein expression using human cell-free extracts • Stergachis, A. & MacCoss, M. (2011) Nature Methods (submitted) . Peptide retention time calibration mixture for 100 80 chromatography
    [Show full text]
  • Human CDK17 / PCTK2 Gene Cdna Clone Plasmid
    Human CDK17 / PCTK2 Gene cDNA clone plasmid Catalog Number: HG11126-M General Information Plasmid Resuspension protocol Gene : PCTAIRE protein kinase 2 1.Centrifuge at 5,000×g for 5 min. Official Symbol : CDK17 2.Carefully open the tube and add 100 l of sterile water to dissolve the DNA. Synonym : PCTAIRE2 3.Close the tube and incubate for 10 minutes at room temperature. 4.Briefly vortex the tube and then do a quick spin to concentrate × Source : Human the liquid at the bottom. Speed is less than 5000 g. 5.Store the plasmid at -20 ℃. cDNA Size: 1572bp The plasmid is ready for: RefSeq : NM_002595.2 • Restriction enzyme digestion • PCR amplification Plasmid: pMD-PCTK2 • E. coli transformation • DNA sequencing Description E.coli strains for transformation (recommended Lot : Please refer to the label on the tube but not limited) Sequence Description : Most commercially available competent cells are appropriate for the plasmid, e.g. TOP10, DH5α and TOP10F´. Identical with the Gene Bank Ref. ID sequence except for the point mutations 1569 T/C not causing the amino acid variation. Vector : pMD18-T Simple Shipping carrier : Each tube contains approximately 10 μg of lyophilized plasmid. Storage : The lyophilized plasmid can be stored at ambient temperature for three months. Quality control : The plasmid is confirmed by full-length sequencing with primers in the sequencing primer list. Sequencing primer list : M13-47 ： 5’ GCCAGGGTTTTCCCAGTCACGAC 3’ RV-M ： 5’ GAGCGGATAACAATTTCACACAGG 3’ Other M13 primers can also be used as sequencing primers. Manufactured By Sino Biological Inc., FOR RESEARCH USE ONLY. NOT FOR USE IN HUMANS.
    [Show full text]
  • Gene Symbol Accession Alias/Prev Symbol Official Full Name AAK1 NM 014911.2 KIAA1048, Dkfzp686k16132 AP2 Associated Kinase 1
    Gene Symbol Accession Alias/Prev Symbol Official Full Name AAK1 NM_014911.2 KIAA1048, DKFZp686K16132 AP2 associated kinase 1 (AAK1) AATK NM_001080395.2 AATYK, AATYK1, KIAA0641, LMR1, LMTK1, p35BP apoptosis-associated tyrosine kinase (AATK) ABL1 NM_007313.2 ABL, JTK7, c-ABL, p150 v-abl Abelson murine leukemia viral oncogene homolog 1 (ABL1) ABL2 NM_007314.3 ABLL, ARG v-abl Abelson murine leukemia viral oncogene homolog 2 (arg, Abelson-related gene) (ABL2) ACVR1 NM_001105.2 ACVRLK2, SKR1, ALK2, ACVR1A activin A receptor ACVR1B NM_004302.3 ACVRLK4, ALK4, SKR2, ActRIB activin A receptor, type IB (ACVR1B) ACVR1C NM_145259.2 ACVRLK7, ALK7 activin A receptor, type IC (ACVR1C) ACVR2A NM_001616.3 ACVR2, ACTRII activin A receptor ACVR2B NM_001106.2 ActR-IIB activin A receptor ACVRL1 NM_000020.1 ACVRLK1, ORW2, HHT2, ALK1, HHT activin A receptor type II-like 1 (ACVRL1) ADCK1 NM_020421.2 FLJ39600 aarF domain containing kinase 1 (ADCK1) ADCK2 NM_052853.3 MGC20727 aarF domain containing kinase 2 (ADCK2) ADCK3 NM_020247.3 CABC1, COQ8, SCAR9 chaperone, ABC1 activity of bc1 complex like (S. pombe) (CABC1) ADCK4 NM_024876.3 aarF domain containing kinase 4 (ADCK4) ADCK5 NM_174922.3 FLJ35454 aarF domain containing kinase 5 (ADCK5) ADRBK1 NM_001619.2 GRK2, BARK1 adrenergic, beta, receptor kinase 1 (ADRBK1) ADRBK2 NM_005160.2 GRK3, BARK2 adrenergic, beta, receptor kinase 2 (ADRBK2) AKT1 NM_001014431.1 RAC, PKB, PRKBA, AKT v-akt murine thymoma viral oncogene homolog 1 (AKT1) AKT2 NM_001626.2 v-akt murine thymoma viral oncogene homolog 2 (AKT2) AKT3 NM_181690.1
    [Show full text]
  • Supplemental Table 3 Two-Class Paired Significance Analysis of Microarrays Comparing Gene Expression Between Paired
    Supplemental Table 3 Two‐class paired Significance Analysis of Microarrays comparing gene expression between paired pre‐ and post‐transplant kidneys biopsies (N=8). Entrez Fold q‐value Probe Set ID Gene Symbol Unigene Name Score Gene ID Difference (%) Probe sets higher expressed in post‐transplant biopsies in paired analysis (N=1871) 218870_at 55843 ARHGAP15 Rho GTPase activating protein 15 7,01 3,99 0,00 205304_s_at 3764 KCNJ8 potassium inwardly‐rectifying channel, subfamily J, member 8 6,30 4,50 0,00 1563649_at ‐‐ ‐‐ ‐‐ 6,24 3,51 0,00 1567913_at 541466 CT45‐1 cancer/testis antigen CT45‐1 5,90 4,21 0,00 203932_at 3109 HLA‐DMB major histocompatibility complex, class II, DM beta 5,83 3,20 0,00 204606_at 6366 CCL21 chemokine (C‐C motif) ligand 21 5,82 10,42 0,00 205898_at 1524 CX3CR1 chemokine (C‐X3‐C motif) receptor 1 5,74 8,50 0,00 205303_at 3764 KCNJ8 potassium inwardly‐rectifying channel, subfamily J, member 8 5,68 6,87 0,00 226841_at 219972 MPEG1 macrophage expressed gene 1 5,59 3,76 0,00 203923_s_at 1536 CYBB cytochrome b‐245, beta polypeptide (chronic granulomatous disease) 5,58 4,70 0,00 210135_s_at 6474 SHOX2 short stature homeobox 2 5,53 5,58 0,00 1562642_at ‐‐ ‐‐ ‐‐ 5,42 5,03 0,00 242605_at 1634 DCN decorin 5,23 3,92 0,00 228750_at ‐‐ ‐‐ ‐‐ 5,21 7,22 0,00 collagen, type III, alpha 1 (Ehlers‐Danlos syndrome type IV, autosomal 201852_x_at 1281 COL3A1 dominant) 5,10 8,46 0,00 3493///3 IGHA1///IGHA immunoglobulin heavy constant alpha 1///immunoglobulin heavy 217022_s_at 494 2 constant alpha 2 (A2m marker) 5,07 9,53 0,00 1 202311_s_at
    [Show full text]
  • Kinome Expression Profiling to Target New Therapeutic Avenues in Multiple Myeloma
    Plasma Cell DIsorders SUPPLEMENTARY APPENDIX Kinome expression profiling to target new therapeutic avenues in multiple myeloma Hugues de Boussac, 1 Angélique Bruyer, 1 Michel Jourdan, 1 Anke Maes, 2 Nicolas Robert, 3 Claire Gourzones, 1 Laure Vincent, 4 Anja Seckinger, 5,6 Guillaume Cartron, 4,7,8 Dirk Hose, 5,6 Elke De Bruyne, 2 Alboukadel Kassambara, 1 Philippe Pasero 1 and Jérôme Moreaux 1,3,8 1IGH, CNRS, Université de Montpellier, Montpellier, France; 2Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel, Brussels, Belgium; 3CHU Montpellier, Laboratory for Monitoring Innovative Therapies, Department of Biologi - cal Hematology, Montpellier, France; 4CHU Montpellier, Department of Clinical Hematology, Montpellier, France; 5Medizinische Klinik und Poliklinik V, Universitätsklinikum Heidelberg, Heidelberg, Germany; 6Nationales Centrum für Tumorerkrankungen, Heidelberg , Ger - many; 7Université de Montpellier, UMR CNRS 5235, Montpellier, France and 8 Université de Montpellier, UFR de Médecine, Montpel - lier, France ©2020 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol. 2018.208306 Received: October 5, 2018. Accepted: July 5, 2019. Pre-published: July 9, 2019. Correspondence: JEROME MOREAUX - [email protected] Supplementary experiment procedures Kinome Index A list of 661 genes of kinases or kinases related have been extracted from literature9, and challenged in the HM cohort for OS prognostic values The prognostic value of each of the genes was computed using maximally selected rank test from R package MaxStat. After Benjamini Hochberg multiple testing correction a list of 104 significant prognostic genes has been extracted. This second list has then been challenged for similar prognosis value in the UAMS-TT2 validation cohort.
    [Show full text]
  • Dissertation Submitted to the Combined Faculties for the Natural
    Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Diplom-Biochemiker Johannes Hermle born in: Offenbach a.M., Germany Oral-examination: July 26, 2017 siRNA SCREEN FOR IDENTIFICATION OF HUMAN KINASES INVOLVED IN ASSEMBLY AND RELEASE OF HIV-1 Referees: Prof. Dr. Hans-Georg Kräusslich Prof. Dr. Dirk Grimm ii Meiner Familie iii Summary Summary The replication of the human immunodeficiency virus type 1 (HIV-1) is as yet not fully understood. In particular the knowledge of interactions between viral and host cell proteins and the understanding of complete virus-host protein networks are still imprecise. An integral picture of the hijacked cellular machinery is essential for a better comprehension of the virus. And as a prerequisite, new tools are needed for this purpose. To create such a novel tool, a screening platform for host cell factors was established in this work. The screening assay serves as a powerful method to gain insights into virus-host-interactions. It was specifically tailored to addressing the stage of assembly and release of viral particles during the replication cycle of HIV-1. It was designed to be suitable for both RNAi and chemical compound screening. The first phase of this work comprised the setup and optimization of the assay. It was shown, that it was robust and reliable and delivered reproducible results. As a subsequent step, a siRNA library targeting 724 human kinases and accessory proteins was examined. After the evaluation of the complete siRNA library in a primary screen, all primary hits were validated in a second reconfirmation screen using different siRNAs.
    [Show full text]