146 results on '"Thomas J. Gonda"'
Search Results
2. Author Correction: Germline mutations in mitochondrial complex I reveal genetic and targetable vulnerability in IDH1-mutant acute myeloid leukaemia
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Mahmoud A. Bassal, Saumya E. Samaraweera, Kelly Lim, Brooks A. Benard, Sheree Bailey, Satinder Kaur, Paul Leo, John Toubia, Chloe Thompson-Peach, Tran Nguyen, Kyaw Ze Ya Maung, Debora A. Casolari, Diana G. Iarossi, Ilaria S. Pagani, Jason Powell, Stuart Pitson, Siria Natera, Ute Roessner, Ian D. Lewis, Anna L. Brown, Daniel G. Tenen, Nirmal Robinson, David M. Ross, Ravindra Majeti, Thomas J. Gonda, Daniel Thomas, and Richard J. D’Andrea
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Science - Published
- 2022
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3. Partial reprogramming of heterologous cells by defined factors to generate megakaryocyte lineage-restricted biomolecules
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Crisbel M. Artuz, Alexander J. Knights, Alister P.W. Funnell, Thomas J. Gonda, Katya Ravid, Richard C.M. Pearson, Kate G.R. Quinlan, and Merlin Crossley
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Biotechnology ,TP248.13-248.65 - Abstract
The ability of transcriptional regulators to drive lineage conversion of somatic cells offers great potential for the treatment of human disease. To explore the concept of switching on specific target genes in heterologous cells, we developed a model system to screen candidate factors for their ability to activate the archetypal megakaryocyte-specific chemokine platelet factor 4 (PF4) in fibroblasts. We found that co-expression of the transcriptional regulators GATA1 and FLI1 resulted in a significant increase in levels of PF4, which became magnified over time. This finding demonstrates that such combinations can be used to produce potentially beneficial chemokines in readily available heterologous cell types. Keywords: Platelet factor 4, Reprogramming, Megakaryocyte, Fibroblast
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- 2018
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4. High-content imaging of neutral lipid droplets with 1,6-diphenylhexatriene
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Max V. Ranall, Brian G. Gabrielli, and Thomas J. Gonda
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high-content imaging ,high-throughput screening ,lipid droplet ,1,6-diphenyl-1,3,5-hexatriene ,DPH ,Nile Red ,Biology (General) ,QH301-705.5 - Abstract
Neutral lipid droplets (LDs) are dynamic lipid storage organelles found in all eukaryotic cells from yeast to mammals and higher plants. LDs are important to many physiological processes that include basic cellular maintenance, metabolism, and diverse medical pathologies. LD accumulation has been studied extensively by a range of methods, but particularly by microscopy with several fluorescent dyes extensively used for qualitative and quantitative imaging. Here, we compared established LD stains Nile Red and BODIPY 493/503 to the 4′, 6-diamidino-2-phenylindole (DAPI)—range dye 1,6-diphenyl-1,3,5-hexatriene (DPH; excitation/emission λmax=350 nm/420 nm) using high-content image analysis. HeLa cells treated with oleic acid or vehicle were used to compare staining patterns between DPH and Nile Red as well as DPH and the LD protein adipophilin. DPH, Nile Red, and BODIPY 493/503 were compared as assay reagents in oleic acid dose-response experiments. Treatment of MCF-7 cells with sodium butyrate was used as a second cellular system for high-content analysis of LD formation. In this experimental context, we demonstrate the compatibility of DPH with GFP, a technical limitation of Nile Red and BODIPY 493/503 dyes. These data show that DPH has comparable sensitivity and specificity to that of Nile Red. Z′-factor analysis of dose-response experiments indicated that DPH and BODIPY 493/503 are well suited for quantitative analysis of LDs for high-throughput screening (HTS) applications.
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- 2011
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5. Adaptation and validation of DNA synthesis detection by fluorescent dye derivatization for high-throughput screening
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Max V. Ranall, Brian G. Gabrielli, and Thomas J. Gonda
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cell-based assay ,ethynyl deoxyuridine ,CuAAC ,Click ,high-content ,high-throughput ,Biology (General) ,QH301-705.5 - Abstract
Cellular proliferation is fundamental to organism development, tissue renewal, and diverse disease states such as cancer. In vitro measurement of proliferation by high-throughput screening allows rapid characterization of the effects of small-molecule or genetic treatments on primary and established cell lines. Current assays that directly measure the cell cycle are not amenable to high-throughput processing and analysis. Here we report the adaptation of the chemical method for detecting DNA synthesis by 5-ethynyl-2′-deoxyuridine (EdU) incorporation into both high-throughput liquid handling and high-content imaging analysis. We demonstrate that chemical detection of EdU incorporation is effective for high-resolution analysis and quantitation of DNA synthesis by high-content imaging. To validate this assay platform we used treatments of MCF10A cells with media supplements and pharmacological inhibitors that are known to affect cell proliferation. Treatments with specific kinase inhibitors indicate that EGF and serum stimulation employs both the mitogen extracellular kinase (MEK)/extracellular-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K)/AKT signaling networks. As described here, this method is fast, reliable, and inexpensive and yields robust data that can be easily interpreted.
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- 2010
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6. Supplementary Methods and Supplementary Figures S1-S13 from Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer
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Nigel A.J. McMillan, Thomas J. Gonda, H. Peter Soyer, Dubravka Skalamera, Paul Leo, Graham Leggatt, Sara McKee, Melinda Christensen, Karin Sedelies, Madison Kelly, Daniel Clarke, Sora Fallaha, Mushfiq Shaikh, Melanie Murrell, Weili Wang, Alexander J. Stevenson, Zay Yar Oo, Max V. Ranall, Fawzi Bokhari, and Brian Gabrielli
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Contains siRNA primary screen analysis and subsequent supplemental analyses; Figure S1 Analysis was performed using customized R scripts and proceeded via a three step process; Figure S2 Summary of Z scores of the top 54 selected target hits from the primary screen were rescreened using OnTarget Plus siRNAs; Figure S3 Mitosis is not a general target in HPV-transformed cancer cell lines; Figure S4A. Dose response for all the cell lines with the Aurora B inhibitor ZM447439; Figure S4B. Flow cytometry of the DNA content of cells were treated with 5 μM ZM447439 and collected at 24, 48, and 72 h, using DMSO as a control; Figure S5: Either wild type or K14E7 transgenic skin grafted onto wild type mice, either treated with Alisertib in vivo or untreated, was stained for mast cells using touluidine blue; Figure S6: Alisertib treatment of HPV-transformed cancer cells promotes apoptosis; Figure S7A. HPV- and non-HPV-transformed cancer cells were treated with 5 μM Alisertib and sampled at intervals over 72 h and analysed by flow cytometry for DNA content; S7B. Immunofluorescence of representative HPV-transformed (HeLa) and non-HPV C33A cells treated with DMSO (Control) or 5μM of Alisertib for 48 h stained for DNA and microtubules (α-tubulin). S7C. Quantitation of bi-nucleated and multinucleated cells in control and Alisertibtreated cells for 1 and 2 days; Figure S8 Representative time-lapse series of HeLa and C33A with and without 5 μM Alisertib treatment transiting mitosis and undergoing apoptosis; Figure S9: The time lines for 50 HeLa and 50 CaSki cells treated with 5 μM Alisertib; Figure S10: The dose response of parental HeLa cells and HeLa cells over expressing either Bcl-2 or Mcl- 1 with etoposide and Taxol, respectively, are shown; Figure S11: HPV-transformed ME180 and non-HPV C33A cells were treated with 5 μM Alisertib for up to 3 days; Figure S12 Dose response curves for HPV16 E7 expressing C33A and SCC25 cell lines determined (one of four replicate experiments); Figure S13: FACS of the GFP levels in the HPV18 E7-IRES-GFP transduced SCC25 cell lines used in Figure 7B, C.
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- 2023
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7. Supplementary Table S1 from Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer
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Nigel A.J. McMillan, Thomas J. Gonda, H. Peter Soyer, Dubravka Skalamera, Paul Leo, Graham Leggatt, Sara McKee, Melinda Christensen, Karin Sedelies, Madison Kelly, Daniel Clarke, Sora Fallaha, Mushfiq Shaikh, Melanie Murrell, Weili Wang, Alexander J. Stevenson, Zay Yar Oo, Max V. Ranall, Fawzi Bokhari, and Brian Gabrielli
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Primary screening daat from kinome siRNA screen
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- 2023
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8. Data from Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer
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Nigel A.J. McMillan, Thomas J. Gonda, H. Peter Soyer, Dubravka Skalamera, Paul Leo, Graham Leggatt, Sara McKee, Melinda Christensen, Karin Sedelies, Madison Kelly, Daniel Clarke, Sora Fallaha, Mushfiq Shaikh, Melanie Murrell, Weili Wang, Alexander J. Stevenson, Zay Yar Oo, Max V. Ranall, Fawzi Bokhari, and Brian Gabrielli
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Human papillomavirus (HPV) is the causative agent in cervical cancer. HPV oncogenes are major drivers of the transformed phenotype, and the cancers remain addicted to these oncogenes. A screen of the human kinome has identified inhibition of Aurora kinase A (AURKA) as being synthetically lethal on the background of HPV E7 expression. The investigational AURKA inhibitor MLN8237/Alisertib selectively promoted apoptosis in the HPV cancers. The apoptosis was driven by an extended mitotic delay in the Alisertib-treated HPV E7–expressing cells. This had the effect of reducing Mcl-1 levels, which is destabilized in mitosis, and increasing BIM levels, normally destabilized by Aurora A in mitosis. Overexpression of Mcl-1 reduced sensitivity to the drug. The level of HPV E7 expression influenced the extent of Alisertib-induced mitotic delay and Mcl-1 reduction. Xenograft experiments with three cervical cancer cell lines showed Alisertib inhibited growth of HPV and non-HPV xenografts during treatment. Growth of non-HPV tumors was delayed, but in two separate HPV cancer cell lines, regression with no resumption of growth was detected, even at 50 days after treatment. A transgenic model of premalignant disease driven solely by HPV E7 also demonstrated sensitivity to drug treatment. Here, we show for the first time that targeting of the Aurora A kinase in mice using drugs such as Alisertib results in a curative sterilizing therapy that may be useful in treating HPV-driven cancers. Mol Cancer Ther; 14(12); 2753–61. ©2015 AACR.
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- 2023
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9. Supplementary Tables S2, S3 from Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer
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Nigel A.J. McMillan, Thomas J. Gonda, H. Peter Soyer, Dubravka Skalamera, Paul Leo, Graham Leggatt, Sara McKee, Melinda Christensen, Karin Sedelies, Madison Kelly, Daniel Clarke, Sora Fallaha, Mushfiq Shaikh, Melanie Murrell, Weili Wang, Alexander J. Stevenson, Zay Yar Oo, Max V. Ranall, Fawzi Bokhari, and Brian Gabrielli
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SiRNA screening data validated gene lists
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- 2023
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10. Data from MYB Is Essential for Mammary Tumorigenesis
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Robert G. Ramsay, Robin L. Anderson, Thomas J. Gonda, Jordane Malaterre, Lloyd Pereira, Sandra Carpinteri, Dane Cheasley, Ryan Stanley Cross, Yvette Drabsch, and Rebecca Yu Miao
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MYB oncogene upregulation is associated with estrogen receptor (ER)-positive breast cancer, but disease requirements for MYB function in vivo have not been explored. In this study, we provide evidence of a critical requirement for MYB functions in models of human and murine breast cancer. In human breast cancer, we found that MYB expression was critical for tumor cell growth both in vitro and in vivo in xenograft settings. In transgenic knockout mice, tissue-specific deletion of the murine MYB gene caused a transient defect in mammary gland development that was reflected in delayed ductal branching and defective apical bud formation. In mouse mammary tumor virus (MMTV)-NEU mice where tumors are initiated by activation of HER2, MYB deletion was sufficient to abolish tumor formation. In the more aggressive MMTV-PyMT model system, MYB deletion delayed tumorigenesis significantly. Together, the findings in these transgenic knockout models implied that MYB was critical during an early window in mammary development when it was essential for tumor initiation, even though MYB loss did not exert a lasting impact upon normal mammary function. Two important MYB-target genes that promote cell survival, BCL2 and GRP78/BIP, were each elevated compared with nontransformed mammary epithelial cells, thereby promoting survival as confirmed in colony formation assays in vitro. Taken together, our findings establish a role for MYB at the hub of ER- and HER2-dependent pathways in mammary carcinogenesis. Cancer Res; 71(22); 7029–37. ©2011 AACR.
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- 2023
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11. Supplementary Figure 4 from MYB Is Essential for Mammary Tumorigenesis
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Robert G. Ramsay, Robin L. Anderson, Thomas J. Gonda, Jordane Malaterre, Lloyd Pereira, Sandra Carpinteri, Dane Cheasley, Ryan Stanley Cross, Yvette Drabsch, and Rebecca Yu Miao
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PDF file - 89K, Epithelial cell gene expression in tissues employed for the analysis of MYB and A-MYB expression in mammary glands at different stages of development contain epithelial cells.
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- 2023
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12. Supplementary Figure 3 from MYB Is Essential for Mammary Tumorigenesis
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Robert G. Ramsay, Robin L. Anderson, Thomas J. Gonda, Jordane Malaterre, Lloyd Pereira, Sandra Carpinteri, Dane Cheasley, Ryan Stanley Cross, Yvette Drabsch, and Rebecca Yu Miao
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PDF file - 360K, Transgene expression driven by the MMTV promoter is not effected by the loss of MYB in the mammary gland.
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- 2023
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13. Supplementary Tables 1-2, Figure Legends 1-5 from MYB Is Essential for Mammary Tumorigenesis
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Robert G. Ramsay, Robin L. Anderson, Thomas J. Gonda, Jordane Malaterre, Lloyd Pereira, Sandra Carpinteri, Dane Cheasley, Ryan Stanley Cross, Yvette Drabsch, and Rebecca Yu Miao
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PDF file - 168K
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- 2023
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14. Supplementary Figure 5 from MYB Is Essential for Mammary Tumorigenesis
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Robert G. Ramsay, Robin L. Anderson, Thomas J. Gonda, Jordane Malaterre, Lloyd Pereira, Sandra Carpinteri, Dane Cheasley, Ryan Stanley Cross, Yvette Drabsch, and Rebecca Yu Miao
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PDF file - 998K, Extent of proliferation, MYB and ER�� expression in wt virgin mammary glands.
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- 2023
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15. Supplementary Figure 1 from MYB Is Essential for Mammary Tumorigenesis
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Robert G. Ramsay, Robin L. Anderson, Thomas J. Gonda, Jordane Malaterre, Lloyd Pereira, Sandra Carpinteri, Dane Cheasley, Ryan Stanley Cross, Yvette Drabsch, and Rebecca Yu Miao
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PDF file - 1.3MB, MYB expression in human breast cancer.
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- 2023
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16. Supplementary Figure 2 from MYB Is Essential for Mammary Tumorigenesis
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Robert G. Ramsay, Robin L. Anderson, Thomas J. Gonda, Jordane Malaterre, Lloyd Pereira, Sandra Carpinteri, Dane Cheasley, Ryan Stanley Cross, Yvette Drabsch, and Rebecca Yu Miao
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PDF file - 170K, MYB mRNA expression is elevated in mammary tumors and tumor cell lines.
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- 2023
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17. MYB deficiency leads to myeloid neoplasia too
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Thomas J. Gonda and Gonda, Thomas J
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Immunology ,MYB ,deficiency ,Cell Biology ,Hematology ,myeloid neoplasia ,Biochemistry - Published
- 2023
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18. C/EBPβ is a MYB- and p300-cooperating pro-leukemogenic factor and promising drug target in acute myeloid leukemia
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Amke Trentmann, Debora A. Casolari, Maria V. Yusenko, Henning D. Mootz, Jan-Henrik Mikesch, Karl-Heinz Klempnauer, Mairin Lenz, Maria Francisca Arteaga, Richard J D'Andrea, Wolfgang Dörner, Stefan Klempnauer, Thomas J. Schmidt, Luca Abdel Ghani, Melanie Horn, Carsten Müller-Tidow, Thomas J. Gonda, Yusenko, Maria V, Trentmann, Amke, Casolari, Debora A, Abdel Ghani, Luca, Lenz, Mairin, Horn, Melanie, Dörner, Wolfgang, Klempnauer, Stefan, Mootz, Henning D, Arteaga, Maria Francisca, Mikesch, Jan Henrik, D'Andrea, Richard J, Gonda, Thomas J, Müller-Tidow, Carsten, Schmidt, Thomas J, and Klempnauer, Karl Heinz
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0301 basic medicine ,Cancer Research ,Biology ,drug target ,Article ,Acute myeloid leukaemia ,03 medical and health sciences ,0302 clinical medicine ,acute myeloid leukemia (AML) ,Genetics ,MYB ,Progenitor cell ,Molecular Biology ,Transcription factor ,Gene ,Cell growth ,CCAAT-Enhancer-Binding Protein-beta ,Myeloid leukemia ,Cell Differentiation ,MYB activity ,Haematopoiesis ,Leukemia, Myeloid, Acute ,030104 developmental biology ,Mechanisms of disease ,030220 oncology & carcinogenesis ,Cancer research ,Transcription factor MYB ,Ectopic expression - Abstract
Transcription factor MYB has recently emerged as a promising drug target for the treatment of acute myeloid leukemia (AML). Here, we have characterized a group of natural sesquiterpene lactones (STLs), previously shown to suppress MYB activity, for their potential to decrease AML cell proliferation. Unlike what was initially thought, these compounds inhibit MYB indirectly via its cooperation partner C/EBPβ. C/EBPβ-inhibitory STLs affect the expression of a large number of MYB-regulated genes, suggesting that the cooperation of MYB and C/EBPβ broadly shapes the transcriptional program of AML cells. We show that expression of GFI1, a direct MYB target gene, is controlled cooperatively by MYB, C/EBPβ, and co-activator p300, and is down-regulated by C/EBPβ-inhibitory STLs, exemplifying that they target the activity of composite MYB-C/EBPβ-p300 transcriptional modules. Ectopic expression of GFI1, a zinc-finger protein that is required for the maintenance of hematopoietic stem and progenitor cells, partially abrogated STL-induced myelomonocytic differentiation, implicating GFI1 as a relevant target of C/EBPβ-inhibitory STLs. Overall, our data identify C/EBPβ as a pro-leukemogenic factor in AML and suggest that targeting of C/EBPβ may have therapeutic potential against AML.
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- 2021
19. Nuclear stabilization of p53 requires a functional nucleolar surveillance pathway
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Katherine M. Hannan, Priscilla Soo, Mei S. Wong, Justine K. Lee, Nadine Hein, Perlita Poh, Kira D. Wysoke, Tobias D. Williams, Christian Montellese, Lorey K. Smith, Sheren J. Al-Obaidi, Lorena Núñez-Villacís, Megan Pavy, Jin-Shu He, Kate M. Parsons, Karagh E. Loring, Tess Morrison, Jeannine Diesch, Gaetan Burgio, Rita Ferreira, Zhi-Ping Feng, Cathryn M. Gould, Piyush B. Madhamshettiwar, Johan Flygare, Thomas J. Gonda, Kaylene J. Simpson, Ulrike Kutay, Richard B. Pearson, Christoph Engel, Nicholas J. Watkins, Ross D. Hannan, and Amee J. George
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Ribosomal Proteins ,Proto-Oncogene Proteins c-mdm2 ,Tumor Suppressor Protein p53 ,General Biochemistry, Genetics and Molecular Biology ,Cell Nucleolus ,Signal Transduction - Abstract
The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity.
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- 2022
20. MYB regulates the DNA damage response and components of the homology-directed repair pathway in human estrogen receptor-positive breast cancer cells
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Partha Mitra, Devathri Nanayakkara, Thomas J. Gonda, Murugan Kalimutho, Kum Kum Khanna, Ren Ming Yang, Eloise Dray, Yang, Ren Ming, Nanayakkara, Devathri, Kalimutho, Murugan, Mitra, Partha, Khanna, Kum Kum, Dray, Eloise, and Gonda, Thomas J
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0301 basic medicine ,Cancer Research ,DNA Repair ,DNA damage ,RAD51 ,Estrogen receptor ,Breast Neoplasms ,Biology ,DNA damage response ,medicine.disease_cause ,Homology directed repair ,Proto-Oncogene Proteins c-myb ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Genetics ,medicine ,Humans ,MYB ,Molecular Biology ,Transcription factor ,BRCA1 Protein ,breast cancers ,Recombinational DNA Repair ,Cancer ,medicine.disease ,030104 developmental biology ,Receptors, Estrogen ,030220 oncology & carcinogenesis ,MCF-7 Cells ,Cancer research ,Female ,Rad51 Recombinase ,cell types ,Carcinogenesis ,DNA Damage - Abstract
Over 70% of human breast cancers are estrogen receptor-positive (ER + ), most of which express MYB. In these and other cell types, the MYB transcription factor regulates the expression of many genes involved in cell proliferation, differentiation, tumorigenesis, and apoptosis. So far, no clear link has been established between MYB and the DNA damage response in breast cancer. Here, we found that silencing MYB in the ER + breast cancer cell line MCF-7 led to increased DNA damage accumulation, as marked by increased γ-H2AX foci following induction of double-stranded breaks. We further found that this was likely mediated by decreased homologous recombination-mediated repair (HRR), since silencing MYB impaired the formation of RAD51 foci in response to DNA damage. Moreover, cells depleted for MYB exhibited reduced expression of several key genes involved in HRR including BRCA1, PALB2, and TOPBP1. Taken together, these data imply that MYB and its targets play an important role in the response of ER + breast cancer cells to DNA damage, and suggest that induction of DNA damage along with inhibition of MYB activity could offer therapeutic benefits for ER + breast cancer and possibly other cancer types Refereed/Peer-reviewed
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- 2019
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21. Childhood acute myeloid leukemia shows a high level of germline predisposition
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David M. Ross, Amanda Smith, Andreas W. Schreiber, Saumya E. Samaraweera, Hamish S. Scott, Amilia Wee, Jonathan Ellis, Christopher N. Hahn, Mark Pinese, Debora A. Casolari, Andrew J. Deans, Paul Leo, Paul Po-Shen Wang, Kyaw Ze Ya Maung, Thomas J. Gonda, Richard J D'Andrea, Anna L. Brown, Kelly Perkins, Mark J. Cowley, Ka Leung Li, Devendra K Hiwase, Andrew S. Moore, Jinghua Feng, Samaraweera, Saumya E, Wang, Paul PS, Li, Ka Leung, Casolari, Debora A, Feng, Jinghua, Maung, Kyaw Ze Ya, Scott, Hamish S, Schreiber, Andreas W, Brown, Anna L, Ross, David M, Gonda, Thomas J, Hahn, Christopher N, and D'Andrea, Richard J
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Oncology ,medicine.medical_specialty ,Immunology ,Population ,Biochemistry ,Germline ,Gene Frequency ,Internal medicine ,Genetic predisposition ,medicine ,Humans ,Genetic Predisposition to Disease ,education ,Child ,Exome ,Germ-Line Mutation ,education.field_of_study ,Whole Genome Sequencing ,business.industry ,Childhood Acute Myeloid Leukemia ,Bone marrow failure ,Cancer ,Cell Biology ,Hematology ,medicine.disease ,Leukemia, Myeloid, Acute ,Germ Cells ,Cohort ,business - Abstract
As germline variants can influence cancer patient treatment decisions, outcomes and counselling, and the level of genetic predisposition for sporadic childhood acute myeloid leukemia (AML) is not clearly established, we undertook a comprehensive analysis of rare germline variants in childhood AML. As childhood AML is rare,1 to date pan-cancer childhood cohorts have included few AML cases and often the germline panels used have not included key genes relevant to myeloid malignancy. We therefore combined data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program together with an Australian childhood AML cohort (Supplemental Tables 1 and 2) to identify the rare germline variants in a large panel of cancer predisposition genes (n=216) compiled from literature review, and including genes involved in familial hematological malignancies (HM) and bone marrow failure (BMF) syndromes (Supplemental Table 3). We analyzed whole genome sequencing (WGS) and whole exome sequence (WES) data available through the TARGET program (n=48) (phs000218.v22.p8.c1) and WES data for the Australian cohort (n=24). Given that damaging and disease-causing variants are predicted to have a low population prevalence we identified extremely rare, potentially deleterious germline variants [VAF >30%; MAF (gnomAD) 10] and classified these as shown in Figure 1. All variants passing initial filtering are listed in Supplemental Table 4. The distribution of germline and somatic variants is shown in Supplemental Table 5, Supplemental Figure 1. Given the small cohort size pairwise comparisons of germline variants and clinico-pathological characteristics did not reveal significant associations after applying multiple correction (Supplemental Table 6).
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- 2021
22. Nuclear stabilisation of p53 requires a functional nucleolar surveillance pathway
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Justine K. Lee, Ulrike Kutay, Sheren Al-Obaidi, Mei S. Wong, Priscilla Soo, Lorena Núñez-Villacís, Christian Montellese, Ross D. Hannan, Rita Ferreira, Kate M Parsons, Amee J George, Gaetan Burgio, Kaylene J. Simpson, Christoph Engel, Nicholas J. Watkins, Katherine M. Hannan, Tobias D. Williams, Johan Flygare, Cathryn M. Gould, Kira D. Wysoke, Maurits Evers, Zhi-Ping Feng, Thomas J. Gonda, Lorey K. Smith, Nadine Hein, Richard B. Pearson, Jeannine Diesch, Perlita Poh, Piyush B. Madhamshettiwar, Megan Pavy, and Jin-Shu He
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biology ,Ribosomal protein ,DNA damage ,Ribonucleoprotein particle ,biology.protein ,Mdm2 ,Ribosome biogenesis ,Gene ,Biogenesis ,Ubiquitin ligase ,Cell biology - Abstract
The nucleolar surveillance pathway (NSP) monitors nucleolar fidelity and responds to nucleolar stresses (i.e., inactivation of ribosome biogenesis) by mediating the inhibitory binding of ribosomal proteins (RPs) to mouse double minute 2 homolog (MDM2), a nuclear-localised E3 ubiquitin ligase, which results in p53 accumulation. Inappropriate activation of the NSP has been implicated in the pathogenesis of collection of human diseases termed “ribosomopathies”, while drugs that selectively activate the NSP are now in trials for cancer. Despite the clinical significance, the precise molecular mechanism(s) regulating the NSP remain poorly understood. Using genome-wide loss of function screens, we demonstrate the ribosome biogenesis (RiBi) axis as the most potent class of genes whose disruption stabilises p53. Furthermore, we identified a novel suite of genes critical for the NSP, including a novel mammalian protein implicated in 5S ribonucleoprotein particle (5S-RNP) biogenesis, HEATR3. By selectively disabling the NSP, we unexpectedly demonstrate that a functional NSP is required for the ability of all nuclear acting stresses tested, including DNA damage, to robustly induce p53 accumulation. Together, our data demonstrates that the NSP has evolved as the dominant central integrator of stresses that regulate nuclear p53 abundance, thus ensuring RiBi is hardwired to cellular proliferative capacity.
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- 2021
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23. Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.
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Dubravka Škalamera, Mareike Dahmer, Amy S Purdon, Benjamin M Wilson, Max V Ranall, Antje Blumenthal, Brian Gabrielli, and Thomas J Gonda
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Medicine ,Science - Abstract
The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors.
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- 2012
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24. Rare variants in Fanconi anemia genes are enriched in acute myeloid leukemia
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Richard J D'Andrea, Andrew J. Deans, Anna L. Brown, James X Gray, Paula Marlton, Thomas J. Gonda, Sarah C Bray, Luen Bik To, Ian D. Lewis, Matthew A. Brown, Tran Nguyen, Debora A. Casolari, Emma L. Duncan, Paul Leo, Vinay Tergaonkar, Gökhan Cildir, Devinder Gill, Stephen Pederson, Saumya E. Samaraweera, Adam D. Ewing, Russell Saal, Mhairi Marshall, Mahmoud A. Bassal, Kyaw Ze Ya Maung, Deepak Singhal, Maung, Kyaw Ze Ya, Leo, Paul J, Bassal, Mahmoud, Casolari, Debora A, Bray, Sarah C, Singhal, Deepak, Samaraweera, Saumya E, Nguyen, Tran, Cildir, Gökhan, Tergaonkar, Vinay, Lewis, Ian, Brown, Anna L, D'Andrea, Richard J, and Gonda, Thomas J
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0301 basic medicine ,Acute Myeloid Leukemia ,Myeloid ,Genotype ,Biology ,gene variants ,lcsh:RC254-282 ,Germline ,03 medical and health sciences ,Germline mutation ,Fanconi anemia ,hemic and lymphatic diseases ,Correspondence ,medicine ,Humans ,Genetic Predisposition to Disease ,Alleles ,Genetic Association Studies ,Bone marrow failure ,Myeloid leukemia ,Genetic Variation ,Hematology ,medicine.disease ,mutations ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Fanconi Anemia Complementation Group Proteins ,FANCB ,Leukemia ,Leukemia, Myeloid, Acute ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Mutation ,Cancer research ,patient - Abstract
Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy caused by somatically acquired changes affecting a well-defined set of genes1. While rare high-risk variants affecting specific transcription factors account for a proportion of myelodysplastic syndrome (MDS) and AML associated with a family history, the contribution of other germline variants conferring low-intermediate risk has not yet been determined, partly because these are more difficult to identify from pedigree analysis. Here we use an Australian AML patient cohort to analyze rare, deleterious variants affecting genes involved in the rare recessive bone marrow failure syndrome Fanconi Anemia (FA). FA is caused by bi-allelic germline mutations in any of the 22 FANC genes (except for FANCB and FANCR which are X-linked and autosomal dominant), and is associated with profoundly increased risk of AML. The proteins encoded by the FANC genes participate in the removal of interstrand crosslinks (ICL) and the protection and resolution of stalled replication forks, an essential step for faithful DNA replication. Deficiency for these genes, combined with other mutations, results in pre-leukemia or leukemia in mouse models.
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- 2018
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25. A high-throughput platform for lentiviral overexpression screening of the human ORFeome.
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Dubravka Škalamera, Max V Ranall, Benjamin M Wilson, Paul Leo, Amy S Purdon, Carolyn Hyde, Ehsan Nourbakhsh, Sean M Grimmond, Simon C Barry, Brian Gabrielli, and Thomas J Gonda
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Medicine ,Science - Abstract
In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.
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- 2011
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26. Ectodermal-Neural Cortex 1 Isoforms Have Contrasting Effects on MC3T3-E1 Osteoblast Mineralization and Gene Expression
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Thomas J. Gonda, Elisabeth J Smith, Simon C. Barry, Leah E. Worton, Yan-Chuan Shi, Edith M. Gardiner, and Jonathan P. Whitehead
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0301 basic medicine ,Gene isoform ,Gene knockdown ,Frizzled ,Chemistry ,Wnt signaling pathway ,ALPL ,Osteoblast ,Cell Biology ,Biochemistry ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,Frzb ,030220 oncology & carcinogenesis ,Gene expression ,medicine ,Molecular Biology - Abstract
The importance of Wnt pathway signaling in development of bone has been well established. Here we investigated the role of a known Wnt target, ENC1 (ectodermal-neural cortex 1; NRP/B), in osteoblast differentiation. Enc1 expression was detected in mouse osteoblasts, chondrocytes and osteocytes by in situ hybridization, and osteoblastic expression was verified in differentiating primary cultures and MC3T3-E1 pre-osteoblast cells, with 57kDa and 67kDa ENC1 protein isoforms detected throughout differentiation. Induced knockdown of both ENC1 isoforms reduced alkaline phosphatase staining and virtually abolished MC3T3-E1 mineralization. At culture confluence, Alpl (alkaline phosphatase liver/bone/kidney) expression was markedly reduced compared with control cells, and there was significant and coordinated alteration of other genes involved in cellular phosphate biochemistry. In contrast, with 67kDa-selective knockdown mineralized nodule formation was enhanced and there was a 2-fold increase in Alpl expression at confluence. There was enhanced expression of Wnt/beta-catenin target genes with knockdown of both isoforms at this time-point and a 5-fold increase in Frzb (Frizzled related protein) with 67kDa-selective knockdown at mineralization, indicating possible ENC1 interactions with Wnt signaling in osteoblasts. These results are the first to demonstrate a role for ENC1 in the control of osteoblast differentiation. Additionally, the contrasting mineralization phenotypes and transcriptional patterns seen with coordinate knockdown of both ENC1 isoforms vs selective knockdown of 67kDa ENC1 suggest opposing roles for the isoforms in regulation of osteoblastic differentiation, through effects on Alpl expression and phosphate cellular biochemistry. This study is the first to report differential roles for the ENC1 isoforms in any cell lineage. This article is protected by copyright. All rights reserved.
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- 2017
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27. Genome-Wide Overexpression Screen Identifies Genes Able to Bypass p16-Mediated Senescence in Melanoma
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Brian Gabrielli, Duncan Lambie, Thomas J. Gonda, Carly Fox, Max V. Ranall, Paul Yaswen, Alexander J. Stevenson, Dubravka Skalamera, Mareike Dahmer-Heath, W. J. Lee, Konstanin Shakhbazov, Lee, Won Jae, Škalamera, Dubravka, Dahmer-Heath, Mareike, Shakhbazov, Konstanin, Ranall, Max V, Fox, Carly, Lambie, Duncan, Stevenson, Alexander J, Yaswen, Paul, Gonda, Thomas J, and Gabrielli, Brian
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0301 basic medicine ,senescence ,Skin Neoplasms ,overexpression screening ,p16 ,Biochemistry ,Genome ,Analytical Chemistry ,CDKN2A ,2.1 Biological and endogenous factors ,Viral ,ORFS ,Melanoma ,Cellular Senescence ,Cancer ,Oncogene Proteins ,Genetics ,Tumor ,biology ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,Melanocytes ,Molecular Medicine ,Human ,Biotechnology ,Senescence ,high-content imaging ,CDK6 ,Genetic Vectors ,Cell Line ,Open Reading Frames ,03 medical and health sciences ,Cell Line, Tumor ,HMGB Proteins ,medicine ,Humans ,Nevus ,neoplasms ,Gene ,Cyclin-Dependent Kinase Inhibitor p16 ,Gene Library ,Neoplastic ,Genome, Human ,Lentivirus ,Cell Cycle Checkpoints ,Cyclin-Dependent Kinase 6 ,Oncogene Proteins, Viral ,medicine.disease ,High-Throughput Screening Assays ,Open reading frame ,HEK293 Cells ,030104 developmental biology ,Gene Expression Regulation ,biology.protein ,Cancer research ,Cyclin-dependent kinase 6 - Abstract
© 2016 Society for Laboratory. Malignant melanomas often arise from nevi, which result from initial oncogene-induced hyperproliferation of melanocytes that are maintained in a CDKN2A/p16-mediated senescent state. Thus, genes that can bypass this senescence barrier are likely to contribute to melanoma development. We have performed a gain-of-function screen of 17,030 lentivirally expressed human open reading frames (ORFs) in a melanoma cell line containing an inducible p16 construct to identify such genes. Genes known to bypass p16-induced senescence arrest, including the human papilloma virus 18 E7 gene (HPV18E7), and genes such as the p16-binding CDK6 with expected functions, as well as panel of novel genes, were identified, including high-mobility group box (HMGB) proteins. A number of these were further validated in two other models of p16-induced senescence. Tissue immunohistochemistry demonstrated higher levels of CDK6 in primary melanomas compared with normal skin and nevi. Reduction of CDK6 levels drove melanoma cells expressing functional p16 into senescence, demonstrating its contribution to bypass senescence.
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- 2017
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28. Genome-wide gain-of-function screen for genes that induce epithelial-to-mesenchymal transition in breast cancer
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Esha T. Shah, Brett G. Hollier, Melissa J. Davis, Nikolas K. Haass, Mareike Dahmer-Heath, Erik W. Thompson, Dubravka Škalamera, Cletus Pinto, Brian Gabrielli, Alexander J. Stevenson, Nur Akmarina B.M. Said, Elizabeth D. Williams, S. M. Daignault, Thomas J. Gonda, Škalamera, Dubravka, Dahmer-Heath, Mareike, Stevenson, Alexander J, Pinto, Cletus, Shah, Esha T, Daignault, Sheena M, Said, Nur Akmarina BM, Davis, Melissa, Haass, Nikolas K, Williams, Elizabeth D, Hollier, Brett G, Thompson, Erik W, Gabrielli, Brian, and Gonda, Thomas J
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0301 basic medicine ,Epithelial-Mesenchymal Transition ,epithelial-mesenchymal transition ,Vimentin ,lentiviral vectors ,Breast Neoplasms ,Metastasis ,03 medical and health sciences ,Open Reading Frames ,vimentin ,breast cancer ,Antigens, CD ,Cell Line, Tumor ,Medicine ,Humans ,Epithelial–mesenchymal transition ,high-content-screening ,Gene ,biology ,business.industry ,Cadherin ,Genome, Human ,Gene Expression Profiling ,Mesenchymal stem cell ,Lentivirus ,Cancer ,Epithelial Cells ,medicine.disease ,Cadherins ,Gene expression profiling ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,Oncology ,Cancer research ,biology.protein ,Disease Progression ,Female ,business ,Research Paper ,Genome-Wide Association Study ,Plasmids - Abstract
Epithelial to mesenchymal transition (EMT) is a developmental program that has been implicated in progression, metastasis and therapeutic resistance of some carcinomas. To identify genes whose overexpression drives EMT, we screened a lentiviral expression library of 17000 human open reading frames (ORFs) using high-content imaging to quantitate cytoplasmic vimentin. Hits capable of increasing vimentin in the mammary carcinoma-derived cell line MDA-MB-468 were confirmed in the non-tumorigenic breast-epithelial cell line MCF10A. When overexpressed in this model, they increased the rate of cell invasion through Matrigel™, induced mesenchymal marker expression and reduced expression of the epithelial marker E-cadherin. In gene-expression datasets derived from breast cancer patients, the expression of several novel genes correlated with expression of known EMT marker genes, indicating their in vivo relevance. As EMT-associated properties are thought to contribute in several ways to cancer progression, genes identified in this study may represent novel targets for anti-cancer therapy. Refereed/Peer-reviewed
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- 2016
29. Abstract P4-07-09: MYB is involved in the DNA damage response in human ER+ breast cancer cells
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R-M Yang, Thomas J. Gonda, P. Mitra, and Eloise Dray
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Cancer Research ,Oncology ,Er breast cancer ,DNA damage ,Cancer research ,MYB ,Biology ,Molecular biology - Abstract
Aims: Over 70% of human breast cancer cells are oestrogen receptor positive (ER+) and express MYB. MYB expression is necessary for the proliferation of ER+ breast cancer cells in vitro and for tumour development in vivo. Our previous studies found that shRNA-mediated MYB knock-down greatly sensitised breast cancer cells to chemically-induced apoptosis by down-regulating the BCL2 (a MYB target gene) (Drabsch et al., 2010). Furthermore, several published studies (Taha et al., 2004; Thomadaki et al., 2006) indicated that actinomycin D and etoposide treatment could induce DNA damage in human ER+ breast cancer (MCF-7) cells. Moreover, silencing MYB increased DNA damage-induced cell death in castration resistant prostate cancer cells by down-regulating DNA damage response genes (Li et al., 2014). However, there is very little information on MYB function in the DNA damage response of ER+ breast cancer cells. Therefore, the aim of this study was to investigate whether silencing MYB affected the DNA damage repair and resulting in cell death in MCF-7 cells. Methods: To achieve down-regulation of MYB expression in human ER+ breast cancer, we used doxycycline inducible shRNA lentiviral vectors (pLV711 (Drabsch et al., 2007; Brown et al., 2010)) in human MCF-7 breast cancer cells. We used PI staining plus FACS analysis for cell death assessment. We also used γ-H2AX protein expression and γ-H2AX foci counting to assess DNA damage. Results: We found that silencing MYB alone did not result in cell death, as reported previously (Drabsch et al., 2010). However, silencing MYB significantly increased actinomycin D-induced cell death. This similar result was also found on etoposide-induced cell death. Furthermore, we found that silencing MYB significantly increased actinomycin D or etoposide-induced γ-H2AX expression. Moreover, anti-apoptotic BCL2 expression, measured by western blotting, was dramatically reduced after the combination of MYB knock down and actinomycin D or etoposide, compared to wild type and nonsilencing controls. Conclusions: Silencing MYB significantly increased DNA damage-induced cell death and D???damage. This may result from down-regulation of BCL2 expression, an effect on DNA damage response genes, or both. This requires further investigation. For example, Rad51 and γ-H2AX colocalization and 53BP1 expression in response to DNA damage will be presented. References: Taha et al., 2004, J Biol Chem, 279, 20546-46. Thomadaki et al., 2006, Biol. Chem., 387, 1081-1086. Drabsch et al., 2007, Proc Natl Acad Sci U S A, 104, 13762-7. Drabsch et al., 2010, Breast Cancer Res, 12:R55. Brown et al., 2010, Hum Gene Ther, 21(8),1005-17. Li et al., 2014, Science Signaling, 7(326). Citation Format: Yang R-M, Dray E, Mitra P, Gonda TJ. MYB is involved in the DNA damage response in human ER+ breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-07-09.
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- 2016
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30. The mechanism of MYB transcriptional regulation by MLL-AF9 oncoprotein
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Lu Cao, Partha Mitra, Thomas J. Gonda, Cao, Lu, Mitra, Partha, and Gonda, Thomas J
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animal structures ,Oncogene Proteins, Fusion ,Transcription, Genetic ,Genes, myb ,lcsh:Medicine ,Biology ,Article ,Acute myeloid leukaemia ,Mediator ,Transcription (biology) ,hemic and lymphatic diseases ,Transcriptional regulation ,Animals ,Humans ,MYB ,Promoter Regions, Genetic ,lcsh:Science ,neoplasms ,Regulation of gene expression ,Multidisciplinary ,lcsh:R ,Promoter ,DOT1L ,Cell biology ,Gene Expression Regulation, Neoplastic ,Leukemia, Myeloid, Acute ,Cyclin-dependent kinase 9 ,lcsh:Q ,Transcription ,Myeloid-Lymphoid Leukemia Protein - Abstract
Acute leukaemias express high levels of MYB which are required for the initiation and maintenance of the disease. Inhibition of MYB expression or activity has been shown to suppress MLL-fusion oncoprotein-induced acute myeloid leukaemias (AML), which are among the most aggressive forms of AML, and indeed MYB transcription has been reported to be regulated by the MLL-AF9 oncoprotein. This highlights the importance of understanding the mechanism of MYB transcriptional regulation in these leukaemias. Here we have demonstrated that the MLL-AF9 fusion protein regulates MYB transcription directly at the promoter region, in part by recruiting the transcriptional regulator kinase CDK9, and CDK9 inhibition effectively suppresses MYB expression as well as cell proliferation. However, MYB regulation by MLL-AF9 does not require H3K79 methylation mediated by the methyltransferase DOT1L, which has also been shown to be a key mediator of MLL-AF9 leukemogenicity. The identification of specific, essential and druggable transcriptional regulators may enable effective targeting of MYB expression, which in turn could potentially lead to new therapeutic approaches for acute myeloid leukaemia with MLL-AF9.
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- 2019
31. Partial reprogramming of heterologous cells by defined factors to generate megakaryocyte lineage-restricted biomolecules
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Merlin Crossley, Richard C. M. Pearson, Alexander J. Knights, Thomas J. Gonda, Crisbel M. Artuz, Kate G. R. Quinlan, Katya Ravid, Alister P. W. Funnell, Artuz, Crisbel M, Knights, Alexander J, Funnell, Alister PW, Gonda, Thomas J, Ravid, Katya, Pearson, Richard CM, Quinlan, Kate GR, and Crossley, Merlin
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0301 basic medicine ,Chemokine ,Cell type ,Lineage (genetic) ,Somatic cell ,lcsh:Biotechnology ,Heterologous ,Platelet factor 4 ,Biology ,Applied Microbiology and Biotechnology ,Article ,fibroblast ,03 medical and health sciences ,Megakaryocyte ,lcsh:TP248.13-248.65 ,medicine ,reprogramming ,Reprogramming ,GATA1 ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,biology.protein ,Fibroblast ,megakaryocyte aplatelet factor 4 ,Biotechnology - Abstract
Highlights • GATA1 and FLI1 switch on the endogenous platelet factor 4 (Pf4) gene in fibroblasts. • Pf4 expression is maintained and increases over time. • PF4 protein is secreted and readily collected. • A new technology for the g0065neration of important biomolecules in heterologous cells., The ability of transcriptional regulators to drive lineage conversion of somatic cells offers great potential for the treatment of human disease. To explore the concept of switching on specific target genes in heterologous cells, we developed a model system to screen candidate factors for their ability to activate the archetypal megakaryocyte-specific chemokine platelet factor 4 (PF4) in fibroblasts. We found that co-expression of the transcriptional regulators GATA1 and FLI1 resulted in a significant increase in levels of PF4, which became magnified over time. This finding demonstrates that such combinations can be used to produce potentially beneficial chemokines in readily available heterologous cell types.
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- 2018
32. TARGETING P53 AND NUCLEOLAR STRESS IN DIAMOND-BLACKFAN ANAEMIA
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Ross D. Hannan, Thomas J. Gonda, Johan Flygare, Brian Gabrielli, Dubravka Skalamera, Melissa D. Ilsley, Adam Stephenson, and Amee J George
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Cancer Research ,Gene knockdown ,Bone marrow failure ,Ribosome biogenesis ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Phenotype ,Cell biology ,medicine.anatomical_structure ,Ribosomal protein ,Apoptosis ,hemic and lymphatic diseases ,Genetics ,medicine ,Bone marrow ,Molecular Biology ,Gene - Abstract
Diamond-Blackfan anaemia (DBA) is a rare congenital erythroid hypoplastic anaemia characterised by severely reduced numbers of erythroid precursors in the bone marrow. This reduction of erythroid progenitors is partly due to p53-induced apoptosis during erythroid differentiation in response to nucleolar stress. Nucleolar stress can be caused by mutations in ribosomal proteins. Mutations in ribosomal proteins are found in over 50% of DBA patients and mutations in RPS19 are found in ∼25% of DBA patients. We hypothesise that preventing p53-induced apoptosis in DBA erythroid cells would lead to the survival and differentiation of erythroid progenitors. We performed a genome-wide, gain-of-function screen to identify factors that can modulate levels of p53 in RPS19 knockdown cells. For screening, we generated a human cell line with doxycycline-inducible shRNA-mediated RPS19 knockdown. Induction leads to a decrease in RPS19 protein and robust induction of p53. Using a lentiviral human ORF expression library containing ∼17000 genes in a high-throughput overexpression assay, we confirmed 36 high-confidence candidates that can modulate levels of p53 in the presence of perturbed ribosome biogenesis. To determine if overexpression of genes that led to a reduction of p53 (p53low hits) can regulate p53 within erythroid cells and thereby rescue the DBA phenotype, we utilised a doxycycline-inducible RPS19 knock-down mouse model. This mouse displays many characteristics of DBA such as bone marrow failure which can be rescued by RPS19 gene transfer or loss of Trp53. We tested if expression of p53low hits can rescue the differentiation and proliferation of shRPS19 erythroid progenitors from foetal livers of 14.5dpc embryos in vitro and bone marrow in vivo. By identifying factors that regulate p53, we aim to identify pathways involved in the disease and targets for novel DBA therapies.
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- 2019
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33. Assessment of CXC ligand 12-mediated calcium signalling and its regulators in basal-like breast cancer cells
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Felicity M. Davis, Iman Azimi, S. J. Roberts‑Thomson, Gregory R. Monteith, Thomas J. Gonda, Erik W. Thompson, S. Y. N. Jamaludin, Amelia A. Peters, Jamaludin, SYN, Azimi, I, Davis, FM, Peters, AA, Gonda, TJ, Thompson, EW, Roberts-Thomson, SJ, and Monteith, GR
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0301 basic medicine ,calcium signalling ,Cancer Research ,Chemokine ,hypoxia-inducible factor 2 alpha ,CXCR4 ,03 medical and health sciences ,breast cancer ,Breast cancer ,0302 clinical medicine ,Epidermal growth factor ,cluster of differentiation 24 ,CXC chemokine receptors ,skin and connective tissue diseases ,biology ,Oncogene ,Chemistry ,Calcium signalling ,Cluster of differentiation 24 ,CXC chemokine receptor type 7 ,Articles ,Cell cycle ,3. Good health ,030104 developmental biology ,Oncology ,Cell culture ,030220 oncology & carcinogenesis ,Cancer cell ,CXC chemokine receptor type 4 ,biology.protein ,Cancer research ,Hypoxia-inducible factor 2α ,hypoxia-inducible factor 2α - Abstract
CXC ligand (L)12 is a chemokine implicated in the migration, invasion and metastasis of cancer cells via interaction with its receptors CXC chemokine receptor (CXCR)4 and CXCR7. In the present study, CXCL12-mediated Ca2+ signalling was compared with two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which demonstrate distinct metastatic potential. CXCL12 treatment induced Ca2+ responses in the more metastatic MDA-MB-231 cells but not in the less metastatic MDA-MB-468 cells. Assessment of mRNA levels of CXCL12 receptors and their potential modulators in both cell lines revealed that CXCR4 and CXCR7 levels were increased in MDA-MB-231 cells compared with MDA-MB-468 cells. Cluster of differentiation (CD)24, the negative regulator of CXCL12 responses, demonstrated increased expression in MDA-MB-468 cells compared with MDA-MB-231 cells, and the two cell lines expressed comparable levels of hypoxia-inducible factor (HIF)2α, a CXCR4 regulator. Induction of epithelial-mesenchymal transition (EMT) by epidermal growth factor exhibited opposite effects on CXCR4 mRNA levels compared with hypoxia-induced EMT. Neither EMT inducer exhibited an effect on CXCR7 expression, however hypoxia increased HIF2α expression levels in MDA-MB-468 cells. Analysis of the gene expression profiles of breast tumours revealed that the highest expression levels of CXCR4 and CXCR7 were in the Claudin-Low molecular subtype, which is markedly associated with EMT features.
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- 2018
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34. Decatenation checkpoint-defective melanomas are dependent on PI3K for survival
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Brian Gabrielli, Kelly Brooks, Loredana Spoerri, Sandra Pavey, Fred Meunier, Alexander J. Stevenson, Thomas J. Gonda, Gency Gunasingh, Max V. Ranall, Brooks, Kelly, Ranall, Max, Spoerri, Loredana, Stevenson, Alex, Gunasingh, Gency, Pavey, Sandra, Meunier, Fred, Gonda, Thomas J, and Gabrielli, Brian
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Skin Neoplasms ,Cell cycle checkpoint ,DNA damage ,Morpholines ,Apoptosis ,Cell Cycle Proteins ,Cell Separation ,Dermatology ,Synthetic lethality ,Biology ,Polymerase Chain Reaction ,PI3K ,Gene Expression Regulation, Enzymologic ,General Biochemistry, Genetics and Molecular Biology ,Phosphatidylinositol 3-Kinases ,Cell Line, Tumor ,melanoma ,Humans ,Kinome ,RNA, Small Interfering ,Melanoma ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Phosphoinositide-3 Kinase Inhibitors ,Triazines ,Kinase ,Stem Cells ,decatenation checkpoint ,Cell Cycle Checkpoints ,G2-M DNA damage checkpoint ,Flow Cytometry ,synthetic lethality ,Gene Expression Regulation, Neoplastic ,DNA Topoisomerases, Type II ,Oncology ,Cancer research ,DNA Damage - Abstract
Melanoma cell lines are commonly defective for the G2-phase cell cycle checkpoint that responds to incomplete catenation of the replicated chromosomes. Here, we demonstrate that melanomas defective for this checkpoint response are less sensitive to genotoxic stress, suggesting that the defective cell lines compensated for the checkpoint loss by increasing their ability to cope with DNA damage. We performed an siRNA kinome screen to identify kinases responsible and identified PI3K pathway components. Checkpoint-defective cell lines were three-fold more sensitive to small molecule inhibitors of PI3K. The PI3K inhibitor PF-05212384 promoted apoptosis in the checkpoint-defective lines, and the increased sensitivity to PI3K inhibition correlated with increased levels of activated Akt. This work demonstrates that increased PI3K pathway activation is a necessary adaption for the continued viability of melanomas with a defective decatenation checkpoint. Refereed/Peer-reviewed
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- 2014
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35. Interaction of c-Myb with p300 is required for the induction of acute myeloid leukemia (AML) by human AML oncogenes
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Ingrid G. Winkler, Warren S. Alexander, Jean-Pierre Levesque, Valerie Barbier, Andrew C. Perkins, Keerthana Krishnan, Jianmin Ding, Crystal McGirr, Ann E.O. Trezise, Peter Papathanasiou, Paula L. Hawthorne, Thomas J. Gonda, Pamela Mukhopadhyay, Diwakar R. Pattabiraman, Konstantin Shakhbazov, Sean M. Grimmond, Pattabiraman, Diwakar R, McGirr, Crystal, Shakhbazov, Konstantin, Barbier, Valerie, Krishnan, Keerthana, Mukhopadhyay, Pamela, Hawthorne, Paula, Trezise, Ann, Ding, Jianmin, Grimmond, Sean M, Papathanasiou, Peter, Alexander, Warren S, Perkins, Andrew C, Levesque, Jean Pierre, Winkler, Ingrid G, and Gonda, Thomas J
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Myeloid ,Oncogene Proteins, Fusion ,Immunology ,Transcription factor complex ,P300-CBP Transcription Factors ,acute myeloid leukemia ,Biology ,Biochemistry ,Mice ,Proto-Oncogene Proteins c-myb ,Proto-Oncogene Proteins ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,p300-CBP Transcription Factors ,MYB ,neoplasms ,Alleles ,Gene Expression Regulation, Leukemic ,Gene Expression Profiling ,Myeloid leukemia ,Oncogenes ,Cell Biology ,Hematology ,medicine.disease ,Mice, Mutant Strains ,DNA-Binding Proteins ,Transplantation ,Leukemia, Myeloid, Acute ,Leukemia ,Cell Transformation, Neoplastic ,HEK293 Cells ,medicine.anatomical_structure ,c-MYB ,Core Binding Factor Alpha 2 Subunit ,Mutation ,Cancer research ,Transcription Factors - Abstract
The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, suggesting that MYB may be a therapeutic target in these diseases. However, realization of this potential requires a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis, and an approach for developing an effective therapeutic. We previously showed that the interactionof c-MybwiththecoactivatorCBP/p300 is essential for its transforming activity. Here, by using cells from Booreana mice which carry a mutant allele of c-Myb, we show that this interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type mice, Booreana cells transduced with AML1-ETO9a or MLL-AF9 retroviruses fail to generate leukemia upon transplantation into irradiated recipients. Finally, we have begun to explore themolecularmechanisms underlying these observations by gene expression profiling. This identified several genes previously implicated in myeloid leukemogenesis and HSC function as being regulated in a c-Myb-p300-dependent manner. These data highlight the importance of the c-Myb-p300 interaction inmyeloid leukemogenesis and suggest disruption of this interaction as a potential therapeutic strategy for acute myeloid leukemia. Refereed/Peer-reviewed
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- 2014
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36. MYB down-regulation enhances sensitivity of U937 myeloid leukemia cells to the histone deacetylase inhibitor LBH589 in vitro and in vivo
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Ping Ye, Liang Zhao, Thomas J. Gonda, Crystal McGirr, Ye, Ping, Zhao, Liang, McGirr, Crystal, and Gonda, Thomas J
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Cancer Research ,Indoles ,Cell Survival ,medicine.drug_class ,p300-CBP coactivator family ,MYB ,Antineoplastic Agents ,Apoptosis ,Mice, SCID ,Hydroxamic Acids ,Mice ,Proto-Oncogene Proteins c-myb ,AML ,Mice, Inbred NOD ,Cell Line, Tumor ,hemic and lymphatic diseases ,Panobinostat ,medicine ,LBH589/Panobinostat ,Animals ,Humans ,xenograft ,Gene knockdown ,U937 cell ,Chemistry ,Cell Cycle ,Histone deacetylase inhibitor ,apoptosis ,Myeloid leukemia ,U937 Cells ,medicine.disease ,Gene Expression Regulation, Neoplastic ,Histone Deacetylase Inhibitors ,Leukemia, Myeloid, Acute ,Leukemia ,Oncology ,Cancer research ,RNA Interference ,K562 Cells ,Neoplasm Transplantation ,K562 cells - Abstract
The effect of combining MYB suppression with the histone deacetylase inhibitor LBH589 was studied inhuman myeloid leukemia cell lines. MYB knockdown inhibited proliferation and induced apoptosis inU937 and K562 cells in vitro, and also sensitized both to the pro-apoptotic effect of LBH589. This was accompanied by enhanced expression of the pro-apoptotic BCL2 family members BOK and BIM. U937 cells carrying inducible MYB shRNA were also transplanted into NOD/SCID mice. The combination of MYB knockdown and LBH589 prolonged survival compared to either treatment alone, suggesting that further development of such combinations might lead to effective and safe leukemia therapies Refereed/Peer-reviewed
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- 2014
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37. The MYB proto-oncogene suppresses monocytic differentiation of acute myeloid leukemia cells via transcriptional activation of its target gene GFI1
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Thomas J. Gonda, Ping Ye, Liang Zhao, Zhao, L, Ye, P, and Gonda, TJ
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Transcriptional Activation ,Cancer Research ,animal structures ,Myeloid ,GFI1 ,p300-CBP coactivator family ,Cellular differentiation ,U937 ,MYB ,Biology ,Proto-Oncogene Mas ,Monocytes ,Oncogene Proteins v-myb ,Genetics ,medicine ,Humans ,Promoter Regions, Genetic ,Molecular Biology ,target gene ,fungi ,Myeloid leukemia ,Cell Differentiation ,U937 Cells ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,Leukemia, Myeloid, Acute ,Haematopoiesis ,medicine.anatomical_structure ,Cancer research ,Ectopic expression ,Transcription Factors ,monocytic differentiation - Abstract
The MYB gene is a master regulator of hematopoiesis and contributes to leukemogenesis in several species including humans. Although it is clear that MYB can promote proliferation, suppress apoptosis and block differentiation, the identities of the MYB target genes that mediate these effects have only been partially elucidated. Several studies, including our own, have collectively identified substantial numbers of MYB target genes, including candidates for each of these activities; however, functional validation, particularly in the case of differentiation suppression, has lagged well behind. Here we show that GFI1, which encodes an important regulator of hematopoietic stem cell (HSC) function and granulocytic differentiation, is a direct target of MYB in myeloid leukemia cells. Chromatin immunoprecipitation and reporter studies identified a functional MYB-binding site in the promoter region of GFI, whereas ectopic expression and small hairpin RNA-mediated knockdown of MYB resulted in concomitant increases and decreases, respectively, in GFI1 expression. We also demonstrate that GFI1, like MYB, can block the induced monocytic differentiation of a human acute myeloid leukemia cell line, and most importantly, that GFI1 is essential for MYB's ability to block monocytic differentiation. Thus, we have identified a target of MYB that is a likely mediator of its myeloid differentiation-blocking activity, and which may also be involved in MYB's activities in regulating normal HSC function and myeloid differentiation. Refereed/Peer-reviewed
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- 2013
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38. Oncosis and apoptosis induction by activation of an overexpressed ion channel in breast cancer cells
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Elena Pera, Michael J. G. Milevskiy, Paraic A. Kenny, Daneth L. Marcial, Kunsala T. D. S. Yapa, Nigel J. Waterhouse, Silke B. Chalmers, Peter J. Cabot, Gregory R. Monteith, S. Y. N. Jamaludin, Thomas J. Gonda, Melissa A. Brown, Eloise Dray, Iman Azimi, Sarah J. Roberts-Thomson, Adrian P. Wiegmans, Kum Kum Khanna, H. Lim, Amelia A. Peters, Korinne Northwood, Felicity M. Davis, Peters, AA, Jamaludin, SYN, Yapa, KTDS, Chalmers, S, Gonda, TJ, and Monteith, GR
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0301 basic medicine ,TRPV4 ,Cancer Research ,Programmed cell death ,Immunoblotting ,Mice, Nude ,TRPV Cation Channels ,Apoptosis ,Breast Neoplasms ,Biology ,Necrosis ,03 medical and health sciences ,Transient receptor potential channel ,Growth factor receptor ,Leucine ,Cell Line, Tumor ,Genetics ,Animals ,Humans ,Molecular Biology ,Calcium signaling ,Mice, Inbred BALB C ,Sulfonamides ,Reverse Transcriptase Polymerase Chain Reaction ,ion channels ,cancer cell proliferation ,Cell cycle ,Xenograft Model Antitumor Assays ,Cell biology ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,Female ,RNA Interference ,Intracellular ,breast cancer cells - Abstract
The critical role of calcium signalling in processes related to cancer cell proliferation and invasion has seen a focus on pharmacological inhibition of overexpressed ion channels in specific cancer subtypes as a potential therapeutic approach. However, despite the critical role of calcium in cell death pathways, pharmacological activation of overexpressed ion channels has not been extensively evaluated in breast cancer. Here we define the overexpression of transient receptor potential vanilloid 4 (TRPV4) in a subgroup of breast cancers of the basal molecular subtype. We also report that pharmacological activation of TRPV4 with GSK1016790A reduced viability of two basal breast cancer cell lines with pronounced endogenous overexpression of TRPV4, MDA-MB-468 and HCC1569. Pharmacological activation of TRPV4 produced pronounced cell death through two mechanisms: apoptosis and oncosis in MDA-MB-468 cells. Apoptosis was associated with PARP-1 cleavage and oncosis was associated with a rapid decline in intracellular ATP levels, which was a consequence of, rather than the cause of, the intracellular ion increase. TRPV4 activation also resulted in reduced tumour growth in vivo. These studies define a novel therapeutic strategy for breast cancers that overexpress specific calcium permeable plasmalemmal ion channels with available selective pharmacological activators. Refereed/Peer-reviewed
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- 2017
39. Ectodermal-Neural Cortex 1 Isoforms Have Contrasting Effects on MC3T3-E1 Osteoblast Mineralization and Gene Expression
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Leah E, Worton, Yan-Chuan, Shi, Elisabeth J, Smith, Simon C, Barry, Thomas J, Gonda, Jonathan P, Whitehead, and Edith M, Gardiner
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Osteoblasts ,Immunoblotting ,Microfilament Proteins ,Neuropeptides ,Nuclear Proteins ,Cell Differentiation ,Alkaline Phosphatase ,Osteocytes ,Mice ,Calcification, Physiologic ,Animals ,Protein Isoforms ,Wnt Signaling Pathway ,In Situ Hybridization ,beta Catenin - Abstract
The importance of Wnt pathway signaling in development of bone has been well established. Here we investigated the role of a known Wnt target, ENC1 (ectodermal-neural cortex 1; NRP/B), in osteoblast differentiation. Enc1 expression was detected in mouse osteoblasts, chondrocytes, and osteocytes by in situ hybridization, and osteoblastic expression was verified in differentiating primary cultures and MC3T3-E1 pre-osteoblast cells, with 57 kDa and 67 kDa ENC1 protein isoforms detected throughout differentiation. Induced knockdown of both ENC1 isoforms reduced alkaline phosphatase staining and virtually abolished MC3T3-E1 mineralization. At culture confluence, Alpl (alkaline phosphatase liver/bone/kidney) expression was markedly reduced compared with control cells, and there was significant and coordinated alteration of other genes involved in cellular phosphate biochemistry. In contrast, with 67 kDa-selective knockdown mineralized nodule formation was enhanced and there was a two-fold increase in Alpl expression at confluence. There was enhanced expression of Wnt/β-catenin target genes with knockdown of both isoforms at this time-point and a five-fold increase in Frzb (Frizzled related protein) with 67 kDa-selective knockdown at mineralization, indicating possible ENC1 interactions with Wnt signaling in osteoblasts. These results are the first to demonstrate a role for ENC1 in the control of osteoblast differentiation. Additionally, the contrasting mineralization phenotypes and transcriptional patterns seen with coordinate knockdown of both ENC1 isoforms vs selective knockdown of 67 kDa ENC1 suggest opposing roles for the isoforms in regulation of osteoblastic differentiation, through effects on Alpl expression and phosphate cellular biochemistry. This study is the first to report differential roles for the ENC1 isoforms in any cell lineage. J. Cell. Biochem. 118: 2141-2150, 2017. © 2016 Wiley Periodicals, Inc.
- Published
- 2016
40. Myb-binding protein 1a (Mybbp1a) regulates levels and processing of pre-ribosomal RNA
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Michael Hölzel, Gernot Längst, Lothar Schermelleh, Thomas J. Gonda, Heinrich Leonhardt, Dirk Eick, Julia Hochstatter, Axel Imhof, Attila Németh, Michaela Rohrmoser, Rebecca Keough, Hochstatter, Julia, Hölzel, Michael, Rohrmoser, Michaela, Schermelleh, Lothar, Leonhardt, Heinrich, Keough, Rebecca, Gonda, Thomas J, Imhof, Axel, Eick, Dirk, Längst, Gernot, and Németh, Attila
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Chromatin Immunoprecipitation ,Nucleocytoplasmic Transport Proteins ,5.8S ribosomal RNA ,Ribosome biogenesis ,RNA polymerase II ,Biology ,Real-Time Polymerase Chain Reaction ,Gene Regulation ,Nucleolus ,Ribosomal RNA (rRNA) ,Ribosomal RNA Processing ,Transcription Regulation ,Biochemistry ,Cell Line ,Mice ,5S ribosomal RNA ,RNA Polymerase I ,Transcription (biology) ,RNA polymerase I ,Animals ,Humans ,RNA Processing, Post-Transcriptional ,RNA, Small Interfering ,Molecular Biology ,General transcription factor ,Nuclear Proteins ,RNA-Binding Proteins ,Cell Biology ,Ribosomal RNA ,Blotting, Northern ,Molecular biology ,Myb-binding ,Cell biology ,DNA-Binding Proteins ,Microscopy, Fluorescence ,RNA, Ribosomal ,biology.protein ,Carrier Proteins ,ribosomal RNA ,Ribosomes ,HeLa Cells ,Transcription Factors - Abstract
Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes. Refereed/Peer-reviewed
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- 2016
41. Adenoid Cystic Carcinoma Can Be Driven by MYB or MYBL1 Rearrangements: New Insights into MYB and Tumor Biology
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Robert G. Ramsay, Thomas J. Gonda, Gonda, Thomas J, and Ramsay, Robert G
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0301 basic medicine ,Oncogene Proteins ,MYB and tumor biology ,Oncogene Proteins, Fusion ,Adenoid cystic carcinoma ,Genes, myb ,Chromosomal translocation ,Biology ,Bioinformatics ,Adenoid ,Translocation, Genetic ,Article ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Carcinoma ,Humans ,MYB ,Gene ,Salivary gland ,fungi ,medicine.disease ,Carcinoma, Adenoid Cystic ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,030220 oncology & carcinogenesis ,adenoid cystic carcinomas ,Cancer research - Abstract
Adenoid Cystic Carcinoma (ACC), the second most common malignancy of salivary glands, is a rare tumor with bleak prognosis for which therapeutic targets are unavailable. We used RNA-sequencing (RNA-seq) to analyze low-quality RNA from archival, formaldehyde-fixed, paraffin-embedded samples. In addition to detecting the most common ACC translocation, t(6;9) fusing the MYB proto-oncogene to NFIB, we also detected previously unknown t(8;9) and t(8;14) translocations fusing the MYBL1 gene to the NFIB and RAD51B genes, respectively. RNA-seq provided information about gene fusions, alternative RNA splicing and gene expression signatures. Interestingly, tumors with MYB and MYBL1 translocations displayed similar gene expression profiles, and the combined MYB and MYBL1 expression correlated with outcome, suggesting that the related Myb proteins are interchangeable oncogenic drivers in ACC. Our results provide important details about the biology of ACC and illustrate how archival tissue samples can be used for detailed molecular analyses of rare tumors.
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- 2016
42. Estrogen receptor-α recruits P-TEFb to overcome transcriptional pausing in intron 1 of the MYB gene
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Robert G. Ramsay, Yvette Drabsch, Thomas J. Gonda, Partha Mitra, Lloyd Pereira, Mitra, Partha, Pereira, Lloyd A, Drabsch, Yvette, Ramsay, Robert G, and Gonda, Thomas J
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Positive Transcriptional Elongation Factor B ,Transcription, Genetic ,Genes, myb ,RNA polymerase II ,Breast Neoplasms ,Biology ,Gene Regulation, Chromatin and Epigenetics ,Proto-Oncogene Mas ,C-MYB ,Transcription (biology) ,Cell Line, Tumor ,Genetics ,Humans ,MYB ,Transcriptional attenuation ,Regulatory Elements, Transcriptional ,Phosphorylation ,P-TEFb ,Estradiol ,Estrogen Receptor alpha ,Estrogens ,Introns ,HEK293 Cells ,Cancer research ,biology.protein ,Cyclin-dependent kinase 9 ,Female ,RNA Polymerase II ,Estrogen receptor alpha ,breast cancer cells - Abstract
The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα+) breast tumors and cell lines. Expression of MYB is controlled, in breast cancer and other cell types, by a transcriptional pausing mechanism involving an attenuation site located ∼1.7kb downstream from the transcription start site. In breast cancer cells, ligand-bound ERα binds close to, and drives transcription beyond this attenuation site, allowing synthesis of complete transcripts. However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation. Using cyclin dependent kinase 9 (CDK9) inhibitors, reporter gene assays and measurements of total and intronic MYB transcription, we show that functionally active CDK9 is required for estrogen-dependent transcriptional elongation. We further show by ChIP and co-immunoprecipitation studies that the P-TEFb complex (CDK9/CyclinT1) is recruited to the attenuation region by ligand-bound ERα, resulting in increased RNA polymerase II Ser-2 phosphorylation. These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy. Refereed/Peer-reviewed
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- 2012
43. Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb
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Matthew A. Brown, Liang Zhao, Ping Zhang, Diwakar R. Pattabiraman, Thomas J. Gonda, Evgeny A. Glazov, Faisal Al-Owaidi, Paul Leo, Zhao, Liang, Glazov, Evgeny A, Pattabiraman, Diwakar R, Al-Owaidi, Faisal, Zhang, Ping, Brown, Matthew A, Leo, Paul J, and Gonda, Thomas J
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Chromatin Immunoprecipitation ,animal structures ,Transcription, Genetic ,p300-CBP coactivator family ,Cellular differentiation ,Myb ,P300-CBP Transcription Factors ,Gene Regulation, Chromatin and Epigenetics ,Biology ,Histones ,Mice ,Proto-Oncogene Proteins c-myb ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Genetics ,Animals ,Gene Regulatory Networks ,p300-CBP Transcription Factors ,MYB ,Cells, Cultured ,Myeloid Progenitor Cells ,030304 developmental biology ,Myelopoiesis ,0303 health sciences ,Binding Sites ,Leukemia ,Gene Expression Profiling ,parallel sequencing ,Genomics ,Chromatin ,Mice, Inbred C57BL ,Repressor Proteins ,Gene Expression Regulation ,RUNX1 ,chemistry ,030220 oncology & carcinogenesis ,Chromatin immunoprecipitation ,Transcription Factors - Abstract
To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb's ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation. Refereed/Peer-reviewed
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- 2011
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44. PG545, a dual heparanase and angiogenesis inhibitor, induces potent anti-tumour and anti-metastatic efficacy in preclinical models
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C Vincent, Edward Hammond, Maree T. Smith, R Brandt, Keith Dredge, Paul Handley, Vito Ferro, Ian Bytheway, Thomas J. Gonda, Dredge, K, Hammond, E, Handley, P, Gonda, TJ, Smith, MT, Vincent, C, Brandt, R, Ferro, V, and Bytheway, I
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Cancer Research ,Pathology ,medicine.medical_specialty ,Angiogenesis ,Mice, Nude ,Angiogenesis Inhibitors ,Antineoplastic Agents ,Mice, SCID ,heparanase ,Metastasis ,Neovascularization ,Mice ,angiogenesis ,chemistry.chemical_compound ,In vivo ,Cell Line, Tumor ,Neoplasms ,medicine ,Animals ,Humans ,Heparanase ,Neoplasm Metastasis ,Glucuronidase ,Mice, Inbred BALB C ,business.industry ,Cancer ,Heparan sulfate ,Saponins ,medicine.disease ,Xenograft Model Antitumor Assays ,Angiogenesis inhibitor ,Mice, Inbred C57BL ,Treatment Outcome ,anti-metastatic ,Oncology ,chemistry ,Cancer research ,heparan sulfate ,anti-tumour ,medicine.symptom ,Translational Therapeutics ,business ,HT29 Cells - Abstract
Background:PG545 is a heparan sulfate (HS) mimetic that inhibits tumour angiogenesis by sequestering angiogenic growth factors in the extracellular matrix (ECM), thus limiting subsequent binding to receptors. Importantly, PG545 also inhibits heparanase, the only endoglycosidase which cleaves HS chains in the ECM. The aim of the study was to assess PG545 in various solid tumour and metastasis models.Methods:The anti-angiogenic, anti-tumour and anti-metastatic properties of PG545 were assessed using in vivo angiogenesis, solid tumour and metastasis models. Pharmacokinetic (PK) data were also generated in tumour-bearing mice to gain an understanding of optimal dosing schedules and regimens.Results:PG545 was shown to inhibit angiogenesis in vivo and induce anti-tumour or anti-metastatic effects in murine models of breast, prostate, liver, lung, colon, head and neck cancers and melanoma. Enhanced anti-tumour activity was also noted when used in combination with sorafenib in a liver cancer model. PK data revealed that the half-life of PG545 was relatively long, with pharmacologically relevant concentrations of radiolabeled PG545 observed in liver tumours.Conclusion:PG545 is a new anti-angiogenic clinical candidate for cancer therapy. The anti-metastatic property of PG545, likely due to the inhibition of heparanase, may prove to be a critical attribute as the compound enters phase I clinical trials. Refereed/Peer-reviewed
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- 2011
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45. A High-Throughput Screening Approach to Identify Therapeutics for the Treatment of Diamond-Blackfan Anaemia
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Ross D. Hannan, Thomas J. Gonda, Jun Chen, Megan Pavy, Amee J George, Piyush B. Madhamshettiwar, Kaylene J. Simpson, Priscilla Soo, Lorena Nunez Villacis, Perlita Poh, Nadine Hein, Richard B. Pearson, Johan Flygare, Sheren Al-Obaidi, and Katherine M. Hannan
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High-Throughput Screening Methods ,Diamond-Blackfan anaemia ,business.industry ,Immunology ,Cancer therapy ,Cell Biology ,Hematology ,Computational biology ,medicine.disease ,Biochemistry ,Cyclic gmp ,medicine ,Diamond–Blackfan anemia ,Erythroid Progenitor Cells ,business ,Throughput (business) ,Protein p53 - Abstract
Diamond-Blackfan Anaemia (DBA) is a rare blood cell aplasia that presents clinically at approximately 2-3 months of age and its main characteristic is reduced erythroid precursors in the bone marrow, i.e. anemia. Mutations in different ribosomal protein (RP) genes have been associated with DBA, with mutations in RPS19 accounting for 20-25% of all cases. It has been proposed that RPS19 deficiency causes perturbations in ribosome biogenesis, thus activation of the p53-dependent Nucleolar Surveillance Pathway (NSP). In this context free RPs (predominantly L5 and L11) in a complex with 5S rRNA sequester the E3 ubiquitin ligase murine double minute 2 (MDM2), leading to the accumulation of p53 and subsequent activation of its transcriptional targets mediating cell cycle arrest or apoptosis. In DBA, one of the molecular mechanisms impairing the proliferation and thus reducing the number of erythroid progenitors that can progress to mature red blood cells is an elevation of p53 protein mediating activation of the NSP. In order to identify potential therapeutics that could be repurposed to prevent the activation of NSP in DBA patients, we have screened compound libraries of clinically approved therapeutics to identify pathways implicated in the p53-dependent NSP due to RPS19 deficiency. We quantitated both cell number and the level of p53 expression, identifying compounds that can result in low and high expression of p53, the latter for potential use in cancer therapy. Using an RPS19 depleted A549 cell line as a model system, the screen successfully identified different therapeutic groups. In the DBA context, we were most interested in the compounds that reduced p53 and had no negative effect on cell number. A selection of 22 molecules were re-evaluated in vitro, again using RPS19 depleted A549 cells, through the quantification of p53 protein expression and densitometry analysis. From this, 10candidates were evaluated ex vivofor their effects on proliferation using bone marrow obtained from an inducible Rps19 knockdown (DBA) mouse model. While we are currently testing a number of compounds in vivo using the Rps19 DBA mouse model (as described), one of the compounds tested thus far has demonstrated a partial rescue of the cKit+ population, no changes in erythroid precursors but interestingly a reversal of the defect in the Granulocyte-Monocyte Progenitor (GMP) population. Impairment in lineage progression in the GMP compartment has also been reported to present in bone marrow failure Shwachman-Diamond Syndrome. We are currently evaluating the mechanism by which this drug is rescuing the c-Kit+ and GMP populations in these mice. In summary, our high-throughput screening approach and follow up studies have identified a suite of novel therapies that may be beneficial for repurposing for the treatment of bone marrow failure by increasing hematopoietic progenitor cells. We plan to evaluate this, and potentially other therapies, in a clinical trial with DBA patients. Disclosures Flygare: LU Holding: Patents & Royalties: Patent.
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- 2018
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46. A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability
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Thomas Sæther, Anne Hege Alm-Kristiansen, Diwakar R. Pattabiraman, L T Vogt-Kielland, Odd S. Gabrielsen, Thomas J. Gonda, Sæther, T, Pattabiraman, DR, Alm-Kristiansen, AH, Vogt-Kielland, LT, Gonda, TJ, and Gabrielsen, OS
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Cancer Research ,Amino Acid Motifs ,Molecular Sequence Data ,Mutant ,SUMO protein ,Bone Marrow Cells ,SUMO binding ,SUMO2 ,Biology ,environment and public health ,Cell Line ,Proto-Oncogene Proteins c-myb ,Transactivation ,Chlorocebus aethiops ,Consensus Sequence ,Genetics ,Animals ,Humans ,Myeloid Cells ,MYB ,Amino Acid Sequence ,Molecular Biology ,Transcription factor ,Psychological repression ,transformation ,Cell Differentiation ,Molecular biology ,hematopoiesis ,Protein Structure, Tertiary ,enzymes and coenzymes (carbohydrates) ,Cell Transformation, Neoplastic ,SUMO ,c-Myb ,COS Cells ,Mutation ,SUMO-interacting motif ,Small Ubiquitin-Related Modifier Proteins ,myeloid ,Protein Binding - Abstract
c-Myb is an essential hematopoietic transcription factor that controls proliferation and differentiation of progenitors during blood cell development. Whereas sumoylation of the C-terminal regulatory domain (CRD) is known to have a major impact on the activity of c-Myb, no role for noncovalent binding of small ubiquitin-like modifier (SUMO) to c-Myb has been described. Based on the consensus SUMO-interacting motif (SIM), we identified and examined putative SIMs in human c-Myb. Interaction and reporter assays showed that the SIM in the in the transactivation domain of c-Myb (V 267 NIV) is functional. This motif is necessary for c-Myb to be able to interact noncovalently with SUMO, preferentially SUMO2/3. Destroying the SUMO-binding properties by mutation resulted in a large increase in the transactivation potential of c-Myb. Mutational analysis and overexpression of conjugation-defective SUMO argued against intramolecular repression caused by sumoylated CRD and in favor of SUMO-dependent repression in trans. Using both a myeloid cell line-based assay and a primary hematopoietic cell assay, we addressed the transforming abilities of SUMO binding and conjugation mutants. Interestingly, only loss of SUMO binding, and not SUMO conjugation, enhanced the myeloid transformational potential of c-Myb. c-Myb with the SIM mutated conferred a higher proliferative ability than the wild-type and caused an effective differentiation block. This establishes SUMO binding as a mechanism involved in modulating the transactivation activity of c-Myb, and responsible for keeping the transforming potential of the oncoprotein in check. Refereed/Peer-reviewed
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- 2010
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47. CDK9 inhibitors selectively target estrogen receptor-positive breast cancer cells through combined inhibition of MYB and MCL-1 expression
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James Sutton, Ren-Ming Yang, Partha Mitra, Thomas J. Gonda, Robert G. Ramsay, Mitra, Partha, Yang, Ren Ming, Sutton, James, Ramsay, Robert G, and Gonda, Thomas J
- Subjects
0301 basic medicine ,Cell Survival ,bcl-X Protein ,Estrogen receptor ,Antineoplastic Agents ,Apoptosis ,Breast Neoplasms ,MYB ,Biology ,03 medical and health sciences ,Proto-Oncogene Proteins c-myb ,0302 clinical medicine ,Breast cancer ,breast cancer ,Cell Line, Tumor ,Cyclin E ,medicine ,Humans ,Cyclin B1 ,RNA, Small Interfering ,Cell Proliferation ,Oncogene Proteins ,Sulfonamides ,Cell growth ,CDK9 inhibitors ,apoptosis ,transcription pausing ,Cell cycle ,medicine.disease ,Bridged Bicyclo Compounds, Heterocyclic ,Molecular biology ,Cyclin-Dependent Kinase 9 ,G2 Phase Cell Cycle Checkpoints ,030104 developmental biology ,Cell killing ,Oncology ,Proto-Oncogene Proteins c-bcl-2 ,Receptors, Estrogen ,030220 oncology & carcinogenesis ,Cancer research ,MCF-7 Cells ,Myeloid Cell Leukemia Sequence 1 Protein ,Female ,RNA Interference ,Research Paper - Abstract
// Partha Mitra 1 , Ren-Ming Yang 1 , James Sutton 2 , Robert G. Ramsay 3,4 and Thomas J. Gonda 1 1 School of Pharmacy, University of Queensland, Brisbane, QLD, Australia 2 Novartis Institute for Biomedical Research, Emeryville, CA, USA 3 Peter MacCallum Cancer Centre, Melbourne, VIC, Australia 4 Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC, Australia Correspondence to: Partha Mitra, email: // Keywords : CDK9 inhibitors, MYB, breast cancer, transcription pausing, apoptosis Received : September 28, 2015 Accepted : January 17, 2016 Published : January 24, 2016 Abstract Our previous studies showed that MYB is required for proliferation of, and confers protection against apoptosis on, estrogen receptor-positive (ER +ve ) breast cancer cells, which are almost invariably also MYB +ve . We have also shown that MYB expression in ER +ve breast cancer cells is regulated at the level of transcriptional elongation and as such, is suppressed by CDK9 i . Here we examined the effects of CDK9 i on breast cancer cells and the involvement of MYB in these effects. ER +ve breast cancer cell lines including MCF-7 were much more sensitive (> 10 times) to killing by CDK9 i than ER -ve /MYB -ve cells. Moreover, surviving cells showed a block at the G2/M phase of the cell cycle. Importantly, ectopic MYB expression conferred resistance to apoptosis induction, cell killing and G2/M accumulation. Expression of relevant MYB target genes including BCL2 and CCNB1 was suppressed by CDK9 inhibition, and this too was reversed by ectopic MYB expression. Nevertheless, inhibition of BCL2 alone either by MYB knockdown or by ABT-199 treatment was insufficient for significant induction of apoptosis. Further studies implied that suppression of MCL-1 , a well-documented target of CDK9 inhibition, was additionally required for apoptosis induction, while maximal levels of apoptosis induced by CDK9 i are likely to also involve inhibition of BCL2L1 expression. Taken together these data suggest that MYB regulation of BCL2 underlies the heightened sensitivity of ER +ve compared to ER -ve breast cancer cells to CDK9 inhibition, and that these compounds represent a potential therapeutic for ER +ve breast cancers and possibly other MYB -dependent cancers.
- Published
- 2016
48. Ribosomal stress induces processing of Mybbp1a and its translocation from the nucleolus to the nucleoplasm
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Thomas J. Gonda, Tomohiro Yamauchi, Rebecca A. Keough, and Shunsuke Ishii
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Nucleophosmin ,Nucleoplasm ,Nucleolus ,Ribosome biogenesis ,Cell Biology ,Biology ,Ribosome ,Molecular biology ,Cell biology ,Cell nucleus ,medicine.anatomical_structure ,Genetics ,medicine ,Nuclear protein ,Nucleolin - Abstract
Myb-binding protein 1a (Mybbp1a) was originally identified as a c-myb proto-oncogene product (c-Myb)-interacting protein, and also binds to various other transcription factors. The 160-kDa Mybbp1a protein (p160(MBP)) is ubiquitously expressed and is post-translationally processed in some types of cells to generate an amino-terminal 67 kDa fragment (p67(MBP)). Despite its interaction with various transcription factors, Mybbp1a is localized predominantly, but not exclusively, in nucleoli. Here, we have purified the two Mybbp1a-containing complexes. The smaller complex contained p67(MBP) and p140(MBP), which lacked the C-terminal region of p160(MBP) containing the nucleolar localization sequences. The larger complex contained the intact p160(MBP) and various ribosomal subunits. Treatment of cells with actinomycin D (ActD), cisplatin or UV, all of which inhibit ribosome biogenesis, induced processing of p160(MBP) into p140(MBP) and p67(MBP). ActD, cisplatin and UV also induced a translocation of Mybbp1a from the nucleolus to the nucleoplasm. Both small and large Mybbp1a complexes contained nucleophosmin and nucleolin. In contrast, nucleostemin was detected only in the large complex, while the cell cycle-regulated protein EBP1 was only in the small complex. These results suggest that Mybbp1a may connect the ribosome biogenesis and the Myb-dependent transcription, which controls cell cycle progression and proliferation.
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- 2007
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49. Directly targeting transcriptional dysregulation in cancer
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Robert G. Ramsay, Thomas J. Gonda, Gonda, Thomas J, and Ramsay, Robert G
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Regulation of gene expression ,Tumour heterogeneity ,Kinase ,Applied Mathematics ,General Mathematics ,Gene Expression Profiling ,Cancer ,Antineoplastic Agents ,Biology ,medicine.disease ,Bioinformatics ,Phenotype ,Signalling ,Gene Expression Regulation ,Neoplasms ,medicine ,Transcriptional regulation ,Cancer research ,Biomarkers, Tumor ,cancer ,Humans ,untreatable cancer ,Transcription factor ,Signal Transduction - Abstract
Drugs that target intracellular signalling pathways have markedly improved progression-free survival of patients with cancers who were previously regarded as untreatable. However, the rapid emergence of therapeutic resistance, as a result of bypass signalling or downstream mutation within kinase-mediated signalling cascades, has curtailed the benefit gained from these therapies. Such resistance mechanisms are facilitated by the linearity and redundancy of kinase signalling pathways. We argue that, in each cancer, the dysregulation of key transcriptional regulators not only defines the cancer phenotype but is essential for its development and maintenance. Furthermore, we propose that, as therapeutic targets, these transcriptional regulators are less prone to bypass by alternative mutational events or clonal heterogeneity, and therefore we must rekindle our efforts to directly target transcriptional regulation across a broad range of cancers. Refereed/Peer-reviewed
- Published
- 2015
50. Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer
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Fawzi Bokhari, Nigel A.J. McMillan, Melanie Murrell, H. Peter Soyer, Graham R. Leggatt, Karin Sedelies, Brian Gabrielli, Zay Yar Oo, Weili Wang, Mushfiq Hassan Shaikh, Melinda E. Christensen, Sora Fallaha, Dubravka Skalamera, Max V. Ranall, Alexander J. Stevenson, Thomas J. Gonda, Madison Kelly, Daniel Clarke, Sara J. McKee, Paul Leo, Gabrielli, Brian, Bokhari, Fawzi, Ranall, Max V, Oo, Zay Yar, Stevenson, Alexander J, Wang, Weili, Murrell, Melanie, Shaikh, Mushfiq, Fallaha, Sora, Clarke, Daniel, Kelly, Madison, Sedelies, Karin, Christensen, Melinda, McKee, Sara, Leggatt, Graham, Leo, Paul, Skalamera, Dubravka, Soyer, H Peter, Gonda, Thomas J, and McMillan, Nigel AJ
- Subjects
Cancer Research ,cervical cancer ,Papillomavirus E7 Proteins ,Aurora A kinase ,Uterine Cervical Neoplasms ,Apoptosis ,chemistry.chemical_compound ,Mice ,Cell Line, Tumor ,Medicine ,Animals ,Humans ,Papillomaviridae ,aurora A ,Mitosis ,Aurora Kinase A ,Cell Proliferation ,Cervical cancer ,biology ,business.industry ,Cell growth ,virus diseases ,Azepines ,biology.organism_classification ,medicine.disease ,Xenograft Model Antitumor Assays ,female genital diseases and pregnancy complications ,Pyrimidines ,Oncology ,chemistry ,Immunology ,Alisertib ,Cancer research ,Myeloid Cell Leukemia Sequence 1 Protein ,Female ,business - Abstract
Human papillomavirus (HPV) is the causative agent in cervical cancer. HPV oncogenes are major drivers of the transformed phenotype, and the cancers remain addicted to these oncogenes. A screen of the human kinome has identified inhibition of Aurora kinase A (AURKA) as being synthetically lethal on the background of HPV E7 expression. The investigational AURKA inhibitor MLN8237/Alisertib selectively promoted apoptosis in the HPV cancers. The apoptosis was driven by an extended mitotic delay in the Alisertib-treated HPV E7–expressing cells. This had the effect of reducing Mcl-1 levels, which is destabilized in mitosis, and increasing BIM levels, normally destabilized by Aurora A in mitosis. Overexpression of Mcl-1 reduced sensitivity to the drug. The level of HPV E7 expression influenced the extent of Alisertib-induced mitotic delay and Mcl-1 reduction. Xenograft experiments with three cervical cancer cell lines showed Alisertib inhibited growth of HPV and non-HPV xenografts during treatment. Growth of non-HPV tumors was delayed, but in two separate HPV cancer cell lines, regression with no resumption of growth was detected, even at 50 days after treatment. A transgenic model of premalignant disease driven solely by HPV E7 also demonstrated sensitivity to drug treatment. Here, we show for the first time that targeting of the Aurora A kinase in mice using drugs such as Alisertib results in a curative sterilizing therapy that may be useful in treating HPV-driven cancers. Mol Cancer Ther; 14(12); 2753–61. ©2015 AACR.
- Published
- 2015
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