title: The Hsp90 inhibitor SNX-7081 is synergistic with fludarabine nucleoside via DNA damage and repair mechanisms in human, p53-negative chronic lymphocytic leukemia.
abstract: Clinical trials of heat shock protein 90 (Hsp90) inhibitors have been limited by high toxicity. We previously showed that the Hsp90 inhibitor, SNX-7081, synergizes with and restores sensitivity to fludarabine nucleoside (2-FaraA) in human chronic lymphocytic leukemia (CLL) cells with lesions in the p53 pathway (Best OG, et al., Leukemia Lymphoma 53:1367-75, 2012). Here, we used label-free quantitative shotgun proteomics and comprehensive bioinformatic analysis to determine the mechanism of this synergy. We propose that 2-FaraA-induced DNA damage is compounded by SNX-7081-mediated inhibition of DNA repair, resulting in enhanced induction of apoptosis. DNA damage responses are impaired in part due to reductions in checkpoint regulators BRCA1 and cyclin D1, and cell death is triggered following reductions of MYC and nucleolin and an accumulation of apoptosis-inducing NFkB2 p100 subunit. Loss of nucleolin can activate Fas-mediated apoptosis, leading to the increase of pro-apoptotic proteins (BID, fas-associated factor-2) and subsequent apoptosis of p53-negative, 2-FaraA refractory CLL cells. A significant induction of DNA damage, indicated by increases in DNA damage marker γH2AX, was observed following the dual drug treatment of additional cell lines, indicating that a similar mechanism may operate in other p53-mutated human B-lymphoid cancers. These results provide valuable insight into the synergistic mechanism between SNX-7081 and 2-FaraA that may provide an alternative treatment for CLL patients with p53 mutations, for whom therapeutic options are currently limited. Moreover, this drug combination reduces the effective dose of the Hsp90 inhibitor and may therefore alleviate any toxicity encountered.
authors.affiliation: School of Molecular Bioscience, University of Sydney, Darlington, NSW 2006, Australia.|Molecular Neuropathology, Brain and Mind Centre, Camperdown, NSW 2050, Australia.
authors.forename: Kimberley L
authors.initials: KL
authors.lastname: Kaufman
authors.affiliation: School of Molecular Bioscience, University of Sydney, Darlington, NSW 2006, Australia.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Jenkins
authors.affiliation: School of Molecular Bioscience, University of Sydney, Darlington, NSW 2006, Australia.
authors.forename: Munther
authors.initials: M
authors.lastname: Alomari
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109, Australia.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Mirzaei
authors.affiliation: Northern Blood Research Centre, Kolling Institute for Medical Research, Royal North Shore Hospital, St Leonards, NSW 2065, Australia.
authors.forename: O Giles
authors.initials: OG
authors.lastname: Best
authors.affiliation: Australian Proteome Analysis Facility, Macquarie University, North Ryde, NSW 2109, Australia.
authors.forename: Dana
authors.initials: D
authors.lastname: Pascovici
authors.affiliation: School of Molecular Bioscience, University of Sydney, Darlington, NSW 2006, Australia.
authors.forename: Swetlana
authors.initials: S
authors.lastname: Mactier
authors.affiliation: Northern Blood Research Centre, Kolling Institute for Medical Research, Royal North Shore Hospital, St Leonards, NSW 2065, Australia.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Mulligan
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109, Australia.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Haynes
authors.affiliation: School of Molecular Bioscience, University of Sydney, Darlington, NSW 2006, Australia.
authors.forename: Richard I
authors.initials: RI
authors.lastname: Christopherson
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D019313:BRCA1 Protein / Q000378:metabolism; D001549:Benzamides / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D019938:Cyclin D1 / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects*; D004357:Drug Synergism; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D052003:NF-kappa B p52 Subunit / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D060066:Protein Interaction Maps / Q000187:drug effects; D040901:Proteomics / Q000379:methods; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D053719:Tandem Mass Spectrometry; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000097796:Nucleolin
chemical_list: D000970:Antineoplastic Agents; D019313:BRCA1 Protein; D001549:Benzamides; D019938:Cyclin D1; D018841:HSP90 Heat-Shock Proteins; D052003:NF-kappa B p52 Subunit; D010750:Phosphoproteins; D016271:Proto-Oncogene Proteins c-myc; D016601:RNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D014740:Vidarabine; D000097796:Nucleolin; C492913:BRCA1 protein, human; C489427:MYC protein, human; C535175:SNX-7081; C024352:fludarabine
keywords: Hsp90 inhibitor; MYC; NFkB2; chronic lymphocytic leukemia (CLL); fludarabine nucleoside