snx-7081 and Leukemia--Lymphocytic--Chronic--B-Cell

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.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Blotting, Western; BRCA1 Protein; Cell Line, Tumor; Chromatography, Liquid; Cyclin D1; DNA Damage; DNA Repair; Drug Synergism; HSP90 Heat-Shock Proteins; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mutation; NF-kappa B p52 Subunit; Nucleolin; Phosphoproteins; Protein Interaction Maps; Proteomics; Proto-Oncogene Proteins c-myc; RNA-Binding Proteins; Signal Transduction; Tandem Mass Spectrometry; Tumor Suppressor Protein p53; Vidarabine

The proteomic effects of the Hsp90 inhibitor, SNX-7081, have been determined on the p53-mutated B-cell chronic lymphocytic leukemia (CLL) cell line, MEC1. Following SNX-7081 treatment (500 nM, 24 h), 51 proteins changed abundance by more than 2-fold (p < 0.05); 7 proteins increased while 44 proteins decreased. Proteins identified as differentially abundant by LC-MS/MS were validated by Western blotting (DDB1, PCNA, MCM2, Hsp90, Hsp70, GRP78, PDIA6, HLA-DR). RT-PCR showed that SNX-7081 unexpectedly modulates a number of these proteins in MEC1 cells at the mRNA level (PCNA, MCM2, Nup155, Hsp70, GRP78, PDIA6, and HLA-DR). Pathway analysis determined that 3 of the differentially abundant proteins (cyclin D1, c-Myc and pRb) were functionally related. p53 levels did not change upon SNX-7081 treatment of p53 wild-type Raji cells or p53-mutated MEC1 and U266 cells, indicating that SNX-7081 has a p53-independent mechanism. The decreases in DDB1, MCM2, c-Myc, and PCNA and increases of pRb and cyclin D1 were confirmed in MEC1, U266, Raji, and p53 null HL60 cells by Western blotting. These data suggest that SNX-7081 arrests the cell cycle and inhibits DNA replication and r epair and provides evidence for the mechanism of the observed synergy between Hsp90 inhibitors and drugs that induce DNA strand breaks.

Topics: Apoptosis; Benzamides; Cell Cycle; Cell Line, Tumor; DNA Repair; DNA Replication; Endoplasmic Reticulum Chaperone BiP; Genes, p53; HSP90 Heat-Shock Proteins; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Proteome; Reproducibility of Results; Tandem Mass Spectrometry

Drug resistance in chronic lymphocytic leukemia (CLL) associated with lesions in the ATM/TP53 pathway represents a major challenge in clinical management. Evidence suggests that heat shock protein-90 (Hsp90) inhibitors may represent a therapeutic option in combination with more conventional therapies. We explored the effects of combining the Hsp90 inhibitor, SNX-7081, with fludarabine in vitro against CLL cells and hematological cell lines. In seven cell lines and 23 patient samples synergy between SNX-7081 and fludarabine (2-FaraA) was apparent in the three TP53 mutated cell lines and at significantly lower concentrations in TP53 or ATM dysfunctional patient cells. In 11/13 2-FaraA-resistant patient samples, SNX-7081 reduced the 50% inhibitory concentration to within a clinically achievable range. Synergy between SNX-7081 and 2-FaraA was evident in both the cell lines and patient samples as a significant decrease in cell viability. Our data suggest that combining SNX-7081 and fludarabine may be effective in the treatment of fludarabine-refractory CLL.

Topics: Antineoplastic Agents; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Benzamides; Cell Cycle; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Cell Survival; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Flow Cytometry; HL-60 Cells; HSP90 Heat-Shock Proteins; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mutation; Protein Serine-Threonine Kinases; Signal Transduction; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Vidarabine

Inhibitors of heat-shockprotein 90 (Hsp90) have been proposed as a novel therapeutic option for Chronic Lymphocytic Leukaemia (CLL), particularly as their mechanism of action appears independent of mutations of ATM or TP53. We investigated the activity of a novel Hsp90 inhibitor, SNX7081, against a panel of eight haematological cell lines and 23 CLL patient samples. SNX7081 displayed significant effects on cell cycle distribution, apoptotic rate and levels of ZAP-70 in the cell lines and in the patient samples, irrespective of TP53 status. Our findings suggest SNX7081 may represent a promising therapeutic option for aggressive CLL.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Benzoquinones; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Genes, p53; Hematologic Neoplasms; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leukemia, Lymphocytic, Chronic, B-Cell; Mutation; Neoplasm Proteins; Tumor Cells, Cultured; ZAP-70 Protein-Tyrosine Kinase