The Hsp90 inhibitor SNX-7081 is synergistic with fludarabine nucleoside via DNA damage and repair mechanisms in human, p53-negative chronic lymphocytic leukemia.

Kimberley L Kaufman,Swetlana Mactier,Richard I Christopherson,Munther Alomari,O Giles Best,Mehdi Mirzaei,Paul A Haynes,Stephen P Mulligan,Dana Pascovici,Yiping Jenkins

doi:10.18632/oncotarget.5715

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.

Highlights

Long term follow up of clinical trials of fludarabine (2-FaraAMP) showed that drug resistance due to mutations in the p53 pathway represents a significant challenge in the clinical management of chronic lymphocytic leukemia (CLL) patients [1]. 2-FaraAMP is dephosphorylated by 5′-nucleotidase at the cell surface to fludarabine nucleoside (2-FaraA), which enters the cell via several nucleoside-specific membrane transporters, and is converted to the cytotoxic triphosphate derivative (2-FaraATP). 2-FaraATP is incorporated into elongating DNA chains and terminates chain synthesis causing double-strand breaks (DSBs) [2]
We previously reported that the HSP90 inhibitor, SNX-7081 synergizes with and restores sensitivity to 2-FaraA, by inducing apoptosis in refractory CLL cells [10], and proposed that this was facilitated by SNX-7081induced inhibition of the cell cycle, DNA replication and repair [16]
Quantitative label-free shotgun LC-MS/MS, employing spectral counting with normalized spectral abundance factor (NSAF), was used to investigate changes in protein levels in human MEC1 CLL cells treated with the heat shock protein 90 (Hsp90) inhibitor SNX-7081, the purine analog 2-FaraA, and dual treatment with both drugs

Summary

Introduction

Long term follow up of clinical trials of fludarabine (2-FaraAMP) showed that drug resistance due to mutations in the p53 pathway represents a significant challenge in the clinical management of CLL patients [1]. 2-FaraAMP is dephosphorylated by 5′-nucleotidase at the cell surface to fludarabine nucleoside (2-FaraA), which enters the cell via several nucleoside-specific membrane transporters, and is converted to the cytotoxic triphosphate derivative (2-FaraATP). 2-FaraATP is incorporated into elongating DNA chains and terminates chain synthesis causing double-strand breaks (DSBs) [2]. Cells with mutated p53 (e.g., MEC1) are generally resistant to DNA damaging agents such as 2-FaraA [7, 8], due to an inability to down-regulate DNA repair proteins and induce apoptosis [9]. Previous work from our laboratory has shown that the Hsp inhibitor SNX-7081 synergizes with and restores sensitivity to 2-FaraA in CLL cells with lesions in the p53 pathway [10]. Our previous study showed that SNX-7081 can induce apoptosis in p53-negative MEC1 CLL cells by deregulating proteins involved with DNA repair and replication, and the cell cycle [16]. Our previous results showed that the Hsp inhibitor SNX-7081 has synergistic effects with 2-FaraA against the p53 mutant cell lines MEC1, MEC2 and U266, and 23 clinical samples of CLL [10]. The results provide an explanation for this drug synergy

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