ATM Mutant Lymphoid Tumour Cells Exhibit Impaired Activation of the Redox-Sensitive Nrf2-ARE Detoxification Pathway and Are Differentially Sensitive to Nrf2-Activating Compounds

Abstract 49

Inactivation of the tumour suppressor gene, Ataxia Telangiectasia Mutated (ATM), is a frequent event in lymphoid malignancies such as chronic lymphocytic leukaemia (CLL), T-prolymphocytic leukaemia (T-PLL), mantle cell lymphoma (MCL) and, to a lesser degree, T-cell acute lymphoblastic leukaemia (ALL). ATM mutant tumour cells exhibit defective p53-mediated apoptosis following DNA damaging agents in vitro and consequently exhibit poor clinical responses. Multiple pro-survival responses are also altered by ATM dysfunction and may reveal novel approaches for treatment. Our previous gene expression and transcription factor array analyses in primary CLL cells indicated impaired binding of the NF-E2-related factor 2 (Nrf2) transcription factor to the antioxidant response element (ARE) in ATM null cells following IR-induced DNA double strand breaks compared to ATM wild type cells. Nrf2 is a stress responsive transcription factor, which is activated by phenolic compounds and electrophilic agents, and induces the transcription of a chemo- and redox-protective ARE gene battery. The function of these gene products include regulation of cellular glutathione levels (glutamate-cysteine ligase subunits), glutathione utilising enzymes (glutathione S-transferases) and drug metabolising enzymes such as NAD(P)H:quinone oxidoreductase-1 (NQO1) and thioredoxin reductase (TXNRD1). To confirm ATM-dependent regulation of the Nrf2-ARE pathway, we first used electromagnetic shift analysis (EMSA) and demonstrated differential binding of Nrf2-ARE in an independent panel of primary CLL cell lysates. Binding patterns of lysates to the ARE probe were reduced in both untreated and in IR-treated ATM null compared to wt cells. We subsequently investigated the impact of impaired binding of Nrf2-ARE on the transcription of target genes. Quantitative RT-PCR revealed that in isogenic MEC1 CLL cell lines with (MEC1-ATMshRNA) and without (MEC1-GFPshRNA) stable ATM knockdown, ATM loss led to significant impairment in the induction of expression of the Nrf2-ARE-dependent genes, NQO1 and TXNRD1, 8h after tert Butylhydroquinone (tBHQ)–induced activation compared with wt cells. Furthermore, primary ATM mutant CLL cells also revealed impaired induction of NQO1 following tBHQ treatment. Finally, 3 isogenic CLL cell line pairs derived from PGA, CII and MEC, either with or without stable ATM knockdown, consistently revealed a 20% reduction in basal total glutathione levels in cells with ATM knockdown compared to controls, indicating impaired Nrf2-mediated glutathione synthesis. Given defective Nrf2-ARE pathway in ATM null cells, we reasoned that ATM mutant lymphoid tumour cells would be differentially sensitive to agents which activate the pathway and are detoxified by Nrf2-ARE gene products. N-acetyl-p-benzoquinoneimine (NAPQI), the metabolite of acetaminophen, activates Nrf2 and is detoxified directly by glutathione. We observed differential sensitivity of CII-ATMshRNA cells with maximal (80%) killing at 150μ M NAPQI compared to CII-GFPshRNA cells which showed no such killing after 4 days of treatment. Furthermore, 4 primary ATM mutant CLLs revealed differential sensitivity to 50μ M NAPQI with maximal (80%) killing achieved at 100μ M NAPQI after 72h. In contrast, 4 ATM wildtype CLLs exhibited no sensitivity even at 150μ M NAPQI. Treatment with N-acetylcysteine (NAC) abolished NAPQI sensitivity indicating a glutathione-dependent mechanism. Finally, Annexin V/PI staining revealed an apoptosis-dependent mechanism which was caspase-independent as pan-caspase inhibitor treatment had no impact on NAPQI-induced cell death. Finally, in IR-resistant CII-ATMshRNA cells, 4h pre-treatment with 50μ M NAPQI resulted in almost complete sensitisation to IR-induced killing. In summary, ATM null lymphoid tumour cells exhibit impaired activation of the NRF2-ARE detoxification pathway. Consequently, ATM mutant cells can be differentially targeted for killing by agents that activate the Nrf2-ARE pathway. This targeted approach may provide novel treatment options for otherwise chemo-resistant ATM mutant tumours and additionally reduce morbidity in elderly patients.