The Gold Compound Auranofin Induces Oxidative Stress and Apoptosis in Primary CLL Cells Independent of Classic Prognostic Markers and the Protective Effect of the Tissue Microenvironment

Abstract 865

Chronic lymphocytic leukemia (CLL), remains incurable, except by allogeneic stem cell transplantation. While multiple chemotherapeutic options are available, relapse and eventually development of chemotherapy resistance are common. Drug resistance of CLL cells is mediated by genetic alterations including deletion of chromosomes 17p (p53 locus) and 11q (ATM locus), and by pro-survival signals emanating from the tissue microenvironment. Thus new therapies are needed that are active against tumor cells in protective tissue sites and those with adverse cytogenetic features. To identify possible new therapies for CLL, we conducted a high throughput screening assay of primary CLL cells exposed to 7 step-wise dilutions of each member of a 2,816 compound library (including FDA-approved drugs and known bio-actives from commercial suppliers). As a control for “unspecific” cytotoxicity, we used PBMCs from normal donors. Of 102 compounds efficacious against CLL cells of all 6 patients tested, 5 were not or minimally toxic in normal lymphocytes. One of these 5 compounds is Auranofin (AF), an FDA-approved oral disease-modifying anti-rheumatic drug.

Next, we investigated the efficacy of AF against an expanded cohort of CLL samples (n=50) and investigated its mechanism of action. Samples were characterized for IGHV mutational status, ZAP70 expression, CD38 expression, and FISH cytogenetics. PBMCs were tested against serial dilutions of AF (final concentrations: 0.125–4μM) for 24 hours in flat bottom 96-well plates. MTS assay was performed to quantify cell viability. A dose-dependent response was observed in all samples. The inhibitory concentration at 50% (IC50) was calculated using GraphPad Prism Software. For a majority of samples, the IC50 was <1μM, a concentration easily achieved in vivo. AF cytotoxicity was independent of the IGHV mutational status, ZAP70 and CD38 expression, and cytogenetic subgroups (including 17p and 11q deletion). To assess whether AF is selectively cytotoxic for CLL cells, we determined drug-induced apoptosis of PBMCs of CLL patients for CLL cells (identified as CD19+/CD3-; >95% of those are CLL cells) and T-cells (CD19-/CD3+) separately using Annexin V staining. AF reduced viability of T-cells on average by 10% and of CLL cells by 50% (n=8; P<.05 for comparison between T and CLL cells). To determine the effect of the microenvironment on AF toxicity, we cultured primary CLL cells in the presence or absence of Nurse-Like Cells (NLC), a model of the protective effect of the microenvironment. NCLs enhance CLL cell viability and confer resistance to chemotherapeutic agents such as fludarabine. However, co-culture on NLCs did not protect CLL cells from the cytotoxic effect of AF. To investigate the effect of AF on tumor biology, total RNA (2.5 μg) from CLL PBMCs treated for 4 and 10 hours with AF in vitro was profiled on Human Genome U133 Plus 2.0 arrays (Affymetrix) and compared to untreated controls. There were 81 genes whose expression changed >2-fold at P<.01, most prominently several genes encoding heat-shock proteins and antioxidant enzymes such as heme oxygenase and thiroredoxin reductase. In Ingenuity Pathway analysis, the most significantly upregulated pathway in response to AF was the NRF2-mediated Oxidative Stress Response (P<.001). Combined, this suggests that AF induced oxidative stress resulting in the upregulation of a detoxifying response. To study the effect of AF on redox balance we used dihydroethidium (DHE) and concomitantly measured cell viability using 3,3′-dihexyloxacarbocyanine iodide (DiOC6) by flow cytometry. AF induced a time- and dose-dependent increase in ROS production in CLL cells that correlated with the onset of apoptosis. In conclusion, AF is selectively cytotoxic for CLL cells and its activity is independent of classic mechanisms of chemotherapy resistance. Based on these observations AF is now being studied in a clinical proof of concept phase IIa clinical trial for patients with relapsed/refractory CLL (NCT01419691).