Targeting the disturbed redox equilibrium in chronic lymphocytic leukemia by novel reactive oxygen species-catalytic ‘sensor/effector’ compounds

Abstract

Precursor transformation, clonal sustenance, and therapeutic resistance in cancer are significantly mediated by deregulated reactive oxygen species (ROS), which primarily act as DNA-stressors. Here, we demonstrate that elevated ROS in chronic lymphocytic leukemia (CLL) may represent a promising therapeutic target. We designed organochalcogens, which, based on a ‘sensor/effector’ principle, would confer selective cytotoxicity through the generation of intolerably high ROS levels preferentially in CLL cells, as these carry a high-level redox burden. Our novel compounds show an encouraging profile of efficient induction of apoptosis, low normal cell toxicity, and promising chemotherapy synergism. These findings warrant further mechanistic and preclinical studies of this therapeutic principle in CLL.

Keywords:

• CLL
• oxidative stress
• ROS
• redox catalysts
• organochalcogens

Introduction

Current chemoimmunotherapy induces high response rates in patients with chronic lymphocytic leukemia (CLL), translating into improved progression-free and overall survival, but no cures [1]. A major challenge remains risk stratification and management of primary refractory or relapsed disease. This requires the broadening of our therapeutic arsenal and justifies the search for new, pathway tailored treatments.

Given their established relevance in cellular stress regulation, especially aging and tumorigenesis, redox metabolic pathways and reactive oxygen species (ROS) have been explored as a therapeutic target [2]. ROS are highly reactive molecules composed of oxygen atom(s), which act as free radicals, e.g. O₂ ^•−, HO^•. They are produced as byproducts during respiration in the electron transport chain. Enhanced accumulation of ROS and their resulting genotoxic stress are recognized mediators of progenitor cell transformation and clonal outgrowth, mainly through promotion of genomic instability [2–4]. However, high ROS levels can also mediate cell death [5]. ROS generating arsenic trioxide in combination with ascorbic acid efficiently kills CLL cells [6]. ROS increase through inhibition of key antioxidant enzymes such as superoxide-dismutase (SOD) also induces CLL cell death [7].

Here, we report on a novel principle to exploit the disturbed redox regulation by a class of organochalcogen-based ROS catalysts [8]. The design of such compounds to potentiate tumor-associated high-level ROS toward toxic levels [Figure 1(A)] contrasts with other redox-based targeting approaches. In fact, we postulate the existence of a certain critical redox threshold, beyond which elevated ROS levels are toxic, even to the malignant cell. This ‘sensor/effector’ principle would turn the pathogenetically ‘beneficial’ ROS elevation of a CLL cell towards its own Achilles' heel. By exploiting pre-existing differences in ROS between normal and CLL cells, one could achieve relevant catalyst specificity. We previously described the chemistry of our novel catalytic compounds and their efficacy in a broad range of tumor models and expand here on their encouraging in vitro activity in CLL [9].

Figure 1. Supporting our principle of selective and efficient targeting of CLL cells by redox catalysis, ROS and glutathione levels in CLL cells differ from those of normal blood lymphocytes. (A) Model of redox catalyst-based aggravation of ROS levels beyond a cytotoxic oxidative stress threshold by capitalizing on pre-existing tumor-to-normal ROS differences. (B) Higher basal levels in ROS (top) and lower levels of glutathione (bottom) between CLL B-cells and healthy donor PBMC are significant (Mann–Whitney U-test). (C) Markedly elevated inherent ROS levels in the CLL (CD19+5+) population as compared to healthy (CD19+5−) B-cells are found within the same patient (two representative cases). (D) Higher ROS levels are found in the CD5-high fraction of CLL cells as compared to the CD5-low compartment in two representative samples. Levels of ROS were investigated by flow cytometry using the ROS dye CM-H₂DCF-DA. Levels of reduced glutathione were analyzed by Thiol Tracker Violet.

Results

Determinants of a disturbed redox equilibrium in CLL cells show ‘neoplastic’ pattern of expression

We compared basal ROS levels in freshly isolated CLL cells from 14 patients with those of peripheral blood mononuclear cells (PBMC) from 11 healthy blood donors. ROS levels were significantly higher in CLL B-cells as compared to healthy-donor PBMC (p = 0.0011) [Figure 1(B)]. Baseline levels of glutathione, which in many ways antagonizes ROS, were lower in CLL cells (p = 0.0014) [Figure 1(B)]. Within five CLL samples in which a prominent normal B-cell population could still be distinguished from the leukemic clone, markedly elevated ROS levels in the CD5+19+ CLL population contrasted with those of the healthy B-cell population [Figure 1(C)].

Based on established concepts of intraclonal heterogeneity with respect to proliferative activity and environmental dependence [10], we analyzed ROS levels within CLL samples along a gradient of CD5 expression, which can serve as a surrogate for such ‘age’ associated features. We found that ROS levels increase with elevated CD5 expression [Figure 1(D)], suggesting that preferentially the CD5-high (proliferating or ‘newborn’) fraction generates markedly increased ROS levels as compared to the resting CD5-low (‘old’) compartment. This supports the concept of growth-mediated genotoxic ROS induction and is of particular interest for treatment designs that could better target this niche, such as the ‘sensor/effector’ principle of toxic ROS catalysis described herein [Figure 1(A)].

Organochalcogen-based redox catalysts increase ROS levels in CLL cells, which show deficiency in antioxidant glutathione response

We designed and synthesized a range of novel organochalcogen-based catalysts [9] and employed them in this proof-of-principle study to exploit the observed elevated ROS levels in CLL and their diminished glutathione content. By turning existing OS into a lethal cocktail of ROS and by catalyzing the oxidation of key cysteine proteins and enzymes, they should be capable of elevating ROS levels preferentially in those cells that already carry an abnormally high redox burden. In our model of a critical redox threshold [Figure 1(A)], this would result in toxic aggravation of the existing redox (dys)balance in CLL. Our in vitro data indicate that redox catalyst treatment increases ROS levels in CLL cells to a much higher degree than in healthy-donor lymphocytes [Figure 2(A)]. In addition, while normal PBMC react toward redox catalyst treatment with increases in intrinsic ROS-antagonistic reduced glutathione, its levels in CLL cells, being already lower at baseline, did not rise upon compound exposure.

Figure 2. Redox catalysts preferentially increase ROS in CLL, which is followed by caspase-mediated apoptosis. (A) ROS levels (top) increase in CLL patient cells more significantly than in healthy donor PBMC after treatment with redox catalyst #6, while levels of the antioxidant glutathione (bottom) behave the opposite way. Levels of ROS were investigated by flow cytometry using the ROS dye CM-H₂DCF-DA. Levels of reduced glutathione were analyzed by Thiol Tracker Violet. (B) Reduction of cell viability as measured by ATPglo in CLL cells vs. healthy-donor cells after treatment with different redox catalysts (# at x-axis). (C) Flow-cytometric annexin V/7-aminoactinomycin D (7AAD) staining providing the percentage of cells having undergone apoptosis after 48 h of compound exposure reveals most marked and selective cell death induction in CLL cells at 0.5 µM for #4, #5, #6, and #7 as compared to PBMC. Asterisks in (B) and (C) indicate significant (p < 0.05, Mann–Whitney U-test) differences; ns stands for not significant. (D) Combinations of redox catalysts with fludarabine increase caspase 3 cleavage over single-compound treatment as analyzed by Western blot. Caspase 3 variants are shown at different exposure times.

Redox ‘sensor/effector’ catalysts preferentially kill CLL cells via apoptosis

We assessed the catalyst efficacy and selectivity in a suspension culture of purified CLL cells as compared to healthy-donor PBMC. After initial screening and chemical modifications, we demonstrated that treatment with selected redox catalysts resulted in a reduction of CLL B-cell viability along with increased apoptosis at low to submicromolar concentrations, whereas normal lymphocytes stayed largely unaffected [Figures 2(B) and 2(C)]. Immunoblot analyses of apoptotic executioners such as cleaved caspase 3 or poly(ADP-ribose) polymerase (PARP) (not shown) and colorimetric caspase 3/7 activity assays confirmed preferential apoptosis induction in CLL 24–48 h after compound treatment [Figure 2(C)]. Overall, we provide evidence of a discriminate increase of ROS levels in CLL cells by our novel redox catalysts over normal PBMC, which is followed by caspase-dependent specific apoptosis. A significant compound activity was seen in the majority of analyzed CLL samples (n = 21/25) and for nearly all of the selected catalysts, however, being of certain interindividual variance. Further studies will address the determinants of such response categories.

Redox catalyst exposure combined with fludarabine treatment is followed by additive/synergistic increase in cell death induction

Tumors of patients who have undergone prior chemotherapy tend to carry higher ROS levels. Therefore, we analyzed cell death induction in CLL cells after in vitro treatment with fludarabine and our redox catalysts. All 10 tested samples exhibited a stronger response to combined fludarabine-plus-catalyst treatment as compared to single-drug exposure, of which the effect was truly additive in 4/10 or even synergistic in 3/10 [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay, not shown; immunoblot, Figure 2(D)]. Overall, we propose a marked effect of combining redox catalysts with conventional DNA-targeting chemotherapeutics (i.e. fludarabine), which can also be extrapolated to a promising sensitivity of relapsing clones.

Discussion

The prevalence of oxidative stress in cancer has received increasing attention [2]. Several strategies have been proposed to therapeutically harness the tumorigenic relevance of redox deregulation whereby increases in ROS levels have already been associated with cell death induction in CLL [5,11,12]. However, many substances increase cellular ROS indiscriminately and are potentially harmful to normal cells, as shown for the cyclooxygenase-2 (COX-2) inhibitor SC-58125 [13]. Here, we provide evidence that modulation of a pre-existing redox (dys)balance by our novel organochalcogen-based ROS catalysts following a ‘sensor/effector’ mode of action and their combination with chemotherapeutics are promising options for selective elimination of CLL cells with low toxicity to normal lymphocytes.

As the supporting rationale for our redox catalysis approach, we show here that ROS levels in CLL are substantially higher than in normal lymphocytes, which in contrast to CLL cells seem to carry a competent glutathione buffer system. Whether catalyst-induced apoptosis in CLL is due to a primarily impaired glutathione response or whether the strong elevation of ROS causes low glutathione levels by oxidizing the reduced glutathione remains to be elucidated. The higher levels of genotoxic ROS in the proposed fraction of proliferating CD5-high CLL cells is of interest as these might be more sensitive to ROS-targeting drugs, which could be extrapolated to the milieu-protected and thus hard-to-target lymph node-based fraction of CLL cells. Our in vitro data indicate that our catalysts indeed push oxidatively stressed CLL cells over a critical redox threshold via ROS accumulation. This results in induction of the otherwise insufficient apoptotic machinery. Whether it also significantly overcomes the strong pro-survival signature of CLL in vivo has to be answered in preclinical models. Importantly, our ROS catalysts seem to affect healthy cells less markedly, as these have a naturally lower ROS burden and an intact glutathione response.

In preliminary analyses, we further indicate here that combination of conventional DNA-targeting chemotherapeutics such as fludarabine with our redox catalysts boosts apoptosis induction and points toward a synergistic effect in a subset of patients. It also implies that repeated application of our compounds in a clinical setting would capitalize on accumulating ROS levels after previous catalyst treatment or prior chemotherapy, or in particularly redox-burdened chemoresistant cells. In fact, as even higher levels of ROS in cancer are induced by chemotherapy and found in the setting of relapse, our concept of catalytic ROS-targeting may especially be valuable in the context of primary or selected chemoresistance. For example, loss of p53 function, as found in about 40–60% of the most treatment-refractory CLL, and its associated promotion of DNA mutations, are paralleled by enhanced ROS production [14]. Furthermore, in first reports, fludarabine resistance in CLL could be overcome by a redox-mediated mechanism, employing β-phenylethyl isothiocyanate (PEITC) with low toxicity to normal lymphocytes [15].

Overall, our findings support the concept that tumorigenically relevant and protective ROS generation comes for the CLL cell with the trade-off of higher and selective vulnerability toward ROS catalysts such as our organochalcogen compounds. Further mechanistic cause–effect studies have to address the link between the aberrant mitochondrial redox metabolism and its significance for specific ROS-mediated apoptosis. Our data also encourage further correlative investigations, e.g. on patient and cell subsets of sensitivity, as redox catalysis holds great promise in chemotherapy combinations and for targeting resistance.

Acknowledgements

N.L., the presenter of the above data at the 5th Young Investigators' Meeting on CLL as part of the 8th International Workshop of the GCLLSG, receives stipend support by a joint pharmacology graduate program of Cologne University and Bayer Health Care AG. M.Ha. is supported by the local CECAD initiative. C.J. is supported by the University of Saarland, the Ministry of Economics and Science of Saarland, DFG grant JA1741/2-1, and the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement 215009 RedCat. M.He. is supported by a DFG grant HE3553/2-1 and the local CECAD initiative, a Max-Eder Junior Group Award of the German Cancer Aid, and the CLL Global Research Foundation.

We thank J Med Chem for permission to present Figures 1(C), 2(B), and 2(C), as they are part of the publication by Doering et al., J Med Chem 2010;53:6954–6963, and protected by copyright law.

Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal .