Arsenic trioxide synergistically potentiates the cytotoxic effect of fludarabine in chronic lymphocytic leukemia cells by further inactivating the Akt and ERK signaling pathways

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Highlights

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    We have identified a synergistic interaction between ATO and fludarabine.

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    ATO sensitize to fludarabine the CLL cases with poor response to this drug.

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    The combination ATO/fludarabine downregulates crucial survival pathways in CLL.

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    The combination of ATO/fludarabine partially overcomes the survival effect of stroma.

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    ATO may be efficient for CLL treatment in combined therapies with fludarabine.

Abstract

CLL remains an incurable disease, making it crucial to continue searching for new therapies efficient in all CLL cases. We have studied the effect of combining arsenic trioxide (ATO) with fludarabine, a frontline drug in CLL. We have found a synergistic interaction between 1 μM ATO and 5 μM fludarabine that significantly enhanced the cytotoxic effect of the individual drugs. Importantly, ATO sensitized fludarabine-resistant cells to the action of this drug. The mechanism behind this effect included the downregulation of phospho-Akt, phospho-ERK, and the Mcl-1/Bim and Bcl-2/Bax ratios. The combination of ATO and fludarabine partially overcame the survival effect induced by co-culturing CLL cells with stromal cells. Therefore, low concentrations of ATO combined with fludarabine may be an efficient therapeutic strategy in CLL patients.

Introduction

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+ B-lymphocytes in peripheral blood and lymphoid tissues [1], [2]. Albeit being a common type of leukemia, CLL remains uncurable. Current therapies include the use of the purine analogue fludarabine, alone or combined with anti-CD20 monoclonal antibodies or kinase inhibitors [3], [4], [5]. Although these treatments control the disease in many cases, patients carrying bad prognostic markers (del17p13, unmutated IgHV) do not respond well to therapy, making it crucial to continue searching for new compounds, efficient in CLL patients who have indication for treatment.

Arsenic trioxide (ATO) is a successful treatment for acute promyelocytic leukemia and it is being trialed for its possible use in other hematologic and non-hematologic malignancies, generally in combined therapies [6], [7], [8]. We and others have shown that ATO, at 2–4 μM concentration or higher, effectively induces in vitro apoptosis of CLL cells, including those cases with unfavorable prognosis [9], [10], [11]. It is not known whether the combination of low doses of ATO with the frontline therapeutic agent fludarabine, could be an efficient treatment for CLL, particularly in those cases with known resistance to fludarabine. In the present report we show that 1 μM ATO synergistically interacts with fludarabine and enhances the cytotoxic effect of fludarabine on all CLL cases studied. We further show that this is due to the down-regulation of important survival pathways and that the combination of ATO and fludarabine partially overcomes the CLL cell survival effect induced by stroma.

Section snippets

Patients, cells and cell cultures

Approval was obtained from the Hospital Puerta de Hierro and the CSIC Bioethics Review Boards for these studies. All patients signed an informed consent before blood was drawn. B-lymphocytes were purified from the 35 CLL samples listed in Table 1 by centrifugation, using Ficoll gradient (Rafer, S.L. Zaragoza, Spain). The resulting B cell population was >90% CD19+, determined on a BD FACScalibur flow cytometer (San Jose, CA, USA). The human stromal cell lines HS-5 (fibroblastoid) and HS-27A

1. ATO enhances the cytotoxic effect of fludarabine in CLL cells

To determine whether ATO enhanced the cytotoxic activity of drugs currently used for the treatment of CLL, CLL cells from previously untreated (UT, n = 14) or treated (T, n = 13) patients (Table 1) were incubated with 1 μM ATO alone or combined with increasing doses of fludarabine. After 48 h, cell viability was measured by flow cytometry using Annexin V and PI. The average constitutive viability of these samples was 86% (UT patients) and 84% (T patients) and was normalized to 100. Fig. 1A

Discussion

CLL remains an incurable disease and current therapies and clinical trials mostly rely on the use of combined therapies [1], [2], [3], [4]. ATO may be a good compound to use in these therapies, due to its demonstrated effectiveness in all CLL cases, regardless of their prognostic markers [9], [10], [11]. Indeed, a previous study showed that ATO, at 2–4 μM, preferentially induced apoptosis in CLL samples from patients with unfavorable prognosis, including those that were resistant to fludarabine

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by grants SAF2012-31613 (AGP) and RTICC (Red Temática de Investigación Cooperativa en Cáncer) RD12/0036/0061 (AGP), from the Ministry of Economy and Competitiveness (MINECO), Spain; S2010/BMD-2314-Neoplasbim (AGP) from the Comunidad de Madrid/European Union; and by a grant from the Fundación para la Investigación Biomédica Hospital Universitario Puerta de Hierro, Madrid (JAGM). CLS was supported by a grant from Roche Pharma and IAJ was supported by the Junta de

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    • 1

    These authors contributed equally to this work.

    2

    These two senior authors contributed equally to this work.

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