Antileukemic activity and cellular effects of the antimalarial agent artesunate in acute myeloid leukemia

doi:10.1016/j.leukres.2017.05.007

Leukemia Research

Volume 59, August 2017, Pages 124-135

https://doi.org/10.1016/j.leukres.2017.05.007 Get rights and content

Highlights

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Artesunate has potent antileukemic activity against AML in vitro and in murine models of AML.
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The cytotoxicity of artesunate against AML is mediated by induction of reactive oxygen species.
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Artesunate augments cytotoxicity of chemotherapy as well as the BCL-2 inhibitor venetoclax in AML.

Abstract

The artimisinins are a class of antimalarial compounds whose antiparasitic activity is mediated by induction of reactive oxygen species (ROS). Herein, we report that among the artimisinins, artesunate (ARTS), an orally bioavailable compound has the most potent antileukemic activity in AML models and primary patients’ blasts. ARTS was most cytotoxic to the FLT3-ITD+ AML MV4-11 and MOLM-13 cells (IC₅₀ values of 1.1 and 0.82 μM respectively), inhibited colony formation in primary AML and MDS cells and augmented cytotoxicity of chemotherapeutics. ARTS lowered cellular BCL-2 level via ROS induction and increased the cytotoxicity of the BCL-2 inhibitor venetoclax (ABT-199). ARTS treatment led to cellular and mitochondrial ROS accumulation, double stranded DNA damage, loss of mitochondrial membrane potential and induction of the intrinsic mitochondrial apoptotic cascade in AML cell lines. The antileukemic activity of ARTS was further confirmed in MV4-11 and FLT3-ITD+ primary AML cell xenografts as well as MLL-AF9 syngeneic murine AML model where ARTS treatment resulted in significant survival prolongation of treated mice compared to control. Our results demonstrate the potent preclinical antileukemic activity of ARTS as well as its potential for a rapid transition to a clinical trial either alone or in combination with conventional chemotherapy or BCL-2 inhibitor, for treatment of AML.

Introduction

The artemisinins are a family of antimalarial compounds derived from the sweet wormwood Artmisia Annua. Among the artemisinins, artesunate (ARTS) appears to be the most active agent with regard to the antimalarial activity and has optimal water solubility and oral bioavailability [1], [2]. The antimalarial activity of artemisinins has been attributed to the generation of reactive oxygen species (ROS) occurring via cleavage of the endoperoxide bond in their structure [3]. Given a relatively high iron content of the cancer cells [4], the iron-catalyzed lysosomal ROS generation appears to be one of the main pro-apoptotic mechanisms mediating the artemisinins’ cytotoxicity [5]. Although artemisinins have shown strong cytotoxic activity against a variety of cancer cell lines in vitro, their mechanisms of action remain to be fully dissected [5], [6], [7], [8], [9].

Although up to 80% of younger patients with acute myeloid leukemia (AML) achieve complete remission with conventional chemotherapy, their 5-year survival is only around 40% [10]. Outcome is particularly poor for older patients as well as those with adverse cytogenetics and molecular abnormalities like FLT3 internal tandem duplication (FLT3-ITD) [10]. Treatment refractoriness and disease relapse are thought to be due to persistence of quiescent leukemia stem cells (LSC) which remain resistant to conventional chemotherapy [11].

Such functionally defined LSCs have been shown to have low levels of ROS as well as overexpression of BCL-2 [12]. Maintenance of low ROS levels appears to be critical to survival and persistence of LSC. Therefore, inducing ROS generation has the potential to selectively target LSC given the sensitivity of the latter to ROS levels, thereby providing the rationale for testing artimisinins in AML. We therefore examined the antineoplastic activity as well as mechanism of activity of artimisinins against AML cell lines and primary cells both in vitro and in animal models. In this paper, we demonstrate that among the artimisinins tested, the compound ARTS has the most potent antileukemic activity that is synergistic with chemotherapeutic agents. We further show that ARTS treatment also led to decrease in BCL-2 expression and strong synergy with the selective BCL-2 inhibitor venetoclax (ABT-199).

Section snippets

Patient samples

Bone marrow (BM) samples from health donors, myelodysplastic syndrome (MDS) patients and AML patients were obtained under a specimen banking protocol approved by the Institutional Review Board of City of Hope Medical Center, in accordance with assurances filed with and approved by the Department of Health and Human Services, and meeting all requirements of the Declaration of Helsinki. Mononuclear cells were isolated using Ficoll (Stem Cell Technologies Inc., Vancouver, BC) density gradient

ARTS exhibits antiproliferative activity and cytotoxicity against AML and augments cytotoxicity of anthracyclines and cytarabine

We examined the antiproliferative activity and cytotoxicity of ARTM, ARTS and DHA against the AML cell lines, MV4-11, MOLM-13, OCI-AML3, NB4 and KG1A. These cells lines were selected as representative of relatively frequent cytogenetic/molecular subsets of AML. Among the artimisinins tested ARTS showed the most antiproliferative activity (Supplementary Fig. S1). The FLT3-ITD+ cell lines MOLM-13 and MV4-11 were the most sensitive to ARTS with IC₅₀ values of 0.82 and 1.1 μM respectively (Table 1,

Discussion

Herein, we first report potent antileukemic activity of ARTS in vitro as well as in three different murine models including a xenograft model using primary AML cells. We also observed in vitro activity against primary MDS cells. We demonstrate that ARTS antileukemia activity depended on generation of ROS and suppression of BCL-2 as also supported by its synergistic activity with the emerging BCL-2 inhibitor ABT-199 (venetoclax). Also interesting to us was the marked synergy between ARTS and

Acknowledgements

The authors also acknowledge support of the Gehr Family Center for Leukemia Research at City of Hope Medical Center. Partly supported by Grant P30CA033572 to City of Hope Comprehensive Cancer Center from National Cancer Institute of the National Institutes of Health.

References (26)

W.E. Ho et al.
Artemisinins: pharmacologic actions beyond anti-malarial
Pharmacol. Ther.
(2014)
A. Hamacher-Brady et al.
Artesunate activates mitochondrial apoptosis in breastcancer cells via iron-catalyzed lysosomal reactive oxygen species production
J. Biol. Chem.
(2011)
N.D. Yang et al.
Artesunate induces cell death in human cancer cells via enhancing lysosomal function and lysosomal degradation of ferritin
J. Biol. Chem.
(2014)
H. Dombret et al.
An update of current treatments for adult acute myeloid leukemia
Blood
(2016)
J. Felipe Rico et al.
AML stem cells: from bench to bedside
Cancer Lett.
(2013)
E. Lagadinou et al.
BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells
Cell Stem Cell
(2013)
M. Paul-Samojedny et al.
Expression of cell survival/death genes: cl-2 and Bax at the rate of colon cancer prognosis
Biochim. Biophys. Acta
(2005)
H.-M. Shen et al.
JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species
Free Radic. Biol. Med.
(2006)
D.H. Hockenbery et al.
Bcl2 functions in an antioxidant pathway to prevent apoptosis
Cell
(1993)
Y. Ozawa et al.
Src family kinases promote AMl cell survival through activation of signal transducers and activators of transcription (STAT)
Leuk. Res.
(2008)

C. Dos Santos et al.

The Src and c-Kit kinase inhibitor dasatanib enhances p53-mediated targeting of human acute myeloid leukemia stem cells by chemotherapeutic agents

Blood

(2013)

L. Cui et al.

Discovery: mechanisms of action and combination therapy of artemisinin

Expert Rev. Anti Infect. Ther.

(2009)

A.M. Gopalakrishnan et al.

Antimalarialmaction of artesunate involves DNA damage mediated by reactive oxygen species

Antimicrobial. Agents Chemother.

(2015)

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In vitro culture with ART838 plus VEN robustly reduced growth and enhanced apoptotic cell death of several human acute leukemia cell lines (5 AMLs, 2 ALLs) and primary acute leukemia samples (5 AMLs, 1 ALL), validating the results of the screen. Previous studies indicated that artemisinins induced high cellular levels of reactive oxygen species (ROS), triggered proapoptotic signaling,18,30,74,76 and altered expression of BCL2 family members in several cancer types.77-79 Notably, a recent study aimed at identifying drugs that synergize with the dual BCL2/BCLxL inhibitor, NAV, against poor-prognosis B-precursor ALLs harboring the BCR-ABL translocation, found that DHA synergized strongly.20
Artemisinins are active against human leukemia cell lines and have low clinical toxicity in worldwide use as antimalarials. Because multiagent combination regimens are necessary to cure fully evolved leukemias, we sought to leverage our previous finding that artemisinin analogs synergize with kinase inhibitors, including sorafenib (SOR), by identifying additional synergistic antileukemic drugs with low toxicity. Screening of a targeted antineoplastic drug library revealed that B-cell lymphoma 2 (BCL2) inhibitors synergize with artemisinins, and validation assays confirmed that the selective BCL2 inhibitor, venetoclax (VEN), synergized with artemisinin analogs to inhibit growth and induce apoptotic cell death of multiple acute leukemia cell lines in vitro. An oral 3-drug "SAV” regimen (SOR plus the potent artemisinin-derived trioxane diphenylphosphate 838 dimeric analog [ART838] plus VEN) killed leukemia cell lines and primary cells in vitro. Leukemia cells cultured in ART838 had decreased induced myeloid leukemia cell differentiation protein (MCL1) levels and increased levels of DNA damage–inducible transcript 3 (DDIT3; GADD153) messenger RNA and its encoded CCATT/enhancer-binding protein homologous protein (CHOP), a key component of the integrated stress response. Thus, synergy of the SAV combination may involve combined targeting of MCL1 and BCL2 via discrete, tolerable mechanisms, and cellular levels of MCL1 and DDIT3/CHOP may serve as biomarkers for action of artemisinins and SAV. Finally, SAV treatment was tolerable and resulted in deep responses with extended survival in 2 acute myeloid leukemia (AML) cell line xenograft models, both harboring a mixed lineage leukemia gene rearrangement and an FMS-like receptor tyrosine kinase-3 internal tandem duplication, and inhibited growth in 2 AML primagraft models.
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In MOLM14 xenograft tumor-bearing mice, ART slowed subcutaneous MOLM14 xenograft tumor growth and extended the mean survival time [68]. ART also increased the survival of mice bearing ML-2 xenograft tumors, MLL-AF9 knock-in transplantation mice, MV4-11 xenograft tumors and human FLT3-ITD AML, but not MV-11 or MOLM-1 xenograft tumors [77,78]. ART exhibited anti-tumor effects against THP-1 xenograft AML by significantly decreasing the expression of STAT3 in tumor tissues in a dose-dependent manner [79].
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Research paperAntileukemic activity and cellular effects of the antimalarial agent artesunate in acute myeloid leukemia

Highlights

Abstract

Introduction

Section snippets

Patient samples

ARTS exhibits antiproliferative activity and cytotoxicity against AML and augments cytotoxicity of anthracyclines and cytarabine

Discussion

Acknowledgements

Pharmacol. Ther.

J. Biol. Chem.

J. Biol. Chem.

Blood

Cancer Lett.

Cell Stem Cell

Biochim. Biophys. Acta

Free Radic. Biol. Med.

Cell

Leuk. Res.

Blood

Discovery: mechanisms of action and combination therapy of artemisinin

Expert Rev. Anti Infect. Ther.

Antimalarialmaction of artesunate involves DNA damage mediated by reactive oxygen species

Antimicrobial. Agents Chemother.

Research paper
Antileukemic activity and cellular effects of the antimalarial agent artesunate in acute myeloid leukemia