Artesunate shows potent anti-tumor activity in B-cell lymphoma

More than 60 different B-cell lymphoma types have been described, with different clinical courses and outcomes [1]. Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are the most common forms of aggressive non-Hodgkin lymphoma (NHL) and indolent NHL, respectively. Current standard of care often involves chemotherapy regimens combined with monoclonal antibodies targeting the B-cell surface molecule CD20 (rituximab) and has led to an improved overall survival for most lymphoma types [2]. A number of small molecule inhibitors are currently under clinical testing, where the majority represents inhibitors of the B-cell receptor signaling (BCR) pathway and downstream signaling molecules [3].

Artesunate is a semisynthetic analogue of artemisinin, extracted from the sweet wormwood plant, Artemisia annua, and is the first-line treatment of severe malaria [4]. Artemisinins have demonstrated cytotoxic effects in vitro against a range of cell lines and also to be effective against drug-resistant cancer cell lines [5, 6], although the underpinning mechanisms remain unclear [7, 8]. Few studies have been reported regarding clinical use of artesunate in cancer, but artesunate has demonstrated anti-tumor effects in a small randomized clinical trial in colon cancer and to transiently decrease tumor size and prostate-specific antigen levels in a patient with advanced prostate cancer [9, 10]. The supply of artesunate has been limited, but semisynthetic production since 2014 has increased the availability [11] and enables additional clinical use of artesunate beyond malaria treatment.

B lymphoma cell lines are representative models with > 80% match with the corresponding primary cancer cells [12, 13] (O. Kallioniemi, pers. com.). Here, we demonstrate a potent induction of apoptosis by artesunate in a broad range of cell lines, representing the most common types of B-cell lymphoma. Artesunate also showed potent anti-tumor efficacy in vivo in a xenograft model, providing a rationale for clinical testing as B-cell lymphoma therapy.

In this study, we tested small molecule inhibitors for potential tumor cell suppressive effects in B-cell lymphoma cell lines and compared with the anti-malaria drug artesunate. The screen identified artesunate as an attractive novel drug with potent activity across different cell lines, representative for various B-cell lymphoma histologies. Artesunate selectively and potently induced apoptosis in the B lymphoma cell lines but had minimal effect in normal B-cells. Of note, artesunate also showed potent anti-tumor effect in a highly aggressive lymphoma xenograft model.

The standard treatment of malaria is artemisinin based combinatorial treatment for 3 days [28], and the experience with longer treatment regimens is limited [9]. In serum, artesunate is converted to dihydroartemisinin within minutes. In malaria patients receiving artesunate, the reported concentration of artesunate and dihydroartemisinin in serum has been measured to approximately 1 and 3 μM, respectively [29]. In healthy volunteers, concentrations of dihydroartemisinin as high as 8 μg/ml (21 μM) have been measured after 10 min exposure to artesunate and were well tolerated [30, 31]. We found IC₅₀ values for artesunate varying from 0.19 to 3.72 μM in B lymphoma cell lines. The strong apoptosis-inducing effect of artesunate at clinical relevant concentrations of artesunate is therefore promising.

Artesunate has earlier been shown to overcome bortezomib resistance in myeloma cells in vitro [32] and to have growth suppressive properties in primary myeloma cells and in myeloma and lymphoma cell lines by inducing apoptosis concomitant with downregulation of MYC [33]. Others have also suggested MYC as a potential target for artesunate [33, 34], and the cell lines used for microarray analysis were chosen based on the MYC status (translocated, non-translocated, and mutated MYC in BL-41, SU-DHL-4, and Oci-Ly-2, respectively). MYC was identified as a central molecule in the network analysis with all three cell lines combined, suggesting MYC involvement, and demonstrated that artesunate had potent effects independent of MYC translocation and mutational status. Furthermore, artesunate also potently induced apoptosis in WILL-2 and Oci-Ly-18 cells, representing “double hit lymphoma,” having aberrant overexpression of MYC and BCL2, and also in U2932, with a subclone with “double hit” aberrations [35]. This is an important observation as double hit lymphomas have dismal outcome [36].

The UPR was identified as the most deregulated pathway in response to artesunate. Additional top pathways activated by artesunate included tRNA charging, protein ubiquitination and amino acid biosynthesis, all connected to changes caused by UPR and ER stress [37, 38]. This suggests that the underpinning mechanism for artesunate-induced apoptosis is induction of ER stress. The UPR is a cellular adaptive response important for re-establishing protein-folding homeostasis by decreasing protein synthesis through phosphorylation of eIF2α and by increasing the ER protein-folding and degradation capacities through transcriptional activation by XBP1 and ATF6α [39‐41]. The UPR is a sensor for ER stress and is activated upon environmental stress or other conditions resulting in accumulation of unfolded proteins, a key step in readjustment of the ER protein folding capacity to meet cellular needs [39, 42]. Importantly, the functional outcome of ER stress depends on intensity and duration, as the UPR is either pro-survival to preserve ER homeostasis or pro-death if the ER stress cannot be resolved [43, 44]. Therefore, in B lymphoma cells, artesunate might directly or indirectly increase the level of ER stress, which ultimately drives the cells into apoptosis. Here, we found artesunate to induce transcriptional upregulation of ATF-4 and DDIT3/CHOP, which also translated into increased protein expression. It has previously been shown that forced expression of ATF-4 in mouse embryo fibroblasts decreased the survival, whereas forced expression of DDIT3 had no effect, suggesting ATF-4 to be the key signal and DDIT3 secondary to ER stress induced cell death [38]. Multiple pathways are involved in ER stress-induced apoptosis, and in most cases, these converge at the mitochondrial level [45]. Artesunate greatly affected metabolism in the B-cell lymphoma cell lines as determined by a systematic diminution of their basal respiration, ATP-linked respiration, maximal respiration capacity, and glycolysis, although at varying degrees. The profound collapse of the maximal respiratory capacity in all cell lines upon artesunate treatment corresponds well with their decreased basal respiration and points to significant and irreversible damage to cell respiration. This is in line with previously reported mitochondrial fragmentation as a consequence of ER stress [27]. Further evidence for this hypothesis comes from the observation that artesunate affected increased cytotoxicity in GSH-depleted malignant cells, but not in normal B-cells. The profound mitochondrial damage which is indicated by the metabolic analysis results could be associated with leaky electron transport chain which in turn may promote the aberrant generation of reactive oxygen species. Since GSH is a key intracellular antioxidant regulating ROS both in the cytosol and the mitochondria, the depletion of intracellular GSH could lead to increased vulnerability to oxidative stress [46].

In general, monotherapy in cancer treatment is unlikely to produce deep and lasting remission [47]. Therefore, combining artesunate with other therapies may suppress multiple oncogenic pathways simultaneously and offer therapeutic synergy. Synergic effects with a new dimeric artemisinin compound, ART-838, with anti-leukemic drugs have recently been reported [48, 49]. ART-838 was found to have an improved bioavailability and increased half-life compared to artesunate. This new compound may therefore give new possibilities in cancer therapy. As induction of ER stress can be a trigger of immunogenic cell death as shown for some of the cytotoxic anti-cancer therapies such as anthracyclines and irradiation [50], artesunate might be used prior to immunotherapeutic maneuvers, such as checkpoint inhibition or T cell-based therapies [51]. Furthermore, immunogenic signals released by dying tumor cells can induce antigen uptake as well as antigen processing and presentation by the antigen presenting cells. In fact, the presence of intratumor T cells may be an important participant in the continued anti-tumor response after the initial treatment with artesunate [51].

Our results indicate that artesunate has potent growth suppressive effects restricted to malignant B-cells that warrant clinical testing in B-cell lymphoma patients.