Repurposing anthelmintic agents to eradicate resistant leukemia

Abstract Despite rapid progress in genomic profiling in acute lymphoblastic leukemia (ALL), identification of actionable targets and prediction of response to drugs remains challenging. To identify specific vulnerabilities in ALL, we performed a drug screen using primary human ALL samples cultured in a model of the bone marrow microenvironment combined with high content image analysis. Among the 2487 FDA-approved compounds tested, anthelmintic agents of the class of macrocyclic lactones exhibited potent anti-leukemia activity, similar to the already known anti-leukemia agents currently used in induction chemotherapy. Ex vivo validation in 55 primary ALL samples of both precursor B cell and T-ALL including refractory relapse cases confirmed strong anti-leukemia activity with IC50 values in the low micromolar range. Anthelmintic agents increased intracellular chloride levels in primary leukemia cells, inducing mitochondrial outer membrane depolarization and cell death. Supporting the notion that simultaneously targeting cell death machineries at different angles may enhance the cell death response, combination of anthelmintic agents with the BCL-2 antagonist navitoclax or with the chemotherapeutic agent dexamethasone showed synergistic activity in primary ALL. These data reveal anti-leukemia activity of anthelmintic agents and support exploiting drug repurposing strategies to identify so far unrecognized anti-cancer agents with potential to eradicate even refractory leukemia.

Introduction Intensive chemotherapy regimens in childhood lymphoblastic leukemia (ALL) have led to substantial improvement in survival, which now overcomes 80%. Despite these advances, relapsed leukemia rank among the most frequent diagnoses in childhood malignancies, and remain a major clinical problem, being often associated with fatal outcome. While powerful new immunotherapeutic approaches are currently being developed in particular in B cell precursor ALL (BCP-ALL), identification of small molecules with anti-leukemic potential that could be incorporated into current treatment regimens represents an appealing possibility to increase efficacy of anti-leukemic therapy. Examples for such a strategy are modulation of survival and cell death pathways through small molecules, such as the BCL-2 mimetic venetoclax, inhibitors of the PI3K/AKT/mTOR pathway such as Torin, or of the MEK pathway such as Trametinib,10. Another strategy exploits the potential of small molecules to activate non-apoptotic cell death pathways, such as the IAP inhibitor birinapant, which we showed to activate necroptosis to potently interfere with leukemia progression. Common to all these strategies is the need for the development of functional approaches to identify the target patient population which is most likely to respond. Compounds that are clinically used for non-cancer indications may represent an appealing source for a novel treatment approach. Repurposing potential anti-leukemic drugs for ALL treatment may accelerate clinical development, and will reduce cost and time required for the introduction in the clinic13,14. We performed a functional imaging-based drug repurposing screen using an extended FDA-approved drug library, which we tested on primary relapsed ALL co-cultured on mesenchymal stroma cells (MSCs) in a model of the bone marrow microenvironment15. Unexpectedly, we identified the anthelmintic agents moxidectin, ivermectin, and milbemycin to have a high anti-leukemic potential as single agents and to exhibit synergistic activity with the BCL-2 inhibitor navitoclax (ABT-263) and standard chemotherapeutic agents of frontline ALL therapy, such as dexamethasone in highly refractory primary ALL.

Materials and methods Human and patient-derived xenograft (PDX) samples Primary human ALL samples were obtained from cryopreserved bone marrow aspirates of patients enrolled in the AIEOP-BFM 2009 and ALL-REZ 2002 studies. Informed consent was obtained in accordance with the Declaration of Helsinki, and approval was granted by the Ethics Commission of the Kanton Zürich (approval no. 2014–0383). Samples were classified as standard risk (SR), medium risk (MR), high risk (HR), very high risk (VHR), and relapse (R) samples according to the clinical criteria used in the ALL-BFM 2000 study16. Primary human ALL cells were transplanted into 5- to 12-week-old immunodeficient NOD/SCID/IL2rγnull (NSG) mice in order to obtain PDX cells17. Leukemia progression was monitored weekly by staining peripheral blood after red blood cell lysis with hCD19-PE and hCD45-Alexa Fluor 647 (Biolegend) and analyzed by flow cytometry (Fortessa LSR, BD Biosciences). Engrafted ALL cells were collected from the spleen. In vivo experiments were approved by the veterinary office of the Canton of Zurich.


Results

Ex vivo drug screen identifies antiparasitic compounds as promising candidates to target refractory leukemia To identify novel compounds with anti-leukemia potential, we used a co-culture model of primary ALL cells on MSCs enabling long-term survival ex vivo (Fig. 1a)15. On this platform, we tested 2487 FDA-approved compounds (Supplementary Table S2) at 1 µM by using a fluorescent cell viability readout combined with automated imaging analysis. After normalization of cell viability to the effect of the highly potent anthracycline idarubicin, we identified 61 compounds with strong anti-leukemia activity (Fig. 1b, Supplementary Fig. S1A, B). As expected, the majority of these drugs were chemotherapeutic agents that are already in clinical use to treat leukemia. Among these, we found DNA-intercalating agents, including mitoxanthrone, daunorubicine, or doxorubicine, which appeared to have the strongest anti-leukemia effect among the screened compounds (Fig. 1c, Supplementary Fig. S1A). Furthermore, agents with known anti-leukemia activity, e.g. steroids, proteasome, and HDAC inhibitors, but also BCL-2 and cIAP antagonists or tyrosine kinase inhibitors also emerged with potent anti-leukemia activity (Supplementary Fig. S1A, B). Unexpectedly, we further identified several agents from drug families that have, so far, not been associated with anti-leukemia activity. For instance, anti-fungal and anti-bacterial agents as well as cardiac glycosides showed substantial anti-leukemia activity (Supplementary Fig. S1A, B). The most potent among these were the anthelmintic drugs ivermectin, moxidectin, and milbemycin, which showed up to 80% maximal cytotoxic effect compared to idarubicin (Fig. 1c).



Repurposing anthelmintic agents to eradicate resistant leukemia

Anthelmintic agents are widely active in precursor B- and T-ALL

Having identified ivermectin, moxidectin, and milbemycin as potential candidates with anti-leukemia activity, we first validated the activity of these three drugs by generating dose response curves on the same ALL sample used for the screen. Interestingly, all three drugs showed a strong cytotoxic effect at low micromolar range concentration with IC50 values between 1.2 and 1.5 µM (Fig. 1d), with moxidectin showing a slightly higher activity as compared to ivermectin and milbemycin. To evaluate the potential of these anthelmintic compounds, we generated dose response curves for these three agents in 47 precursor B-ALL and 8 T-ALL PDXs (Fig. 2a). The tested T- and B-ALL samples responded to the three anthelmintic drugs with comparable sensitivities among the two leukemia types, with IC50 values between 300 nM and 2.5 µM for milbemycin and moxidectin, and a somewhat lower ivermectin sensitivity, with some of the tested cases showing IC50 values higher than 3 µM (Fig. 2a, Supplementary Table S3). Interestingly, most of the screened samples responded to moxidectin at IC50 values of 1–1.5 µM, while the responses to milbemycin were more spread among the samples (Fig. 2a, Supplementary Table S3). We next addressed whether we could identify differential sensitivities in samples from different risk groups, including SR, MR, HR, and VHR, according to the classification used in the ALL-BFM 2000 study as well as relapse samples16. With the exception of one relapsed T-ALL sample that showed very high sensitivity to ivermectin and milbemycin, we could not detect relevant differences in the sensitivity to all three anthelmintic agents across the samples. (Fig. 2b). It is worth noting though that most of the relapse samples responded to anthelmintic agents in the same sensitivity range as compared to the samples collected at diagnosis (Fig. 2b), suggesting that these agents target a mechanism that is not being selected during progression from diagnosis to relapse.


Repurposing anthelmintic agents to eradicate resistant leukemia

Discussion

Different approaches may be applied to identify new treatment strategies for resistant disease. While direct targeting of oncogenic lesions represents an appealing approach, its clinical translation has remained challenging frequently leading to only transient responses. By contrast, targeting specific pathways, on which leukemia cells rely, has been more promising for drug development, exemplified by venetoclax targeting BCL-2 or PI3K/AKT/mTOR inhibitors710. We here report on a drug repurposing screen that we conducted in refractory primary human ALL cells, using an extended FDA-approved drug library. We identified the anthelmintic agents, ivermectin, moxidectin, and milbemycin, as novel agents with high anti-leukemic potential for refractory ALL. These compounds had a strong anti-leukemic effect ex vivo in primary leukemia cells at low micromolar concentrations, extending earlier studies that described activity of ivermectin in acute myeloid leukemia (AML) and other primarily solid tumors Next to anthelmintic agents, we also identified other compound families with potential anti-leukemic agents, such as bactericidal and fungicidal agents. However, these compounds were active in concentrations that could never be reached in vivo, while the vast experience with anthelmintic agents indicates that these could be applied at active anti-leukemic concentrations in vivo (NCT03012828 (ref. )). The anthelmintic agent ivermectin has been widely used to eliminate ectoparasites, while moxidectin has been recently approved by the FDA to treat onchocerciasis in humans. Among the anthelmintic agents, moxidectin has earlier been shown to have a safer profile and a better efficiency when applied as antiparasitic treatment compared to ivermectin44,45. Furthermore, several clinical trials involving moxidectin did not report severe adverse effects at doses four time higher than the one used to treat human onchocerciasis (NCT03012828 (ref. 46)). Moreover, the chemical structure of moxidectin with high lipophilicity increases the half-life of the drug47, an observation that may be linked to its higher drug efficiency, as compared to other anthelmintic agents. Ivermectin on the other hand has been safely used in immunocompromised patients to treat scabies48,49. This attractive safety profile, together with the sensitizing activity towards steroids, warrants further clinical development of anthelmintics, possibly not as single agents but in combination with standard chemotherapy to eradicate minimal residual disease and to prevent relapse. Yet another aspect supporting clinical development arises from activity of anthelmintics across samples from different risk groups including SR ALL. The potential of these agents to improve the response to steroids also in good responding cases and potentially to decrease toxicity could be tested in a clinical trial.


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