Altered cancer cell metabolism, described nearly a century ago by Warburg (1), has gone a long way from an intriguing scientific observation to a validated drug target in the clinic. Initially believed to serve as an alternative energy source for the rapidly dividing cancer cells, “Warburg’s effect” of oxidative glycolysis is now considered an effective means of metabolic rewiring which provides multiple precursors for key biosynthetic pathways (2–4). Excellent examples showing alterations in cell bioenergetics and the clinical use of certain metabolic vulnerabilities come from acute lymphoblastic leukaemia (ALL) (4).

Childhood ALL (cALL) accounts for about one third of all paediatric cancers. The disease is characterised by undifferentiated highly proliferative lymphoid cells of B-cell (~85%), T-cell (10–15%) or mixed lineage (<5%). The genetic and epigenetic aberrations underlying this malignancy are marked by great heterogeneity among patients with a recent study highlighting that there may be over 20 sub-types of B-cell cALL (5). Furthermore, there are a number of metabolic alterations in cALL present already at diagnosis. Some examples include aberrant transcriptional profile involving upregulation of glycolysis and downregulation of citric acid cycle genes (6), mutations in mitochondrial DNA affecting Oxidative Phorphorylation (OxPhos) (7), activation of the AKT pathway in T-ALL, which can cause upregulation of glycolysis and metabolic rewiring (8, 9), and others. Many of the standard chemotherapeutics can target cell metabolism. Glucocorticoids (GCs), the backbone of induction therapy, are effectively decreasing glucose uptake and shifting metabolism towards glutamine synthesis and fatty acid oxidation (10, 11). Anthracyclins inhibit topoisomerase II, but can also block Complex I of the electron transport chain (ETC) decreasing mitochondrial function (11, 12). L-Asparaginase is another critical part of standard treatment. It depletes extracellular asparagine levels, but also leads to glutamine deamination (13), glycolysis, and pyrimidine synthesis (14). Lastly, Methotrexate inhibits the folic acid cycle, thus the synthesis of nucleotides (15).

Refined risk group stratification and treatment regimens, including targeting of metabolic vulnerabilities in blast cells, have improved overall survival to 85–90% (16). Nevertheless, 5-year event-free survival after relapse for certain sub-groups of patients (e.g. with isolated bone marrow relapse, or deletion of IKZF1) may be less than 30% (17). Therefore, novel therapeutic approaches are urgently required especially for high risk groups, refractory disease, and relapsed patients.

Drug repurposing is a strategy whereby a drug, which is already approved for the treatment of a certain condition, is transferred into a different disease context. The advantage of this approach is saving considerable amount of time (at least 2–3 times quicker (18)) and money (3–10 times less on average (19)), ultimately translating into saving more lives faster and at a lower cost. Encouraging examples of drug repositioning already in use or in clinical trials range from Aspirin in colorectal cancer (20), Metformin (for diabetes) and Daunorubicin (antibiotic) for various malignancies (20, 21), retinoic acid (vitamin A) in acute promyelocytic leukaemia (APL) (22), a combination of retinoic acid and Tranylcypromine (TCP—anti-depressant) in acute myeloid leukaemia (23), and others.

Atovaquone (Ato) is an analogue of ubiquinone (the oxidised form of coenzyme Q10) making it an effective inhibitor of complex III of the electron transport chain (ETC) (24). Due to its high specificity and excellent tolerability Ato has been used against protozoa like Plasmodium spp and Toxoplasma gondii, turning it into a key component of standard anti-malarial therapy including in children (25). Interestingly, in recent years Ato has been highlighted as a potent anti-proliferative and apoptotic agent in different malignancies. For instance, Ato has proved effective in vitro in breast cancer (26), in pharyngeal and colorectal cancer cell lines in enhancing the effect of radiotherapy (27), in retinoblastoma cells (28), and in haematologic malignancies like chronic lymphoblastic leukaemia (29) and acute myeloid leukaemia (30). Nevertheless, the effect of Ato on cALL has not been studied.

Therefore, given the body of evidence in literature that cALL cells have alterations in their mitochondrial and metabolic function, together with encouraging results using Ato in other malignancies, the aim of our study was to investigate if Ato may be effective against cALL cells as well.

In summary, we have investigated the anti-proliferative activity of Atovaquone on two established B-cell precursor cALL cell lines—REH and Sup-B15, as well as on a Prednisolone-resistant sub-clone of Sup-B15. We found that Ato elicited a very similar effect on mitochondrial respiration and ATP production in all three cell lines at concentrations of ~30 μM. These findings are in line with previous in vitro studies [10 μM in breast cancer (26) and cervical cancer cells (36), 30 μM in colorectal, pharyngeal and lung cancer cell lines (27, 37), ~15–50 μM for glioblastoma cells (38), 20 μM in retinoblastoma (28) and thyroid cancer cells (39)]. In particular, Ato reduced the levels of basal and maximal respiration, decreased the respiratory capacity of the cells and ATP production as measured by the Seahorse Mito Stress test. Even if we could carry out these experiments on a limited number of patient samples, we observed that Ato can induce similar changes of mitochondrial respiration in primary lymphoblasts as well.

GSEA and GO analyses in REH cells confirmed some of the phenotypic observations in our study—alterations in cell metabolism, induction of apoptosis, and cell cycle arrest ( Figure 1 and Supplementary Figure 2 ), and pointed to other interesting concomitant changes that may be due to Ato. Several papers from the past 5 years have investigated the anti-cancer effect of Ato. Most of them have focussed on inhibition of OxPhos, which we confirm herein as well, whilst two publications show that Ato may work through targeting the AKT–mTOR pathway (28). The interplay between mTOR complex 1 (mTORC1) and mitochondrial function and cell metabolism is well-documented (40). Activation of the AKT–mTOR pathway, which is altered in up to one third of children with T-ALL (41), may lead to considerable changes in cell metabolism and upregulation of glycolysis (4, 42). Investigating the potential effect of Ato on this pathway was beyond the scope of our study. Nevertheless, one of the most downregulated genes we found (log2 = −2.1, adjusted p-value = 0.0018) is PTEN and GSEA from our RNA-seq showed significant and strong downregulation of mTORC1 genes ( Supplementary Table 2 and Supplementary Figure 3 ). Of note, there are links between mTORC1 and the MYC oncoprotein, as well as between overexpression of MYC and shifts in cALL metabolism (9). Therefore, it is noteworthy that our GSEA also revealed downregulation of MYC targets in Ato-treated cells ( Supplementary Figure 3 ).

Ato has been shown to alleviate hypoxia and sensitise cancer cells to radiotherapy (27). Our GSEA analysis highlights alterations of hypoxia as one of the top hits and indeed we see downregulation of the direct HIF1-α target VEGFα after treatment ( Supplementary Table 2 ). There is evidence that radiation therapy for high-risk T-ALL patients may reduce rates of relapse (43). Given that hypoxia is a major factor for the efficacy of radiation therapy, that Ato reduces hypoxia in pre-clinical models (27, 44) and in our gene set enrichment analysis ( Figure 1 ), Ato treatment may be worthy of further investigation in this clinical context of B-cell cALL too.

The combination of Ato with Idarubicin is another interesting aspect of our study. There is an independent confirmation of the synergy between Ato and anthracyclins coming from work on thyroid cancer cells. Lv and colleagues found that Ato and Doxorubicin are an effective strategy working through ETC and consequent STAT3 inhibition (39). Of note, Ato and Idarubicin were most effective in REH cells and Sup-PR cells, both of which are proliferating faster than Sup-B15. Therefore, it may be interesting to test this combination on matched patient samples at diagnosis and relapse, but this is beyond the scope of our current work.

GC-resistance is believed to be the main cause of drug-failure and relapse in cALL (45). Therefore, it was interesting to find that Sup-PR cells remain sensitive to Ato and that there was an additive effect of Ato and Prednisolone in this GC-resistant line, but not in the parental cells. Investigating the molecular mechanisms of resistance (e.g. why Sup-PR cells have significantly lower oxygen consumption rates than Sup-B15 cells— Figure 2 ) and of re-sensitisation to Prednisolone by Ato was another important aspect which fell out of the scope of our study. However, the effectiveness of Ato alone and in combination with Idarubicin or Prednisolone in GC-resistant cells is worthy of further pre-clinical investigations with matched samples and may add another context to a potential clinical trial.

Importantly, there are several key points related to the patient samples’ data and the potential clinical applicability of this study that need to be addressed. Due to the small number of available patient samples, which all belong to the most common type of cALL and its most frequent molecular subtype, one of the main limitations of our work is the lack of depth and representativity of different entities within this heterogeneous disease. Even though the experimental data with these four patient samples cannot be over-interpreted and directly translated to the clinic, we show as a proof-of-concept that Ato can hit its target in primary lymphoblasts as well. We observe significant decrease in mitochondrial respiration and ATP production ( Figure 4B and Supplementary Table 6 ), similarly to what we have in REH, Sup-B15, and Sup-PR cells. Nevertheless, the effect of Ato on cell viability in the patient samples ( Figure 4A ) is not as prominent as in the cell lines. This modest decrease in cell proliferation, however, may be due to either sub-optimal concentrations of Ato or to the very slow cell division of cultured primary cells, which would not allow for the detection of potential cell cycle arrest (as seen in the fast proliferating REH cells) and would also account for the low OCR readings especially after Ato treatment. We also found that the combination of Ato and Idarubicin in patient samples elicits a range of responses—from antagonistic to synergistic. Besides potential differences in proliferation rates ex vivo, which may account for the lack of uniformity in the response to Ato and Idarubicin, the small number of patient samples does not allow for any speculations other than that there may be unidentified molecular and/or cytogenetic factors which may determine either advantageous or adverse results of the double treatments. Therefore, a larger cohort of patients including relapsed samples may be worth testing.

Lastly, the IC₅₀ concentration we found for our cell lines of 30 μM (~11 ug/ml) ( Supplementary Figures 1A, E ) and the concentrations of maximum 90 μM (~33 ug/ml) that we managed to test on some of the patient samples are within the clinically recorded levels for Ato. The steady-state plasma concentration in children achieved by daily administration of 30 mg/kg for 12 days is 37.1 ± 10.9 ug/ml (as described in the prescribing information for Mepron^®). Other literature data suggests similar values—Cmax and Cmin of respectively 40.5 ± 12.2 and 34.0 ± 11.7 ug/ml (of 30 mg/kg daily) (46). Furthermore, there is an interesting finding coming from a retrospective study of over 200 patients with AML who had undergone bone marrow transplantation. What Xiang et al. found was that administration of Ato for nearly two months as a part of standard anti-infection prophylaxis led to significant reduction of relapse rates (13 vs 23%, p = 0.039) (30). Therefore, considering the number of robust pre-clinical studies in other cancers, the good safety data for Ato and the clear effect on proliferation due to ETC inhibition, it is tempting to speculate that Ato would be worthy of further pre-clinical in vivo investigation and perhaps clinical trials as the ongoing one on Ato and radiation therapy (NCT02628080).

In conclusion, we present novel data demonstrating the anti-leukaemic effect of Atovaquone, the mechanism of action of the drug and the concomitant gene expression changes that may underpin the phenotypic changes observed. Furthermore, our results suggest that this FDA-approved ETC inhibitor may have additive or synergistic effect together with Idarubicin. GC-resistant cells may be re-sensitised to Prednisolone when treated with Atovaquone. Therefore, the findings of this study may present a promising new therapeutic approach targeting cell metabolism, which may be worthy of further investigation.

The original contributions presented in the study are publicly available. This data can be found here: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE161959.

The studies involving human participants were reviewed and approved by the Ethics committee at Medical University of Plovdiv. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

YS, TI, TS, KP, and VS designed the study. YS, TI, HB, and KG developed the methodology. YS, TI, TS, KP, and VS analysed the data. YS, VS, TS, and KP wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.

This project was funded by the Bulgarian National Science Fund (project No. KP-06-M21/9 /19.12.18), and supported by the National University Complex for Biomedical and Applied Research with participation in BBMRI-ERIC, NUCBPI-BBMRI.BG, (contracts No. D01-285/ 17.12.2019 and DO1-395/ 18.12.2020) within the National Road Map For Research Infrastructure.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.