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Antimetabolic cooperativity with the clinically approved l-asparaginase and tyrosine kinase inhibitors to eradicate CML stem cells

Objective Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. Methods Using complementary cell-based techniques, metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. Results Although TKI suppressed glycolysis, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However, clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. Conclusion Targeting glutamine metabolism with the FDA-approved drug, Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells.


INTRODUCTION
Because of the development of tyrosine kinase inhibitor (TKI) therapy, the outcome of chronic myeloid leukemia (CML) patients has drastically improved. Unfortunately, TKI, such as Imatinib mesylate and other second or third generation BCR-ABL1 inhibitors, preferentially target differentiated cells and leave some of the CML stem cells alive [1e4]. A fraction of LSC can survive independently from BCR-ABL1 signaling and as such are totally insensitive to Imatinib [2,5]. TKIs are effective in inducing a longterm response and treatment discontinuation is proposed to patients with persistent deep molecular response [1,3]. The STIM study (Multicenter, nonrandomized Stop Imatinib Trial) underlined that 40% of patients successfully reached treatment-free remission with no disease recurrence. However, resuming treatment is necessary for patients exhibiting BCR-ABL1 transcript increase following Imatinib discontinuation. The inability of TKI to kill LSCs and/or progenitor cells is at the origin of those relapses [5]. There is a strong need for strategies targeted at improving TKI efficacy against LSC in CML in order to definitely eradicate the disease and enable long-term definite TKI discontinuation in most patients. Cancer cell metabolism has opened up a new avenue in cancer treatment because it has become possible to target specific metabolic features of cancer cells, thus providing a potential therapeutic window [6]. Many oncogenes and oncogenic pathways driving cancer development also drive metabolism. Classically, it is assumed that cancer metabolism depends on glycolysis, which is predominant even in normoxic conditions (a phenomenon called the Warburg effect). The aberrant activation of the MAPK pathway via the BRAFV600E mutation increases glucose uptake and glycolysis enabling intense cell proliferation (for review [6e8]). Consistent activation of the PI3K/Akt/mTOR pathway by BCR-ABL1 also increases glucose metabolism in leukemic cells [9e11]. In light of these observations, it is not surprising that the exposure to oncogenic "driver" inhibitors such as BRAFV600E inhibitors [7,8] or BCR-ABL1 inhibitors [11,12] dramatically reduces glucose uptake and glycolysis to promote cell cycle arrest. It has become evident that cancer metabolism cannot be reduced solely to the Warburg effect and it consists of a more complex network linking glucose metabolism and others nutrients like glutamine. A large body of evidence indicates that mitochondrial oxidative metabolic pathways play a crucial role in cancer development, particularly to immediately compensate for glucose deprivation. This dependence on mitochondrial oxidative metabolism allows cells to avoid cell death induced by MAPK inhibitors [7,13] or TKI [6,12]. Moreover, CML stem cells are particularly sensitive to mitochondrial oxidative metabolism inhibitors [13,14]. One interesting therapeutic strategy in cancer could be the combination of molecular-targeted drugs with antiglycolytic activities and inhibitors of the compensatory mitochondrial oxidative pathways thereby creating an "antimetabolic cooperativity" [6,15]. Herein, our objective was to develop a preclinical proof of concept for antimetabolic cooperativity against CML stem cells. We demonstrated that the combination of Kidrolase, a L-asparaginase used as treatment of acute lymphoblastic leukemia, with Imatinib possesses antimetabolic cooperativity and acts synergistically to eradicate CML stem cells in vitro and ex vivo.

Chemicals
Imatinib mesylate was purchased from SigmaeAldrich, Nilotinib and Dasatinib were purchased from Selleckchem. Kidrolase and Erwinase were provided by CHU Lille, [ 13 C 6 ] Glucose and [ 13 C 5 ] Glutamine were obtained from Professor Bart Ghesquière.
2.2. Patient samples CML samples consisted of blood or bone marrow samples obtained from individuals in chronic or acute CML stages recruited from the Hematology Department (Lille CHU, France), after having obtained a signed informed consent in accordance with the Declaration of Helsinki and approval of the institutional ethics committee (CPP Lille). CD34þ cells were isolated from umbilical cord blood using the EasySepÔ Human CD34 Positive Selection Kit II (Stemcell Technologies). Cytapheresis of CML patients were kindly provided by Pr. François-Xavier Mahon (CHU Bordeaux, France). CD34þ cells were cultivated at 37 C and 5% CO 2 in RPMI medium (Gibco) supplemented with 10% fetal calf serum (Gibco), 50 U/ml penicillin, 50 mg/ml streptomycin, and a growth factor cocktail containing 10 ng/ml of interleukins (IL)-3, (IL)-6, (IL)-7 and granulocyte colony-stimulating factor (G-CSF), 5 ng/ml of granulocyte macrophage colony-stimulating factor (GM-CSF), and 25 ng/ml of stem cell factor (SCF) (Peprotech).

Cell lines
The leukemic DA1-3b cell line was generated by stable transfection of BCR-ABL1 [14e16]. Isolation of tumor cells d60 and d365 were previously described [14e17]. K562, KCL-22 and KU812 cell lines were grown in the same conditions. MS-5 mesenchymal cells were grown in a-MEM medium w/o nucleosides (Gibco) supplemented with 10% fetal calf serum, 50 U/ml penicillin, 50 mg/ml streptomycin, 2 mM L-glutamine and 2 mM pyruvate. The identity of HBL, LND, and Mel-4M was also confirmed by karyotyping and array comparative genomic hybridization testing.
2.4. OCR and ECAR measurement, determination of cellular ATP Extracellular acidification rate measurements (ECAR) and oxygen consumption rate (OCR) were measured using the Seahorse XFe24 analyzer (Seahorse Bioscience, Billerica, MA, USA). Detailed methods are provided in Supplementary Information files.

Colony forming cell (CFC) assay
Clonogenic assay was realized with cells seeded into 35 mm petri dish in semi-solid methylcellulose medium (MethocultÔ M3231 for murin cells or MethocultÔ H4230 for human cells, Stemcell Technologies). Cells were treated with the drugs for 72 h and then centrifugated. The pellet was resuspended in Iscove's modified Dulbecco medium (Lonza) supplemented with 2% fetal calf serum and 50 U/ml penicillin, 50 mg/ml streptomycin at 10,000 cells/ml, and cell suspension was added to methylcellulose medium (1000 cells/1ml/dish) and left at 37 C and 5% CO 2 . Colony forming efficiency was determined after 7 days using Leica DMI8 inverted microscope (Leica Microsystems) and quantified using Image J software.

Metabolite flux
Two hundred thousand cells were supplemented with media containing uniformly labelled U-13C 6 glucose (25 mM) or 13C-glutamine (2 mM) for 24 h. Detailed methods are provided in the Supplementary Information files.
2.7. Immunoblot analysis and real-time quantitative reverse transcription For immunoblot, cell lysates were prepared as described previously [14,17]. mRNA quantititaive detection was performed with real-time PCR using the Lightcycler 480 detector (Roche Applied Science, Manheim Germany) as previously published [15,17].

Amino acid measurements
Amino acids concentration assay (mmol/l) was performed via highperformance liquid chromatography (Shimadzu C18 column, Kyoto, Japan) associated with tandem mass spectrometry (Sciex 3200 Qtrap, Framingham, MA) using the aTRAQ kit for amino acid analysis of physio-logical fluids (Sciex). Acquisition in the mass spectrometer was achieved by multiple reaction monitoring. Data recording and analysis were performed with Analyst software, v.1.6 (Sciex). Internal controls were systematically analyzed for each sample series.
Original Article 2.10. Statistical analysis All data points are represented as means AE SD. A two-tailed Student's t test was used to compare mean values between two groups. Oneway or two-way analyses of variance (ANOVA) followed by Dunnett's or Sidak post hoc testing were used to compare mean values between multiple groups. The statistical analysis was performed using Prism version 6.0f (GraphPad Software, La Jolla, CA). P < 0.05 was considered statistically significant.  . Leukemic cells were treated by sub-lethal concentration of TKI of BCR-ABL1 (Imatinib 0.2 mM, PD180970 0.01 mM, Nilotinib 5 nM, Dasatinib 2 nM) for 24 h, and basal and maximal ECAR were assessed. Basal glycolysis ans maximal glycolysis are measured following glucose or oligomycin injection respectively (mean þ/-SD, n ¼ 3. *p < 0.05). (D) DA1-3b leukemic cells were treated with Imatinib (0.2 mM) for 72 h or with oligomycin A (1 mM) or/and 2-DG (10 mM) for 4 h. ATP levels were then measured by luminescence (mean þ/-SD, n ¼ 3. * or #P < 0.05 respectively compared to control). (E) Isotopolog quantification of TCA cycle intermediates levels by liquid chromatography-mass spectrometry analysis in DA1-3b cells grown in media containing U-13C 5 glutamine and treated or not with Imatinib (0.2 mM for 24 h) (mean AE SD, n ¼ 3, *p < 0.05). (F) DA1-3b cells were cultured in medium containing a combination as indicated of Imatinib (0.5 mM), glutamine (2 mM) and asparagine (4 mM) for 48 h. Necrosis and apoptosis were determined by flow cytometry analysis of Annexin V and Sytox blue staining (mean AE SD, n ¼ 3, *p < 0.05).

Metabolic organization of CML involves both glycolysis and glutamine dependent mitochondrial OXPHOS
We first compared the metabolism of DA1-3b leukemic cells expressing BCR-ABL1 to the isogenic cell line DA1 not expressing BCR-ABL1 ( Figure 1AeC). There was a significant increase in glycolysis (as validated by ECAR and gene expression) ( Figure 1A,B) as well as mitochondrial respiration ( Figure 1C) in cells transfected with p210 BCR-ABL1 compared to control cells. In BCR-ABL1-expressing DA1-3b cells, the mitochondrial respiration was largely sustained by glutamine ( Figure 1D), indicating that CML cells consume both glucose and glutamine. In the presence of glutamine, glucose, and pyruvate, DA1-3b exhibited the highest proliferative rates ( Figure 1E). Under glucose or glutamine deprivation conditions, DA1-3b cells were still able to proliferate, albeit at a lower rate than in conditions with both nutrients ( Figure. 1E). Glucose or glutamine starvation did not trigger an obvious increase in cell death but promoted G0/G1 cell cycle arrest ( Figure. 1F). This result is consistent with the observation that inhibition of either glycolysis with 2DG or mitochondrial respiration with oligomycin A was insufficient to totally deplete DA1-3b ( Figure 1G, left panel) or primary CD34þ leukemic ( Figure 1G, right panel) cells in ATP. This is probably due to the development of compensatory mechanisms to prevent energy collapse. These results suggest that CML cells have a metabolic flexibility, enabling them to survive during carbon source deprivation periods. Oonly the inhibition of the two metabolic pathways was able to deplete cells in ATP and kill BCR-ABL1 expressing cells. Overall, these results strongly suggest that the depletion of carbon sources, glucose and glutamine, is required for energy crisis and subsequent CML killing.
3.2. Upon Imatinib exposure BCR-ABL1þ cells are dependent on mitochondrial metabolism for survival Previous studies showed that Imatinib displays a sustained inhibitory effect on glucose uptake and glycolysis via a reduction in the expression of key proteins such as GLUT-1 or PKM2 [10,18e20]. In this study, we specifically examined the metabolism of leukemic cells that survive Imatinib exposure at sub-lethal (Supplementary Figs. S1AeD), and clinically relevant anti-proliferative concentrations [11,21]. At concentrations below 0.2 mM, Imatinib induced a strong antiproliferative effect in DA1-3b cells ( Supplementary Fig. S1A) but did not induce mitochondrial apoptosis as evidenced by the absence of translocation of phosphatidyl-serines, the retained high mitochondrial membrane potential (Djm) and low level of mitochondrial ROS ( Supplementary Fig. S1 B-E). We performed metabolic flux analysis using 13C-labeled glucose in CML cells treated with vehicle or Imatinib for 24 h before the incubation with labeled glucose. Cells treated with Imatinib exhibited a major impairment of glucose uptake and glycolysis as judged by the decrease in labeled glycolytic intermediates and labeled lactate ( Figure 2A). Moreover, it was associated with a reduction in the glucose flux via the nonoxidative pentose phosphate pathway (Figure 2A). In agreement with the inhibition of glucose uptake and lactate production (Supplementary Figs. S2A and S2B), we also observed a decrease in glycolysis-associated protein expression ( Supplementary Fig. S2C), as well as a decrease in ECAR in BCR-ABL1þ leukemic cells exposed to Imatinib (Fig. 2B) or to other clinical BCR-ABL1 inhibitors ( Figure 2C). Despite the glycolysis decrease, Imatinib exposure did not deplete in the high-energy molecule, ATP indicating that cells that survive to Imatinib can maintain energy state in the absence of efficient glycolysis ( Figure 2D). This situation is compatible with mitochondrial metabolism maintenance despite Imatinib exposure, and a slight decrease in basal OCR under TKI treatment ( Supplementary Fig. 2E). To explore whether glutamine was used to fuel mitochondrial activity in such conditions, we cultured cells in [13C5] L-glutamine in the presence or absence of Imatinib and analyzed intracellular metabolites via mass spectrometry. As shown in Figure 2E, cells that survived Imatinib continued to use glutamine to produce the TCA intermediates alphaKetoGlutarate in the absence of efficient glycolysis ( Figure 2B). We observed that leukemic cells exposed to Imatinib used glutamine-dependent mitochondrial activity to survive. These cells became highly sensitive to the withdrawal of glutamine but not asparagine ( Figure 2F). To survive Imatinib, BCR-ABL1þ cells require glutamine-derived carbon maintaining the TCA cycle in the absence of glycolysis.
3.3. L-asparaginases inhibit glutamine metabolism and reduce leukemic cell growth but are insufficient to eradicate BCR-ABL1þ cells We previously demonstrated that targeting glutamine metabolism bears potential anti leukemic effects on myeloid leukemia cells [19,20,22]. We sought to target glutamine addiction in Imatinibsurviving BCR-ABL1þ cells. L-asparaginases (e.g. the E.coli-asparaginase, Kidrolase) are used to treat pediatric and adult forms of acute lymphoblastic leukemia and are also used in pediatric AML. These therapeutically relevant components are able to deaminate Lasparagine into aspartate. These enzymes also deplete glutamine and the antileukemic activity correlates with their ability to deplete extra cellular asparagine better than glutamine at lower doses, and to deplete both amino acids at higher doses [21,23]. We studied the metabolic effects of the FDA-approved Kidrolase in BCR-ABL1 þ leukemic cells (Figure 3). In conditions depleting extracellular glutamine and asparagine (Figure 3AeB and Supplementary  Fig. S3A), the flux experiment indicates that L-asparaginase depleted drastically the anaplerotic flux of glutamine into the TCA in BCR-ABL1þ cells ( Figure 3C). This was accompanied by a significant reduction in OCR (Figure 3DeE), confirming that glutamine is a major energy source to fuel mitochondrial respiration in BCR-ABL1þ cell lines, but also in CD34þ CD38-leukemic cells from CML patients. As a result of mitochondrial inhibition, Kidrolase displayed strong antiproliferative effects without predominant cytotoxic activity in BCR-ABL1þ cells ( Supplementary Fig. S3B and Fig. S3G). This absence of important cell death in BCR-ABL1þ cells was compatible with the maintenance of high ATP level in Kidrolase-treated cells ( Figure 3F). L-asparaginase increased intracellular glycolytic intermediates in BCR-ABL1þ cells ( Figure 3G). We tested whether the glycolysis increase with Kidrolase treatment allowed leukemic cells to cope with metabolic stress. We found that the compensatory increase in glycolysis supported the survival of cells exposed to Kidrolase since glycolysis inhibition with 2DG did synergize with Kidrolase to induce BCR-ABL1þ cell death ( Figure 3H). To determine whether Kidrolase sensitivity is a general feature of leukemic cells, we tested a large panel of BCR-ABL1 þ or -leukemic myeloid cell lines. The antileukemic effects of Kidrolase were dose dependent and occurred in all tested cell lines. However, the antileukemic responses were highly heterogeneous in term of cell death observed up to 72 h ( Supplementary Fig. S3C). We discovered a reverse correlation between glycolysis and Kidrolase-induced leukemic cell death ( Figure 3I). The FDA-approved glutamine inhibitor Kidrolase hinders glutamine-dependent mitochondrial metabolism but is insufficient to eradicate BCR-ABL1þ cells due to glycolytic compensation. (E) Assessment of mitochondrial respiration in CD34þ CD38þ/À patient primary leukemic cell sub-population from 2 CML patients by measuring OCR with XFe24 Seahorse. Molecules were injected as indicated: vehicle or Kidrolase. Percentages OXPHOS inhibition were calculated after 60, 120 and 180 min of exposure to kidrolase (mean AE SD, n ¼ 3, *p ¼ 0.05). F) Intracellular ATP level of DA1-3b cells exposed to oligomycin (oli.), 2-deoxy-glucose (2DG) or a combination of the two inhibitors for 4 h, with or without 72 h pre-incubation with Kidrolase 1 UI/mL (mean AE SD, n ¼ 3, *p ¼ 0.05). (G) Isotopolog quantification of glycolysis intermediates measured by LC-MS analysis in DA1-3b cells treated with Kidrolase 0.5 UI/mL for 24 h and grown in media containing U-13C 6 glucose (mean AE SD, n ¼ 3, *p ¼ 0.05). (H) DA1-3b cells were cultured with combination of 2-DG (0.033e100 mM) and Kidrolase (0.00033e10 UI/mL) for 72 h and cell proliferation inhibition was quantified by fluorescence using CyQUANT Cell Proliferation Assay. The response of the combination was compared with its single agents against the widely used Loewe model for drug-withitself dose additivity using Chalice software and presented as an isobologram. (mean AE SD, n ¼ 3, 48hrs exposure to Imatinib (0.5 mM) and Kidrolase treatments as indicated (mean AE SD, n ¼ 3, * or #p < 0.05 respectively compared to control). (C) DA1-3b cells were cultured with combination of Imatinib (0.0001e100 mM) and Kidrolase (0.00033e10 UI/mL) for 72 h and cell proliferation inhibition was quantified by fluorescence using CyQUANT Cell Proliferation Assay). Isobologram was determined as seen in Figure 3(D) Immunoblotting of pro-and anti-apoptotic proteins (as indicated) in DA1-3b cells treated with Imatinib  Previously, mice were immunized with irradiated IL12etransduced DA1-3b cells, challenged with wild-type DA1-3b cells and randomly killed after 60 days or 365 days follow-up [2,24]. Leukemic residual cells were collected from bone marrow of sacrificed mice and used to generate stable cell lines (DA1-3b/d60 or DA1-3b/d365). (B) Lethal leukemia developped in mice injected intraperitoneally with DA1-3b wt, DA1-3b/d60 or DA1-3b/d365 cells. (CeD) DA1-3b, DA1-3b/d60 and DA1-3b/d365 cells were treated by Imatinib and Kidrolase for 48 h and cell death was assessed using flow cytometry analysis of annexin V and PI stainings.

Dual treatment with imatinib and kidrolase significantly induces death of BCR-ABL1þ leukemic cells
Given the metabolic flexibility of BCR-ABL1þ cells observed above, we hypothesized that the combination of Imatinib and L-asparaginases such as Kidrolase could be therapeutically relevant by blocking both glycolysis and mitochondrial metabolism. Metabolic flux analyses indicate that cells treated with the Imatinib and Kidrolase combination caused much more impairment of the carbon flux into the TCA cycle, than either of these drugs alone ( Figure 4A right panel). This decreased TCA activity was not linked to changes in the respiratory chain protein expression as evidenced by immunoblot of several proteins of each complex (Supplementary Fig. 3E). This drug combination also effectively impeded the glycolytic flux ( Figure 4A left panel). In BCR-ABL1þ cell lines, Kidrolase synergized with Imatinib to induce cell death ( Figure 4B-C and supplementary 3F). The combination of Imatinib with Kidrolase enhanced cell death by potentiating the intrinsic pathway of apoptosis through the downregulation of Bcl-2 and Bcl-XL protein levels and Bim upregulation ( Figure 4D). The combination of Kidrolase and Imatinib showed the highest killing effect compared to the association of Kidrolase and other anti-leukemic drugs (daunorubicin or idarubicin) ( Figure 4E). Kidrolase acted in synergy with Imatinib in BCR-ABL1 þ leukemia cells even when cells were cultured on the MS5 stroma cell line ( Figure 4F) or in hypoxic conditions ( Figure 4G), situations known to protect leukemia cells from L-asparaginase [22] or Imatinib effects [23,24]. These results suggest that Imatinib plus Kidrolase elicit death synergistically in BCR-ABL1 þ leukemic cells.

Imatinib synergized with kidrolase to eradicate BCR-ABL1þ persistent stem cells and leukemia-initiating cells in CML patients
We explored the potential of the synergistic combination of Imatinib plus Kidrolase against subpopulations of short-term relapse-inducing CML cells. First, the subpopulation of cells that survived in the presence of Imatinib for 7 days was enriched in persistent CML ( Figure 5A) and Imatinib inhibition led to the proliferation of blast cell clones ( Figure 5B). This persistent population, which survived during TKI exposure, showed a high level of mitochondrial metabolism as determined by their mitochondrial potential, mitochondrial mass and the overproduction of mitochondrial ROS ( Figure 5C). To confirm the predominant role of mitochondrial metabolism, we isolated two populations characterized by low (Dj m low) or high mitochondrial (Dj m high) membrane potential using cell sorting. As expected, the percentage of persistent leukemia cells was higher in Dj m high DA1-3b cells in comparison with Dj m low cells, whereas the combination of Imatinib and Kidrolase efficiently eradicated both subpopulations. Finally, we showed that Erwinase, another L-asparaginase, also synergized with Imatinib to kill persistent BCR-ABL1þ cells ( Figure 5E). We studied the effect of this drug combination on long-term persistent BCR-ABL1þ cells originated from a mouse model of in vivo leukemia dormancy [15]. In our model, the DA1-3b/d60 and DA1-3b/d365 cells were derived from the BCR-ABL1 þ DA1-3b cells injected in mice and isolated after 2 months or 1 year of tumor dormancy, respectively [16,24] (Figure 6A). DA1-3b/d60 and DA1-3b/d365 were injected intraperitoneally in mice to develop a lethal leukemia ( Figure 6B). As expected, death of DA1-3b/60 and DA1-3b/ d365-bearing mice was delayed in comparison with DA1-3b WT mice, confirming the maintenance in vivo of the dormant phenotype ( Figure 6B). Persistent-leukemic DA1-3b/d60 and DA1-3b/d365 cells were completely refractory to Kidrolase monotherapy and also partially resistant to treatment with Imatinib ( Figure 6C,D). However, the combination of both drugs eradicated long-term persistent cells as evidenced by the induction of apoptosis (annexin V staining) ( Figure 6D) and loss of colony-forming potential ( Figure 6E). Kidrolase in combination with Imatinib reduced colony formation of DA1-3b cells by more than 90% compared to Imatinib alone ( Figure 5E). We assessed the ex vivo effects of Kidrolase plus Imatinib in different CD34þ and CD38-subpopulations of primary CML CD34þ progenitor cells (freshly collected from newly diagnosed patients) (n ¼ 4; Figure 6G and Supplementary Fig. S4) and from 6 cryopreserved primary CML cells from patients at diagnosis ( Supplementary  Fig. S5). We underlined that the killing effect was more pronounced on the stem cell-enriched CD34þ CD38-subpopulation as compared to more differentiated CML cells ( Figure 6G and Supplementary Fig. S4). As expected, the combination had no effect on CD34þ progenitor cells from healthy individuals (n ¼ 2; Figure 6F). We used LTC-IC assays to confirm that cells surviving BCR-ABL inhibition retained the in vitro functional capacity of stem cells. As seen in Figure 6H and Supplementary Fig. S6, we confirmed the efficacy of Kidrolase in combination with Imatinib in reducing the stem cell potential of primary CML cells. The combined use of Imatinib and Kidrolase synergistically increase selective cytotoxicity in CML stem cells.

DISCUSSION
We characterized the metabolic effects of the FDA-approved drug Kidrolase in association with TKI in myeloid leukemia and demonstrated that they displayed synergistic antimetabolic effects eradicating CML stem cells. It is well established that TKI such as Imatinib possess a potent anti-Warburg effect. Imatinib efficiently hampers glucose metabolism through the reduction of GLUT-1 surface localization [10], inhibition of BCR-ABL1-mediated PKM2 phosphorylation or the modulation of PKM isoforms [18]. Glycolysis inhibition alone is often insufficient to eradicate cells due to compensatory activating metabolic pathways [6,13]. Upon Imatinib exposure, mitochondrial oxidative metabolism is maintained at high levels due to fatty acid [18] and/or glutamine oxidation (Figure 2). This suggests that remaining TKItolerant cells become addicted to mitochondrial activity for survival [13,25]. In agreement with our results, mitochondrial metabolism is spared by FLT3 ITD TK inhibitors in myeloid cells [25,26]. The mechanisms that result from glycolytic metabolism shift toward oxidative metabolism remain largely unknown. However, we and others [25,27] observed a significant increase in mitochondrial mass upon TKI exposure suggesting important changes in mitochondrial biogenesis. As a consequence of mitochondrial addiction, TKI-tolerant cells were highly sensitive to the antileukemia effects of mitochondrial-targeting drugs. Myeloid leukemia presents numerous mitochondrial-druggable targets [25,26,28e30]. CPT1 (carnitine O-palmitoyltransferase 1) inhibitors, which reduce fatty acid oxidation and mitochondrial OXPHOS, significantly decrease the number of quiescent leukemia progenitor cells [27,31]. The combination of mitochondrial drugs and TKI consists in a rational approach where the therapeutic objective is to consider complementary mechanisms of action. TKI and mitochondrial inhibitors address the two compensatory aspects of the metabolism, glycolysis and mitochondrial oxidation, which neither of the monotherapy is capable of achieving in order to kill leukemia cells. Several preclinical studies evidenced that TKI, such as FLT3 ITD inhibitors, can synergize with mitochondrial inhibitors for potent antileukemia effects [13,25,28e30]. Inhibition of the mitochondrial "booster" STAT3 [25,31], or the mitochondrial translation [13,29] can synergize with BCR-ABL1 inhibitors in CML. We completed the frame including the inhibition of glutamine-dependent mitochondrial metabolism. Genetic and pharmacological inhibition of the first enzyme in glutamine metabolism is synthetically lethal with FLT3-ITD-TKI in myeloid leukemia cells [25,32]. This effect seems related to glutathione depletion and enhancement of mitochondrial oxidative stress resulting in leukemia cell death [29,30]. We used L-asparaginase to deplete extracellular glutamine and therefore mitochondrial metabolism. To best translate in vitro results into clinical application, we selected the existing FDAapproved drug Kidrolase. However, L-asparaginase clinical use has been associated with a number of dose-dependent toxicities [32,33]. When combined with TKI, L-asparaginase should be less toxic since exposure to BCR-ABL1 inhibitors specifically increases the sensitivity of BCR-ABL1þ cells to mitochondrial targeting drugs (results and [30,34]). Imatinib has already been added to intensive combination chemotherapy, which included asparaginase, to treat newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia [35]. Toxicities of the combined treatment were manageable and included grade 4 myelosuppression (with severe neutropenia) and reversible grade 3 hyperbilirubinemia, which was more likely due to treatment with L-asparaginase alone. The toxicities of the combination could probably be largely attributed to the L-asparaginase effects. As demonstrated ( Figure 3A), L-asparaginase depleted the extracellular AA, glutamine and asparagine. Several studies uncovered a crucial role of asparagine in cancer cell growth and proliferation [33,36] as well as cell survival to glutamine deprivation [34,37]. Thus, asparagine is required for anabolism and cell proliferation in the absence of glutamine [13,36,38,39]. For these reasons, the synergistic effect of L-Asparaginase observed here is probably based on the depletion of both extracellular asparagine and glutamine. It is noteworthy that LSCs relies on AA including glutamine and glutamate to maintain OXPHOS for survival [37]. Targeting mitochondrial metabolism was suggested as a therapeutic approach against CML stem cells [13,38,39]. Recently a therapeutic drug combination targeting mitochondrial metabolism demonstrated its efficacy against LSCs in patients with AML [2,40]. We evidenced that both glycolysis and glutamine-dependent mitochondrial metabolism had to be impaired to eradicate LSCs. Targeting compensatory pathways of glutamine metabolism in CML stem cells can improve the efficacy of cancer treatments that impair glucose utilization. We provided preclinical evidence that the antimetabolic cooperativity by the combination of oncogene tyrosine kinase inhibitors and mitochondrial inhibitors constitutes a novel interesting therapeutic approach to eradicate LSC. This antimetabolic strategy can target CML stem cells and could therefore limit therapeutic failure in patients following the discontinuation of TKI therapy.