Targeting glutaminolysis in chondrosarcoma in context of the IDH1/2 mutation

Introduction Chondrosarcoma is a malignant cartilage-forming bone tumour in which mutations in IDH1 and IDH2 frequently occur. Previous studies suggest an increased dependency on glutaminolysis in IDH1/2 mutant cells, which resulted in clinical trials with the drugs CB-839, metformin and chloroquine. In this study, the preclinical rationale for using these drugs as a treatment for chondrosarcoma was evaluated. Methods Expression of glutaminase was determined in 120 cartilage tumours by immunohistochemistry. Ten chondrosarcoma cell lines were treated with the metabolic compounds CB-849, metformin, phenformin (lipophilic analogue of metformin) and chloroquine. Results A difference in glutaminase expression levels between the different tumour grades (p = 0.001, one-way ANOVA) was identified, with the highest expression observed in high-grade chondrosarcomas. Treatment with CB-839, metformin, phenformin or chloroquine revealed that chondrosarcoma cell lines are sensitive to glutaminolysis inhibition. Metformin and phenformin decreased mTOR activity in chondrosarcoma cells, and metformin decreased LC3B-II levels, which is counteracted by chloroquine. Conclusion Targeting glutaminolysis with CB-839, metformin, phenformin or chloroquine is a potential therapeutic strategy for a subset of high-grade chondrosarcomas, irrespective of the presence or absence of an IDH1/2 mutation.


INTRODUCTION
Chondrosarcoma is the second most common primary bone malignancy in humans. It represents a heterogeneous collection of cartilage-forming tumours, which can be divided in several subtypes and histological grades. 1 The most common subtype is conventional chondrosarcoma (85%), which arises centrally in the medulla of the bone. Conventional chondrosarcoma is histologically graded to determine treatment strategy and the patient's prognosis. The atypical cartilaginous tumour (ACT, previously known as chondrosarcoma grade 1), accounts for 61% of cases. First-line treatment consists of curettage with local adjuvant treatment, resulting in a 5-year survival rate of 95%. Grade II (36%) and grade III (3%) chondrosarcomas have a worse 5-year survival of 86% and 58%, respectively, due to the occurrence of metastases. [1][2][3] These tumours are treated with en bloc resection. Dedifferentiated chondrosarcoma is a highly malignant subtype with an overall survival rate of 7-24%. 4 Mesenchymal chondrosarcoma has a 10-year survival rate between 44 and 54%. 5,6 It is a rare aggressive subtype in which distant metastasis can be identified even after 20 years. [5][6][7] Chondrosarcoma patients with inoperable disease, due to tumour location, tumour size or extensive metastatic disease benefit from a doxorubicin-based chemotherapeutic regimen, which increases the 3-year survival from 8 to 26%. 8 As the overall efficacy of chemotherapy is limited, new treatment options are needed, which can be identified by further unravelling the essential driver genes and pathways of these tumours.
Potential driver mutations of central conventional and dedifferentiated chondrosarcoma are gain of function mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), which have been identified in 38-70% of the cases. 9,10 Its occurrence in the benign precursors lesions (enchondromas), of which 52-87% harbour an IDH1/2 mutation, 11,12 further demonstrates that IDH1/2 mutations are an early event in chondrosarcoma genesis. IDH1 and IDH2 are essential enzymes in cell metabolism, as they convert isocitrate to α-ketoglutarate (α-KG) in respectively the cytoplasm and the mitochondria. The mutant enzyme acquires the activity to convert α-KG to D-2-hydroxyglutarate (D-2-HG), an oncometabolite that competitively inhibits the α-KG dependent enzymes by the high structural similarities. 13 Processes involved in chondrosarcoma progression make these cells independent of the mutant IDH enzymes, as treatment with AGI-5198, a specific IDH1 mutant inhibitor, did not influence the tumourigenic properties of these cells. 14 Therefore, we propose to exploit the metabolic vulnerability caused by the IDH1/2 mutations as therapeutic strategy for chondrosarcoma. IDH1/2 mutant cells need α-KG for the production of D-2-HG, which can be generated via glycolysis and glutaminolysis. It has been suggested that IDH1/2 mutated tumours depend on glutaminolysis for their α-KG supply, [15][16][17] which led to two clinical trials that were recently started in IDH1/2 mutated solid tumours, including chondrosarcomas. The first one is a phase I trial with the drug CB-839 (NCT02071862 clinicaltrials.gov), an inhibitor of glutaminase (Fig. 1). The second one is a phase IB/II trial with the drugs metformin and chloroquine (NCT02496741 clinicaltrails.gov), after which the feasibility of phenformin may be explored as an alternative to metformin in case of lack of effect of metformin. 18 Metformin is a first-in-line drug used for the treatment of type II diabetes mellitus that inhibits gluconeogenesis in the liver. It has several effects on cellular proteins, among which it (1) activates adenosine monophosphate activated protein kinase (AMPK), thereby inhibiting the mammalian target of rapamycin (mTOR); 19 (2) inhibits complex 1 of the electron transport chain; 20 and (3) indirectly inhibits glutaminase, the enzyme that converts glutamine to glutamate, via c-Myc; (Fig. 1). 21,22 Phenformin is a lipophilic analogue of metformin with similar working mechanisms, but in contrast to metformin it does not depend on solute carrier (SLC) 22A1-3 transport to get into cells; 20,23 The anti-malaria drug chloroquine, in addition to its well-known anti-autophagy potency, is able to inhibit glutamate dehydrogenase, an enzyme converting glutamate to α-KG (Fig. 1). 24,25 In this study, we evaluate whether there is preclinical rationale to target glutaminolysis as a treatment for chondrosarcoma by determining the expression levels of glutaminase in chondrosarcoma primary tumours and by evaluating the effect of metformin, phenformin, chloroquine and CB-839 on chondrosarcoma cells.

MATERIALS AND METHODS
Immunohistochemistry on tissue microarrays Glutaminase monoclonal antibody (AB156776, Abcam) (1:400) was used for immunohistochemical stainings on previously generated and published formalin-fixed, paraffin-embedded tissue microarrays 26 as described. 27 Hundred and twenty cartilage tumours could be scored, consisting of 12 benign (enchondromas or osteochondromas), 56 ACTs, 36 grade II and 16 grade III tumours.
Statistical analysis Statistical analysis on immunohistochemistry data was performed using Statistical Package for the Social Sciences 23 (SPSS Statistics, IBM). One-way ANOVA with the Fisher's least significant difference (LSD) post-hoc analysis was used to compare glutaminase expression levels between different tumour grades. The difference in glutaminase protein expression between high-grade cartilage tumours (grade II and grade III cartilage tumours) and low-grade cartilage tumours (enchondromas, osteochondromas and ACT) was determined using independent-samples T test. Results were considered significant at the α = 0.05 level.
Cell culture Five IDH1 or IDH2 mutated (JJ012, 28 SW1353 (ATCC #HTB-94), L2975, 29 L835 29 and HT1080 30 ) and five IDH1/2 wildtype (CH2879, 31 MCS170, 32 CH3573, 33 NDCS1 34 and L3252b 29 ) chondrosarcoma cell lines were analysed. Five of these originate from conventional chondrosarcoma (JJ012, SW1353, L835, CH2879 and CH3573), three from dedifferentiated chondrosarcoma (L2975, NDCS1 and L3252b) and one from mesenchymal chondrosarcoma (MCS170). HT1080 was originally reported as a fibrosarcoma of bone. As this is a diagnosis of exclusion and this cell line is now known to harbour an IDH1 mutation, this tumour most probably reflects a dedifferentiated chondrosarcoma. 14  bovine serum (FBS) (F7524, Sigma Aldrich). MCS170 was cultured in IMDM (Gibco) with 15% FBS. The authenticity of the cells was confirmed by STR profiling with the GenePrint10 (Promega Benelux BV) and cells were tested for mycoplasma using MycoAlert (Lonza, Switzerland) before the start of the experiments. Cell lines were never cultured for more than three months, and were tested for mycoplasma every 4 weeks (using RT-PCR).
qRT-PCR RNA isolation of chondrosarcoma cells and the anonymised controls (growth plate and articular cartilage) was performed using TRIzol (Ambion biosystems, Invitrogen) followed by a standard RNA isolation protocol and cDNA synthesis. 35 Product size and sequence were validated using Qiaxcel (Qiagen) and Sanger sequencing (Applied Biosystems 48-or 96-cappilary 3730 system, Leiden genome technology centre), respectively. Standard qRT-PCR analyses were performed as described previously 36 to determine glutaminase, SLC22A1, SLC22A2 and SLC22A3 expression levels. GPR108, CYPa and CPSF6 were used as housekeeping genes for normalisation. 37  For the analyses in which the effect of FBS on CB-839 sensitivity was evaluated, the medium with or without FBS and the corresponding concentrations of the metabolic compounds were added at the same time, so after the cells were allowed to adhere overnight.
Cell count assay As the PrestoBlue assay measures mitochondrial activity, we confirmed that the effects of metformin, phenformin and chloroquine on cell viability were caused by an absolute decrease in cell number by fixing the cells in 4% paraformaldehyde for 15 min, followed by nuclear staining using Hoechst 33342 (Fischer Scientific). The plates were imaged using a BD Pathway 855 imager (Becton Dickinson), after which the images were processed using an Image-Pro Analyser 7.0 algorithm. Hoechst area was used as a read out to quantify the amount of cells in each well.
Analysis of apoptosis For analysis of apoptosis, the caspase-glo 3/7 assay (Promega) was used according to manufacturer's instructions. Cells were seeded in white walled 96-wells plates (Corning BV Life Sciences) in densities which resulted in 70% confluence after 24 h as described previously. 39 HT1080, JJ012, SW1353, NDCS1 and CH2879 cells were treated with their IC 75 of metformin, phenformin, chloroquine and CB-839 (based on 72 h dose response curves). The concentration of compounds used was 10 mM metformin, 100 μM phenformin, 50 μM chloroquine and 6 μM CB-839 if IC 75 , were above these concentrations. CH2879 cells treated with 1 μM doxorubicin (obtained from the in-house hospital pharmacy) and 50 μM ABT-737 (Catalog No. S1002, Selleckchem) were used as positive control. For the negative control doxorubicin and ABT-737 were combined with Z-vad-FMK (550377, BD Biosciences). After 24 h the caspase-glo substrate was added 1:1 followed by incubation of 60 min at room temperature. Wells were analysed using Wallac 1420 VICTOR2.

RESULTS
Glutaminase is a potential therapeutic target in a subset of chondrosarcomas By immunohistochemistry, a difference in glutaminase expression levels between the different tumour grades was identified (ANOVA, p = 0.001), and the post-hoc analysis revealed that specifically the grade II tumours and the grade III tumours had higher expression levels compared to the ACTs (both p = 0.001) (Fig. 2a-e). As no difference between central and peripheral cartilage tumours was observed, these were combined in the analyses. Grouping the high-grade cartilage tumours (grade II and grade III chondrosarcomas) and the low-grade cartilage tumours (ACTs and enchondromas/osteochondromas) further demonstrated the significant difference in glutaminase expression levels between high-grade and low-grade cartilage tumours (p < 0.0001, independent-samples T test) (Fig. 2a). No difference in glutaminase expression between IDH1/2 mutant and IDH1/2 wildtype central cartilage tumours was observed (Fig. 2b). Therefore, glutaminase is higher expressed in high-grade compared to lowgrade cartilage tumours but does not correlate to IDH1/2 mutation status.
Using qRT-PCR analyses, we demonstrate that all cell lines have higher expression levels of glutaminase compared to the controls (growth plate and cartilage), although expression levels are variable (Fig. 3a). Inhibition of glutaminase using CB-839 in ten chondrosarcoma cell lines revealed that HT1080 (IDH1 R132C ), SW1353 (IDH2 R172S ) and, to a lesser extent, JJ012 (IDH1 R132G ), were very sensitive for glutaminase inhibition, with IC 50 values below 5 μM (Fig. 3b, Table 1). L2975 (IDH2 R172W ), NDCS1 (IDH1/2 WT ) and CH3573 (IDH1/2 WT ) had IC 50 values of 10.2, 13.5 and 17.5 μM, while the remaining four cell lines (one IDH1 R132C , three IDH1/2 WT ) had IC 50 values above 50 μM. Interestingly, absence of FBS, increased the sensitivity to CB-839 especially in the IDH1/2 mutant cell lines, while there was no clear difference in the cell lines with wildtype IDH1/2 (Fig. 3c). In conclusion, these experiments demonstrate that a subset of chondrosarcoma cell lines is dependent on glutaminase-mediated glutaminolysis to maintain cell viability.
Metformin, phenformin and chloroquine inhibit chondrosarcoma cell viability Treating the chondrosarcoma cell line panel for 72 h with metformin, phenformin or chloroquine demonstrated that sensitivity for these compounds differed between the different chondrosarcoma cell lines (Fig. 4a)  to the other cell lines (Table 1). Treating the chondrosarcoma cell lines for 7 days increased the effect of metformin, phenformin and chloroquine on cell viability (Fig. 4b). Hoechst quantification confirmed that the effects on cell viability were caused by an absolute decrease in cell amount (Supplementary Figure 1). No difference between IDH1/2 mutant and IDH1/2 wildtype chondrosarcoma cell lines in sensitivity for metformin, phenformin and chloroquine was observed. To further demonstrate that the IDH1 mutant enzyme does not influence sensitivity to these compounds, the inhibitors were combined with AGI-5198, a specific inhibitor of the mutant IDH1 enzyme, in IDH1 mutant JJ012 and HT1080 cells. Cell viability (Fig. 4c and Supplementary Figure 2) was unaffected by cotreatment with AGI-5198. These results demonstrate that chondrosarcoma cell lines can be targeted by metformin, phenformin and chloroquine and further demonstrates the dependency of chondrosarcoma cell lines on glutaminolysis independent of the presence of the IDH1/2 mutant enzyme.

Cellular effects of glutaminolysis inhibition
To investigate the effect on apoptosis, caspase-glo 3/7 assays were performed. Chloroquine slightly increased caspase 3/7 activity in three out of five cell lines tested (Fig. 5a). While the other compounds did impact cell viability after 24 h (Fig. 5b), no effect on caspase 3/7 activity was observed. Next, we evaluated the effect of the four metabolic compounds on phosphorylated S6 protein levels, as this is an indicator of mTOR activity. As shown in Fig. 5c, metformin and phenformin decreased phosphorylated S6 levels in four out of five and three out of five cell lines respectively. However, metformin and phenformin did not affect phosphorylated S6 levels in HT1080 cells, the cell line that is most sensitive to metformin. Interestingly, metformin decreased LC3B-II levels in four out of five cell lines, which indicates an increase in autophagy. 40 As expected, chloroquine greatly increased LC3B-II levels. CB-839 did not affect phosphorylated S6 or LC3B protein levels. Thus, while the induction of apoptosis is very limited, metformin and phenformin decreased mTOR activity in chondrosarcoma cells, and metformin decreased autophagy, an effect that is counteracted by chloroquine.
Metformin is sufficiently transported into chondrosarcoma cells to completely inhibit mitochondrial respiration Next, expression levels of SLC22A1-3 were determined by qRT-PCR analyses, as these transporters are essential for the cellular uptake of metformin and might explain the variability in sensitivity for metformin. Although expression is variable, all cell lines express SLC22A1 (Fig. 6a). Interestingly, the two IDH2 mutated cell lines had the highest expression of SLC22A1. SLC22A2 is only expressed by L2975 (Fig. 6b) and SLC22A3 is only expressed by SW1353, MCS170 and CH2879 (Fig. 6c). This demonstrates that all cell lines express transporters for the cellular uptake of metformin, but expression levels differ.
To evaluate the effect of metformin on chondrosarcoma cell metabolism, seahorse experiments with three chondrosarcoma cell lines (one IDH1 mutated, one IDH2 mutated and one IDH wildtype) were performed. Strikingly, metformin completely inhibited mitochondrial respiration in all cell lines tested independent of IDH1/2 mutation status or SLC22A1 levels (Fig. 6d). The small increase in OCR observed after the addition of FCCP and increased ECAR levels after glucose injection demonstrate that the metformin treated cells are still viable. Interestingly, metformin treated cells showed higher levels of glycolysis when glucose is present compared to the controls. However, oligomycin A injection increased glycolytic levels in controls but not in treated cells, indicating a maximum in glycolytic energy production is achieved in treated cells after glucose addition (Fig. 6e). The small difference in glycolytic activity between treated and untreated cells cannot compensate for the observed loss in ATP production through mitochondrial respiration, suggesting that chondrosarcoma cells utilise other pathways for energy production.

DISCUSSION
In this study, we demonstrated a difference in glutaminase expression levels between the different chondrosarcoma grades, with the highest expression observed in high-grade tumours. We therefore examined whether glutaminolysis could be exploited as a therapeutic target for high-grade chondrosarcoma. Based on current ongoing clinical trials targeting glutaminolysis, to which chondrosarcoma patients can be enrolled, we interfered with glutaminolysis in chondrosacoma cell lines at different levels ( Fig. 1).
First, we inhibited glutaminase using the glutaminase inhibitor CB-839, and indeed, six out of ten chondrosarcoma cell lines showed IC 50 values below 20 μM. CB-839 did not induce apoptosis, autophagy or mTOR activity, suggesting that it likely impacts cell viability via other mechanisms.
Second, we used the widely used anti-diabetic drug metformin, which, among other effects, indirectly inhibits glutaminase via c-Myc; (Fig. 1), 21,22 inhibits complex 1 of the electron transport chain, 20 and inhibits mTOR signalling. 19 Indeed, a subset of chondrosarcoma cell lines was sensitive to metformin, especially when treated for a longer time period. mTOR signalling was previously shown to be important in chondrosarcoma 41 and we confirmed that metformin decreased mTOR activity in all but one chondrosarcoma cell lines tested, which is in line with findings in other studies. 19 Interestingly, mTOR activity was not inhibited in HT1080 which is the most sensitive for metformin treatment, suggesting that mTOR inhibition alone cannot explain the impact of metformin on chondrosarcoma cell viability. Furthermore, while apoptosis was absent, metformin seemed to induce autophagy in the majority of cell lines tested, which can likely be linked to the effect of metformin on AMPK. 42 In this study, we further demonstrate that metformin completely blocks mitochondrial respiration in chondrosarcoma cells, likely caused by its effect on complex I of the electron transport chain. However, blocking complex I of the electron transfer chain (and therefore oxidative glutaminolysis) alone was not sufficient to inhibit cell viability, as an even higher concentration of metformin only had a small impact on cell viability within the 24-h time frame of the mitochondrial respiration experiments (Fig. 5b). The small difference in glycolysis observed upon treatment with metformin is likely insufficient to compensate for the total los of ATP production through oxidative means, suggesting that chondrosarcoma cells are not dependent on oxidative metabolism and require other sources, in addition to the increased glycolysis, to supply the necessary energy. In prostate cancer cell lines, it was demonstrated that metformin treatment increased the dependency on reductive glutaminolysis. 43 Further identifying these pathways might provide interesting targets for combination treatment with metformin.
Third, as an alternative to metformin, we used its lipophilic analogue phenformin. In contrast to metformin, phenformin does not need SLC22A1-3 transport to get into cells.
As expected, the effect of phenformin on cell viability, mTOR activity, and apoptosis was very similar to metformin. Contrary to metformin, phenformin is not used in the clinic due to an increased risk of lactic acidosis. Moreover, we show that all chondrosarcoma cell lines sufficiently express the SLC22A1 transporter that is necessary for metformin uptake, suggesting that there is limited rationale to move to phenformin trials for chondrosarcomas when the metformin trial demonstrates limited efficacy.
Fourth, we evaluated the anti-malaria drug chloroquine, which, in addition to inhibiting glutamate dehydrogenase, is a wellknown inhibitor of autophagy and thereby inhibits many other Targeting glutaminolysis in chondrosarcoma E. F. P. Peterse et al.
metabolic and signal transduction pathways. 44,45 The chondrosarcoma cell lines were sensitive to chloroquine. Also, we confirmed that chloroquine inhibited autophagy in all chondrosarcoma cell lines tested. Moreover, a slight induction of apoptosis was seen in three out of five chondrosarcoma cell lines. Thus, we used four different drugs to evaluate whether the increased dependence on glutaminolysis could be therapeutically exploited using repurposing of existing drugs, and confirmed that a subset of chondrosarcoma cell lines is indeed sensitive to glutaminolysis inhibition. We could not identify a correlation between levels of glutaminase expression and sensitivity for any of these metabolic compounds in the panel of ten chondrosarcoma cell lines. Of note, there was also no correlation of the IDH1/ 2 mutation status of chondrosarcoma cells with sensitivity to these compounds, or with the expression levels of glutaminase in primary tumours. We could therefore not confirm the prevailing hypothesis that IDH1/2 mutant chondrosarcoma cells rely on glutaminolysis to generate sufficient α-KG for D-2-HG production, [15][16][17] as it seems that also high-grade chondrosarcomas that are wildtype for IDH1/2 depend on glutaminolysis. To explain this dependency of IDH1/2 wildtype chondrosarcoma on glutaminolysis, it is tempting to speculate that the hypoxic microenvironment, which is a characteristic of chondrosarcoma, 46 is equally important or even overrates the effect of the IDH1/2 mutation in chondrosarcoma. HIF-1α activates pyruvate dehydrogenase kinase, which inactivates pyruvate dehydrogenase, thereby inhibiting the influx of pyruvate into the TCA cycle. 47 In this context, TCA cycle anaplerosis is required for the synthesis of fatty acids, which is then primarily mediated via glutaminolysis. 48 Therefore, the hypoxic microenvironment of chondrosarcomas potentially explains why chondrosarcomas depend on glutaminolysis irrespective of the presence or absence of an IDH1/2 mutation.
Our results indicate that there is limited preclinical rationale to select chondrosarcoma patients for treatment with these compounds based on their IDH1/2 mutation status. This is in contrast to the studies by Cuyas et al. 16 and Molenaar et al., 49 where, respectively, an increased sensitivity for metformin was identified in an engineered IDH1 mutant breast cancer cell line, and in an engineered IDH1 mutant colorectal cancer cell line compared to their wildtype parental cells. The differences in tumour types and the fact that these cell lines harbour an engineered instead of an endogenous IDH1/2 mutation likely explain the differences in experimental findings.
In conclusion, our results demonstrate that glutaminase is higher expressed in high-grade compared to low-grade chondrosarcomas. High-grade chondrosarcomas are dependent on glutaminolysis which is independent of IDH1/2 mutation status. This dependence can be therapeutically exploited by repurposing existing drugs that inhibit glutaminolysis, including CB-839, metformin, phenformin and chloroquine.