3.1 Breast cancer cell lines display differences in sensitivity to pharmacological glutaminase inhibition depending on culture medium composition
MDA-MB-231 cells grown in αMEM + 5% FBS (hereafter αMEM) were treated with CB-839 and drug sensitivity was assessed 3 days later by IC50 analysis. The observed IC50 of 3.3 ± 0.71 μM (mean ± sem, n = 4) was >100-fold higher than the reported value of 26 nM  (Figure 1A), albeit using a different endpoint method (3H-thymidine incorporation versus Promega Cell Titer Glo assay). To understand this discrepancy we treated MDA-MB-231 cultures with CB-839 in RPMI 1640 + 5% FBS (hereafter RPMI 1640) as used by Gross et al., 2014 and this change in culture medium resulted in an IC50 of 19.3 ± 9.3 nM (n = 3), in strong agreement with the published value (Figure 1A). MDA-MB-231 cultures were also grown and treated with CB-839 in DMEM + 5% FBS (hereafter DMEM) and in this case were also relatively insensitive to CB-839 with an IC50 of 5.5 ± 0.71 μM (n = 2) (Figure 1A). These findings suggest that the culture medium composition significantly influences the potency of CB-839.
To explore this relationship further, a panel of 10 additional breast cancer cell lines were treated with CB-839 in either RPMI 1640 or αMEM (Figure 1B). In general, the TNBC cell lines were more sensitive than the receptor-positive cell lines, with the exception of MCF7, in agreement with published reports . CB-839 IC50 values were lower in RPMI 1640 compared with αMEM culture medium in many cell lines tested, with statistically significant effects observed in four cases; BT549, MDA-MB-468, Hs578T and SKBR3, and a trend toward an effect in HCC1143 and MCF7. T47D and BT474 displayed no difference in CB-839 IC50 when cultured in either RPMI 1640 or αMEM.
The expression of 12 genes involved in pyruvate uptake, mitochondrial transport and metabolism was compared for the 11 breast cancer cell lines using data from the Cancer Cell Line Encyclopedia . The cell lines that did not respond to exogenous pyruvate (T47D and BT474) expressed low levels of MCT genes (Supplementary Table S1). In contrast, the other cell lines that did respond to extracellular pyruvate expressed high levels of either SLC16A1 (MCT1) or SLC16A3 (MCT4). This suggests that MCT expression may be a key determinant that allows extracellular pyruvate to suppress CB-839 response.
BPTES, a related allosteric glutaminase inhibitor, was then investigated using the same 11 cell line panel (Figure 1C). In agreement with the CB-839 observations, BPTES IC50 values were generally lower in RPMI 1640 compared with αMEM culture medium, with statistically significantly lower values observed in BT549, MDA-MB-468 and Hs578T.
3.2 Extracellular pyruvate concentrations influence CB-839 potency
The import and metabolism of extracellular glutamine serves as a key TCA cycle anaplerotic substrate in proliferating cancer cells. Previous reports have highlighted the potential of oxaloacetate, pyruvate, glutamate or cell permeable α-KG (dimethyl α-KG) to act as alternative anaplerotic substrates that can rescue cell viability during glutamine deprivation and in some cases antagonise the activity of glutaminase inhibitors . We reviewed the composition of the RPMI 1640, αMEM and DMEM formulations used in our studies to identify different components that may be responsible for the difference in sensitivity observed (Supplementary Table S2). Of the numerous different components, one of the key differences noted was sodium pyruvate. While αMEM and DMEM contain 1 mM sodium pyruvate, RPMI 1640 does not contain added pyruvate. We hypothesised that utilisation of extracellular pyruvate by TNBC cells may support mitochondrial anaplerosis, resulting in decreased dependence on glutamine and rendering cells less sensitive to glutaminase inhibition.
To test this hypothesis MDA-MB-231 cultures were treated with CB-839 in RPMI 1640 supplemented with increasing concentrations of sodium pyruvate (Figure 2A). The introduction of sodium pyruvate resulted in a concentration-dependent increase in the CB-839 IC50 value from 20.7 ± 8.7 nM (n = 3) in the unsupplemented RPMI 1640 to 2.4 ± 0.2 µM (n = 5) in the culture medium supplemented with 1 mM sodium pyruvate (mean ± sem). All pyruvate concentrations above 31 µM resulted in statistically significant increases in CB-839 IC50 compared with unsupplemented RPMI 1640.
BT549 and Hs578T also demonstrated pyruvate concentration-dependent changes in glutaminase inhibitor sensitivity (Figure 2B and 2C). In BT549 cultures CB-839 IC50 increased from 0.74 ± 0.37 µM (n = 7) in the unsupplemented culture medium to 4.5 ± 2.0 µM (n = 4) in the culture medium supplemented with 1 mM sodium pyruvate (mean ± sem). CB-839 IC50 was significantly increased at pyruvate concentrations ≥100 µM. In Hs578T cultures CB-839 IC50 increased from 1.2 ± 0.2 µM in the unsupplemented culture medium to 3.3 ± 0.4 µM in the culture medium supplemented with 1 mM sodium pyruvate (n = 4). In this cell line the CB-839 IC50 was significantly increased at pyruvate concentrations ≥500 µM.
In sensitive cells glutaminase inhibition using CB-839 was reported to decrease the concentration of many TCA cycle intermediates, including malate, citrate and fumarate [6, 23]. To determine whether exogenous pyruvate can restore TCA cycle intermediates during glutaminase inhibition we assessed steady-state fumarate levels, as a measure of TCA cycle anaplerosis. Cellular fumarate levels were significantly decreased following CB-839 treatment (Figure 2C and 2D), in agreement with published findings . Addition of 1 mM sodium pyruvate to RPMI 1640 culture medium prevented this decrease, suggesting that exogenous pyruvate can act as a TCA cycle anaplerotic substrate in TNBC cells when glutamine metabolism is pharmacologically inhibited.
Further studies were conducted to determine whether these effects were dependent to pyruvate carboxylase (PC) activity (Supplementary Figure S1). Treatment with phenylacetic acid (PAA) to inhibit PC was unable to sensitise MDA-MB-231, Hs578T and MCF7 cells to CB-839 in pyruvate-containing culture medium, with a small effect observed in BT549 cells. This finding suggests that in these cell line models replenishment of TCA cycle intermediates by pyruvate during conditions of glutaminase inhibition is largely dependent on the pyruvate dehydrogenase complex and not on PC activity.
3.3 Production and paracrine secretion of pyruvate by TNBC cells impairs CB-839 potency
The pyruvate concentration in RPMI 1640 + 5% FBS culture medium was quantified at 2.8 ± 0.2 µM (mean ± sem, n = 3, Figure 3A). As RPMI 1640 does not contain sodium pyruvate the pyruvate detected likely comes from the 5% (v/v) serum, in line with an approximate concentration of 50-70 µM in the undiluted FBS. We compared pyruvate secretion by three cell lines Hs578T, SUM159PT and a metastatic variant of MDA-MB-231 (MDA-MB-231-luc-D3H2LN). After 48 h culture the resulting pyruvate concentrations in the conditioned culture medium were 58.8 ± 4.8, 61.9 ± 3.1 and 83.9 ± 1.0 µM for Hs578T, MDA-MB-231-luc-D3H2LN and SUM159PT cells, respectively (Figure 3A). When treated with the MCT1 inhibitor AZD3965 at 1 µM the concentration of pyruvate in the conditioned culture medium was decreased to 28.9 ± 2.9, 56.7 ± 4.1 and 62.1 ± 0.1 µM for Hs578T, MDA-MB-231-luc-D3H2LN and SUM159PT cells, respectively (Figure 3A). Thus, pharmacological MCT1 inhibition can decrease the secretion of pyruvate by TNBC cells into the extracellular environment.
These samples of conditioned (or unconditioned) culture medium were then supplemented with various concentrations of CB-839 and used to culture drug-naïve MDA-MB-231 cells. The conditioned culture medium with the highest concentrations of pyruvate provided resistance to CB-839, in agreement with studies using sodium pyruvate supplementation (Figure 3B & 3C). Indeed, strong linear correlations were observed between the relative % of 3H-thymidine incorporation and pyruvate concentration in the samples supplemented with CB-839 at all four concentrations, suggesting that higher levels of pyruvate impair CB-839 activity (Figure 3B). Similarly, when CB-839 IC50 was calculated a strong linear correlation was observed between CB-839 IC50 and pyruvate concentration, also supporting the hypothesis that higher extracellular levels of pyruvate impair CB-839 activity (Figure 3C). Notably, the AZD3965-treated samples from all three cell lines (Hs578T, MDA-MB-231-luc-D3H2LN and SUM159PT) provided lower CB-839 IC50 in recipient MDA-MB-231 cells, confirming that MCT1 inhibition can increase CB-839 sensitivity in this in vitro setting.
3.4 TNBC cells grown as 3D spheroids display reduced sensitivity to CB-839 compared with 2D cultures
To investigate the possibility of impaired CB-839 activity due to paracrine environmental effects we used an in vitro 3D spheroid culture model. Following 4 days establishment spheroid cultures were treated with 0.01, 0.1, 1 or 10 µM CB-839. MDA-MB-231 and SUM159PT formed regular slow-growing 3D structures (Figure 4A and 4B). Hs578T formed regular spherical clusters that displayed minimal increase in size from day 4 to day 8 (Figure 4C). Microscopic imaging demonstrated minimal effects of CB-839-treatment on spheroid growth/integrity, even after 4 days exposure to 10 µM of drug. However, when cell proliferation in these cultures was assessed using 3H-thymidine incorporation (16 h overnight incubation from day 7 to day 8), CB-839 caused a clear concentration-dependent decrease in cell proliferation in all three cell lines (Figure 4D-F). In MDA-MB-231 spheroids the CB-839 concentration needed to halve the relative amount of thymidine incorporation (i.e. IC50) was 0.88 ± 0.26 µM. This change represents a loss in sensitivity of >100-fold compared with the 2D monolayer IC50 of 8.4 ± 0.17 nM (n = 4, P = 0.015, t test). This 2D IC50 for a 4 day CB-839 exposure was slightly lower than the 19.3 and 20.7 nM IC50 values previously observed with 3 day drug exposure (Figure 1A and 2A).
A 32-fold loss of CB-839 activity was observed in SUM159PT cells when they were grown as 3D spheroids. The IC50 of CB-839 was 5.0 ± 0.96 µM for 3D cultures compared with 0.15 ± 0.041 µM for 2D monolayers (n = 4, P = 0.003). Similarly, a 14-fold loss of CB-839 activity was observed in Hs578T with 3D IC50 of 6.6 ± 2.0 µM compared with 2D IC50 0.45 ± 0.022 µM (n = 4, P = 0.019). Again, the 4 day drug exposure produced a modestly lower IC50 than the 3 day IC50 of 1.0 and 1.2 µM observed previously (Figures 1B and 2C). Thus, in the three TNBC cell lines studied CB-839 sensitivity was significantly impaired when cells were grown as 3D spheroids.
The plasma AUC (0-8 h) in patients treated with CB-839 in a 600 mg BID (fed) schedule was approximately 11 µM*h with Cmax of 2.3 µM and Cmin of 0.65 µM . Thus, continuous treatment of the spheroids with CB-839 at 1 µM represents an approximate exposure which may be achievable in patient tumours. At this concentration there is a substantial decrease in CB-839 sensitivity in all of the spheroid models tested compared with regular 2D cell culture conditions.