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Modulation of intermediary metabolism in cancer therapy
Since chemoresistance is an emerging problem in BH3 mimetic therapy, we extended these studies to more selective BH3 mimetics, such as ABT-199, which has replaced navitoclax owing to the dose-limiting thrombo- cytopenia associated with BCL-XL inhibition.3 Moreover, other selective BH3 mimetics that target BCL-XL (A- 1331852) and MCL-1 (A-1210477 and S63845) have been introduced for use in several other malignancies.5-7 Using these BH3 mimetics and relevant cancer cell lines, we tried to mimic the rapid resistance observed in CLL patients following navitoclax treatment (Figure 1A,B), in order to identify ways to tackle chemoresistance, as it emerges. For this, we chose the BCL-2-dependent MAVER-1, BCL-XL-dependent K562 and MCL-1-depen- dent H929 cell lines and exposed them to ABT-199, A- 1331852 and A-1210477, respectively, to generate differ- ent models of resistance (Figure 1C and Online Supplementary Figure S2). Initial exposure of the relevant cell lines to the corresponding BH3 mimetic resulted in a rapid, time-dependent induction of apoptosis as assessed by the activation of caspase-9 and caspase-3 as well as cleavage of the canonical caspase substrate, PARP (Online Supplementary Figure S2A). Resistance to BH3 mimetics in these cells was generated by following the scheme pre- sented in Figure 1C, when the initially sensitive cells [A] became relatively resistant [E], after four exposures (with- in 8 weeks) to their respective BH3 mimetic (Figure 1C). Similarly, a rapid resistance to the different BH3 mimetics was also observed using the other three resistance models (Online Supplementary Figure S2B-D). The rapid and mod- est resistance to the different BH3 mimetics in these cell lines was comparable to the extent of resistance observed in CLL cells during navitoclax therapy (Figure 1B).
Resistance to BH3 mimetics can be overcome by inhibiting multiple BCL-2 family members
Since resistance to BH3 mimetics has often been attrib- uted to elevated expression levels of one or more anti- apoptotic BCL-2 family members, we wanted to identify whether such changes could be responsible for the observed resistance. Comparison of the sensitive [A], intermediate [C] and resistant [E] cells from the different cell lines did not reveal any consistent differences in BCL- 2 family expression to explain the resistance (Online Supplementary Figure S3). We, therefore, sought to identify whether changes in protein-protein interactions among different pro-apoptotic BH3-only members and their anti- apoptotic counterparts could explain the resistance to BH3 mimetics. To do this, we performed immunoprecip- itation studies to isolate the anti-apoptotic proteins bound to BIM and PUMA, which were abundantly expressed in the three different cell types. However, in the sensitive [A] and resistant [E] MAVER-1 cells, immunoprecipitation of BIM and PUMA revealed similar binding of BCL-2 and BCL-XL and little or no binding to MCL-1 (Online Supplementary Figure S4). Likewise, no dif- ferences were observed in the binding of BIM and PUMA to BCL-XL and MCL-1 in sensitive and resistant K562 or H929 cells (Online Supplementary Figure S4).
Although the protein expression levels and immuno- precipitation studies did not support an involvement of other BCL-2 family proteins in the observed resistance, the resistance to ABT-199 observed in MAVER-1 cells was completely overcome by a combination of ABT-199 with either A-1331852 or A-1210477, but not by either A-
1331852 or A-1210477 alone, suggesting that the resistant cells depend not only on BCL-2 but also on BCL-XL and/or MCL-1 for survival (Figure 1D). Furthermore, a combination of all three BH3 mimetics induced apoptosis in all the resistant cells, emphasizing the importance of all three anti-apoptotic BCL-2 family members in chemore- sistance in these cells (Figure 1D). In K562 and H929 cells, the resistance was overcome by the combination of A- 1331852 and A-1210477, but not ABT-199, thus implicat- ing primary roles for BCL-XL and MCL-1 in chemoresis- tance (Figure 1E,F). Similar to the MAVER-1 cells, the chemoresistant K562 cells also exhibited enhanced apop- tosis following treatment with a combination of all three BH3 mimetics (Figure 1E), suggesting that some contribu- tion of BCL-2 could not be totally excluded in these cells. These observations were almost entirely reproducible in the other three models of resistance (Online Supplementary Figure S5), supporting the notion that BCL-XL and/or MCL-1 contributed significantly to the observed chemoresistance in the different models.
Modulation of glutamine uptake and/or metabolism enhances sensitivity to BH3 mimetics
Although the above results demonstrate that a combi- nation of BH3 mimetics can overcome resistance, such an approach targeting multiple members of the BCL-2 fami- ly requires careful evaluation of the therapeutic index, as these proteins perform redundant functions in the main- tenance of normal cellular homeostasis. An alternative strategy to overcome chemoresistance to BH3 mimetics could be achieved by altered metabolism, as depriving cells of glutamine has recently been shown to overcome MCL-1-mediated chemoresistance in multiple myelo- ma.19 In our experiments, glutamine deprivation for 16 h alone did not exhibit any effect on overall cell survival and yet sensitized both the sensitive [A] and resistant [E] cells to BH3 mimetic-mediated apoptosis (Figure 2A). The increase in apoptosis observed in both sensitive and resistant cells indicates that glutamine deprivation most likely provides an additional cytotoxic cue that induces apoptosis in the sensitive and resistant cells, but could also bypass the resistance mechanism in the resistant cells. Nevertheless, our results suggest that targeting the glutamine metabolic pathway could enhance apoptosis and circumvent chemoresistance to BH3 mimetics in all our resistance models (Figure 2A and Online Supplementary Figure S6). To investigate the therapeutic potential of this approach, we wished to further under- stand how changes in glutamine metabolism might alter BH3 mimetic-mediated apoptosis.
Glutamine is transported into cells primarily via the SLC1A5 transporter and metabolized to glutamate, pri- marily via glutaminase (GLS)-mediated glutaminolysis.22 Alternatively, glutamate can be generated from glutamine as a by-product of the hexosamine biosynthetic pathway, during the conversion of fructose-6-phosphate to glu- cosamine-6-phosphate, catalyzed by the enzyme, gluta- mine:fructose-6-phosphate-amidotransferase (GFAT) (Figure 2B).22 Glutamate can then generate a-ketoglu- tarate (a-KG) either via glutamate dehydrogenase (GLUD)-mediated oxidative deamination or a series of aminotransferase reactions (Figure 2B).14,22 Downregulation by RNA interference or pharmacological inhibition of key players involved in both glutamine uptake and its subsequent metabolism restored sensitivi-
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