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A. Al-Zebeeby et al.
bafilomycin A1 failed to revert torin-1-mediated chemosensitization, suggesting that this effect could be independent of autophagy (Figure 5B). Furthermore, genetic silencing of autophagy proteins, ATG5 and ATG7, which are critical for the induction of autophagy, also failed to revert torin-1-mediated sensitization (Figure 5C), confirming our finding that mTOR inhibi- tion circumvented resistance and enhanced sensitivity to BH3 mimetics independently of autophagy. In summary, our findings demonstrate that modulation of glutamine metabolism and its downstream signaling pathways, namely reductive carboxylation, lipogenesis and choles- terogenesis, as well as inhibition of mTOR signaling could enhance the therapeutic efficacy of BH3 mimetic therapy thereby circumventing chemoresistance to BH3 mimetics (Figure 5D).
Targeting intermediary metabolism enhances sensitivity to navitoclax in primary samples
from patients with chronic lymphocytic leukemia
Our results indicate that targeting various facets of intermediary metabolism enhanced sensitivity to differ- ent BH3 mimetics in cell lines derived from relevant hematologic malignancies. To further extend our observa-
AB
tions in cell lines to primary samples from patients, we used CLL cells isolated from patients during the lead-in period (L1D1) as well as cells from the same patients after five cycles of navitoclax therapy (C5D1), as previously detailed in Figure 1. Using these samples, we wanted to determine whether modulating glutamine metabolism would enhance apoptosis mediated by navitoclax. For this, we exposed CLL cells to CB-839 and simvastatin for 24 h followed by navitoclax for 4 h and assessed the extent of apoptosis. In agreement with our cell line data, both CB-839 and statins overcame the resistance to navi- toclax-mediated apoptosis in primary CLL cells (Figure 6), supporting the therapeutic translatability of our data from cell lines to patients.
Discussion
Anti-apoptotic BCL-2 family members are attractive drug targets both because of their high expression levels in several cancers and because of their well-characterized pro-survival roles. Even with extensive supportive in vitro data, the use of BH3 mimetics in treating cancer patients is still in its infancy, with venetoclax, a BCL-2-specific
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Figure 4. Inhibition of lipogenesis and cholesterogenesis enhances sensitivity to BH3 mimetics. (A) Scheme representing reductive carboxylation, lipogenesis and cholesterogenesis. (B) Apoptotic sensitivity of K562 resistant [E] cells exposed to A-1331852 (10 nM) for 4 h was restored following genetic knockdown for 72 h of key enzymes in fatty acid synthesis. Western blots confirmed the knockdown efficiency of the different short interfering (si) RNA. (C) Apoptotic sensitivity of K562 resistant [E] cells exposed to A-1331852 (10 nM) for 4 h was restored following pharmacological inhibition of key enzymes in fatty acid synthesis using SB204990 (1 mM) for 72 h or GSK2194069 (100 nM) for 48 h. (D) Metabolic supplementation of K562 sensitive [A] and resistant [E] cells with palmitate (50 mM) for 48 h prior to the exposure of cells to GSK2194069 (100 nM) overcame the sensitizing effect of GSK2194069 on A-1331852-mediated apoptosis. (E) Genetic knockdown for 72 h of HMGR or (F) pharmacological inhibition of HMGR by simvastatin (250 nM) for 72 h, atorvastatin (10 mM) for 48 h or pitavastatin (1 mM) for 72 h. ***P⩽0.001; **P⩽0.01. Error bars = mean ± standard error of mean (n=3). PS: phosphatidylserine; DMSO: dimethylsulfoxide.
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haematologica | 2019; 104(5)