Alterations in expression of specifically BCL-XL and MCL-1 dictate sensitivity of CLL cells to the Bcl-2 specific inhibitor venetoclax (VEN). We and others have shown upregulation of these anti-apoptotic proteins by interaction of CLL cells with CD4+ T helper cells within their lymph node microenvironment (LN-ME) mediated by CD40 signalling. We also reported significant metabolic changes of LN-ME activated CLL cells but whether metabolic alterations can be linked to VEN resistance remains unclear. As VEN is increasingly used in early stages of CLL, better understanding and tools to circumvent VEN resistance are highly needed. We aim to reveal the metabolic adaption of CLL to CD40 signalling in connection with VEN resistance.
After in vitro CD40 signalling stimulation of peripheral blood (PB) CLL cells, mitochondrial mass and glucose uptake were measured by flow cytometry, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured on Seahorse XF Analyser. The result demonstrated that CD40 stimulation enhances both oxidative phosphorylation (OXPHOS) and glycolysis. This was also confirmed by microarray and metabolomics analyses, as genes and metabolites involved in these two metabolic pathways are significantly upregulated by CD40 stimulation. To find out whether these pathways are linked to VEN resistance, PB CLL cells were treated with OXPHOS or glycolysis inhibitors during CD40 stimulation. Remarkably, OXPHOS inhibition by electron transport chain (ETC) inhibitors (rotenone, antimycin A and oligomycin) counteracted strongly for VEN resistance, while glycolysis inhibition by 2-Deoxy-D-glucose (2DG) did not. The three ETC inhibitors also attenuated CLL activation, ATP production and NAD levels. Interestingly, complex II inhibition of the ETC (TTFA and DMM) did not affect VEN resistance. Regarding BCL-2 family members induced by CD40 ligation, both MCL-1 and BCL-XL were downregulated by these ETC inhibitors. In addition, OXPHOS inhibition strongly elevates glycolysis, and vice versa, which illustrates a strong metabolic plasticity of CLL cells.
To further investigate the cross-talk between CD40 signalling, VEN resistance and mitochondrial metabolism, the three main fuels of the TCA cycle were inhibited: pyruvate (by UK5099), glutamine (by DON) and fatty acids (by etomoxir). Even though the OCR and ECAR were slightly decreased by (combinations of) these fuel inhibitors, neither CD40 signalling nor VEN sensitivity was affected. Next, we inhibited PI3K by idelalisib, BTK by ibrutinib and mTOR by rapamycin, which are three downstream targets of CD40 signalling. The results showed that only rapamycin inhibited CD40 activation and metabolic activities, and none of the three inhibitors counteracts VEN resistance. Lastly, we investigated CD40 splicing and overall expression. Interestingly, CD40 stimulation has a huge impact on CD40 expression itself, and these changes were blocked by ETC inhibition. These data indicate that ETC inhibition affects CD40 signals to counteract VEN resistance, by directly affecting the expression of CD40 protein on the cell membrane.
In conclusion, after CD40 stimulation, CLL cells become metabolically activated and highly flexible in the use of mitochondrial fuels. The enhanced OXPHOS but not glycolysis contributes to VEN resistance, while ETC inhibition reverses CLL VEN resistance by directly suppressing CD40 expression on CLL. These findings link CLL metabolism directly to CD40 transcription and signalling, which may contribute to clinical VEN resistance.
Disclosures
van der Windt: genmab: Current Employment. Kater:Abbvie: Research Funding; Roche: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Genentech: Research Funding. Eldering:Genentech: Research Funding; Celgene: Research Funding; Janssen: Research Funding.
Mitochondrial heteroplasmy is responsible for Atovaquone drug resistance in Plasmodium falciparum