Effects of Ibrutinib on Metabolic Alterations and Micro-environmental Signalling in Chronic Lymphocytic Leukaemia
CLL cells circulate between peripheral blood (PB) and lymph nodes (LN) which necessitates high metabolic plasticity. In LN, CLL cells receive signals from surrounding cells, and become metabolically activated. However, insight into the altered metabolism of LN CLL and how it relates to therapeutic responses is lacking. As it is technically difficult to obtain direct insight into CLL LN metabolism, we applied a two-tiered strategy. By using PB samples from patients before/after treatment with the Bruton's tyrosine kinase inhibitor ibrutinib (IBR), which drives CLL cells out of the LN, combined with in vitro re-stimulation of TME signals, we indirectly mapped the metabolism of CLL in their TME, also the effects of IBR treatment.
PB samples from 7 CLL patients before or after 3 months of ibrutinib treatment were in vitro stimulated via CD40 and B cell receptor (BCR). Seahorse extracellular flux analyses, expression of activation markers (CD95, pS6 by FACS) and metabolomics by mass-spec were performed; RNA was isolated for expression of Myc and its target genes.
CLL cells stimulated by CD40 showed a higher increase of oxygen consumption rate and extracellular acidification rate than by BCR, and a prominent effect on OXPHOS and glycolytic activity was confirmed in direct LN samples. Subsequent metabolomics analyses showed metabolic reprogramming following CD40 or BCR stimulation revealed both shared and distinct responses. The affected metabolic pathways, predicted by significantly changed metabolites, were compared in a pairwise fashion; upregulated by CD40 and BCR but downregulated by IBR, respectively. The results demonstrated 5 upregulated pre-defined pathways by both CD40 and BCR triggering: purine metabolism, Warburg effect, lysine degradation, glucose-alanine cycle and glutamate metabolism. Contrastly, the following pathways indicated the two signals had distinct functions on regulating metabolism: CD40 signalling mostly regulates pathways related to OXPHOS and energy production, while BCR signalling mainly involves glucose metabolism, usually contributes to biosynthesis. Metabolomics analyses also demonstrated that CD40 responses decreased but BCR response increased after IBR. Additionally, Myc and its target genes were induced after BCR and CD40 stimulation. Effects of IBR on Myc (target) expression were variable for BCR and reduced for CD40 stimulation.
In vivo IBR treatment suppresses CD40 expression and activation and enhances BCR responsiveness. Metabolic changes of CLL in LN are recapitulated by these two signals, while IBR treatment shows opposite effects, together providing indirect insight into the LN metabolism. In LN, CD40 may play a prominent role to enhance most of the key metabolic pathways, particularly OXPHOS. This is the first study to describe the metabolic network of CLL cells in LN, and the long-term effects of IBR may yield new clues to therapy response and resistance.