Effects of Ibrutinib on Metabolic Alterations and Micro-Environmental Signalling in Chronic Lymphocytic Leukaemia

Introduction. Altered metabolism is one of the hallmarks of cancer. CLL cells circulate between peripheral blood (PB) and lymph nodes (LN) which necessitates high metabolic plasticity. In LN, CLL cells receive proliferative and pro-survival signals from surrounding cells, and become metabolically activated. However, detailed insight into the altered metabolism of LN CLL and how this may be related to therapeutic responses is lacking. As it is technically difficult to obtain direct insight into CLL LN metabolism, we have applied a two-tiered strategy. By using PB samples taken from patients before/after treatment with the Bruton's tyrosine kinase (BTK) 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, as well as the effects of IBR treatment. We hypothesized that the overlapping/distinct metabolites affected by IBR and in vitro stimulations would reflect the actual CLL metabolism in LN.

Methods. PB samples were obtained from 7 CLL patients before or after 3 months of ibrutinib treatment. These paired samples were in vitro stimulated via CD40 and B cell receptor (BCR), which are potential key signals within the tumour microenvironment (TME). Seahorse extracellular flux (ECF) analyses, expression of activation markers (CD95, pS6 by FACS), RNA was isolated for expression of Myc (major driver of metabolic reprogramming) and its target genes, and metabolomics by mass-spec was performed.

Results. ECF analyses showed that in comparison to BCR stimulated PB CLL cells, stimulation by CD40 resulted in a high increase of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). A prominent effect on OXPHOS and glycolytic activity was confirmed in direct LN samples, and indirectly by marker analyses in LN emigrants using CXCR4/CD5 staining [1]. Subsequent metabolomics analyses showed that 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 (KEGG) by both CD40 and BCR triggering: purine metabolism, Warburg effect, lysine degradation, glucose-alanine cycle and glutamate metabolism. In contrast, the following pathways indicated the two signals had distinct functions on regulating metabolism: CD40 signalling mostly regulates amino acid metabolism, tricarboxylic acid cycle (TCA) and mitochondrial metabolism related to oxidative phosphorylation (OXPHOS) and energy production. BCR signalling mainly involves glucose and glycerol metabolism, which are usually related to biosynthesis.

CLL cells from IBR-treated patients showed enhanced BCR responsiveness, in line with the increased in surface IgM expression upon IBR [2]. In contrast, IBR treatment suppressed in vitro CD40 activation, which was accompanied by a lower CD40 expression. Metabolomics analyses also demonstrated that CD40 responses decreased but BCR response increased after IBR. Additionally, analyses of Myc and its target genes showed that they are induced after BCR as well as CD40 stimulation. Effects of IBR on Myc (target) expression were variable for BCR and reduced for CD40 stimulation.

Conclusions. 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.

References

1. Calissano, Carlo, et al. "Intraclonal complexity in chronic lymphocytic leukemia: fractions enriched in recently born/divided and older/quiescent cells." Molecular Medicine 17.11 (2011): 1374-1382.

2. Drennan, Samantha, et al. "Ibrutinib therapy releases leukemic surface IgM from antigen drive in chronic lymphocytic leukemia patients." Clinical Cancer Research 25.8 (2019): 2503-2512.