Altered metabolism is one of the hallmarks of cell division and of cancer. CLL cells circulate between peripheral blood (PB) and lymph nodes (LN), where they receive proliferative and pro-survival signals from surrounding cells. Yet insight into the metabolism of LN CLL and how this may relate to therapeutic responses is lacking. To obtain insight into CLL LN metabolism, we applied a two-tiered strategy. First, we sampled PB from 8 patients at baseline, and after 3-month ibrutinib (IBR) treatment, which forces egress of CLL cells from LNs. Second, we applied in vitro B-cell receptor (BCR) or CD40 stimulation to mimic the LN microenvironment, and performed metabolomics and transcriptomics. The combined analyses indicated prominent changes in purine, glucose and glutamate metabolism occurring in the LN. CD40 signaling mostly regulated amino acid metabolism, tricarboxylic acid cycle (TCA) and energy production. BCR signaling preferably engaged glucose and glycerol metabolism, and several biosynthesis routes. Pathway analyses demonstrated opposite effects of in vitro stimulation versus IBR treatment. In agreement, the metabolic regulator MYC and its target genes were induced after BCR/CD40 stimulation and suppressed by IBR. Next, 13C-fluxomics performed on CD40/BCR-stimulated cells confirmed a strong contribution of glutamine as fuel for the TCA cycle while glucose was mainly converted into lactate and ribose-5-phosphate. Finally, inhibition of glutamine import with V9302 attenuated CD40/BCR-induced resistance to venetoclax. Altogether, these data provide insight into crucial metabolic changes driven by CLL LN microenvironment. The prominent use of amino acids as fuel for the TCA cycle suggests new therapeutic vulnerabilities.

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