TRAF3-Inactivated Chronic Lymphocytic Leukemia Cells Show an Enhanced Metabolic Plasticity That Can be Attenuated By Glutaminolysis and Mitochondrial Pyruvate Import Inhibition

I ntroduction:TRAF3 gene is located in 14q32.32 and has been identified as a tumor suppressor gene involved in B-cell survival, immune response, and cellular metabolism. TRAF3 is frequently encompassed within 14q deletion and affected by inactivating mutations in 4% of chronic lymphocytic leukemia (CLL) patients. Recently, our group found that CLL patients harboring biallelic TRAF3 inactivation (deletion and mutation) showed a dismal prognosis, with a short time to first treatment (Pérez Carretero, AJH 2022). However, the biological role of TRAF3 alterations in CLL pathogenesis remains to be elucidated. Thus, the aims of this study wereto assess the functional consequences of these alterations and to determine their impact on therapy response.

Methods: Previously reported TRAF3 mutations were mimicked in a CRISPR/Cas9-edited CLL model by using the PGA1 cell line (PGA1- TRAF3^mut). Metabolomic (liquid chromatography-mass spectrometry), transcriptomic (RNAseq) and functional analyses were carried out. To evaluate the metabolic specificities of PGA1- TRAF3 ^mut cells, oxidative phosphorylation (OXPHOS) and glycolytic capacity were studied by assessing the mitochondrial oxygen consumption rate (OCR) and extracellular acidification (ECAR), respectively. Additionally, we assessed the metabolite profile of 25 primary CLL cells (n=3 TRAF3^mut cells, n=22 TRAF3^wt cells) and their response to a library of drugs (CLL drugs and metabolic inhibitors), in the presence and absence of microenvironment (with and without HS-5 co-culture and IL-2 and CpG stimulation).

Results: Metabolomic analysis of CRISPR/Cas9-edited CLL models showed an enrichment of metabolites involved in Warburg effect (pyruvate, phosphoenolpyruvate, and lactate), citric acid cycle (TCA- acetyl-CoA, α-ketoglutarate, succinate) and glutamate metabolism (α-ketoglutarate) in PGA1- TRAF3 ^mut cells. Moreover, PGA1- TRAF3 ^mut cells exhibited higher basal and maximal respiration levels and a more important spare capacity and generation of ATP-linked to mitochondrial respiration. Glycolysis was also slightly enhanced in the TRAF3 ^mut cells. Remarkably, primary CLL cells with TRAF3 mutations presented a higher concentration of phosphoenolpyruvate, glutamate and the TCA intermediates fumarate and malate, as well as higher levels of NADH and ATP, proving an increased energy production in these cells. Altogether, these results reveal that TRAF3 ^mut cells showed an enhanced mitochondrial glycolytic metabolism.

Given the metabolic changes identified in TRAF3 ^mut cells, we subsequently assessed their OCR and ECAR after treatment with metabolic inhibitors. Unlike in WT cells, UK5099 administration (inhibitor of the mitochondrial pyruvate transporter that regulates mitochondrial glycolysis) induced an increase in the maximal respiration of TRAF3 ^mut cells, suggesting a metabolic reprogramming towards an alternative metabolic way fueling mitochondrial metabolism. Therefore, we tested the effect of C968 (glutaminolysis inhibitor), finding the same effect on TRAF3^mut cells, which reveals a specific metabolic plasticity (mitochondrial glycolysis/glutaminolysis). Notably, the transcriptomic analysis revealed an upregulation of the glutamine transporter SLC1A4 (p-adj=0.007), what could be favoring glutamine uptake for an enhanced glutaminolysis.

Interestingly, by blocking pyruvate import and glutaminolysis simultaneously, a decrease in TRAF3 ^mut CLL cells proliferation was observed (p=0.03), with equal levels of the previously dysregulated metabolites to those of the WT, which may indicate that this treatment sensitizes TRAF3 ^mut cells through its relieving effect on metabolic reprogramming. Moreover, primary CLL cells with TRAF3 mutations were also sensitive to the combination, in the presence and absence of microenvironment (p=0.01, p=0.04 respectively), demonstrating the potential of the combination of metabolic inhibitors as a new therapeutic approach in TRAF3 ^mut patients.

Conclusion: Our work identifies a novel role of TRAF3 alterations in the CLL cellular metabolism. Specifically, TRAF3 inactivation induced an increased mitochondrial glycolytic activity and distinct metabolic dependencies in CLL cells, contributing to an enhanced metabolic plasticity (mitochondrial glycolysis/glutaminolysis) potentially targetable in CLL.

Funding: PI21/00983 FEHH-GELLC(CPC), JYCL(ARS)