Ultrahigh-Resolution NMR with Water Signal Suppression for a Deeper Understanding of the Action of Antimetabolic Drugs on Diffuse Large B-Cell Lymphoma.

Ultrahigh-resolution NMR has recently attracted considerable attention in the field of complex samples analysis. Indeed, the implementation of broadband homonuclear decoupling techniques has allowed us to greatly simplify crowded 1H spectra, yielding singlets for almost every proton site from the analyzed molecules. Pure shift methods have notably shown to be particularly suitable for deciphering mixtures of metabolites in biological samples. Here, we have successfully implemented a new pure shift pulse sequence based on the PSYCHE method, which incorporates a block for solvent suppression that is suitable for metabolomics analysis. The resulting experiment allows us to record ultrahigh-resolution 1D NOESY 1H spectra of biofluids with suppression of the water signal, which is a crucial step for highlighting metabolite mixtures in an aqueous phase. We have successfully recorded pure shift spectra on extracellular media of diffuse large B-cell lymphoma (DLBCL) cells. Despite a lower sensitivity, the resolution of pure shift data was found to be better than that of the standard approach, which provides a more detailed vision of the exo-metabolome. The statistical analyses carried out on the resulting metabolic profiles allow us to successfully highlight several metabolic pathways affected by these drugs. Notably, we show that Kidrolase plays a major role in the metabolic pathways of this DLBCL cell line.

The Alternative RelB NF-kB Subunit Exerts a Critical Survival Function upon Metabolic Stress in Diffuse Large B-Cell Lymphoma-Derived Cells.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma in adults and reveals distinct genetic and metabolic signatures. NF-κB transcription factor family is involved in diverse biological processes enabling tumor development and resistance to anticancer-therapy through activation of its two main pathways, the canonical and the alternative NF-κB pathways, the main actor of the latter being the RelB NF-kB subunit. RelB DNA binding activity is frequently activated in DLBCL patients and cell lines. RelB activation defines a new DLBCL subgroup with dismal outcome upon immunochemotherapy, and RelB confers DLBCL cell resistance to DNA damage. However, whether RelB can impact on DLBCL cell metabolism and survival upon metabolic stress is unknown. Here, we reveal that RelB controls DLBCL oxidative energetic metabolism. Accordingly, RelB inhibition reduce DLBCL mitochondrial ATP production, and sensitizes DLBCL cells to apoptosis induced by Metformin and L-asparaginase (®Kidrolase), two FDA approved antimetabolic drugs targeting mitochondrial metabolism. RelB also confers DLBCL cell resistance to glutamine deprivation, an essential amino acid that feeds the TCA cycle. Taken together, our findings uncover a new role for RelB in the regulation of DLBCL cell metabolism and DLBCL cell survival upon metabolic stress.

The alternative RelB NF-κB subunit is a novel critical player in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid malignancy affecting adults. The NF-κB transcription factor family is activated by 2 main pathways, the canonical and the alternative NF-κB activation pathway, with different functions. The alternative NF-κB pathway leads to activation of the transcriptionally active RelB NF-κB subunit. Alternative NF-κB activation status and its role in DLBCL pathogenesis remain undefined. Here, we reveal a frequent activation of RelB in a large cohort of DLBCL patients and cell lines, independently of their activated B-cell-like or germinal center B-cell-like subtype. RelB activity defines a new subset of patients with DLBCL and a peculiar gene expression profile and mutational pattern. Importantly, RelB activation does not correlate with the MCD genetic subtype, enriched for activated B-cell-like tumors carrying MYD88L265P and CD79B mutations that cooperatively activate canonical NF-κB, thus indicating that current genetic tools to evaluate NF-κB activity in DLBCL do not provide information on the alternative NF-κB activation. Furthermore, the newly defined RelB-positive subgroup of patients with DLBCL exhibits a dismal outcome after immunochemotherapy. Functional studies revealed that RelB confers DLBCL cell resistance to DNA damage-induced apoptosis in response to doxorubicin, a genotoxic agent used in the front-line treatment of DLBCL. We also show that RelB positivity is associated with high expression of cellular inhibitor of apoptosis protein 2 (cIAP2). Altogether, RelB activation can be used to refine the prognostic stratification of DLBCL and may contribute to subvert the therapeutic DNA damage response in a segment of patients with DLBCL.