LAUSR

Chronic lymphocytic leukemia (CLL) has focused a strong research effort over the last years due to its high incidence, that will likely increase due to the progressive ageing of the population, and the lack of truly curative therapies. Genome-wide screenings in CLL patients revealed a group of mutations in genes covering divergent cellular pathways [1]. Unfortunately, the mutations found did not affect to more than 13% of the patients at its best. New genome-wide studies widen the search of mutations to cover untranslated and regulatory regions of the genome [2]. At the sight of all of these absolutely necessary but enormously dispersed genomic and gene expression data, some researchers have started to point to post-translational mechanisms as the unifying defect explaining the common cellular characteristics of CLL. Noteworthy, several preclinical studies have tested the use of inhibitors of some of these post-translational modifications. This is the case of MLN4924-pevonedistat, an investigational inhibitor of protein NEDDylation that is being tested in clinical trials for the treatment of different neoplasias (https://clinicaltrials. gov/ct2/results?term=MLN4924&Search=Search). We wanted to investigate the real extension of the NEDDylation-related alterations in B-CLL cells. Our hypothesis is that a transversal process like this posttranslational modification may be able to provide a unifying explanation to the wide array of alterations related to the development of CLL, thus providing molecular support to the use of NEDDylation inhibitors in the treatment of this disease. Current whole-cell profiling techniques do not allow the specific identification of NEDDylated proteins, so we used a method of immunoaffinity enrichment of proteins containing a K-GG remnant left by ubiquitin or ubiquitin-like (UBL) peptides after digestion with trypsin. The peptides thus purified were then identified by mass spectrometry. The application of this method over samples of B-CLL cells and CD19+ cells obtained from healthy donors allowed us to have a wide view of the differentially ubiquitin or UBLmodified proteins in this pathology. However, the diGly remnant left on ubiquitinated peptides after tryptic digestion is shared by ubiquitin, NEDD8 and ISG15 modifications. In order to discriminate those peptides originally conjugated to NEDD8 from those modified by ubiquitination or other UBLs, we analyzed the same B-CLL samples but treated with the NEDDylation inhibitor MLN4924. The procedure identified a total of 3384 differentially modified peptides in CLL corresponding to 1746 proteins. 651 site modifications were unreported at the date of the screening. Of those K-GG remnant modifications, 2142 (63%) corresponded to a ≥2.5-fold increase in B-CLL cells with respect to the healthy controls, while only 401 (12%) were reduced ≥2.5-fold. Of those 2142 augmented modifications, 353 were notably reverted by the incubation with MLN4924 (threshold 2.5-fold) (Fig. 1). Noteworthy, 76% of these sites were not detected by the UbiSite antibody in the work by Akimov et al. [3]. Overall, MLN4924 affected the amount of the K-GG remnant in 1666 sites of 1008 proteins (threshold 2.5-fold); post-translational modifications induced by MLN4924 were found in 609 proteins, while modifications impaired by the inhibitor were found in 399 proteins. Interestingly, 37 proteins displayed modifications of both types. Although these data suggest an increment of NEDDylated proteins in CLL, we found no differences in the * Carlos Pipaón [email protected]