Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.     

Biotransformation of furfural and 5-hydroxymethyl furfural by enteric bacteria

Summary A survey was conducted with seventeen enteric bacterial strains (including the generaKlebsiella, Enterobacter, Escherichia, Citrobacter, Edwardsiella andProteus) to examine their ability to transform furfural and 5-hydroxymethyl furfural (5-MHF). The enteric bacteria were able to convert furfural to furfuryl alcohol under both aerobic and anaerobic conditions in a relatively short incubation time of 8 h. 5-HMF was transformed by all the enteric bacteria studied to an unidentified compound postulated to be 5-hydroxymethyl furfuryl alcohol, which had an absorbance maximum of 222 nm. These bacteria did not transform furfuryl alcohol or 2-furoic acid. The enteric bacteria did not use furfural, 5-HMF, furfuryl alcohol or 2-furoic acid as sole source of carbon and energy. Biotransformation of furfural and 5-HMF was accomplished by co-metabolism in the presence of glucose and peptone as main substrates. The rate of transformation was similar under both aerobic and anaerobic conditions. These transformations are likely to be of value in the detoxification of furfurals, and in their ultimate conversion to methane and CO₂ by anaerobic digestion.     

A highly multiplexed quantitative phosphosite assay for biology and preclinical studies

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho‐signaling in drug‐treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.