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

<div><p>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.</p></div

Mino/FR cell are highly sensitive to ABT-199.

<p>Proliferation of Mino and Mino/FR cells in presence of of 0.01–10 μM Bcl-2 inhibitor ABT199. Cells were grown for 6–8 days in presence ABT199. Relative toxicity of the drugs was determined by the WST-8 cell proliferation assay. Dashed curves and open circles or triangles indicate cell proliferation in absence of ABT199. Maximum absorbance (highest number of viable cells) of cells grown without ABT199 experiment was set as 100%. Other curves represent the cells grown in increasing concentrations (indicated by the associated number) of ABT199. Standard deviations were < 5% for all measurements.</p

Differentially expressed proteins in Mino/FR cells identified by the proteomic analysis.

<p>Differentially expressed proteins with fold-change >1.5 are listed. Gene names are shown, proteins with fold-change >3 fold are present in bold letters).</p

Proliferation of Mino and Mino/FR cells in presence of fludarabine and other anti-lymphoma agents.

<p>Cells were grown for 6–8 days in presence of increasing concentrations of <b>(A)</b> fludarabine, <b>(B)</b> cladribine, <b>(C)</b> cytarabine, <b>(D)</b> gemcitabine, <b>(E)</b> ibrutinib, <b>(F)</b> bortezomib, <b>(G)</b> doxorubicin, <b>(H)</b> cisplatin, <b>(I)</b> bendamustine and <b>(J)</b> methotrexate. Relative toxicity of the drugs was determined by the WST-8 cell proliferation assay, Dashed lines with open circles or triangles indicate cell proliferation in absence of an anti-lymphoma drug. Other curves represent the cells grown in increasing concentrations (indicated by the associated number) of the tested drug. Maximal absorbance (highest number of viable cells) of cells grown without an anti-lymphoma agent in each experiment was set as 100%. Standard deviations were < 5% for all measurements.</p

Correlation of protein expression ratios in forward and reverse SILAC experiments.

<p>Heavy/Light protein ratios (log values) from the forward experiment were plotted against log values of Light/Heavy protein ratios obtained from the reverse labeling experiment.</p

Verification of differential expression of the key proteins identified by proteomics.

<p><b>(A)</b> Relative expression of four differentially expressed proteins—deoxycytidine kinase (dCK), phoshatase SHP-1 (alias PTPN6), Bcl-2 and phoshoserine aminotransferase (PSAT-1)–was determined by Western Blotting using specific antibodies in Mino and Mino/FR cells. GAPDH was used as a loading control. <b>(B)</b> Relative expression of two surface CD markers (CD20 and CD38) determined by flow cytometry using specific antibodies. Open histograms represent Mino/FR cells, full histograms show Mino cells. Histograms demonstrate approximately 2-fold decreased expression of CD20 and CD38 in Mino/FR cells as indicated by decreased median fluorescence intensity.</p

Decreased levels of total BTK and its activated p-BTKY²³³ form in Mino/FR cells.

<p>Relative expression of total and phosphorylated (active) p-BTK233 was determined by Western blotting using specific antibodies in Mino and Mino/FR cells. GAPDH was used as the loading control.</p

Dimethyl fumarate induces ferroptosis and impairs NF-κB/STAT3 signaling in DLBCL

Despite the development of novel targeted drugs, the molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) still poses a major therapeutic challenge. DLBCL can be classified into at least two major subtypes, i.e. germinal center B-cell-like (GCB) and the aggressive activated B-cell-like (ABC) DLBCL, each characterized by specific gene expression profiles and mutation patterns. Here we demonstrate a broad anti-tumor effect of dimethyl fumarate (DMF) on both DLBCL subtypes, which is mediated by the induction of ferroptosis, a form of cell death driven by the peroxidation of phospholipids. Due to high expression of arachidonate 5-lipoxygenase in concert with low glutathione and glutathione peroxidase 4 levels, DMF induces lipid peroxidation and thus ferroptosis particularly in GCB DLBCL. In ABC DLBCL cells, which are addicted to NF-κB and STAT3 survival signaling, DMF treatment efficiently inhibits the activity of the IKK complex and JAK kinases. Interestingly, the BCL-2 specific BH3 mimetic ABT-199 and an inhibitor of ferroptosis suppressor protein 1 synergize with DMF in inducing cell death in DLBCL. Collectively, our findings identify the clinically approved drug DMF as a promising novel therapeutic option in the treatment of both GCB and ABC DLBCL

Schematic illustration of the processes associated with fludarabine resistance in Mino/FR cells summarizes the landmark observations in our analyses.

<p>Schematic illustration of the processes associated with fludarabine resistance in Mino/FR cells summarizes the landmark observations in our analyses.</p