Integrative Gene Expression Profiling Reveals G6PD-Mediated Resistance to RNA-Directed Nucleoside Analogues in B-Cell Neoplasms

<div><p>The nucleoside analogues 8-amino-adenosine and 8-chloro-adenosine have been investigated in the context of B-lineage lymphoid malignancies by our laboratories due to the selective cytotoxicity they exhibit toward multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL) cell lines and primary cells. Encouraging pharmacokinetic and pharmacodynamic properties of 8-chloro-adenosine being documented in an ongoing Phase I trial in CLL provide additional impetus for the study of these promising drugs. In order to foster a deeper understanding of the commonalities between their mechanisms of action and gain insight into specific patient cohorts positioned to achieve maximal benefit from treatment, we devised a novel two-tiered chemoinformatic screen to identify molecular determinants of responsiveness to these compounds. This screen entailed: 1) the elucidation of gene expression patterns highly associated with the anti-tumor activity of 8-chloro-adenosine in the NCI-60 cell line panel, 2) characterization of altered transcript abundances between paired MM and MCL cell lines exhibiting differential susceptibility to 8-amino-adenosine, and 3) integration of the resulting datasets. This approach generated a signature of seven unique genes including <em>G6PD</em> which encodes the rate-determining enzyme of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase. Bioinformatic analysis of primary cell gene expression data demonstrated that G6PD is frequently overexpressed in MM and CLL, highlighting the potential clinical implications of this finding. Utilizing the paired sensitive and resistant MM and MCL cell lines as a model system, we go on to demonstrate through loss-of-function and gain-of-function studies that elevated G6PD expression is necessary to maintain resistance to 8-amino- and 8-chloro-adenosine but insufficient to induce <em>de novo</em> resistance in sensitive cells. Taken together, these results indicate that G6PD activity antagonizes the cytotoxicity of 8-substituted adenosine analogues and suggests that administration of these agents to patients with B-cell malignancies exhibiting normal levels of <em>G6PD</em> expression may be particularly efficacious.</p> </div

Increased G6PD expression is observed in MM and CLL patient samples.

<p>(A) G6PD expression was interrogated in clinical myeloma samples through querying the dataset ‘Zhan Myeloma 3’ for G6PD probe set ‘202275_at’ in the Oncomine™ database. MM samples exhibit a 4.745 fold increase in G6PD expression relative to normal plasma cells (NPC). MGUS: Monoclonal Gammopathy of Undetermined Significance. According to the authors Zhan et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041455#pone.0041455-Zhan1" target="_blank">[31]</a>, the diagnosis of MGUS included the following criteria: levels of monoclonal protein ≤30 g/L, plasma cell bone marrow infiltration <10%, and no incidence of related organ or tissue impairment. (B) G6PD expression was interrogated in clinical CLL samples through querying the dataset ‘Haslinger Leukemia’ for G6PD probe set ‘38043_at’ in the Oncomine™ database. CLL samples exhibit a 2.319 fold increase in G6PD expression relative to normal B lymphocytes (NBL). Data in parts (A) and (B) are log₂ transformed and median-centered. Boxes indicate 95 and 5 percentiles and median expression values. Whiskers represent minimum and maximum expression values. * <i>P</i><.05 ** <i>P</i><.01 *** <i>P</i><.005.</p

Gene expression patterns positively correlated with 8-Cl-Ado resistance.

<p>Gene expression patterns positively correlated with 8-Cl-Ado resistance.</p

Elevated G6PD expression in resistant cell lines translates to greater enzymatic activity.

<p>(A) G6PD transcript abundance was evaluated through real time RT-PCR and normalized to the MM.1S cell line (n = 4). (B) G6PD protein abundance was evaluated through immunoblot analysis. GAPDH serves as a loading control. Representative blot from three independent experiments is shown. (C) Cell extracts were evaluated for 6PGD and G6PD activities via incubation with 6-phosphogluconate (6PG), glucose-6-phosphate (G6P) or both metabolites together. Activity was determined by measurement of NADP⁺ to NADPH conversion rates (n = 5 for MM cell lines, n = 4 for MCL cell lines). (D) G6PD-specific, 6PGD-independent activity was more stringently analyzed through subtraction of 6PG-induced NADPH generation from G6P/6PG-induced NADPH generation from data in part (C). Data in parts (A), (C), and (D) are means ± SEM. * P<.05 ** P<.01 *** P<.005.</p

Transcriptomic microarray datasets independently identify seven mRNAs highly associated with responsiveness to 8-substituted adenosine analogues.

<p>(A) Paired sensitive and resistant MCL (JeKo and Granta 519, respectively) and MM (MM.1S and U266, respectively) cell lines were treated with a range of concentrations of 8-amino-adenosine for 24 hours before flow cytometric quantification of cell viability was performed using both DAPI and AnnexinV-FITC staining. Data are means ± SEM (<i>n</i> = 3). (B) Cell lines from part (A) under basal conditions were subjected to transcriptomic microarray analysis. Expression of genes significantly under- and over-expressed by at least a 1.5 fold change in sensitive cell lines relative to resistant paired cell lines are depicted and fold changes represented in the heatmap. (C) Integration of the list of genes highly expressed in sensitive cell lines in part (B) with genes highly expressed in sensitive constituent cell lines of the NCI-60 panel (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041455#pone-0041455-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041455#pone.0041455.s006" target="_blank">Table S2</a>) reveals overlap of one gene. (D) Integration of the list of genes highly expressed in resistant cell lines in part (B) with genes highly expressed in resistant constituent cell lines of the NCI-60 panel (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041455#pone-0041455-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041455#pone.0041455.s005" target="_blank">Table S1</a>) reveals overlap of six genes.</p

Ectopic expression of G6PD with and without GLUT1 is insufficient to abrogate 8-NH₂-Ado sensitivity.

<p>(A) Sensitive cell lines were transduced with lentivirus harboring an empty vector control (EV) or G6PD cDNA (G6PD) and stable cell lines were generated. G6PD protein expression was analyzed by immunoblotting. A representative blot from three independent experiments is shown. (B) JeKo and (C) MM.1S stable cell lines were treated for 17 hours with the indicated concentrations of 8-NH₂-Ado. Cell death was assessed solely by DAPI staining (n = 3 for JeKo, n = 5 for MM.1S). (D) MM.1S empty vector- and G6PD-expressing stable cell lines were transduced with GFP or GLUT1 cDNAs. G6PD and GLUT1 expression was analyzed by immunoblotting. A representative blot from three independent experiments is shown. (E) Cells from part (D) were treated for 17 hours with the indicated concentrations of 8-NH₂-Ado. Cell death was assessed solely by DAPI staining (n = 3). (F) JeKo and (G) MM.1S stable cell lines were treated for 48 hours with the indicated concentrations of 8-Cl-Ado. In parts (F)–(G), cell death was assessed solely by DAPI staining (n = 5 for JeKo, n = 3 for MM.1S) and data represent the increase in cell death relative to untreated cells (normalized to 0%). Data in parts (B), (C), and (E)–(G) are means ± SEM. Statistically insignificant differences are indicated by n.s. except in part (E) in which all differences between cells receiving identical treatments were not statistically significant. * P<.05 ** P<.01 *** P<.005.</p