miR-125b and miR-532-3p predict the efficiency of rituximab-mediated lymphodepletion in chronic lymphocytic leukemia patients. A French Innovative Leukemia Organization study

Anne-Laure Gagez, Isabelle Duroux-Richard, Stéphane Leprêtre, Frédérique Orsini-Piocelle, Rémi Letestu, Sophie De Guibert, Edouard Tuaillon, Véronique Leblond, Olfa Khalifa, Valérie Gouilleux-Gruart, Anne Banos, Olivier Tournilhac, Jehan Dupuis, Christian Jorgensen, Guillaume Cartron, Florence Apparailly, Anne-Laure Gagez, Isabelle Duroux-Richard, Stéphane Leprêtre, Frédérique Orsini-Piocelle, Rémi Letestu, Sophie De Guibert, Edouard Tuaillon, Véronique Leblond, Olfa Khalifa, Valérie Gouilleux-Gruart, Anne Banos, Olivier Tournilhac, Jehan Dupuis, Christian Jorgensen, Guillaume Cartron, Florence Apparailly

Abstract

The underlying in vivo mechanisms of rituximab action remain incompletely understood in chronic lymphocytic leukemia. Recent data suggest that circulating micro-ribonucleic acids correlate with chronic lymphocytic leukemia progression and response to rituximab. Our study aimed at identifying circulating micro-ribonucleic acids that predict response to rituximab monotherapy in chronic lymphocytic leukemia patients. Using a hierarchical clustering of micro-ribonucleic acid expression profiles discriminating 10 untreated patients with low or high lymphocyte counts, we found 26 micro-ribonucleic acids significantly deregulated. Using individual real-time reverse transcription polymerase chain reaction, the expression levels of micro-ribonucleic acids representative of these two clusters were further validated in a larger cohort (n=61). MiR-125b and miR-532-3p were inversely correlated with rituximab-induced lymphodepletion (P=0.020 and P=0.001, respectively) and with the CD20 expression on CD19+ cells (P=0.0007 and P<0.0001, respectively). In silico analyses of genes putatively targeted by both micro-ribonucleic acids revealed a central role of the interleukin-10 pathway and CD20 (MS4A1) family members. Interestingly, both micro-ribonucleic acids were negatively correlated with MS4A1 expression, while they were positively correlated with MS4A3 and MSA47 Our results identify novel circulating predictive biomarkers for rituximab-mediated lymphodepletion efficacy in chronic lymphocytic leukemia, and suggest a novel molecular mechanism responsible for the rituximab mode of action that bridges miR-125b and miR-532-3p and CD20 family members. (clinicaltrials.gov Identifier: 01370772).

Trial registration: ClinicalTrials.gov NCT01370772.

Copyright© Ferrata Storti Foundation.

Figures

Figure 1.
Figure 1.
MicroRNAs expression profile discriminates CLL patients with low or high lymphocyte counts before treatment. (A) The profiles of 26 microRNAs significantly differently expressed (P<0.05) between high and low lymphocyte concentration samples isolated from CLL patients (n=5/group) were visualized using a supervised heatmap (average linkage and Pearson’s correlation). Relative miRNA expression was calculated using the comparative threshold cycle (Ct) method. For normalization, the mean Ct value of all miRNA targets was used. Dendrograms indicated the correlation between groups of samples and miRNAs. Samples are in columns and miRNAs in rows. Each column represents an individual sample and each row represents a single miRNA. The heat map shows relative levels of miRNA expression in a green (low relative expression) to red (high relative expression) scale. (B–C) Expression levels of 4 miRNAs representative of each cluster, miR-193 band miR-125b for cluster 1 (B), and miR-532-3p and miR-652 for cluster 2 (C), were measured for 123 CLL patients included in the CLL2010FMP protocol, using RT-qPCR. A significant inverse correlation was observed depending on the lymphocyte counts for miR-193b r=−0.19), miR-125b (r=−0.39), miR-652 (r=−0.30) and miR-532-3p (r=−0.34) (Spearman’s correlation test).
Figure 2.
Figure 2.
Efficacy of lymphodepletion after rituximab treatment inversely correlates with miR-125b and miR-532-3p expression levels. miR-125b (r=−0.42) (A) and miR-532-3p (r=−0.49) (B) expression levels were quantified by RT-qPCR. The lymphodepletion was measured 22 days after 4 doses of rituximab infusion and correlated with miRNAs (Spearman’s correlation test) (n=61).
Figure 3.
Figure 3.
Target gene prediction for miR-125b and miR-532-3p. (A) To identify putative miR-125b and miR-532-3p target genes, we used miRWalk software. VENNY, an interactive tool for comparing lists with Venn Diagrams, was used to predict common genes between human regulatory B cells IL-10+ and IL-10−, and putative miR-125b and miR-532-3p target genes. 26 genes putatively targeted by miR-125b and miR-532-3p, and specifically dysregulated in IL-10+ B cells transcriptome analysis are listed. (B) The layout of these 26 putative targets was built in the context of rituximab treatment (MS4A1 (CD20 gene)) using Ingenuity Pathway Analysis (IPA). 9 genes revealed a central role for the IL-10 pathway.
Figure 4.
Figure 4.
Inverse correlation between miR-125b and miR-532-3p expression levels and circulating CD20 surface antigen expression on CD19+ cells in CLL patients blood. CD20 expression levels on CD19+ lymphocytes were quantified using flow cytometry. PBMCs were collected and miR-125b (r=−0.37) (A) and miR-532-3p (r=−0.29) (B) were quantified using RT-qPCR. Circulating CD20 antigen was correlated to miRNA expression (Spearman’s correlation test) (n=61).
Figure 5.
Figure 5.
miR-125b and miR-532-3p expression levels correlate with the expression of three MS4A family members. (A) Spearman’s correlation between miR-125b or miR-532-3p and MS4A mRNA family expression levels in CLL patients blood (n=61). (B) Putative miR-125b and miR-532-3p target binding sites on MS4A family members. CDS: coding DNA sequence; rho: correlation coefficient; UTR: untranslated region.
Figure 6.
Figure 6.
Schematic representation of the potential mechanisms of action of miR-125b and miR-532-3p on the modulation of rituximab activity in CLL cells. In CLL patients displaying low expression levels of miR-125b and miR-532-3p, the MS4A1 mRNA and CD20 surface receptor are upregulated, while the expression of MS4A3 and MS4A7 mRNA are downregulated (MS4A3 and MS4A7 protein expressions were unknown). Consequently, rituximab (RTX) efficacy is optimum for lymphodepletion (Lymphodepletion +++). In contrast, CLL patients with high expression levels of miR-125b and miR-532-3p display low levels of MS4A1 mRNA and CD20 surface receptor, and high levels of MS4A3 and MS4A7 mRNA (MS4A3 and MS4A7 protein expressions were unknown), which might form hetero-oligomers with CD20 and impede optimal lymphodepletion (Lymphodepletion +) by rituximab. Interrogation marks indicate data that were not experimentally confirmed in the present study.

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