Ezrin Is a Potential Druggable Target in Chronic Lymphocytic Leukemia

Despite development in therapeutic strategies for chronic lymphocytic leukemia (CLL), most patients remain incurable, relapse, or are refractory to current treatments, indicating the need to expand the antineoplastic repertoire for this disease. Ezrin (EZR) is a known oncogene in solid tumors, but its role in hematological neoplasms is still poorly explored. There is evidence that EZR plays a key function in cell survival and activation of BCR-mediated signaling in B-cell lymphomas. Here, we uncover the expression of EZR in samples from CLL and healthy donors, and the cellular and molecular effects of the NSC305787, a pharmacological EZR inhibitor, in CLL cellular models. For gene expression studies, EZR mRNA expression data from healthy donors (normal lymphocytes, n = 11) and CLL patients (n = 103) samples were derived from the publicly accessible AmaZonia! database 2008 (discovery cohort). Purified CD19⁺ cells from peripheral blood samples were collected from 56 CLL patients and 10 age-matched healthy donors from the University Hospital of the Medical School of Ribeirão Preto (validation cohort). For functional and molecular assays, peripheral blood mononuclear cells of 24 CLL patients from the Instituto do Câncer do Estado de São Paulo were used. All samples were obtained after informed consent and protocol approved by the Ethical Committee of the Institution. For functional transcriptomics, RNA-seq data published by Landau et al. were obtained from cBioPortal, and all transcripts were pre-ranked according to their differential expression by comparing samples with high versus low EZR expression using limma-voom package in Galaxy and used for gene set enrichment analysis (GSEA). The MEC-1 cell line was cultured following the recommendations of DSMZ. PBMCs from CLL patients were cultured in RPMI Media 1640 and 10% autologous serum. NSC305787 was used as an EZR inhibitor. Cell viability was determined by MTT assay, apoptosis rates were determined by annexin V/PI labeling and flow cytometry, DNA content was performed by PI labeling and flow cytometry, protein expression was determined by Western Blot assay, and gene expression was evaluated by quantitative PCR. Statistical analyses were performed using GraphPad Prism 8; Mann-Whitney test, Student's t-test, or ANOVA and Bonferroni post-test were used for measurable factors, as appropriate. A value of p < 0.05 was considered statistically significant. EZR was highly expressed in lymphocytes derived from CLL patients (discovery cohort: p = 0.003; validation cohort: p = 0.005). GSEA indicated that high EZR expression was positively associated with relevant signaling pathways associated with CLL development and progression, including p53, PI3K/AKT/mTOR, NFkB, and MAPK (all p < 0.05 and FDR q < 0.05). Of note, pharmacological inhibition of EZR had remarkable antineoplastic effects in primary cells derived from CLL patients, corroborating the potential relevance of this target in disease maintenance. In a CLL cell line, EZR inhibition reduced viability, clonogenicity, cell cycle progression, and induced apoptosis (all p < 0.05). Pharmacological EZR inhibition also reduced activation of the ERK, S6RP, and NFkB, indicating that EZR not only associates, but participates in the activation of these signaling pathways in CLL. In conclusion, our results indicate that EZR is high expressed in CLL and associated with molecular signatures relevant to the development and maintenance of the disease. Pharmacological EZR inhibitions attenuate various cellular and molecular behaviors associated with high risk in CLL and may present a new class of drugs to expand the treatment repertoire of this disease. Supported by: FAPESP, CNPQ, and CAPES.

No relevant conflicts of interest to declare.