The Role Of Mir-155 In Controlling Key Growth and Survival Pathways In ABC-Subtype DLBCL

The incidence of diffuse large B-cell lymphoma (DLBCL) is rising, a situation that highlights the need for improved therapy with greater efficacy and fewer side effects. The fundamental clinical problem is that standard frontline combination chemotherapy for DLBCL (R-CHOP) achieves lasting remissions but is not curative. Development of a new, effective, low-toxicity frontline therapy against DLBCL that incorporates targeted agents is much needed. Constitutive activation of the NF-kB, NFAT, and STAT3 (NNS) pathways is a hallmark of DLBCL, particularly the activated B-cell–like (ABC) subtype. Although these pathways have been studied intensively recently, the precise mechanisms controlling these pathways in DLBCL is still uncertain. Elucidating the key mechanism(s) controlling these pathways should improve our understanding of the pathophysiology of DLBCL as well as the therapeutic strategy against this deadly disease. MicroRNAs (miRNA) are short 18-23 nucleotide long noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA. Aberrant expression of miRNAs has been implicated in numerous diseases, including cancers. MiRNA-155 (miR- 155) has shown to be overexpressed in aggressive hematologic malignancies, including DLBCL. The oncogenic nature of this miRNA was confirmed in Em-miR-155 transgenic mice, where the mice develop aggressive pre B cell neoplasms within 3-4 weeks. However, the role miR-155 in the pathophysiology of DLBCL is still unclear. We performed gene expression profiling (GEP) in 24 representative DLBCL cell lines (17 GCB- and 7 ABC-subtype). Our data show that miR-155 expression is significantly higher in ABC vs. GCB DLBCL cell lines (p-value<0.0001). We then analyzed the expression levels of miR-155 in primary DLBCL cells using Oncomine (https://www.oncomine.org), a publicly available cDNA cancer microarray database. Analysis of a representative data set (from Lenz G. et al) indicated that miR-155 levels were 3.12-fold higher in primary ABC-DLBCL (167 cases) than in GCB-DLBCL (183 cases). Since ABC-DLBCL subtype has been shown to have active B-cell receptor (BCR) signaling mediating NNS pathways and previous studies have shown that activation of mir-155 in normal and malignant B cells is through BCR stimulation, we examined whether miR-155 is involved in these pathways in DLBCL. Our results indicated that inhibition of miR-155 by transiently transfecting an anti-miR-155 into ABC-DLBCL cell lines harboring high expression of miR-155, suppresses NNS activation, leading to cell growth inhibition as wells as reducing DLBCL clonogenic activity. Next, we examined whether blocking the BCR pathway, using a novel small molecule Btk inhibitor, ibrutinib, can lead to miR-155 inhibition. Ibrutinib at low drug concentrations can effectively inhibit miR-155 expression, leading to NNS inhibition. To test the efficacy and specificity of ibrutinib in DLBCL, we screened ibrutinib in 24 DLBCL cell lines using proliferation assays. Our data showed that ibrutinib preferentially suppress proliferation of most ABC-DLBCL cell lines, whereas most GCB DLBCL cell lines were not highly responsive with drug concentrations less than 10 uM. Our result indicated that miR-155 plays a critical role in the BCR-mediated NNS growth survival pathways in ABC-DLBCL subtype and represents a potential target for therapy in personalized medicine.