B-cell development is punctuated by developmental stages that rely on precise expression of regulatory genes and gene rearrangements necessary to advance the developing cell. MicroRNAs (miRNAs) function at the post-transcriptional level by targeting mRNA transcripts adding a layer of gene expression modulation previously unappreciated. miRNAs have been demonstrated to be intricately involved in each of the developmental stages of the B-cell. These small non-coding RNAs have been shown to either promote malignant transformation or suppress it. Hence, some miRNAs have been dubbed oncomiRs while others are called tumor suppressors. In samples of B-cell lymphoma dysregulated expression of numerous miRNAs is well documented, but functional roles for many of these remain to be elucidated.
miR-146a is an NF-κB responsive gene that modulates the inflammatory response. Early studies identified IRAK1, and TRAF6 as targets of this miRNA. Since these two proteins are adaptor molecules of the NF-κB pathway they complete a negative feedback loop between miR-146a and NF-κB. Studies on the effects of deficiency of miR-146a demonstrated a role in myeloid, T- and B-cell malignancy as these animals developed all three given enough time. Several malignancies including breast cancer, papillary thyroid carcinoma, prostate cancer, and pancreatic cancer models have demonstrated the functional relevance of this miRNA suppressing proliferation, invasion, and migration. In B-cell malignancies the question remained does miR-146a modulate lymphomagenesis and if so is it through NF-κB. In our study using animals overexpressing c-Myc and deficient for miR-146a we observed reduced survival, increased lymph node involvement, differential involvement of the spleen, and malignancies of a mature B-cell phenotype. High throughput sequencing identified differentially expressed genes enriched for targets of the EGR1 transcription factor. Furthermore ectopic miR-146a expression in B-cell malignancies showed reduced proliferation with a concomitant decrease of EGR1 at the transcriptional as well as the translational level. Finally, EGR1 was identified as a bona fide target of miR-146a.
In response to cellular insults p53 induces various genes involved in cell cycle control, DNA repair, and apoptosis. Initial high throughput sequencing studies identified numerous miRNAs upregulated upon induction of p53. Among the miRNAs upregulated upon p53 induction is miR-34a. miR-34a can modulate genes involved in cell cycle progression, cell growth, and apoptosis. Developmental studies show miR-34a ectopic expression can arrest B-cells at the pro-B stage. Additionally, osteosarcoma and ovarian cancer samples demonstrate reduced expression of miR-34a. Given miR-34a’s role in B cell development and its dysregulation in numerous malignancies we were interested in characterizing its role in B-cell lymphomagenesis. To answer this question we opted to use the well-established Eμ-Myc transgenic mouse line. High-throughput sequencing of c-Myc driven B-cell malignancies sufficient/deficient for miR-34a identified novel targets Csf1r, Jhy and Fgfr1. GSEA analysis of c-Myc driven B-cell malignancies revealed enrichment of miR-34a putative targets. miR-34a ectopic expression in B-cell lymphoma cell lines demonstrated reduced proliferation, as well as novel targets Csf1r, Jhy, and Fgfr1. These findings identify novel targets of miR-34a in the context of B-cell malignancies.
This thesis identifies novel targets for both miR-146a and miR-34a that modulate B-cell oncogenesis demonstrated their roles as tumor suppressors. These studies pave the way for novel therapeutic approaches for c-Myc driven B-cell malignancies.