Suppression of RISC-Independent Decoy and RISC-Mediated mRNA Base-Pairing Activities of MicroRNA-328 Is Required for Differentiation-Arrest and Enhanced Survival of Blast Crisis CML Progenitors.
Authors: Eiring Anna M , Harb Jason , Neviani Paolo , Oaks Joshua , Liu Shujun , Spizzo Riccardo , Schwind Sebastian , Santhanam Ramasamy , Hickey Christopher , Becker Heiko , Chandler Jason C. , Andino Raul , Cortes Jorge , Hokland Peter , Huettner Claudia , Bhatia Ravi , Roy Denis-Claude , Liebhaber Stephen , Caligiuri Michael A. , Marcucci Guido , Garzon Ramiro , Croce Carlo M. , Calin George A , Perrotti Danilo 
Published: October 2019

Abstract
Abstract 855
MicroRNAs (miRs) and heterogeneous ribonucleoproteins (hnRNPs) are post-transcriptional gene regulators that bind mRNA in a sequence-specific manner. We have reported that a) hnRNP-E2 suppresses CEBPA mRNA translation and inhibits myeloid maturation of bone marrow (BM) progenitors from chronic myelogenous leukemia patients in myeloid blast crisis (CML-BCCD34+; Perrotti et al, Nat Genet 2002); and b) miR-328 expression is lost in myeloid CML-BCCD34+ progenitors (n=6) and its restored expression at physiological levels rescues granulocytic differentiation and impairs clonogenic potential of primary BCR/ABL+ blasts (Eiring et al, ASH 2007). Here we show by Northern blot, real-time PCR, and microarray analyses that miR-328 levels increase during granulocytic differentiation of normal human CD34+ and mouse Lin− BM progenitors, but not during differentiation towards erythroid, megakaryocytic or monocytic lineages. BCR/ABL uses the same MAPKERK1/2-hnRNP-E2 signaling pathway to suppress both C/EBPα and miR-328, as pharmacologic or shRNA-mediated inhibition of these molecules restored miR-328 expression in BCR/ABL+ cells. In fact, two functional C/EBPα binding sites are present in the miR-328 promoter region and C/EBPα interacts in vivo with these regulatory elements to enhance miR-328 transcription.
Importantly, we also show that restored maturation of BCR/ABL+ blasts requires direct interaction of hnRNP-E2 with the C-rich regions of miR-328. Indeed, RNA-immunoprecipitation (RIP) assays demonstrated that miR-328 directly binds to hnRNP-E2 independent of the RNA-induced silencing complex (RISC). Furthermore, ectopic miR-328, but not miR-181b, resulted in decreased in vivo binding of hnRNP-E2 to the uORF/spacer region of CEBPA mRNA, thereby releasing CEBPA from hnRNP-E2 translation inhibition and rescuing C/EBPa-driven neutrophil maturation (decoy activity). Differentiation of miR-328-expressing CML-BCCD34+ blasts (88.8±2.4% post-mitotic cells) correlated with induction of C/EBPa protein expression, whereas CEBPA mRNA and hnRNP E2 protein levels remained unchanged. The existence of a direct miR-328/hnRNP-E2/CEBPA interplay was formally demonstrated in vitro using RRL-directed translation assays and in vivo using the 6.15 clone of 32D-BCR/ABL cells that do not express endogenous CEBPA mRNA and require ectopic C/EBPα (wt-uORF-CEBPA) for differentiation. Addition of miR-328, but not miR-330, to hnRNP-E2-containing RRL reactions increased newly synthesized 35S-C/EBPa levels by >100%. Likewise, forced miR-328 expression in vivo resulted in decreased hnRNP-E2 binding to CEBPA mRNA, induction of C/EBPa protein but not mRNA and rescued granulocytic differentiation of 6.15-wt-uORF-CEBPA but not vector-transduced 6.15 cells.
While hnRNP-E2 was not found in complex with basic RISC components (Dicer, TRBP2 and Ago2), RIP assays detected miR-328 associated to Dicer and Ago2 in miR-328-expressing cells, suggesting that it also acts through canonical RISC-dependent base-pairing with mRNA targets. Indeed, we identified the BCR/ABL-regulated PIM1 serine-threonine kinase as a bona fide miR-328 target in BCR/ABL+ cells. Ectopic miR-328 suppressed PIM1 protein but not mRNA levels, and this effect required integrity of the miR-328 binding site present in the PIM1 3'UTR. Forced expression of a wild-type but not kinase-deficient PIM1 lacking the 3'UTR into miR-328-expressing cells fully rescued BCR/ABL clonogenicity, suggesting that miR-328-induced PIM1 suppression accounts for reduced survival of miR-328-infected BCR/ABL+ blasts. To show that miR-328 acts on PIM1 in a RISC-dependent manner, we mutated the miR-328 seed sequence (miR-328-Mut) while retaining its C-rich character. Similar to wild-type miR-328, miR-328-Mut efficiently interacted with hnRNP-E2, restored C/EBPa protein expression and rescued granulocytic differentiation, but was unable to silence PIM1 in 32D-BCR/ABL cells, indicating that the C-rich character of miR-328 is essential for its decoy activity, while its seed sequence integrity is necessary for RISC-dependent pairing to mRNA targets.
Thus, the discovery of dual activities for miR-328 not only adds a new layer of complexity to the mechanisms regulating CML disease progression, but also highlights the ability of miRNAs to alter mRNA metabolism by acting as molecular decoys for RNA-binding proteins.

Disclosures:
Cortes: Novartis: Research Funding.