Abstract 1950: Suppression of RISC-independent decoy and RISC-mediated RNA-pairing activities of microRNA-328 is required for maturation-arrest and enhanced survival of blast crisis CML progenitors

Abstract MicroRNAs (miRs) and heterogeneous ribonucleoproteins (hnRNPs) are post-transcriptional gene regulators that bind to mRNA in a sequence-specific manner. We showed that hnRNP-E2 inhibits myeloid maturation of bone marrow (BM) progenitors from chronic myelogenous leukemia patients in myeloid blast crisis (CML-BC) by suppressing CEBPA mRNA translation. We report here that loss of miR-328 is induced by BCR/ABL and specifically occurs in CML-BC, and its restored expression rescues differentiation and impairs clonogenic potential of BCR/ABL+ BM progenitors. Accordingly, miR-328 increases during granulocytic differentiation of human CD34+ and mouse LSK BM stem/progenitor cells. Mechanistically, BCR/ABL uses the MAPK-hnRNP-E2 pathway to suppress C/EBPα and miR-328 expression as pharmacologic inhibition of and/or shRNAs against these molecules efficiently restore miR-328 expression. Interestingly, two functional C/EBPα binding sites are present in the miR-328 promoter and positively regulate its transcription. We also show that maturation of differentiation-arrested BCR/ABL+ blasts requires direct interaction of hnRNP-E2 with the miR-328 C-rich regions. Moreover, imatinib treatment restores miR-328 expression, thus allowing its direct binding to hnRNP E2 independent from the RISC complex. Importantly, physiological miR-328 expression decreased hnRNP E2 binding to the uORF/spacer region of endogenous CEBPA mRNA (decoy activity). This, in turn, releases CEBPA mRNA from hnRNP E2 translation inhibition and allows in vitro and in vivo BCR/ABL+ cell differentiation. Although hnRNP E2 was not found in complex with the basic RISC components in BCR/ABL+ cells, miR-328 was found associated to Dicer and Ago2, suggesting that miR-328 also acts through base-pairing with the 3′UTR of mRNA targets in a RISC-dependent manner. In fact, miR-328 suppresses PIM1 protein but not mRNA expression and this effect requires the integrity of the PIM1 3′UTR. Indeed, forced expression of a wild type, but not a kinase-deficient, PIM1 lacking the 3′UTR into miR-328-expressing cells fully rescues BCR/ABL clonogenicity, suggesting that miR-328-induced inhibition of PIM1 accounts for reduced survival of miR-328-infected CML-BCCD34+ blasts. To demonstrate that miR-328 acts on PIM1 in a RISC-dependent manner, we mutated the miR-328 in the seed sequence (miR-328-Mut) while retaining its C-rich character. As expected, miR-328-Mut interacted with hnRNP-E2 and rescued C/EBPα-mediated differentiation, but did not silence PIM1 expression. Thus, the discovery of dual activities for miR-328 which affect myeloid differentiation and survival not only adds a layer to the complexity of mechanisms regulating CML-BC but also highlights the ability of miRNAs to alter mRNA metabolism by acting as molecular decoys for RNA binding proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1950.

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