Mir-9 Is Aberrantly Expressed In MPN Patients and Accelerates Erythropoietic Cell Growth and Differentiation

Abstract 1983

The myeloproliferative neoplasms (MPNs), PV, ET and IMF, harbor a common gain-of-function mutation of JAK2V617F but in the past several years other molecular lesions have been noted as well such as mutation of TET2, chromsomal gains and losses, aberrant mRNA expression and aberrant microRNA expression. Since miRNAs are important regulators of hematopoiesis, we measured miRNA expression levels in CD34+ cells isolated from 8 patient samples (4 PV with JAK2V617F, 3 ET with wild-type JAK2 and 1 IMF with unknown JAK2 status) and 4 healthy controls using a Taqman Low Density Array (TLDA) representing 667 known miRNAs. We identified 28 miRNAs that were significantly deregulated in MPN patients compared to controls (p<0.05). Many of these miRNAs (18/24; 75%) were also deregulated in an independent miRNA array study using MPN patient granulocytes (n=39, 25 PV, 14 ET). Among these miRNAs, miR-9 expression was significantly upregulated in MPN patient CD34+ cells as well as in mature granulocytes. Furthermore, miR-9 expression was responsive to modulation of JAK2 activity in both gain of function (overexpression of JAK2V617F in TF-1 cells) and loss of function (inhibition of JAK2 activity in HEL cells) systems, suggesting that miR-9 may be dysregulated by aberrant JAK2V617F signaling. Analysis of miR-9 expression in ex-vivo, uni-lineage human CD34+ cultures, revealed that miR-9 is expressed at low levels during erythropoiesis, while levels gradually increase during myeloid differentiation. To determine how deregulated expression of miR-9 affected erythropoiesis, we transduced human bone marrow CD34+ cells (AllCells) with empty vector or miR-9 lentivirus, and examined erythroid differentiation and proliferation in liquid culture over 12 days. miR-9 expressing cells displayed a consistent growth advantage (∼30%), and accelerated erythroid differentiation (CD71+, GlyA+) at days 7 and 9. HITS-CLIP is a high throughput miRNA target prediction method developed in the Darnell lab based on crosslinking Argonaute protein to miRNA and target mRNA, providing highly specific identification of direct miRNA targets. We previously identified genes differentially expressed in PV patient CD34+ cells compared to normal CD34+ cells using Affymetrix HG-U133A arrays (117 genes, p<0.05). A comparison of predicted miR-9 mRNA targets identified using HITS-CLIP in P13 mouse brain, with genes downregulated in PV patient microarray studies, identified KLF4 which encodes a zinc finger protein involved in pluripotency and YWHAZ a 14-3-3 protein involved in signaling pathways regulating proliferation and survival as potential miR-9 targets relevant to aberrant hematopoiesis in MPN. Dysregulated cell growth in MPN may result in part from aberrant miRNA expression.