Mir-21 Mediates Hematopoietic Suppression in MDS by Activating TGF-b Signaling,
Authors: Bhagat Tushar D. , Zhou Li , Sokol Lubomir , Mantzaris Ioannis , Gundabolu Krishna , Gordon Shanisha A. K. , Yu Yiting , Nischal Sangeeta , Polineni Rahul , Schinke Carolina , Chrysofakis Grigorios , Wickrema Amittha , Pellagatti Andrea , Boultwood Jacqueline , Steidl Ulrich G , Liu Gang , List Alan F , Bitzer Markus , Verma Amit 
Published: October 2019

Abstract
Abstract 3813
Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. TGF-b is a hematopoietic inhibitory cytokine that has been indirectly linked to the pathogenesis of some subsets of MDS. We have shown that smad2, a component of the TGF- signaling pathway, is constitutively activated and upregulated in MDS progenitors (Blood, 112(8):3434; 2008). Since there is conflicting data about upregulation of TGF- b levels in MDS, we next sought to determine the molecular basis of TGF- b pathway activation in this disease. We observed that smad-7, a negative regulator of TGF- b receptor-I kinase, is markedly down regulated in MDS and leads overactivation of the receptor and subsequent smad2 phosphorylation / activation in this disease (Cancer Res, 71(3):955–63). In the present study we wanted to determine the cause of smad7 reduction in MDS. Since microRNA dysregulation has been reported in many malignancies, we explored the 3'UTR of the smad7 gene for putative microRNA binding sites and observed predicted mir-21 and mir-15/16 binding sites that were conserved across species. mir-21 was found to be elevated in a microarray screen in MDS (British J. Haem. 153(1):24–32) and was subsequently found by us to be significantly elevated by qPCR in MDS marrow samples when compared with age matched controls (TTest, N=11 in each group, P Value= 0.02). Luciferase reporters containing wild type and mutant 3' UTR of the smad7 gene were then used to determine whether mir21 was able to directly bind to the predicted sequence in the gene. Enforced expression of mir-21 was able to inhibit the wild type smad7 reporter expression and a mutation of 4 complementary residues in the 3'UTR led to abrogation of this effect, thus demonstrating direct effects of mir-21 on smad7 gene. To test the role of mir-21 in regulating TGF-b signaling in vivo, we used chemically modified, locked nucleic acid (LNA) inhibitors of mir-21. These were used in a TGF-overexpressing transgenic mouse model that develops progressive anemia and dysplasia and thus serves as a model of human bone marrow failure. Treatment with the mir-21 inhibitor led to significant increases in RBC counts when compared to placebo (P value<0.01, T test) and led to increase in smad7 expression and decrease in smad2 phosphorylation in bone marrow progenitors of the treated mice. Finally, mir-21 inhibitor treatment led to increases in erythroid and myeloid colony formation from human MDS bone marrow stem cells, demonstrating its ability in stimulating hematopoiesis in vitro. Taken together, these studies demonstrate that mir-21 mediated reduction in smad-7 contributes to ineffective hematopoiesis in MDS by activating TGF-beta signaling in bone marrow progenitors. Most importantly, these studies illustrate the therapeutic potential of mir-21 inhibitors in this disease.

Disclosures:
List: Celgene: Consultancy.