Knockdown (KD) of Mir-126 Expression Enhances Tyrosine Kinase Inhibitor (TKI)-Mediated Targeting of Chronic Myelogenous Leukemia (CML) Stem Cells
Authors: Zhang Bin , Li Ling , Chen Ching-Cheng , Stein Anthony S. , Ali Haris , Snyder David S. , Lin Allen , Dorrance Adrienne , Swiderski Piotr , Bhatia Ravi , Perrotti Danilo , Forman Stephen J. , Kuo Ya-Huei , Kortylewski Marcin , Marcucci Guido 
Published: October 2019

Abstract
BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in inducing remission and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. The long-term goal of our research is to improve the understanding of molecular mechanisms regulating LSC growth and develop effective mechanism-based therapeutic strategies to target LSC in CML. MicroRNAs (miRNAs) are short non-coding RNAs that regulate translation of target protein-coding messenger RNAs (mRNAs) and levels of the corresponding encoded proteins. Among distinct miRNAs, miR-126-3p (miR-126) is expressed in normal hematopoietic stem cells (HSCs) and early hematopoietic progenitor cells (HPCs) and plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo (Cell Stem Cell, 2012). We previously reported that miR-126 expression varies among AML patients; higher level of miR-126 associated with poor outcome, a LSC-associated gene expression signature and increased LSC quiescence; and miR-126 down-regulation decreased LSC self-renewal activity in serial transplant experiments (Leukemia, 2015). Here, we report that the more primitive LSK (Lin-Sca-1+Kit+) from SCL-tTA-BCR/ABL mouse model of CML had higher miR-126 expression compared to more differentiated subpopulations, i.e. common myeloid progenitors (CMP) (3.2 fold) and granulocyte-macrophage progenitors (GMP) (6.4 fold). Among LSK subpopulations, LSC (Flt3-CD150+CD48- LSK) demonstrated the highest miR-126 levels (P<0.01). Furthermore, quiescent (Hoechst-Pyronin-) LSC demonstrated higher miR-126 expression (2.6 fold, p<0.01), generated significantly higher long-term engraftment of donor CML cells in recipient mice (58.6±4.4% vs 33±3.4% in BM at 16 wks after transplanted with 100 LSC/mouse, p=0.003) and enhanced leukemogenic capacity (5 out of 9 mice receiving quiescent LSC developed CML within 14 wks after transplantation vs. none of 10 mice receiving proliferating LSC) compared with proliferating (Hoechst+/-Pyronin+) LSC. Human primary untreated chronic phase (CP) CML CD34+CD38- primitive progenitors also showed higher miR-126 expression compared with CD34+CD38+ committed progenitors. KD of miR-126 in LSK cells of CML mouse model and in primary CML CD34+CD38- cells by using either miRZip-126-3p anti-miR lentivirus (System Biosciences) or a novel, myeloid cell-specific CpG-miR-126 oligonucleotide (ODN) inhibitor increased LSC cell-cycle entry as demonstrated by increased Ki67 staining (p<0.05) and EdU labeling (p<0.05), but affected apoptosis only slightly compared to scramble-treated controls. More importantly, miR-126 KD either by lentivirus or CpG-miR-126 ODN inhibitor combined with Nilotinib (NIL) significantly increased apoptosis of LSC compared with NIL alone (34% vs 19%; p=0.01 for lentivirus KD; and 40% vs 35%; p=0.03 for CpG-miR-126 inhibitor KD), resulting in significant reduction of cell growth and colony forming cell (CFC) frequency in methylcellulose progenitor assays. Conversely, enforced miR-126 expression by miR-126 precursor lentivirus (System Biosciences) impaired cell-cycle entry (28% vs 33%; p=0.05) and led to significantly reduced apoptosis (27% vs 19%; P=0.01) in NIL (5µM) treated CML CD34+CD38- cells compared to cells treated with control lentivirus and NIL. We further investigated the effect of miR-126 KD on growth of primary CML LSC and therapeutic response in vivo. CP CML CD34+ cells were cultured with CpG-scrambled RNA or CpG-miR-126 ODN inhibitor (500nM), in the presence or absence of NIL (5µM) for 4 days, and then transplanted into irradiated NSG mice. While the experiment is still ongoing, we have already observed a significantly reduced engraftment in blood at 4 wks in recipient mice receiving cells treated with CpG-miR-126 inhibitor and NIL compared with those receiving cells treated with CpG-scrambled RNA and NIL (0.16±0.03% vs 0.4±0.04%; P=0.01). Final results of this in vivo experiment and in vivo treatment of SCLtTA/BCR-ABL mice with CpG-miR-126 inhibitor and NIL will be reported at the meeting. Altogether, these observations suggest that miR-126 play a role in CML LSC homeostasis and down-regulation of miR-126 may decrease CML LSC quiescence, increase LSC proliferation and in turn enhance their sensitivity to TKI. miR-126 therefore may represent a novel therapeutic target in CML.

Disclosures
Stein: Amgen: Speakers Bureau. Snyder:BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Forman:Mustang: Research Funding; Amgen: Consultancy.