Abstract 4788: MiR-509 inhibits human leukemia cell growth
Authors: Tan Yee Sun , Cheng Wen-Chih , Civin Curt I. 
Published: May 2015

Even though the majority of children with precursor B-cell acute lymphoblastic leukemia (B-ALL) can be cured, ALL is still the most frequent cause of cancer-related deaths and causes significant treatment-related toxicity in pediatrics. Thus, there is a need for more effective and less toxic therapies for B-ALL. Specific microRNAs (miRs) may be useful as therapeutics, or to identify downstream therapeutic targets, since miRs regulate essentially all cellular processes. We identified miR-509 via a human genome-wide screen (Cheng WC et al. BioTechniques 2013;54:77. PMC3671589) for miRs that inhibit growth of the NALM6 human B-ALL cell line (harboring the ETV6-PDGFRB fusion gene). MiR microarray expression analysis revealed low endogenous levels of miR-509 in multiple acute leukemia cell lines, CD34+ hematopoietic stem-progenitor cells from normal human donors, and normal B and T lymphocytes. Therefore, enforced expression of miR-509 was used for our mechanistic studies in leukemia cells. Since NALM6 cannot be easily transfected, the precursor miR-509 sequence was cloned downstream of green fluorescence protein (GFP) into a lentiviral vector construct. Enforced lentivector-mediated miR-509 expression was confirmed using RT-qPCR. In a confirmatory growth competition assay measuring the percent GFP-expressing cells by flow cytometry, NALM6 cells transduced with lentiviral miR-509 were progressively outnumbered by mock-transduced cells over five weeks. MiR-509's growth inhibitory effect in NALM6 was further confirmed by orthogonal assays employing trypan blue dye exclusion and alamarBlue fluorescence. MiR-509 similarly inhibited growth of another B-ALL cell line, RCH-ACV (harboring the TCF3-PBX1 fusion gene). In addition, NOD-SCID IL2rg-/- (NSG) mice transplanted miR-509-transduced NALM6 cells had increased survival as compared to empty vector. In order to elucidate the cellular mechanism by which miR-509 exerts its growth inhibitory effects, we performed cell cycle analysis using BrdU/7-AAD staining. MiR-509-transduced NALM6 cells had reduced numbers of S-phase cells, as compared to empty vector-transduced cells. We also observed an increase in apoptotic cell death in miR-509-transduced NALM6 cells, as compared to empty vector-transduced cells. To identify potential targets of miR-509, we used TargetScan to predict targets. We then focused on mRNAs that were expressed in NALM6 cells, as determined by gene expression data in the Cancer Cell Line Encyclopedia. RAB5C was reduced in miR-509 transduced cells as evidenced by qPCR and western blot; luciferase assays indicate that RAB5C is a direct miR-509-3p target. Knockdown of RAB5C by shRNA lentiviral constructs recapitulated the growth inhibitory phenotype induced by miR-509 overexpression in NALM6 cells. In summary, miR-509 suppresses B-ALL cell growth by inhibiting cell proliferation and inducing apoptosis, and miR-509 and RAB5C may be new therapeutic targets for B-ALL.

Citation Format: Yee Sun Tan, Wen-Chih Cheng, Curt I. Civin. MiR-509 inhibits human leukemia cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4788. doi:10.1158/1538-7445.AM2014-4788