Therapeutic antagonists of microRNAs deplete leukemia-initiating cell activity
Chinavenmeni S. Velu, … , Brian Gebelein, H. Leighton Grimes
Chinavenmeni S. Velu, … , Brian Gebelein, H. Leighton Grimes
Published December 16, 2013
Citation Information: J Clin Invest. 2014;124(1):222-236. https://doi.org/10.1172/JCI66005.
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Therapeutic antagonists of microRNAs deplete leukemia-initiating cell activity

Abstract

Acute myelogenous leukemia (AML) subtypes that result from oncogenic activation of homeobox (HOX) transcription factors are associated with poor prognosis. The HOXA9 transcription activator and growth factor independent 1 (GFI1) transcriptional repressor compete for occupancy at DNA-binding sites for the regulation of common target genes. We exploited this HOXA9 versus GFI1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of HOX-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b inhibited in vitro leukemic colony forming activity and depleted in vivo leukemia-initiating cell activity of HOX-based leukemias, which led to leukemia-free survival in a murine AML model and delayed disease onset in xenograft models. These data establish microRNA as functional effectors of endogenous HOXA9 and HOX-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML.

Authors

Chinavenmeni S. Velu, Aditya Chaubey, James D. Phelan, Shane R. Horman, Mark Wunderlich, Monica L. Guzman, Anil G. Jegga, Nancy J. Zeleznik-Le, Jianjun Chen, James C. Mulloy, Jose A. Cancelas, Craig T. Jordan, Bruce J. Aronow, Guido Marcucci, Balkrishen Bhat, Brian Gebelein, H. Leighton Grimes

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Figure 1

GFI1 antagonizes HOXA9 transformation, and HOXA9-PBX1-MEIS1 directly regulates Mir21 and Mir196b.

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GFI1 antagonizes HOXA9 transformation, and HOXA9-PBX1-MEIS1 directly reg...
(A) Hierarchical clustering of gene expression array data from pediatric AML samples (columns) versus 365 class discriminating genes (rows) whose expression is similar to either GFI1 or HOXA9. Color key of AML subtypes. (B) Graphic representation of experimental scheme. (C) CFU assay of Lin– BM cells cotransduced with retroviral vectors encoding NUP98-HOXA9 and either empty vector control, GFI1 or GFI1N382S (n = 2). (D) CFU assay of either Gfi1+/+ or Gfi1+/– Lin– BM cells transduced with a retroviral vector encoding NUP98-HOXA9 and sorted for GFP (n = 2). *P < 0.05; **P < 0.01 (E) Composite GFI1-HOXA9-PBX1-MEIS1 DNA-binding site on Mir196b promoter. (F) EMSA with purified GFI1 and HOXA9-PBX1-MEIS1 proteins on a synthetic oligonucleotide encoding a DNA sequence in the Mir196b locus. (G) EMSA identical to that shown in part B, except the oligonucleotide encodes a TC→GA mutation in the putative GFI1 binding site. (H) Quantitation of competitive EMSA results from the HOXA9-PBX1-MEIS1 complex bound to either the WT or mutant oligonucleotide, alone or at several time points after addition of purified GFI1 protein. (I and J) ChIP analysis with a GFI1-specific monoclonal antibody (2.5D.17) with isotype control IgG (Con IgG) (I) or HA-epitope tag antibody for HOXA9 (J). (K) Diagram showing the composite HOXA9-PBX1-MEIS1 DNA-binding site on Mir21 promoter. (L) EMSA with purified HOXA9-PBX1-MEIS1 proteins on a synthetic oligonucleotide encoding a DNA sequence in the Mir21 locus. (M) ChIP analysis with an HA-epitope tag specific antibody from cells expressing HA-tagged HOXA9.