Abstract 1117: Dissection of the major hematopoietic quantitative trait locus in chromosome 6q23.3 identifies miR-3662 as a player in hematopoiesis and AML
Authors: Maharry Sophia E. , Walker Christopher J. , Liyanarachchi Sandya , Mehta Sujay , Blachly James S. , Guttridge Denis C. , Bloomfield Clara D. , de la Chapelle Albert , Eisfeld Ann-Kathrin 
Published: July 2016

Background: Over 15 genome-wide association studies (GWASs) have identified a narrow region in chromosome 6q23.3 as a major hematopoietic quantitative trait locus (QTL) for many hematologic traits. The associated phenotypes have yet to be fully explained by genes in the region, including MYB. We noted a newly annotated microRNA within the QTL, miR-3662, and sought to characterize its role in the QTL by exploring its function in normal and malignant hematopoiesis, specifically acute myeloid leukemia (AML).

Results: To assess the association between the QTL-defining SNPs and miR-3662 abundance, we genotyped 10 GWAS SNPs in 200 blood samples from non-leukemic individuals. The risk alleles of all 10 SNPs associated with higher miR-3662 abundance. Homozygosity for rs66650371, a three base pair (bp) deletion, showed the strongest association (P&lt;.0001). Reinserting the three bp in cells homozygous for rs66650371 with CRISPR/Cas9 technology lowered miR-3662 abundance by 20%, suggesting rs66650371 directly affects miR-3662 transcription. MiR-3662 was nearly absent in primitive hematopoietic progenitor cells (HPCs) cells, whereafter its abundance increased throughout differentiation. MiR-3662 overexpression increased cell growth and colony formation of HPCs, particularly of the erythroid lineage.

To explore the role of miR-3662 in malignant hematopoiesis, we overexpressed miR-3662 in AML cell lines (Kasumi1, KG1a, MV4-11) and primary AML patient blasts (n = 12). Overexpression of miR-3662 reduced cell growth and viability in vitro, and significantly reduced tumor size in a murine xenograft model (scramble vs. miR-3662; n = 9 mice/group).

We used targeted RNA sequencing to identify differentially expressed genes between forced miR-3662-expresssing- and control AML patient blasts using a panel of 361 candidate genes (TruSeq RNA platform, Illumina). IKBKB was identified as a promising target, and direct regulation of IKBKB's 3′-UTR by miR-3662 was confirmed via luciferase assay. MiR-3662 overexpression lowered IKBKB mRNA and protein expression in both HPCs and AML patient blasts. As IKBKB promotes activation and nuclear localization of NF-κB, overexpression of miR-3662 led to decreased NF-κB transactivation potential via luciferase assay. Confocal microscopy of HPCs and AML cells, as well as subcellular fraction immunoblots, revealed decreased NF-κB nuclear localization upon miR-3662 overexpression. Re-introduction of IKBKB partly rescued miR-3662's phenotype by increasing nuclear localization of NF-κB, decreasing growth of HPCs, and increasing viability of AML cells.

Conclusion: The risk alleles of the 6q23.3 region associated with higher miR-3662 abundance, and miR-3662 abundance was functionally linked to rs66650371. We characterized miR-3662 as an agent in normal and malignant hematopoiesis, and demonstrated that miR-3662 reduces NF-κB activity by targeting IKBKB.

Citation Format: Sophia E. Maharry, Christopher J. Walker, Sandya Liyanarachchi, Sujay Mehta, James S. Blachly, Denis C. Guttridge, Clara D. Bloomfield, Albert de la Chapelle, Ann-Kathrin Eisfeld. Dissection of the major hematopoietic quantitative trait locus in chromosome 6q23.3 identifies miR-3662 as a player in hematopoiesis and AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1117.