AML-018 Exploring Potential Molecular Dependency of t(8;21)(q22;q22.1) Positive Acute Myeloid Leukemia on the Non-Coding RNA HOTAIRM1-miR222 Axis

Abstract

Introduction Acute myeloid leukemia (AML) is a heterogenous disease categorized into subtypes based on characteristic chromosomal translocations, each having distinct biology. Patients with translocation t(8;21)(q22;q22.1) undergoing conventional chemotherapy show an overall survival of around 50%. Studying subtype-specific biology is crucial to developing targeted therapies and improving patient outcomes. HOTAIRM1, a long non-coding RNA regulates key genes during hematopoiesis, and its expression increases as myeloid cells differentiate. In AML, HOTAIRM1 is deregulated, and we aimed to study the significance of this aberrant expression. Materials and Methods We performed quantitative polymerase chain reaction (qPCR) to study HOTAIRM1 expression in the bone marrow of patients with AML (n=48), and six AML cell lines (Kasumi-1, THP1, HL60, KG1, MOLM13, and MOLM14). Overexpression was achieved by cloning HOTAIRM1 into pcDNA3.1+ vector followed by transfection. Cell cycle and apoptosis assays were performed using flow cytometry. MTT assay was used to calculate inhibitory concentration 50 (IC50) values. Results We found that HOTAIRM1 was significantly downregulated in patients with AML (P=.0005) and in AML cell lines (P<.01), especially in the t(8;21) positive subgroup. Overexpressing HOTAIRM1 led to cell cycle arrest (P<.01) and increased apoptosis (P<.01) in t(8;21) positive Kasumi-1 cells, indicating specific regulation of HOTAIRM1 in this subtype. We identified over-expressed microRNA miR-222 as a potential regulator of HOTAIRM1 expression in Kasumi-1. High miR-222 expression was associated with significantly lower overall survival (P=.02). Inhibiting miR-222 in Kasumi-1 mirrored the effect of HOTAIRM1 overexpression, resulting in cell cycle arrest (P<.001) mediated by p27 protein upregulation and increased apoptosis (P<.001) via BCL2, BCLXL, and MCL1 proteins. Inhibiting miR-222 however, did not increase HOTAIRM1 expression. Interestingly, HOTAIRM1 overexpression in Kasumi-1 led to reduced miR-222 (P<.05) indicating HOTAIRM1’s role in regulating miR-222, not vice versa. Treating Kasumi-1 cells with the hypomethylating drug azacytidine increased HOTAIRM1 expression (P<.0001) suggesting that aberrant promoter hypermethylation contributes to low HOTAIRM1 expression in the t(8;21) positive subtype. Conclusion Our data indicate that rescuing HOTAIRM1 expression in Kasumi-1 cells with drugs such as azacytidine, leads to inhibition of leukemic phenotypes driven by oncogenic miR-222. This suggests that targeting the non-coding RNA HOTAIRM1-miR-222 pathway could be a novel therapeutic approach in t(8;21) AML.