Time Sequential Transcriptome Analysis Identifies Mir-126 As an Early Biomarker for Inv(16) Acute Myeloid Leukemia (AML) Disease Progression

Inv(16)(p13q22)/t(16;16)(p13.1;q22) is one of the most common chromosomal mutations found in human acute myeloid leukemia (AML). This inversion on chromosome 16 creates a fusion gene CBFb-MYH11 and leads to the expression of a fusion protein CBFβ-SMMHC. Expression of Cbfβ-SMMHC (CM) in a conditional Cbfb-MYH11 knock-in mouse model ( Cbfb +/56M /Mx1-Cre ) leads to development of spontaneous AML with the acquisition of additional genetic and epigenetic alterations. In order to further understand the underlying mechanism(s) driving leukemogenesis and identify predictive biomarkers during disease progression, we performed RNA-seq to track changes of transcriptiome in time series peripheral blood samples using illumina HiSeq sequencer. A cohort of 16 mice were included in the experiment, 9 out of which are CM conditional knock-in mice and the remaining 7 are controls. Peripheral blood samples were collected before the induction of CM expression as the "0" time point samples. After induction, peripheral blood samples were collected at 1, 2, 3.5, 4.5, 5.5 and 6.5 month (experimental end point) after induction, and leukemic samples were collected when mice were moribund as the "end" time point samples. Between 3.5 and 6.5 months, 6 out of 9 CM knock-in mice in this cohort had succumbed to lethal leukemia. Total RNA was isolated for mRNA and microRNA (miR) sequencing library preparation. The mapped sequencing read counts were annotated to genes and differential expression was compared using edgeR. False discovery rate (FDR) were used to adjust for multiple comparisons. Genes with adjusted P value (FDR) 2 were considered differentially expressed genes. Unsupervised clustering analysis revealed that all diseased mice showed unique disease-related mRNA and miRNA signatures at the end point. The disease-related signatures were absent in all control mice and the three CM conditional knock-in mice that did not develop leukemia. We found that 2,032 mRNA genes and 106 miRs were significantly up-regulated whereas 2,926 mRNA genes and 121 miRs were significantly down-regulated at the leukemia end point. Principal component analysis revealed that the disease-related changes occur quite early, at 1-2 month after induction. Among the earliest changes, miR-126-3p and miR-126-5p, previously reported to be associated with human inv(16) AML, were significantly up-regulated (log fold change =2.09 and 2.36 respectively; p + AML and treated with with anti-miR126 (5 mg/kg, daily, 30 days) had a significant delay in leukemia progression and enhanced survival (control 42% survival v.s. anti-miR-126 80% survival at 70 days; n=7-10), supporting the notion that high miR-126 contributes to disease growth. Further mechanistic investigation of miR-126 function during inv(16) AML progression are ongoing. In conclusion, transcriptome analysis of time series samples during spontaneous leukemia progression allows identification of disease-related changes and early increase of miR-126 as a potential biomarker. Further functional validation is expected to provide mechanistic insights and rational for targeting miR-126 in inv(16) AML. Disclosures Stein: Seattle Genetics: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Stemline Therapeutics: Consultancy, Research Funding; Argios: Research Funding; Celgene: Research Funding.