Characterization of the Post-Transcriptional Regulation of AML1-ETO Expression in t(8;21) Leukemia Cells

The translocation between chromosomes 8q22 and 21q22 [t(8;21)(q22;q22)] is among the most common recurrent cytogenetic abnormalities associated with acute myeloid leukemia (AML), appearing in 8-15% of AML patients. The t(8;21) results in a fusion of the AML1 ( RUNX1 ) locus on chromosome 21 with the ETO ( RUNX1T1 ) locus on chromosome 8, creating the AML1-ETO (AE) fusion protein. The AE fusion contains the N-terminal AML1 runt homology domain and nearly the entire ETO protein. Therefore, AE RNA transcripts have the ETO 3' untranslated region (3'UTR). Recent evidence suggests that AE expression level is an important factor in t(8;21) leukemogenesis. Single cell qPCR of t(8;21) AML patient samples shows greatly increased expression of AE from t(8;21)+ leukemia stem cells at diagnosis versus t(8;21)+ hematopoietic stem cells during remission (Shima T, et al. Exp. Hematol. 2014). Furthermore, cells highly expressing the AE9a truncation are required to establish disease in the AE9a transplantation mouse model (Link KA, et al. PNAS . 2016). Unpublished data from our lab indicates that the level of AE transcripts is substantially higher in t(8;21)+ leukemic blasts versus t(8;21)+ differentiated monocytes/granulocytes. More importantly, this enhanced expression is associated with shortening of the AE 3'UTR. Therefore, identifying the factors that control AE expression via its 3'UTR may provide valuable insight into t(8;21) leukemogenesis. Post-transcriptional regulation of mRNA occurs primarily through sequence specific interactions between cis -elements in the 3'UTR and trans -factors, such as micro-RNAs (miRNA). We sought to determine the specific AE 3'UTR cis -elements and trans -factors involved in the regulation of AE expression.

The full-length AE 3'UTR is 5.2kb long. However, we detected several shorter-length isoforms in t(8;21) patients samples. To determine the effect on expression contributed by these 3'UTR isoforms, we made a series of luciferase reporters containing the different AE 3'UTR isoform sequences. In transient transfection assays in the t(8;21)+ Kasumi-1 and SKNO-1 cell lines, we observed an up to 5-fold difference in luciferase activities between the AE 3'UTR isoforms, with shorter 3'UTR isoforms having significantly higher activity. This result suggests that cis -elements within the longer AE 3'UTRs negatively affect AE expression. To characterize the complete set of cis -regulatory elements within the 5.2kb AE 3'UTR, we made luciferase reporters with a series of progressive 600bp fragments of the AE 3'UTR. Luciferase assays with this set of reporters in two t(8;21) AML cell lines Kasumi-1 and SKNO-1 showed significant differences in luciferase activity between the fragments (up to 5-fold). Interestingly, both t(8;21)+ cell lines had a similar pattern of luciferase activity, with a consistently down-regulated region that is only present in longer AE 3'UTR isoforms. Furthermore, comparing to results in non-t(8;21) cell lines (K562, U937, 293T, HL-60, and THP-1), we found t(8;21)+ cell type specific patterns in regulation.

We further sought to elucidate the role of miRNAs in regulating AE expression via the 3'UTR. Normal miRNA biogenesis requires a multi-step process involving the RNAase DICER. To assess the contribution of miRNAs in AE expression, we inhibited miRNA biogenesis by knocking down DICER in the t(8;21)+ Kasumi-1 and SKNO-1 cell lines. These cells showed increased AE expression by qPCR and western-blot, compared to controls. Furthermore, DICER knockdown significantly increased luciferase reporter activity in a subset of the 600bp AE 3'UTR fragments corresponding to the down-regulated region of the 3'UTR. Together these data suggest that there is a miRNA targeted regulatory region that is only present in longer AE 3'UTR isoforms. This supports a model where shortened 3'UTRs avoid miRNA targeted down-regulation by eliminating their binding sites from AE transcripts to increase the fusion gene expression.

Collectively, we examined the influence of AE 3'UTR isoforms on expression, mapped the regulatory elements within the AE 3'UTR, and identified the region that is involved in miRNA mediated regulation. Combining these results with miRNA target prediction programs and publicly available t(8;21) patient miRNA expression data, we have generated a list of potential miRNAs that target this region and are examining their role in regulating AE expression.