The TAL1 Complex Represses the FBXW7 Tumor Suppressor Through Mir-223 in Human T-Cell Acute Lymphoblastic Leukemia

Abstract 1296

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T-cell acute lymphoblastic leukemia (T-ALL) and causes T-ALL in murine transgenic models, emphasizing its importance in the molecular pathogenesis of this disease. However, the mechanism by which TAL1 leads to transformation of thymocytes is unclear. Dysregulation of miRNAs play an important role in tumorigenesis in diverse cancer types. A recent study identified miR-223 as the most abundant miRNA in T-ALL patient samples and was oncogenic by virtue of its ability to accelerate Notch-induced T-ALL in a murine model (Mavrakis et al. Nature Genetics 2011). However, the underlying mechanisms leading to dysregulated miRNA expression in T-ALL remain poorly understood.

In order to explore the hypothesis that aberrant expression of miRNAs is mediated by the TAL1 oncogene in T-ALL, we generated high-resolution maps of the genome-wide occupancy of the TAL1 complex, including E2A, HEB, GATA3, LMO2 and RUNX1 by chromatin immunoprecipitation coupled to massively parallel DNA sequencing (ChIP-seq). Analysis of binding sites in two TAL1-positive T-ALL cell lines (Jurkat and CCRF-CEM cells) and two primary T-ALL samples identified 54 miRNAs where binding of the TAL1 complex was within 10 kb of either the transcriptional start sites or the start sites of genes that contain miRNAs in their intronic regions. To determine which of these miRNAs were not only directly bound, but also regulated by the TAL1 complex, we analyzed global changes in miRNAs after knockdown of TAL1 in Jurkat cells using two independent shRNAs. By miRNA expression profiling, we identified significant changes in expression of 25 miRNAs, of which nine were down-regulated on TAL1 knockdown (and thus positively regulated by TAL1) and 16 were up-regulated on TAL1 knockdown (and thus negatively regulated by TAL1). Of these 25 miRNAs, four (miR-223, miR181a*, miR-26a and miR-29c) were shown to be direct targets of the TAL1 complex based on our ChIP-seq data.

We chose to focus on miR-223 because it exhibited the most dynamic down-regulation after TAL1 knockdown. ChIP-qPCR validated binding of the TAL1 complex to a region within 4 kb of the miR-223 transcriptional start site. Analysis of RNA polymerase II and CBP binding showed significant enrichment, and high levels of H3K4M3 and H3K79M2 modification were detected indicative of transcriptional initiation and elongation of this locus. Furthermore, expression of miR-223 was significantly higher in the TAL1-positive cell lines (n=13) as compared to the TAL1-low cells (n=10) (P<0.0001). miR-223 levels also closely mirrored TAL1 levels in murine thymic subsets, with marked down-regulation after the DN2 stage, suggesting miR-223 is a physiological target of TAL1 during normal thymic development, and that its overexpression in TAL1-positive T-ALL cells, arrested at the double-positive (DP) stage, is aberrant compared to their normal DP counterpart.

To test the hypothesis that the growth inhibition observed after TAL1 knockdown is mediated by decreases in miR-223 expression, we retrovirally infected Jurkat and RPMI-8402 T-ALL cell lines with a miR-223 construct, such that miR-223 expression was no longer under the control of TAL1 in these cells. Forced expression of miR-223 partially rescued the growth inhibitory effects induced by TAL1 knockdown, in both a lentiviral and doxycycline-inducible shRNA system. Additionally, inhibition of mature miR-223 by lentiviral infection of a miR-223 shRNA construct led to significant growth inhibition of TAL1-positive cell lines through the induction of apoptosis. Thus, maintenance of miR-223 expression is required for optimal growth of TAL1-positive T-ALL cells.

The highest ranked predicted target of miR-223 by Targetscan is the FBXW7 tumor suppressor, a ubiquitin ligase that is mutated in a significant proportion of T-ALL patients and targets oncogenes such as c-MYC, NOTCH and mTOR for degradation. Accordingly, overexpression of miR-223 in TAL1-low miR-223-low T-ALL cells markedly down-regulated FBXW7 protein expression. Furthermore, the up-regulation of FBXW7 protein expression observed on knockdown of TAL1 in TAL1-positive cell lines could be prevented by retroviral miR-223 expression. Thus, miR-223 is an important target of TAL1 and links the TAL1 oncogene to repression of the FBXW7 tumor suppressor.