Alternative Splice Variants of IRAK4 That Activate Innate Immune Signaling Are Associated with U2AF1 Mutations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

Alternative RNA splicing and mutations in spliceosome genes are common features of human cancer. For example, recurring mutations of spliceosome genes in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) most frequently involve U2AF1, SF3B1, and SRSF2. Although aberrant RNA splicing is implicated in the pathogenesis of human cancers, it is less understood which misspliced genes contribute the malignant state. A global analysis of alternatively spliced genes and RNA isoforms in breast cancer, lung cancer, and AML revealed enrichment of alternatively spliced genes associated with inflammatory and immune pathways in the cancer cells as compared to the respective normal tissues. One such example is cancer-specific isoform expression of Interleukin Receptor Associated Kinase 4 (IRAK4), a serine/threonine kinase downstream of toll-like receptor (TLR) signaling and implicated in the pathogenesis of MDS/AML (Rhyasen et al., Cancer Cell 2013). Examination of the spliced isoforms by RNA sequencing showed that normal tissues preferentially express an alternatively spliced isoform of IRAK4 resulting from exclusion of the exon 4 (IRAK4-Short). In contrast, the majority of MDS and AML samples exclusively express an IRAK4 isoform that includes exon 4 (IRAK4-Long). Skipping of IRAK4 exon 4 results in an in-frame deletion of the N-terminal death domain, which is required for IRAK4 oligomerization and efficient TLR signaling, yet retains its C-terminal kinase domain. Immunoblotting confirmed that MDS/AML samples predominantly express the IRAK4-Long protein, while normal hematopoietic BM cells express the IRAK4-Short protein lacking the N-terminal domain. IRAK4-Long expression is significantly associated with increased NF-kB and innate immune signaling and correlates with poor AML patient outcome. Functional characterization of the IRAK4 isoforms in human AML cell lines revealed that IRAK4-Long induces NF-kB activation. In contrast, IRAK4-Short is less efficient at activating NF-kB (via phosphorylation of IKKbeta), yet it activates p38/MAPK signaling. To gain insight into the alternative splicing regulation of IRAK4 exon 4, we examined IRAK4 isoform expression and associated spliceosome gene mutations in MDS/AML patients. Of all examined genetic associations, mutation of U2AF1 (S34F) significantly correlated with inclusion of exon 4 and expression of IRAK4-Long, suggesting that mutations in U2AF1 instruct expression of IRAK4 RNA isoforms with maximal functional potential. To explore the direct regulation of IRAK4 by U2AF1, wildtype or mutant (S34F) U2AF1 were expressed in CD34+ cord blood cells, and IRAK4 isoform expression and exon usage was determined by RNA-sequencing. Expression of U2AF1-S34F resulted in significant retention of IRAK4 exon 4 (i.e. IRAK4-Long), while expression of wildtype U2AF1 correlated with exclusion of IRAK4 exon 4 (i.e., IRAK4-Short). Moreover, exon 4 and flanking intron sequences were cloned into a splicing reporter. Overexpression of U2AF1-S34F induced retention of the cassette exon 4, while wild-type U2AF1 mediated exclusion of exon 4. Taken together, these findings illustrate the importance of cancer-associated RNA splicing alterations and their consequences on downstream molecular networks required for cancer pathogenesis. In addition, we find that U2AF1 mutations result in expression of IRAK4 isoforms providing a genetic link to chronic innate immune signaling and IRAK1/4 activation in MDS and AML.