Abstract 616: AF4-AF9 interaction as a potential therapeutic target for MLL-rearranged mixed-lineage leukemias

Chromosomal translocations involving the mixed-lineage leukemia (MLL) gene lead to acute myeloid or lymphoid leukemias that are often associated with poor prognosis. The translocations result in in-frame fusion between MLL and one of the over sixty known partner genes that are heterogeneous in nature. AF4 and AF9 are two most common MLL fusion partners that interact specifically with each other in subnuclear foci. An AF4-mimetic peptide which disrupts the AF4-AF9 interaction is able to cause necrotic cell death in leukemic cell lines harboring MLL-AF4 or MLL-AF9 fusion oncogenes, emphasizing the importance of the AF4-AF9 interaction in leukemias caused by these mutations. AF4 and AF9 are found in a multi-subunit complex called AEP (AF4 family/ENL family/pTEFb complex) along with the transcription elongation factor pTEFb. The AEP complex is normally recruited to MLL target loci, where it functions to facilitate transcription elongation. One possible mechanism of MLL target gene misregulation is through the fusion of MLL to an AEP component protein, resulting in dysregulation of MLL target genes. To address the role AF4-AF9 interactions in MLL leukemias, mutations in AF9 that abolish the interaction with AF4 have been identified. A small AF9 interacting motif in AF4 designated RI3A- which upon overexpression display a whole cell expression pattern, co-localizes with GFP-taggedAF9 in the nucleus. Intriguingly, the AF9 D544R mutant does not co-localize with RI3A- but can be rescued by the K764E complimentary mutation in RI3A-. The critical role of the AF4-AF9 interaction in MLL fusion leukemias is further supported by a dual luciferase assay which assesses the promoter activity of a well characterized MLL target gene Hoxa9. Consistent with previous reports, MLL-AF9 activates Hoxa9 reporter gene expression. Hoxa9 promoter activity is enhanced when MLL-AF9 is co-expressed with full length AF4 but is ablated by the AF4 RI3A- fragment. This suggests that the domains outside of the AF9-binding motif of AF4 have an essential role in Hoxa9 transcriptional activation and also pose the AF4 RI3A- motif as a potent dominant negative inhibitor of the MLL-AF9 oncogene. Mouse hematopoietic progenitor cells can be immortalized by MLL-AF9 and form colonies in semi-solid media. The colony forming potential of the MLL-immortalized cells is significantly attenuated by the D544R as well as the V508E mutations in AF9, which showed impaired interaction with AF4 RI3A- in co-localization studies. Quantitative RT-PCR demonstrated that the lower colony forming ability correlated with lower gene expression of the MLL target gene, Hoxa9. We are currently investigating if overexpression of AF4 augments colony forming potential of MLL-AF9 and whether the AF4 RI3A- would have an inhibitory effect. These data indicate that binding of AF4 to MLL-AF9 plays an important role thus can be a potential therapeutic target for MLL fusion leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 616. doi:10.1158/1538-7445.AM2011-616