The role of the MLL/AF4 fusion oncogene in acute lymphoblastic leukaemia maintenance

PhD ThesisThe chromosomal rearrangement t(4;11)(q21;q23) marks an aggressive acute lymphoblastic leukaemia (ALL) subtype particularly prevalent in infants and associated with poor outcome. This cytogenetic abnormality encodes the fusion oncogene MLL/AF4, which plays a pivotal role in cell death resistance; however, the underlying molecular processes are not fully understood. Therefore, to gain further insight, RNAi-mediated ablation of MLL/AF4 in the t(4;11)-positive ALL cell line SEM was combined with global gene expression profiling and concomitant apoptosis inhibitor studies. Phenotypically, MLL/AF4 depletion impaired proliferation, cell cycle progression, clonogenicity and caused a strong apoptosis induction. Global transcriptome analysis found up-regulation of proapoptotic and anti-proliferative genes, while mitogenic signalling mediators and stemness-related markers were down-regulated. Supplementing MLL/AF4-depleted cells with the pan-caspase inhibitor zVAD suppressed apoptosis, but failed to abrogate cell death. Subsequent gene expression profiling showed induction of genes implicated in the necroptotic cell death pathway. Interestingly, addressing this using established necroptosis inhibitors did not rescue the phenotype. Finally, the cytokine ANGIOPOIETIN-1 (ANGPT1) was identified as a novel MLL/AF4-modulated gene; MLL/AF4 knock-down correlated with a substantial decrease of ANGPT1 levels. Concordantly, screening an B-precursor ALL patient cohort found ANGPT1 to be highly overexpressed in t(4;11)-positive ALL. Although no correlation with clinical prognostic factors could be established, ANGPT1 was found to contribute to the leukaemic phenotype, as RNAi-mediated ANGPT1 depletion impaired proliferation and viability in vitro and impinged on disease development in vivo. Concluding, it was found that t(4;11)-positive ALL cells display a high degree of oncogene addiction towards MLL/AF4, since depletion strongly perturbed the leukaemic phenotype, compromising survival and self-renewal. Furthermore, ANGPT1 was identified as a novel proleukaemic factor cooperating with MLL/AF4 in maintaining the disease