C/EBPa mutations in AML
Nine percent of patients with Acute Myeloid leukemia (AML) have mutations in the gene encoding the transcription factor CCAAT-enhancer-binding protein alpha (C/EBPa, CEBPA). Although it is known that these mutations induce a block in myeloid differentiation and lead to increased self renewal, the molecular mechanisms underlying these changes are still poorly understood. In the recent past, we have established a number of cell line models that allow functional studies of CEBPA mutations. We are now employing shRNA- and drug screening approaches to identify critical vulnerable points in CEBPA-mutant AML development and progression.
In the recent past, we have established a number of cell line models that allow functional studies of CEBPA mutations. Through affinity-purification coupled to Mass Spectrometry, we found that the C/EBPa p30 preferably interacts with Wdr5, a component of MLL histone methyltransferase complexes (Figure 1A). It has been previously demonstrated that the presence of Wdr5 in MLL complexes is critical for the activity of MLL to catalyse H3K4 methylations. We could show that shRNA-mediated downregulation of Wdr5 was able to overcome the p30-induced differentiation block in vitro (Figure 1B) and led to a decrease in leukemia-initiating cell (LIC) frequencies in vivo (Figure 1C). Through a collaboration with the Structural Genomics Consortium (SGC) in Toronto, we got access to OICR-9429, a newly developed antagonist of the WDR5-MLL interaction. OICR-9429 bound to the MLL-interacting pocket ofWdr5 with high affinity, thereby disrupting the interaction between WDR5 and MLL. OICR-9429-treatment of C/EBPa p30-expressing cells led to the up-regulation of myeloid surface markers and the attainment of a differentiated cellular morphology (Figure 1D). Finally, global gene expression profiling flowed by Gene Set Enrichment Analysis (GSEA) revealed that Wdr5 antagonism by OICR-9429 treatment led to a down-regulation of gene sets that are highly expressed in C/EBPa p30/p30 LICs (Figure 1E). Overall, these data indicate that Wdr5 is a critical mediator of p30-dependent cellular functions in C/EBPa-mutated AML. Furthermore, these results highlight Wdr5 and the MLL complex as potential therapeutic targets in leukemia.
In summary, our data indicate that CEBPA-mutated AML is highly sensitive to perturbation of the MLL/SET complex, either via genetic ablation of MLL or through pharmacological targeting. Our data indicate that leukemic mutations of C/EBPα selectively cooperate with the SET/MLL complex to regulate gene expression. These findings expand our understanding of and may inform new therapeutic strategies for N-terminal CEBPA mutated AML. Currently, we are investigating the effect of MLL-targeting inhibition on primary human AML cells from patients with CEBPA mutations. In parallel, we are trying to characterize functional consequences of targeting the C/EBPα p30-MLL interaction using genetic and biochemical approaches.
