Investigating the Role of Cdc73 in T-cell Acute Lymphoblastic Leukemia (T-ALL)

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PROJECT SUMMARY About 60% of T-cell acute lymphoblastic leukemia (T-ALL) patients have activating NOTCH1 mutations, making NOTCH1 the most prevalent oncogene in this cancer. In early clinical trials, pan-Notch inhibitors like gamma- secretase inhibitors (GSI) caused excessive toxicity because Notch has essential functions in tissue homeostasis. To circumvent this toxicity, work by my lab and others supports an alternative strategy of targeting context-dependent transcriptional cofactors that are required for Notch to activate its enhancers. In theory, inhibiting specific Notch cofactors might have less toxicity than inhibiting all Notch functions. Cell division cycle 73 (Cdc73) is a scaffold component of the polymerase-associated factor complex (PAF1c). Like flies with mutant Notch, flies with mutant PAF1c display notched wings and impaired expression of Notch target genes, suggesting that PAF1c is linked to the Notch pathway. My preliminary data suggests that Cdc73 interacts with the Notch1 complex at the T-cell specific Notch-dependent MYC enhancer and promotes Notch-induced leukemogenesis in vivo. MYC is a critical Notch-induced oncogene in T-ALL. The canonical function of PAF1c is to activate mRNA synthesis at promoters by recruiting histone modifier enzymes and transcriptional machinery to a small subset of transcription start sites. Its non-canonical function at enhancers is poorly understood. My proposal examines the role of Cdc73 in enhancer activation and proliferation of primary human T-ALL cells. I hypothesize that Cdc73 facilitates the activation of enhancers that drive Notch-dependent oncogenic gene expression programs in T- ALL. Therefore, deletion of Cdc73 will inhibit leukemic cell proliferation by inactivating enhancers known to drive T-ALL. My impact is to shift the T-ALL field away from pan-Notch inhibitors by targeting the CDC73 cofactor to impair oncogenic Notch transcriptional complexes. My first aim is to determine the role of Cdc73 in T-ALL proliferation in vivo by measuring the anti-leukemic effects of Cdc73 withdrawal in Notch-activated primary human patient samples and in a clinically relevant transgenic mouse model of Notch-activated T-ALL. My second aim is to determine the mechanism by which Cdc73 enhances Notch1 target gene transcription by quantifying the effects of Cdc73 deletion on mRNA/eRNA transcription and enhancer activation through proteomic and transcriptional genomic approaches. I expect to show that enhancer-bound Cdc73/PAF1c activates a subset of enhancers enriched for functional eRNAs, thus facilitating future identification of eRNAs that support T-ALL. I also expect to establish Cdc73 as a new vulnerability of Notch-driven leukemic cells that connects Notch to the transcriptional machinery and chromatin modifying enzymes that activate enhancers. Successful completion of this project will show proof-of-principle that the Drosophila model of Notch as a cofactor-dependent transactivator can be applied to develop human anti-cancer strategies. I will also show the first instance that Paf1C, an important epigenetic regulator that has been well established to activate mRNA transcription, might engage canonical factors at non-canonical sites to promote enhancer RNA transcription and enhancer activation.