Cdc73 protects Notch-induced T-cell leukemia cells from DNA damage and mitochondrial stress [Bru-seq and BruUV-seq]

Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL). But in clinical trials, pan-Notch inhibitors caused excessive toxicity. To find alternative ways to target Notch signals, we investigated Cell division cycle 73 (Cdc73), which is a component of the RNA polymerase-associated transcriptional machinery and has been previously described as a Notch cofactor. Emerging evidence also suggests that transcriptional machinery might be an attractive vulnerability in T-ALL. In this setting, we show that CDC73 co-binds a subset of Notch-occupied regulatory elements in an ETS1-dependent context. In mouse models, Cdc73 is important for Notch-induced T-cell development and maintenance of Notch-induced T-ALL. Mechanistically, Cdc73, Ets1, and Notch activate genes that promote DNA repair and oxidative phosphorylation. Cdc73 induces these pathways through canonical functions in mRNA synthesis rather than non-canonical functions in enhancer activation. Our study suggests that Cdc73 acts through context-dependent mechanisms to promote a gene expression program that mitigates the genotoxic and metabolic stress of supraphysiological Notch signaling. We also provide mechanistic support for testing inhibitors of DNA repair, oxidative phosphorylation, and transcriptional machinery as anti-leukemic therapy while highlighting strategies that disable pathways that intersect with Notch at chromatin to target Notch signals without directly targeting the Notch complex. Conditional Cdc73 knockout murine Notch-induced T-ALL cell lines (969 and 970) and a control murine Notch-induced T-ALL cell line (708) were treated with EtOH vehicle or OHT. Three biological replicates were prepared per condition and subject to both metabolic labeling with bromouridine and sequencing (Bru-seq) and Bru-seq with ultraviolet irradiation (BruUV-seq). 36 libraries were produced for Bru-seq and BruUV-seq (18 each).