ETS1 Promotes Context-Dependent Activation of Notch Signals in T-Cell Leukemia

Abstract

Notch is a signaling pathway involved in cellular growth and development. Aberrant activation of the Notch pathway is prevalent in cancer, including more than 60% of cases of T-cell acute lymphoblastic leukemia (T-ALL). Clinical trials using pan-Notch inhibitors to treat T-ALL patients have revealed excessive toxicities; Notch is required for tissue homeostasis in organs like the intestines. Intermittent dosing with pan-Notch inhibitors is tolerable but has weak anti-cancer effects. An alternative strategy for inhibiting Notch signaling in cancer would be to target tissue-specific cofactors required for Notch’s transcriptional activity. Notch cannot activate response elements in the nucleus of the cell on its own; it requires a favorable nuclear context produced by transcriptional cofactors. Furthermore, Notch often utilizes tissue-specific enhancers to selectively drive transcription of its target genes in particular cell types; others have demonstrated that deletion of a T-cell specific Notch-dependent enhancer for the MYC oncogene impairs T-cell development and T-ALL proliferation without effects in other tissues. Targeting a cofactor required for Notch’s function at T-cell specific enhancers might inhibit leukemic cell growth without the toxicities of pan-Notch inhibition. One such tissue-specific cofactor might be Ets1, a hematopoietic transcription factor implicated in the pathogenesis of other cancers. Ets1 is an attractive candidate as a Notch cofactor in T-ALL since it is expressed primarily in lymphoid cells and binds to the majority of Notch1 response elements in a T-ALL cell line. To investigate the importance of Ets1 in T-cell development and T-ALL, I used a conditional Ets1 knockout mouse and RNAi knockdown of Ets1. Not only is Ets1 important for the same stages of T-cell development as Notch1, but deletion of Ets1 in Notch-driven T-ALL cells reduced leukemia cell burden and prolonged survival in mice. In order to identify the mechanism by which Ets1 inhibits the growth of T-ALL cells, I performed RNA-seq and ChIP-seq studies in a human T-ALL cell line. Ets1 co-regulates the expression of a subset of NOTCH1 target genes, including known T-ALL oncogenes. Ets1 is also important for Notch complex binding and activation of Notch-driven enhancers essential for T-cell development and T-ALL proliferation. Finally, silencing Ets1 sensitizes human T-ALL cell lines to pan-Notch inhibitors, suggesting that targeting both Ets1 and Notch might be an effective therapeutic strategy. This thesis work supports an emerging model in which transcription factors assist Notch in activating enhancers; exploiting this dependency might combat Notch activity in cancer with less toxicity than inhibiting Notch directly.