TARGETING NOTCH TRAFFICKING IN HUMAN CANCER CELLS: A. PHARMACOLOGIC INHIBITION OF THE VACUOLAR H+ ATPASE REDUCES PHYSIOLOGIC AND ONCOGENIC NOTCH SIGNALING. B. HIGH CONTENT SCREEN FOR NOVEL MODULATORS OF THE NOTCH PATHWAY.

Abstract: Notch signaling is prominently involved in cell fate decision and growth regulation in metazoan tissues. Because of this, Notch is often upregulated in cancer and current efforts point to developing drugs that block its activation. Notch receptor endocytosis towards acidic compartments is a recently appreciated determinant of signaling activation. The Vacuolar H+ ATPase (V-ATPase) is responsible for acidification of endocytic organelles and recently it has been shown that mutants in V-ATPase subunit encoding genes in model organisms display loss of Notch signaling phenotypes. In the first part of my graduate studies, we aimed at discovering whether pharmacologic reduction of V-ATPase activity affected Notch signaling. We found that administration of BafilomycinA1 (BafA1), a highly specific V-ATPase inhibitor decreases Notch signaling during Drosophila and Zebrafish development, and in human cells in culture. In normal breast cells, we have found that BafA1 treatment leads to accumulation of Notch in the endo-lysosomal system, and reduces its processing and signaling activity. In Notch-addicted breast cancer cells, BafA1 treatment reduces growth in cells expressing membrane tethered forms of Notch, while sparing cells expressing cytoplasmic forms. In contrast, V-ATPase inhibition reduces growth of leukemia cells, without affecting Notch activating cleavage. However, consistent with the emerging roles of V-ATPase in controlling multiple signaling pathways, in these cells Akt activation is reduced, as it is also the case in BafA1-treated breast cancer cells. Our data support V-ATPase inhibition as a novel therapeutic approach to counteract tumor growth sustained by signaling pathways regulated at the endo-lysosomal level. The functions of Notch throughout the life of an individual are varied and complex. This complexity is not sufficiently accounted for by the limited core of known Notch signaling components and a growing body of evidence attributes it to additional factors that determine whether, when and how Notch functions within a given context. Considering this, in the second part of my graduate work, we sought to identify novel genes that might influence Notch. Thus, we performed a high content immunofluorescence-based RNA interference screen of a pharmacologically-relevant subset of the human genome. To this end, we monitored how knockdown of specific genes perturbs the localization of the Notch-1 receptor in human breast cells under resting and signaling conditions. Here we present the screen setup, the primary screen results and the candidate follow-up strategy.