Role of Notch3 in sustaining UPR signaling in Notch3-overexpressing T-cell acute lymphoblastic leukemia.

Introduction. Aberrant Notch signaling has been implicated in the development of several diseases, including T-cell acute lymphoblastic leukemia (T-ALL), a malignant disorder that originates from hematopoietic precursors committed to T-cell lineage. Survival rates in T-ALL patients have greatly improved in the last decades but still a substantial number of patients will relapse and die. An increased understanding of T-ALL biology has opened opportunities for the development of targeted therapies for the treatment of this disease. Recently, several studies suggest the role of the unfolded protein response (UPR) in acute leukemias. UPR is a conserved adaptive signaling pathway which tries to restore protein homeostasis mainly after Endoplasmatic Reticulum (ER) stress. Since it has been demonstrated that cancer cells are able to maintain malignancy by acquiring therapy resistance through its UPR signaling, the disruption of UPR could be considered a selective anti-leukemia therapy. Our main aim is to demonstrate that Juglone, a naturally-occurring naphtoquinone, is able to induce ER stress/UPR perturbation, thus having therapeutic effects on T-ALL cells. Methods. Notch3-positive and Notch3-negative human TALL leukemia cells; pharmacological treatments; PI/Annexin staining and cytofluorimetric analysis; RNA interference; RT-qPCR; Western blot. Results. Here we show the mechanism whereby Juglone induced apoptosis in T-cell acute lymphoblastic leukemia (T-ALL) cells. Time course studies indicate that initial apoptotic events are inhibition of proteasome activity, concomitant with an increased endoplasmic reticulum (ER) stress signaling. Moreover, Juglone induced Notch downregulation in leukemic cells: however, while Notch3 inhibition contributed to amplify Juglone-induced T-ALL cell apoptosis, Notch1 did not. We futher demostrate that Notch3 downregulation during Juglone treatment prevents leukemia cells from engaging a functional UPR in order to buffer the Juglone-mediated proteotoxic stress in the ER, thus finally inducing T-ALL apoptosis. Conclusions. Our findings provide a rationale to target the ER stress/UPR signaling through Notch3 inhibition, Juglone-dependent, in a T-ALLs subset displaying upregulation of Notch3.