Nutlin-3 upregulates the expression of Notch1 in both myeloid and lymphoid leukemic cells, as part of a negative feed-back anti-apoptotic mechanism Running title: Nutlin-3 upregulates Notch1 expression

The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 significantly up-regulated the steady-state mRNA and protein levels of Notch1 in p53 wild-type (OCI, SKW6.4) but not in p53 deleted (HL-60) or p53 mutated (BJAB) leukemic cell lines. A direct demonstration that Notch1 was a transcriptional target of p53 in leukemic cells was obtained in experiments carried out with siRNA for p53. Moreover, inhibition of Notch1 expression by using Notch1 specific siRNA significantly increased cytotoxicity in p53 wild-type leukemic cells. Of note, Nutlin-3 up-regulated Notch1 expression also in primary p53 wild-type B-chronic lymphocytic leukemia (B-CLL) cells and the combined use of Nutlin-3 plus pharmacological γ-secretase inhibitors of the Notch signaling, showed a synergistic cytotoxicity in both p53 wild-type leukemic cell lines and primary B-CLL. A potential drawback of γ-secretase inhibitors was their ability to enhance osteoclastic maturation of normal circulating preosteoclasts induced by RANKL+M-CSF. Notwithstanding, Nutlin-3 completely suppressed osteoclastogenesis irrespective of the presence of γ-secretase inhibitors. Taken together, these data indicate that the p53-dependent up-regulation of Notch1 in response to Nutlin-3 represents an anti-apoptotic feedback mechanism able to restrain the potential therapeutic efficacy of Nutlin-3 in hematological malignancies. Therefore, therapeutic combinations of Nutlin-3+γ-secretase inhibitors might potentiate the cytotoxicity of Nutlin-3 in p53 wild-type leukemic cells.