Nutlin-3 up-regulates the expression of Notch1 in both myeloid and lymphoid leukemic cells, as part of a negative feedback antiapoptotic mechanism

The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 significantly upregulated the steady-state mRNAand protein levels of Notch1 in TP53^wild-type (OCI, SKW6.4) but not in TP53^deleted (HL-60) or TP53^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 using Notch1-specific siRNA significantly increased cytotoxicity in TP53^wild-type leukemic cells. Of note, Nutlin-3 up-regulated Notch1 expression also in primary TP53^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 TP53^wild-type leukemic cell lines and primary B-CLL cells. 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 antiapoptotic feedback mechanism able to restrain the potential therapeutic efficacy of Nutlin-3 in hematologic malignancies. Therefore, therapeutic combinations of Nutlin-3 + γ-secretase inhibitors might potentiate the cytotoxicity of Nutlin-3 in p53^wild-type leukemic cells.