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

Abstract

The small molecule inhibitor of the MDM2/ p53 interaction Nutlin-3 significantly upregulated the steady-state mRNA and 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. IntroductionThe activation of p53 is tightly regulated by the human homolog of murine double minute 2 (MDM2) gene, 1 which is an E3 ubiquitin ligase for p53 and itself and controls p53 half-life mainly via ubiquitindependent degradation. In response to a variety of stimuli, such as cellular stress, the p53-MDM2 interaction is disrupted and p53 rapidly accumulates within the cell. 1 Potent and selective small molecule inhibitors of the p53-MDM2 interaction, the Nutlins, have been recently reported. 2,3 These compounds bind MDM2 in the p53 binding pocket with high selectivity and can release p53 from negative control, leading to effective stabilization of p53 and activation of the p53 pathway. 2,3 It has been demonstrated that treatment with the active enantiomer Nutlin-3a results in rising levels of p53 protein and subsequent induction of cell cycle arrest and apoptosis in a variety of tumor cells. 2 In contrast to most solid tumors, TP53 is mutated in approximately 10% to 15% of both myeloid and lymphoid leukemias at diagnosis, 4 and several recent studies have demonstrated that Nutlin-3 induces ex vivo cytotoxic cell death of most TP53 wild-type primary hematologic malignancies, including acute myeloid leukemias, multiple myeloma, B-chronic lymphocytic leukemias (B-CLL), and B-cell lymphomas. [5][6][7][8][9][10][11][12][13][14][15] Although it is well established that p53 mediates a variety of cellular functions, such as cell cycle arrest, cellular senescence, and, only as ultimate choice, apoptosis, 16,17 some experimental evidence suggests that inhibition of the transcriptional activity of p53 might paradoxically result in an increase of the p53-mediated proapoptotic activity, 18 also in B-CLL cells. 19 Therefore, the aim of this study was to further investigate the relationship between the transcriptional and cytotoxic activities induced by Nutlin-3 and to elucidate whether Nutlin-3 promotes the transcription of antiapoptotic genes in leukemic cells, which might hamper and/or reduce its potential therapeutic efficacy. In this respect, it has recently emerged that a potential target gene of the p53 pathway is NOTCH1, 20 which belongs to an evolutionarily conserved pathway that profoundly impacts mammalian development. Of note, a recent study published while this article was under preparation has demonstrated that circulating B-CLL cells overexpress Notch1 and Notch2 family members with respect to circulating normal B lymphocytes. 21 The same study suggested that constitutively...

Trp53 regulates Notch 4 signaling through Mdm2

“…Beverly and colleagues observed suppression of Trp53 levels by NICD1 and suggested that this might reflect Mdm2-dependent events (Beverly et al, 2005). Secchiero and colleagues (Secchiero et al, 2009) found that, in cells lacking active Trp53, Notch1 protein levels were stable in the presence or absence of the Mdm2 and Trp53 inhibitor Nutlin, consistent with the notion that binding of Trp53 to Notch 1 is important for Mdm2-mediated degradation of Notch1.…”

Gliotoxin is a potent NOTCH2 transactivation inhibitor and efficiently induces apoptosis in chronic lymphocytic leukaemia (CLL) cells

“…Besides the key role of NOTCH2 in CLL, NOTCH1 gain of function mutations are found in a small group of CLL patients (Fabbri et al, 2011;Puente et al, 2011). NOTCH1 has been identified as a direct target gene of TP53 and might counteract the apoptotic effect of TP53 in CLL cells (Secchiero et al, 2009).…”

p53 and Notch signaling in chronic lymphocytic leukemia: clues to identifying novel therapeutic strategies

“…An intriguing link between p53 and the Notch 1 pathway has been revealed in a study by Secchiero et al 27 Treatment of primary CLL cells with nutlin 3 resulted in elevation of both p53 and Notch 1. Furthermore, g-secretase inhibitors, which block the generation of the active Notch proteins (Figure 2), augmented the cytotoxicity of nutlin 3, compatible with the interpretation that induction of Notch 1 expression by p53 initiated an antiapoptotic feedback mechanism that limited the cytotoxic actions of nutlin 3.…”

“…24 The Notch signaling pathway Recent evidence suggests that the Notch signaling pathway is a downstream target of p53. 25 The p53/Notch interaction may account for some recent observations in CLL cells, 26,27 which are described in detail below. We therefore review briefly the molecular regulation of the Notch pathway.…”

“…The p53 dependence of nutlin-induced Notch 1 expression was demonstrated by the observation that Notch 1 elevation occurred in p53 wild-type myeloid and lymphoid leukemic cell lines but not in lines lacking a functional p53 gene. 27 Furthermore, silencing of p53 expression by transfection of small interfering RNA abrogated induction of Notch 1 by nutlin. Silencing of Notch 1 by small interfering RNA enhanced the cytotoxic actions of nutlin 3, elegantly demonstrating the antiapoptotic role of Notch 1 induction.…”