High Frequency of p53/MDM2/p14ARF Pathway Abnormalities in Relapsed Neuroblastoma

Abstract

Purpose: Most neuroblastomas initially respond to therapy but many relapse with chemoresistant disease. p53 mutations are rare in diagnostic neuroblastomas, but we have previously reported inactivation of the p53/MDM2/p14 ARF pathway in 9 of 17 (53%) neuroblastoma cell lines established at relapse. Hypothesis: Inactivation of the p53/MDM2/p14 ARF pathway develops during treatment and contributes to neuroblastoma relapse. Methods: Eighty-four neuroblastomas were studied from 41 patients with relapsed neuroblastoma including 38 paired neuroblastomas at different stages of therapy. p53 mutations were detected by automated sequencing, p14 ARF methylation and deletion by methylation-specific PCR and duplex PCR, respectively, and MDM2 amplification by fluorescent in situ hybridization.Results: Abnormalities in the p53 pathway were identified in 20 of 41 (49%) cases. Downstream defects due to inactivating missense p53 mutations were identified in 6 of 41 (15%) cases, 5 following chemotherapy and/or at relapse and 1 at diagnosis, postchemotherapy, and relapse. The presence of a p53 mutation was independently prognostic for overall survival (hazard ratio, 3.4; 95% confidence interval, 1.2-9.9; P = 0.02). Upstream defects were present in 35% of cases: MDM2 amplification in 3 cases, all at diagnosis and relapse and p14 ARF inactivation in 12 of 41 (29%) cases: 3 had p14 ARF methylation, 2 after chemotherapy, and 9 had homozygous deletions, 8 at diagnosis and relapse. Conclusions: These results show that a high proportion of neuroblastomas which relapse have an abnormality in the p53 pathway. The majority have upstream defects suggesting that agents which reactivate wild-type p53 would be beneficial, in contrast to those with downstream defects in which p53-independent therapies are indicated. Clin Cancer Res; 16(4); 1108-18. ©2010 AACR.Neuroblastoma is the most common extracranial pediatric solid tumor. It remains one of the most difficult cancers to cure, with <40% of patients with high-risk disease (stage 4 over 18 months of age or MYCN-amplified disease) becoming long-term survivors. Most high-risk neuroblastomas initially respond to cytotoxic therapy, however, over half relapse with chemoresistant disease and this often correlates with the intensity of therapy (1).The p53 gene is inactivated by mutation in >50% of human malignancies (2). p53 is a key regulator of cell cycle checkpoints and apoptosis, which upon activation by cellular stress, particularly DNA damage, binds DNA in a sequence-specific manner to activate the transcription of a large number of downstream genes, including p21 and MDM2, which results in apoptosis, cell cycle arrest, differentiation, and DNA repair (reviewed in ref.3). MDM2 functions upstream of p53 as a ubiquitin ligase that targets p53 for proteosome-mediated degradation, forming an autoregulatory feedback loop which tightly regulates p53 cellular levels (4). MDM2 amplification has been shown in some tumors and could suppress the activity of p53 by increasing its degradation.The INK4...

Checkpoint kinase inhibitor synergizes with DNA‐damaging agents in G1 checkpoint‐defective neuroblastoma

“…Recent studies found that, about half of the relapsed neuroblastoma tumors had aberrations in the p53/MDM2/p14 ARF pathway, with one-third being p53 mutations and two-third being MDM2/ p14 ARF abnormalities. 13 Thus, even if not all MDM2/p14 ARF abnormalities were responsive to Chk1 inhibition as our findings suggested, it is conceivable about one-third of all relapsed neuroblastoma patients could potentially benefit from a combination therapy utilizing Chk1 inhibitor and chemotherapeutic drug. Moreover, since recent studies suggest that most p53 mutations found in neuroblastomas are acquired after chemotherapy, while MDM2/p14 ARF abnormalities and MDM2 polymorphism are found primarily at diagnosis, 8,10,11,13 it seems reasonable to exploit Chk1 inhibitors either early on at diagnosis in patients with documented MDM2/p14 ARF abnormalities, or after recurrence when tumors have acquired p53 mutations.…”

“…10,11 Deregulation of the p53/MDM2/p14 ARF pathway has also been linked to TWIST1, BMI1, PPM1D, and loss of CHD5, as well as cytoplasmic sequestration of p53. 12 The high frequency ($ 50%) of aberrations in the p53/MDM2/ p14 ARF pathway found in both neuroblastoma cell lines established at relapse and in relapsed neuroblastoma tumors 7,13 raises the possibility that p53 pathway defect could be a key de novo and acquired chemoresistance mechanism. 12 In this study, we utilized a representative panel of neuroblastoma cell lines with aberrations in their p53/MDM2/ p14 ARF pathway, to study their G 1 checkpoint status and chemoresistance.…”

Functional Analysis of the p53 Pathway in Neuroblastoma Cells Using the Small-Molecule MDM2 Antagonist Nutlin-3

“…A remarkable feature is that p53 is rarely mutated at diagnosis and only in a minority of neuroblastoma tumors at relapse, as shown by a recent study that found mutation rates of 2% and 15%, respectively (3). Conflicting data exist about p53 pathway signaling in neuroblastoma cells.…”

Small Molecule Drugs and Targeted Therapies for Neuroblastoma

“…Recently, a higher incidence of p53 mutations was found in a study on 84 neuroblastomas from 41 patients with relapsed disease, including 38 paired neuroblastomas at different stages of therapy. Inactivating missense p53 mutations were identified in 6/41 (15%) cases, 5 following chemotherapy and/or at relapse and only 1 at both diagnosis and relapse (Carr-Wilkinson, O'Toole et al, 2010). Studies on neuroblastoma cell lines demonstrated that loss of p53 function due to mutations of p53 gene or gene silencing by p53 shRNA can confer multidrug resistance to neuroblastoma cells (Keshelava, Zuo et al, 2000;Keshelava, Zuo et al, 2001;Xue, Haber et al, 2007).…”

“…In the majority of neuroblastoma tumours maintaining wild-type p53 gene, an increasing amount of data shows that the p53 pathway may not be functional due to non-mutational mechanisms. For example, a high incidence of abnormalities in the p53/MDM2/p14 ARF pathway was found in human neuroblastoma cell lines established at relapse (53%) (Carr, Bell et al, 2006) and patient samples at relapse (49%) (Carr-Wilkinson, O'Toole et al, 2010). MDM2, the essential negative regulator of p53, is transcriptionally regulated by the MYCN oncogene in neuroblastoma (Slack, Chen et al, 2005).…”