Abstract
Neuroblastoma is the most common childhood extracranial solid tumor. In high-risk cases, many of which are characterized by amplification of MYCN, outcome remains poor. Mutations in the p53 (TP53) tumor suppressor are rare at diagnosis, but evidence suggests that p53 function is often impaired in relapsed, treatment-resistant disease. To address the role of p53 loss of function in the development and pathogenesis of high-risk neuroblastoma, we generated a MYCN-driven genetically engineered mouse model in which the tamoxifen-inducible p53ER(TAM) fusion protein was expressed from a knock-in allele (Th-MYCN/Trp53(KI)). We observed no significant differences in tumor-free survival between Th-MYCN mice heterozygous for Trp53(KI) (n = 188) and Th-MYCN mice with wild-type p53 (n = 101). Conversely, the survival of Th-MYCN/Trp53(KI/KI) mice lacking functional p53 (n = 60) was greatly reduced. We found that Th-MYCN/Trp53(KI/KI) tumors were resistant to ionizing radiation (IR), as expected. However, restoration of functional p53ER(TAM) reinstated sensitivity to IR in only 50% of Th-MYCN/Trp53(KI/KI) tumors, indicating the acquisition of additional resistance mechanisms. Gene expression and metabolic analyses indicated that the principal acquired mechanism of resistance to IR in the absence of functional p53 was metabolic adaptation in response to chronic oxidative stress. Tumors exhibited increased antioxidant metabolites and upregulation of glutathione S-transferase pathway genes, including Gstp1 and Gstz1, which are associated with poor outcome in human neuroblastoma. Accordingly, glutathione depletion by buthionine sulfoximine together with restoration of p53 activity resensitized tumors to IR. Our findings highlight the complex pathways operating in relapsed neuroblastomas and the need for combination therapies that target the diverse resistance mechanisms at play. Cancer Res; 76(10); 3025-35. ©2016 AACR.
Highlights
Neuroblastoma is the most common childhood extracranial solid tumor
The p53 pathway was induced by ionizing radiation (IR) as evidenced by increased levels of transcriptionally active p53 phosphorylated at serine 15 (p-p53Ser15) in tumors, spleen, and thymus (Supplementary Fig. S1B), as well as an increase in the p53 target gene Cdkn1a (p21Cip1/Waf1; Supplementary Fig. S1C)
Induction of DNA damage was demonstrated by the presence of phosphorylated histone gamma-H2AX (g-H2AX), primarily in the spleen and the gut (Supplementary Fig. S2). These results suggest that Th-MYCN mice are representative of p53 WT human neuroblastoma
Summary
Neuroblastoma is the most common childhood extracranial solid tumor. In high-risk cases, many of which are characterized by amplification of MYCN, outcome remains poor. To address the role of p53 loss of function in the development and pathogenesis of high-risk neuroblastoma, we generated a MYCN-driven genetically engineered mouse model in which the tamoxifen-inducible p53ERTAM fusion protein was expressed from a knock-in allele (Th-MYCN/Trp53KI). In contrast with other MYC-driven cancers, such as medulloblastoma [5] and lymphoma, mutation of genes in the p53 pathway is rare at diagnosis [6], implying that selection for p53 pathway deficiency occurs following treatment Additional mechanisms such as epigenetic alterations and metabolic adaptation have been www.aacrjournals.org implicated in aggressive neuroblastoma [7,8,9,10,11]. To better understand the role of p53 in the development of high-risk therapyresistant neuroblastoma, we generated a MYCN-driven genetically engineered mouse (GEM) model with inducible p53 loss of function
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