Journal of the National Cancer Institute Advance Access published online on November 10, 2009
JNCI Journal of the National Cancer Institute, doi:10.1093/jnci/djp376
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© The Author 2009. Published by Oxford University Press.
EDITORIAL |
Targeted Molecular Therapy for Neuroblastoma: The ARF/MDM2/p53 Axis
Affiliations of authors: Micheal E. Debakey Department of Surgery (EK) and Department of Pediatrics (JS), Baylor College of Medicine, Houston, TX
Correspondence to: Jason Shohet, MD, PhD, Texas Children's Cancer Center, Department of Pediatrics, Baylor College of Medicine, Feigin Building, Room 750.01, 1102 Bates St, Houston, TX 77030 (e-mail: jmshohet{at}texaschildrenshospital.org).
Neuroblastoma remains a major therapeutic challenge in pediatric oncology despite decades of intensive research and therapeutic trials. This aggressive embryonal malignancy of neural crest origin has a peak age of onset of 22 months and accounts for approximately 11% of all pediatric cancers and 15% of all pediatric cancer deaths (1). With current treatment protocols, including high-dose chemotherapy with autologous stem cell transplantation, radiation, and surgery, about 80% of high-risk patients will go into remission (2). However, the majority of these patients relapse and succumb to therapy-resistant tumors and have long-term survival rates of less than 50%. As illustrated by the work presented by Van Maerken et al. (3), studies using both cell culture and animal models of neuroblastoma are producing exciting new discoveries into its pathogenesis and molecular biology. These insights provide hope that rationally designed and targeted molecular therapeutics can be translated into less toxic and more effective treatments for neuroblastoma.
A key feature of neuroblastoma is that it is uniformly p53 wild-type at diagnosis (4) with intact intrinsic and extrinsic apoptotic mechanisms, including mitochondrial-mediated cytochrome c release and caspase activation. Although altered in vitro expression of mitochondrial-associated proapoptotic and antiapoptotic Bcl-2 family members, for example, Mcl-1 (5) and the inhibitor of apoptosis proteins [eg, survivin (6)] are found in neuroblastoma cell lines, in vivo transgenic neuroblastoma and human xenograft models respond to genotoxic stress with robust wild-type p53-mediated responses. These include stabilization of nuclear p53, increased p53 transcriptional activity, cell cycle arrest, and apoptosis (7,8). Clinically, genotoxic chemotherapy induces a rapid reduction in tumor volume in more than 80% of high-risk patients (2). In those patients with chemoresistant disease at diagnosis, mutant p53 is exceptionally rare because more than 98% of de novo neuroblastomas have wild-type p53 by sequence analysis (9).
As a central modulator of numerous essential cellular processes, p53 is positioned at the nexus of many upstream regulators [reviewed in (10,11)]. The primary inhibitor of p53 activity is MDM2, an E3 ligase, which, in complex with MDM4 and the E2 ligase UBcH5a, acts to both ubiquitinate p53 and repress its transcriptional activity (12). MDM2 is in turn inhibited by the tumor suppressor p14ARF, which can bind to and prevent MDM2-mediated ubiquitination of p53 and activate p53 responses (13). In normal cells, tight feedback regulation of the ARF/MDM2/p53 axis controls p53 activity through a host of protein modifications (eg, phosphorylation, acetylation, sumoylation) as well as ubiquitination and deubiquitination and limits the effects of p53 activation (Figure 1) [reviewed in (14,15)].
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In cancer cells with intact p53 activity such as neuroblastoma, MDM2 inhibition and subsequent rapid increases in nuclear p53 levels potently "re-activate" dormant apoptotic pathways and rapidly induce apoptotic cell death (16). Detailed insight into control of p53 in normal and malignant cells has created an opportunity to develop rationally designed drugs such as Nutlin-3a (17), which specifically blocks MDM2-p53 interactions with high affinity at low micromolar concentrations. Such novel therapeutic approaches exploit the intact apoptotic machinery found in neuroblastoma and potentially other p53 wild-type malignancies.
Clues to the mechanism of resistance to p53-mediated apoptosis come from the embryonal origins of neuroblastoma. Neuroblastoma arises from a subset of neural crest cells, which migrate from the central nervous system and are destined to form the enteric sympathetic nervous system and sympathetic ganglia (18,19). Rapid apoptosis within the ganglia microenvironment is a normal component of the final differentiation and modeling of the sympathetic nervous system (20). This process is partially regulated in normal neural crest cells via PIK/AKT and transient MYCN signaling (21). In the well-characterized MYCN-transgenic mouse model of neuroblastoma, targeted expression of MYCN within the neural crest lineage leads to hyperproliferative "rests" of PHOX2b/Nestin-positive neuroblasts in the maturing ganglia (22). These neuroblasts proliferate and transform despite the presence of wild-type p53 and strong developmentally programmed differentiation and apoptotic stimuli within the microenvironment (18,23).
Recent data from a compound transgenic mouse model demonstrate that this process is markedly restrained by Mdm2 haploinsufficiency, suggesting that aberrant regulation of MDM2 by the MYCN transgene is an important component of overall suppression of p53 in neuroblastoma (23). A similar requirement for two functional alleles of Mdm2 to elicit maximal C-Myc–driven tumorigenesis is also demonstrated in the eu-Myc–driven mouse lymphoma model (24,25). Thus, in both normal and transformed neuroblasts, MYCN and MDM2 work together to inhibit apoptosis. This process is likely retained within neuroblastoma tumor stem cells (which share many attributes with their neural crest precursors), further supporting the therapeutic concept of using small molecule inhibitors to disrupt the MDM2/p53 interaction.
Because the majority of children with neuroblastoma succumb to chemoresistant relapse, Van Maerken et al. sought to determine the efficacy of targeting MDM2/p53 interaction in chemoresistant and sensitive cell lines using the small molecule MDM2 inhibitor Nutlin-3a. In vitro, they demonstrate comparable IC50 values for both p53 wild-type parental cells and a doxorubicin-resistant subclone (between 3 and 4 um) and a 10-fold higher IC50 for a p53 mutant (missense mutant 404T>G) vincrinstine-resistant subclone. These results were corroborated with in vivo tumor growth studies using a subcutaneous xenograft model system and oral administration of Nutlin-3a. Interestingly, they also showed that MDM2 inhibition with Nutlin-3a reduces metastases from the subcutaneous xenografts. These elegant preclinical data are the first to demonstrate the efficacy of p53 reactivation in a chemoresistant model of neuroblastoma and set the stage for further clinical and preclinical investigations.
Their results should be extended to additional animal models of neuroblastoma and further study should include combining MDM2 inhibition with additional molecular targets that are synergistic with the effects of Nutlin-3a in other tumor systems [ie, histone deacetylase inhibitors (26,27), mammalian target of rapamycin inhibitors (28), and Aurora kinase inhibitors (29)]. Van Maerken et al. demonstrated a reduction in metastasis with MDM2 inhibition in their subcutaneous xenografts. In vivo studies on tumor metastasis and angiogenesis that involve tumor–microenvironment interactions are ideally modeled in an orthotopic setting. A well-characterized orthotopic site for neuroblastoma is direct injection into the perirenal capsule (30,31). The treatment of this type of neuroblastoma xenograft with MDM2 inhibitors alone and in combination with other therapies may well reveal additional synergistic sensitivities to this therapeutic approach and support rapid translation of this approach into the clinic.
An important clinical correlation is the rate of p53 mutations found in human neuroblastoma at relapse or after major chemotherapy treatment. Available evidence from tumor cell lines derived from patient samples suggest that less than 15% of relapsed samples harbor mutant p53 (32). However, the upstream regulators of p53 appear to be frequently altered. In particular, suppression of the MDM2 inhibitor p14ARF through multiple mechanisms (ie, deletions, epigenetic silencing, etc.), amplification of MDM2, and elevated expression of ARF inhibitors BMI-1 (33) and TWIST-1 (34) are all found in subsets of relapsed samples (Figure 1) (3,35). Of note, these chemoresistant relapsed tumors with suppressed p14ARF and increased MDM2 activity should maintain sensitivity to MDM2 inhibition, as suggested by Van Maerken et al. (3). Additional mechanisms of p53 suppression are likely prevalent in chemoresistant neuroblastoma, underscoring the need for detailed molecular analysis (including p53 sequencing) of relapsed tumors so that therapy can be tailored to these patients.
Modern chemotherapy-based treatment for neuroblastoma has reached the maximum safe limits on dose intensity and incurs a high cost in terms of long-term and late side effects (36). Despite these aggressive treatment regimens, approximately half of the high-risk patients still relapse some time after completing therapy, and about 20% of tumors are refractory to genotoxic treatment at diagnosis. The fact that direct inactivating p53 mutations are rare regardless of stage of treatment suggests that neuroblastoma has an innate requirement for baseline p53 activity (perhaps to resist oncogenic stress). As modeled by Van Maerken et al., the chemoresistant relapsed tumors may still be sensitive to MDM2 inhibition and can be combined with other nongenotoxic-targeted therapies. Further in vivo modeling to optimize the best clinical use of MDM2 inhibition and phase I trials of this approach is urgently needed to define the safety and efficacy targeting the ARF/MDM2/p53 axis in children suffering from this aggressive cancer.
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