Treatment of Ewing sarcoma family of tumours through the pharmacological activation of p53.

Abstract

Sarcomas constitute a diverse heterogeneous group of solid bone and soft tissue malignancies of mesenchymal origin. It is estimated that sarcomas account for approximately 15% of all paediatric and 1% of all adult cancers, with 200,000 new cases reported worldwide each year. To date 60 distinct histological subtypes have been described, ranging from indolent to highly invasive and metastatic. Clinical management primarily consists of wide excisional surgery in conjunction with adjuvant therapies (radiotherapy or chemotherapy) depending on the subtype. Despite significant strides in understanding the cytogenetic profiles of sarcomas, limited improvement in overall survival rates has been achieved over the past few decades for most sarcoma subtypes. The use of multi-agent schedules and dose intensification in patients with chemo-sensitive subtypes has yielded some improvement in survival but at the expense of significantly increased toxicity and risk of developing secondary malignancies. In light of the limitations of systemic chemotherapy, particularly for those sarcoma subtypes that are intrinsically chemo-resistant, new targeted therapeutic modalities are urgently required. Tumourigenesis is a multifaceted process that requires dysregulation of several pathways that are essential for cellular growth and survival. One such pathway critical for the prevention of oncogenic transformation is mediated by the tumour suppressor p53. The TP53 gene located at 17p13 encodes a 53-kDa nuclear phosphoprotein with sequence-specific DNA-binding properties. In response to various cellular and oncogenic insults, p53 drives the expression of specific target genes required for the initiation of cell cycle arrest, apoptosis, DNA damage repair, and senescence pathways. Underscoring its pivotal role against tumour development, the p53 gene (TP53) is mutated in at least 50% of all human malignancies. In the remaining wild-type p53 tumours, p53 function is suppressed through various mechanisms. In the quest for more effective cancer therapeutics, considerable research has been undertaken to reinstate p53 function in wild-type p53 tumour cells through the use of small targeted agents. As sarcomas are predominately of wild-type p53 status with less than 20% TP53 mutations, this unique tumour group presents an ideal model system for the pre-clinical testing of p53-based therapies. One mechanism frequently employed by wild-type p53 tumours to circumvent the tumour surveillance function of p53 is through overexpression or amplification of MDM2 (Murine Double Minute 2) or MDM4 (structural homologue of MDM2). MDM2 is a key E3 ubiquitin ligase that targets p53 for ubiquitin-dependent degradation, thereby tightly regulating the stability and subcellular localization of p53. In contrast, MDM4 primarily regulates the transcriptional activity of p53 as it possesses no intrinsic E3 ligase activity and therefore cannot directly promote the degradation of p53. Crystallization studies of the MDM2–p53 complex revealed that three residues within the transactivation domain of p53 (Phe¹⁹, Trp²³ and Leu²⁶) were responsible for binding the hydrophobic cleft located on the N-terminal surface of MDM2. The well-defined, small interface of MDM2-p53 has led to the design of numerous small-molecule inhibitors to target the MDM2-p53 interaction. The most well-known and extensively studied MDM2-p53 antagonist is Nutlin-3a. Identified by Vassilev and colleagues (Hoffmann- La Roche), this cis-imidazoline compound effectively binds the p53-binding groove of MDM2 by mimicking the interactions of the three key p53 amino acids. Promising results from several preclinical studies have demonstrated the therapeutic potential of Nutlin-3a in various solid and haematological malignancies with wild-type p53. As the clinical translation of MDM2 inhibitors is relatively advanced with Nutlin-3a (RG7112) entering phase II trials, the principal focus of the research detailed in this thesis was to evaluate whether pharmacological activation of the p53 pathway can provide a new therapeutic means for the targeted treatment of sarcomas, in particular Ewing sarcoma. In addition to Nutlin-3a, the ability of low dose actinomycin D and SJ-172550 (MDM4 inhibitor) to restore p53 function has also been assessed.