Thesis (Ph. D.)--State University of New York at Binghamton, Department of Biological Sciences, 2009. / Includes bibliographical references (leaves 128-145).

Lung cancer is the most common cause of cancer-related mortality worldwide with current treatments providing limited therapeutic benefit in most cases. TP53 (Trp53, p53) mutations occur in approximately 50% of lung adenocarcinoma cases and are associated with poor prognosis and so novel therapies that target these p53 mutant lung tumours are urgently needed. Despite the high frequency of p53 mutations in lung tumours, the impact these mutations have on response to therapy remains unclear in this cancer type. The aim of my project is to characterise the gain-of-function and dominant-negative effects of p53 mutations in lung tumours and to identify ways of therapeutically targeting these p53 mutant tumours based on dependencies and susceptibilities that our analysis uncovers. To characterise the gain-of-function and dominant-negative effects of p53 mutations I compared p53 mutant murine lung tumour cells that endogenously express either a contact (R270H, equivalent to R273H in humans) or conformational (R172H, equivalent to R175H in humans) p53 mutant protein and p53 null lung tumour cell lines; both in the presence and absence of wild-type p53. Interestingly, transcriptional and functional analysis uncovered metabolic gain-of-functions that are specific to the type of p53 mutation. Upregulation of mevalonate pathway expression was observed only in R270H lung tumours and consequently R172H and R270H lung tumours displayed distinct sensitivities to simvastatin, a mevalonate pathway inhibitor widely used in the clinic. Furthermore, the transcriptional signature underlying this sensitivity to simvastatin was also present in human lung tumours with contact p53 mutations, indicating that these findings may be clinically relevant. On the other hand, our analysis of the potential dominant-negative effects of the p53 mutants on wild-type p53 demonstrated that wild-type p53 was able to induce typical p53 target genes to a similar level in p53 null and mutant cells. Furthermore, wild-type p53 restoration resulted in comparable tumour suppressive responses in p53 mutant and null tumours and thus, p53-restoration therapy will likely be of benefit to patients with p53 mutations in lung cancer. Hence, I have demonstrated that lung tumours harbouring contact and conformational p53 mutations display common and distinct therapeutic susceptibilities.

TP53 est un gène suppresseur de tumeur muté dans la moitié des cancers humains. La plupart des mutations de TP53 affectent le domaine de liaison à l’ADN de la protéine, et sont situées au niveau de sept « hotspot » mutationnels. Parmi elles, la mutation Y220C est retrouvée dans 100 000 nouveaux cas de cancer par an et dans 10 familles Li-Fraumeni de prédisposition au cancer. Pour étudier cette mutation in vivo, j’ai créé au cours de ma thèse le modèle murin exprimant une protéine p53 portant la mutation Y217C équivalente à la Y220C humaine. J’ai pu démontrer que non seulement la mutation inactive la fonction de la protéine mais qu’elle semble également conférer de nouvelles fonctions oncogéniques. Plus intéressant encore, ce modèle constitue un très bon outil pour le test d’une nouvelle stratégie thérapeutique spécifique de cette mutation. Par ailleurs, le modèle murin p53∆31/∆31 a été créé dans notre laboratoire et a permis de démontrer in vivo le rôle de régulateur négatif du domaine C-terminal de p53 sur son activité. Au cours de mes travaux de recherche, j’ai pu montrer que p53 joue un rôle dans la régulation de nouvelles voies cellulaires chez la souris et chez l’Homme, et établi le mécanisme en jeu dans cette régulation. De façon surprenante, ces fonctions insoupçonnées sont impliquées dans la promotion de l’instabilité génétique et une activité accrue de p53 peut entraîner des maladies d’insuffisance médullaire prédisposant au cancer. Nos observations ont donc des retombées fondamentales mais également cliniques pour le diagnostic de ces maladies ainsi que pour les stratégies anti-tumorales selon le statut de p53. / TP53 is a tumor suppressor gene mutated in half of human cancers. Most of TP53 mutations affect the core DNA binding domain of the protein, and are located on seven « hotspot » residues. Among them, the Y220C mutation is found in 100 000 new cancer cases per year and in 10 Li-Fraumeni families of cancer predisposition. To study this mutation in vivo, I created a mouse model carrying the Y217C mutation, equivalent to the human Y220C. I showed that not only this mutation inactivates the function of the protein, but that it also confers new oncogenic functions. Moreover, this model is a great tool to evaluate the efficiency of a novel anti-tumoral strategy in vivo. Furthermore, the mouse p53∆31/∆31 model was created in our team and allowed us to demonstrate the negative regulatory role of the C-terminus of p53 in vivo. I used this model to discover the implication of p53 in the regulation of unsuspected cellular pathways in mice but also in humans, and to establish the mechanism of regulation. Surprisingly, these functions are responsible for the promotion of the genomic instability and p53’s high activity can cause bone marrow failure syndromes that predispose to cancer. Our observations have both fundamental and clinical impact for the diagnosis of these diseases and for the therapeutic anti-tumoral strategies according to p53’s status.

Le cancer est responsable de plus de 15% des décès. L’activation d’oncogènes et l’inactivation de gènes suppresseurs de tumeur contribuent à la transformation des cellules. Dans 50% des cas de cancers le gène TP53 est muté. C’est pourquoi comprendre les conséquences de ces mutations est indispensable pour développer des tests permettant de cibler p53 dans le cadre de thérapies. Dans cette étude nous avons utilisé la nouvelle technique de modification de génomes, CRISPR-Cas9. Cette technique a été utilisée dans le but d’introduire des mutations spécifiques de TP53 dans le génome de fibroblastes non tumoraux. Nous avons alors analysé les effets de ces mutations au niveau transcriptionnel et protéomique. Ces analyses aideront à identifier les effets spécifiques de chaque mutation de p53. Ces résultats seront utilisés pour établir des lignées cellulaires permettant de cribler et d’identifier des composés capables de restaurer la fonction sauvage de p53. / Cancer is responsible for more than 15% of human deaths. Activation of oncogenes and inactivation of tumor suppressor genes contribute to malignant transformation of cells. Mutations of the tumor suppressor gene TP53 are observed in about 50% of human cancers. Therefore, it is of high interest to understand functional consequences of TP53 mutations in order to develop biological tests that allow targeting mutant p53 for oncotherapy. In this study we use CRISPR-Cas9, the latest genome editing technique, for introducing specific TP53 mutations into the genome of a non-tumoral fibroblast cell line. We analyze the effects of p53 mutations at the transcriptomic and proteomic level. These analyses will help identifying gene- and pathway-specific effects of distinct p53 mutations. These results will be used for establishing cell lines that allow high throughput screening, in order to discover new chemical compounds that are able to restore crucial functions of mutant p53 proteins.

Doxorubicin (Dox) is a chemotherapeutic agent that causes significant cardiotoxicity. We showed previously that Dox activates p53 and induces apoptosis in mouse hearts. This study was designed to elucidate the molecular events that lead to p53 stabilization, to examine the pathways involved in Dox-induced apoptosis, and to evaluate the effectiveness of pifithrin-α (PFT-α), a p53 inhibitor, in blocking apoptosis of rat H9c2 myoblasts. H9c2 cells that were exposed to 5 μM Dox had elevated levels of p53 and phosphorylated p53 at Ser15. Dox also triggered a transient activation of p38, p42/p44ERK, and p46/p54JNK MAP kinases. Caspase activity assays and Western blot analysis showed that H9c2 cells treated with Dox for 16 h had marked increase in the levels of caspases-2, -3, -8, -9, -12, Fas, and cleaved poly(ADP ribose) polymerase (PARP). There was a concomitant increase in p53 binding activity, cytochrome c release, and apoptosis. These results suggest that Dox can trigger intrinsic, extrinsic, and endoplasmic reticulum-associated apoptotic pathways. Pretreatment of cells with PFT-α followed by Dox administration attenuated Dox-induced increases in p53 levels and p53 binding activity and partially blocked the activation of p46/p54JNK and p42/p44ERK. PFT-α also led to decreased levels of caspases-2, -3, -8, -9, -12, Fas, PARP, cytochrome c release, and apoptosis. Our results suggest that p53 stabilization is a focal point of Dox-induced apoptosis and that PFT-α interferes with multiple steps of Dox-induced apoptosis.

Apesar do excelente prognóstico dos tumores de Wilms (TW) localizados (estádios I e II) e de histologia favorável (HF), 10% deles recidivam. Em 122 pacientes com TW com essas características, diagnosticados de 1976 e 2001, analisamos alguns fatores clínicos, como a idade por ocasião do diagnóstico e peso do tumor, em todos os pacientes; fatores biológicos, como o TP53 e a glicoproteína-p, em 40 deles; e variáveis histológicas de microestadiamento (invasão de seio renal, cápsula tumoral, vasos intra-renais e pseudocápsula inflamatória) em 28 com TW em estádio I. Correlacionando todos esses fatores com a presença de recidiva, observamos que a chance maior de recidiva estatisticamente significativa somente foi verificada em pacientes com duas ou mais variáveis de microestadiamento e/ou peso tumoral maior que 550 g / In spite of the excellent prognosis of localized favorable histology (FH) of Wilms' tumor (WT), 10% of them will relapse. In 122 TW patients with these characteristics, diagnosed between 1976 and 2001, some clinical factors have been analyzed, such as age at diagnosis and tumor weight in all patients; biological factors, like TP53 and p-glycoprotein, in 40 of them; and microsubstaging histological variables (invasion of renal sinus, tumor capsule, intrarenal vessels, and inflammatory pseudocapsule). Correlating all of those factors with relapse, we have observed that only patients with the association of two or more microsubstaging variables and/or tumor weight over 550 g showed a statistically significant higher chance of relapse

The DNA damage response is an important barrier to tumorigenesis. Impairment of p53 function is crucial to tumorigenesis by allowing evasion of p53 dependent responses. The mechanisms involve either (i) missense mutations, (ii) partial abrogation of signaling pathways or effector molecules that regulate p53, (iii) epigenetic deregulation. The tyrosine to cysteine mutation, Y220C, in p53 is found in around 100,000 new cancer cases per annum. This mutation destabilizes the core domain by 4 kcal mol-1 and destabilizes p53 under physiological conditions. The large to small mutation results in the fusing of two shallow pockets to create an extended surface cleft that a number of different fragments bind. The small molecule PK083, 1-(-ethyl-9H-carbazol-3-yl)-N-methanamine, binds the mutant-specific crevice with a KD = 150 μM and raised the protein mutant's half-life to over 15 minutes vs. 4 minutes in the absence of the ligand. This presents an ideal starting point towards the design of a p53 rescuing drug. A library of carbazoles was designed and synthesized, guided by SAR studies, crystallographic information and computational chemistry, with the aim of optimizing the structure toward a more potent PK083 analogue. Affinity gains were achieved by exploitation of direct fluorine-protein interactions between PK9255 (N-methyl-1-(9- (2,2,2-trifluoroethyl)-9H-carbazol-3-yl)methanamine), and the backbone carbonyls of Leu145 and Trp146 and the thiol of Cys220, resulting in a Kd = 28 μM. Further affinity gains were achieved through SAR studies targeting the proline-rich subsite II. Chemistry was optimized to allow a diversity-oriented synthesis toward 2,6,9- substituted carbazoles. A small library of PK083 analogues, where the subsite II targeting group was a halogen, ether, ester, amide or heterocycle were synthesized, identifying the heterocyclic compounds as most potent. A scan of heterocyclic compounds was carried out to identify the most potent heterocyclic substitution.

Apesar do excelente prognóstico dos tumores de Wilms (TW) localizados (estádios I e II) e de histologia favorável (HF), 10% deles recidivam. Em 122 pacientes com TW com essas características, diagnosticados de 1976 e 2001, analisamos alguns fatores clínicos, como a idade por ocasião do diagnóstico e peso do tumor, em todos os pacientes; fatores biológicos, como o TP53 e a glicoproteína-p, em 40 deles; e variáveis histológicas de microestadiamento (invasão de seio renal, cápsula tumoral, vasos intra-renais e pseudocápsula inflamatória) em 28 com TW em estádio I. Correlacionando todos esses fatores com a presença de recidiva, observamos que a chance maior de recidiva estatisticamente significativa somente foi verificada em pacientes com duas ou mais variáveis de microestadiamento e/ou peso tumoral maior que 550 g / In spite of the excellent prognosis of localized favorable histology (FH) of Wilms' tumor (WT), 10% of them will relapse. In 122 TW patients with these characteristics, diagnosed between 1976 and 2001, some clinical factors have been analyzed, such as age at diagnosis and tumor weight in all patients; biological factors, like TP53 and p-glycoprotein, in 40 of them; and microsubstaging histological variables (invasion of renal sinus, tumor capsule, intrarenal vessels, and inflammatory pseudocapsule). Correlating all of those factors with relapse, we have observed that only patients with the association of two or more microsubstaging variables and/or tumor weight over 550 g showed a statistically significant higher chance of relapse

p53 is a pivotal tumour suppressor in mammalian cells. It protects the integrity of a number of cellular pathways, preventing the malignant transformation of cells. There is however perhaps nothing more efficient at disrupting cellular pathways than a virus. Viruses infiltrate cells commandeering the normal growth and survival pathways for their narcissistic needs. While the association between viral infections and the induction of p53 has long been recognised, there is controversy surrounding the ultimate role of p53 during a virus infection. The classical model of p53 in an adenovirus infection is that p53 is a formidable obstacle which needs to be overcome. Adenoviruses overcome p53 by degrading the protein and removing its ability to transactivate its target genes. However the degradation is not immediate and there is increasing evidence which would suggest p53 is actually beneficial to an adenovirus infection. In the introductory chapter, I review what is known about p53 and virus infections. What emerges from this review is the sheer number of interactions that occur between viruses and p53, indicating its importance in an infection. Additionally it shows that adenoviruses are not the only virus shown to benefit from the presence of p53. What beneficial role p53 may be fulfilling in an adenovirus infection is unclear. The experiments reported in this thesis investigate the functions of p53 in an adenovirus infection. In Chapter Three, immunoblots on a panel of adenovirus infected cells reveal that p53 levels do not correlate with the level of the classical p53 target proteins. This indicates that p53 is disconnected from its target genes during an infection. Promoter/reporter assays carried out on infected cells show that adenovirus can directly regulate p53 target genes independently of p53. In Chapter Five, I show this regulation is dependent on E1a, with transient transfection of E1a resulting in the marked activation of p53 target promoters. E1a mediated transactivation appears to be reliant on the largest splice variant of E1a (E1a-289R) and the presence of pRB. Electrophoresis mobility shift assays reveal that the transcription factor Sp1 is involved. In Chapter Four, p53 transcription in an adenovirus infection was directly assayed by using an artificial p53 consensus response element. The results show that p53 is unable to activate its consensus response element during an infection. However, I show that p53 is transcriptionally competent in an infection, and is able to transactivate a mutant derivative of the p53 consensus sequence. This shows that p53 is not only transcriptional competent but has a gain-of-function in an infection. This gain-of-function requires E1a, and appears not to be due to a change in the DNA binding affinity of p53. The data in this thesis show that adenoviruses not only appear to inhibit and control the normal transcriptional profile of p53 but appear to modify p53, giving it a new transcriptional profile. This provides a possible mechanism by which p53 could aid an adenovirus infection.

In the studies described in this dissertation, we investigated the roles of p53 in normal development, teratogen-induced apoptosis, and birth defects. In the first study, the activation of p53 and its target genes, p21, NOXA, and PUMA, were examined during neural tube closure in mouse embryos exposed to hyperthermia (HS) or 4- peroxycyclophosphamide (4CP), teratogens known to induce neural tube defects (NTDs). In the second study, using p53-deficient mice, we examined the expression of mRNAs and microRNAs (miRNAs) during neural tube closure. In the third study, the incidence of NTDs was investigated in p53- and p21-deficient mouse embryos exposed to HS. Finally, we examined the induction of apoptosis in p53-deficient mouse embryos exposed to HS. HS and 4CP induced the activation of p53 by phosphorylation and accumulation of the protein, leading to an increase in p21 proteins and mRNAs. Although HS and 4CP also induced the expression of Noxa and Puma mRNAs, no significant increases in NOXA and PUMA proteins were observed, suggesting a possible role of transcriptionindependent apoptosis. In the second study, we showed that the expression of 388 genes and 5 miRNAs were significantly altered in p53 -/- compared to p53 +/+ embryos. Finally, we showed that 10% of p53 -/- pups exhibit exencephaly, spina bifida, and/or preaxial polydactyly, whereas no malformations were observed among p21 -/- offspring in the absence of HS. HS resulted in an increased incidence of exencephaly in both p53 and p21 null mice indicating that these two proteins act as teratogen suppressors. Our preliminary data additionally showed that a decreased level of apoptosis was observed in HS-treated embryos lacking a p53 allele, suggesting that too little apoptosis may be causally linked to NTDs observed in embryos exposed to HS. Taken together, these studies suggest that precise control of apoptosis and cell cycle arrest pathways are critical for neural tube development and the prevention of teratogen-induced NTDs.