To reactivate or not to reactivate : Control of KSHV lytic replication is essential for apoptosis in response to p53 restoration

1 REVIEW OF THE LITERATURE. 2 1. TUMOR SUPPRESSOR PROTEIN p53. 2 1.1 Discovery of p53 . 2 1.2 p53 structure . 2 1.3 The p53 family . 3 1.4 Regulation of p53. 4 1.4.1 Control of p53 stability by MDM2 . 4 1.4.2 Role of ARF in p53 regulation. 6 1.4. 3 MDM2-MDMX network in p53 regulation . 6 1.4. 4 Regulation of p53 activity by phosphorylation. 7 1.5 Functions of p53. 8 1.5. 1 Role of p53 in cell cycle arrest. 8 1.5. 2 Role of p53 in apoptosis . 10 1.6 Restoration of p53 function . 11 1.6. 1 MDM2 antagonists. 12 1.6. 2 Nutlin-3 as an innovative therapeutic tool in hematological malignancies.14 2. NUCLEOPHOSMIN (NPM), A MULTIFUNCTIONAL PHOSPHOPROTEIN. 15 2.1 NPM in centrosome duplication. 16 2.2 NPM involvement in transcriptional regulation. 18 2.3 Biological role of NPM in tumorigenesis. 20 2.4 NPM and viruses . 22 3. KAPOSI’S SARCOMA HERPESVIRUS (KSHV). 23 3.1 KSHV-associated malignancies . 23 3.1.1 Kaposi’s sarcoma (KS) . 23 3.1.2 Primary effusion lymphoma (PEL). 24 3.1.3 Other KSHV-induced disorders . 26 3.1.4 Treatment modalities against KSHV-associated diseases. 27 3.1.4.1 Anti-viral therapies . 27 3.1.4.2 Therapies under investigation . 28 3.2 KSHV latency program . 30 3.2.1 KSHV latency-associated nuclear antigen . 32 3.2.1.1 LANA and episome maintenance . 32 3.2.1.2 LANA and regulation of viral and cellular gene transcription . 33 3.2.1.3 LANA and repression of lytic replication . 35 3.2.2 KSHV-encoded cyclin (v-cyclin). 35 3.3 Molecular events involved in the switch from latency to lytic KSHV replication . 37 3.3.1 Lytic replication phase. 37 3.3.2 ORF50/RTA: master regulator of the latent-lytic switch . 39 3.3.2.1 Downstream targets of RTA . 40 3.3.3 Diverse cellular factors involved in KSHV reactivation . 41 3.4 Key aspects of lytic replication for KSHV pathogenesis . 43 AIMS OF THE STUDY.45 MATERIALS AND METHODS. 46 RESULTS AND DISCUSSION. 57 1. RESTORATION OF p53 FUNCTION BY MDM2 INHIBITION AS A NOVEL THERAPEUTIC MODALITY AGAINST PEL CELLS (I). 57 1.1 Inhibition of MDM2 leads to cell cycle arrest and apoptosis in PEL cells . 57 1.2 Restoration of the p53 pathway selectively kills KSHV-infected cells . 59 1.2.1 Active DNA-damage signaling in PEL cells augments cytotoxicity of Nutlin-3 . 60 1.2.2 Disruption of the p53-MDM2-LANA complex sensitizes PEL cells to apoptosis upon p53 restoration . 61 1.2.3 High levels of MDM2 and co-infection with EBV may sensitize PEL cells to Nutlin-3-induced cytotoxicity . 62 2. RESTORATION OF THE p53 PATHWAY AS A TREATMENT FOR KSHV LYMPHOMAS IN VIVO (I, II) . 63 2.1 MDM2 inhibition by Nutlin-3 eradicates subcutaneous PEL tumors in mice . 63 2.2 Anti-tumor activity of Nutlin-3 occurs in intraperitoneal PEL tumors in mice . 64 2.3 The p53 pathway can be efficiently restored in clinical PEL tumors ex vivo . 66 2.4. Viral lytic replication impairs the apoptotic potential of Nutlin-3 in vivo . 67 2.4.1 KSHV viral reactivation attenuates the p53-dependent apoptotic response to MDM2 inhibition. 68 2.5 Chemical and physiological induction of viral reactivation recapitulates resistance to MDM2 inhibition in vitro . 68 2.6 Decrease in p53 protein level in lytic PEL cells is proteasome-mediated. 70 3. THE SWITCH FROM LATENCY TO LYTIC REPLICATION A CRITICAL STEP IN THE KSHV PATHOGENESIS (III, IV; UNPUBLISHED DATA) . 72 3.1 Nucleophosmin is a novel regulator of the latent-lytic switch . 72 3.1.1 Nucleophosmin is phosphorylated by v-cyclin-CDK6 in vivo in PEL cells . 72 3.1.2 Phosphorylation of NPM promotes the NPM-LANA association, which is further regulated by acetylation . 74 3.1.3 Silencing of NPM leads to viral reactivation . 77 3.2. Phosphorylation of NPM on Thr199 correlates with KSHV latency . 78 3.3 NPM phosphorylation is required for efficient establishment of latency (unpublished data) . 79 CONCLUSIONS AND FUTURE PROSPECTS. 82 ACKNOWLEDGEMENTS . 84 BIBLIOGRAPHY . 86 ORIGINAL PUBLICATIONS This thesis is based on the following original publications, which are referred to in the text by their Roman numerals. I. Sarek G,