p53 Park Hyunmi Awakening guardian angels : drugging the p53 pathway p53 and E2F : partners in life and death

Evolution of Cancer & Tumor suppressor substantial regenerative capacity 1> Cancer is a genetic pathology that arises in the adult tissues of long-lived organisms, such as vertebrates, whose tissues retain a substantial regenerative capacity throughout somatic cells accumulate mutations life and, consequently, whose somatic cells accumulate mutations. 2> both the genesis and evolution of cancers are actively restrained by various tumor suppressive mechanisms. sensors that accurately discriminate between normal and neoplastic cell growth. 3> sensors that accurately discriminate between normal and neoplastic cell growth.

p53 inactivation in tumors p53 inactivation in tumours occurs through two general mechanisms. point mutations in p53 itself 1> First, the inactivation of p53 function by point mutations in p53 itself. abrogation of signalling pathways or effector molecules 2> second, through the partial abrogation of signalling pathways or effector molecules that regulate p53 activity. 3> Currently, around 11 million people are living with a tumour that contains an inactivating mutation of TP53 (the human gene that encodes p53) and another 11 million have tumours in which the p53 pathway is partially abrogated through the inactivation of other signalling or effector components. The p53 pathway is therefore a prime target for new cancer drug development

Mammalian p53 family proteins – p53, p63, p73 SAM – sterile alpha motif : Protein-protein interaction domain Structure of p53

Physiological functions of p53 transcription factor 1> transcription factor that has an essential role in guarding the cell in response to various stress signals through the induction of cell cycle arrest, apoptosis or senescence. Non-transcriptional activities 2> Non-transcriptional activities These include direct effects on survival proteins in the mitochondria, regulation of microRNA processing and reports of possible direct p53 involvement in DNA repair pathways.

P S15,20/T18 Regulation of p53 1> direct transcriptional squelching & unleashes its transcriptional activity. p53 degradation & p53 accumulation 2> p53 degradation & p53 accumulation : by the MDM2 E3-ubiquitin ligase and the related protein MDM4 (also known as MDMX)

Regulation of p53 feedback loop, p53 activates transcription of MDM2. 1> In an important feedback loop, p53 activates transcription of MDM2. : The increased expression of MDM2 leads to a decrease in p53 levels and the inactivation of p53, which in turn leads to a decrease in the rate of MDM2 transactivation by p53. ARF (also known as p14ARF in humans and p19ARF in mice) 2> ARF (also known as p14ARF in humans and p19ARF in mice) also have an important role in controlling p53 stability.

MDM4 - MDM4 has no measurable E3-ligase activity binds to the same site on p53 as does MDM2 p53 transactivation (ubiquitylation of p53) - structural homologue of MDM2 (50%) - form a heterocomplex with MDM2 - can be ubiquitylated by MDM2 and is consequently degraded - overexpression has been found in 40% (13 of 31) of the tumour cell lines

Regulation of p53 ( p53 inactivation in tumors ) abrogation of signalling pathways or effector molecules partial abrogation of signalling pathways or effector molecules MDM2 and MDM4. 1> increased expression of the p53-negative regulators MDM2 and MDM4. ARF 2> deletion or epigenetic inactivation of the p53-positive regulator and MDM2 inhibitor ARF.

Regulation of p53 ( p53 inactivation in tumors ) point mutations in p53 itself core DNA-binding domain In the other p53-defective tumours TP53 is mutated, and approximately 95% of these mutations lie in the core DNA-binding domain. 1> DNA contact mutations 2> structural mutations

to develop molecules that can reactivate p53 function in human tumours. bind to either full-length p53or the core DNA-binding domain 1> small molecules have been sought that will bind to either full-length p53 or the core DNA-binding domain of mutant p53 and restore its normal activity. inhibitors of the protein–protein interaction 2> inhibitors of the protein–protein interaction of p53 with the negative regulator MDM2 have been developed that show clear antitumour activity in preclinical animal models. forward chemical genetics (FCG) cell-based methods 3> molecules and drug combinations have been sought using forward chemical genetics (FCG) cell-based methods to identify molecules that selectively kill tumour cells by activating mutant or wild-type p53 Routes to drug discovery traditional high-throughput and computational design strategies 1> Direct against a purified target 2> Indirect against a pathway

restoring its binding function Strategies for reactivating mutant p53 - restoring its binding function destabilize the protein The tumour associated mutations thermally destabilize the protein further at body temperature abrogation of DNA binding and the impairment of the p53 Response. ( Inactivation of p53) ∴ stabilize p53 in its active biological conformation restoring its binding function potentially rescue wild-type p53 function. Small molecule

restoring its binding function Strategies for reactivating mutant p53 - restoring its binding function Ex. 2> non-peptide molecules : CP identified by Pfizer on the basis of an in vitro assay of mutant p53 unfolding interacts directly with DNA expression of both reporter and endogenous p53 target genes following transfection of TP53-null cells with two different mutant TP53 genes. Ex. 1> antibodies & peptide : bind the p53 carboxy-terminus (such as pAb421) a synthetic peptide (p53C) derived from the C-terminal domain effect on the DNA binding ability peptide stability / transport into tumour cells

restoring its binding function Strategies for reactivating mutant p53 - restoring its binding function Ex. 3> structural data and taking advantage of computational techniques library of probable p53 stabilizers crystal structure of a series of tumour-associated mutant p53 proteins in silico screening identification of small molecules that could bind to this pocket compound PhiKan083 difficult to develop as not all mutations in the p53 DNA-binding domain

inhibitors of the protein–protein interaction Reactivating wild-type p53 - inhibitors of the protein–protein interaction increased expression of MDM2 ∴ therapeutic index for MDM2 inhibition inhibition the p53-MDM2 interaction potentially rescue wild-type p53 function. Small molecule Inhibition of p53 activity in tumours

inhibitors of the protein–protein interaction Reactivating wild-type p53 - inhibitors of the protein–protein interaction Ex. 1> nutlins : small-molecule MDM2 antagonists with in vivo activity bind to the p53 pocket of MDM2 inhibit the p53–MDM2 interaction leading to the stabilization of p53 Ex. 2> benzodiazepenes and spiro-oxindole Benzodiazepene derivative TDP spiro-oxindole derivative MI-219 activation of the p53 pathway MDM2 is induced by p53 activation as part of an inducible feedback loop that negatively regulates the p53 response. MDM4.

inhibitors of the protein–protein interaction Reactivating wild-type p53 - inhibitors of the protein–protein interaction Ex. 3 > The interplay of MDM2 and MDM4 : Dual-specific peptides Ex. 4> ARF : inhibit the E3 ubiquitin ligase activity of MDM2 activation of p53

forward chemical genetics (FCG) cell-based methods induction of a p53 reporter screens typically use the induction of a p53 reporter in a cell line as a readout FCG : Forward chemical genetics : Libraries of small molecules are screened for their ability to induce a particular phenotype in cells or cellular extracts. three components : a collection of compounds a biological assay with a quantifiable phenotypic output a method to identify the target(s) of the active compounds.

forward chemical genetics (FCG) cell-based methods 1> Compounds that interact with DNA or affect topoisomerase function. quinacrine, Chloroquine induction of DNA damage induced p53 phosphorylation at ser15 or ser20 ( p53 activation) 2> Inhibitors of nucleic acid synthesis. NCI188491, NCI154829, actinomycinD, DRB, PALA, mycophenolic acid Inhibitors of the ribonucleotide synthesis pathway increase p53 levels and transcriptional activity

3> Compounds that disrupt mitosis. taxol, the vinca alkaloids forward chemical genetics (FCG) cell-based methods in response to mitotic poisons p53 is stabilized and activated ? 4> Compounds that directly interact with p53. RITA directly bind to p53 inhibit the interaction of p53 with MDM2 5> Compounds that target p53 regulatory factors tenovin-1, tenovin-6 : inhibit the NAd+-dependent deacetylase activity of SIRT1 and SIRT2 p53 deacetylation by SIRT1 p53 stability and transcriptional activity

p53 & E2F p53 & E2F - transcription factors that affect cell fate

E2f Family 1> In mammals, the E2f family comprises eight genes (E2F1–8), which give rise to nine distinct proteins. 2> Activator E2fs : E2F1, E2F2 and E2F3A interact only with Rb 3> repressor E2fs : E2F4–8 Repression of gene expression

Regulation of E2F - Play a major role during the G1/S transition in the mammalian cell cycle. - cyclin D1 - INK4A (also known as p16), a cyclin-dependent kinase (CDK) inhibitor

In many human tumours INK4A–Rb–E2f ARF–MDM2–p53 deregulation or hyperactivation of E2f. inactivation of p53. extensive crosstalk between the INK4A–Rb–E2f and the ARF–MDM2–p53 pathways and in particular between the transcription factors E2F1 and p53. Extensive crosstalk between E2F1 and p53.

Common upstream regulators of p53 and E2F1 * regulators affect both p53 and E2F1 Checkpoint kinases(phosphorylation). 1> Checkpoint kinases(phosphorylation). : ATM, CHK1/CHK2 acetylation. 2> acetylation. : p300/PCAF, SIRT1 MDM2. 3> MDM2. :E2f degradation by the F-box protein SKP2 proteasome dependent ubiquitin-independent manner The CdKN2A locus. 4> The CdKN2A locus. * well-known regulators 1> p53 : MDM2, post-translational modifications, subcellular localization, DNA binding, transactivation 2> E2f : RB, post-translational modifications

E2f activates p53 lack functional Rb in most human tumours INK4A–Rb cascade deregulated and hyperactive E2f p53 activation

E2f activates p53 ARF 1> p53 – ARF – E2f module (two negative feedback loops) E2f ↑ ARF ↑ p53 ↑ ARF ↓

E2f activates p53 PIN1 3> PIN1 : prolyl isomerase / orchestrates p53 acetylation SIRT1 SIRT1 : deacetylase SKP2 SKP2 : F-box protein interaction between p300 and p53 suppressing p300-mediated acetylation of p53 ASPPs ASPPs : Apoptosis stimulating of p53 protein / pro-apoptotic cofactors of p53 p73 p73 : p53 family member / serves as a pro-apoptotic cofactor of p53 ATM and CHK2 2> ATM and CHK2 : in ARF-independent / phosphorylation of p53

PIN1

E2f and p53 cooperate in apoptosis 1> E2F1 induces stabilization and activation of p53 2> transactivate of pro-apoptotic genes negatively regulate the anti- apoptotic genes lack functional Rb in most human tumours INK4A–Rb cascade deregulated and hyperactive E2f

E2f and p53 in cell cycle arrest and senescence 1> Activator E2fs can function as suppressors of p53. 2> Repressor E2fs are effectors of p53-induced growth 3> Potential inhibition of activator E2fs by p53-regulated genes.

miR34a