Outline lecture 2

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Outline lecture 2 Regulators of the p53
pathway positive regulators proteins that
interact with p53 to act tx negative
regulators Mdm2 Mdm4 Pirh2 Cop1 Arf as a
regulator of the p53 pathway Rb regulates the
pathway Summary mechanisms to derail p53
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The p53 pathway
Positive effectors
Atm
Chk2
CKII
Puma Noxa Apaf1 Perp1 Bax
p53
p19Arf
Mdm2
Positive effector
Pirh2
Mdm4
Cop1
p21
Apoptosis
Cell cycle arrest
Negative regulators
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Discovery of Mdm2
Immunoprecipitation with p53 antibodies brought
down a protein of 90 KDa Purified, sequenced,
match a known cDNA, the murine double minute 2
gene A gene amplified in mouse cells with double
minute chromosomes that gave the cells a
growth advantage Mdm2 inhibited p53 transcription
function
Cell 691237
4
Mdm2 amplification in human tumors

• Osteosarcomas
• Soft tissue sarcomas
• Glioblastomas
• Breast carcinoma

Momand et al, NAR 26 3453
5
Mdm2-/- mice are embryo lethal mice die due to
apoptosis. Due to p53 constitutive
activity? Mdm2-/-p53-/- mice are viable.
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Mdm2 inhibits p53 function

• Interacts with and inhibits p53 transactivation
• Amplified in tumors
• Loss of function phenotype rescued by
• loss of p53
• 4. Mdm2 transgenic mouse devlops tumors

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MDM2 domains
p53-binding
NLS
RF
ACIDIC
ZF
(19-102)
305
322
223
274
438
478
181
-185
TFIIE-binding (1-222)
RNA- binding
L5-binding (159-383)
E2F1-binding (1-220)
TBP-binding
(120-222)
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Ubiquination
p53
mdm2
E1
E2
p53
mdm2
UB
p53
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Mdm2
Tetramerization domain
p53
Activation domain
DNA binding domain
NLS
Atm Chk2
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MDM2
NLS
ACIDIC
p53-binding
ZINC
RING
50 identity
50
50
MDM4
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Mdm2 and Mdm4 mouse models
Mdm2/
Mdm2/ p53/
Mdm4/ p53/
Mdm4/
Mdm2/ Mdm4/ p53/
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MDM4
MDM2
High levels of MDM2, and MDM4in HNSCC lacking p53
mutations
13
Mdm4 increased in retinoblastoma
Nature 44461
14
Mdm2/? and Mdm4/? mice are normal
15
Mdm2 and Mdm4 haploinsufficiency
increases sensitivity to ionizing radiation
IR 6 Gy
IR 4 Gy
125
125
100
100
Percent survival
75
75
50
50
p 0.001
25
25
0
0
0
10
20
30
40
50
0
10
20
30
40
50
Time (days post IR)
Time (days post IR)
WT
M4/-
M2/-
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p53 loss rescues Mdm2 and Mdm4 Haploinsufficient
phenotypes
IR 6Gy
IR 6Gy
125
125
100
100
75
Percent survival
75
50
50
25
25
0
0
0
10
20
30
40
50
0
10
20
30
40
50
Time (days post IR)
Time (days post IR)
M2/-
M4/-p53KO
M2/-p53KO
M4/-
p53KO
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Mdm2/? Mdm4/? MEFs show decreased
transformation potential
WT
Mdm2/- Mdm4/-
Mdm2/- Mdm4/-
Vector Ras Myc
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Em-Myc mice
EMBO 221442
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Mdm4 haploinsufficiency delays lymphomagenesis
Em-myc Mdm4/-
Em-myc Mdm4/
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SNP309 in the Mdm2 promoter extends an Sp1
Binding Site
SNP309
Ets/AP1
p53-REs
Exon 2
Exon 1
1
524
261
WT
CGGGGGCCGGGGGCTGCGGGGCCGCTTCGGCGCGGGAGGTCCGGATGATC
GCAGG
Sp1
Sp1
Sp1
Sp1
SNP309
CGGGGGCCGGGGGCTGCGGGGCCGCTGCGGCGCGGGAGGTCCGGATGATC
GCAGG
Sp1
Sp1
Sp1
Sp1
Bond et al. Cell 119591
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SNP 309 associates with an Accelerated Onset of
Breast Cancer
p 0.01
Cumulative of Individuals With Breast Cancer
Age of Onset
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Mdm2/? Mdm4/?
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Mdm2/? Mdm4/? embryo lethal phenotypes
13.5 dpc
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Mdm2/? Mdm4/? are small
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Mdm2/? Mdm4/? mice lack hematopoesis
Wt
Mutant
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Mdm2/? Mdm4/? p53 /? mice are normal
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Multiple proteins negatively regulate p53
Mdm4
Nature Gen 2992
p53
Cop1
Mdm2
Nature 378203
Nature 42986
Pirh2
Cell 112779
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p53
Mdm2
ARF
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p16INK4a and p19ARF/p14ARF Locus
E2
E3
E1b
E1a
p16INK4a
AUG
AUG
p19ARF

• Second most frequently inactivated locus in
  cancer
• Deletions
• CpG Methylation
• Point mutations (p16)

Cell 83993
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Arf null mice are tumor prone Cell 91649 Arf
null MEFS have no p53 mutations Arf binds
mdm2 Cell 92725 Cell 92713 Hypothesis
Increased Arf levels sequester Mdm2
Mdm2
p53
Arf
Arf
Mdm2
p53
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One locus, two hits
p16
Cdk4/cyclin D
Rb
E2F
p19Arf
Mdm2
p53
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Two pathways meet
p16
cycD
cdk4
Rb
E2F1
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Two pathways meet
p16
cycD
cdk4
Rb
E2F1
Arf
p53
Arf
Arf
Mdm2
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Mechanisms of inactivating p53
Mutate the p53 DNA binding domain
Overexpress inhibitors MDM2 or MDM4 (Possibly
Pirh2 and Cop1)
Deletion of Arf
Express HPV E6
Mislocalization
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Tumors disrupt the same pathway by different
means
Molecular changes
treatment
Delete p53 Mutate p53 Increase Mdm2 Increase Mdm4
Reintroduction of p53 Drugs that activate
p53 Drugs that disrupt the interaction Drugs that
disrupt the interaction