Varda Rotter - Weizmann Institute of Science.

Varda Rotter
Department of Molecular Cell Biology,
The Weizmann Institute of Science,
Rehovot, Israel
Can mutant p53 change faces into
guardian of the genome?

Mutant p53: oncogenic gain of function
Brosh and Rotter, Nature Cancer Rev 2009

Expression of mutant p53 in human colon carcinoma
Rotter, V. (1983). p53, a transformation-related cellular encoded protein, can be used as a
biochemical marker for the detection of primary tumor cells.
Proc. Natl. Acad. Sci. 80:2613-2617

p53 somatic mutations:
prevalence of appearance in cancer types
5

Germ-line p53 mutations:
The Li-Fraumeni Syndrome (LFS)

• The Li-Fraumeni Syndrome
(LFS): Human heterozygous
carrier are born apparently
normal developed variety of
tumors later in life!
• Mostly associated with LOH.
• Connection to Cancer stem cell
activityl!

Experimental model:
p53 heterozygote mice develop
normally
Yet, develop tumors late in life

Experimental mouse stem cells models:
wtp53, mutp53 and HZp53
• Adult stem cells: MSCs and C-FUCs
multipotent; lower genomic fidelity; important for
tissue regeneration and are associated with
cancer development.
UNDER GO LOH
• Embryonic stem cells: iPSCs and ES
pluripotent; important for development; high
genomic fidelity.
DO NOT UNDERGO LOH

What is the mechanism
that suppress mutant
p53 in mutp53 knock-in
ES?
Proteomic control??

(human&mouse)
(mouse)
(human & mouse)
WT p53 Mutant p53
Conformation specific antibodies:

Mutant p53 shifts into Wild type p53
conformation in ES

Identifying potential stabilizers of WT p53
conformation
Mass Spectrometry
Collaboration with
T. Geiger at TAU

Binders bound to WT form in WT ESCs:
Validation… WT p53 binds known Binders:
MDM2, MDM4, p53BP1, Trim24
Mass Spectometry:

Interactome network of binders:
Mass Spectrometry:
binders of WT conformation of Mut p53
Post-
translational
modifications
Ubiquitin-
related
Chaperones
Other
binders

Conclusion:
Mutant p53 protein is “refolded”
into a wild type p53 protein
conformation in embryonic stem
cells
Rivlin et al., PNAS 2014

p53 based therapy:
Small peptide

Development of p53 based
therapy
Develop a ways to “convert” mutant p53 into
wild type p53
Dr. Perry Tal
Prof. Varda Rotter
Prof. Moshe Oren
23

Conversion of mutant (M) p53 protein conformation
into wild type (W) p53 protein conformation:
Novel mutant p53-based
personalized cancer therapy:
for cancers that express a mutant p53
M
M
Treatment of various p53 mutant types with
combinations of small peptides

Tumor suppressor Oncogenic
Folded correctly Partially Denatured
Binds to consensus DNA Does not bind DNA
Low protein levels High protein levels
MM
p53 folding conformation is in a state of equilibrium
An example of equilibrium state are p53 temperature
sensitive mutations E285, V143
37°C
32°C
p53 conformation equilibrium
25

Phage Display
26
Synthesis of DNA coding
for a peptide library
NEB PhD-7 and PhD-12
libraries each displaying
109-1011 peptides
Packaging and display of the
peptide library fused to
pIII phage protein
Reaction of phage with
an immobilized target
Elution of bound phageInfection of bacteria
Washing of unbound phage
Amplification
of phage in
bacteria
Deep Sequencing

Ab 1620
Peptide Phage
Library
screening
Conformational shift of p53
28

Protocol for developing mutant
p53 “conversion”
• Screening of phage display peptide libraries for
peptides capable of p53 modulation
• Deep sequencing of pools of positive clones from
the libraries
• Computational deduction of optimized peptide
sequences
• Peptide synthesis
• Functional validation in several in vitro assays
• Functional validation in animal cancer models
29

Peptide Phage Library screening help
identifying small peptides that can
“educate” mutant p53 to function like
the wild type p53 tumor suppressor!

N ame P eptide Seq
pCAP201 HPTHPIRLRDNLTR 14-3-3 3 3
pCAP202 DEDAKFRIRILMRR APAF1 3 2 3 8
pCAP203 RRRHDSCHNQLQNYDHSTE ASPP1 3 2 4 6 4 6 6 4 4 42
pCAP204 MSTESNMPRLIQNDDRRR ASPP2 2 4 4 5 5 20
pCAP205 RCRNRKKEKTECLQKESEK ATF3 2 5
pCAP206 RRRSHSQENVDQDTDE BAK 3 3 2 1 2 6 19
pCAP207 RRSRSNEDVEDKTEDE BAK 0
pCAP208 RRIRSGGKDHAWTPLHENH BARD1 0
pCAP209 HTPHPPVARTSPLQTPRR BCL2 0
pCAP210 DEFERYRRFSTSRRR BCL-XL 3 2 2 2 6 3 3 6 29
pCAP211 PDSEPPRMELRRR BCR 0
pCAP212 myr-REEETILIIRRR BRG1 3
pCAP213 RRIKMIRTSESFIQHIVS BTF 3 5
pCAP214 RRRESEQRSISLHHHST C-ABL 2 2
pCAP215 RRDTFDIRILMAF CARM1 0
pCAP216 myr-HFNHYTFESTCRRRRC CAS 2 2
pCAP217 HSTPHPPQPPERRR CCDC8 2 2
pCAP218 RREVTELHHTHEDRR CEP72 0
pCAP219 SYRHYSDHWEDRRR CETD2 1 1
pCAP220 STTHPHPGTSAPEPATRRR CHD6 2 2 2 6
pCAP221 myr-RRKHNKHRPEPDSDER CTF2 3 5 3 4 2 5 6 5 2 5 4 6 6 60
pCAP222 RYEENNGVNPPVQVFESRTR CUL7 4
pCAP223 SPWTHERRCRQR CYP27B1 0
pCAP224 RRKSEPHSLSGGYQTGAD DIABLO 2 2
pCAP225 HTIHSISDFPEPPDRRRR DMP1 3 3
pCAP226 DDSDNRIIRYRR G3BP2 3 3 6
pCAP227 myr-RRKILFIRLMHNKH GAS2 4 6 5 5 5 6 5 6 3 5 5 3 4 67
pCAP228 DEDAAHSTGHPHNSQHRRRRHIPK1 3 0
pCAP229 myr-PRVLPSPHTIHPSQYP HIPK2 2 3 3 3 3 3 3 3 3 29
pCAP230 RKRGKSYAFFVPPSESKERW HMGB1 3 5 5 4 3 6 5 5 2 6 4 6 6 63
pCAP231 HRTQSTLILFIRRGRET HTRA2 3 9
pCAP232 RSRSSHLRDHERTHT HZF 0
pCAP233 SHYHTPQNPPSTRRR IFI16 3 3 3 2 11
pCAP234 HRTGHYTRCRQRCRSRSHNRHKLF4 2 2
pCAP235 RSYSKLLCLLERLRISP MIF 2 2 2 11
pCAP236 RRRSTNTFLGEDFDQ MORTALIN 0
pCAP237 HTIHVHYPGNRQPNPPLILQR MULE 3 5 2 12
pCAP238 TSPHPSLPRHIYPRR NFAT 2 4 6
pCAP239 REGFYGPWHEQRRR OGA 2 2 4
pCAP240 TEQHHYIPHRRR OSGIN2 3 3
pCAP241 LIGLSTSPRPRIIR PAR3 0
pCAP242 myr-RRLIVRILKLPNPPER PARC 4 3 6 6 6 6 3 4 6 6 5 6 6
pCAP243 RRCRSILPLLLLSR PERP 2 2
pCAP244 RRVSELQRNKHGRKHEL PIAS1 2 5
pCAP245 NHITNGGEEDSDCSSRRRRL PIN1 3 2 8
pCAP246 RRRLDDEDVQTPTPSEYQN PIRH2 2 5
pCAP247 RRITEIRGRTGKTTLTYIED RAD51 3 6
pCAP248 EIYGESGKTDEHALDTEYRR RAD51 0
pCAP249 myr-DERTGKTRRYIDTRDIRR RAD51 3 3 6
pCAP250 myr-RRHSTPHPD RAD9 4 6 5 5 5 5 5 6 3 6 6 6 6 72
pCAP251 RLRRVILRSYHE RAD9 3 3
pCAP252 RRVILRSYDGGHSTPHPD RAD9 0
pCAP253 TGKTFVKRHLTEFEKKYR RAN 0
pCAP254 NHFDYDTIELDTAGEYSRRR RAS 0
pCAP255 DPEPPRYLPPPPERR RASSF5 0
pCAP256 RTLHGRRVILHEGGHSISDLK RPA70 2 2
pCAP257 myr-HSSHHHPTVQHRR SIN3A 4 4 8
pCAP258 FLIGPDRLIRSR SIVA 6 4 5 2 2 4 23
pCAP259 myr-RTLIGIIRSHHLTLIRR SMG1 5 4 5 3 2 2 25
pCAP260 RRTFIRHRIDSTEVIYQDED STK11 0
pCAP261 RRRQPLPSAPENEE STK15 2 3 5
pCAP262 ESKTGHKSEEQRLRRYR TBP 0
pCAP263 YDDEHNHHPHHSTHRRR TSC22 0
pCAP264 RRRREVHTIHQHGIVHSD TTK 0
4
3
3
3
4
6
3
5
2
5
3
3
6
4
4
2
3
2
3
3
2
Name Sequence Confor
mation
DNA
binding
Viability PCR p53
binding
pCAP-
p53 Conformation
Activating Peptide
Lead peptides were screened
using a variety of assays
• p53 conformation
• Binding of mutp53 to p53RE
• Viability of mutp53 cancer
cells
• p53 target gene activation
• Binding to p53
Lead pCAP
pCAP-250
myr-RRHSTPHPD
Summary of functional validation
31

4-10 lead peptides were identified as
functionally effective in a verity of assays;
p53 conformation
Binding of mutp53 to p53RE
Viability of mutp53 cancer cells
p53 target genes activation
Binding to p53
pCAP- p53 Conformation Activating Peptide
Lead pCAPs
pCAP-250 myr-RRHSTPHPD
pCAP-227 myr-RRKILFIRLMHNKH
pCAP-325 myr-RRIRDPRILLLHFD
pCAP-242 myr-RRLIVRILKLPNPPER
pCAP-144 SFILFIRRGRLGRRRRRRRRR
Peptides functional screening summary
Tal P, Eizenberger S, Cohen E, Goldfinger N,
Pietrokovski S, Oren M, Rotter V. Oncotarget. 2016

Predicted model of peptide
pCAP-250 and p53 binding
“Anchor-Dock” algorithm produced a
model of pCAP-250 peptide folding
and its interaction with p53
33Avi Ben-Shimon

TP53 Mutations in the Genome Era
Exonic mutation frequencies from WES/WGS studies
(COSMIC v65)
SomaticMutation
Frequency
TP53
M. Olivier IARC
34

In vitro
Lead pCAP-250 effect on apoptosis and p53 targets
Ovarian Cancer model ES2 cells p53S241F
Time treatment pCAP-250
FoldmRNAexpression
Btg2/GAPDH
FoldmRNAexpression
p21/GAPDH
Annexin-PI assay (pCAP-250 12μg/ml)
p53 target genes expression
FoldmRNAexpression
PUMA/GAPDH
FoldmRNAexpression
CD95/GAPDH
Apoptosis Growth arrest
Peptid
e

In vivo
Pre-clinical trial - Mouse #3 as a function of time
Ovarian Cancer ES2 cells p53S241F
Peptide I.D - pCAP-250
Administration- Intra-tumor tumor injection
Dose – 10 μg/tumor, 3 times a week
Tal et al, Oncotarget, 2016

Intra tumor (IT)
injection of
10μg/tumor 3
times a week
Control peptide
pCAP-250
Day 27 Control
peptide
pCAP-250
p - 0.014
8.4E+06 5.1E+07 1.8E+08
4.8E+08
9.7E+08
1.7E+09
2.7E+09
4.0E+09
4.9E+09
0.E+00
2.E+09
4.E+09
6.E+09
0 3 7 10 13 17 20 24 27
Time (Days)
Con peptide n=10
pCAP-250 n=10
8.4E+06 6.0E+07
2.1E+08
5.8E+08
4.3E+08
2.8E+08
1.9E+08
1.2E+08 9.6E+07
0.E+00
5.E+08
1.E+09
2.E+09
2.E+09
0 3 7 10 13 17 20 24 27
Time (Days)
Con peptide n=10
pCAP-250 n=10
ES2 cells Luminescence over time (in-vivo)
Treatment
IVISRead(photons) Ovarian Cancer Animal Model: ES2
cells p53S241F
28

Ovarian cancer ES2 cells:
daily SC injections of pCAP
38
Con pCAP SC 0.05mg/day
14 days Average 9.12 108
pCAP-250 SC 0.05mg/day
14 days Average 3.54 108
2 5 9 12 15 2 5 9 12 15
Control SC pCAP 250 SC
0.00E+000
1.00E+009
2.00E+009
Time Days Time Days

Programmable release 1d-6weeks
2 µg/ml
IC50 ES2 Cells
4 – 6 µg/ml
20mg/Kg0
500
1000
1500
2000
2500
0 1 2 3 4 5 6 7 8
Plasmaconcentration(ng/ml)
Time (day)
pCAP 250 @ 3 mg/kg sc (minipump)
pCAP 250 @ 10 mg/kg sc (minipump)
pCAP-250 PK sub-cutaneous release by ALZET mini-pump
Peptide Controlled
release with ALZET mini-pump

pCAP-250
ALZET pump
20mg/Kg
14 Days
1.0E+07 2.8E+07 6.8E+07 1.3E+08
6.9E+08
8.2E+08
1.1E+09
1.7E+09
2.0E+09
2.3E+09
1.5E+08
5.8E+08
9.7E+08
7.9E+08
4.5E+08
2.7E+08
2.2E+08
2.1E+08 4.6E+07
0.0E+00
5.0E+08
1.0E+09
1.5E+09
2.0E+09
2.5E+09
3.0E+09
0 4 7 10 14 17 21 24 28 32
Time (Days)
Control peptide n=10
pCAP-250 mini-pumps n=10
pCAP-250 intra-tumor n=10
pCAP-250
intra-tumor
10µg/tumor
3 times/week
Treatment
IVISRead(photons)
105 cells injection
Control
intra-tumor
10µg/tumor
3 times/week
Control
ALZET pump
20mg/Kg
14 Days
Peptide delivery in-vivo ALZET mini-pumps
Ovarian Cancer ES2 cells Luminescence over time (in-vivo)

Breast cancer animal model
MDA-MB231 cells p53E280K triple negative
Day 18 Day 29
Day 18 Day 29
Intra tumor injection 6μg/tumor 3 times a week
pCAP-
250,154,242
Control
41

Control
peptides
pCAP
Treatment
Day 34
Day 35
IVISRead(photons)
5*105cells injection
Treatment
Intra tumor injection
6μg/tumor 3 times a week
Colon carcinoma SW-480 p53R273H
SW-480 cells Luminescence over time (in-vivo)
42
2.23E+07
2.79E+08
9.25E+08
1.76E+09
2.41E+09
3.27E+09
3.58E+09
4.22E+09
4.44E+09
4.67E+09
1.07E+09 1.06E+09
9.91E+08
7.52E+08
3.11E+08 2.30E+08 7.81E+07
0.E+00
2.E+09
4.E+09
6.E+09
0 4 8 11 15 19 23 27 31 34
Time (Days)
Con peptides n=10
pCAP-325,250,154 n=10

• The p53 pCAPs “convert”
mutant p53 conformation

Acknowledgments:
Alina Molchadsky
Noa Rivlin*
Yoav Shetzer
Gabriela Koiefman
Meital Charni
Yan Stein
Hilla Solomon
Giusepo Lonetto
Tom Kaufman
Perry Tal*
Shay Eizenberger*
Naomi Goldfinger
Collaborators:
Moshe Oren
Tami Geiger

Varda Rotter - Weizmann Institute of Science.

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