Download `WAKING UP` SILENCED p53 IN CANCER

‘WAKING UP’ SILENCED p53 IN CANCER
Min Lu1, Hilde Breyssens1, Victoria Salter1, Luca Tordella1, Mario Notara1, Shan Zhong1, Ying Hu1, Caroline Baer1, Indrika Ratnayaka1, Alex Sullivan1, Nicholas R. Brown2, Jane Endicott2, Stefan Knapp3,10,
Benedikt M. Kessler4,10, Mark R. Middleton5, Christian Siebold6, E. Yvonne Jones6, Elena V. Sviderskaya7, Jonathan Cebon8, Thomas John8, Otavia L. Caballero9, Colin R. Goding1, and Xin Lu1
1Ludwig
Institute for Cancer Research, 3Structural Genomics Consortium, 4Centre of Cellular and Molecular Physiology, 6Division of Structural Biology, 10Target Discovery Institute, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom; 2Department of
Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom, 5Department of Oncology, Churchill Hospital, University of Oxford, Oxford OX3 7LE, United Kingdom, 7Cell Signaling Research Centre, Division of Biomedical Sciences, St George’s, University of London,
SW17 0RE, United Kingdom, 8Ludwig Institute for Cancer Research, Austin Health, University of Melbourne, Victoria 3084, Australia, 9Ludwig collaborative laboratory, Johns Hopkins University School of Medicine, MD 21231, USA
ASPP: Apoptosis Stimulating Protein of p53
SUMMARY
p53 is a protein that is important for mediating cell death after DNA damage and consequently plays an
important role in cancer cell death after chemotherapy. Currently, around 12 million people live with cancers
containing wild-type (WT) but silenced p53. ‘Waking up’ silenced p53 represents a strategy with huge clinical
potential but with significant challenges. We investigated whether in melanoma, a cancer type that typically
retains WT p53 yet notoriously doesn’t respond to conventional chemotherapy, we could ‘wake-up’ p53 and kill
cancerous cells.
α
α
Pro
Ankyrin SH3 ASPP1
Pro
Ankyrin SH3
Pro
We identify that cyclin B1/CDK1 phosphorylates iASPP 1, leading to iASPP nuclear localization 2 and p53
inhibition. Nuclear iASPP is enriched in melanoma metastasis and associates with poor patient survival. Most
WT p53-expressing melanoma cell lines co-express high levels of phosphorylated nuclear iASPP and MDM2.
iASPP
Senescence
ASPP2
(53BP2)
Cell cycle
arrest
Ankyrin SH3 iASPP
Apoptosis
p53
Others…
Bind partners: p53/p63/p73,
ASPP1/2
NF-kB, YAP, Bcl2, PP1…
Inhibition of nuclear iASPP and MDM2 with small molecules 3 restored p53 pro-apoptotic function 4.
Concurrent p53 restoring and BRAFV600E inhibition achieved additive suppression in vitro and in vivo,
presenting an alternative for melanoma therapy.
Function
Domains
Why is nuclear iASPP bad news? It hijacks p53 !
iASPP in the nucleus is bad news in melanoma
Cancer
Only 2-15% patients
survive in 5 years if
melanoma cells
transfer to other
organs (metastasis)
p53 is destroyed because of genetic mutations in 12 million people
Transferred
melanoma cells
(metastasis) have
more nuclear iASPP
Healthy
p53 is a failsafe commander that sits in the cell nucleus. It sends out various
instructions to protect against cancer
Patients with more
nuclear iASPP have
decreased survival
rates
Cancer
p53 is not destroyed, but instead kept silent in 12 million people
Rescue p53 by locking iASPP outside the nucleus and kill melanoma
How does iASPP get into the nucleus and hijack p53?
1
R86
P85
R86
S84
Kill melanoma
outside the body
S84
Screen drugs
from library
Locked
1. iASPP is self-locked and
can not enter nucleus.
P85
iASPP
locked
Unlocked by phosphate
Y814
Cell
suicide
Y814
Co-crystal shows interaction between N- and C-terminus of iASPP;
Ser84 phosphorylation disrupts interaction
* It is confirmed by IP that N-iASPP binds C-iASPP; phospho N-iASPP (also
mimic N-iASPP-S84D/S113D) fails to binds C-iASPP (* data not shown).
p53 causes cancer cells to commit
suicide (apoptosis).
Culture melanoma cells in dish
Locked iASPP
outside nucleus
Healthy cell
p53 wakes up, cells commit suicide
iASPP outside
the nucleus
iASPP in
the nucleus
3
2
Add drug to lock iASPP outside the nucleus
Kill melanoma
inside the body
Unlocked iASPP
inside nucleus
Locked
iASPP
Unlocked
iASPP
2. cyclin B1/CDK1
unlocks iASPP.
3. Nuclear driver
is released.
iASPP dedimerizes upon S84/S113
phospho
iASPP locates in cytoplasm; iASPPS84D/S113D locates in nucleus
* It is confirmed by kinase assay that
it is cyclin B1/CDK1 phosphorylating
iASPP on S84/S113.
* It is confirmed that endogenous
iASPP also trans-located into nucleus
upon S84/S113 phospho.
Mice with melanoma tumors
p53 “wakes up” and tumors shrink
An alternative for
melanoma therapy
4
4. iASPP moves into nucleus and
hijacks p53. p53 is kept silent.
Free drug to lock iASPP
iASPP
Unlocked iASPP
hijacks p53
p53
Melanoma cell
References
1. Lu M., Breyssens H., et al.
2. Lu M., Zak J., et al.
3. Lu X, Lu M. et al.
4. Mccarthy N.
p53 selectively binds phosphorylated slower-migrating iASPP (* also
mimic iASPP-S84D/S113D) in melanoma lysates
* Luciferase assay shows iASPP-S84D/S113D is more active than iASPP in
inhibiting p53’s transcriptional activity on BAX, PIG3 and PUMA targets;
* iASPP K.O. MEFs reintroduced with iASPP-S84DS113D is more resistant
than iASPP to apoptosis by expressing less BAX, PIG3 and PUMA proteins.
WT p53 is co-inhibited by p-iASPP and MDM2
: co-expression of p-iASPP, WT p53, high MDM2
: co-expression of iASPP, mutant p53, low MDM2
: no association among above proteins
Restoring p53 Function in Human Melanoma Cells by Inhibiting MDM2 and Phosphorylated Nuclear iASPP. Cancer Cell. 2013, 23: 618-633.
A code in Ankyrin repeats Domain Defines a Novel Nuclear import pathway. Cell. 2014 (Accepted in Feb. In press).
IASPP Phosphorylation and Metastatic potential. World Intellectual Property Organization (Patent number: WO 2013131019). 2012.
Tumour suppressors: Restoring function. Nat Rev Cancer (2013 Research Highlight). 2013, 13: 379.
* Synergy of p-iASPP inhibitor and MDM2 inhibitor (Nutlin3) in
reactivating p53 in melanoma in vitro and in vivo.
* Addition of p53 reactivation and BRAFV600E inhibition (by first-line
drug vemurafenib) in suppressing melanoma in vitro and in vivo.
* Data not shown.
Correspondence
Min Lu
Ludwig Institute for Cancer Research
University of Oxford
[email protected]