This document discusses replicative stress (RS), which is DNA damage that occurs during DNA replication. RS can lead to aging and can be induced by oncogenes in cancer cells. The receptor protein ATR plays a key role in the cellular response to RS. The document describes various studies investigating ATR, including how depleting the protein CDC25A can render cells resistant to ATR inhibitors. It also discusses the development of a screening method using CRISPR/Cas9 to identify genes that can confer resistance or sensitivity to ATR inhibitors when overexpressed or deleted. This screening approach could help identify cancers most vulnerable to ATR inhibition and potential resistance mechanisms.

2. Telomeres ROS Radiation
…
Replicative Stress
WHY do we accumulate DNA damage?

3. Replication Stress = Excess of ssDNA
ssDNA
Copy Number Variants Anaphase Bridges
Sister Chromatid Exchanges
(mitotic recombination)
Translocations

4. Replicative Stress
ATR
Chk1
The RS-response in a nutshell

5. Replicative Stress
ATR
Chk1
Tricks to study the RSR in mammals
ATR
ATRii
ATRS/S
Super-mice

6. (1) RS induces ageing.
(2) RS in utero accelerates ageing
later in
life.
(3) Strategies to alleviate ageing.

7. RS-induces aging
ATR
Murga et al Nat Genet (2009)

8. Intrauterine Programming of Ageing
RS in utero accelerates ageing later in life

9. Strategies to alleviate ageing
ATRSeckel
SUPER-RNR
X
By increasing nucleotide production!
SUPER-RNR
WT
Lopez-Contreras et al Genes Dev (2015)

10. Can we exploit the presence of
oncogene-induced RS in cancer for
tumor therapy?

11. ATR
ONCOGENE
REPLICATIVE STRESSREPLICATIVE STRESS
ORIGINAL HYPOTHESIS
IF oncogenes generate replicative stress, can we exploit
this property for the targeting of cancer cells?

12. FROM BASIC SCIENCE TO THE CLINIC
2008
Acellsystemto
activateATRatwill
2009
ATR-Seckelmice
2011
ATR-Seckelmice
donotdevelopBurkitt
2011
Development
ofATRinhibitors
2013
LicensedtoMerck
forclinicaldevelopment
Toledoetal
Murgaetal
Toledoetal
Murgaetal
Without good basic science, there is nothing to translate!

13. (1)Identifying which tumors are
most sensitive to ATR inhibitors
(2) Mechanisms of resistance to
ATR inhibition
AND WHAT AFTER 2013…?

14. How do we identify those tumors that
are most sensitive to ATR inhibitors?
Tumors with high levels of replication
stress select for high levels of CHK1
(which they need to be able to replicate
with high levels of RS)
Lopez-Contreras et al J Exp Med (2012)

15. Classification of human tumors by Chk1 levels
HEMATOLOGICAL

16. MLL-AF9
1 month
AML
Focusing on pediatric leukemias of poor prognosis
AML
10 days
IRES
RASV12 GFP LUCIFERASE
C57/BL6
Morgado et al Sci Sig (2016)

17. UNTREATED

18. ATRi

19. (1)Identifying which tumors are most sensitive to
ATR inhibitors
(Burkitt Lymphoma; Ewing Sarcoma; AML)
(2) Mechanisms of resistance to ATR inhibition
What are we up to now?

20. CAS9
A Dox-inducible CAS9 mouse for in vivo CRISPR
Cas9ind

21. No DOX DOX 48 hours DOX 120 hoursNeg control
Exogenousgene
(GFP)
Efficient gene deletions in Cas9ind
mouse ES cells
GFP
BFP
Cells are infected with lentiviruses expressing the sgRNA sequences and BFP
Endoenousgene
(p53)
GFP infected

22. mESCCas9ind
sgRNA-LIBRARY/BFP+
87897 sgRNAs
Target 20000 genes
+DOX
BFP+
BFP+
MOI: 0.3
BFP+
Sorting
KO-LIBRARY of
mES cells
Koike-Yusa et al, Nat Biotech 2014
“Genomewide” library of KO Cas9ind
mouse ES cells

23. Proof-of principle screening: 6-Thioguanine

24. - 6-TG (20 µM)/7 days: 5.106
cells.
5 resistant clones:
1 clone (Pde2a?)
4 clones (HPRT)
- ATRi (0.9 µM)/9 days: 5.106
cells.
7 resistant clones:
5 clones (cdc25a)
1 clone (TMEM123)
1 clone (unknown yet)
SUMMARY OF OUR SCREENINGS
NOTE: We choose doses at which NO wild type cell survives
1st library
*
DRUG
9 days
Clonal
isolation
5.106
cells

25. SUMMARY OF OUR SCREENINGS
2nd library
- 6-TG (20 µM)/7 days: 5.106
cells.
- ATRi (0.9 µM)/9 days: 5.106
cells.
16 resistant clones:
6 clones (cdc25a)
6 clones (CNOT8)
1 clone (Clca1, IL20)
1 clone (Dgcr8)
1 clone (Olfr877)
1 clone (Pcdhb2, Hoxb5)
6 resistant clones:
6 clones (HPRT): 3 different sgRNAs
*
*
C
ontrol
TUBULIN
CDC25A
CDC25A-KO
(clone#4)
CDC25A-KO
(clone#5)
CDC25A-KO
(clone#8)

26. CDC25A deficient cells are (HIGHLY) resistant to ATRi

ATRi 300nM
ATRi 900nM
ATRi 2µM
ATRi 3µM
Control
CDC25A-KO
(clone #4)
CDC25A-KO
(clone #5)
CDC25A-KO
(clone #8)
Validated with multiple sgRNAs

27. CDC25A depletion renders human cancer cells resistant to ATRi
Ruiz, Mayor-Ruiz et al Mol Cell (2016)

28. And what about
GAIN OF FUNCTION?

29. SAM strategy
Konnerman et al, Nature 517: 583-8

30. SAM-ready mESC cells
EF1-a dCas9VP64
SV40 pABlasticidin-R
2A
dCas9
VP64
MS2 p65HSF1
sgRNA
EF1-a MS2-p65-HSF1 SV40 pAEGFP
2A
Lentiviral library containing 69,225 sgRNA for 23,439 RefSeq isoforms
U6 Prom sgRNA-MS2 SV40 pAEF1-a Prom Puromycin-R
Collaboration with F Zhang

31. We can force the expression of silent genes in mESC
-
sgRNA-IL2R1-2sgRNA-Hbb-h1-3
DOX DOX-
Hbb-h1
Relativeexpressionlevels

32. We can force the expression of silent genes in mESC
-
sgRNA-IL2R1-2sgRNA-Hbb-h1-3
DOX DOX-
IL2R1Relativeexpressionlevels

33. Example of GOF screening in our SAM-mESC
NOTE: We choose doses at which NO wild type cell survives
ATRi (0.9 µM)
4 days
Clonal
isolation
5.106
cells
5 resistant clones:
1 clone (ctxn1)
1 clone (???)
1 clone (Pcolce2, Cyslt22)
1 clone (Mpp6)
1 clone (Sf3b2, olfr482)
1st library1st library (G4 mES cells)
7 resistant clones:
1 clone (Mns1)
1 clone (Sftpc, Ostn, Klf2)
1 clone (TopoRS1)
1 clone (Asah1, TopoRS1)
1 clone (Asb16)
1 clone (Uvssa, cd180, Sin3a)
1 clone (Sirbp1, Rab39)
2nd library (R1 mES cells)

34. Why ATR? The WHO
Why ATR? The HOW
Matilde Murga
Emilio Lecona
Sergio Ruiz
Vanesa Lafarga
Isabel Morgado
Federica Schiavoni
Cristina Mayor
Julia Specks
Teresa Olbrich
Sara Rodrigo
Marta Anton
Maria Vega
Alicia Gonzalez