A clinically relevant heterozygous ATR mutation sensitizes colorectal cancer cells to replication stress.
Colorectal cancer (CRC) ranks third among the most frequent malignancies and represents the second most common cause of cancer-related deaths worldwide. By interfering with the DNA replication process of cancer cells, several chemotherapeutic molecules used in CRC therapy induce replication stress (RS). At the cellular level, this stress is managed by the ATR-CHK1 pathway, which activates the replication checkpoint. In recent years, the therapeutic value of targeting this pathway has been demonstrated. Moreover, MSI + (microsatellite instability) tumors frequently harbor a nonsense, heterozygous mutation in the ATR gene. Using isogenic HCT116 clones, we showed that this mutation of ATR sensitizes the cells to several drugs, including SN-38 (topoisomerase I inhibitor) and VE-822 (ATR inhibitor) and exacerbates their synergistic effects. We showed that this mutation bottlenecks the replication checkpoint leading to extensive DNA damage. The combination of VE-822 and SN-38 induces an exhaustion of RPA and a subsequent replication catastrophe. Surviving cells complete replication and accumulate in G2 in a DNA-PK-dependent manner, protecting them from cell death. Together, our results suggest that RPA and DNA-PK represent promising therapeutic targets to optimize the inhibition of the ATR-CHK1 pathway in oncology. Ultimately, ATR frameshift mutations found in patients may also represent important prognostic factors.
Pubmed ID: 35361811 RIS Download
Research resources used in this publication
Additional research tools detected in this publication
None foundAntibodies used in this publication
- RPA32/RPA2 antibody [9H8] (RRID:AB_302873)
- GAPDH antibody [6C5] (RRID:AB_2107448)
- KAP1 antibody [20C1] - ChIP Grade (RRID:AB_447151)
- Rat anti-Mouse IgG1 Secondary Antibody, PE-Cyanine7, eBioscience (RRID:AB_11150243)
- Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555 (RRID:AB_2535844)
- Goat Anti-Rabbit IgG (H+L) Antibody, Alexa Fluor 488 Conjugated (RRID:AB_143165)
- Goat Anti-Rat IgG-FITC (RRID:AB_2795818)
- Goat Anti-Rabbit IgG(H+L)-HRP (RRID:AB_2795980)
- Goat Anti-Mouse IgG, Human ads-HRP (RRID:AB_2619742)
- KAP1 (phospho S143) antibody (RRID:AB_11157160)
- Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb (RRID:AB_2080501)
- ATM (D2E2) Rabbit mAb (RRID:AB_2062659)
- Anti-53BP1, phospho (Ser1778) Antibody, Unconjugated (RRID:AB_490917)
- beta Tubulin antibody - Loading Control (RRID:AB_727043)
- Phospho RPA32 (S4/S8) Antibody (RRID:AB_210547)
- Mouse Anti-DNA, single stranded Monoclonal antibody, Unconjugated, Clone 16-19 (RRID:AB_94645)
- Chk1 (2G1D5) Mouse mAb (RRID:AB_2080320)
- Phospho-Chk1 (Ser345) (133D3) Rabbit mAb (RRID:AB_331212)
- ATR Antibody (RRID:AB_2227860)
- ATR (phospho Thr1989) antibody (RRID:AB_2687562)
- Phospho-ATM (Ser1981) (D25E5) Rabbit mAb (RRID:AB_2798100)
- Anti-DNA-PK Antibody, Unconjugated (RRID:AB_10692482)
- DNA PKcs (phospho S2056) antibody - ChIP Grade (RRID:AB_869495)
- BrdU (RRID:AB_609568)
- Chk2 Antibody (RRID:AB_2080793)
- Phospho-Chk2 (Ser19) Antibody (RRID:AB_330057)
- Phospho-TIF1-beta (Ser824) Antibody (RRID:AB_2209906)
- Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (RRID:AB_2118009)
- Cleaved Caspase-3 (Asp175) Antibody (RRID:AB_2341188)
Associated grants
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This is a list of tools and resources that we have found mentioned in this publication.
RPA32/RPA2 antibody [9H8] (antibody)
RRID:AB_302873
This monoclonal targets RPA32/RPA2
View all literature mentionsGAPDH antibody [6C5] (antibody)
RRID:AB_2107448
This monoclonal targets GAPDH antibody [6C5]
View all literature mentionsKAP1 antibody [20C1] - ChIP Grade (antibody)
RRID:AB_447151
This monoclonal targets KAP1
View all literature mentionsRat anti-Mouse IgG1 Secondary Antibody, PE-Cyanine7, eBioscience (antibody)
RRID:AB_11150243
This monoclonal secondary targets IgG1
View all literature mentionsGoat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555 (antibody)
RRID:AB_2535844
This polyclonal secondary targets IgG (H+L)
View all literature mentionsGoat Anti-Rabbit IgG (H+L) Antibody, Alexa Fluor 488 Conjugated (antibody)
RRID:AB_143165
This polyclonal targets IgG (H+L)
View all literature mentionsGoat Anti-Rat IgG-FITC (antibody)
RRID:AB_2795818
This unknown targets IgG
View all literature mentionsGoat Anti-Rabbit IgG(H+L)-HRP (antibody)
RRID:AB_2795980
This unknown targets IgG(H+L)
View all literature mentionsGoat Anti-Mouse IgG, Human ads-HRP (antibody)
RRID:AB_2619742
This polyclonal targets mouse IgG
View all literature mentionsKAP1 (phospho S143) antibody (antibody)
RRID:AB_11157160
This polyclonal targets KAP1 (phospho S143) antibody
View all literature mentionsPhospho-Chk2 (Thr68) (C13C1) Rabbit mAb (antibody)
RRID:AB_2080501
This monoclonal targets CHEK2
View all literature mentionsATM (D2E2) Rabbit mAb (antibody)
RRID:AB_2062659
This monoclonal targets ATM (D2E2) Rabbit mAb
View all literature mentionsAnti-53BP1, phospho (Ser1778) Antibody, Unconjugated (antibody)
RRID:AB_490917
This unknown targets 53BP1, phospho (Ser1778)
View all literature mentionsbeta Tubulin antibody - Loading Control (antibody)
RRID:AB_727043
This polyclonal targets beta Tubulin antibody - Loading Control
View all literature mentionsPhospho RPA32 (S4/S8) Antibody (antibody)
RRID:AB_210547
This unknown targets Phospho-RPA32 (Ser4, Ser8)
View all literature mentionsMouse Anti-DNA, single stranded Monoclonal antibody, Unconjugated, Clone 16-19 (antibody)
RRID:AB_94645
This monoclonal targets DNA, single stranded
View all literature mentionsChk1 (2G1D5) Mouse mAb (antibody)
RRID:AB_2080320
This monoclonal targets Chek1
View all literature mentionsPhospho-Chk1 (Ser345) (133D3) Rabbit mAb (antibody)
RRID:AB_331212
This monoclonal targets Chk1, phospho (Ser345)
View all literature mentionsATR (phospho Thr1989) antibody (antibody)
RRID:AB_2687562
This polyclonal targets ATR (phospho Thr1989)
View all literature mentionsPhospho-ATM (Ser1981) (D25E5) Rabbit mAb (antibody)
RRID:AB_2798100
This monoclonal targets ATM
View all literature mentionsAnti-DNA-PK Antibody, Unconjugated (antibody)
RRID:AB_10692482
This unknown targets DNA-PK
View all literature mentionsDNA PKcs (phospho S2056) antibody - ChIP Grade (antibody)
RRID:AB_869495
This polyclonal targets Human DNA PKcs (phospho S2056)
View all literature mentionsPhospho-Chk2 (Ser19) Antibody (antibody)
RRID:AB_330057
This unknown targets Chk2, phospho (Ser19)
View all literature mentionsPhospho-TIF1-beta (Ser824) Antibody (antibody)
RRID:AB_2209906
This polyclonal targets TRIM28
View all literature mentionsPhospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (antibody)
RRID:AB_2118009
This monoclonal targets Histone H2A.X, phospho (Ser139)
View all literature mentionsCleaved Caspase-3 (Asp175) Antibody (antibody)
RRID:AB_2341188
This polyclonal targets Cleaved Caspase-3 (Asp175)
View all literature mentionsHCT 116 (cell line)
RRID:CVCL_0291
Cell line HCT 116 is a Cancer cell line with a species of origin Homo sapiens (Human)
View all literature mentions
