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Kinase-independent role for CRAF-driving tumour radioresistance via CHK2.

Advani SJ, Camargo MF, Seguin L, Mielgo A, Anand S, Hicks AM, Aguilera J, Franovic A, Weis SM, Cheresh DA - Nat Commun (2015)

Bottom Line: Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance.CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response.Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Medicine and Applied Sciences at the UC San Diego Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Although oncology therapy regimens commonly include radiation and genotoxic drugs, tumour cells typically develop resistance to these interventions. Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance. CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response. Accordingly, a phospho-mimetic mutant of CRAF (S338D) is sufficient to induce the CRAF/CHK2 association enhancing tumour radioresistance, while an allosteric CRAF inhibitor sensitizes tumour cells to ionizing radiation or genotoxic drugs. Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

No MeSH data available.


Related in: MedlinePlus

CRAF kinase activity is not required to drive radioresistance.(a) HCT-116 cells were stably transfected to express wild-type CRAF (WT) or the phospho-mimetic CRAF S338D mutant. Cells treated with or without 6 Gy were analysed for DNA damage using clonogenic (*P=0.012, n=3 wells per group, two-sided t-test) and comet tail assays (P<0.0001, n=100+ cells per group, two-sided t-test). Bars represent mean±s.e.m. Data are representative of two independent experiments. (b) Immune-compromised nu/nu mice were implanted s.c. with tumour cells to each thigh, and only the right thigh received three fractions of 6 Gy on Days 5, 7 and 9. Graph shows mean tumour volume±s.e.m, *P=0.04 from two-sided t-test comparing WT+IR (n=10) versus S338D+IR (n=9) at the endpoint on Day 15. (c) Stably transfected U87 cells expressing wild-type CRAF (WT) or the CRAF kinase-dead, phospho-mimetic double mutant (S338D/K375M) were exposed to 6 Gy. DNA damage was assessed by clonogenic (P=0.002, n=3 wells per group), comet tail (*P=0.0005, n=100+ cells per group) and γH2AX assays (*P=0.0007, n=6 fields per group). All bar graphs show mean±s.e.m. P values from two-sided t-tests comparing WT versus each CRAF mutant. Data are representative of two independent experiments. (d) CRAF−/− MEFs were transfected with GFP-tagged WT, S338A, S338D or K375M CRAF for 72 h and then given 6 Gy. DNA damage was assessed using γH2AX staining. Graph shows mean γH2AX foci per cell±s.e.m. for n=50+ cells analysed per group. *P<0.05 from two-sided t-test comparing WT and CRAF−/−. Data are representative of three independent experiments.
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f2: CRAF kinase activity is not required to drive radioresistance.(a) HCT-116 cells were stably transfected to express wild-type CRAF (WT) or the phospho-mimetic CRAF S338D mutant. Cells treated with or without 6 Gy were analysed for DNA damage using clonogenic (*P=0.012, n=3 wells per group, two-sided t-test) and comet tail assays (P<0.0001, n=100+ cells per group, two-sided t-test). Bars represent mean±s.e.m. Data are representative of two independent experiments. (b) Immune-compromised nu/nu mice were implanted s.c. with tumour cells to each thigh, and only the right thigh received three fractions of 6 Gy on Days 5, 7 and 9. Graph shows mean tumour volume±s.e.m, *P=0.04 from two-sided t-test comparing WT+IR (n=10) versus S338D+IR (n=9) at the endpoint on Day 15. (c) Stably transfected U87 cells expressing wild-type CRAF (WT) or the CRAF kinase-dead, phospho-mimetic double mutant (S338D/K375M) were exposed to 6 Gy. DNA damage was assessed by clonogenic (P=0.002, n=3 wells per group), comet tail (*P=0.0005, n=100+ cells per group) and γH2AX assays (*P=0.0007, n=6 fields per group). All bar graphs show mean±s.e.m. P values from two-sided t-tests comparing WT versus each CRAF mutant. Data are representative of two independent experiments. (d) CRAF−/− MEFs were transfected with GFP-tagged WT, S338A, S338D or K375M CRAF for 72 h and then given 6 Gy. DNA damage was assessed using γH2AX staining. Graph shows mean γH2AX foci per cell±s.e.m. for n=50+ cells analysed per group. *P<0.05 from two-sided t-test comparing WT and CRAF−/−. Data are representative of three independent experiments.

Mentions: To validate the role of CRAF pS338 in radioresistance, HCT-116 cells expressing a phospho-mimetic mutant of CRAF (S338D) or full-length wild-type (WT) CRAF were exposed to IR and examined for cell survival and DNA damage. Expression of CRAF S338D protected cells from IR-induced damage compared with cells expressing WT CRAF (Fig. 2a, Supplementary Fig. 5), suggesting that CRAF pS338 is sufficient to promote radioresistance. To validate this finding in vivo, mice were implanted with HCT-116 tumours (expressing either WT or S338D CRAF) on bilateral flanks, and only the right flank was subjected to localized 6 Gy radiation on days 5, 7 and 9 after tumour implantation. While irradiation inhibited the growth of tumours expressing WT CRAF, tumours expressing CRAF S338D continued to grow (Fig. 2b) indicating that the phospho-mimetic CRAF mutant is sufficient to protect tumours from radiation damage.


Kinase-independent role for CRAF-driving tumour radioresistance via CHK2.

Advani SJ, Camargo MF, Seguin L, Mielgo A, Anand S, Hicks AM, Aguilera J, Franovic A, Weis SM, Cheresh DA - Nat Commun (2015)

CRAF kinase activity is not required to drive radioresistance.(a) HCT-116 cells were stably transfected to express wild-type CRAF (WT) or the phospho-mimetic CRAF S338D mutant. Cells treated with or without 6 Gy were analysed for DNA damage using clonogenic (*P=0.012, n=3 wells per group, two-sided t-test) and comet tail assays (P<0.0001, n=100+ cells per group, two-sided t-test). Bars represent mean±s.e.m. Data are representative of two independent experiments. (b) Immune-compromised nu/nu mice were implanted s.c. with tumour cells to each thigh, and only the right thigh received three fractions of 6 Gy on Days 5, 7 and 9. Graph shows mean tumour volume±s.e.m, *P=0.04 from two-sided t-test comparing WT+IR (n=10) versus S338D+IR (n=9) at the endpoint on Day 15. (c) Stably transfected U87 cells expressing wild-type CRAF (WT) or the CRAF kinase-dead, phospho-mimetic double mutant (S338D/K375M) were exposed to 6 Gy. DNA damage was assessed by clonogenic (P=0.002, n=3 wells per group), comet tail (*P=0.0005, n=100+ cells per group) and γH2AX assays (*P=0.0007, n=6 fields per group). All bar graphs show mean±s.e.m. P values from two-sided t-tests comparing WT versus each CRAF mutant. Data are representative of two independent experiments. (d) CRAF−/− MEFs were transfected with GFP-tagged WT, S338A, S338D or K375M CRAF for 72 h and then given 6 Gy. DNA damage was assessed using γH2AX staining. Graph shows mean γH2AX foci per cell±s.e.m. for n=50+ cells analysed per group. *P<0.05 from two-sided t-test comparing WT and CRAF−/−. Data are representative of three independent experiments.
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f2: CRAF kinase activity is not required to drive radioresistance.(a) HCT-116 cells were stably transfected to express wild-type CRAF (WT) or the phospho-mimetic CRAF S338D mutant. Cells treated with or without 6 Gy were analysed for DNA damage using clonogenic (*P=0.012, n=3 wells per group, two-sided t-test) and comet tail assays (P<0.0001, n=100+ cells per group, two-sided t-test). Bars represent mean±s.e.m. Data are representative of two independent experiments. (b) Immune-compromised nu/nu mice were implanted s.c. with tumour cells to each thigh, and only the right thigh received three fractions of 6 Gy on Days 5, 7 and 9. Graph shows mean tumour volume±s.e.m, *P=0.04 from two-sided t-test comparing WT+IR (n=10) versus S338D+IR (n=9) at the endpoint on Day 15. (c) Stably transfected U87 cells expressing wild-type CRAF (WT) or the CRAF kinase-dead, phospho-mimetic double mutant (S338D/K375M) were exposed to 6 Gy. DNA damage was assessed by clonogenic (P=0.002, n=3 wells per group), comet tail (*P=0.0005, n=100+ cells per group) and γH2AX assays (*P=0.0007, n=6 fields per group). All bar graphs show mean±s.e.m. P values from two-sided t-tests comparing WT versus each CRAF mutant. Data are representative of two independent experiments. (d) CRAF−/− MEFs were transfected with GFP-tagged WT, S338A, S338D or K375M CRAF for 72 h and then given 6 Gy. DNA damage was assessed using γH2AX staining. Graph shows mean γH2AX foci per cell±s.e.m. for n=50+ cells analysed per group. *P<0.05 from two-sided t-test comparing WT and CRAF−/−. Data are representative of three independent experiments.
Mentions: To validate the role of CRAF pS338 in radioresistance, HCT-116 cells expressing a phospho-mimetic mutant of CRAF (S338D) or full-length wild-type (WT) CRAF were exposed to IR and examined for cell survival and DNA damage. Expression of CRAF S338D protected cells from IR-induced damage compared with cells expressing WT CRAF (Fig. 2a, Supplementary Fig. 5), suggesting that CRAF pS338 is sufficient to promote radioresistance. To validate this finding in vivo, mice were implanted with HCT-116 tumours (expressing either WT or S338D CRAF) on bilateral flanks, and only the right flank was subjected to localized 6 Gy radiation on days 5, 7 and 9 after tumour implantation. While irradiation inhibited the growth of tumours expressing WT CRAF, tumours expressing CRAF S338D continued to grow (Fig. 2b) indicating that the phospho-mimetic CRAF mutant is sufficient to protect tumours from radiation damage.

Bottom Line: Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance.CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response.Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Medicine and Applied Sciences at the UC San Diego Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Although oncology therapy regimens commonly include radiation and genotoxic drugs, tumour cells typically develop resistance to these interventions. Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance. CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response. Accordingly, a phospho-mimetic mutant of CRAF (S338D) is sufficient to induce the CRAF/CHK2 association enhancing tumour radioresistance, while an allosteric CRAF inhibitor sensitizes tumour cells to ionizing radiation or genotoxic drugs. Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

No MeSH data available.


Related in: MedlinePlus