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Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors.

Jaiswal BS, Janakiraman V, Kljavin NM, Eastham-Anderson J, Cupp JE, Liang Y, Davis DP, Hoeflich KP, Seshagiri S - PLoS ONE (2009)

Bottom Line: We find that in colon cancer cells harboring a KRAS(G13D) mutant allele, knocking down KRAS alone or the RAFs in combination or the RAF effectors, MEK1 and MEK2, together is effective in delaying tumor growth in vivo.In melanoma cells harboring an NRAS(Q61L) or NRAS(Q61K) mutant allele, we find that targeting NRAS alone or both BRAF and CRAF in combination or both BRAF and PIK3CA together showed efficacy.Our data indicates that targeting oncogenic NRAS-driven melanomas require decrease in both pERK and pAKT downstream of RAS-effectors for efficacy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Genentech Inc., South San Francisco, California, United States of America.

ABSTRACT

Background: Oncogenic RAS is a highly validated cancer target. Attempts at targeting RAS directly have so far not succeeded in the clinic. Understanding downstream RAS-effectors that mediate oncogenesis in a RAS mutant setting will help tailor treatments that use RAS-effector inhibitors either alone or in combination to target RAS-driven tumors.

Methodology/principal findings: In this study, we have investigated the sufficiency of targeting RAS-effectors, RAF, MEK and PI3-Kinase either alone or in combination in RAS mutant lines, using an inducible shRNA in vivo mouse model system. We find that in colon cancer cells harboring a KRAS(G13D) mutant allele, knocking down KRAS alone or the RAFs in combination or the RAF effectors, MEK1 and MEK2, together is effective in delaying tumor growth in vivo. In melanoma cells harboring an NRAS(Q61L) or NRAS(Q61K) mutant allele, we find that targeting NRAS alone or both BRAF and CRAF in combination or both BRAF and PIK3CA together showed efficacy.

Conclusion/significance: Our data indicates that targeting oncogenic NRAS-driven melanomas require decrease in both pERK and pAKT downstream of RAS-effectors for efficacy. This can be achieved by either targeting both BRAF and CRAF or BRAF and PIK3CA simultaneously in NRAS mutant tumor cells.

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Combined targeting of BRAF and CRAF in NRASQ61K-mutant SK-MEL-30 melanoma cell delays tumor formation in vivo.(A–B) Immunoblot analysis of SK-MEL-30 cells expressing control luciferase shRNA or shRNAs targeting NRAS, BRAF, CRAF or both BRAF and CRAF reveals respective protein knock-down and their effects on phosphorylation of downstream targets. (C) Proliferation of SK-MEL-30 cells expressing control luciferase shRNA or shRNAs targeting NRAS, BRAF, CRAF or both BRAF and CRAF. (D–H) shRNA targeting NRAS (E) or both BRAF and CRAF in SK-MEL-30 (H) delays tumor growth in vivo, whereas, induction of BRAF (F), CRAF (G) targeting shRNA or control luciferase (D) shRNA did not affect tumor growth. Each data point is the mean±SEM tumor volume of 10 mice. Dotted line in (D–H) represents data from dox treated animals.
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pone-0005717-g006: Combined targeting of BRAF and CRAF in NRASQ61K-mutant SK-MEL-30 melanoma cell delays tumor formation in vivo.(A–B) Immunoblot analysis of SK-MEL-30 cells expressing control luciferase shRNA or shRNAs targeting NRAS, BRAF, CRAF or both BRAF and CRAF reveals respective protein knock-down and their effects on phosphorylation of downstream targets. (C) Proliferation of SK-MEL-30 cells expressing control luciferase shRNA or shRNAs targeting NRAS, BRAF, CRAF or both BRAF and CRAF. (D–H) shRNA targeting NRAS (E) or both BRAF and CRAF in SK-MEL-30 (H) delays tumor growth in vivo, whereas, induction of BRAF (F), CRAF (G) targeting shRNA or control luciferase (D) shRNA did not affect tumor growth. Each data point is the mean±SEM tumor volume of 10 mice. Dotted line in (D–H) represents data from dox treated animals.

Mentions: In order to understand if other NRAS mutant lines employ similar signaling cascades downstream of NRAS, we chose to study SK-MEL-30, another melanoma line with an NRASQ61K mutant allele. We generated SK-MEL-30 cells bearing inducible shRNA that target NRAS, BRAF, CRAF, PIK3CA alone, or combinations of BRAF and CRAF, or BRAF and PIK3CA. Inducible knock-down of NRAS, BRAF, CRAF individually or BRAF and CRAF or BRAF and PIK3CA together showed a decrease in the appropriate target protein with concomitant changes in downstream signaling as indicated by decreases in pERK and/or pAKT levels (Figure 6A, 6B, 7A). As with IPC298, knock-down of NRAS or the combined knock-down of BRAF and CRAF or BRAF and PI3KCA was effective in decreasing both the pERK and pAKT (Figure 6A, 6B, 7A). In in vitro proliferation studies, induction of shRNA targeting NRAS, BRAF, CRAF, dual targeting of BRAF/CRAF, or BRAF/PIK3CA had a reduction in proliferation of ∼20%, ∼12%, ∼20%, ∼40% and ∼28%, respectively (Figure. 6C and 7B), while the control shLUC or shRNAs targeting PIK3CA did not have a significant effect on proliferation compared to un-induced cells (Figure. 6C and 7B). In in vivo tumor growth studies, tumors carrying shRNAs that target NRAS alone, or combinations of BRAF and CRAF, or BRAF and PIK3CA, showed a significant delay in tumor growth (Figure 6E, 6H and 7D) compared to the corresponding sucrose controls. As observed in IPC298 cells, targeting BRAF, CRAF or PIK3CA individually in SK-MEL-30 cells did not effect in vivo tumor growth (Figure 6F, 6G and 7C). These results further confirm the requirement for sustained decrease in pERK and pAKT mediated signaling achieved either through combined targeting of BRAF and CRAF or BRAF and PIK3CA effector arms to be a more general requirement for effective therapy in NRAS mutant tumors.


Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors.

Jaiswal BS, Janakiraman V, Kljavin NM, Eastham-Anderson J, Cupp JE, Liang Y, Davis DP, Hoeflich KP, Seshagiri S - PLoS ONE (2009)

Combined targeting of BRAF and CRAF in NRASQ61K-mutant SK-MEL-30 melanoma cell delays tumor formation in vivo.(A–B) Immunoblot analysis of SK-MEL-30 cells expressing control luciferase shRNA or shRNAs targeting NRAS, BRAF, CRAF or both BRAF and CRAF reveals respective protein knock-down and their effects on phosphorylation of downstream targets. (C) Proliferation of SK-MEL-30 cells expressing control luciferase shRNA or shRNAs targeting NRAS, BRAF, CRAF or both BRAF and CRAF. (D–H) shRNA targeting NRAS (E) or both BRAF and CRAF in SK-MEL-30 (H) delays tumor growth in vivo, whereas, induction of BRAF (F), CRAF (G) targeting shRNA or control luciferase (D) shRNA did not affect tumor growth. Each data point is the mean±SEM tumor volume of 10 mice. Dotted line in (D–H) represents data from dox treated animals.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683562&req=5

pone-0005717-g006: Combined targeting of BRAF and CRAF in NRASQ61K-mutant SK-MEL-30 melanoma cell delays tumor formation in vivo.(A–B) Immunoblot analysis of SK-MEL-30 cells expressing control luciferase shRNA or shRNAs targeting NRAS, BRAF, CRAF or both BRAF and CRAF reveals respective protein knock-down and their effects on phosphorylation of downstream targets. (C) Proliferation of SK-MEL-30 cells expressing control luciferase shRNA or shRNAs targeting NRAS, BRAF, CRAF or both BRAF and CRAF. (D–H) shRNA targeting NRAS (E) or both BRAF and CRAF in SK-MEL-30 (H) delays tumor growth in vivo, whereas, induction of BRAF (F), CRAF (G) targeting shRNA or control luciferase (D) shRNA did not affect tumor growth. Each data point is the mean±SEM tumor volume of 10 mice. Dotted line in (D–H) represents data from dox treated animals.
Mentions: In order to understand if other NRAS mutant lines employ similar signaling cascades downstream of NRAS, we chose to study SK-MEL-30, another melanoma line with an NRASQ61K mutant allele. We generated SK-MEL-30 cells bearing inducible shRNA that target NRAS, BRAF, CRAF, PIK3CA alone, or combinations of BRAF and CRAF, or BRAF and PIK3CA. Inducible knock-down of NRAS, BRAF, CRAF individually or BRAF and CRAF or BRAF and PIK3CA together showed a decrease in the appropriate target protein with concomitant changes in downstream signaling as indicated by decreases in pERK and/or pAKT levels (Figure 6A, 6B, 7A). As with IPC298, knock-down of NRAS or the combined knock-down of BRAF and CRAF or BRAF and PI3KCA was effective in decreasing both the pERK and pAKT (Figure 6A, 6B, 7A). In in vitro proliferation studies, induction of shRNA targeting NRAS, BRAF, CRAF, dual targeting of BRAF/CRAF, or BRAF/PIK3CA had a reduction in proliferation of ∼20%, ∼12%, ∼20%, ∼40% and ∼28%, respectively (Figure. 6C and 7B), while the control shLUC or shRNAs targeting PIK3CA did not have a significant effect on proliferation compared to un-induced cells (Figure. 6C and 7B). In in vivo tumor growth studies, tumors carrying shRNAs that target NRAS alone, or combinations of BRAF and CRAF, or BRAF and PIK3CA, showed a significant delay in tumor growth (Figure 6E, 6H and 7D) compared to the corresponding sucrose controls. As observed in IPC298 cells, targeting BRAF, CRAF or PIK3CA individually in SK-MEL-30 cells did not effect in vivo tumor growth (Figure 6F, 6G and 7C). These results further confirm the requirement for sustained decrease in pERK and pAKT mediated signaling achieved either through combined targeting of BRAF and CRAF or BRAF and PIK3CA effector arms to be a more general requirement for effective therapy in NRAS mutant tumors.

Bottom Line: We find that in colon cancer cells harboring a KRAS(G13D) mutant allele, knocking down KRAS alone or the RAFs in combination or the RAF effectors, MEK1 and MEK2, together is effective in delaying tumor growth in vivo.In melanoma cells harboring an NRAS(Q61L) or NRAS(Q61K) mutant allele, we find that targeting NRAS alone or both BRAF and CRAF in combination or both BRAF and PIK3CA together showed efficacy.Our data indicates that targeting oncogenic NRAS-driven melanomas require decrease in both pERK and pAKT downstream of RAS-effectors for efficacy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Genentech Inc., South San Francisco, California, United States of America.

ABSTRACT

Background: Oncogenic RAS is a highly validated cancer target. Attempts at targeting RAS directly have so far not succeeded in the clinic. Understanding downstream RAS-effectors that mediate oncogenesis in a RAS mutant setting will help tailor treatments that use RAS-effector inhibitors either alone or in combination to target RAS-driven tumors.

Methodology/principal findings: In this study, we have investigated the sufficiency of targeting RAS-effectors, RAF, MEK and PI3-Kinase either alone or in combination in RAS mutant lines, using an inducible shRNA in vivo mouse model system. We find that in colon cancer cells harboring a KRAS(G13D) mutant allele, knocking down KRAS alone or the RAFs in combination or the RAF effectors, MEK1 and MEK2, together is effective in delaying tumor growth in vivo. In melanoma cells harboring an NRAS(Q61L) or NRAS(Q61K) mutant allele, we find that targeting NRAS alone or both BRAF and CRAF in combination or both BRAF and PIK3CA together showed efficacy.

Conclusion/significance: Our data indicates that targeting oncogenic NRAS-driven melanomas require decrease in both pERK and pAKT downstream of RAS-effectors for efficacy. This can be achieved by either targeting both BRAF and CRAF or BRAF and PIK3CA simultaneously in NRAS mutant tumor cells.

Show MeSH
Related in: MedlinePlus