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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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Effect of BRAF and PIK3CA knock-down on KRASG13D mutant HCT116 cell growth.(A) Western blot analysis of PIK3CA or BRAF knock-down in HCT116 at 72 h post dox induction of relevant shRNAs. The effect of knock-down on the phosphorylation status of relevant downstream targets is shown. (B) Proliferation of BRAF and PIK3CA shRNA expressing cells 4 days post dox treatment. (C) shRNA targeting PIK3CA when induced in mice bearing HCT116 tumors did not delay tumor growth. (D) BRAF knock-down in HCT116 derived tumors shows a trend towards delayed tumor growth. Each data point is the mean±SEM tumor volume derived from 10 mice. Dotted line in (C, D) represents data from dox treated animals.
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pone-0005717-g002: Effect of BRAF and PIK3CA knock-down on KRASG13D mutant HCT116 cell growth.(A) Western blot analysis of PIK3CA or BRAF knock-down in HCT116 at 72 h post dox induction of relevant shRNAs. The effect of knock-down on the phosphorylation status of relevant downstream targets is shown. (B) Proliferation of BRAF and PIK3CA shRNA expressing cells 4 days post dox treatment. (C) shRNA targeting PIK3CA when induced in mice bearing HCT116 tumors did not delay tumor growth. (D) BRAF knock-down in HCT116 derived tumors shows a trend towards delayed tumor growth. Each data point is the mean±SEM tumor volume derived from 10 mice. Dotted line in (C, D) represents data from dox treated animals.

Mentions: HCT116-BRAF knockdown line, upon dox treatment showed reduction in BRAF protein levels and a concomitant decrease in phospho ERK levels relative to the total ERK levels (Figure 2A). Similarly, HCT116-PIK3CA knockdown lines when treated with dox showed a reduction in the levels of PIK3CA and a decrease in phospho AKT compared to the control un-induced cells. In proliferation studies, both the dox treated PIK3CA knockdown and BRAF knockdown HCT116 lines showed a 20–25% reduction in proliferation compared to non-dox treated cells (Figure 2B). However, in vivo, mice bearing tumors derived from PIK3CA knock-down HCT116-cells, showed no effect on tumor growth following dox treatment (Figure 2C). These results are consistent with the fact that the mutant PIK3CA in HCT116 did not functionally substitute for KRAS in experiments where KRAS was silenced, both in vitro and in vivo (Figure 1B and 1F). In contrast to knock-down of PIK3CA in HCT116, nude mice bearing subcutaneous tumors resulting from injected HCT116 BRAF-inducible shRNAs cells, when treated with dox showed a delay in tumor growth (Figure 2D). The delay was not statistically significant, in part due to the outlier tumor volumes observed towards the end of this study. However, this could also be in part due to the fact that the oncogenic RAS could engage other RAF family members like CRAF and continue to promote tumor growth and survival. To fully evaluate the contributions of the RAF arm that could result from engagement of other RAFs, we generated HCT116 inducible shRNA knock-down lines where we conditionally silenced CRAF or both CRAF and BRAF together. As expected, knock-down of BRAF and CRAF together decreased pMEK level completely, while knock-down of CRAF alone showed some residual pMEK levels (Figure 3A). Consistent with this finding, the BRAF/CRAF double knock-down line showed a decrease in pERK levels while the CRAF knock-down alone did not substantially affect pERK levels in vitro. However, knock-down of either CRAF or BRAF and CRAF together decreased pAKT levels (Figure 3A). In proliferation studies, CRAF knock-down lines showed a ∼25% decrease in proliferation, while the combined CRAF and BRAF knock down had ∼40% reduction in proliferation (Figure 3C). In vivo, combined CRAF and BRAF knock-down showed a significant delay in tumor growth compared to the un-induced control (Figure 3E). Although not as significant as the dual RAF knock-down, CRAF knock-down alone significantly delayed tumor growth (Figure 3D). These results suggest that KRAS in HCT116 cells engages both CRAF and BRAF for promoting tumor growth with CRAF being the dominant effector arm.


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)

Effect of BRAF and PIK3CA knock-down on KRASG13D mutant HCT116 cell growth.(A) Western blot analysis of PIK3CA or BRAF knock-down in HCT116 at 72 h post dox induction of relevant shRNAs. The effect of knock-down on the phosphorylation status of relevant downstream targets is shown. (B) Proliferation of BRAF and PIK3CA shRNA expressing cells 4 days post dox treatment. (C) shRNA targeting PIK3CA when induced in mice bearing HCT116 tumors did not delay tumor growth. (D) BRAF knock-down in HCT116 derived tumors shows a trend towards delayed tumor growth. Each data point is the mean±SEM tumor volume derived from 10 mice. Dotted line in (C, D) represents data from dox treated animals.
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Related In: Results  -  Collection

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pone-0005717-g002: Effect of BRAF and PIK3CA knock-down on KRASG13D mutant HCT116 cell growth.(A) Western blot analysis of PIK3CA or BRAF knock-down in HCT116 at 72 h post dox induction of relevant shRNAs. The effect of knock-down on the phosphorylation status of relevant downstream targets is shown. (B) Proliferation of BRAF and PIK3CA shRNA expressing cells 4 days post dox treatment. (C) shRNA targeting PIK3CA when induced in mice bearing HCT116 tumors did not delay tumor growth. (D) BRAF knock-down in HCT116 derived tumors shows a trend towards delayed tumor growth. Each data point is the mean±SEM tumor volume derived from 10 mice. Dotted line in (C, D) represents data from dox treated animals.
Mentions: HCT116-BRAF knockdown line, upon dox treatment showed reduction in BRAF protein levels and a concomitant decrease in phospho ERK levels relative to the total ERK levels (Figure 2A). Similarly, HCT116-PIK3CA knockdown lines when treated with dox showed a reduction in the levels of PIK3CA and a decrease in phospho AKT compared to the control un-induced cells. In proliferation studies, both the dox treated PIK3CA knockdown and BRAF knockdown HCT116 lines showed a 20–25% reduction in proliferation compared to non-dox treated cells (Figure 2B). However, in vivo, mice bearing tumors derived from PIK3CA knock-down HCT116-cells, showed no effect on tumor growth following dox treatment (Figure 2C). These results are consistent with the fact that the mutant PIK3CA in HCT116 did not functionally substitute for KRAS in experiments where KRAS was silenced, both in vitro and in vivo (Figure 1B and 1F). In contrast to knock-down of PIK3CA in HCT116, nude mice bearing subcutaneous tumors resulting from injected HCT116 BRAF-inducible shRNAs cells, when treated with dox showed a delay in tumor growth (Figure 2D). The delay was not statistically significant, in part due to the outlier tumor volumes observed towards the end of this study. However, this could also be in part due to the fact that the oncogenic RAS could engage other RAF family members like CRAF and continue to promote tumor growth and survival. To fully evaluate the contributions of the RAF arm that could result from engagement of other RAFs, we generated HCT116 inducible shRNA knock-down lines where we conditionally silenced CRAF or both CRAF and BRAF together. As expected, knock-down of BRAF and CRAF together decreased pMEK level completely, while knock-down of CRAF alone showed some residual pMEK levels (Figure 3A). Consistent with this finding, the BRAF/CRAF double knock-down line showed a decrease in pERK levels while the CRAF knock-down alone did not substantially affect pERK levels in vitro. However, knock-down of either CRAF or BRAF and CRAF together decreased pAKT levels (Figure 3A). In proliferation studies, CRAF knock-down lines showed a ∼25% decrease in proliferation, while the combined CRAF and BRAF knock down had ∼40% reduction in proliferation (Figure 3C). In vivo, combined CRAF and BRAF knock-down showed a significant delay in tumor growth compared to the un-induced control (Figure 3E). Although not as significant as the dual RAF knock-down, CRAF knock-down alone significantly delayed tumor growth (Figure 3D). These results suggest that KRAS in HCT116 cells engages both CRAF and BRAF for promoting tumor growth with CRAF being the dominant effector arm.

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