Preclinical evidence shows that hitting a single point along the RAF/MEK/ERK cascade disrupts intra-pathway negative feedback loop, may cause paradoxical pathway activation and may lead to functional resistance. Thus, the “vertical” combination of agents simultaneously inhibiting RAF and MEK has been proposed as a strategy to synergistically inhibit tumor growth and delay resistance. Experimental procedures: Molecular and functional effects of single and combined MEK (using trametinib, T), BRAF (using dabrafenib, D), and RAF (using the pan-RAF inhibitor, RAF265, R) inhibition were dissected by WB analysis and conservative isobologram analysis to assess functional synergism, using a fixed dose-ratio experimental design. Summary data: We examined the in vitro molecular and functional consequences of D and T, alone or in combination, in a panel of different human BRAFV600E melanoma cell lines; both drugs inhibited cell growth and inactivated the MAPK pathway, but little or no synergistic growth inhibition was observed with their combination (CI: 0.7 - 1.3x106). Conversely, combined D+T suppressed malignant growth with highly synergistic effects in KRAS-mutant lung (2/5 cell lines tested) and pancreatic (4/6 cell lines tested) cancer models (CI: 0.1 - 0.7), in which selective BRAF inhibition induced hyperphosphorylation of MEK, ERK, and p90RSK (paradox effect). At concentrations inhibiting both BRAF and CRAF, R did not induce paradox MAPK activation and did not result in growth inhibitory synergism when combined with T. To define the role of RAS gene status in determining sensitivity/resistance to single and combined RAF and MEK blockade, we analyzed two isogenic tumor cell line models: H1299 expressing individual codon 12 KRAS mutants and isogenic HCT116 clones differing for the presence of a homo or heterozygous G13D KRAS mutation. Conversely, in lung cancer models driven by either EGFR mutations (HCC827, H1650) or HER-2 overexpression (Calu-3), selective BRAF inhibition also induced a paradox MAPK activation, which could be blocked using a reversible EGFR/HER-2 inhibitor (Lapatinib); in this context, combination (D+T) resulted in a non-synergic growth inhibitory effects. Conclusions: Overall, our data indicate that, in appropriate cellular contexts, vertical RAF/MEK inhibition-based combination strategies exert highly synergistic antitumor effects across different cancer models.

Del Curatolo, A., Cesta Incani, U., Ciuffreda, L., Falcone, I., Shirasawa, S., Broggini, M., Sperduti, I., Eramo, A., De Maria, R., Cognetti, F., Milella, M., (Abstract) A vertical combination strategy hitting multiple steps along the MAPK cascade: Molecular mechanisms of action and putative genetic determinants of synergism, <<CANCER RESEARCH>>, 2014; (74): N/A-N/A. [doi:10.1158/1538-7445.AM2014-803] [http://hdl.handle.net/10807/116530]

A vertical combination strategy hitting multiple steps along the MAPK cascade: Molecular mechanisms of action and putative genetic determinants of synergism

De Maria, R.;
2014

Abstract

Preclinical evidence shows that hitting a single point along the RAF/MEK/ERK cascade disrupts intra-pathway negative feedback loop, may cause paradoxical pathway activation and may lead to functional resistance. Thus, the “vertical” combination of agents simultaneously inhibiting RAF and MEK has been proposed as a strategy to synergistically inhibit tumor growth and delay resistance. Experimental procedures: Molecular and functional effects of single and combined MEK (using trametinib, T), BRAF (using dabrafenib, D), and RAF (using the pan-RAF inhibitor, RAF265, R) inhibition were dissected by WB analysis and conservative isobologram analysis to assess functional synergism, using a fixed dose-ratio experimental design. Summary data: We examined the in vitro molecular and functional consequences of D and T, alone or in combination, in a panel of different human BRAFV600E melanoma cell lines; both drugs inhibited cell growth and inactivated the MAPK pathway, but little or no synergistic growth inhibition was observed with their combination (CI: 0.7 - 1.3x106). Conversely, combined D+T suppressed malignant growth with highly synergistic effects in KRAS-mutant lung (2/5 cell lines tested) and pancreatic (4/6 cell lines tested) cancer models (CI: 0.1 - 0.7), in which selective BRAF inhibition induced hyperphosphorylation of MEK, ERK, and p90RSK (paradox effect). At concentrations inhibiting both BRAF and CRAF, R did not induce paradox MAPK activation and did not result in growth inhibitory synergism when combined with T. To define the role of RAS gene status in determining sensitivity/resistance to single and combined RAF and MEK blockade, we analyzed two isogenic tumor cell line models: H1299 expressing individual codon 12 KRAS mutants and isogenic HCT116 clones differing for the presence of a homo or heterozygous G13D KRAS mutation. Conversely, in lung cancer models driven by either EGFR mutations (HCC827, H1650) or HER-2 overexpression (Calu-3), selective BRAF inhibition also induced a paradox MAPK activation, which could be blocked using a reversible EGFR/HER-2 inhibitor (Lapatinib); in this context, combination (D+T) resulted in a non-synergic growth inhibitory effects. Conclusions: Overall, our data indicate that, in appropriate cellular contexts, vertical RAF/MEK inhibition-based combination strategies exert highly synergistic antitumor effects across different cancer models.
Inglese
Del Curatolo, A., Cesta Incani, U., Ciuffreda, L., Falcone, I., Shirasawa, S., Broggini, M., Sperduti, I., Eramo, A., De Maria, R., Cognetti, F., Milella, M., (Abstract) A vertical combination strategy hitting multiple steps along the MAPK cascade: Molecular mechanisms of action and putative genetic determinants of synergism, <<CANCER RESEARCH>>, 2014; (74): N/A-N/A. [doi:10.1158/1538-7445.AM2014-803] [http://hdl.handle.net/10807/116530]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10807/116530
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