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BRAF inhibitors suppress apoptosis through off-target inhibition of JNK signaling.

Vin H, Ojeda SS, Ching G, Leung ML, Chitsazzadeh V, Dwyer DW, Adelmann CH, Restrepo M, Richards KN, Stewart LR, Du L, Ferguson SB, Chakravarti D, Ehrenreiter K, Baccarini M, Ruggieri R, Curry JL, Kim KB, Ciurea AM, Duvic M, Prieto VG, Ullrich SE, Dalby KN, Flores ER, Tsai KY - Elife (2013)

Bottom Line: The prevailing explanation for this is drug-induced paradoxical ERK activation, resulting in hyperproliferation.Here we show an unexpected and novel effect of vemurafenib/PLX4720 in suppressing apoptosis through the inhibition of multiple off-target kinases upstream of c-Jun N-terminal kinase (JNK), principally ZAK.Our results implicate suppression of JNK-dependent apoptosis as a significant, independent mechanism that cooperates with paradoxical ERK activation to induce cSCC, suggesting broad implications for understanding toxicities associated with BRAF inhibitors and for their use in combination therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, United States.

ABSTRACT
Vemurafenib and dabrafenib selectively inhibit the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) kinase, resulting in high response rates and increased survival in melanoma. Approximately 22% of individuals treated with vemurafenib develop cutaneous squamous cell carcinoma (cSCC) during therapy. The prevailing explanation for this is drug-induced paradoxical ERK activation, resulting in hyperproliferation. Here we show an unexpected and novel effect of vemurafenib/PLX4720 in suppressing apoptosis through the inhibition of multiple off-target kinases upstream of c-Jun N-terminal kinase (JNK), principally ZAK. JNK signaling is suppressed in multiple contexts, including in cSCC of vemurafenib-treated patients, as well as in mice. Expression of a mutant ZAK that cannot be inhibited reverses the suppression of JNK activation and apoptosis. Our results implicate suppression of JNK-dependent apoptosis as a significant, independent mechanism that cooperates with paradoxical ERK activation to induce cSCC, suggesting broad implications for understanding toxicities associated with BRAF inhibitors and for their use in combination therapies. DOI: http://dx.doi.org/10.7554/eLife.00969.001.

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Paradoxical MEK and ERK activation require intact Craf.Wild-type (WT) and isogenic Craf−/− MEFs were retrovirally transduced with HRASG12V and adenovirus E1A thereby enabling anchorage-independent growth for soft agar assays. (A) WT MEFs exhibit paradoxical MEK and ERK activation, effects that are significantly reduced in Craf−/− MEFs, particularly for MEK activation. (B) HRASG12V–transformed HaCaT cells with (‘TKD’) and without (‘SCR’) triple knockdown of ZAK, MAP4K5, and MAP2K4 show equivalent paradoxical ERK activation. (C) Transformed WT and Craf−/− MEFs show equivalent expression of E1A (sc-25, Santa Cruz) and RAS (sc-32, Santa Cruz). (D) Transformed HaCaT cells show equivalent expression of RAS.DOI:http://dx.doi.org/10.7554/eLife.00969.023
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fig6s1: Paradoxical MEK and ERK activation require intact Craf.Wild-type (WT) and isogenic Craf−/− MEFs were retrovirally transduced with HRASG12V and adenovirus E1A thereby enabling anchorage-independent growth for soft agar assays. (A) WT MEFs exhibit paradoxical MEK and ERK activation, effects that are significantly reduced in Craf−/− MEFs, particularly for MEK activation. (B) HRASG12V–transformed HaCaT cells with (‘TKD’) and without (‘SCR’) triple knockdown of ZAK, MAP4K5, and MAP2K4 show equivalent paradoxical ERK activation. (C) Transformed WT and Craf−/− MEFs show equivalent expression of E1A (sc-25, Santa Cruz) and RAS (sc-32, Santa Cruz). (D) Transformed HaCaT cells show equivalent expression of RAS.DOI:http://dx.doi.org/10.7554/eLife.00969.023

Mentions: While the effects of these BRAFi on JNK-dependent apoptosis is clear and independent of ERK activity, the relative contribution of paradoxical ERK activation vs JNK pathway inhibition to tumorigenesis has not been precisely addressed (Figure 6A). To accomplish this, we took advantage of the fact that paradoxical ERK activation requires intact CRAF (Hatzivassiliou et al., 2010; Heidorn et al., 2010; Poulikakos et al., 2010) (Figure 6A). We used isogenic, matched WT and Craf-deficient (Craf−/−) mouse embryonic fibroblasts (MEFs) and transformed them with adenovirus E1A and human HRASG12V to enable anchorage-independent growth (Figure 6—figure supplement 1). These Craf−/− cells do not exhibit strong paradoxical MEK or ERK activation, consistent with previous reports (Poulikakos et al., 2010) (Figure 6—figure supplement 1A). Wild-type and matched Craf-deficient MEFs were plated in soft agar assays (Su et al., 2012) and treated with PLX4720. Both WT and Craf-deficient MEFs exhibited a significant colony formation advantage in the presence of drug (Figure 6B–D). Based upon this analysis, we estimated that the effect of paradoxical ERK activation to be 60% and other effects, including inhibition of JNK activity, to account for the rest (40%) of the total colony growth advantage (Figure 6D). To assess the role of JNK signaling directly, we used HRASG12V-transformed HaCaT cells with (‘TKD’) and without (‘SCR’) triple lentiviral knockdown of ZAK, MAP4K5, and MKK4 (Figure 3D,E), to perform similar colony formation assays to assess responses to PLX4720 treatment (Figure 6E). Drug treatment conferred a significant colony formation advantage in both sets of cells, which exhibit equivalent paradoxical ERK activation (Figure 6—figure supplement 1B). Yet, untreated TKD HaCaT cells produced more colonies than SCR HaCaT cells suggesting that JNK pathway suppression results in an advantage in the absence of drug and paradoxical ERK activation (Figure 6E). Drug-treated SCR and TKD HaCaT cells, had elevated colony counts to similar levels, as expected, because both lines would experience similar degrees of both paradoxical ERK activation and JNK inhibition, and TKD cells (knocked down for ZAK, MAP4K5, MKK4) are unlikely to experience any further suppression of JNK signaling (Figure 3D). Based on this, we estimated the effect of JNK pathway inhibition to be 17.6% (Figure 6E). When combined with the MEF experiment, we estimate that the effect of JNK inhibition contributes approximately 17.6–40% of the total effect of PLX4720-accelerated colony formation (Figure 6D,E). Importantly, although we can quantify these individual contributions, it is clear in many contexts in cancer that hyperproliferation and inhibition of apoptosis are highly cooperative (Hanahan and Weinberg, 2011), and our data do not preclude the possibility that one or both are individually required.10.7554/eLife.00969.022Figure 6.Paradoxical ERK activation and JNK pathway inhibition make significant and separable contributions to BRAFi-induced growth.


BRAF inhibitors suppress apoptosis through off-target inhibition of JNK signaling.

Vin H, Ojeda SS, Ching G, Leung ML, Chitsazzadeh V, Dwyer DW, Adelmann CH, Restrepo M, Richards KN, Stewart LR, Du L, Ferguson SB, Chakravarti D, Ehrenreiter K, Baccarini M, Ruggieri R, Curry JL, Kim KB, Ciurea AM, Duvic M, Prieto VG, Ullrich SE, Dalby KN, Flores ER, Tsai KY - Elife (2013)

Paradoxical MEK and ERK activation require intact Craf.Wild-type (WT) and isogenic Craf−/− MEFs were retrovirally transduced with HRASG12V and adenovirus E1A thereby enabling anchorage-independent growth for soft agar assays. (A) WT MEFs exhibit paradoxical MEK and ERK activation, effects that are significantly reduced in Craf−/− MEFs, particularly for MEK activation. (B) HRASG12V–transformed HaCaT cells with (‘TKD’) and without (‘SCR’) triple knockdown of ZAK, MAP4K5, and MAP2K4 show equivalent paradoxical ERK activation. (C) Transformed WT and Craf−/− MEFs show equivalent expression of E1A (sc-25, Santa Cruz) and RAS (sc-32, Santa Cruz). (D) Transformed HaCaT cells show equivalent expression of RAS.DOI:http://dx.doi.org/10.7554/eLife.00969.023
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6s1: Paradoxical MEK and ERK activation require intact Craf.Wild-type (WT) and isogenic Craf−/− MEFs were retrovirally transduced with HRASG12V and adenovirus E1A thereby enabling anchorage-independent growth for soft agar assays. (A) WT MEFs exhibit paradoxical MEK and ERK activation, effects that are significantly reduced in Craf−/− MEFs, particularly for MEK activation. (B) HRASG12V–transformed HaCaT cells with (‘TKD’) and without (‘SCR’) triple knockdown of ZAK, MAP4K5, and MAP2K4 show equivalent paradoxical ERK activation. (C) Transformed WT and Craf−/− MEFs show equivalent expression of E1A (sc-25, Santa Cruz) and RAS (sc-32, Santa Cruz). (D) Transformed HaCaT cells show equivalent expression of RAS.DOI:http://dx.doi.org/10.7554/eLife.00969.023
Mentions: While the effects of these BRAFi on JNK-dependent apoptosis is clear and independent of ERK activity, the relative contribution of paradoxical ERK activation vs JNK pathway inhibition to tumorigenesis has not been precisely addressed (Figure 6A). To accomplish this, we took advantage of the fact that paradoxical ERK activation requires intact CRAF (Hatzivassiliou et al., 2010; Heidorn et al., 2010; Poulikakos et al., 2010) (Figure 6A). We used isogenic, matched WT and Craf-deficient (Craf−/−) mouse embryonic fibroblasts (MEFs) and transformed them with adenovirus E1A and human HRASG12V to enable anchorage-independent growth (Figure 6—figure supplement 1). These Craf−/− cells do not exhibit strong paradoxical MEK or ERK activation, consistent with previous reports (Poulikakos et al., 2010) (Figure 6—figure supplement 1A). Wild-type and matched Craf-deficient MEFs were plated in soft agar assays (Su et al., 2012) and treated with PLX4720. Both WT and Craf-deficient MEFs exhibited a significant colony formation advantage in the presence of drug (Figure 6B–D). Based upon this analysis, we estimated that the effect of paradoxical ERK activation to be 60% and other effects, including inhibition of JNK activity, to account for the rest (40%) of the total colony growth advantage (Figure 6D). To assess the role of JNK signaling directly, we used HRASG12V-transformed HaCaT cells with (‘TKD’) and without (‘SCR’) triple lentiviral knockdown of ZAK, MAP4K5, and MKK4 (Figure 3D,E), to perform similar colony formation assays to assess responses to PLX4720 treatment (Figure 6E). Drug treatment conferred a significant colony formation advantage in both sets of cells, which exhibit equivalent paradoxical ERK activation (Figure 6—figure supplement 1B). Yet, untreated TKD HaCaT cells produced more colonies than SCR HaCaT cells suggesting that JNK pathway suppression results in an advantage in the absence of drug and paradoxical ERK activation (Figure 6E). Drug-treated SCR and TKD HaCaT cells, had elevated colony counts to similar levels, as expected, because both lines would experience similar degrees of both paradoxical ERK activation and JNK inhibition, and TKD cells (knocked down for ZAK, MAP4K5, MKK4) are unlikely to experience any further suppression of JNK signaling (Figure 3D). Based on this, we estimated the effect of JNK pathway inhibition to be 17.6% (Figure 6E). When combined with the MEF experiment, we estimate that the effect of JNK inhibition contributes approximately 17.6–40% of the total effect of PLX4720-accelerated colony formation (Figure 6D,E). Importantly, although we can quantify these individual contributions, it is clear in many contexts in cancer that hyperproliferation and inhibition of apoptosis are highly cooperative (Hanahan and Weinberg, 2011), and our data do not preclude the possibility that one or both are individually required.10.7554/eLife.00969.022Figure 6.Paradoxical ERK activation and JNK pathway inhibition make significant and separable contributions to BRAFi-induced growth.

Bottom Line: The prevailing explanation for this is drug-induced paradoxical ERK activation, resulting in hyperproliferation.Here we show an unexpected and novel effect of vemurafenib/PLX4720 in suppressing apoptosis through the inhibition of multiple off-target kinases upstream of c-Jun N-terminal kinase (JNK), principally ZAK.Our results implicate suppression of JNK-dependent apoptosis as a significant, independent mechanism that cooperates with paradoxical ERK activation to induce cSCC, suggesting broad implications for understanding toxicities associated with BRAF inhibitors and for their use in combination therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, United States.

ABSTRACT
Vemurafenib and dabrafenib selectively inhibit the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) kinase, resulting in high response rates and increased survival in melanoma. Approximately 22% of individuals treated with vemurafenib develop cutaneous squamous cell carcinoma (cSCC) during therapy. The prevailing explanation for this is drug-induced paradoxical ERK activation, resulting in hyperproliferation. Here we show an unexpected and novel effect of vemurafenib/PLX4720 in suppressing apoptosis through the inhibition of multiple off-target kinases upstream of c-Jun N-terminal kinase (JNK), principally ZAK. JNK signaling is suppressed in multiple contexts, including in cSCC of vemurafenib-treated patients, as well as in mice. Expression of a mutant ZAK that cannot be inhibited reverses the suppression of JNK activation and apoptosis. Our results implicate suppression of JNK-dependent apoptosis as a significant, independent mechanism that cooperates with paradoxical ERK activation to induce cSCC, suggesting broad implications for understanding toxicities associated with BRAF inhibitors and for their use in combination therapies. DOI: http://dx.doi.org/10.7554/eLife.00969.001.

Show MeSH
Related in: MedlinePlus