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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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p53 does not respond to stress in cSCC and HaCaT cell lines.cSCC cell lines SRB1, SRB12, and COLO16 were either unirradiated or irradiated with 1 kJ/m2 of UVB in the absence (‘o’, 1:2000 DMSO) or presence (‘+’) of 1 μM PLX4720 and isolated for protein extracts 24 hr later. (A) Western blots of total p53 reveal that none of the cell lines upregulate p53 in response to UV irradiation. SRB12 cells do not express p53. (B) HaCaT cells are known to be mutant for p53 and the presence of p53 in unstressed cells, combined with the failure to upregulate levels following UV radiation, is a hallmark of functionally inactive p53 in cell lines. Loading controls are the same as those in Figure 1F.DOI:http://dx.doi.org/10.7554/eLife.00969.009
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fig2s1: p53 does not respond to stress in cSCC and HaCaT cell lines.cSCC cell lines SRB1, SRB12, and COLO16 were either unirradiated or irradiated with 1 kJ/m2 of UVB in the absence (‘o’, 1:2000 DMSO) or presence (‘+’) of 1 μM PLX4720 and isolated for protein extracts 24 hr later. (A) Western blots of total p53 reveal that none of the cell lines upregulate p53 in response to UV irradiation. SRB12 cells do not express p53. (B) HaCaT cells are known to be mutant for p53 and the presence of p53 in unstressed cells, combined with the failure to upregulate levels following UV radiation, is a hallmark of functionally inactive p53 in cell lines. Loading controls are the same as those in Figure 1F.DOI:http://dx.doi.org/10.7554/eLife.00969.009

Mentions: We next examined the responses of primary normal human epidermal keratinocytes (NHEKs) to vemurafenib. UV-induced apoptosis was significantly suppressed (approximately 70%) by vemurafenib in these cells (Figure 2A, Figure 1—figure supplement 1D), and the UV-induced upregulation of phospho-JNK and phospho-p38 was likewise suppressed most significantly at 6 and 24 hr (Figure 2B). As in the cSCC and HaCaT cell lines, activation of ERK was observed following exposure to vemurafenib (Figure 2B). The presence of cleaved caspase-3 correlated with high levels of apoptosis in the UV-treated cells and its absence with rescue by vemurafenib at 24 hr post-irradiation (Figure 2C). In probing members of the BCL2 family, we found similar results to those in the cSCC and HaCaT cell lines. BIM and MCL1 were unaffected by vemurafenib but NOXA induction at 24 hr post-UV irradiation was diminished by vemurafenib (Figure 2C). The advantage of using primary cells is that p53 is intact. In NHEKs, p53 is stabilized by 24 hr post-UV irradiation and this is unaffected by vemurafenib (Figure 2C). However, since BCL2 family members can be modulated by JNK (Tournier et al., 2000; Haeusgen et al., 2011) and p53 (Oda et al., 2000) in apoptosis, the inhibition of NOXA expression by PLX4720 and vemurafenib (Figures 1G and 2C) likely reflects p53-independent regulation of NOXA given that p53 is mutant in HaCaT (Lehman et al., 1993) cells, p53 is undetectable in SRB12 cells, and p53 levels do not change with radiation in SRB1, COLO16, or HaCaT cells, (Figure 2—figure supplement 1). PUMA, BAX, BCL2, BCL-XL, and BCL2A1 expression were unchanged following irradiation and were unchanged by PLX4720 or vemurafenib exposure (data not shown, Figure 2—figure supplement 2). We conclude from our results that vemurafenib and PLX4720 suppress UV-induced apoptosis by inhibiting JNK signaling and NOXA induction in BRAF and RAS WT cells.10.7554/eLife.00969.008Figure 2.Vemurafenib and PLX4720 suppress apoptosis and JNK signaling in primary human keratinocytes and cSCC cells independently of MEK/ERK signaling.


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)

p53 does not respond to stress in cSCC and HaCaT cell lines.cSCC cell lines SRB1, SRB12, and COLO16 were either unirradiated or irradiated with 1 kJ/m2 of UVB in the absence (‘o’, 1:2000 DMSO) or presence (‘+’) of 1 μM PLX4720 and isolated for protein extracts 24 hr later. (A) Western blots of total p53 reveal that none of the cell lines upregulate p53 in response to UV irradiation. SRB12 cells do not express p53. (B) HaCaT cells are known to be mutant for p53 and the presence of p53 in unstressed cells, combined with the failure to upregulate levels following UV radiation, is a hallmark of functionally inactive p53 in cell lines. Loading controls are the same as those in Figure 1F.DOI:http://dx.doi.org/10.7554/eLife.00969.009
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2s1: p53 does not respond to stress in cSCC and HaCaT cell lines.cSCC cell lines SRB1, SRB12, and COLO16 were either unirradiated or irradiated with 1 kJ/m2 of UVB in the absence (‘o’, 1:2000 DMSO) or presence (‘+’) of 1 μM PLX4720 and isolated for protein extracts 24 hr later. (A) Western blots of total p53 reveal that none of the cell lines upregulate p53 in response to UV irradiation. SRB12 cells do not express p53. (B) HaCaT cells are known to be mutant for p53 and the presence of p53 in unstressed cells, combined with the failure to upregulate levels following UV radiation, is a hallmark of functionally inactive p53 in cell lines. Loading controls are the same as those in Figure 1F.DOI:http://dx.doi.org/10.7554/eLife.00969.009
Mentions: We next examined the responses of primary normal human epidermal keratinocytes (NHEKs) to vemurafenib. UV-induced apoptosis was significantly suppressed (approximately 70%) by vemurafenib in these cells (Figure 2A, Figure 1—figure supplement 1D), and the UV-induced upregulation of phospho-JNK and phospho-p38 was likewise suppressed most significantly at 6 and 24 hr (Figure 2B). As in the cSCC and HaCaT cell lines, activation of ERK was observed following exposure to vemurafenib (Figure 2B). The presence of cleaved caspase-3 correlated with high levels of apoptosis in the UV-treated cells and its absence with rescue by vemurafenib at 24 hr post-irradiation (Figure 2C). In probing members of the BCL2 family, we found similar results to those in the cSCC and HaCaT cell lines. BIM and MCL1 were unaffected by vemurafenib but NOXA induction at 24 hr post-UV irradiation was diminished by vemurafenib (Figure 2C). The advantage of using primary cells is that p53 is intact. In NHEKs, p53 is stabilized by 24 hr post-UV irradiation and this is unaffected by vemurafenib (Figure 2C). However, since BCL2 family members can be modulated by JNK (Tournier et al., 2000; Haeusgen et al., 2011) and p53 (Oda et al., 2000) in apoptosis, the inhibition of NOXA expression by PLX4720 and vemurafenib (Figures 1G and 2C) likely reflects p53-independent regulation of NOXA given that p53 is mutant in HaCaT (Lehman et al., 1993) cells, p53 is undetectable in SRB12 cells, and p53 levels do not change with radiation in SRB1, COLO16, or HaCaT cells, (Figure 2—figure supplement 1). PUMA, BAX, BCL2, BCL-XL, and BCL2A1 expression were unchanged following irradiation and were unchanged by PLX4720 or vemurafenib exposure (data not shown, Figure 2—figure supplement 2). We conclude from our results that vemurafenib and PLX4720 suppress UV-induced apoptosis by inhibiting JNK signaling and NOXA induction in BRAF and RAS WT cells.10.7554/eLife.00969.008Figure 2.Vemurafenib and PLX4720 suppress apoptosis and JNK signaling in primary human keratinocytes and cSCC cells independently of MEK/ERK signaling.

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