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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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cSCC and papillomas arising in Hairless mice treated with PLX4720 do not have Ras mutations.(A and B) cDNA was reverse-transcribed from total RNA, PCR-amplified with the above primers (B) and analyzed by Sanger sequencing for mutations in both directions. No mutations in Hras, Kras, or Nras were detected in any of the papillomas (n = 5) or carcinomas (n = 3) isolated from PLX4720-treated mice. One of the papillomas from untreated mice had a heterozygous point mutation (A) in Hras (G35A, G12E) among 14 samples (12 papillomas, 2 cSCC).DOI:http://dx.doi.org/10.7554/eLife.00969.021
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fig5s1: cSCC and papillomas arising in Hairless mice treated with PLX4720 do not have Ras mutations.(A and B) cDNA was reverse-transcribed from total RNA, PCR-amplified with the above primers (B) and analyzed by Sanger sequencing for mutations in both directions. No mutations in Hras, Kras, or Nras were detected in any of the papillomas (n = 5) or carcinomas (n = 3) isolated from PLX4720-treated mice. One of the papillomas from untreated mice had a heterozygous point mutation (A) in Hras (G35A, G12E) among 14 samples (12 papillomas, 2 cSCC).DOI:http://dx.doi.org/10.7554/eLife.00969.021

Mentions: We also used the Hairless mouse model of squamous cell carcinoma to assess whether PLX4720 would affect UV-driven tumor development. This is particularly relevant since it appears that UV exposure is an important initiating event in BRAFi-accelerated cSCC (Su et al., 2012). Unlike the DMBA/TPA model, in which lesions almost universally harbor Hras mutations (Brown et al., 1990), the Hairless model has a very low frequency of Ras mutation in papillomas and carcinomas (van Kranen et al., 1995), more similar to sporadic human cSCC. The cohorts (n = 5 each) were identically irradiated thrice weekly (12.5 kJ/m2 per week UVB) for 72 days before starting on PLX4720 treatment vs vehicle control. Within 20 days of administration of drug, hyperkeratotic papules were visible on the backs of PLX4720-treated animals (Figure 5A,B), which steadily grew into cSCC over the following several weeks (Figure 5C,D). Within this period of 150 days (78 days of drug treatment), control-treated mice had not yet developed any visible lesions (Figure 5E). When we quantified the effects of each of these drug treatments, we found significant decreases in both phospho-JNK expression (p=0.046; Figure 5F,G,J) and cleaved caspase 3 expression (p=0.019; Figure 5H–J) in PLX4720-treated mice as compared to control-treated mice. Importantly, we sequenced the entire coding regions for Ras (Hras, Kras, Nras) and found no mutations in any of the tumors in PLX4720-treated mice, as compared to one of 14 papillomas and carcinomas in a cohort of control-treated chronically-irradiated Hairless mice (Figure 5—figure supplement 1).10.7554/eLife.00969.020Figure 5.PLX4720 and JNK inhibition dramatically accelerate cSCC development in the UV-driven Hairless mouse model.


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)

cSCC and papillomas arising in Hairless mice treated with PLX4720 do not have Ras mutations.(A and B) cDNA was reverse-transcribed from total RNA, PCR-amplified with the above primers (B) and analyzed by Sanger sequencing for mutations in both directions. No mutations in Hras, Kras, or Nras were detected in any of the papillomas (n = 5) or carcinomas (n = 3) isolated from PLX4720-treated mice. One of the papillomas from untreated mice had a heterozygous point mutation (A) in Hras (G35A, G12E) among 14 samples (12 papillomas, 2 cSCC).DOI:http://dx.doi.org/10.7554/eLife.00969.021
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814616&req=5

fig5s1: cSCC and papillomas arising in Hairless mice treated with PLX4720 do not have Ras mutations.(A and B) cDNA was reverse-transcribed from total RNA, PCR-amplified with the above primers (B) and analyzed by Sanger sequencing for mutations in both directions. No mutations in Hras, Kras, or Nras were detected in any of the papillomas (n = 5) or carcinomas (n = 3) isolated from PLX4720-treated mice. One of the papillomas from untreated mice had a heterozygous point mutation (A) in Hras (G35A, G12E) among 14 samples (12 papillomas, 2 cSCC).DOI:http://dx.doi.org/10.7554/eLife.00969.021
Mentions: We also used the Hairless mouse model of squamous cell carcinoma to assess whether PLX4720 would affect UV-driven tumor development. This is particularly relevant since it appears that UV exposure is an important initiating event in BRAFi-accelerated cSCC (Su et al., 2012). Unlike the DMBA/TPA model, in which lesions almost universally harbor Hras mutations (Brown et al., 1990), the Hairless model has a very low frequency of Ras mutation in papillomas and carcinomas (van Kranen et al., 1995), more similar to sporadic human cSCC. The cohorts (n = 5 each) were identically irradiated thrice weekly (12.5 kJ/m2 per week UVB) for 72 days before starting on PLX4720 treatment vs vehicle control. Within 20 days of administration of drug, hyperkeratotic papules were visible on the backs of PLX4720-treated animals (Figure 5A,B), which steadily grew into cSCC over the following several weeks (Figure 5C,D). Within this period of 150 days (78 days of drug treatment), control-treated mice had not yet developed any visible lesions (Figure 5E). When we quantified the effects of each of these drug treatments, we found significant decreases in both phospho-JNK expression (p=0.046; Figure 5F,G,J) and cleaved caspase 3 expression (p=0.019; Figure 5H–J) in PLX4720-treated mice as compared to control-treated mice. Importantly, we sequenced the entire coding regions for Ras (Hras, Kras, Nras) and found no mutations in any of the tumors in PLX4720-treated mice, as compared to one of 14 papillomas and carcinomas in a cohort of control-treated chronically-irradiated Hairless mice (Figure 5—figure supplement 1).10.7554/eLife.00969.020Figure 5.PLX4720 and JNK inhibition dramatically accelerate cSCC development in the UV-driven Hairless mouse model.

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