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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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Double staining of sporadic cSCC confirms phospho-JNK and cleaved caspase-3 expression within keratinocytes of tumors.Sections were processed for standard immunohistochemistry and stained with primary antibodies against phospho-JNK, cleaved caspase-3 (Cell Signaling; peroxidase–DAB) as before, together with antibodies against cytokeratins 5/6 (clone D5/16 B4—Thermo; peroxidase–AEC). Results of the double staining show that in all cases, phospho-JNK staining and cleaved caspase 3 staining was observed exclusively in keratinocytes within tumors. Keratinocytes (CK5/6 +) are significantly larger and have more cytoplasm than macrophages or lymphocytes. Scale bar is 50 μM.DOI:http://dx.doi.org/10.7554/eLife.00969.019
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fig4s1: Double staining of sporadic cSCC confirms phospho-JNK and cleaved caspase-3 expression within keratinocytes of tumors.Sections were processed for standard immunohistochemistry and stained with primary antibodies against phospho-JNK, cleaved caspase-3 (Cell Signaling; peroxidase–DAB) as before, together with antibodies against cytokeratins 5/6 (clone D5/16 B4—Thermo; peroxidase–AEC). Results of the double staining show that in all cases, phospho-JNK staining and cleaved caspase 3 staining was observed exclusively in keratinocytes within tumors. Keratinocytes (CK5/6 +) are significantly larger and have more cytoplasm than macrophages or lymphocytes. Scale bar is 50 μM.DOI:http://dx.doi.org/10.7554/eLife.00969.019

Mentions: We then explored whether vemurafenib or PLX4720-mediated suppression of JNK and apoptosis is relevant in vivo. We first examined cSCC arising in patients treated with vemurafenib and compared them to sporadic cSCC that were histologically similar, arising in individuals never treated with vemurafenib (Figure 4A–E). Phospho-JNK and cleaved caspase-3 expression were assessed by immunohistochemistry and then quantified following normalization by unit area (mm2) of tumor tissue (malignant keratinocytes) only (Figure 4A–D, Figure 4—figure supplement 1). Sporadic cSCC arising in patients never treated with vemurafenib (n = 15) contained substantially greater expression of phospho-JNK (p=0.013; Figure 4A,E) and cleaved caspase-3 (p=0.042; Figure 4C,E) as compared to lesions arising in vemurafenib-treated patients (n = 16; Figure 4B,D,E). Therefore, we found significant reductions in phospho-JNK and cleaved caspase-3 expression in human cSCC suggesting that suppression of JNK activity and apoptosis occur in vivo in patients treated with vemurafenib.10.7554/eLife.00969.018Figure 4.Vemurafenib and PLX4720 suppress apoptosis and JNK signaling in vivo.


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)

Double staining of sporadic cSCC confirms phospho-JNK and cleaved caspase-3 expression within keratinocytes of tumors.Sections were processed for standard immunohistochemistry and stained with primary antibodies against phospho-JNK, cleaved caspase-3 (Cell Signaling; peroxidase–DAB) as before, together with antibodies against cytokeratins 5/6 (clone D5/16 B4—Thermo; peroxidase–AEC). Results of the double staining show that in all cases, phospho-JNK staining and cleaved caspase 3 staining was observed exclusively in keratinocytes within tumors. Keratinocytes (CK5/6 +) are significantly larger and have more cytoplasm than macrophages or lymphocytes. Scale bar is 50 μM.DOI:http://dx.doi.org/10.7554/eLife.00969.019
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4s1: Double staining of sporadic cSCC confirms phospho-JNK and cleaved caspase-3 expression within keratinocytes of tumors.Sections were processed for standard immunohistochemistry and stained with primary antibodies against phospho-JNK, cleaved caspase-3 (Cell Signaling; peroxidase–DAB) as before, together with antibodies against cytokeratins 5/6 (clone D5/16 B4—Thermo; peroxidase–AEC). Results of the double staining show that in all cases, phospho-JNK staining and cleaved caspase 3 staining was observed exclusively in keratinocytes within tumors. Keratinocytes (CK5/6 +) are significantly larger and have more cytoplasm than macrophages or lymphocytes. Scale bar is 50 μM.DOI:http://dx.doi.org/10.7554/eLife.00969.019
Mentions: We then explored whether vemurafenib or PLX4720-mediated suppression of JNK and apoptosis is relevant in vivo. We first examined cSCC arising in patients treated with vemurafenib and compared them to sporadic cSCC that were histologically similar, arising in individuals never treated with vemurafenib (Figure 4A–E). Phospho-JNK and cleaved caspase-3 expression were assessed by immunohistochemistry and then quantified following normalization by unit area (mm2) of tumor tissue (malignant keratinocytes) only (Figure 4A–D, Figure 4—figure supplement 1). Sporadic cSCC arising in patients never treated with vemurafenib (n = 15) contained substantially greater expression of phospho-JNK (p=0.013; Figure 4A,E) and cleaved caspase-3 (p=0.042; Figure 4C,E) as compared to lesions arising in vemurafenib-treated patients (n = 16; Figure 4B,D,E). Therefore, we found significant reductions in phospho-JNK and cleaved caspase-3 expression in human cSCC suggesting that suppression of JNK activity and apoptosis occur in vivo in patients treated with vemurafenib.10.7554/eLife.00969.018Figure 4.Vemurafenib and PLX4720 suppress apoptosis and JNK signaling in vivo.

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