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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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Vemurafenib and PLX4720 inhibit multiple kinases upstream of JNK and p38.The schematic shows MAP kinases upstream of JNK and p38 that are inhibited by these BRAF inhibitors (gray-shaded). Vemurafenib and PLX4720 inhibit ZAK (principally) and MKK4 (MEK4/MAP2K4), resulting in inhibition of MKK7 and MKK4 and, ultimately, JNK. p38 activation was diminished by drug exposure in some contexts , but not to the degree that JNK activation was. Vemurafenib and PLX4720 also inhibit MAP4K5, which has been shown to be upstream of MKK4 and JNK.DOI:http://dx.doi.org/10.7554/eLife.00969.017
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fig3s4: Vemurafenib and PLX4720 inhibit multiple kinases upstream of JNK and p38.The schematic shows MAP kinases upstream of JNK and p38 that are inhibited by these BRAF inhibitors (gray-shaded). Vemurafenib and PLX4720 inhibit ZAK (principally) and MKK4 (MEK4/MAP2K4), resulting in inhibition of MKK7 and MKK4 and, ultimately, JNK. p38 activation was diminished by drug exposure in some contexts , but not to the degree that JNK activation was. Vemurafenib and PLX4720 also inhibit MAP4K5, which has been shown to be upstream of MKK4 and JNK.DOI:http://dx.doi.org/10.7554/eLife.00969.017

Mentions: To examine the requirements for ZAK, MAP4K5, and MKK4 in activating JNK activation and apoptosis more directly, we performed lentiviral shRNA knockdown experiments in HaCaT cells. HaCaT cells with knockdown of ZAK (‘shZAK2’) showed a strong suppression of ZAK protein expression (Figure 3—figure supplement 1A) and of UV-induced apoptosis, showing 70% suppression of apoptosis relative to that achieved by PLX4720 in control (‘SCR’) cells (Figure 3D, Figure 3—figure supplement 1A). An additional clone of shRNA against ZAK (‘shZAK1’) showed similar results (Figure 3—figure supplement 1B,C), demonstrating that even greater knockdown of ZAK can account for nearly the entire effect of PLX4720 on JNK activation and apoptosis. Western blots show significant suppression of phospho-MKK4/MKK7 in shZAK2 knockdown cells (Figure 3E). Triple knockdown cells (‘TKD’) with combined shRNA knockdown of ZAK, MKK4, and MAP4K5 kinases, as confirmed by Western (Figure 3E, Figure 3—figure supplement 1A), showed comparable suppression of apoptosis to that of drug-treated control cells (Figure 3D, Figure 3—figure supplement 1B) and substantial suppression of phospho-MKK4/MKK7 induction (Figure 3E). Furthermore, single knockdown of MKK4 and MAP4K5 (Figure 3—figure supplement 2A), only partially suppresses UV-induced apoptosis or phospho-JNK induction in HaCaT cells (Figure 3—figure supplement 2B,C). Knockdown of ZAK alone was able to account for 91.3% of the suppression of UV-induced apoptosis in a distinct cell line, SRB1 (Figure 3—figure supplement 3A,B), with corresponding suppression of phospho-JNK induction (Figure 3—figure supplement 3C). As knockdown of ZAK alone can account for up to 93.7% of the effect of PLX4720 treatment, we conclude that the potent inhibition of JNK activation and resultant apoptosis by PLX4720 and vemurafenib is due to the off-target inhibition of ZAK principally, with smaller additional contributions from inhibition of MKK4 and MAP4K5, which abrogates the activation of the two kinases essential for JNK phosphorylation and activation: MKK4 and MKK7 (Figures 2H–I,3D–E, Figure 3—figure supplement 4). Consistent with our findings, ZAK has been shown to be critically important for JNK activation upstream of MKK4 and MKK7 (Wang et al., 2005) and doxorubicin-induced apoptosis (Sauter et al., 2010; Wong et al., 2013).


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)

Vemurafenib and PLX4720 inhibit multiple kinases upstream of JNK and p38.The schematic shows MAP kinases upstream of JNK and p38 that are inhibited by these BRAF inhibitors (gray-shaded). Vemurafenib and PLX4720 inhibit ZAK (principally) and MKK4 (MEK4/MAP2K4), resulting in inhibition of MKK7 and MKK4 and, ultimately, JNK. p38 activation was diminished by drug exposure in some contexts , but not to the degree that JNK activation was. Vemurafenib and PLX4720 also inhibit MAP4K5, which has been shown to be upstream of MKK4 and JNK.DOI:http://dx.doi.org/10.7554/eLife.00969.017
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3s4: Vemurafenib and PLX4720 inhibit multiple kinases upstream of JNK and p38.The schematic shows MAP kinases upstream of JNK and p38 that are inhibited by these BRAF inhibitors (gray-shaded). Vemurafenib and PLX4720 inhibit ZAK (principally) and MKK4 (MEK4/MAP2K4), resulting in inhibition of MKK7 and MKK4 and, ultimately, JNK. p38 activation was diminished by drug exposure in some contexts , but not to the degree that JNK activation was. Vemurafenib and PLX4720 also inhibit MAP4K5, which has been shown to be upstream of MKK4 and JNK.DOI:http://dx.doi.org/10.7554/eLife.00969.017
Mentions: To examine the requirements for ZAK, MAP4K5, and MKK4 in activating JNK activation and apoptosis more directly, we performed lentiviral shRNA knockdown experiments in HaCaT cells. HaCaT cells with knockdown of ZAK (‘shZAK2’) showed a strong suppression of ZAK protein expression (Figure 3—figure supplement 1A) and of UV-induced apoptosis, showing 70% suppression of apoptosis relative to that achieved by PLX4720 in control (‘SCR’) cells (Figure 3D, Figure 3—figure supplement 1A). An additional clone of shRNA against ZAK (‘shZAK1’) showed similar results (Figure 3—figure supplement 1B,C), demonstrating that even greater knockdown of ZAK can account for nearly the entire effect of PLX4720 on JNK activation and apoptosis. Western blots show significant suppression of phospho-MKK4/MKK7 in shZAK2 knockdown cells (Figure 3E). Triple knockdown cells (‘TKD’) with combined shRNA knockdown of ZAK, MKK4, and MAP4K5 kinases, as confirmed by Western (Figure 3E, Figure 3—figure supplement 1A), showed comparable suppression of apoptosis to that of drug-treated control cells (Figure 3D, Figure 3—figure supplement 1B) and substantial suppression of phospho-MKK4/MKK7 induction (Figure 3E). Furthermore, single knockdown of MKK4 and MAP4K5 (Figure 3—figure supplement 2A), only partially suppresses UV-induced apoptosis or phospho-JNK induction in HaCaT cells (Figure 3—figure supplement 2B,C). Knockdown of ZAK alone was able to account for 91.3% of the suppression of UV-induced apoptosis in a distinct cell line, SRB1 (Figure 3—figure supplement 3A,B), with corresponding suppression of phospho-JNK induction (Figure 3—figure supplement 3C). As knockdown of ZAK alone can account for up to 93.7% of the effect of PLX4720 treatment, we conclude that the potent inhibition of JNK activation and resultant apoptosis by PLX4720 and vemurafenib is due to the off-target inhibition of ZAK principally, with smaller additional contributions from inhibition of MKK4 and MAP4K5, which abrogates the activation of the two kinases essential for JNK phosphorylation and activation: MKK4 and MKK7 (Figures 2H–I,3D–E, Figure 3—figure supplement 4). Consistent with our findings, ZAK has been shown to be critically important for JNK activation upstream of MKK4 and MKK7 (Wang et al., 2005) and doxorubicin-induced apoptosis (Sauter et al., 2010; Wong et al., 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.

Show MeSH
Related in: MedlinePlus