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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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Dabrafenib fails to suppress apoptosis and phospho-JNK upregulation following UV irradiation at bioequivalent doses as compared to PLX4720.Based upon human pharmacokinetic data and in vitro experiments, dabrafenib and PLX4720 were compared in multiple settings at bioequivalent doses (0.05 μM and 1.0 μM, respectively). (A) Both BRAFi suppress the growth of A375 and WM35 BRAFV600E melanoma cell lines to the same degree at these doses. (B and C) At these doses, dabrafenib fails to suppress UV-induced apoptosis significantly in HaCaT and SRB1 cells. (D and E) Likewise, dabrafenib fails to suppress phospho-JNK induction, whereas PLX4720 potently suppresses phospho-JNK induction as shown earlier. (F) Dabrafenib inhibits ZAK kinase with an estimated IC50 of 28.92 ± 2.23 nM, with no significant inhibition of MAP4K5 or MKK4 up to 1 μM. At 0.01 μM of dabrafenib, the retained activity of ZAK kinase is over 64%.DOI:http://dx.doi.org/10.7554/eLife.00969.024
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fig6s2: Dabrafenib fails to suppress apoptosis and phospho-JNK upregulation following UV irradiation at bioequivalent doses as compared to PLX4720.Based upon human pharmacokinetic data and in vitro experiments, dabrafenib and PLX4720 were compared in multiple settings at bioequivalent doses (0.05 μM and 1.0 μM, respectively). (A) Both BRAFi suppress the growth of A375 and WM35 BRAFV600E melanoma cell lines to the same degree at these doses. (B and C) At these doses, dabrafenib fails to suppress UV-induced apoptosis significantly in HaCaT and SRB1 cells. (D and E) Likewise, dabrafenib fails to suppress phospho-JNK induction, whereas PLX4720 potently suppresses phospho-JNK induction as shown earlier. (F) Dabrafenib inhibits ZAK kinase with an estimated IC50 of 28.92 ± 2.23 nM, with no significant inhibition of MAP4K5 or MKK4 up to 1 μM. At 0.01 μM of dabrafenib, the retained activity of ZAK kinase is over 64%.DOI:http://dx.doi.org/10.7554/eLife.00969.024

Mentions: The recently updated combination trial of the BRAFi dabrafenib and MEKi trametinib shows a low 7% cSCC rate in 54 patients, (Flaherty et al., 2012) suggesting that combined MEK inhibition can reduce, but not eliminate cSCC formation, nevertheless reinforcing a role for paradoxical ERK activation (Su et al., 2012). However, clinical trial data on dabrafenib alone at 150 mg PO BID, shows an overall aggregated cSCC rate of 6.1% (Falchook et al., 2012; Hauschild et al., 2012; Long et al., 2012) vs 22% for vemurafenib at 960 mg PO BID in several hundred patients (Flaherty et al., 2010; Chapman et al., 2011; Sosman et al., 2012; Menzies et al., 2013). We interpreted this as being reflective of differences between vemurafenib and dabrafenib, as opposed to unequivocal proof that paradoxical ERK activation is the only mechanism involved. To explore this further, we used similar assays to assess the effects of dabrafenib on apoptosis, JNK signaling, and colony formation. In stark contrast to vemurafenib, dabrafenib has little effect on apoptosis and JNK signaling at doses that are biologically equivalent based upon growth inhibition of BRAFV600E melanoma cells and human pharmacokinetic data (Falchook et al., 2012; Gowrishankar et al., 2012) (Figure 6—figure supplement 2A). Peak serum concentrations of dabrafenib at 150 mg PO BID in humans (Falchook et al., 2012) are over 50-fold lower (1.55 μM) than mean sustained serum levels of vemurafenib (86 μM) at 960 mg PO BID (Flaherty et al., 2010), and the GI50 for the A375 melanoma cell line is less than 0.01 μM for dabrafenib (Greger et al., 2012) vs 0.50 μM for PLX4720 (Tsai et al., 2008). Even at 0.05 μM, dabrafenib did not significantly impact UV-induced phospho-JNK upregulation or apoptosis in HaCaT and SRB1 cells (Figure 6—figure supplement 2B–E). We profiled dabrafenib activity against ZAK, MKK4, and MAP4K5, and found that ZAK is a significant off-target kinase for dabrafenib as well, but at 0.01 μM, over 64% of activity is retained (Figure 6—figure supplement 2F). Neither MKK4 nor MAP4K5 is substantially inhibited by dabrafenib up to 1 μM (Figure 6—figure supplement 2F).


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)

Dabrafenib fails to suppress apoptosis and phospho-JNK upregulation following UV irradiation at bioequivalent doses as compared to PLX4720.Based upon human pharmacokinetic data and in vitro experiments, dabrafenib and PLX4720 were compared in multiple settings at bioequivalent doses (0.05 μM and 1.0 μM, respectively). (A) Both BRAFi suppress the growth of A375 and WM35 BRAFV600E melanoma cell lines to the same degree at these doses. (B and C) At these doses, dabrafenib fails to suppress UV-induced apoptosis significantly in HaCaT and SRB1 cells. (D and E) Likewise, dabrafenib fails to suppress phospho-JNK induction, whereas PLX4720 potently suppresses phospho-JNK induction as shown earlier. (F) Dabrafenib inhibits ZAK kinase with an estimated IC50 of 28.92 ± 2.23 nM, with no significant inhibition of MAP4K5 or MKK4 up to 1 μM. At 0.01 μM of dabrafenib, the retained activity of ZAK kinase is over 64%.DOI:http://dx.doi.org/10.7554/eLife.00969.024
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fig6s2: Dabrafenib fails to suppress apoptosis and phospho-JNK upregulation following UV irradiation at bioequivalent doses as compared to PLX4720.Based upon human pharmacokinetic data and in vitro experiments, dabrafenib and PLX4720 were compared in multiple settings at bioequivalent doses (0.05 μM and 1.0 μM, respectively). (A) Both BRAFi suppress the growth of A375 and WM35 BRAFV600E melanoma cell lines to the same degree at these doses. (B and C) At these doses, dabrafenib fails to suppress UV-induced apoptosis significantly in HaCaT and SRB1 cells. (D and E) Likewise, dabrafenib fails to suppress phospho-JNK induction, whereas PLX4720 potently suppresses phospho-JNK induction as shown earlier. (F) Dabrafenib inhibits ZAK kinase with an estimated IC50 of 28.92 ± 2.23 nM, with no significant inhibition of MAP4K5 or MKK4 up to 1 μM. At 0.01 μM of dabrafenib, the retained activity of ZAK kinase is over 64%.DOI:http://dx.doi.org/10.7554/eLife.00969.024
Mentions: The recently updated combination trial of the BRAFi dabrafenib and MEKi trametinib shows a low 7% cSCC rate in 54 patients, (Flaherty et al., 2012) suggesting that combined MEK inhibition can reduce, but not eliminate cSCC formation, nevertheless reinforcing a role for paradoxical ERK activation (Su et al., 2012). However, clinical trial data on dabrafenib alone at 150 mg PO BID, shows an overall aggregated cSCC rate of 6.1% (Falchook et al., 2012; Hauschild et al., 2012; Long et al., 2012) vs 22% for vemurafenib at 960 mg PO BID in several hundred patients (Flaherty et al., 2010; Chapman et al., 2011; Sosman et al., 2012; Menzies et al., 2013). We interpreted this as being reflective of differences between vemurafenib and dabrafenib, as opposed to unequivocal proof that paradoxical ERK activation is the only mechanism involved. To explore this further, we used similar assays to assess the effects of dabrafenib on apoptosis, JNK signaling, and colony formation. In stark contrast to vemurafenib, dabrafenib has little effect on apoptosis and JNK signaling at doses that are biologically equivalent based upon growth inhibition of BRAFV600E melanoma cells and human pharmacokinetic data (Falchook et al., 2012; Gowrishankar et al., 2012) (Figure 6—figure supplement 2A). Peak serum concentrations of dabrafenib at 150 mg PO BID in humans (Falchook et al., 2012) are over 50-fold lower (1.55 μM) than mean sustained serum levels of vemurafenib (86 μM) at 960 mg PO BID (Flaherty et al., 2010), and the GI50 for the A375 melanoma cell line is less than 0.01 μM for dabrafenib (Greger et al., 2012) vs 0.50 μM for PLX4720 (Tsai et al., 2008). Even at 0.05 μM, dabrafenib did not significantly impact UV-induced phospho-JNK upregulation or apoptosis in HaCaT and SRB1 cells (Figure 6—figure supplement 2B–E). We profiled dabrafenib activity against ZAK, MKK4, and MAP4K5, and found that ZAK is a significant off-target kinase for dabrafenib as well, but at 0.01 μM, over 64% of activity is retained (Figure 6—figure supplement 2F). Neither MKK4 nor MAP4K5 is substantially inhibited by dabrafenib up to 1 μM (Figure 6—figure supplement 2F).

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