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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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PLX4720 does not confer a proliferative advantage to cSCC and HaCaT cell lines.(A) SRB1, (B) SRB12, (C) COLO16, and (D) HaCaT cells were treated with DMSO (1:2000) or the indicated concentrations of PLX4720 for at least 28 days during which cells were serially passaged and counted. Over that time frame there was a slight decrement in the proliferation of SRB12 and HaCaT cells in the presence of 1 μM PLX4720. All cells treated at 5 μM PLX4720 exhibited decreased proliferation.DOI:http://dx.doi.org/10.7554/eLife.00969.006
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fig1s3: PLX4720 does not confer a proliferative advantage to cSCC and HaCaT cell lines.(A) SRB1, (B) SRB12, (C) COLO16, and (D) HaCaT cells were treated with DMSO (1:2000) or the indicated concentrations of PLX4720 for at least 28 days during which cells were serially passaged and counted. Over that time frame there was a slight decrement in the proliferation of SRB12 and HaCaT cells in the presence of 1 μM PLX4720. All cells treated at 5 μM PLX4720 exhibited decreased proliferation.DOI:http://dx.doi.org/10.7554/eLife.00969.006

Mentions: We performed our initial studies using cSCC (SRB1, SRB12, COLO16) and keratinocyte (HaCaT) cell lines. Cells treated with 1 kJ/m2 of UVB (FS40 lamp) undergo apoptosis within 24 hr (Figure 1A–D). Surprisingly, this apoptosis was suppressed by at least 70% in cells concomitantly treated with 1 μM PLX4720 (Figure 1A–D) compared to control DMSO-treated cells as measured by FACS for Annexin V+; TMRE (tetramethylrhodamine)-low cells (Figure 1E, Figure 1—figure supplement 1A–C). Similar results were obtained using doxorubicin as the inducer of apoptosis, and similar suppression of apoptosis was obtained using 1 μM PLX4720 in all cells (Figure 1—figure supplement 2A,B). Importantly, these cells have no oncogenic RAS or BRAF mutations (Table 1), and PLX4720 conferred no significant proliferative advantage to the tested cells (Figure 1—figure supplement 3) even when used at concentrations that inhibit the proliferation of BRAFV600E melanoma cell lines (Tsai et al., 2008).10.7554/eLife.00969.003Figure 1.PLX4720 suppresses UV-induced apoptosis.


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)

PLX4720 does not confer a proliferative advantage to cSCC and HaCaT cell lines.(A) SRB1, (B) SRB12, (C) COLO16, and (D) HaCaT cells were treated with DMSO (1:2000) or the indicated concentrations of PLX4720 for at least 28 days during which cells were serially passaged and counted. Over that time frame there was a slight decrement in the proliferation of SRB12 and HaCaT cells in the presence of 1 μM PLX4720. All cells treated at 5 μM PLX4720 exhibited decreased proliferation.DOI:http://dx.doi.org/10.7554/eLife.00969.006
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1s3: PLX4720 does not confer a proliferative advantage to cSCC and HaCaT cell lines.(A) SRB1, (B) SRB12, (C) COLO16, and (D) HaCaT cells were treated with DMSO (1:2000) or the indicated concentrations of PLX4720 for at least 28 days during which cells were serially passaged and counted. Over that time frame there was a slight decrement in the proliferation of SRB12 and HaCaT cells in the presence of 1 μM PLX4720. All cells treated at 5 μM PLX4720 exhibited decreased proliferation.DOI:http://dx.doi.org/10.7554/eLife.00969.006
Mentions: We performed our initial studies using cSCC (SRB1, SRB12, COLO16) and keratinocyte (HaCaT) cell lines. Cells treated with 1 kJ/m2 of UVB (FS40 lamp) undergo apoptosis within 24 hr (Figure 1A–D). Surprisingly, this apoptosis was suppressed by at least 70% in cells concomitantly treated with 1 μM PLX4720 (Figure 1A–D) compared to control DMSO-treated cells as measured by FACS for Annexin V+; TMRE (tetramethylrhodamine)-low cells (Figure 1E, Figure 1—figure supplement 1A–C). Similar results were obtained using doxorubicin as the inducer of apoptosis, and similar suppression of apoptosis was obtained using 1 μM PLX4720 in all cells (Figure 1—figure supplement 2A,B). Importantly, these cells have no oncogenic RAS or BRAF mutations (Table 1), and PLX4720 conferred no significant proliferative advantage to the tested cells (Figure 1—figure supplement 3) even when used at concentrations that inhibit the proliferation of BRAFV600E melanoma cell lines (Tsai et al., 2008).10.7554/eLife.00969.003Figure 1.PLX4720 suppresses UV-induced apoptosis.

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