BRAF inhibitors suppress apoptosis through off-target inhibition of JNK signaling
- University of Texas MD Anderson Cancer Center, United States
- University of Texas, United States
- Max F Perutz Laboratories, Austria
- Feinstein Institute for Medical Research, United States
Figures
Figure 1 with 3 supplements
PLX4720 suppresses UV-induced apoptosis.
The cSCC and HaCaT cell lines were either unirradiated or irradiated with 1 kJ/m2 of UVB in the absence (‘o’, 1:2000 DMSO) or presence (‘+’) of 1 μM PLX4720 and isolated for FACS analysis and …
Figure 1—figure supplement 1
PLX4720 potently suppresses apoptosis in cSCC, HaCaT cell lines, and NHEK cells.
Representative FACS plots of Annexin V vs TMRE in SRB1 (A), SRB12 (B), HaCaT (C), and NHEK (D) cells demonstrated low levels of apoptosis (Annexin V+, TMRE-low in quadrant 1) in unirradiated cells …
Figure 1—figure supplement 2
PLX4720 suppresses doxorubicin-induced JNK activation and apoptosis in cSCC and HaCaT cell lines.
COLO16 and HaCaT cell lines were either treated with doxorubicin or PBS and lysed 24 hr later in the absence (‘o’, 1:2000 DMSO) or presence (“+”) of 1 μM PLX4720. (A) COLO16 and (B) HaCaT cells …
Figure 1—figure supplement 3
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 …
Figure 2 with 2 supplements
Vemurafenib and PLX4720 suppress apoptosis and JNK signaling in primary human keratinocytes and cSCC cells independently of MEK/ERK signaling.
Normal human epidermal keratinocytes (NHEKs) were irradiated with 1 kJ/m2 of UVB in the absence (‘o’, 1:2000 DMSO) or presence (‘+’) of 1 μM vemurafenib and isolated for FACS analysis and protein …
Figure 2—figure supplement 1
p53 does not respond to stress in cSCC and HaCaT cell lines.
cSCC cell lines SRB1, SRB12, and COLO16 were either unirradiated or irradiated with 1 kJ/m2 of UVB in the absence (‘o’, 1:2000 DMSO) or presence (‘+’) of 1 μM PLX4720 and isolated for protein …
Figure 2—figure supplement 2
BCL2 family members BCL2, BCL-XL, and BCL2A1 are not modulated by acute UV exposure or PLX4720.
HaCaT cells were either unirradiated or irradiated with 1 kJ/m2 of UVB in the absence (‘o’, 1:2000 DMSO) or presence (‘+’) of 1 μM PLX4720 and isolated for protein extracts 24 hr later. Western …
Figure 3 with 4 supplements
PLX4720 and vemurafenib suppress apoptosis and JNK signaling through inhibition of off-target kinases.
(A–C) In-vitro kinase assays for ZAK, MKK4, and MAP4K5 were performed across a 10-point concentration range from 0.05 to 1000 nM in triplicate, revealing significant inhibition of kinase activity …
Figure 3—figure supplement 1
Knockdown of ZAK potently inhibits JNK activation and UV-induced apoptosis.
(A) Western blot of HaCaT cells expressing two shRNA clones and HaCaT TKD cells (containing shZAK2) all show significant knockdown of ZAK protein, though shZAK2 produces slightly less knockdown. …
Figure 3—figure supplement 2
Single knockdown of MKK4 or MAP4K5 partially inhibits JNK activation and UV-induced apoptosis.
(A) Western blot of HaCaT cells expressing two shRNA clones against MKK4 and MAP4K5 show significant knockdown of targets proteins (shMKK4-1: 89.3%, shMKK4-2: 71.9%, shMAP4K5-1: 86.4%, shMAP4K5-2: …
Figure 3—figure supplement 3
Knockdown of ZAK potently inhibits JNK activation and UV-induced apoptosis in SRB1 cells.
(A) Western blot of SRB1 cells expressing two shRNA clones (shZAK1, shZAK2) all show significant knockdown of ZAK protein. (B) SRB1 cells, expressing non-silencing scramble-shRNA (‘SCR’), shZAK1, or …
Figure 3—figure supplement 4
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 …
Figure 4 with 1 supplement
Vemurafenib and PLX4720 suppress apoptosis and JNK signaling in vivo.
(A–D) cSCC samples from vemurafenib-treated patients and non-treated patients were analyzed by immunohistochemistry for phospho-JNK and cleaved caspase-3 expression. cSCC arising in …
Figure 4—figure supplement 1
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 …
Figure 5 with 1 supplement
PLX4720 and JNK inhibition dramatically accelerate cSCC development in the UV-driven Hairless mouse model.
(A–E) Chronically-irradiated Hairless mice were treated with PLX4720 (n = 5), or vehicle (n = 5) starting at day 72 (arrow, E). Tumors were induced within 20 days of PLX-4720 treatment (B), whereas …
Figure 5—figure supplement 1
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…
Figure 6 with 3 supplements
Paradoxical ERK activation and JNK pathway inhibition make significant and separable contributions to BRAFi-induced growth.
(A) We envision two separable, parallel mechanisms by which PLX4720 and vemurafenib contribute to cSCC development. Drug-induced paradoxical ERK activation and inhibition of JNK signaling occur in …
Figure 6—figure supplement 1
Paradoxical MEK and ERK activation require intact Craf.
Wild-type (WT) and isogenic Craf−/− MEFs were retrovirally transduced with HRASG12V and adenovirus E1A thereby enabling anchorage-independent growth for soft agar assays. (A) WT MEFs exhibit …
Figure 6—figure supplement 2
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 …
Figure 6—figure supplement 3
Dabrafenib produces a colony formation advantage only in WT MEFs.
At 0.05 μM, dabrafenib produce a significant growth advantage in E1A-HRASG12V- transformed WT MEFs. In E1A-HRASG12V-transformed Craf−/− MEFs, dabrafenib fails to confer a significant growth …
Tables
Table 1
Lack of BRAF and RAS mutations in cSCC and HaCaT cell lines
| ALK_F1174LIV_T3520CAG | ALK_F1245C_T3734G |
| ALK_F1245VI_T3733GA | BRAF_G464EVA_G1391ATC |
| BRAF_G466R_G1396CA | BRAF_K601E_A1801G |
| BRAF_V600EAG_T1799ACG_F | CTNNB1_S45APT_T133GCA |
| CTNNB1_T41APS_A121GCS | EGFR_Y813C_A2438G |
| GNAS_R201SC_C601AT | KRAS_G12SRC_G34ACT |
| KRAS_Q61EKX_C181GAT | MET_H1112_A3335GT |
| MET_Y1248HD_T3742CG | PIK3CA_A1046V_C3137T |
| PIK3CA_C420R_T1258C | PIK3CA_E110K_G328A |
| PIK3CA_E418K_G1252A | PIK3CA_F909L_C2727G |
| PIK3CA_H1047RL_A3140 GT | PIK3CA_H701P_A2102C |
| PIK3CA_N345K_T1035A | PIK3CA_Q060K_C178A |
| PIK3CA_R088Q_G263A | PIK3CA_S405F_C1214T |
| TNK2_R99Q_G296A | BRAF_G466EVA_G1397ATC |
| BRAF_V600LM_G1798 TA | CTNNB1_S37APT_T109GCA |
| CTNNB1_S45CFY_C134GTA | EGFR_G719_G2155TA |
| EGFR_L858R_T2573G | EGFR_T790M_C2369T |
| EPHA3_K761N_G2283 | FGFR2_S252W_C755G |
| FOXL2_C134W_C402G | KIT_K642E_A1924G |
| KIT_R634W_C1900T | KIT_V560D_T1679A |
| KIT_V825A_T2474C | KIT_Y553N_T1657A |
| KRAS_G12DAV_G35ACT | MET_N375S_A1124G |
| NRAS_G12SRC_G34ACT | PIK3CA_E453K_G1357A |
| PIK3CA_E545AGV_A1634CGT | PIK3CA_H1047RL_A3140 GT..1. |
| PIK3CA_K111N_G333C | PIK3CA_M1043V_A3127G |
| PIK3CA_P539R_C1616G | BRAF_E586K_G1756A |
| BRAF_G469EVA_G1406ATC | CTNNB1_S33APT_T97GCA |
| CTNNB1_S37CFY_C110GTA | EGFR_L861_T2582AG |
| EGFR_T854I_C2561T | FGFR2_N549KK_T1647GA |
| FRAP_R2505P_G7514C | FRAP_S2215Y_C6644T |
| IDH2_R172MK_G515 TA | JAK2_V617F_G1849T |
| KIT_L576P_T1727C | KIT_N566D_A1696 G |
| KRAS_A146PT_G436CA | NRAS_G12DAV_G35ACT |
| KRAS_Q61HHE_A183CTG | KRAS_G13SRC_G37ACT |
| NRAS_G13DAV_G38ACT | NRAS_Q61HHQ_A183TCG |
| PDGFRA_N659Y_A1975T | PDGFRA_V561D_T1682A |
| PIK3CA_E545KQ_G1633AC | PIK3CA_H1047Y_C3139T |
| PIK3CA_Q546EK_C1636 GA | PIK3CA_Y1021HN_T3061CA |
| RET_M918T_T2753C | AKT1_G173R_G517C |
| AKT2_E17K_G49A | BRAF_G469R_G1405CA |
| BRAF_L597R_T1790G | BRAF_V600_G1800 |
| CTNNB1_G34EVA_G101ATC | EGFR_S720P_T2158C |
| GNA11_Q209LP_A626 TC | IDH1_R132CGS_C394TGA |
| IDH2_R140LQ_G419 TA | IDH2_R140W_C418T |
| IDH2_R172S_G516T | KIT_D816HNY_G2446CAT |
| KIT_V559ADG_T1676CAG | KRAS_G10R_G28A |
| KRAS_Q61LPR_A182TCG | MET_H1112Y_C3334T |
| MET_M1268T_T3803C | MET_T1010I_C3029T |
| NRAS_A146T_G436A | NRAS_Q61EKX_C181GAT |
| PDGFRA_D842V_A2525T | PDGFRA_D842_G2524TA |
| PDGFRA_N659K_C1977A | PIK3CA_E542KQ_G1624AC |
| PIK3CA_G1049R_G3145C | PIK3CA_M1043I_G3129ATC |
| PIK3R1_D560Y_G1678T | PRKAG2_N488I_A1463T |
| AKT2_G175R_G523C | AKT3_G171R_G511A |
| ALK_F1174L_C3522AG | ALK_I1171N_T3512A |
| ALK_R1275QL_G3824AT | BRAF_D594GV_A1781 GT |
| CTNNB1_D32HNY_G94CAT | FBWX7_R465C_C1393T |
| FBWX7_R479QL_G1436AT | FBWX7_R505HLP_G1514ATC |
| FGFR3_G370C_G1108T | GNAQ_Q209H_A627T |
| IDH2_R140W_C419T | IDH2_R172GW_A514 GT |
| KIT_N822KNK_T2466GCA | KRAS_G13DAV_G38ACT |
| PDPK1_D527E_C1581G | PIK3CA_E542VG_A1625TG |
| PIK3CA_E545D_G1635CT | PIK3CA_T1025SA_A3073TG |
| PIK3CA_Y1021C_A3062G | PIK3R1_N564K_C1693AG |
| PRKAG1_R70Q_G209A | AKT1_E17K_G49A |
| AKT1_K179M_A536T | BRAF_V600EAG_T1799ACG_R |
| CDK4_R24C_C70T | CDK4_R24H_G71A |
| CTNNB1_D32AGT_A95CGV | FBWX7_R465HL_G1394AT |
| FGFR3_G697C_G2089T | FGFR3_K650MT_A1949 TC |
| FGFR3_R248C_C742T | FGFR3_S371C_A1111T |
| FGFR3_Y373C_A1118G | GNAS_R201H_G602A |
| IDH1_R132HL_G395AT | KIT_N822YHD_A2464TCG |
| MET_R988C_C2962T | MET_Y1253D_T3757 G |
| NRAS_G13SRC_G37ACT | NRAS_Q61RPL_A182GCT |
| PIK3CA_Q546LPR_A1637TCG | TNK2_E346K_G1036A |
| PIK3CA_H1047RL_A3140 GT | ALK_F1245C_T3734G |
-
The listed gene mutations were screened by Sequenom INT16/20 panel (Characterized Cell Line Core, MD Anderson Cancer Center) and HRAS was sequenced by Sanger sequencing. All examined loci were wild-type in the cSCC cell lines SRB1, SRB12, COLO16, and keratinocyte cell line HaCaT. The PIK3R1_M326I_G978 polymorphism was found in the SRB12 cell line.
Table 2
Quantitative competitive binding assays reveal additional kinase targets of PLX4720
| Gene Name | Entrez gene Symbol | Percent control (50 nM) | Percent control (200 nM) | Percent control (1000 nM) | Percent control (10 μM) | Calculated estimate of IC50 (nM) | Published biochemical IC50 (nM) |
|---|---|---|---|---|---|---|---|
| ASK1 | MAP3K5 | 89 | 98 | 97 | 100 | 14,179.29 | |
| ASK2 | MAP3K6 | 94 | 100 | 100 | 100 | ||
| BLK | BLK | 91 | 78 | 32 | 1 | 446.56 | |
| BRAF(V600E) | BRAF | 38 | 19 | 3.9 | 0.1 | 32.04 | 13 |
| BRK | PTK6 | 47 | 14 | 2.4 | 0.2 | 30.38 | 130 |
| DLK | MAP3K12 | 95 | 98 | 100 | 92 | ||
| FGR | FGR | 69 | 38 | 11 | 2.5 | 153.47 | |
| HPK1 | MAP4K1 | 100 | 100 | 100 | 47 | ||
| LZK | MAP3K13 | 94 | 100 | 96 | 75 | ||
| MAP3K1 | MAP3K1 | 96 | 100 | 92 | 84 | ||
| MAP3K15 | MAP3K15 | 94 | 97 | 91 | 59 | ||
| MAP3K2 | MAP3K2 | 100 | 93 | 87 | 41 | ||
| MAP3K3 | MAP3K3 | 94 | 97 | 98 | 75 | ||
| MAP3K4 | MAP3K4 | 100 | 100 | 100 | 65 | ||
| MAP4K2 | MAP4K2 | 98 | 100 | 99 | 67 | ||
| MAP4K3 | MAP4K3 | 100 | 95 | 90 | 56 | ||
| MAP4K4 | MAP4K4 | 92 | 99 | 100 | 46 | ||
| MAP4K5 | MAP4K5 | 96 | 100 | 63 | 8 | 1257.42 | |
| MEK3 | MAP2K3 | 100 | 100 | 100 | 64 | ||
| MEK4 | MAP2K4 | 48 | 27 | 2.6 | 0.05 | 37.96 | |
| MEK6 | MAP2K6 | 82 | 100 | 100 | 47 | ||
| MINK | MINK1 | 89 | 100 | 98 | 55 | ||
| MKK7 | MAP2K7 | 100 | 100 | 100 | 84 | ||
| MLK1 | MAP3K9 | 100 | 100 | 100 | 100 | >10,000 | >5000 |
| MLK2 | MAP3K10 | 100 | 82 | 100 | 76 | ||
| MLK3 | MAP3K11 | 100 | 100 | 100 | 100 | ||
| MST1 | STK4 | 100 | 93 | 84 | 55 | 6709.79 | >5000 |
| OSR1 | OXSR1 | 100 | 94 | 95 | 42 | ||
| PAK1 | PAK1 | 93 | 97 | 83 | 22 | ||
| RIPK1 | RIPK1 | 99 | 87 | 85 | 50 | ||
| SRMS | SRMS | 1.9 | 0.55 | 0.05 | 0 | 0.64 | |
| STK39 | STK39 | 100 | 100 | 100 | 59 | ||
| TAK1 | MAP3K7 | 90 | 88 | 85 | 49 | ||
| TAOK1 | TAOK1 | 87 | 94 | 89 | 65 | 7532.57 | >5000 |
| TAOK2 | TAOK2 | 92 | 100 | 93 | 51 | ||
| TAOK3 | TAOK3 | 100 | 98 | 96 | 58 | ||
| TNIK | TNIK | 97 | 89 | 79 | 24 | ||
| ZAK | ZAK | 20 | 4 | 0.7 | 0.1 | 9.47 |
-
Quantitative competitive binding assays were performed for a group of kinases previously tested against PLX4720 as well as a group of MAP kinases upstream of JNK. Published biochemical IC50s for PLX4720 are listed (see main text) for comparison and demonstrate good quantitative correspondence between estimated Kd from binding assays and biochemical IC50s. ZAK and MKK4 (MAP2K4) were very tightly bound by PLX4720 with estimated Kd below 50 nM. Bold text indicates the kinases tested for inhibition by PLX4720 with in-vitro kinase assays.
Table 3
Quantitative competitive binding assays reveal additional kinase targets of vemurafenib
| Percent control (50 nM) | Percent control (200 nM) | Percent control (1000 nM) | Percent control (10 μM) | Calculated estimate of IC50 (nM) | Published biochemical IC50 (nM) | ||
|---|---|---|---|---|---|---|---|
| ASK1 | MAP3K5 | 90 | 94 | 97 | 100 | 11,972.22 | >1000 |
| ASK2 | MAP3K6 | 94 | 98 | 100 | 74 | ||
| BLK | BLK | 96 | 66 | 30 | 0.55 | 518.03 | 547 |
| BRAF(V600E) | BRAF | 63 | 25 | 5.4 | 0.5 | 64.78 | 31 |
| BRK | PTK6 | 63 | 28 | 6.9 | 0.35 | 68.04 | 213 |
| DLK | MAP3K12 | 98 | 97 | 66 | 92 | ||
| FGR | FGR | 65 | 49 | 13 | 1.6 | 149.26 | 63 |
| HPK1 | MAP4K1 | 95 | 88 | 67 | 15 | ||
| LZK | MAP3K13 | 100 | 99 | 93 | 74 | ||
| MAP3K1 | MAP3K1 | 98 | 84 | 89 | 81 | ||
| MAP3K15 | MAP3K15 | 84 | 100 | 84 | 91 | ||
| MAP3K2 | MAP3K2 | 91 | 91 | 89 | 83 | ||
| MAP3K3 | MAP3K3 | 87 | 97 | 100 | 94 | ||
| MAP3K4 | MAP3K4 | 95 | 92 | 87 | 46 | ||
| MAP4K2 | MAP4K2 | 99 | 82 | 95 | 46 | ||
| MAP4K3 | MAP4K3 | 80 | 90 | 82 | 24 | ||
| MAP4K4 | MAP4K4 | 96 | 92 | 83 | 23 | 2842.34 | >1000 |
| MAP4K5 | MAP4K5 | 62 | 33 | 4.1 | 0.1 | 58.21 | 51 |
| MEK3 | MAP2K3 | 100 | 96 | 98 | 54 | ||
| MEK4 | MAP2K4 | 19 | 4.1 | 0.2 | 0.05 | 6.82 | |
| MEK6 | MAP2K6 | 91 | 97 | 87 | 21 | 4080.69 | >1000 |
| MINK | MINK1 | 100 | 100 | 91 | 66 | 14,761.44 | >1000 |
| MKK7 | MAP2K7 | 97 | 95 | 94 | 85 | ||
| MLK1 | MAP3K9 | 100 | 93 | 97 | 41 | 13,979.88 | >1000 |
| MLK2 | MAP3K10 | 92 | 96 | 87 | 78 | ||
| MLK3 | MAP3K11 | 98 | 100 | 100 | 77 | ||
| MST1 | STK4 | 99 | 83 | 51 | 12 | ||
| OSR1 | OXSR1 | 100 | 100 | 89 | 98 | ||
| PAK1 | PAK1 | 99 | 98 | 91 | 46 | ||
| RIPK1 | RIPK1 | 92 | 100 | 99 | 73 | ||
| SRMS | SRMS | 24 | 9.6 | 0.75 | 0 | 11.15 | 18 |
| STK39 | STK39 | 100 | 100 | 97 | 66 | ||
| TAK1 | MAP3K7 | 93 | 88 | 86 | 88 | ||
| TAOK1 | TAOK1 | 91 | 100 | 97 | 79 | ||
| TAOK2 | TAOK2 | 98 | 92 | 95 | 70 | 11,770.83 | >1000 |
| TAOK3 | TAOK3 | 92 | 98 | 92 | 80 | 15,468.75 | >1000 |
| TNIK | TNIK | 95 | 94 | 66 | 11 | ||
| ZAK | ZAK | 9 | 1.8 | 0.25 | 0.05 | 4.03 |
-
Quantitative competitive binding assays were performed for a group of kinases previously tested against vemurafenib as well as a group of MAP kinases upstream of JNK. Published biochemical IC50s for vemurafenib are listed (see main text) for comparison and demonstrate good quantitative correspondence between estimated Kd from binding assays and biochemical IC50s. ZAK and MKK4 (MAP2K4) were very tightly bound by vemurafenib with estimated Kd below 50 nM. Bold text indicates the kinases tested for inhibition by vemurafenib with in-vitro kinase assays.
