Abstract
Background/Aim: The B-raf proto-oncogene, serine/threonine kinase (BRAF) V600E mutation is frequent in patients with advanced melanoma. PLX4032, an inhibitor of BRAFV600E kinase, is effective for the treatment of melanoma in BRAF V600E-positive patients; however, resistance eventually develops due to paradoxical activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinases (ERK) pathway resulting from RAF dimerization. In this study, we investigated the inhibitory effects of a novel imidazothiazole-based compound, KS28, on RAF dimerization and resistance to PLX4032 in melanoma. Materials and Methods: The effects of KS28 were examined by immunoblotting, cell viability, terminal deoxynucleotidyl transferase dUTP nick-end labeling, reporter-gene, and soft-agar assays. Results: KS28 treatment inhibited RAF dimerization in PLX4032-resistant A375 (A375R) cells, leading to suppression of the MEK/ERK pathway. In addition, KS28 reduced activator protein 1 transactivation in A375R cells, reduced cell viability, and increased DNA fragmentation. Moreover, treatment with KS28 suppressed anchorage-independent growth of A375R cells. Similarly, in an orthotopic tumor xenograft model, KS28 treatment suppressed the growth of tumors formed by A375R cells in BALB/c mice. Conclusion: KS28 plays a vital role in overcoming PLX4032 resistance in melanoma by down-regulating the MEK/ERK pathway.
Melanoma is a highly metastatic skin cancer that arises from melanin-producing cells called melanocytes (1). The prevalence of melanoma has continued to rise during the past few decades more rapidly than almost any other cancer type and has become a significant health risk worldwide (2, 3). The key risk factors predisposing individuals to melanoma include alterations to genes, as well as lifestyle and environmental factors (4). According to the presence of activating gene mutations, melanoma is categorized into four types: RAS viral oncogene (RAS)-mutant, B-raf proto-oncogene, serine/threonine kinase (BRAF)-mutant, neurofibromin (NF1)-mutant, and triple wild-type melanoma (5). About one-half of all patients with metastatic melanoma harbor a BRAF mutation, whereas 15-20% of cases possess NRAS mutations (6, 7). The most commonly occurring mutation in melanoma is BRAF V600E, which comprises 90% of BRAF mutations, resulting from the substitution of Glu for Val at the second position of codon 600 (BRAF nucleotide 1799 T>A; codon GTG>GAG) (8). In addition, BRAF V600E mutations occur in other types of human cancer, for instance, thyroid, colon, and lung cancer (9). BRAF V600E mutant kinase leads to the constitutive activation of oncogenic signaling pathways, for instance, RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinases (ERK) and nuclear factor kappa B, which are involved in distinct mechanisms of melanoma progression, including cell proliferation, angiogenesis, and apoptosis (10, 11). These observations have guided the establishment of several RAF kinase and MEK inhibitors, such as sorafenib, vemurafenib (PLX4032), dabrafenib, and trametinib (12, 13).
Sorafenib was first used as an inhibitor of BRAF in patients with BRAF V600E mutation or advanced metastatic melanoma (14). Sorafenib activity against CRAF was higher than that against those with wild-type BRAF and BRAF V600E mutant. Several clinical trials of sorafenib monotherapy have shown the limited anticancer activity in patients with advanced melanoma harboring BRAF mutations (15-17). Moreover, combination therapy with sorafenib failed to improve the clinical efficacy in patients with melanoma (18). PLX4032 and dabrafenib were developed as a targeted drugs to inhibit BRAF V600E protein kinase directly (19, 20) and were subsequently approved by the Food and Drug Administration, for treating patients with advanced metastatic melanoma with BRAF V600E mutation (21). These inhibitors show remarkable effects in inhibiting ERK phosphorylation, cell proliferation, and the growth of melanoma tumors carrying BRAF V600E mutation. In addition, clinical trials of PLX4032 and dabrafenib treatment for BRAF V600E in melanoma showed improvements in patient survival (14). Nevertheless, the development of acquired resistance restrained the clinical efficacy of PLX4032 and dabrafenib in patients with advanced metastatic melanoma (22-25). Thus, the mechanisms involved in acquired resistance should be considered in order to generate effective BRAF inhibitors with durable effects in patients with advanced or metastatic melanoma.
Several known mechanisms explain resistance to PLX4032 in melanoma, such as alternative mRNA splicing and copy-number amplification of the BRAF gene, NRAS mutations, and up-regulation of mitogen-activated protein kinase kinase 8 (COT) (24, 26, 27). In melanoma cells having NRAS mutations or increased expression of CRAF, PLX4032 treatment paradoxically activates the mitogen-activated protein kinase (MAPK) pathway (28, 29). The exact mechanisms underlying this paradoxical activation remain unclear, but PLX4032-induced dimerization of RAF seems to be an essential step (23). BRAF V600E is constitutively active in its monomeric form (30). PLX4032 binds to BRAF V600E monomers and inhibits the MAPK pathway by inhibiting its activity but fails to attenuate the formation of homo-, and hetero-dimers of BRAF V600E. More specifically, the binding of PLX4032 to one subunit in the dimer significantly reduces the affinity for binding to the following subunit, resulting in activation of the MAPK pathway (31). Pan-RAF inhibitors can inhibit all three forms of RAF kinases (i.e., ARAF, BRAF, and CRAF). Inhibition of monomeric BRAF V600E and RAF dimers is thought to be essential for minimizing the paradoxical activation of the MAPK pathway (32).
In this study, we examined the anticancer effects of a novel imidazo[2,1-b]oxazole derivative, N-(2-((4-(6-(4-fluoro-3-hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)ethyl) benzene sulfonamide (KS28), as a potent inhibitor of BRAF V600E kinase, as well as BRAF and CRAF kinases.
Materials and Methods
Reagents and antibodies. Dulbecco’s modified Eagle’s medium (DMEM), Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), L-glutamine, and gentamicin were purchased from Invitrogen (Carlsbad, CA, USA). Cell proliferation enzyme-linked immunosorbent assay and BrdU were purchased from Roche Applied Science (Indianapolis, IN, USA). Polyvinylidene difluoride membranes were obtained from Millipore (Billerica, MA, USA). The jetPEI® cationic polymer transfection reagent was purchased from Polyplus Transfection (New York, NY, USA). A dual-luciferase reporter-gene assay kit was purchased from Promega (Madison, WI, USA). Antibodies against MEK1/2, ERK1/2, p90RSK, phospho-MEK1/2 (Ser217/221), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), phospho-p90RSK (Ser380), BRAF, CRAF and cleaved-caspase 3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against BRAF V600E and β-actin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibody against cleaved poly (ADP-ribose) polymerase (PARP) and rabbit IgG was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). PLX4032 was obtained from Advanced ChemBlock (San Diego, CA, USA). Epidermal growth factor (EGF) was obtained from Calbiochem-Novabiochem (San Diego, CA, USA). KS28 was kindly provided by Dr. Chang-Hyun Oh (Center for Biomaterials, Korea Institute of Science and Technology, Seoul, Republic of Korea).
Cell culture and establishment of resistant cell lines. A375 human melanoma and JB6 Cl41 mouse epidermal cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The A375R cells were established as described previously (33), and maintained in DMEM supplemented with 10% FBS and 5 μM PLX4032. JB6 Cl41 cells were maintained in Eagle’s MEM supplemented with 5% FBS, and A375 cells were cultured in DMEM supplemented with 10% FBS. All cell lines were cultured and maintained at 37°C in a humidified atmosphere containing 5% CO2.
In vitro enzyme assay. The enzymatic assays of BRAF WT, BRAF V600E, and CRAF were performed as described previously (35). Briefly, the Kinase HotSpotSM service in Reaction Biology Corp. (http://www.reactionbiology.com) was used for the enzyme assays at a 1 μM concentration of ATP and 3-fold dilution factor.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. MTT assay was performed as described previously (34). Briefly, 1×104 cells grown in 96-well plates were treated with 0.01, 0.1, 1, 3, or 5 μM of PLX4032 or KS28 for 24 h, followed by treatment with EZ-Cytox Cell viability reagent (Daeli Lab Service, Seoul, Republic of Korea). The absorbance produced by the water-soluble formazan was measured at 450 nm.
Cell proliferation assay via 5-bromo-2’-deoxyuridine (BrdU) incorporation. Cells were seeded (5×103 cells/well) into 96-well plates in 100 μl of MEM supplemented with 5% FBS. After 24 h, the cells were treated with 0.5 or 1 μM KS28 for 48 h, in the presence or absence of 10 ng/ml EGF, labeled with 10 μl/well BrdU labeling solution, and then incubated for 4 h at 37°C in an atmosphere with 5% CO2. Cell proliferation was estimated by measuring the absorbance at 370 nm.
Protein immunoblot analysis. Cells were harvested and disrupted in RIPA lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.25% sodium deoxycholate, 1 mM ethylenediamine tetra-acetic acid, 1% NP40, 1 mM NaF, 0.2 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium orthovanadate, and protease inhibitor cocktail (Roche Life Science, Indianapolis, IN, USA). Proteins in the whole cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted as described previously (34).
Reporter-gene assay. Reporter-gene assays were performed using lysates from c-Fos- or activator protein 1 (AP1)-Luc-transfected JB6 Cl41, A375, and A375R cells to detect firefly luciferase activity. The reporter-gene vector pRL-TK-luciferase plasmid (Promega) was co-transfected into each cell line, and Renilla luciferase activity generated by this vector was used to normalize transfection efficiency. The luciferase activity was measured using Promega Dual-Glo luciferase assay kit (Promega) as described previously (36).
Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay. The induction of apoptosis after treatment with 1 μM PLX4032 or 3 μM KS28 alone for 24 h was assessed by TUNEL staining and detected with an in situ Cell Death detection kit (Roche Life Science), according to the manufacturer’s instructions. The detailed method was described previously (31).
Anchorage-independent growth assay (soft-agar assay). The effects of PLX4032 and KS28 on cell transformation were investigated in JB6 Cl41, A375, and A375R cells. 8×103 cells with passage number less than 10 were exposed to 0.5 or 1 μM PLX4032 and KS28 in 1 ml of 0.3% basal medium Eagle’s medium containing 10% FBS, 2 mM L-glutamine, and 25 μg/ml gentamicin. The cultures were maintained at 37°C in an incubator with 5% CO2 for 14 days. Cell colonies were scored using an Axiovert 200M fluorescence microscope and Axio Vision software (Carl Zeiss, Thornwood, NY, USA).
Tumorigenicity assay in mice. Male BALB/c mice (6 weeks old) were obtained from Orient (Seongnam, Republic of Korea) and maintained in cages in a light- and temperature-controlled room. A375 or A375R cells were subcutaneously injected (2×106 cells) into the left flank of mice. After 5 days, the mice were randomly divided into different groups. Mice were intraperitoneally injected with saline, PLX4032 (20 mg/kg), or KS28 (10 mg/kg) three times each week. Mice were euthanized 14 days after the first injection, and tumor volume was calculated, and weight measured for all groups. Tumor diameters were measured using calipers (Mitutoyo, Kawasaki, Japan) and the volume was calculated as follows: Tumor volume=0.5× [(large diameter) × (small diameter)2]. All animal care and experimental procedures complied with local guidelines and were approved by the Animal Experiments Committee of Chosun University (approval number: CIACUC 2020-S0022).
Statistical analysis. Data were analyzed statistically using one-way analysis of variance, and values of p<0.05 were considered significant. Statistical calculations were performed using Prism v.8.4.2 (GraphPad Software, San Diego, CA, USA). The results are expressed as means±standard error of triplicate measurements from three independent experiments.
Results
Potent imidazothiazole-based inhibitor of BRAF, CRAF, and BRAF V600E kinases. Based on our previous knowledge of the inhibitory effect of imidazothiazole derivatives on V600E-BRAF and CRAF (35), we performed a biological investigation by testing KS28 (Figure 1) against BRAF, CRAF, and BRAF V600E kinases. The target compound was tested at a concentration of 1 μM, and the compounds that produced promising inhibitory effects, with >75% inhibition, were further tested in a 10-dose mode to measure their half-maximal inhibitory concentrations (IC50). The IC50 values are presented in Table I. Therefore, KS28 was characterized as an inhibitor of BRAF, CRAF, and BRAF V600E kinases.
Chemical structure of novel imidazo[2,1-b]oxazole derivative used in this study. Structure of N-(2-((4-(6-(4-fluoro-3-hydroxyphenyl) imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)benzene-sulfonamide, KS28.
Half-maximal inhibitory concentrations (IC50) for KS28 (C23H19FN6O3S2, MW: 510.56) and C-Raf proto-oncogene, serine/threonine kinase (CRAF) inhibitor GW5074, against BRAF V600E, BRAF, and CRAF kinases, as determined from the in vitro kinase assay.
KS28 suppressed cell proliferation and epithelial cell transformation in JB6 Cl41 cells. Since we characterized KS28 as a dual BRAF V600E and RAF inhibitor, we next analyzed whether this compound inhibited the MAPK signaling pathway induced by EGF. JB6 Cl41 cells were pretreated with KS28 then with EGF. Treatment with KS28 attenuated EGF-induced phosphorylation of MEK1/2, ERK1/2, and p90RSK (Figure 2A). AP1 is a heterodimer composed of JUN and FOS proteins, whose activity is regulated by the MAPK pathway in response to growth factors such as EGF. To investigate whether KS28 can suppress c-Fos and Ap1 transcriptional activity in JB6 Cl41 cells, we used reporter plasmids carrying the luciferase gene under the control of the c-Fos promoter or Ap1 response elements. Treatment with KS28 resulted in a dose-dependent reduction in c-Fos transcription and Ap1 transactivation induced by EGF (Figure 2B and C). Next, we evaluated the effects of KS28 on JB6 Cl41 cell growth using BrdU incorporation and soft-agar assays. KS28 displayed potent antiproliferative activity in EGF-treated JB6 Cl41 cells (Figure 2D). Similarly, KS28 significantly and dose-dependently reduced neoplastic cell transformation of JB6 Cl41 cells induced by EGF, as evident by reduced colony formation in soft agar matrix. (Figure 2E).
Effects of KS28 on cell proliferation and epithelial cell transformation induced by epidermal growth factor (EGF) in JB6 Cl41 cells. A: JB6 Cl41 cells were serum-starved for 24 h, pre-treated with 0.5 or 1 μM KS28 for 24 h, and then exposed to 10 ng/ml EGF for 15 min, harvested, lysed, and immunoblotted using specific antibodies against mitogen-activated protein kinase kinase 1/2 (MEK1/2), extracellular regulated kinase 1/2 (ERK1/2), p90 ribosomal S6 kinase (p90RSK), β-actin and their phosphorylated forms. JB6 Cl41 cells were co-transfected with luciferase reporter-gene constructs Fos proto-oncogene, AP-1 transcription factor subunit (c-Fos)-Luc (B) or activator protein 1 (Ap1)-Luc (C), and pRL-TK vector. At 24 h after transfection, cells were serum-starved for 24 h, pre-treated with 0.5 or 1 μM KS28, and then exposed to 10 ng/ml EGF for 24 h before luciferase assays were performed. D: JB6 Cl41 cells were pre-treated with 0.5 or 1 μM KS28 and exposed to 10 ng/ml EGF for 48 h, after which cell proliferation was estimated using BrdU incorporation assays. E: JB6 Cl41 cells were treated with 0.5 or 1 μM KS28 in the presence or absence of 10 ng/ml EGF in a soft-agar matrix and incubated at 37°C in 5% CO2 for 14 days. Representative colonies from three separate experiments were photographed (left), followed by calculating the average colony numbers and sizes (right). Data represent the means±standard deviation of triplicate measurements. Statistical analyses were conducted using one-way analysis of variance. Significantly different at: ##p<0.01 compared to the vehicle-treated control (dimethyl sulfoxide: DMSO); *p<0.05 and **p<0.01 compared to cells treated with EGF only.
KS28 suppressed MEK/ERK pathway via inhibition of RAF dimerization in A375R cells. PLX4032 was shown to induce hyperactivation of the MEK/ERK pathway via dimerization of RAF, which can lead to the development of acquired resistance (31). As pan-RAF inhibitors can overcome such paradoxical activation, we hypothesized that KS28 might overcome PLX4032 resistance in advanced melanoma. Therefore, we initially examined RAF dimerization in wild-type A375 and PLX4032-resistant A375R cells. We found increased formation of CRAF–BRAF V600E and CRAF– CRAF dimers in PLX4032-resistant cells compared to that in parental A375 cells (Figure 3A). Furthermore, phosphorylation levels of MEK, ERK, and p90RSK were highly elevated in A375R cells (Figure 3B). The formation of BRAF–CRAF, BRAF V600E–CRAF, CRAF–CRAF hetero-, and homo-dimers was induced in PLX4032-treated A375R cells, whereas KS28 treatment considerably reduced such dimerization (Figure 3C). Next, we examined the effects of KS28 on activation of the MEK–ERK signaling pathway in A375R cells. Consistent with previous findings (34), MEK1/2 and ERK1/2 phosphorylation levels remained unchanged, even after treatment of A375R cells with PLX4032, compared to parental cells. In contrast, treatment with KS28 profoundly inhibited the phosphorylation of MAPK signaling components in A375R cells (Figure 3D). Next, we examined the effects of KS28 on c-Fos and Ap1 activity in PLX-4032-resistant A375R cells. The transcriptional activity of c-Fos and the transactivational activity of Ap1 were significantly reduced by KS28 compared to PLX4032 in A375R cells (Figure 3E and F). Taken together, these results suggest that KS28 may overcome PLX4032 resistance in advanced melanoma by inhibiting RAF dimerization.
Effects of KS28 on raf proto-oncogene, serine/threonine kinase (RAF) dimerization and mitogen activated protein kinase signaling pathway in PLX4032-resistant A375R cells. A: Immunoprecipitation (IP) of A375 and A375R cells with anti-CRAF antibody, followed by immunoblotting with specific antibodies against endogenous B-raf proto-oncogene serine/threonine kinase (BRAF), C-raf proto-oncogene serine/threonine kinase (CRAF), and BRAF V600E. B: A375 and A375R cells were seeded and cultured for 48 h, harvested, then lysed. Proteins in whole-cell lysates (WCL) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted using specific antibodies against kinase 1/2 (MEK1/2), extracellular regulated kinase 1/2 (ERK1/2), p90 ribosomal S6 kinase (p90RSK), β-actin, and their phosphorylated forms. C: A375R cells were treated with or without 0.5 or 1 μM PLX4032 and 0.5 or 1 μM KS28 for 24 h. Proteins were immunoprecipitated with anti-CRAF, followed by immunoblotting with specific antibodies against endogenous BRAF, CRAF, and BRAF V600E. D: A375 and A375R cells were seeded and treated with or without 0.5 or 1 μM PLX4032 or 0.5 or 1 μM KS28 for 24 h. Cells were harvested, and proteins in the whole-cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted using specific antibodies against MEK1/2, ERK1/2, p90RSK, β-actin, and their phosphorylated forms. A375R cells were co-transfected with the luciferase reporters Fos proto-oncogene, AP-1 transcription factor subunit c-Fos-Luc (E) or activator protein 1 Ap1-Luc (F), along with pRL-TK vector. At 24 h after transfection, cells were serum-starved for 24 h then treated with or without 0.5 or 1 μM PLX4032 and 0.5 or 1 μM KS28 for 24 h before luciferase assays were performed. Data are the means±standard deviation of triplicate measurements. Statistical analyses were conducted using one-way analysis of variance. Significantly different at **p<0.01 compared to the vehicle-treated control (dimethyl sulfoxide: DMSO).
KS28 induces apoptosis and DNA fragmentation in A375R cells. As KS28 inhibited the MEK/ERK signaling pathway, we next investigated the effects of KS28 on apoptosis in A375R cells. Given that the cleavage of PARP and caspase-3 is a hallmark of apoptosis, we treated A375R cells with KS28 and measured the levels of cleaved caspase-3 and cleaved PARP by immunoblotting. Upon treatment with PLX4032, cleaved PARP and caspase-3 levels dose-dependently increased in parental A375 cells, whereas KS28 treatment induced greater cleavage of PARP and caspase-3 in A375R cells (Figure 4A). Next, we evaluated the effects of KS28 on the viability of A375R cells. The results showed that KS28 treatment of A375R cells effectively reduced cell viability compared to PLX4032 (Figure 4B). We next performed TUNEL staining to detect nuclear fragmentation of cells. The results showed more extensive DNA fragmentation in KS28-treated A375R cells than in PLX4032-treated A375R cells (Figure 4C).
Effects of KS28 on the apoptotic signaling pathway in PLX4032-resistant A375R cells. A: A375 and A375R cells were seeded and treated with or without 0.5 or 1 μM PLX4032 and 0.5 or 1 μM KS28 for 24 h. Cells were harvested, and proteins in the whole-cell lysates (WCL) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted using specific antibodies against cleaved-caspase 3, cleaved-poly (ADP-ribose) polymerase (PARP) and β-actin. B: A375 and A375R cells were treated with the 0.01, 0.1, 1, 3, and 5 μM of either PLX4032 or KS28 for 24 h, then cell viability was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Data are the means±standard deviation of triplicate measurements. Statistical analyses were conducted using one-way analysis of variance. Significantly different at **p<0.01 compared to the vehicle-treated control (dimethyl sulfoxide: DMSO). C: A375 and A375R cells were treated with 1 μM PLX4032 or 3 μM KS28 alone for 24 h. Cells were fixed and stained with TdT-mediated dUTP nick-end labeling (TUNEL) solution, and DNA fragmentation induced (green) by both compounds was measured. DAPI: 4’,6-Diamidino-2-phenylindole (blue).
KS28 suppressed colony formation and tumorigenicity of A375R cells. Given that activation of the transcription factor AP1 by various tumor promoters plays the most important role in neoplastic transformation and tumorigenesis, we examined the inhibitory effects of KS28 on anchorage-independent growth of PLX4032-resistant cells using a soft-agar assay. The results showed that treatment with PLX4032 was ineffective in terms of dose-dependent inhibition of colony formation by A375R cells. However, KS28 profoundly reduced the number and size of A375R cell colonies in a dose-dependent manner (Figure 5A and B).
Inhibitory effects of KS28 on neoplastic transformation and tumorigenicity of A375R cells. A375 and A375R cells were exposed to 0.5 or 1 μM PLX4032 and 0.5 or 1 μM KS28 in a soft-agar matrix and incubated at 37°C in 5% CO2 for 14 days. A: Representative colonies from three separate experiments. B: Average colony numbers and sizes. BALB/c mice were injected subcutaneously with A375 and A375R cells. On day 3 post-injection, mice were injected intraperitoneally with 10 mg/kg KS28 or 20 mg/kg PLX4032 alone and tumor growth was measured. C: Representative images of tumors. D: Tumor weight and tumor volume at the time of sacrifice (day 14). Data represent the means±standard deviation of triplicate measurements. Statistical analyses were conducted using one-way analysis of variance. Significantly different at: *p<0.05 and **p<0.01 compared to the untreated control.
Finally, we studied the antitumorigenic effects of KS28 in vivo. In agreement with the findings above, KS28 treatment significantly reduced the size and weight of tumors formed by A375R cells in BALB/c mice (Figure 5C and D). These data suggest that KS28 has a potential therapeutic role in overcoming PLX4032 resistance in patients with advanced melanoma.
Discussion
Approximately 50% of patients with advanced metastatic melanoma harbor a mutation in the gene that encodes BRAF (37). Over 90% of BRAF mutations involve the substitution of glutamic acid for valine at codon 600 (8). The current approach to treating advanced melanoma is mainly focused on the discovery of selective inhibitors of mutant BRAF V600E (14). PLX4032, a potent inhibitor of BRAF V600E, is effective against metastatic melanoma. However, resistance to PLX4032 develops within 6-8 months in patients with metastatic melanoma (25). Several studies are currently underway on how to overcome PLX4032 resistance (12, 29, 33, 38). Here, we investigated a novel imidazothiazole-based compound, KS28, as a dual BRAF V600E and RAF inhibitor, which inhibited the kinase activity of BRAF and CRAF. KS28 reduced cell viability, anchorage-independent growth, and induced cleavage of PARP and DNA fragmentation in PLX4032-resistant A375R cells. Collectively, our results suggest that KS28 can overcome acquired resistance to PLX4032 in patients with advanced melanoma.
BRAF V600E melanoma cells frequently develop resistance to PLX4032 through reactivation of the MEK/ERK signaling pathway (39). To our knowledge, several mechanisms have been implicated in such reactivation, including alterations to the BRAF gene, overexpression of BRAF RNA splice variants, overexpression of CRAF and COT kinases, and increased formation of RAF dimers (24, 26, 27). Indeed, dimerization of the spliced form of BRAF V600E, known as p65 BRAF V600E, mediates acquired resistance to PLX4032 (23). In the present study, we observed an increase in BRAF V600E–CRAF hetero- and CRAF–CRAF homo-dimerization in A375R cells. Consistent with this, the MEK/ERK signaling pathway was also up-regulated in PLX4032-resistant A375R cells. According to previous findings (40), blocking all RAF isoforms, known as pan-RAF inhibition, can overcome paradoxical activation of the MEK/ERK cascade followed by RAF dimerization. Since we identified KS28 as an inhibitor of BRAF, CRAF, and BRAF V600E kinases, we tested whether KS28 can combat dimerization-mediated PLX4032 resistance. In this study, we found that KS28 treatment inhibited RAF dimer formation and attenuated MEK/ERK signaling in PLX4032-resistant A375R cells. Taken together, our results suggest that KS28 overcomes PLX4032 resistance by inhibiting RAF dimer formation and activation of the MEK/ERK pathway.
The RAF/MEK/ERK cascade in normal cells is a key signaling pathway that controls cell proliferation, survival, and differentiation (41). It has been reported that BRAF V600E induces MAPK signaling and activates an important target of MAPKs, namely, transcription factor AP1 (42). AP1 exists as a dimeric complex consisting of FOS, JUN, activating transcription factors, and musculoaponeurotic fibrosarcoma protein family members, which have been implicated in melanoma cell proliferation, progression, and tumor development (43). In addition, the AP1 subunit FOSL1 is involved in enolase 2 (ENO2) transcription, the inhibition of which can enhance sensitivity to PLX4032 (44). In addition, inhibition of ENO2 suppressed the proliferation and migration of BRAF V600E-mutated colorectal cancer cells (44). Interestingly, the BRAF V600E mutation induces PLX4032 resistance in B-cells through the activation of AP1 (42). Here, we hypothesized that inhibition of AP1 activity by KS28 might effectively overcome PLX4032 resistance in BRAF V600E melanoma cells. We found that KS28 suppressed EGF-induced MAPK activation via MEK1/2, ERK1/2, and p90RSK in JB6 Cl41 and A375R cells. We further demonstrated that KS28 reduced EGF-induced transcriptional activity of c-Fos and the transactivation activity of Ap1 in JB6 Cl41 and A375R cells. KS28 consequently inhibited the proliferation and transformation of JB6 Cl41 and A375R cells induced by EGF. KS28 treatment reduced the growth of tumors formed by PLX4032-resistant A375R cells in BALB/c mice. These data suggest that KS28 inhibits AP1 activity and plays an essential role in overcoming acquired resistance.
Current strategies to overcome resistance to PLX4032 are focused on the development of pan-RAF inhibitors (32). Pan-RAF inhibitors have demonstrated anticancer activity in preclinical models of colon cancer, multiple myeloma, and melanoma (40, 45). LY3009120, a pan-RAF inhibitor, demonstrated antitumor effects in patients with melanoma and colon cancer with BRAF V600E (32). BRAF V600E inhibits the expression of pro-apoptotic BCL2-like 11, an essential initiator of cell death, through MEK/ERK signaling in melanoma cells (46). The pan-RAF inhibitor INU-152 attenuated the MEK/ERK signaling pathway in BRAF V600E melanoma cells and suppressed tumor growth in a mouse model of human melanoma (40).
In summary, we demonstrated that inhibition of RAF dimer formation using a potent, novel imidazothiazole-based inhibitor of BRAF V600E kinase inhibited melanoma tumor growth in PLX4032-resistant A375R cells. RAF inhibitors are often used in combination with MEK1/2 or AKT serine/threonine kinase 1 (AKT) inhibitors to improve their efficacy and prevent acquired resistance. Therefore, further studies are required to evaluate the efficacy of KS28 in combination with other compounds, such as MEK or AKT inhibitors, so as to achieve maximum clinical benefit for patients with melanoma.
Acknowledgements
This work was supported by research funds from Chosun University (2021).
Footnotes
Authors’ Contributions
M.P. and G.K. P.Y.B, and S.S. designed and performed experiments, analyzed, and interpreted data, and wrote the article. S.Z. and CH.O. Carried out the design and synthesis of novel biologically active compounds. H.S.C. conceived the study, designed experiments, interpreted data, and wrote the article. All Authors reviewed the article.
Conflicts of Interest
The Authors declare they have no conflicts of interest.
- Received February 14, 2022.
- Revision received May 12, 2022.
- Accepted May 13, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
