Targeting TBK1 inhibits migration and resistance to MEK inhibitors in mutant NRAS melanoma

Abstract

Melanoma is a devastating form of skin cancer with limited therapeutic options. Fifteen to 20% of patients with melanoma have an activating mutation in the GTPase, NRAS. The major downstream effectors of RAS are RAFs (ARAF, BRAF, and CRAF), phosphoinositide 3-kinase (PI3K), and the Ral guanine exchange factors (RalGEF). TANK-binding kinase 1 (TBK1) is an atypical IκB kinase family member that acts downstream of RalGEFs. Whereas many studies have analyzed RAF and PI3K signaling in mutant NRAS melanoma, the role of RalGEF/Ral is understudied and TBK1 has not been examined. To address this, TBK1 was modulated with knockdown approaches and targeted therapies to determine the role of TBK1 in motility, apoptosis, and signaling. In melanoma, NRAS overexpression increased TBK1 phosphorylation. TBK1 depletion inhibited migration and invasion, whereas its constitutive overexpression led to an increase in invasion. In three-dimensional systems that mimic the dermal microenvironment, TBK1 depletion or inhibition cooperated with MEK inhibitors to promote apoptosis, particularly in the context of MEK-insensitive mutant NRAS. This effect was absent in melanoma cells that are wild-type for NRAS. These results suggest the utility of TBK1 inhibitors as part of a treatment regimen for patients with mutant NRAS melanoma, for whom there are no current effective therapies.

Implications: TBK1 promotes the malignant properties of NRAS-mutant melanoma and its targeting, in combination with MEK, promotes apoptosis, thus providing a potential novel targeted therapeutic option.

Figure 1. TBK1 is expressed in mutant NRAS cell lines

(A) Cell lysates from mutant NRAS melanoma cells, a primary human keratinocyte culture, and mutant BRAF melanoma cells were analyzed by Western blot for TBK1, phospho-TBK1, NRAS and actin (loading control). (B) WM3211 melanoma cells wild-type for BRAF and NRAS were non-transduced (−) or transduced with constitutively active NRAS^Q61K. After selection, cells were lysed and lysates analyzed by Western blot for the proteins indicated. (C) Mutant NRAS melanoma cell lines were transfected with non-targeting or TBK1-targeting siRNA for 72 hours. Cells were serum-starved overnight and treated with serum-free medium, full serum medium, or 1 μM insulin for 20 min. Cells were lysed and lysates analyzed by Western blot analysis. (D) WM1366 and WM136A cells with a tetracycline-inducible system (TR) expressing a TBK1-myristoylated construct (myr) were treated with or without doxycycline for 48 hours; and parental SKMel2 and SKMel2 constitutively expressing TBK1-myr cells were lysed. Lysates were analyzed by Western blot analysis. (E) Immunofluorescence image of WM1366 TR-TBK1-myr with or without doxycycline for 24 hours stained for DAPI (blue) and TBK1 (green). Scale bar = 25 μm.

Figure 2. TBK1 promotes migration and invasion of melanoma cells

(A) Immunofluorescence image of WM1361A stained for DAPI (blue), FAK (red) and TBK1 (green). Scale bar = 25 μm. (B) WM1366, SBcl2, WM1346, WM1361A and SKMel2 cells were transfected with non-targeting or TBK1-targeting siRNA for 72 hours and lysates were analyzed by Western blotting. (C) Mutant NRAS cells were transfected as in (B). Cells were allowed to migrate through Boyden chambers toward an attractant of full serum medium for 24 hours. Counts taken (in triplicate fields of view) from the control siRNA (average set at 100% migration) were used to calculate percent migration (n=3; errors bars, S.E.; *, p<0.05). Representative 10× images of migrated cells are shown. Scale bar = 100 μm. (D) Mutant NRAS cells were transfected as in (B). Cells were allowed to invade through Boyden chambers coated with Matrigel toward an attractant of full serum medium for 24 hours. Counts taken (in triplicate fields of view) from the control siRNA (average set at 100% invasion) were used to calculate percent invasion (n=3; errors bars, S.E.; *, p<0.05). Representative 10× images of migrated cells are shown. Scale bar = 100 μm. (E) WM1366 TR TBK1-myr and WM136A TR TBK1-myr cells were treated with or without doxycycline for 24 hours; and parental SKMel2 and SKMel2 constitutively expressing TBK1-myr were seeded for migration assay as in (C). (F) WM1366 TR TBK1-myr and WM136A TR TBK1-myr cells were treated with or without doxycycline for 24 hours; and parental SKMel2 and SKMel2 constitutively expressing TBK1-myr were seeded for invasion assay as in (D).

Figure 3. Knockdown of TBK1 enhances apoptosis in combination with AZD6244 specifically in mutant NRAS cell lines

(A) Mutant NRAS cells were transfected with non-targeting or TBK1-targeting siRNA #1 and #2, and, after 72 hours in 2D conditions, were analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05). (B) Mutant NRAS cells were transfected with non-targeting or TBK1-targeting siRNA and plated at low density for a total of 7 days. Full-sized image, top, and 4× magnification, bottom. (C) Mutant NRAS cells were transfected as in (A), cultured in 3D collagen in the presence or absence of AZD6244 (3.3 μM). After 24 hours, cells were extracted and analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05). (D) WM1366 cells were transfected as in (A) and cultured in 3D collagen as in (C). After 24 hours, cells were lysed and lysates analyzed by Western blotting. (E) WM1361A TR TBK1-myr pretreated with or without doxycycline, SKMel2 and SKMel2 TBK1-myr were cultured in 3D collagen in the presence or absence of AZD6244 (3.3 μM). After 48 hours, cells were released from the gel and analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05). (F) WM3211, WM3211 transduced with NRAS^Q61K, and WM983A cells were transfected as in (A), then cultured and treated in 3D collagen as in (C). After 24 hours, cells were extracted and analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05).

Figure 4. BX795 in combination with AZD6244 enhances apoptosis in MEK inhibitor-resistant mutant NRAS melanoma cells in 3D

(A) SKMel2 and doxycycline-induced WM1366 TR TBK1-myr cells were treated with increasing concentrations of the TBK1 inhibitor, BX795, for 24 hours. Cells were lysed and lysates analyzed by Western blotting. (B) Mutant NRAS cells were plated at low density and treated with DMSO or BX795 (1 μM) for 1 week. Full-sized image, top, and 4× magnification, bottom. (C) WM1366, SBcl2, WM1346, WM1361A, and SKMel2 cells were cultured in 3D collagen in the presence or absence of BX795 (1 μM) and/or AZD6244 (3.3 μM). After 48 hours, cells were extracted and analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05). (D) WM1366 cells were placed in 3D collagen and treated as in (C). After 24 hours, cells were lysed and lysates analyzed by Western blotting. (E) WM793-Res #5 and WM793-Res #12 cells grown in 5 μM PLX4720 were cultured in 3D collagen and treated as in (C). After 48 hours, cells were extracted and analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05).

Figure 5. AZ245 in combination with AZD6244 enhances apoptosis in MEK inhibitor-resistant lines in 3D

(A) Structure of the TBK1 inhibitor AZ13102909 (AZ909). (B) SKMel2 and doxycycline-induced WM1366 TR TBK1-myr cells were treated with increasing concentrations of AZ909 for 24 hours. Cells were lysed and lysates analyzed by Western blotting. (C) Mutant NRAS cells were plated at low density and treated with DMSO or AZ909 (1 μM) for 1 week. Full-sized image, top, and 4× magnification, bottom. (D) Mutant NRAS cells were cultured in 3D collagen in the presence or absence of AZ909 (1 μM) and/or AZD6244 (3.3 μM). After 48 hours, cells were extracted and analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05). (E) WM1366 cells were placed in 3D collagen and treated as in (D). After 48 hours, cells were lysed and lysates analyzed by Western blotting. (F) WM793-Res #5 and WM793-Res #12 cells grown in 5 μM PLX4720 were cultured in 3D collagen and treated as in (D). After 48 hours, cells were extracted and analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05).