NL-103, a novel dual-targeted inhibitor of histone deacetylases and hedgehog pathway, effectively overcomes vismodegib resistance conferred by Smo mutations

Abstract

Misregulation of hedgehog (Hh) signaling has been implicated in the pathogenesis of basal cell carcinoma (BCC) and medulloblastoma. Vismodegib, an orally bioavailable Hh signal pathway inhibitor targeting Smo, has been approved for the treatment of advanced BCC. However, acquired drug resistance to vismodegib induced by point mutation in Smo is emerging as a major problem to vismodegib treatment. In this study, we designed and synthesized a novel chimeric compound NL-103, which comprises structural elements of Hh pathway inhibitor vismodegib, and histone deacetylase (HDAC) inhibitor vorinostat. NL-103 simultaneously and significantly inhibited both HDACs and Hh pathway. Importantly, NL-103, as well as vorinostat, effectively overcame vismodegib resistance induced by Smoothened point mutations. Moreover, NL-103 and vorinostat, but not vismodegib, significantly downregulated the expression of Gli2 which plays an important role in Hh pathway. These results indicate that HDAC inhibitory activity is essential for NL-103 to overcome vismodegib resistance and that dual inhibition of HDAC and Hh signaling pathway may be a rational strategy for overcoming vismodegib resistance. Our findings suggest that NL-103 may be a promising compound for clinical development as a more effective Hh pathway inhibitor.

Keywords: Drug resistance; NL-103; hedgehog pathway inhibitor; histone deacetylase inhibitor; smoothened mutation.

Figure 1

Schematic representation of the design and chemical structure of NL-103. To create NL-103, we introduced hydroxamic acid (functional group for HDAC inhibition) with a flexible side chain onto the pyridine backbone of a putative Smo inhibitor.

Figure 2

NL-103 increases the acetylation levels of α-tubulin and total histones, and effectively inhibits Hh signaling by targeting Smo. (A) NIH3T3 cells were treated with varying concentrations of NL-103 or vorinostat. After 6 h, cells were harvested and the acetylation levels of α-tubulin and total histones were measured using immunoblotting. (B) Vismodegib, NL-103 and vorinostat dose-dependently inhibit Shh-N-induced firefly luciferase expression in NIH3T3-12Gli cells. Seeded cells were divided into two groups. One group was cultured in HEK293 control medium and the other was maintained in Shh-N conditioned medium. Both groups were treated with various concentrations of each compound. For cells treated with the same compound at the same concentration, reporter activity in Shh-N was normalized to that in control medium. Empirically, this calculation method can effectively filter out the nonspecific effects of compound on reporter activity. Data are the average of three independent experiments ± SD. (C) GDC-0449 and NL-103 compete for the binding of BODIPY-cyclopamine to Smo-WT-expressing HEK293T cells, but not SAHA. Nonspecific binding as defined by BODIPY-cyclopamine levels of cells treated with mock transfection. (D) Flow cytometric quantitation of specific BODIPY-cyclopamine binding to Smo-expressing cells was used to determine the affinities of Smo antagonists through binding competitions.

Figure 3

NL-103 prevents ciliary localization of Smo induced by Hh pathway agonists. (A–G) Monoclonal cells expressing Smo-WT-EYFP were treated with indicated compounds or conditions (DMSO, 0.1%; Shh-N, 20%; SAG, 100 nmol/L). Smo-WT-EYFP (green) was visualized by direct detection of EYFP. Primary cilia (red) were marked with acetylated-α-tubulin antibody by immunofluorescence. Nuclei (blue) were visualized by DAPI staining. Arrows point to cilia with weak (white) cilia staining. Representative images are provided (Scale bars: 5 μm). At least two monoclonal cell lines were applied and similar results were obtained.

Figure 4

Effects of vismodegib, NL-103 or vorinostat on Hh signaling mediated by exogenous human wildtype Smo and two clinically relevant Smo mutants. (A) Luciferase reporter activity after transfection of Smo variants in the absence (blank bars) or presence (gray bars) of Shh-N. (B–D) Luciferase reporter activity in NIH3T3-12Gli cells expressing Smo-WT (B), Smo-M2 (C), or Smo-DH (D) after treatment with various doses of vismodegib (circles), NL-103 (triangles) or vorinostat (squares) in the absence (blank) or presence (black) of Shh-N. Reporter activity is normalized to DMSO-treated cultures. Data are the average of three independent experiments ± SD. (E) Flow cytometric determination of specific BODIPY-cyclopamine binding to HEK293T cells expressing Smo-DH, and the competitions of vismodegib and NL-103 for the binding of BODIPY-cyclopamine to Smo mutants.

Figure 5

Effects of Hh pathway stimulators or inhibitors on ciliary localization of Smo-M2. (A–F) Monoclonal cells expressing Smo-M2-EYFP were treated with indicated compounds or conditions (DMSO, 0.1%; Shh-N, 20%; SAG, 100 nmol/L). Smo-M2-EYFP (green), primary cilia (red), and nuclei (blue) were visualized as before. Representative images are provided (Scale bars: 5 μm). At least two monoclonal cell lines were applied and similar results were obtained.

Figure 6

Effects of vismodegib, NL-103 or vorinostat on the transcription of Gli-1 and Gli-2. (A, B) Gli-1 mRNA levels after treatment with various doses of compounds in the presence (A) or absence of Shh-N (B). (C, D) Gli-2 mRNA levels after treatment with various doses of compounds in the presence (C) or absence of Shh-N (D). NIH3T3-12Gli cells were maintained in either HEK293 control medium (blank bars) or Shh-N conditioned medium (gray bars). Cells were treated with DMSO control, vismodegib, NL-103 or vorinostat in triplicate. After 24 h, samples were collected and total RNA was extracted. Levels of mRNA were assessed by quantitative real-time PCR. Data indicate mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test.