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. 2013 Jan 14;23(1):23-34.
doi: 10.1016/j.ccr.2012.11.017. Epub 2013 Jan 3.

Itraconazole and arsenic trioxide inhibit Hedgehog pathway activation and tumor growth associated with acquired resistance to smoothened antagonists

James Kim  1 , Blake T Aftab, Jean Y Tang, Daniel Kim, Alex H Lee, Melika Rezaee, Jynho Kim, Baozhi Chen, Emily M King, Alexandra Borodovsky, Gregory J Riggins, Ervin H Epstein Jr, Philip A Beachy, Charles M Rudin
Affiliations

Itraconazole and arsenic trioxide inhibit Hedgehog pathway activation and tumor growth associated with acquired resistance to smoothened antagonists

James Kim et al. Cancer Cell. .

Abstract

Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors. Leading inhibitors in clinical development act by binding to a common site within Smoothened, a critical pathway component. Acquired Smoothened mutations, including SMO(D477G), confer resistance to these inhibitors. Here, we report that itraconazole and arsenic trioxide, two agents in clinical use that inhibit Hedgehog signaling by mechanisms distinct from that of current Smoothened antagonists, retain inhibitory activity in vitro in the context of all reported resistance-conferring Smoothened mutants and GLI2 overexpression. Itraconazole and arsenic trioxide, alone or in combination, inhibit the growth of medulloblastoma and basal cell carcinoma in vivo, and prolong survival of mice with intracranial drug-resistant SMO(D477G) medulloblastoma.

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Figures

Figure 1
Figure 1. A simplified view of Hh signaling
Binding of a Hh ligand to Patched (PTCH) de-represses SMO, causing activation and translocation to the nucleus of GLI2, which initiates the transcription of target genes including PTCH and GLI1. Cyclopamine, its derivative, IPI-926, and its mimics GDC-0449 and NVP-LDE225 all bind competitively to a site within the transmembrane portion of SMO; the SMO missense mutations indicated by yellow asterisks decrease binding and confer resistance to these drugs. Itraconazole inhibits SMO by a distinct mechanism and ATO inhibits the pathway at the level of GLI.
Figure 2
Figure 2. Itraconazole inhibits the Hh pathway in the context of GDC-0449 resistant SMOD477G
(A & B) Relative Hh pathway activity as determined by expression of 8×-GLI-luciferase reporter in SHHN stimulated Smo−/− MEFs expressing SMOWT (blue) or SMOD477G (red) treated with (A) GDC-0449 or (B) itraconazole. Data represent mean of triplicates ± SD. (C & D) Proliferation of Ptch−/+; p53−/− MB tumorspheres expressing endogenous SMOWT (blue) or SMOD477G (red) treated with increasing doses of (C) GDC-0449 and (D) itraconazole. Data represent mean of quadruplicates ± SEM. (E & F) Relative Gli1 mRNA transcription in (C) GDC-0449-treated and (D) itraconazole-treated MB tumorspheres expressing endogenous SMOWT (blue) or SMOD477G (red). Data represent mean of quadruplicates ± SEM. See also Figure S1.
Figure 3
Figure 3. Itraconazole combines with cyclopamine to inhibit GDC-0449-resistant SMO function
(A) Relative 8×-GLI-luciferase expression in SHHN-stimulated Smo−/− MEFs expressing SMOWT or SMOD477G, treated with GDC-0449, itraconazole, or both. (B) Effect of KAAD-cyclopamine 100 nM, itraconazole 2 μM, or both on SHHN-stimulated 8×-GLI-luciferase expression in Smo−/− MEFs expressing SMOD477G. (C) Dose-response curves of KAAD-cyclopamine for SHHN activated signaling in Smo−/− MEFs expressing SMOD477G, in the presence or absence of itraconazole. Data represent mean of triplicates ± SD. See also
Figure 4
Figure 4. ATO and itraconazole combine to inhibit Hh pathway activation and tumor growth by SMOWT
(A & B) Effect of combination therapy on the IC50 concentrations of itraconazole (A) or ATO (B) in NIH-3T3 cells expressing endogenous SMOWT stimulated with SHHN in 8×-GLI-luciferase signaling assays. Data represent mean of triplicates ± SD. (C & D) Nude mice with SMOWT hind-flank MB allografts were treated with vehicle control (40% cyclodextrin PO bid, N=7 tumors; black), GDC-0449 (100 mg/kg PO bid, N=7; red) ATO (7.5 mg/kg IP qd, N=7 tumors; green), itraconazole (75 mg/kg PO bid, N=7 tumors; blue), or both ATO and itraconazole (N=7 tumors; blue-green). Effect of GDC-0449, ATO, itraconazole, or the combination of ATO and itraconazole on (C) tumor growth and (D) Gli1 mRNA expression compared to vehicle control. Data represent group means ± SEM. *p < 0.01 vs. Vehicle; ** p < 0.01 vs. ATO alone; *** p < 0.01 vs. itraconazole alone. See also Figure S3, Table S2 and S3.
Figure 5
Figure 5. Combination of ATO and itraconazole inhibits tumor growth of Hh-dependent basal cell carcinoma
NOD/SCID mice with established K14-CreER/+; Ptch+/−; p53fl/fl BCC allografts were treated with vehicle control (40% cyclodextrin po bid, N=4 tumors; black), ATO (7.5mg/kg IP qd, N=6 tumors; green), itraconazole (75 mg/kg po bid, N=10 tumors; blue), or both ATO and itraconazole (N=16 tumors; blue-green; p<0.01 for the combination compared to either itraconazole or ATO alone). Data represent group means ± SEM.
Figure 6
Figure 6. ATO and itraconazole combine to inhibit SMOD477G activity and tumor growth, and improves survival in an orthotopic medulloblastoma model
(A–C) Smo−/− MEFs were transfected with either SmoWT or SmoD477G and stimulated with SHHN. Data represent mean of triplicates ± SD. (A) Relative Hh pathway activity as assessed by relative 8×-GLI-luciferase expression in the presence or absence of itraconazole, ATO, or the combination. Dashed line represents the level of pathway inhibition of SMOWT by itraconazole 1.5 μM. (B) Effect of the addition of increasing doses of ATO to itraconazole in cells expressing SMOD477G. (C) Effect of increasing doses of itraconazole on the IC50 of ATO in cells expressing SMOD477G. (D & E) Nude mice with established SMOD477G MB allografts were treated with vehicle control (40% cyclodextrin PO bid, N=8 tumors; black), GDC-0449 (100 mg/kg PO bid, N=8; red); ATO (7.5mg/kg IP qd, N=8 tumors; green), itraconazole (75 mg/kg PO bid, N=8 tumors; blue), or both ATO and oral itraconazole (N=8 tumors; blue-green); (D) tumor growth over time and (E) relative Gli1 mRNA expression. Data represent group means ± SEM. *p < 0.01 vs. Vehicle; ** p < 0.01 vs. ATO alone; *** p < 0.01 vs. ITRA alone. (F) Kaplan-Meier survival analysis of an orthotopic model of SMOD477G MB treated with vehicle control (N=9), GDC-0449 (N=9), itraconazole (N=9), ATO (N=9), or both ATO and itraconazole (N=9) using same doses as in (D). See also Figure S4 and Table S4.
Figure 7
Figure 7. Combination of ATO and itraconazole inhibits all other known drug-resistant SMO mutants and GLI2 overexpression
SHHN stimulated Smo−/− MEFs were transfected with (A) SmoWT or mutant Smo constructs resistant to (B) GDC-0449 or (C–G) NVP-LDE225. The effects of itraconazole, ATO, and the combination of both agents on Hh pathway activity was assessed by relative 8×-GLI-luciferase activity. Labels of panels (B–G) indicate the mutant SMO that was expressed. (H) Effect of itraconazole, ATO, or the combination on relative 8×-GLI-luciferase activity in NIH-3T3 cells transfected with Gli2 +/− SHHN stimulation. Data represent mean of triplicates ± SD. See also Figure S5.
See this image and copyright information in PMC

Comment in

  • Smoothing out drug resistance.
    Kasper M, Toftgård R. Kasper M, et al. Cancer Cell. 2013 Jan 14;23(1):3-5. doi: 10.1016/j.ccr.2012.12.011. Cancer Cell. 2013. PMID: 23328478

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