Melika Rezaee

Itraconazole and Arsenic Trioxide Inhibit Hedgehog Pathway Activation and Tumor Growth Associated with Acquired Resistance to Smoothened Antagonists

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.

Inhibition of the hedgehog pathway in patients with basal-cell nevus syndrome: final results from the multicentre, randomised, double-blind, placebo-controlled, phase 2 trial

BACKGROUND Aberrant hedgehog signalling underlies the development of basal-cell carcinomas. We previously reported the interim analysis of a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial in patients with the basal-cell nevus (Gorlin) syndrome indicating that the smoothened inhibitor vismodegib reduces basal-cell carcinoma tumour burden and prevents new basal-cell carcinoma growth in patients with basal-cell nevus syndrome. We report the final results of this 36 month trial. METHODS In our multicentre, randomised, double-blind, placebo-controlled, phase 2 trial we enrolled patients aged 35-75 years with basal-cell nevus syndrome with at least ten surgically eligible basal-cell carcinomas at the Childrens Hospital Oakland, Columbia University outpatient dermatology clinic (NY, USA) and a private practice outpatient dermatology office in Newport Beach (CA, USA). Patients were assigned to vismodegib or placebo (2:1) according to a randomisation sequence generated by computer code. The primary endpoint of the trial of 41 patients was to compare the effect of oral vismodegib (150 mg/day) versus placebo on the incidence of new surgically eligible basal-cell carcinomas after 3 months of treatment. In the subsequent, open-label phase (n=37) patients continued vismodegib at two sites for as long as month 36 (n=25) and at the third site were monitored up to month 36 (n=12). Additional endpoints for this phase were: whether continuous versus interrupted dosing differentially affected tumour burden; time to reach various levels of reduction in tumour burden; reduction in tumour size in patients who took less than 50% of the expected number of vismodegib tablets; reduction in the number of surgical excisions required per year before, during, and after treatment; and the effect of vismodegib on hedgehog target gene expression. We monitored patients at visits every 3 months for up to 36 months. The primary endpoint was analysed on a modified intention-to-treat basis. This trial is registered with ClinicalTrials.gov, number NCT00957229. FINDINGS Between Sept 22, 2009, and Jan 24, 2011, 41 patients were monitored for a median of 36 months (IQR 36-36). Patients treated with vismodegib (n=26) had a mean reduced rate of new surgically eligible basal-cell carcinomas compared with patients randomly assigned to placebo (n=15; two [SD 0·12] new surgically eligible basal-cell carcinomas per patient per year vs 34 [1·32] new surgically eligible basal-cell carcinomas per patient per year, p<0·0001). In the 11 patients initially assigned to placebo, mean cross over to vismodegib reduced the development of new surgically eligible basal-cell carcinomas compared with placebo (0·4 [SD 0·2] new surgically eligible basal-cell carcinomas per patient per year vs 30·0 [7·8] new surgically eligible basal-cell carcinomas per patient per year, p<0·0001). Only three (17%) of 18 patients tolerated vismodegib continuously for the full 36 months. Fewer new surgically eligible basal-cell carcinomas developed in patients receiving vismodegib continuously than in those who interrupted dosing (mean 0·6 [0·72] new surgically eligible basal-cell carcinomas per patient per year vs 1·7 [1·8] new surgically eligible basal-cell carcinomas per patient per year, p<0·0001). Treatment-related grade 3-4 adverse events included weight loss of 20% or more (n=6) and muscle cramps (n=2). Two patients died during the course of the trial, one each from laryngeal and metastatic prostate cancer, deemed probably unrelated to drug. INTERPRETATION Vismodegib reduces basal-cell carcinoma tumour burden in patients with basal-cell nevus syndrome. Adverse events associated with vismodegib frequently led to interruption of treatment, which is followed by basal-cell carcinoma recurrence. FUNDING Genentech investigator-initiated trial funding, Clinical and Translational Science Award from the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Cancer Institute, Damon Runyon Cancer Research Foundation Clinical Investigator Award, Swim across America Foundation, and Michael J Rainen Family Foundation.

Effects of Combined Treatment With Arsenic Trioxide and Itraconazole in Patients With Refractory Metastatic Basal Cell Carcinoma

IMPORTANCE Tumor resistance is an emerging problem for Smoothened (SMO) inhibitor-treated metastatic basal cell carcinoma (BCC). Arsenic trioxide and itraconazole antagonize the hedgehog (HH) pathway at sites distinct from those treated by SMO inhibitors. OBJECTIVE To determine whether administration of intravenous arsenic trioxide and oral itraconazole in patients with metastatic BCC is associated with a reduction in GLI1 messenger RNA expression in tumor and/or normal skin biopsy samples. DESIGN, SETTING, AND PARTICIPANTS Five men with metastatic BCC who experienced relapse after SMO inhibitor treatment underwent intravenous arsenic trioxide treatment for 5 days, every 28 days, and oral itraconazole treatment on days 6 to 28. Data were collected from April 10 to November 14, 2013. Follow-up was completed on October 3, 2015, and data were analyzed from June 5 to October 6, 2015. MAIN OUTCOMES AND MEASURES The primary outcome was the change in messenger RNA levels of the GLI family zinc finger 1 (GLI1) gene (HH-pathway target gene) in biopsy specimens of normal skin or BCC before and after treatment. Secondary objectives were evaluation of tumor response and tolerability. RESULTS Of the 5 patients (mean [SD] age, 52 [9] years; age range, 43-62 years), 3 completed 3 cycles of treatment and 2 discontinued treatment early owing to disease progression or adverse events. Adverse effects included grade 2 transaminitis and grade 4 leukopenia with a grade 3 infection. Overall, arsenic trioxide and itraconazole reduced GLI1 messenger RNA levels by 75% from baseline (P < .001). The best overall response after 3 treatment cycles was stable disease in 3 patients. CONCLUSIONS AND RELEVANCE Targeting the HH pathway with sequential arsenic trioxide and itraconazole treatment is a feasible treatment for metastatic BCC. Although some patients experienced stable disease for 3 months, none had tumor shrinkage, which may be owing to transient GLI1 suppression with sequential dosing. Continuous dosing may be required to fully inhibit the HH pathway and achieve clinical response.

Effect of Calcium Channel Blockade on Vismodegib-Induced Muscle Cramps

Author Contributions: Dr S. C. Chen had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Aphivantrakul, K.-H. Chen, S. C. Chen. Acquisition, analysis, or interpretation of data: All authors. Drafting of the manuscript: Martin. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: K.-H. Chen. Administrative, technical, or material support: S. C. Chen. Study supervision: S. C. Chen.

Pilot study on the bioactivity of vitamin d in the skin after oral supplementation.

Laboratory studies suggest that vitamin D (VD) supplementation inhibits skin carcinogenesis. However, epidemiologic studies report mixed findings in the association between circulating VD levels and skin cancer risk. We conducted a clinical study to determine whether oral cholecalciferol supplementation would exert direct bioactivity in human skin through modulation of the VD receptor (VDR). We enrolled 25 individuals with serum 25-hydroxyvitamin-D levels <30 ng/mL and with skin photodamage to take 50,000 IU of cholecalciferol biweekly for 8 to 9 weeks. Then, we obtained baseline and end-of-study skin biopsies from photodamaged (PD) and photoprotected (PP) skin, and from benign nevi (BN) and tested for mRNA expression of VDR and cytochrome P450-24 (CYP24), and markers of keratinocytic differentiation. High-dose cholecalciferol supplementation significantly elevated circulating levels of 25-hydroxyvitamin-D (P < 0.0001) and 1,25-dihydroxyvitamin-D (P < 0.0001). VDR expression in PD- and PP-skin showed minimum changes after supplementation. CYP24 expression in PD- and PP-skin was increased after supplementation by 186%, P = 0.08, and 134%, P = 0.07, respectively. In BNs from 11 participants, a trend for higher VDR and CYP24 expression was observed (average of 20%, P = 0.08, and 544%, P = 0.09, respectively). Caspase-14 expression at the basal layer in PD skin samples was the only epidermal differentiation marker that was significantly increased (49%, P < 0.0001). High-dose cholecalciferol supplementation raised serum VD metabolite levels concurrently with CYP24 mRNA and caspase-14 levels in the skin. Our findings of significant variability in the range of VDR and CYP24 expression across study samples represent an important consideration in studies evaluating the role of VD as a skin cancer chemopreventive agent. Cancer Prev Res; 8(6); 563–9. ©2015 AACR.

Abstract 3248: Pilot study on the bioactivity of vitamin D in the skin after oral supplementation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Experimental studies suggest that vitamin D (VD) plays an important role in skin carcinogenesis. In humans, epidemiologic studies have reported mixed findings in the association between circulating vitamin D levels and skin cancer risk. We conducted a pilot clinical study to determine whether oral VD supplementation would exert any bioactivity in human skin. Methods: The study accrued twenty-five healthy individuals with serum 25-hydroxyvitamin D levels <30 ng/mL and with moderate to severe photodamage on the forearms. Participants took 50,000 IU of VD3 capsules twice a week for 8-9 weeks. Baseline and end-of study skin biopsies were obtained from photodamaged (PD) and photoprotected (PP) skin, and benign nevi (BN), when available, for assessment of changes in putative biomarkers of VD activity. Biomarkers evaluated include the mRNA expression of vitamin D receptor (VDR) and cytochrome P450 24 (CYP24) in keratinocytes and available BN. In addition, molecular markers of keratinocytic differentiation (caspase 14 and loricrin protein expression), epidermal thickness, serum levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were assessed. Results: High dose VD supplementation significantly elevated the circulating levels of 25-hydroxyvitamin D from 21.6 ± 5.2 to 70.5 ± 18.2 ng/mL (p < 0.0001) and 1,25-dihydroxyvitamin D from 31.1 ± 12.4 to 51.4 ± 13.5 pg/mL (p < 0.0001). VDR expression in PD- and PP-skin showed minimum changes after VD supplementation. CYP24 expression in PD- and PP-skin showed a non-statistically significant increase after VD supplementation (average of 186% increase, p = 0.08, and 134% increase, p=0.07, respectively). Higher VDR and CYP24 expression was observed in BNs collected at post-intervention than that at baseline from eleven participants, but the difference did not reach statistical significance (average of 20% higher, p = 0.08, and 544% higher, p= 0.09, respectively). The large inter-subject variation in VDR and CYP24 expression may have limited the statistical evaluation. The epidermal differentiation markers did not change significantly after VD supplementation with the exception of a 49% increase (p< 0.0001) in caspase 14 expression at the basal layer in PD skin samples. When epidermal thickness was analyzed, the only significant change was identified when the analysis was stratified by the baseline median thickness. Samples with baseline thickness below or equal to the median exhibited a significant increase in thickness at the end of the intervention. Conclusion: The study showed that following effective oral supplementation based on VD serum levels, subtle indicators of increased keratinocytic differentiation and CYP24 activation can be observed. Future studies evaluating the role of VD as a skin cancer chemopreventive agent with biomarker modulation as an endpoint should be considered before larger intervention studies are implemented in at risk populations. Citation Format: Clara Curiel-Lewandrowski, Jean Tang, Janine Einspahr, Yira Bermudez, Chiu-Hsieh Hsu, Melika Rezaee, Alex Lee, Joe Tangrea, Howard Parnes, David Alberts, H-H. Sherry Chow. Pilot study on the bioactivity of vitamin D in the skin after oral supplementation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3248. doi:10.1158/1538-7445.AM2014-3248