Abstract 4669: Targeting hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells
Authors: Faiao-Flores Fernanda , Alves-Fernandes Debora Kristina Magalhaes , Pennacchi Paula Comune , Sandri Silvana , Vicente Anna Luiza Silva Almeida , Scapulatempo-Neto Cristovam , Vazquez Vinicius de Lima , Reis Rui Manuel , Smalley Keiran S. , Maria-Engler Silvya Stuchi 
Published: July 2016

Abstract
Melanoma represents the deadliest of all skin cancers being responsible for more than 75% of skin cancer-related deaths. Although the results with BRAF inhibitors have been very promising, practically all of the patients treated thus far have developed resistance to these drugs. The Hedgehog (Hh) signaling pathway has been implicated in a variety of malignancies, including melanoma. However, there are no reports on the activation of this pathway in the setting of vemurafenib-resistance. Herein, we first identified that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, showed increased levels of Hh pathway component genes glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared to naïve cells. We also observed these findings in melanoma samples from patients. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with non-canonical Hh signaling, involving TGFβ/SMAD signaling. Knockdown of GLI1 and GLI2 restored the vemurafenib-resistant cells to sensitivity, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a 3D skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphtalmia transcription factor (MITF) upregulation. Our findings also demonstrated that GLI1/GLI2 modulation could be a useful strategy to prevent drug resistance. Alternating pre-treatment with vemurafenib and Gant61 significantly reduced IC50 of subsequent vemurafenib treatment in naïve melanoma cells, demonstrating a promising approach for the control of vemurafenib-resistance in patients with unresectable melanoma. The modulation of vemurafenib chemosensitivity was triggered by GLI1/GLI2 repression during the acquisition of resistance, which did not occur in the treatment protocols with only vemurafenib continuously or in alternate days. The alternating treatment regimen with vemurafenib and Gant61 was able to suppress GLI expression, delaying or decreasing vemurafenib resistance. Taken together, our data demonstrated an unprecedented mechanism of vemurafenib resistance by GLI1/GLI2 upregulation, shedding light on the development of Hh pathway inhibitors as a promising strategy for melanoma treatment.
Citation Format: Fernanda Faiao-Flores, Debora Kristina Magalhaes Alves-Fernandes, Paula Comune Pennacchi, Silvana Sandri, Anna Luiza Silva Almeida Vicente, Cristovam Scapulatempo-Neto, Vinicius de Lima Vazquez, Rui Manuel Reis, Keiran S. Smalley, Silvya Stuchi Maria-Engler. Targeting hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4669.