Targeting GLI1 and GLI2 with small molecule inhibitors to suppress GLI-dependent transcription and tumor growth.

Aberrant activation of Hedgehog (HH) signaling in cancer is the result of genetic alterations of upstream pathway components (canonical) or other oncogenic mechanisms (noncanonical), that ultimately concur to activate the zinc-finger transcription factors GLI1 and GLI2. Therefore, inhibition of GLI activity is a good therapeutic option to suppress both canonical and noncanonical activation of the HH pathway. However, only a few GLI inhibitors are available, and none of them have the profile required for clinical development due to poor metabolic stability and aqueous solubility, and high hydrophobicity. Two promising quinoline inhibitors of GLI were selected by virtual screening and subjected to hit-to-lead optimization, thus leading to the identification of the 4-methoxy-8-hydroxyquinoline derivative JC19. This molecule impaired GLI1 and GLI2 activities in several cellular models interfering with the binding of GLI1 and GLI2 to DNA. JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in several cancer cell lines in vitro. Specificity towards GLI1 and GLI2 was demonstrated by lower activity of JC19 in GLI1- or GLI2-depleted cancer cells. JC19 showed excellent metabolic stability and high passive permeability. Notably, JC19 inhibited GLI1-dependent melanoma xenograft growth in vivo, with no evidence of toxic effects in mice. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2.

Combined Inhibition of Smoothened and the DNA Damage Checkpoint WEE1 Exerts Antitumor Activity in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is characterized by resistance to chemotherapy and a poor prognosis. Therefore, treatments that can effectively suppress tumor growth are urgently needed. Aberrant activation of hedgehog (HH) signaling has been implicated in several cancers, including those of the hepatobiliary tract. However, the role of HH signaling in intrahepatic CCA (iCCA) has not been completely elucidated. In this study, we addressed the function of the main transducer Smoothened (SMO) and the transcription factors (TFs) GLI1 and GLI2 in iCCA. In addition, we evaluated the potential benefits of the combined inhibition of SMO and the DNA damage kinase WEE1. Transcriptomic analysis of 152 human iCCA samples showed increased expression of GLI1, GLI2, and Patched 1 (PTCH1) in tumor tissues compared with nontumor tissues. Genetic silencing of SMO, GLI1, and GLI2 inhibited the growth, survival, invasiveness, and self-renewal of iCCA cells. Pharmacologic inhibition of SMO reduced iCCA growth and viability in vitro, by inducing double-strand break DNA damage, leading to mitotic arrest and apoptotic cell death. Importantly, SMO inhibition resulted in the activation of the G2-M checkpoint and DNA damage kinase WEE1, increasing the vulnerability to WEE1 inhibition. Hence, the combination of MRT-92 with the WEE1 inhibitor AZD-1775 showed increased antitumor activity in vitro and in iCCA xenografts compared with single treatments. These data indicate that combined inhibition of SMO and WEE1 reduces tumor burden and may represent a strategy for the clinical development of novel therapeutic approaches in iCCA.

Emerging Roles of Hedgehog Signaling in Cancer Immunity.

Hedgehog-GLI (HH) signaling plays an essential role in embryogenesis and tissue homeostasis. Aberrant activation of the pathway through mutations or other mechanisms is involved in the development and progression of numerous types of cancer, including basal cell carcinoma, medulloblastoma, melanoma, breast, prostate, hepatocellular and pancreatic carcinomas. Activation of HH signaling sustains proliferation, suppresses cell death signals, enhances invasion and metastasis, deregulates cellular metabolism and promotes angiogenesis and tumor inflammation. Targeted inhibition of the HH pathway has therefore emerged as an attractive therapeutic strategy for the treatment of a wide range of cancers. Currently, the Smoothened (SMO) receptor and the downstream GLI transcriptional factors have been investigated for the development of targeted drugs. Recent studies have revealed that the HH signaling is also involved in tumor immune evasion and poor responses to cancer immunotherapy. Here we focus on the effects of HH signaling on the major cellular components of the adaptive and innate immune systems, and we present recent discoveries elucidating how the immunosuppressive function of the HH pathway is engaged by cancer cells to prevent immune surveillance. In addition, we discuss the future prospect of therapeutic options combining the HH pathway and immune checkpoint inhibitors.

Quinolines and Oxazino-quinoline Derivatives as Small Molecule GLI1 Inhibitors Identified by Virtual Screening.

A virtual screening approach based on a five-feature pharmacophoric model for negative modulators of GLI1 was applied to databases of commercially available compounds. The resulting quinoline derivatives showed significant ability to reduce the GLI1 protein level and were characterized by submicromolar antiproliferative activity toward human melanoma A375 and medulloblastoma DAOY cell lines. Decoration of the quinoline ring and chemical rigidification to an oxazino-quinoline scaffold allowed us to deduce SAR considerations for future ligand optimization.