br SMO antagonists pitfalls and limitations The first

SMO antagonists: pitfalls and limitations The first Hh inhibitor discovered was cyclopamine, a naturally occurring alkaloid isolated from Veratrum californicum[21], which inactivates SMO by directly binding to its hepathelical bundle. Nevertheless, cyclopamine has shown several limitations as a drug candidate, such as toxicity and teratogenicity, poor oral bioavailability, and suboptimal pharmacokinetics with relatively short elimination half-life [22]. In recent years, drug discovery efforts directed against the Hh pathway have been focused predominantly on the development of SMO antagonists and a remarkable number of small molecules of natural, semisynthetic or synthetic origin have been developed and extensively reviewed in recent reports 23, 24, 25. Several SMO antagonists have demonstrated efficacy in mouse xenografts and, most notably, have been investigated in clinical trials against a large range of metastatic and advanced cancers 26, 27. Among them, vismodegib (GDC-0449/Erivedge) became the first Hh inhibitor to receive approval from the USA FDA in January 2012 for the treatment of locally advanced or metastatic BCC [28]. Despite the initial enthusiasm, clinical development of SMO antagonists has ultimately proved disappointing, due to scarce pharmacokinetics, low selectivity on CSCs, severe side effects, and the emergence of drug resistance. Indeed, after an initial clinical response to treatment with vismodegib, a patient with metastatic MB showed tumor regrowth within 3 months due to D473H point mutation that rendered SMO insensitive to the drug [29]. Furthermore, acquired resistance has been also observed in BCC patients under treatment with vismodegib and in a Phase I study of saridegib (IPI-926). In this study, nine patients with BCC, previously progressed on vismodegib, failed to respond to saridegib, suggesting the existence of overlapping mechanisms of resistance [30]. Recently, genomic analysis of SMO resistance to vismodegib in BCC patients has revealed a number of additional SMO mutations and variants that confer constitutive activity and drug resistance 31, 32. All variants have shown partial or complete resistance to vismodegib, while the aPKC-ι/λ/GLI inhibitor PSI and the GLI2 antagonist arsenic trioxide (ATO) were both able to suppress Hh pathway activation in the presence of any SMO variants [31]. Several lines of evidence also suggest that cancer STF-118804 can acquire resistance to SMO antagonists via SMO-independent hyperactivation of the powerful downstream GLI transcription factors, or mutations at different nodal points of the Hh pathway. Indeed, preclinical and clinical trials have shown that SMO drug resistance can be the consequence of (i) GLI2 amplification during vismodegib or sonidegib (LDE-225) treatment; (ii) upregulation of noncanonical and synergistic GLI signaling [e.g., phosphoinositide 3-kinase (PI3K) pathway, observed during sonidegib treatment]; and (iii) increase of the expression of ATP-binding cassette transporters (ABCs), such as P-glycoprotein (Pgp), which diminishes drug efficacy by increasing its cellular clearance 29, 33, 34, 35, 36. Moreover, the onset and progression of some types of Hh-driven cancers are related to Hh-pathway-activating mutations downstream of SMO, such as loss of SUFU or GLI1 amplification, thus rendering SMO antagonists ineffective in these scenarios. Not least, studies investigating systemic treatments with SMO antagonists have revealed several side effects including dysgeusia, alopecia, fatigue, nausea, diarrhea, decreased appetite, hyponatremia, weight loss, and especially muscle cramping due to noncanonical SMO signaling (SMO–AMP-activated protein kinase axis) and Ca2+ influx 37, 38.
GLI factors: new attractive targets in Hh-dependent tumors GLI transcription factors are the final effectors of the Hh pathway and share common structural features, such as five highly conserved tandem zinc fingers (ZFs), a fairly conserved N-terminal domain, several potential protein kinase A (PKA) binding sites, and additional conserved regions at the C terminus. Nevertheless, GLI proteins exert different functions in vivo: GLI1 acts only as a transcriptional activator, whereas GLI2 and GLI3 can act both as transcriptional activators and as repressors, depending on the specific cell context and on the activation state of Hh signaling. In absence of upstream Hh signaling, some protein kinases [PKA, glycogen synthase kinase (GSK)3β and casein kinase (CK)1] phosphorylate GLI proteins, leading to ubiquitylation/proteosome-dependent GLI1 degradation or GLI2 and GLI3 proteolytic cleavage into repressor forms (GLI2R and GLI3R). These events are mediated by Cullin1/beta-transducin repeats-containing proteins (β-TrCP) E3 ubiquitin ligase complex 1, 39. In contrast, the activation of Hh signaling inhibits this processing, resulting in full-length GLI2 and GLI3, which have activator function (GLI3A and GLI2A) [38]. The balance between activator and repressor functions of GLI transcription factors determines the status of the Hh transcriptional program and consequently the behavior of responding cells.