Mutations Affecting Smoothened Drive Resistance to Hedgehog Pathway Inhibitors in Treating Advanced Basal Cell Carcinoma
Introduction. Basal cell carcinoma (BCC) is the most frequently diagnosed cancer in the world.1 BCC’s arise from constant activation of the Hedgehog pathway (HH) in hair follicles and intrafollicular epidermis.2 This drives oncogenesis not only in basal cell carcinoma, but also in medulloblastoma and cancers of the pancreas, colon, lung, breast, prostate, and blood.3 Most BCC’s are surgically treated, but this is not effective for advanced BCC’s.4 Oral HH inhibitors are being investigated for treatment of advanced BCC’s.1 These inactivate signaling by inhibiting Smoothened (SMO), a Frizzled G protein-coupled receptor that is a component of the HH pathway and is conserved from flies to humans. However, a significant problem with this treatment is drug resistance.1 Methods. RNA sequencing and transcriptional analysis of resistant BCC biopsies were performed.3,5 GLI1 levels were quantified using luciferase assays.3 Binding energetics were measured through molecular dynamic simulations6 and GBSA binding energy calculations.7 3D homology modeling of SMO was constructed to assess pyridine ring substitutions of the drug.7 DYRK1B inhibition was studied to assess an alternative HH inactivation method.8 Results. GLI1 expression and activity were higher in resistant BCC’s, indicating primary drug resistance through restoration of HH signaling.3 This is achieved through heterozygous single nucleotide mutations in the ligand binding pocket (LBP) residues of SMO.5 Allele fractions indicated that mutant tumor cells gain a growth advantage and are selected for survival after exposure to the drug.3 IC50 values of the mutant complexes were significantly higher than that of wildtype.5 Mutated residues decrease the strength of interactions between the drug and LBP.6 Binding energy is significantly increased in mutant complexes, while strength and number of bonds are decreased.7 These interactions change the physical orientation of the drug in the binding site based on elevated RMSF values.7 Two categories of mutations exist: those directly inside the LBP and those allosterically affecting the LBP.7 Both types shift the pyridine ring of the drug. Specific mutations were identified that confer partial vs. complete drug resistance.3 DYRK1B inhibition was effective in both SMO-dependent and SMO-independent signaling.8 Conclusion. Genetic screening of patients may help predict response to oral HH inhibitors. Higher drug dosage may overcome partial resistance, while complete resistance or de novo resistance suggest pursuing treatment targeting outside of the SMO LBP. Pre-existing mutations may serve as biomarkers of prognosis in oral HH inhibitor therapy. Future research may substantiate insight into alternative or combination treatments for BCC and many other cancers.
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