Development of novel Hedgehog pathway inhibitors for cancer treatment

Hedgehog (HH) signaling is a conserved pathway that plays a pivotal role during embryonic development, tissue homeostasis and regeneration. Aberrant activation of the HH pathway has been reported to drive tumor progression in numerous cancers, including melanoma. The key signaling components of the HH signaling are the ligands, Sonic, Indian and Desert Hedgehog, the transmembrane receptor Patched 1 (PTCH1), the cell membrane receptor Smoothened (SMO) and the three transcription factors GLI1, GLI2 and GLI3, which upon activation of the HH signaling translocate into the nucleus and activate the transcription of specific target genes. In the last few years, HH pathway has emerged as a promising therapeutic target for the development of anti-cancer therapies. However, despite significant progress, current SMO antagonists show several limitations and few GLI inhibitors are available. In the first part of this study, we characterized the biological effect of three quinolines, as promising GLI inhibitors. In particular, the 4-methoxy-8- hydroxyquinoline derivative JC19 impaired GLI1 and GLI2 activities in several melanoma cell lines, interfering with the binding of GLI1 and GLI2 to DNA. Moreover, JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in vitro and in vivo. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2. In the second part, we studied the biological activity of a new class of Antibody drug-conjugated (ADC), structures composed by a monoclonal antibody, a cytotoxic payload and a linker-unit, that connects antibody and payload. We synthesized three novel ADCs made by a SMO inhibitor (Cyclopamine) and Cetuximab, a monoclonal antibody against EGFR, a receptor tyrosine kinase overexpressed in different types of cancer, including melanoma. In melanoma cell lines, ADCs showed a more robust reduction of melanoma cell viability and proliferation compared to single components (Cetuximab and SMOi). Moreover, all ADCs decreased protein expression of markers belonging to both HH and EGFR signaling cascades. Notably, the ADC B48 Bis showed a stronger antiproliferative effect compared to the coadministration of Cetuximab+SMOi, confirming its ability to effectively deliver the payload to melanoma cells. Moreover, B48 Bis suppressed cell proliferation by increasing apoptosis. Finally, we confirmed that B48 Bis is internalized in melanoma cell lines.