The PROTAC selectively degrading Bcl-xL represents a novel Hedgehog pathway inhibitor with capacity of combating resistance to Smoothened inhibitors while sparing bone growth.

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Rationale: Primary and acquired resistance to Smoothened (Smo) inhibitors largely hampered their clinical efficacy. Given the important functions of hedgehog (Hh) pathway in bone formation and development, the permanent defects in bone growth caused by Smo inhibitors further restrict the use of Smo inhibitors for pediatric tumor patients. Anti-apoptotic Bcl-2 proteins regulate Hh activity by engaging a Bcl-2 homology (BH) domain sequence found in suppressor of fused (Sufu). In this study, we tested the effect of SIAIS361034, a Proteolysis Targeting Chimera (PROTAC) specifically targeting B-cell lymphoma extra large (Bcl-xL) to the celeblon (CRBN) E3 ligase for degradation, on combating the resistance and reducing the toxicity of bone growth caused by Hh inhibition. Methods: Fluorescence polarization, homogeneous time-resolved fluorescence (HTRF) assay, immunoblot, and immunoprecipitation (IP) were used to evaluate whether SIAIS361034 is an appropriate Bcl-xL PROTAC. Dual luciferase reporter assay, real-time quantitative PCR (RT-qPCR), depilatory model, and SmoA1 model were established to assess the effect of SIAIS361034 on the activity of Hh signaling pathway and its ability to overcome drug resistance in vitro and in vivo. Molecular mechanisms of SIAIS361034 for inhibiting Hh activity were demonstrated by dual luciferase reporter assay, immunoblot, and immunofluorescence staining. PET-CT and histopathology of bone tissues were used to assess the effects of SIAIS361034 on bone growth. Results: We observed that SIAIS361034 efficiently and selectively inhibits the activity of the Hh pathway in vitro and in vivo, by interrupting Bcl-xL/Sufu interaction, therefore, promoting the interaction of Sufu with Gli1. Moreover, SIAIS361034 possesses the ability of combating resistance to current Smo inhibitors caused by Smo mutations and Gli2 amplification and remarkably inhibits the growth of SmoA1 tumors in vivo. In contrast to von Hippel-Lindau (VHL) E3 ligase, our result further reveals little detectable expression of CRBN in two types of cells critical for bone development, human articular chondrocytes and human fetal osteoblastic cells. Moreover, treatment with SIAIS361034 results in no impairment on the bone growth of young mice, accompanying no alteration of the expression of Bcl-xL and Gli1 proteins. Conclusion: Our findings demonstrate that selectively targeting Bcl-xL by PROTAC is a promising strategy for combating resistance to Smo inhibitors without causing on-target drug toxicities of bone growth.