Targeting resistance to Smoothened antagonists by FACT inhibition

Resistance to clinically available targeted drugs has become a critical issue in hedgehog-driven cancer treatment. Our previous studies have demonstrated two epigenetic/transcriptional targeted therapeutic strategies, BET inhibition and CDK7 inhibition, could overcome both primary and acquired resistance to Smoothened inhibitor (SMOi) drugs, providing a promising direction for novel anti-hedgehog drug development. In this study, we performed CRISPR-Cas9 screening of epigenetic/transcriptional targeted sgRNA library in hedgehog-driven medulloblastoma (SHH-MB) cells and combined with tumor dataset analyses to identify other potential epigenetic/transcriptional targeted strategies for treating aberrant hedgehog pathway and overcoming SMOi-resistance. Our results demonstrated structure specific recognition protein 1 (SSRP1), a subunit of Facilitates Chromatin Transcription (FACT) complex, was a hedgehog-induced essential oncogene and therapeutic target of hedgehog-driven cancer. FACT inhibitor CBL0137, which has entered human clinical trials against cancer, could effectively suppress multiple mouse and human hedgehog-driven cancer models that are either SMOi-responsive or -resistant both in vitro and in vivo. Mechanistically, CBL0137 exerted its anti-hedgehog activity mainly through targeting the transcription of GLI1/2, which are core transcription factors of hedgehog pathway. ChIP-qPCR analyses further revealed SSRP1 could bind to the promoter regions of GLI1/2, while CBL0137 treatment substantially disrupted these interactions. Moreover, CBL0137 could work synergistically with BET inhibitor or CDK7 inhibitor on antagonizing aberrant hedgehog pathway and growth of either SMOi-responsive or -resistant hedgehog-driven cancer models. Taken together, our study identified FACT inhibition as another promising epigenetic/transcriptional targeted therapeutic strategy for treating hedgehog-driven cancer and overcoming SMOi-resistance. mRNA profiling:RNA was prepared from vehicle or CBL0137 (1μM) treated mouse SHH-subtype medulloblastoma cells (SmoWT) derived from spontaneous medulloblastoma of Ptch1+/-, Trp53-/- mice for 24 h. Trim galore was used to automatically detect and trim adapters. Reads were mapped to mm10 reference genome using Hisat2. Read count was generated using HTSeq (version 0.11.1). Differentially expressed genes (DEGs) were calculated using DESeq2 (version 1.26.0). Absolute value of fold change (FC) > 1.5 and false discovery rate (FDR) < 0.05 was used as cut-off value to select significant target genes. FPKM (Fragments per Kilobase Million) was calculated from the number of reads that mapped to each particular gene sequence and the gene length and the sequencing depth were taken into account.