Suppressing Aberrant Hedgehog Pathway and Overcoming Resistance to Smoothened Antagonists via Targeting Super-enhancer-driven Transcriptional Dependencies [ChIP-seq]

Aberrant activation of Hedgehog (Hh) signaling pathway plays important roles in both oncogenesis and targeted therapy of many cancers. The clinical application of FDA-approved Hh-targeted Smoothened inhibitor (SMOi) drugs is hindered due to the emergence of various primary or acquired drug resistance, indicating the need of novel anti-Hh therapies. Our previous studies demonstrate that epigenetic/transcriptional targeted therapies represent a promising direction for anti-Hh drug development. In this study, we identified CDK9 and CDK12, two transcription elongation regulators, as novel therapeutic targets for antagonizing the aberrant Hh pathway and overcoming SMOi resistance. CDK9 inhibition and CDK12 inhibition exhibited similarly potent anti-Hh activities when treating various SMOi responsive or resistant Hh-driven tumor models as previously reported BET inhibition or CDK7 inhibition. We also utilized SHH-subtype medulloblastoma (SHH-MB) as the representative Hh-driven cancer model to perform Super-enhancer (SE) analysis and elucidate the crucial roles of SE in Hh-driven oncogenesis and above-mentioned anti-Hh epigenetic/transcriptional targeted therapies. Furthermore, we identified IRS1, encoding a critical component and cytoplasmic adaptor protein of the IGF pathway, as an oncogenic Hh-driven SE target gene and effective therapeutic target of multiple Hh-driven tumor models, including the SMOi-resistant ones. Collectively, our study demonstrates that the SE-driven transcriptional dependencies represent promising therapeutic vulnerabilities for suppressing the aberrant Hh pathway and overcoming the SMOi resistance. As CDK9 inhibitor and IRS inhibitor drugs have already entered human clinical trials for cancer treatment, our study provides comprehensive preclinical support for expanding their trials to Hh-driven cancers in near future. Overall design: We utilize mouse SHH-MB models SmoWT-MB and SMB56 as representative Hh-driven tumor models to investigate the potential roles of SE in Hh-driven oncogenesis and epigenetic/transcriptional targeted anti-Hh therapy. To do so, we generated H3K27ac ChIP-seq data of SmoWT-MB and SMB56 to profile their active enhancer landscape. The samples were collected under two different conditions, in vitro cultured (SmoWT-1 and SMB56-1) or freshly separated from subcutaneous tumor tissues (SmoWT-2 and SMB56-2). Also, we performed similar SE profiling in two SHH-MB patient-derived xenograft (PDX) models SHH-MB_PDX1 and SHH-MB_PDX2. Meanwhile, we performed anti-HA ChIP-seq analysis of SmoWT-MB cells stably expressing an active form of HA-tagged human GLI2 without the N-terminal region (HA-GLI2-DeltaN) to investigate direct Hh target gene in mouse SHH-MB.