Targeting Super-Enhancer-Driven Transcriptional Dependencies Suppresses Aberrant Hedgehog Pathway Activation and Overcomes Smoothened Inhibitor Resistance.

Aberrant activation of the Hedgehog (Hh) signaling pathway plays important roles in oncogenesis and therapeutic resistance in several types of cancer. The clinical application of FDA-approved Hh-targeted Smoothened inhibitors (SMOi) is hindered by the emergence of primary or acquired drug resistance. Epigenetic and transcriptional targeted therapies represent a promising direction for developing improved anti-Hh therapies. In this study, we integrated epigenetic/transcriptional-targeted small-molecule library screening with CRISPR/Cas9 knockout library screening and identified CDK9 and CDK12, two transcription elongation regulators, as therapeutic targets for antagonizing aberrant Hh activation and overcoming SMOi resistance. Inhibition of CDK9 or CDK12 potently suppressed Hh signaling and tumor growth in various SMOi responsive or resistant Hh-driven tumor models. Systemic epigenomic profiling elucidated the Hh-driven super-enhancer (SE) landscape and 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 in Hh-driven tumor models. Collectively, this study identifies SE-driven transcriptional dependencies that represent promising therapeutic vulnerabilities for suppressing the Hh pathway and overcoming SMOi resistance. As CDK9 and IRS inhibitors have already entered human clinical trials for cancer treatment, these findings provide comprehensive preclinical support for developing trials for Hh-driven cancers.

Therapeutic targeting the oncogenic driver EWSR1::FLI1 in Ewing sarcoma through inhibition of the FACT complex.

EWS/ETS fusion transcription factors, most commonly EWSR1::FLI1, drives initiation and progression of Ewing sarcoma (EwS). Even though direct targeting EWSR1::FLI1 is a formidable challenge, epigenetic/transcriptional modulators have been proved to be promising therapeutic targets for indirectly disrupting its expression and/or function. Here, we identified structure-specific recognition protein 1 (SSRP1), a subunit of the Facilitates Chromatin Transcription (FACT) complex, to be an essential tumor-dependent gene directly induced by EWSR1::FLI1 in EwS. The FACT-targeted drug CBL0137 exhibits potent therapeutic efficacy against multiple EwS preclinical models both in vitro and in vivo. Mechanistically, SSRP1 and EWSR1::FLI1 form oncogenic positive feedback loop via mutual transcriptional regulation and activation, and cooperatively promote cell cycle/DNA replication process and IGF1R-PI3K-AKT-mTOR pathway to drive EwS oncogenesis. The FACT inhibitor drug CBL0137 effectively targets the EWSR1::FLI1-FACT circuit, resulting in transcriptional disruption of EWSR1::FLI1, SSRP1 and their downstream effector oncogenic signatures. Our study illustrates a crucial role of the FACT complex in facilitating the expression and function of EWSR1::FLI1 and demonstrates FACT inhibition as a novel and effective epigenetic/transcriptional-targeted therapeutic strategy against EwS, providing preclinical support for adding EwS to CBL0137's future clinical trials.