Interrogation of cancer gene dependencies reveals paralog interactions of autosome and sex chromosome-encoded genes.

Genetic networks are characterized by extensive buffering. During tumor evolution, disruption of functional redundancies can create de novo vulnerabilities that are specific to cancer cells. Here, we systematically search for cancer-relevant paralog interactions using CRISPR screens and publicly available loss-of-function datasets. Our analysis reveals >2,000 candidate dependencies, several of which we validate experimentally, including CSTF2-CSTF2T, DNAJC15-DNAJC19, FAM50A-FAM50B, and RPP25-RPP25L. We provide evidence that RPP25L can physically and functionally compensate for the absence of RPP25 as a member of the RNase P/MRP complexes in tRNA processing. Our analysis also reveals unexpected redundancies between sex chromosome genes. We show that chrX- and chrY-encoded paralogs, such as ZFX-ZFY, DDX3X-DDX3Y, and EIF1AX-EIF1AY, are functionally linked. Tumor cell lines from male patients with loss of chromosome Y become dependent on the chrX-encoded gene. We propose targeting of chrX-encoded paralogs as a general therapeutic strategy for human tumors that have lost the Y chromosome.

Iterative Design and Optimization of Initially Inactive Proteolysis Targeting Chimeras (PROTACs) Identify VZ185 as a Potent, Fast, and Selective von Hippel-Lindau (VHL) Based Dual Degrader Probe of BRD9 and BRD7.

Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a target protein within cells can be a lengthy and unpredictable process, and it remains unclear whether any combination of E3 and target might be productive for degradation. We describe a probe-quality degrader for a ligase-target pair deemed unsuitable: the von Hippel-Lindau (VHL) and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by systematically varying conjugation patterns and linkers and monitoring cellular degradation activities, kinetic profiles, and ubiquitination, as well as ternary complex formation thermodynamics. The emerged structure-activity relationships guided the discovery of VZ185, a potent, fast, and selective degrader of BRD9 and of its close homolog BRD7. Our findings qualify a new chemical tool for BRD7/9 knockdown and provide a roadmap for PROTAC development against seemingly incompatible target-ligase combinations.

Highly Selective PTK2 Proteolysis Targeting Chimeras to Probe Focal Adhesion Kinase Scaffolding Functions.

Focal adhesion tyrosine kinase (PTK2) is often overexpressed in human hepatocellular carcinoma (HCC), and several reports have linked PTK2 depletion and/or pharmacological inhibition to reduced tumorigenicity. However, the clinical relevance of targeting PTK2 still remains to be proven. Here, we present two highly selective and functional PTK2 proteolysis-targeting chimeras utilizing von Hippel-Lindau and cereblon ligands to hijack E3 ligases for PTK2 degradation. BI-3663 (cereblon-based) degrades PTK2 with a median DC50 of 30 nM to >80% across a panel of 11 HCC cell lines. Despite effective PTK2 degradation, these compounds did not phenocopy the reported antiproliferative effects of PTK2 depletion in any of the cell lines tested. By disclosing these compounds, we hope to provide valuable tools for the study of PTK2 degradation across different biological systems.

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis.

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.