Most encouragingly, we observed robust TRIM21 molecular glue activity against pancreatic cancer xenografts and patient-derived PDAC organoid models. 14/18

How does blocking the nuclear pore trigger apoptosis? One factor is that cancer cells are addicted to a constant stream of short-lived pro-survival mRNAs. Starving the cell of key survival signals results in rapid cell death. 13/18

We then confirmed the crucial link. Using multiple assays (NanoBiT in cells, TR-FRET in vitro), we demonstrated that the compound induces physical proximity between TRIM21 and NUP98. 11/18

We went back to our original CRISPR KO screen and realized that a single guide (out of 4) against NUP98 conferred strong resistance to drug activity. This was missed at the gene summary level! The active guide uniquely targeted the NUP98 autoproteolytic domain (APD). 10/18

TRIM21 is a ubiquitin ligase that labels other proteins for destruction. To identify its substrates, we performed extensive proteomic profiling and proximity labeling which demonstrated degradation of nuclear pore complex proteins. 9/18

With TRIM21 as our likely molecular target, we performed a tiled base editing screen, which helped map the compound binding site to the PRYPSRY domain. Direct compound binding to recombinant TRIM21 PRYSPRY was confirmed via SPR and crystallography. 8/18

In a fantastic collaboration with Nathanael Gray's lab, we developed new derivatives that were >10-fold more potent, while still being highly selective and completely dependent on TRIM21 for their activity. 7/18

Next, we conducted genome-scale CRISPR KO and activation modifier screens yielding TRIM21 and IRF genes as the top functional mediators of drug response across the entire genome. 6/18

The compound was re-tested in full dose-response across ~900 cell lines, yielding TRIM21 mRNA expression as the top predictive biomarker. Activity was also enriched against pancreatic and head & neck cancers. 5/18

Our story begins with cell line viability profiling via the PRISM Drug Repurposing Project @CancerDepMap. We found that a former clinical drug candidate, an erastin derivative (PRLX-93936), showed highly selective anticancer activity. 4/18

In work led by Linjie Yuan, Wenzhi Ji, and Brendan Dwyer, we employed large-scale phenotypic profiling, genetic modifier screens, and proteomics to discover that a prior clinical-stage drug acts as a TRIM21 molecular glue to degrade the nuclear pore complex. 3/18

Excited to share our discovery of potent TRIM21 molecular glues with anticancer activity, online today @CD_AACR: "Defining the antitumor mechanism of action of a clinical-stage compound as a selective degrader of the nuclear pore complex". 1/18