Targeting oncoproteins with a positive selection assay for protein degraders

Abstract

Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal (“down”) assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal (“up”) assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9–based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.

Unifying Catalysis Framework to Dissect Proteasomal Degradation Paradigms

“…For example, three independent studies identified distinct chemical scaffolds that stabilize DDB1-CDK12 interaction and thus lead to the enhanced degradation of Cyclin K, a CDK12 interactor. − These findings represent a novel mechanism by which a molecular glue can induce POI degradation by a distant PPI rather than directly reprogramming the POI-E3 interface. Phenotypic screens that capitalize on degradation nodes, such as hyponeddylated cells, or exploit cancer vulnerabilities − are elegant strategies to discover and enhance the druggability of novel degrader biology in relevant cellular contexts. Notably, Koduri et al devised a creative screening strategy for novel IKZF1 degraders, which led to the identification of Spautin-1, a novel molecular glue that does not require CRBN, and subsequently deployed this strategy to uncover CDK2’s role in regulating the abundance of the oncogenic transcription factor ASCL1 pertinent to small cell lung cancers .…”

“…Phenotypic screens that capitalize on degradation nodes, such as hyponeddylated cells, or exploit cancer vulnerabilities − are elegant strategies to discover and enhance the druggability of novel degrader biology in relevant cellular contexts. Notably, Koduri et al devised a creative screening strategy for novel IKZF1 degraders, which led to the identification of Spautin-1, a novel molecular glue that does not require CRBN, and subsequently deployed this strategy to uncover CDK2’s role in regulating the abundance of the oncogenic transcription factor ASCL1 pertinent to small cell lung cancers . Additionally, genetic screens have also provided early insights into potential resistance mechanisms emerging from multiple proteasomal degrader modalities. ,, This collection of studies has embraced the tractability of genetic screens and translatability of phenotypic approaches to intentionally identify novel molecular glues and deconvolute underlying mechanisms of proteasomal degradation.…”

PROTAC targeted protein degraders: the past is prologue

“…The first rational discovery of molecular glues between a ligase and a substrate involved a series of compounds that restored binding affinity between the β-TRCP ligase and its mutated phospho-degron, discovered in a biochemical screen 234 . More recently, targeted discovery of degrader compounds against oncoproteins in a cell-based system has enabled the discovery of novel cereblon (CRBN)-dependent and CRBN-independent IKAROS family zinc finger 1 (IKZF1) degraders 235 , suggesting that screening for degraders against a target of choice is a strategy that can yield a bounty of phenotypically relevant degrader molecules. Lastly, multiple recent papers have described agnostic screening approaches for degrader molecules 84 – 86 (as described in more detail in the main text) that were also highlighted by a comprehensive review, which offers guidelines for discovering E3 ligase modulators by phenotypic screening strategies 76 .…”

High-Throughput CRISPR Screening in Hematological Neoplasms

“…Other algorithms such as the Bayesian Analysis of Gene EssentiaLity algorithm (BAGEL) [ 126 ] and STARS [ 28 ], which are based in supervised learning and gene ranking methods, respectively, have been also used in CRISPR screenings of hematological neoplasms ( Table 2 ). BAGEL has been only applied in leukemia screenings [ 128 , 129 ] whereas STARS has been used with leukemia [ 125 ], myeloma [ 56 ] and lymphoma [ 63 ] models.…”