Profiling the Landscape of Drug Resistance Mutations in Neosubstrates to Molecular Glue Degraders

Abstract

Targeted protein degradation (TPD) holds immense promise for drug discovery but mechanisms of acquired resistance to degraders remain to be fully identified. Here we used CRISPR-suppressor scanning to identify mechanistic classes of drug resistance mutations to molecular glue degraders in GSPT1 and RBM39, neosubstrates targeted by E3 ligase substrate receptors cereblon and DCAF15, respectively. While many mutations directly alter the ternary complex heterodimerization surface, distal resistance sites were also identified. Several distal mutations in RBM39 led to modest decreases in degradation yet can enable cell survival, underscoring how small differences in degradation can lead to resistance. Integrative analysis of resistance sites across GSPT1 and RBM39 revealed varying levels of sequence conservation and mutational constraint that control the emergence of different resistance mechanisms, highlighting that many regions co-opted by TPD are inessential. Altogether, our study identifies common resistance mechanisms for molecular glue degraders and outlines a general approach to survey neosubstrate requirements necessary for effective degradation.

CRISPR‐Suppressor Scanning for Systematic Discovery of Drug‐Resistance Mutations

“…We typically define enriched hit sgRNAs as those with resistance scores >2 standard deviations above the mean of negative controls-these correspond to sgRNAs whose mutational outcomes confer resistance to the drug used in the screen. The example results in Figure 4C show a collection of hit sgRNAs targeting the RBM39 gene conferring resistance to indisulam, a molecular glue that binds to RBM39 and DCAF15 and induces degradation of RBM39 (Gosavi et al, 2022). A key point of validation for a CRISPR-suppressor scanning screen is the identification of known or expected resistance mechanisms.…”

“…As an example of this application, our lab has investigated differences between resistance profiles comparing indisulam to a related drug, E7820 (Fig. 4C, right) (Gosavi et al, 2022). Testifying to similarities between the two drugs, the results of the two screens are well correlated and most enriched hits are shared, including the aforementioned sgL266, sgL266/R267, and sgD151/A152 hits.…”

“…Cas9 predominately generates indel mutations, which are often sufficient for experiments in which the exact types and sizes of the induced mutations are not significant concerns. Such approaches using Cas9 mutagenesis have been highly successful at identifying in-frame indel resistance alleles (Donovan et al, 2017;Gosavi et al, 2022;Ipsaro et al, 2017;Neggers et al, 2018;Shi et al, 2015;Vinyard et al, 2019). However, there may be specific applications in which it is advantageous to use other CRISPR-based technologies.…”

“…Cas9 cleavage forms double-stranded breaks, whose subsequent repair by the error-prone non-homologous end-joining and microhomology-mediated end-joining pathways generates mutations upon which drug selection can act. Our laboratory and others have applied this approach to uncover mechanistic insights across a diverse number of protein targets and cell lines (Donovan et al, 2017;Gosavi et al, 2022;Ipsaro et al, 2017;Neggers et al, 2018;Vinyard et al, 2019), demonstrating the versatility of CRISPR-suppressor scanning as a general approach. Crucially, Cas9 mutagenesis produces a diverse spectrum of mutations, many of which are indel mutations.…”

“…Thus, by challenging this pool of mutated cells with drug treatment, it is possible to systematically identify mutations conferring resistance. This approach, termed CRISPR-suppressor scanning, has been demonstrated to uncover deep biological insights into drug mechanism of action as well as target protein function (Gosavi et al, 2022;Ipsaro et al, 2017;Vinyard et al, 2019). Notably, resistance mutations nominated by CRISPR-suppressor scanning can indeed exist far from the drug-binding pocket, even in interacting proteins that do not directly bind the drug (Kwok et al, 2022;Ngan et al, 2022).…”