(d) Scatter plots showing resistance scores ( y axis) in MOLM-13 under E7820 (left) or indisulam (right) treatment at four weeks. Protein domains and the structural degron site are demarcated by the colored panels. Data points represent mean values across three replicate treatments. When the sgRNA cut site falls between two amino acids, both amino acids are denoted. The GSPT1-targeting sgRNAs ( n = 239) are arrayed by amino acid position in the GSPT1 CDS on the x axis corresponding to the position of the predicted cut site. Resistance scores were calculated as the log 2(fold-change sgRNA enrichment under drug treatment) normalized to the mean of the negative control sgRNAs ( n = 22).
(c) Scatter plot showing resistance scores ( y axis) in MOLM-13 under CC-885 (left) or ZXH-1-161 (right) treatment at four weeks. (b) Schematic showing the CRISPR-suppressor scanning workflow applied to molecular glue degraders. (a) Chemical structures of degraders used in this study. CRISPR-suppressor scanning identifies regions of GSPT1 and RBM39 that mediate targeted protein degradation by molecular glue degraders. (10,12,13) New IMiD derivatives tailored to degrade novel neosubstrates, including GSPT1 and IKZF2, have entered clinical trials for oncology applications. (10) Structural studies on the CC-885-CRBN-GSPT1 (11,12) ternary complex were critical in determining a β-hairpin structural degron, a unifying motif across the diverse array of IMiD-targeted neosubstrates necessary for CRL4 CRBN-mediated degradation. (10,11) As a leading example, CC-885 ( Figure 1a), an analogue of lenalidomide, was shown to gain the ability to induce degradation of GSPT1 (also known as eRF3A), a translation termination factor essential for acute myeloid leukemia (AML) cell proliferation. (6−9) Mechanistic studies of IMiDs revealed that the selectivity of the CRBN-IMiD recognition surface and their targeted neosubstrates could be broadly modulated through even subtle chemical changes to the IMiD structure. Immunomodulatory drugs (IMiDs), including thalidomide and its analogues lenalidomide and pomalidomide, bind to cereblon (CRBN), a substrate receptor for the CUL4-RING (CRL4) E3 ubiquitin ligase, and induce its complexation with various neosubstrates that are subsequently degraded.
Altogether, our study identifies common resistance mechanisms for molecular glue degraders and outlines a general approach to survey neosubstrate requirements necessary for effective degradation. 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 nonessential. 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.
While many mutations directly alter the ternary complex heterodimerization surface, distal resistance sites were also identified. Here, we used clustered regularly interspaced short palindromic repeats (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. Targeted protein degradation (TPD) holds immense promise for drug discovery, but mechanisms of acquired resistance to degraders remain to be fully identified.
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