Discovery of a Novel Mechanism of Resistance to Thalidomide Derivatives

Pharmacologic agents that induce protein degradation represent an emerging and important class of therapeutic agents. Thalidomide and thalidomide derivatives (TDs) function by bringing target proteins and the E3 ubiquitin ligase substrate adaptor CRBN into proximity leading to ubiquitination and proteasomal degradation of critical oncoproteins. While highly effective, most patients eventually develop resistance to these agents, though the mechanisms by which this occurs and why some patients respond to different drugs within the class remains largely unknown. One major impediment to monitoring these drugs' activity is the need to quantitatively measure multiple protein targets simultaneously. To overcome this issue, we have developed a novel targeted mass spectrometry (MS) assay to measure the levels of CRBN, 8 substrate proteins that are degraded in the presence of TDs (IKZF1, IKZF3, CK1α, ZFP91, RNF166, ZNF692, GSPT1 and GSPT2), and 2 housekeeping control proteins (β-actin and GAPDH). This assay exhibits several advantages over currently used methodologies in that it is quantitatively precise and multiplexed for high throughput, allowing for sensitive detection of a panel of proteins from a single sample.

The precision of quantitation of our MS assay enabled us to investigate the relationship between substrate level and drug-mediated degradation by measuring all known substrates in dense time course experiments following treatment of MM1S cells with lenalidomide. These experiments revealed that substrates are degraded in an ordered fashion with IKZF1 and IKZF3 being degraded before CK1α and ZFP91, suggesting that substrates may compete for access to CRBN. To test this directly we overexpressed either RNF166 or ZNF692, two recently identified substrates, in MM1S cells, which led to lenalidomide resistance. This resistance was not mediated by any direct cellular dependence on these proteins but instead was due to their competition for binding to CRBN leading to diminished degradation of other essential lenalidomide targets, IKZF1, IKZF3, and CK1α. Thus, increased expression of proteins capable of interacting with CRBN represents a novel potential mechanism of resistance to this class of drugs.

CRBN is essential for lenalidomide-mediated degradation of substrates and sensitivity to drug. Quantitative detection of CRBN in our MS assay enabled us to study the effect of changes in CRBN levels on the activity of TDs. Loss of CRBN led to decreased degradation of all substrates and resistance, whereas overexpression of CRBN augmented degradation of all substrates leading to increased sensitivity to lenalidomide in MM1S cells. In a panel of ten MM cell lines CRBN level correlated with increased substrate degradation and was a significant determinant of differential sensitivity to lenalidomide. These findings demonstrate that CRBN is a limiting factor for both degradation of substrates and sensitivity to TDs.

Finally, we took advantage of the high throughput nature of our MS assay and generated dense dose response and kinetic curves for 3 additional TDs (pomalidomide, avadomide and CC-885) in MM1S cells. By combining this with in vivo binding data we have created a detailed map of substrate binding, degradation and order of interaction for each TD. Among other findings, these data demonstrate that lenalidomide differs from pomalidomide and avadomide in that it is a superior degrader of CK1α but inferior at degrading IKZF3 and ZFP91, features that likely underlie some of the differential clinical activity and disease specificity of these agents.

In sum, we have developed a highly sensitive, multiplexed quantitative MS assay allowing us to characterize the biochemical activity of TDs in unprecedented detail. Using this assay we show that substrates compete for access to a limiting pool of CRBN and identify a novel mechanism of resistance. The modularity of our assay allows for the detection of new substrates as they are discovered or reconfiguration of the assay to study other degraders. Future translation of this assay to clinical samples will facilitate accurate monitoring of pharmacodynamic activity and elucidation of mechanisms of resistance in vivo.