Patterns of substrate affinity, competition, and degradation kinetics underlie biological activity of thalidomide analogs

Abstract

Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.

Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

“…dTAG V -1 effectively combined with THAL-SNS-032 and co-treatment led to pronounced degradation of both LACZ-FKBP12 F36V and CDK9 at levels comparable to those with treatment of each degrader alone (Fig. 1e, lanes 8-12), avoiding potential substrate competition effects 28 . To confirm the utility of dTAG V -1 for target-specific validation studies, we evaluated KRAS G12V degradation, an oncogenic driver of PDAC, in PATU-8902 FKBP12 F36V -KRAS G12V ; KRAS −/− cells 15 .…”

Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

“…Pronounced degradation of LACZ-FKBP12 F36V and CDK9 was observed to levels comparable to those with treatment of each degrader alone, avoiding potential substrate competition effects. 22 To confirm the utility of dTAG V -1 for target validation, we evaluated KRAS G12V degradation, an oncogenic driver of PDAC, in PATU-8902 FKBP12 F36V -KRAS G12V ; KRAS -/- cells. 17 dTAG V -1 treatment led to rapid KRAS G12V degradation, which was rescued by use of dTAG V -1-NEG, pre-treatment with proteasome-inhibitor (carfilzomib) or Nedd8 activating enzyme inhibitor (MLN4924), and VHL knockout, consistent with the mechanism of action of a VHL-recruiting degrader (Fig.…”

Increased mitochondrial apoptotic priming with targeted therapy predicts clinical response to re‐induction chemotherapy

“…Finally, to determine whether LEN's effect was on target, we measured substrate protein levels before and after ex vivo and in vivo LEN‐based therapy. Significant relative decreases in IKZF1 (ex vivo, P = .024; in vivo, P = .003) and CSNK1a1 protein levels (ex vivo, P = .003; in vivo , P = .007) were observed in all cases, including responders and non‐responders, suggesting LEN‐induced priming is mediated by substrate degradation in the malignant cells (Figure S3A,B).…”

“…Frozen cell pellets were analyzed using immuno‐multiple reaction monitoring mass spectrometry as previously described …”