Using stability-based proteomics, this study shows how protein folding stability-based profiling can expand the actionable target landscape of small molecules beyond canonical covalent interactions, uncovering noncovalent off-targets that may underlie response heterogeneity and treatment-associated toxicity.
Abstract
KRASG12C inhibitors have demonstrated meaningful clinical benefit in KRASG12C-mutant non-small cell lung cancer (NSCLC), yet responses remain heterogeneous and treatment-associated toxicities persist for reasons that are incompletely understood. Cysteine profiling indicates that these covalent inhibitors are highly selective for mutant KRAS; however, such approaches cannot detect noncovalent engagement of additional non-RAS proteins. Here, we used a protein-folding stability profiling technique, stability of proteins from rates of oxidation (SPROX), to identify protein targets of the clinical KRASG12C inhibitor, divarasib (GDC-6036), in KRAS-mutant NSCLC lysates. SPROX revealed a focused set of candidate interactors, including the essential splicing factor RBM39, which was reproducibly stabilized at both divarasib concentrations tested. We subsequently confirmed that divarasib directly and noncovalently binds to RBM39 protein. In NSCLC cells, divarasib increased RBM39 protein abundance and antagonized RBM39 degradation induced by the aryl-sulfonamide molecular glue indisulam through a post-transcriptional mechanism. Divarasib and RBM39 degraders reciprocally antagonized each other’s cytotoxicity, and RBM39 knockdown modestly reduced divarasib-induced cell death. Mechanistically, divarasib-mediated RBM39 stabilization regulated both INSR expression and alternative splicing, altered downstream insulin receptor signaling, and contributed to divarasib-associated cytotoxicity. Consistent with these findings, RBM39 and INSR expression were positively correlated across multiple human cancer types. Collectively, these findings identify RBM39 as a previously unrecognized noncovalent target of divarasib and uncover an RBM39–INSR signaling axis that modulates cellular responses to both divarasib and RBM39 degraders. Significance Covalent KRASG12C inhibitors are widely considered highly target-selective because adduct-based chemoproteomics approaches almost exclusively identify mutant KRAS as their target, detecting few proteins beyond it. However, these methods cannot capture noncovalent interactions and can sometimes miss covalent interactors. Using stability-based proteomics, we show that the clinical KRASG12C inhibitor divarasib targets RBM39, an essential splicing factor degraded by anticancer aryl-sulfonamide molecular glues. Divarasib opposes degrader-induced RBM39 loss, attenuates degrader cytotoxicity, and preserves RBM39-dependent regulation of the insulin receptor. These findings uncover a previously unrecognized non-KRAS axis of divarasib activity that is invisible to conventional target-deconvolution methods. They also suggest potential liability associated with combining RBM39 degraders with divarasib or other KRASG12C inhibitors that engage RBM39. More broadly, our study illustrates how protein folding stability-based profiling can expand the actionable target landscape of small molecules beyond canonical covalent interactions, uncovering noncovalent off-targets that may underlie response heterogeneity and treatment-associated toxicity. HIGHLIGHTS Protein folding stability profiling identifies RBM39 as a noncovalent target of divarasib. Divarasib stabilizes RBM39 and blocks its degradation by aryl-sulfonamide glues. Divarasib and RBM39 degraders reciprocally antagonize each other’s cytotoxicity. RBM39 supports insulin receptor expression and divarasib-induced cell death. eTOC BLURB Chen et al. apply protein folding stability-based proteomics to the covalent KRASG12C inhibitor divarasib and identify the splicing factor RBM39 as a noncovalent target of divarasib. Divarasib stabilizes RBM39, antagonizes aryl-sulfonamide degraders, and preserves RBM39-dependent insulin receptor signaling, revealing a non-KRAS axis invisible to adduct-based target deconvolution.
BACKGROUND AND PURPOSE
Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer and a leading cause of cancer-related mortality worldwide. Current targeted therapies are limited by acquired resistance, highlighting the urgent need for novel therapeutic agents and targets. The aim of this study is to investigate the anti-tumor efficacy of oxypeucedanin (OPD) against LUAD and elucidate its underlying molecular mechanism.
EXPERIMENTAL APPROACH
In vitro functional assays, bioinformatics prediction, molecular docking, and binding validation experiments were employed to identify the direct target of OPD. Ubiquitination assays, co-immunoprecipitation coupled with mass spectrometry, and proteomic analysis were used to delineate the downstream pathway. Genetic knockdown/ overexpression rescue experiments were performed, and in vivo efficacy was evaluated using patient-derived organoids and mouse xenograft models.
RESULTS
OPD potently suppressed LUAD cell proliferation and induced apoptosis both in vitro and in vivo. Mechanistically, OPD directly bound to the motor protein KIF11 and promoted its ubiquitin-proteasomal degradation via the E3 ligase TRIM21. KIF11 degradation led to destabilization of its interacting partner, MCM2, consequently downregulating the CDK1/ Cyclin B1 complex and ultimately triggering G2/M phase cell cycle arrest.
CONCLUSION AND IMPLICATIONS
OPD functions as a novel molecular degrader of KIF11, exerting its anti-LUAD effects by promoting KIF11 ubiquitination and subsequent proteasomal degradation. These results highlight OPD as a promising therapeutic strategy for LUAD.
KRAS is one of the most frequently mutated oncogenes in cancer, driving tumorigenesis and resistance to therapy. However, its smooth, shallow surface and lack of deep binding pockets have historically made it difficult to target with small molecules. Our lab identified Merodantoin (C1), a small molecule that selectively induces cell death in KRAS-mutant cancer cells by hyperactivating KRAS. This activation leads to stimulation of the mTORC2–AKT signalling pathway, elevated reactive oxygen species (ROS) levels, and subsequent cell death via mitochondrial fission and mitophagy. To further elucidate the upstream mechanisms underlying KRAS hyperactivation, we employed mass spectrometry- and Western blot-based Cellular Thermal Shift Assays (MS-CETSA and WB-CETSA). In KRAS G13D mutant cells, C1 treatment resulted in thermal stabilization of KRAS, its upstream adaptor GRB2, and the prenylation enzymes Farnesyltransferase-α/β, which are essential for KRAS membrane localization and function. Functional knockdown studies showed that GRB2 silencing partially rescued both C1-induced cytotoxicity and impaired spheroid formation, suggesting a contributory—but not exclusive—role in modulating the KRAS-driven response. In KRAS wild-type cells, C1 induced minimal cell death and exhibited stability in GRB2 but not KRAS. Autophagy-inducing treatments (Rapamycin, EBSS) stabilized KRAS in mutant cells but not GRB2, and neither protein in wild-type cells, indicating that KRAS stabilization is mutation-dependent, while GRB2 stabilization is specific to C1. These results suggest that C1-induced lethality involves coordinated stabilization of multiple upstream KRAS regulators, with GRB2 acting as one component that may amplify signalling in a mutant-specific context. Together, this reveals a novel mechanistic axis of synthetic lethality in KRAS-driven cancers.
Benedict J. Leong, Kartini Iskandar, Liang Ying Yu, Shazib Pervaiz. SELECTIVE CYTOTOXICITY IN KRAS-MUTANT CANCER CELLS THROUGH COORDINATED STABILIZATION OF KRAS AND ITS UPSTREAM REGULATORS USING SMALL MOLECULE DRUG [abstract]. In: Proceedings of Frontiers in Cancer Science 2025; 2025 Nov 5-7; Singapore. Philadelphia (PA): AACR; Cancer Res 2026;86(13_Suppl):Abstract nr P106.
Benedict J. Leong, K. Iskandar, Liang Ying Yu et al.· Cancer Research· 0 citations
Proper subcellular protein localization is fundamental for protein function, yet cancer cells often exploit this machinery for survival. While the nuclear import receptor KPNB1 (importin β1) is overexpressed in various cancers, its oncogenic mechanisms remain incompletely understood and its therapeutic targeting remains in infancy. Through screening of an FDA-approved compound library, we uncovered simeprevir (Sim) and lusutrombopag (Lus) as direct KPNB1 inhibitors that disrupt its import complex assembly. These compounds blocked KPNB1-mediated nuclear import of p65, thereby suppressing Bcl-2 transcription and activating mitochondrial apoptosis. Overexpression of KPNB1, p65, or Bcl-2, but not nuclear localization signal (NLS)-deleted p65, reversed the effect of Sim/Lus. Sim induced apoptosis in multiple cancer cell lines by downregulating Bcl-2 expression and suppressed A549 xenografts growth without observable toxicity. Analysis of The Cancer Genome Atlas (TCGA) datasets substantiated the clinical relevance of the KPNB1-p65-Bcl-2 axis. Our study elucidates a therapeutically targetable KPNB1-p65-Bcl-2 survival axis in multiple cancers and establishes a foundation for developing next-generation KPNB1 inhibitors.
Cong Li, Qiaohua Yan, Muhammad Muddasar Saeed et al.· Cell Chemical Biology· 0 citations
KRAS is a major oncogenic driver, but therapeutic targeting remains limited by restricted druggability, pathway reactivation, and resistance to allele-specific inhibitors. Targeted protein degradation (TPD) offers a complementary approach enabling catalytic, sustained suppression of KRAS signaling. We developed panKRAS degraders using a structure-guided strategy combining reversible KRAS binders with VHL-recruiting PROTACs. High-affinity binders derived from a fragment-first approach were translated into degraders through optimization of linker design and ternary complex formation. The degrader ACBI3 showed broad activity but limited efficacy against GTP-loaded (KRAS ON) mutants due to insufficient ternary complex stability. Biophysical and cooperativity-driven optimization led to ACBI4, a next-generation degrader forming highly stable ternary complexes independent of nucleotide state. ACBI4 enables potent pan-allelic degradation, including ON-biased mutants, establishing KRAS degradation as a viable therapeutic modality and positioning ACBI4 as a pioneering panKRAS ON/OFF chemical probe.
Andreas Antonius. Mantoulidis. Title of the talk: KRAS - Degrading the Undruggable [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr IA06.
A. Mantoulidis· Clinical Cancer Research· 0 citations
Mutant KRAS inhibition has revolutionized the treatment of lung adenocarcinoma. Unfortunately, responses to this form of targeted therapy are often of limited duration because of the development of resistance. Targeted protein degradation, including using PROTACs (PROteolysis-TArgeting Chimeras), presents an alternative approach to targeting oncogenic drivers in cancer. In this issue of Cancer Research, Martín and colleagues developed a dTAG-KRASG12V syngeneic mouse model that allows for the study of the effects of degrading the KRASG12V oncoprotein in vivo. The authors discovered that degrading the KRASG12V oncoprotein leads to regression of the resulting lung adenocarcinoma tumors. Most of the regression was based on cancer cell-intrinsic responses, although the tumor microenvironment also underwent substantial remodeling. Despite the initial efficacy of the treatment, the authors found that prolonged PROTAC KRAS degrader treatment eventually resulted in relapse. Resistance to PROTAC treatment seemed to be driven by dysregulation of the ubiquitin-proteasome system that is required for the activity of the PROTAC degraders. Despite developing resistance to the PROTAC degraders, the resulting tumors were still dependent on the KRAS oncoprotein, meaning that they were still sensitive to conventional KRAS inhibitors. Thus, PROTACs that degrade the KRAS oncoprotein are a promising modality for the treatment of lung adenocarcinomas. Resistance to PROTACs may differ from conventional KRAS inhibitors, suggesting potential strategies for overcoming such resistance. See related article by Martín et al., p. 4115.
S. Garcia Borrego, S. Misale· Cancer Research· 0 citations