EGFR inhibitor-resistant lung cancers exhibit collateral sensitivity to a covalent, cysteine-independent KEAP1 oligomerizing molecular bridge

Domain: chemistry
Published: 2026-01-14
Editorial outcome: major_revision

Abstract
Targeted therapies have revolutionized cancer care. Unfortunately, most patients develop refractory, multifocal resistance to these therapies within a matter of months. Here, we demonstrate that the evolution of resistance to EGFR inhibitors in EGFR-mutant non-small cell lung cancer endows cells with hypersensitivity to a PAINS-like small molecule, MCB-613. Systematic proteomic, functional genomic, and biochemical studies revealed that MCB-613 binds KEAP1 in a covalent, cysteine-independent fashion, acting as a divalent molecular bridge that relies upon lysine residues in the KEAP1 dimerization domain to join monomers of KEAP1 together. Oligomerization of KEAP1 by MCB-613 sets into motion a fatal cascade of KEAP1 dysfunction, ROS accumulation, and ATF4/CHOP-dependent cell death. Together, these findings demonstrate that diverse models of EGFR inhibitor-resistant NSCLC share the common feature of elevated integrated stress response activity, and that a covalent molecular bridge which activates non-canonical KEAP1-ATF4 signaling can exploit this feature to select against resistance evolution.

Introduction
Although the use of targeted therapies to treat cancer has resulted in encouraging clinical responses, these benefits are, unfortunately, often short-lived. In the case of non-small cell lung cancers (NSCLC) driven by activating mutations in the epidermal growth factor receptor (EGFR), for instance, most patients will develop resistance to first-line targeted therapy within 24 months1,2. This problem is complicated by two interrelated considerations: (1) EGFR-mutant NSCLC cells can develop resistance to EGFR inhibitors through a wide variety of distinct mechanisms; and, (2) these mechanisms can coevolve simultaneously in the same patient or tumor3. This presents clinicians and researchers with a difficult quandary: on the one hand, treatments which use one drug to target an individual resistance mechanism are insufficient and unlikely to prove curative; on the other hand, treatments which use multiple drugs to target many different mechanisms at the same time are demanding, and in many cases infeasible. Thus, new approaches to the problem of “multifocal” drug resistance are needed.

One strategy is to identify, target, and exploit vulnerabilities which emerge as a consequence of drug resistance itself. These acquired, or “collateral,” sensitivities—which are distinct from chemical synthetic lethalities in that they persist even after removal of the original, selecting drug—have long been documented in the microbial literature4. Recently, for instance, it was shown that clinical isolates taken from the lungs of cystic fibrosis patients suffering from chronic, drug-resistant Pseudomonas aeruginosa infections consistently harbored mutations in the nfxB gene5. These mutations, though pathoadaptive in the setting of conventional anti-pseudomonal therapy with fluoroquinolones, nevertheless render bacterial populations collaterally sensitive to the commonly used and readily available aminoglycoside antibiotic amikacin. In point of fact, drug resistance often promotes targetable collateral sensitivities to commonplace, existing drugs. In the case of the recently approved fluorocycline antibiotic eravacycline, for example, drug resistance in Klebsiella pneumoniae promotes sensitivity to common used antibiotics like aztreonam and ceftazidime6.

In recent years, this concept has increasingly been applied to the study of drug resistance in cancer as well. It has been demonstrated, for example, that the acquisition of BRAF inhibitor resistance in BRAF-mutant melanoma engenders an upregulation of reactive oxygen species (ROS) which in some cells promotes a stable and clinically relevant collateral sensitivity to histone deacetylase (HDAC) inhibitors7. Separately, previous work has shown that diverse pathways of drug resistance in this setting also converge upon the activation of the c-MYC transcription factor; this, in turn, gives rise to shared, actionable collateral sensitivities to drugs which target MYC synthetic lethal partners such as certain tyrosine kinase families and metabolic pathways8. Since at least some of these targetable collateral sensitivities to cancer drug resistance are both stable and predictable, moreover, it is conceivable that future first-line therapies could be designed specifically to guide tumor evolution toward targetable, collaterally sensitive states. Indeed, previous work has shown that BET bromodomain inhibitors can be used to guide acute myeloid leukemia (AML) cells into an “evolutionary trap” which collaterally sensitizes them to existing, clinically approved BCL2 inhibitors9. Finally, these effects are manifold: with examples cited in a growing number of cases, it appears increasingly likely that numerous such collateral sensitivities may in fact be hidden throughout the landscape of drug-resistant cancers10–14. Indeed, the identification of such a collateral sensitivity, were it to be shared across clones driven by distinct mechanisms of resistance to the same selecting agent, would represent an opportunity to design therapies with the unique potential to select against resistance evolution15.

Here, we report on the discovery of a collateral sensitivity shared across multiple, distinct models of EGFR inhibitor resistance in EGFR-mutant non-small cell lung cancer. Specifically, we show that the acquisition of EGFR inhibitor resistance sensitizes EGFR-mutant NSCLC cells to a small molecule with PAINS-like features known as MCB-613, an effect that relies upon a unique, cysteine-independent mode of interaction with Kelch-like ECH associated protein 1 (KEAP1). More precisely, MCB-613 acts as a molecular bridge: through two distinct and symmetrically situated unsaturated sites, this electrophilic small molecule relies upon a lysine residue (lysine 97) situated within the protein dimerization domain and covalently tethers together monomers of KEAP1 to induce the formation of dysfunctional oligomers. Unlike typical, non-covalent dimers of KEAP1, these dysfunctional complexes then dissociate from the canonical repression substrate nuclear factor erythroid 2-related factor 2 (NRF2); this effect, however, is not required for MCB-613-induced cell death. Instead, MCB-613 selectively targets drug-resistant, EGFR-mutant NSCLC cells by taking advantage of their elevated integrated stress response (ISR) activity, promoting the accumulation of ROS and downstream ATF4- and CHOP-dependent cell death.

Results
A pharmacologic screen to identify shared collateral sensitivities in EGFR-mutant, EGFR inhibitor-resistant NSCLC
To identify targetable collateral sensitivities shared across clones bearing distinct mechanisms of EGFR inhibitor resistance, we compiled a panel of four well-characterized EGFR-mutant non-small cell lung cancer cell lines with varying degrees of resistance to EGFR inhibition: PC9, PFR3, GR4, and WZR12. The “parental” PC9 cell line is drug-naïve and retains exquisite sensitivity to EGFR inhibition. The previously characterized PFR3, GR4, and WZR12 cell lines—which are all derived from PC9s and driven by diverse, clinically relevant resistance mechanisms including IGF1R activation, EGFR T790M secondary site mutation, and downstream MAPK1 amplification, respectively—display increasing degrees of resistance to the first-generation EGFR inhibitor gefinitib (Fig. 1a)16–18. We screened all four of these cell lines against a library of 2100 small molecules in a three-day viability assay at two separate drug doses (Fig. 1b, Supplementary Data 1 and Supplementary Table 1). As expected, we detected numerous collateral resistances and sensitivities in EGFR inhibitor-resistant cells (Fig. 1c). Predictably, receptor tyrosine kinase inhibitors, including numerous other EGFR inhibitors (gefitinib, erlotinib, and afatinib, among others), scored as shared collateral resistances, providing an important internal positive control. Of the collateral sensitivities, however, one in particular stood out across all three drug-resistant derivatives: a relatively understudied, PAINS-like molecule called MCB-613 (Fig. 1d). Indeed, a follow-up, 3-day validation assay confirmed that the drug-resistant PFR3, GR4, and WZR12 derivatives were collaterally sensitive to treatment with MCB-613 (Fig. 1e).

We next sought to assess the breadth of this shared collateral sensitivity to EGFR inhibitor resistance. First, we gathered together two drug-naïve, EGFR-mutant NSCLC cell lines—PC9 and HCC827—which are known to develop EGFR inhibitor resistance through diverse mechanisms19–22. We used increasing doses of gefitinib to cultivate EGFR inhibitor resistance in these cell lines before selecting individual clones and querying them for sensitivity to MCB-613 (Supplementary Fig. 1a, b). In all clones tested, resistance to EGFR inhibition was associated with collateral sensitivity to MCB-613 (Fig. 1f, g). Finally, to assess whether this finding was limited to gefitinib or extended to other clinically relevant EGFR inhibitors, we evolved MGH134, a previously described, patient-derived cell line model of EGFR-mutant NSCLC, to resistance against the first-line, third-generation small molecule inhibitor osimertinib; in accordance with the above findings, resistance to this standard-of-care targeted therapy also conferred increased sensitivity to MCB-613 (Fig. 1h, Supplementary Fig. 1c)23.

Lastly, to evaluate whether the broad, shared collateral sensitivity of EGFR-mutant, EGFR inhibitor-resistant NSCLC cells to MCB-613 represents a viable preclinical strategy for overcoming multifocal drug resistance, we administered MCB-613 to tumor bearing mice. Despite the suboptimal solubility and pharmacokinetic properties of some molecules belonging to the chalcone class, we observed significant growth inhibition by MCB-613 in gefitinib-resistant GR4 tumors but not gefitinib-sensitive, parental PC9 tumors (Fig. 1i)24–26. Given the strong correlation between resistance to EGFR inhibition and sensitivity to MCB-613 observed above, we hypothesized that the combination of these two therapeutic approaches in a single EGFR-mutant NSCLC population would suffice to successfully suppress all cellular outgrowth. To test this hypothesis, we mixed drug-naïve and drug-resistant PC9 cells together and then exposed this heterogeneous population to either gefitinib alone, MCB-613 alone, or a combination of the two. Although neither molecule on its own suppressed outgrowth, the combination of the two was enough to completely eradicate the population (Fig. 1j). When considered together, these findings establish MCB-613 as a bonafide collateral sensitivity shared across diverse models of EGFR inhibitor resistance.

Proteomic and functional genomics screens reveal that MCB-613 targets KEAP1
MCB-613 (4-ethyl-2,6-bis-pyridin-3-yl-methylene cyclohexanone) is a reactive, PAINS-like electrophile belonging to the bis-chalcone family and boasting multiple previously described protein targets, including steroid receptor coactivator 3 (SRC3) and ubiquitin-specific peptidase 15 (USP15), which controls selective depletion of mutant p53 (Fig. 2a)27,28. In our hands, however, the depletion of these previously described targets failed to rescue the selective cell death effect of MCB-613, suggesting that they may not be relevant in the setting of drug-resistant EGFR-mutant NSCLC (Supplementary Fig. 2a–f). Acknowledging that MCB-613 is not drug-like, that it has structural elements that resemble those found in common pan-assay interference compounds (PAINS), and that the mechanism through which MCB-613 drives cell death in EGFR inhibitor-resistant cells could involve multiple functional targets or general electrophilic stress, we nevertheless set out to determine whether this agent may function through a previously unidentified defined target and mechanism-of-action. To do this, we devised a comprehensive strategy leveraging synthetic chemistry, proteomics, and functional genomics.

First, we developed an approach for identifying all protein interactors of MCB-613 in parental and EGFR inhibitor-resistant cells. Using MCB-613 and C151-0598, a molecule bearing close structural similarity but showing severely attenuated activity, we designed a “click chemistry,” azide-alkyne cycloaddition (CuAAC) approach to differential affinity purification and target identification (Fig. 2b and Supplementary Fig. 2g). We first generated “clickable” derivatives of MCB-613 and C151-0598 by replacing the ethylene units at the 4-positions of their central cyclohexanone moieties with propargyl ethers (Fig. 2c, d, Supplementary Note). We called these active and inactive clickable derivatives “ARM-3-124” and “ARM-3-115,” respectively. Next, we verified that the derivatives phenocopied their parent compounds in EGFR inhibitor-sensitive and -resistant EGFR-mutant NSCLC cells (Fig. 2e). Finally, we deployed them for target identification. Specifically, we exposed parental and drug-resistant cells to either compound at a dose (2 μM) which only showed activity in the ARM-3-124/WZR12 arm before collection, lysis, and copper-catalyzed azide-alkyne cycloaddition to install a biotin handle. Samples were analyzed by mass spectrometry to identify bound peptides. Across all conditions, this approach identified >1000 unique peptides corresponding to 610 protein interactors. After applying various filters, the list was further refined to a total of 478 putative interactors (Supplementary Data 2).

Next, we sought to evaluate the functional contributions of each of the 478 protein interactors to the collateral sensitivity of EGFR inhibitor-resistant NSCLC cells to MCB-613. Thus, we designed a targeted, loss-of-function CRISPR/Cas9 library containing single-guide RNA (sgRNA) sequences against each of the 478 interactors and screened it in parental and drug-resistant cell lines. We reasoned that the target of MCB-613 may be found among the genes which, when lost, conferred a greater growth disadvantage upon resistant derivatives than upon parental cells. Of all such genes, it was loss of KEAP1, encoding the E3 ubiquitin ligase substrate adaptor Kelch-like ECH associated protein 1 (KEAP1), that most closely phenocopied the effects of MCB-613 and scored as the greatest collateral sensitivity in the screen (Fig. 2f). (We were reassured by this, as it is consistent with the fact that MCB-613—like other α, β-unsaturated electrophiles—interacts widely with members of the ubiquitin-proteasome system (UPS) (Supplementary Fig. 2h, Supplementary Data 2)29.) Indeed, follow-up validation in parental PC9 and gefitinib-resistant PFR3, GR4, and WZR12 cells confirmed that KEAP1 is modestly differentially essential in the setting of EGFR inhibitor resistance (Fig. 2g and Supplementary Fig. 2i), albeit less so than MCB-613 treatment. We took this as evidence that KEAP1 is the protein interactor responsible for the selective cell death effect of MCB-613 in the setting of EGFR inhibitor resistance in NSCLC. Furthermore, we reasoned that, if MCB-613 works through KEAP1 inhibition, then KEAP1 knockout from a bulk population of resistant derivatives should eradicate all cells sensitive to KEAP1 loss and leave behind a reservoir of cells that no longer demonstrate sensitivity to this molecule. Indeed, genetic knockout of KEAP1 rescued the effect of MCB-613 across each of the resistant derivatives tested (Fig. 2h and Supplementary Fig. 2i). Taken together, these results suggest that the interaction between MCB-613 and KEAP1 is required for this agent’s selective effect in EGFR inhibitor-resistant NSCLC cells and suggest that the molecule’s effects on KEAP1 may be more complex than simple loss-of-function.

MCB-613 acts as a covalent molecular bridge
To further explore the interaction between MCB-613 and KEAP1, we redeployed our clickable probes in parental and drug-resistant cells before collection, lysis, and CuAAC. Gel electrophoresis and immunoblotting confirmed that the active probe, ARM-3-124, bound to KEAP1 (Fig. 3a). Furthermore, a thermal shift assay confirmed direct interaction between MCB-613 and purified KEAP1 (Fig. 3b). Specifically, we observed a dose-dependent decrease in Tm, which occurs when a ligand stabilizes a non-native protein conformational state and can indicate protein destabilization30. Indeed, follow-up cellular thermal shift assays (CETSA) in drug-resistant WZR12 cells confirmed that MCB-613 binds directly to and destabilizes both ectopic and endogenous KEAP1 (Fig. 3c and Supplementary Fig. 3a–c)31,32. Because protein instability often promotes degradation, we hypothesized that MCB-613 influences KEAP1 protein levels. Indeed, exposure of parental PC9 and gefitinib-resistant WZR12 cells to increasing doses of MCB-613 revealed a dose-dependent decrease in KEAP1 protein levels at the expected molecular weight. To our surprise, however, we also noticed a concomitant accumulation of KEAP1 at higher molecular weight (Fig. 3d). This suggested that, rather than degradation, MCB-613 might promote KEAP1 oligomerization. Although prior reports have shown that a variety of structurally diverse electrophiles can oligomerize KEAP1, the majority of these act in a non-covalent fashion. Certain other compounds, such as methylglyoxal, have been shown to form covalent adducts between KEAP1 monomers33. MCB-613 seems to be unusual in that it falls into the latter category, inducing the formation of a complex which persists even in the face of stringent reducing conditions, suggesting that it does not arise as a result of non-covalent protein-protein interactions or disulfide bridges (Supplementary Fig. 3d)34.

Small molecules that act at protein-protein interfaces to promote direct interaction are known as molecular glues or bridges35. To assess whether MCB-613 indeed causes KEAP1 oligomerization, we expressed V5-KEAP1 in drug-resistant WZR12 cells before treatment with MCB-613, co-immunoprecipitation, and electrophoresis. Subsequent gel excision and MS analysis of the drug-induced, high molecular weight V5-KEAP1 band confirmed that it was substantially enriched for KEAP1 above all other protein species, suggesting that it consisted of the expected oligomer (Fig. 3e and Supplementary Data 3). An in vitro binding assay involving two purified and differentially tagged KEAP1 species treated with increasing doses of MCB-613 produced a similar transition from monomeric to high molecular weight KEAP1, providing us with further, direct evidence that MCB-613 induces KEAP1 oligomerization (Fig. 3f). Taken together, these data suggest a model in which MCB-613 uses its symmetrically situated α, β-unsaturated bonds to form a covalent bridge between distinct monomers of KEAP1 (Fig. 3g).

To test this model, we synthesized ARM-143, a closely related structural analogue of MCB-613 that harbors only one α, β-unsaturated bond (Supplementary Fig. 3e). As anticipated, treatment with ARM-143 failed to convert KEAP1 from its monomeric to its oligomeric form (Fig. 3h). To validate this finding, we next designed an assay capable of detecting addition of either of these molecules to monomers of KEAP1. Because such an addition would equip the protein with a rare functional ketone moiety, we reasoned we could use a click chemistry, hydrazone-mediated biotinylation as a readout for target engagement (Supplementary Fig. 3f, g). Indeed, this assay confirmed that although ARM-143 interacts covalently with monomers of KEAP1, it fails to reproduce the oligomers seen with MCB-613 (Supplementary Fig. 3h). Furthermore, ARM-143 also showed severely attenuated potency and failed to reproduce the collateral sensitivity to EGFR inhibitor resistance that is seen with MCB-613, suggesting that the molecular bridge-like features of the active parent compound are essential for selective cell death in this setting (Fig. 3i). To further investigate this final point, we tested a panel of structurally diverse, known KEAP1 inhibitors—including seven reactive electrophiles as well as one recently identified non-covalent inhibitor—against both parental and drug-resistant EGFR-mutant NSCLC cells34,36–41. Of these, curcumin alone—which bears significant structural similarity to MCB-613—effected a collateral sensitivity which was shared across all resistant derivatives (Supplementary Fig. 4a, b). These findings suggest that electrophilic stress and KEAP1 inhibition alone are not enough to cause collateral sensitivity, but instead that certain symmetric electrophiles act as covalent molecular bridges to oligomerize KEAP1 and cause selective cell death in drug-resistant cells.

Molecular bridging by MCB-613 relies on the KEAP1 dimerization domain
KEAP1 is recognized for its ability to sense and ligand a plethora of structurally diverse, thiol-reactive xenobiotics with its network of 27 cysteine residues34,42–44. We employed activity-based protein profiling (ABPP) to explore proteome-wide cysteine engagement with MCB-613, particularly focusing on KEAP1. We achieved extensive proteome coverage, quantifying 8,373 peptides, aligning with previous studies45. Contrary to expectations, and despite the fact that MCB-613 demonstrated rapid labeling of GSH in a classic GSH reactivity assay (Supplementary Fig. 5a), we observed only a mild level of cysteine engagement by MCB-613 across the proteome. (Unsurprisingly, ARM-143, which we have shown above labels KEAP1 (Supplementary Fig. 3h), showed decreased activity in the GSH assay, which was expected due to its lack of a dienone structure. The fact that MCB-613, on the other hand, rapidly labels GSH in an in vitro assay further speaks to its tool-like and non-drug-like nature.) Remarkably, only 8 cysteines showed an engagement score exceeding 10% (p value ≤ 0.05), none of which were associated with KEAP1 (Supplementary Data 4 and Supplementary Fig. 5b, c). To validate this finding, we substituted each of the 27 cysteine residues in KEAP1 with isostructural but less nucleophilic serine residues; however, none of these modifications rescued oligomerization of KEAP1 induced by MCB-613 (Supplementary Fig. 5d). One explanation for these findings is that MCB-613 might covalently engage cysteine residues through a reversible mechanism, thus rendering detection difficult in assays such as those described here. Although this cannot be ruled out on the base of these data alone, the fact that competitive displacement is often detectable even with non-covalent inhibitors led us to speculate instead that MCB-613’s activity might depend upon alternative residues within KEAP146.

To identify which sites within KEAP1 might be required by MCB-613, we used an HA-Clover tag system to design a series of increasingly truncated forms of KEAP1. The first fragment, HA-Clover KEAP160, was limited to the first sixty amino acids of KEAP1, which comprise the protein’s N-terminal domain. The next fragment, HA-Clover KEAP1178, further incorporates the protein’s BTB domain. Similarly, the third and fourth fragments, HA-Clover KEAP1314 and HA-Clover KEAP1597, stretch to include the IVR and then Kelch domains, respectively. Also included was a full-length KEAP1 construct spanning the full 624 amino acids (HA-Clover KEAP1WT) (Fig. 4a). We transfected these fragments into 293T cells before treating with an active dose of MCB-613 for 1h, preparing for immunoblot, and probing with an anti-GFP antibody. As expected, MCB-613 caused the accumulation of high molecular weight bands in cells expressing the HA-Clover KEAP1WT construct, again reflecting oligomerization of KEAP1. Indeed, similar bands appeared in the samples that were transfected with HA-Clover KEAP1178, HA-Clover KEAP1314, and HA-Clover KEAP1597 as well; the sole fragment which failed to reproduce this finding was HA-Clover KEAP160 (Fig. 4b). We therefore concluded that KEAP1 oligomerization by MCB-613 likely relies upon residues between positions 60 and 178, which unsurprisingly represents the protein’s dimerization domain.

To narrow down our region of interest, we next repeated the approach described above by serially truncating HA-Clover KEAP1178 in increments of ten amino acids apiece (Fig. 4c). After treatment with MCB-613 and immunoblotting, we observed that drug-induced oligomerization remained intact so long as the first 100 amino acids of KEAP1 were expressed; with the expression of HA-Clover KEAP190, however, the oligomerization effect was severely attenuated (Fig. 4d). We were encouraged by this finding, since the region between KEAP1 residues Gln74 and Met120 is known to form a hydrophobic surface cavity which has been implicated in interaction with electrophiles such as pubescenoside A (PBA)47. In accordance with the ABPP findings presented above, moreover, the primary sequence of KEAP1 situated between amino acid positions 90 and 100 contains no cysteine residues. It does, however, contain a lysine at position 97, which stands out as a strong candidate for nucleophilic participation in Michael addition with MCB-613. Indeed, MCB-613 showed high lysine reactivity in an in vitro assay (again, and expectedly, unlike ARM-143), and computational modeling suggests that MCB-613 could even fit neatly into this previously described surface cavity, participating in hydrogen bonding with surrounding amino acid residues such as Arg116 and forming irreversible bonds bilaterally with the Lys97 residues of distinct KEAP1 monomers (Supplementary Fig. 6a, Fig. 4e). Unfortunately, lysine ABPP remains technically challenging, and an attempt to query this residue was ultimately unsuccessful. In fact, to our knowledge, no previously described lysine-reactive ABPP probe has ever been shown to successfully detect this specific peptide48–51.

To test the hypothesis that Lys97 is required for MCB-613-induced KEAP1 oligomerization, we compared wild-type KEAP1 with a mutant version of the protein in which the nucleophilic lysine residue at position 97 was replaced with a non-reactive alanine residue (K97A). In comparison with wild-type KEAP1, the K97A mutant exhibited dramatically diminished oligomerization when treated with MCB-613 (Fig. 4f). Consistent with this finding, expression of the full-length KEAP1 K97A mutant in EGFR inhibitor-resistant WZR12 cells completely blocked their sensitivity to MCB-613 (Fig. 4g). Thus, MCB-613 relies upon lysine 97 to oligomerize KEAP1.

MCB-613 promotes cell death through a NRF2-independent, ROS- and ATF4-dependent mechanism
KEAP1 plays a canonical role as an adaptor subunit of the Cullin 3-based E3 ubiquitin ligase machinery. Under homeostatic conditions, KEAP1 homodimerizes and binds its substrates—most notably, the master transcription factor nuclear factor erythroid 2-related factor 2 (NRF2)—to facilitate ubiquitination and degradation. In the presence of oxidative or electrophilic stress, however, direct modification of KEAP1 by oxidants and/or electrophiles disables the protein’s substrate adaptor function and allows for the rapid cellular accumulation of KEAP1 substrates. In the case of NRF2, this broadly results in activation of antioxidant transcriptional programs52. Interestingly, previous work has shown that the acquisition of resistance to first-generation EGFR inhibitors suppresses NRF2 activity53. We therefore wondered whether MCB-613-mediated KEAP1 perturbation influenced NRF2 protein and/or activity levels. Indeed, and as expected, treatment of EGFR inhibitor-resistant cells with MCB-613 led to rapid accumulation of NRF2 at the protein level and subsequent activation of NRF2 transcriptional programs (Fig. 5a, b and Supplementary Fig. 6b). Although NRF2 transcriptional programs are conventionally thought of as cytoprotective, recent work has shown that they can produce cytotoxicity in a subset of lung cancer cell lines through an NADH-mediated phenomenon known as “reductive stress.”54 Thus, we hypothesized that MCB-613-mediated KEAP1 inhibition might selectively target drug-resistant NSCLC cells through activation of NRF2 and initiation of reductive stress. To the contrary, however, the genetic knockout of NRF2 not only failed to rescue the collateral sensitivity of drug-resistant NSCLC cells to MCB-613, but instead further sensitized them to it (Fig. 5c and Supplementary Fig. 6c). Importantly, the KEAP1 K97A mutant (which blocks MCB-613-mediated KEAP1 oligomerization and cellular toxicity) does not affect basal NRF2 expression (Supplementary Fig. 6d). Although WZR12 cells bearing the KEAP1 K97A mutant do show some NRF2 accumulation with MCB-613 treatment, presumably due to the presence of endogenous wild-type KEAP1, this is blunted in comparison with the accumulation observed with overexpression of wild-type KEAP1 (Supplementary Fig. 6e). Thus, the effect of MCB-613 cannot be explained by its effects on NRF2, which appears to provide a limited buffering function against MCB-613-mediated stress.

Given the findings above, we next wondered whether our diverse, EGFR inhibitor-resistant models held in common any other features which might explain their shared collateral sensitivity to treatment with MCB-613. It is known, for example, that gefitinib resistance promotes the accumulation of reactive oxygen species, and recent studies have shown that before EGFR-mutant NSCLC cells acquire stable drug resistance, they first pass through an initial, drug-tolerant persister state characterized by elevated levels of ATF455,56. As expected, and in accordance with these prior literature reports, our drug-resistant derivatives showed elevated baseline levels of reactive oxygen species, as well as elevated protein levels of ISR pathway members ATF4 and CHOP (Fig. 5d, e). In the latter case, in fact, we observe a direct correlation between the degree of resistance to gefitinib on the one hand and basal levels of protein expression of ATF4 and CHOP on the other (R2=0.9085 and 0.8713, respectively) (Supplementary Fig. 6f). Treatment of drug-resistant cells with MCB-613, moreover, exacerbated both of these phenotypes (Fig. 5f, g), but showed no effect on similar pathways, such as the heat shock response (as measured by levels of HSP60, HSP70, and HSP90 proteins, Supplementary Fig. 6g). MCB-613’s effects on both cell viability and ATF4 levels were rescued by pre-treatment with ROS scavengers such as N-acetylcysteine (NAC) and α-tocopherol (Fig. 5h, i and Supplementary Fig. 7a), and the induction of ROS by MCB-613 depended upon KEAP1 (Supplementary Fig. 7b). (Although it cannot be definitively ruled out by these studies alone that NAC is working by directly interfering with MCB-613, the data shown here suggest against that possibility.) Further, the effects of MCB-613 on ATF4 levels were, like cell viability (Fig. 2h), also KEAP1-dependent (Fig. 5j and Supplementary Fig. 7c). Together, these results suggest that diverse EGFR inhibitor-resistant models share features of ISR hyperactivation. Further, they are consistent with a model wherein KEAP1 oligomerization by MCB-613 leads to ROS induction and ATF4/CHOP activation in drug-resistant cells.

The ISR is a bivalent pathway: although moderate pathway engagement leads to stress amelioration and cellular recovery, excessive ISR signaling leads to enhanced cytotoxicity and, ultimately, cellular demise57. We therefore wondered whether the ISR hyperactivation, which is triggered in response to MCB-613-induced ROS in EGFR inhibitor-resistant cells, was primarily cytoprotective or cytotoxic. To answer this question, we knocked out the ISR pathway members ATF4 or CHOP in WZR12 cells before treating with MCB-613. Impressively, the loss of either of these ISR effectors was sufficient to rescue the effect of MCB-613 on cell viability (Fig. 5k, l and Supplementary Fig. 7d). Consistent with evidence that MCB-613’s activity requires KEAP1 oligomerization, ARM-143 failed to induce ATF4 expression (Supplementary Fig. 7e). Together, these findings suggest that, in the setting of EGFR inhibitor resistance, MCB-613 acts through a unique, KEAP1-dependent, NRF2-independent mechanism to selectively induce ROS and trigger an ATF4- and CHOP-mediated form of cell death.

Methods
This study complies with all relevant ethical regulations. Animal protocols were approved by the Animal Care and Use Committee of the Baylor College of Medicine. All newly synthesized reagents (e.g., oligonucleotides and plasmids) are available upon request.

Cell lines and reagents
All cell lines were maintained in a humidified incubator at 37°C with 5% CO2. 293FT cells were cultured in DMEM high glucose medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% non-essential amino acids, 1% GlutaMAX, and 1% sodium pyruvate. All other cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. The PC9 cell line was obtained from Sigma (90071810). The 293FT cell line was obtained from the Duke Cell Culture Facility (2832-R700-07). The PFR3, GR4, and WZR12 cell lines were a generous gift from Pasi Janne. The patient-derived MGH134 cell line was a generous gift from Aaron Hata. Drug-resistant cell lines were derived as previously described8,90. MCB-613 was a generous gift from Bert O’Malley. All other drugs were purchased from Cayman Chemical (gefitinib, osimertinib) and ChemDiv (C151-0598), or synthesized (Supplementary Note).

High-throughput pharmacologic screen
The Selleck Bioactives Compound Library was used in conjunction with the Duke Functional Genomics Core. In brief, 2100 bioactive compounds were stamped onto 384-well plates in duplicate using an Echo Acoustic Dispenser. Cell culture media containing PC9, PFR3, GR4, or WZR12 cells was added to wells using a Matrix WellMate for a final density of 300 cells per well and final concentrations of 2 and 10µM for each drug. After 72h, cell viability was assessed using the CellTiterGlo Luminescent Viability Assay (Promega) on a BMG Clariostar plate reader. The duplicate treatment wells for each compound were averaged and normalized to averages for duplicate control wells. Normalized values were then analyzed by calculating the log2 (resistant derivative/parental) for each compound at both doses, and in parallel by subtracting the score for the parental cell line from the scores of the resistant derivatives. Hits that scored across multiple resistant derivatives and at the lower dose were prioritized. For further details, please refer to Supplementary Data 1 and Supplementary Table 1.

Dose-response (GI50) assays
Cells were seeded into 96-well plates at a density of 2000 cells/well and allowed to adhere overnight. The following day, cells were treated in triplicate with either vehicle (DMSO) or an eight-point drug dilution and returned to the incubator. After 72h, luminescence was quantified with CellTiterGlo (Promega). Relative cell viability was estimated by normalizing the raw luminescence values for the drug-treated wells to the DMSO controls.

In vivo studies
Animal protocols were approved by the Animal Care and Use Committee of the Baylor College of Medicine. Female NOD SCID mice (NOD.CB17-Prkdcscid/NCrCrl) were obtained commercially from Charles River Laboratories and housed in a strict sterile housing environment with temperatures around 22°C and relative humidity of 40–60%. A standard light/dark cycle [12-h light, 12-h dark (12L:12D) cycle] condition was used. At the time of the study, 5×106 PC9 or GR4 cells were bilaterally implanted via subcutaneous injection into the flanks of 5–6 week-old female mice and allowed to grow for 2 weeks. After two weeks, tumors were measured, mice were weighed, and treatment was initiated. Mice were treated with either saline or MCB-613 (20 mg/kg) in saline three times weekly via intraperitoneal (i.p.) injection. During the treatment period, mice were weighed, and their tumors were measured weekly. The Animal Care and Use Committee of the Baylor College of Medicine permits a maximal tumor size of 2.0 cm in diameter in any direction for single, externally visible tumors. The maximal tumor size in this study did not exceed 1.5 cm in diameter in any direction for any mouse.

Synthesis of ARM-3-124, ARM-3-115, and ARM-143
Briefly, the preparation of clickable analogs (ARM-3-115, ARM-3-124) of C151-0598 and MCB-613 was performed in a one-step synthesis starting from 4-propargylcyclohexanone and benzaldehyde or nicotinaldehyde. Using these starting materials, this step consisted of a double aldol condensation with benzaldehyde. It was achieved in ethanol at room temperature using potassium hydroxide as the base. Therey, ARM-3-115, the analog of C151-0598, was obtained in modest yield (21%). The analog of MCB-613 was also obtained through a double aldol condensation of the adequate 4-propargylcyclohexanone with nicotinaldehyde, but under acidic conditions (HCl in acetic acid). The target compound ARM-3-124 was obtained in 37% yield. Finally, ARM-143 was also prepared by aldol condensation, in acidic media, between nicotinaldehyde and commercially available 4-ethyl-2-(pyridine-3-ylmethyl)cyclohexanone, in 27% yield.
For more detailed characterization of chemical synthesis, yield, and purity, please see Supplementary Note.

Copper-catalyzed azide-alkyne cycloaddition (CuAAC)-“click” reaction
The click chemistry experiments were conducted as previously described91. Briefly, cells were seeded into six biological replicates and exposed for 1h to a 2µM dose of either the active ARM-3-124 (3 samples) or inactive ARM-3-115 (3 samples). After treatment, cells were washed three times in PBS, and the pellet was resuspended in 1330µL of 1% NP40 buffer completed with protease inhibitor. Resuspended cells were sonicated for 30s three times and incubated 30min on ice before centrifugation at 20,000×g for 10min. After cell lysis, proteins were extracted and quantified using the Bradford method. For each condition, up to 10 mg of protein in 9.4ml 1% NP40 buffer was used to perform the biotin addition using 100µL of biotin azo-azide at 5mM, 200µL TCEP at 50mM, 100µL TBTA at 10mM, and 200µL CuSO4 at 50mM. Protein was then incubated for 1h in the dark at room temperature. After incubation, 40ml ice-cold methanol was added. Protein was precipitated overnight in methanol at −20°C. Protein was then centrifuged at 5200×g for 30min at 0°C and washed 3 times in the same conditions using ice-cold methanol. After drying, protein was resuspended in resuspension buffer (6M urea, 2M thiourea, 10mM HEPES) and sonicated. Protein was supplemented with 40µL DTT and incubated for 40min at room temperature. Forty microliters of iodoacetamide was added to the samples and incubated 30min at room temperature in the dark. After pre-washing of the streptavidin beads, resuspended proteins were added to the beads in a 15ml tube and incubated on a rotator for 2h at room temperature. Beads were collected by centrifugation at 200×g for 3min at room temperature and then washed with resuspension buffer (PBS and 1% SDS, PBS) solution twice. After washing, elution was performed using sodium dithionite solution. Eluted proteins were incubated overnight at −20°C with ice-cold methanol. Proteins were then pelleted and dried before being resuspended in 4% SDS buffer and 2X SDS free loading buffer. Samples were then separated by one-dimensional electrophoresis and stained with Instant Blue® dye. Gels were divided into pieces measuring approximately 1mm3 and sent to Ross Tomaino at the Taplin Biological Mass Spectrometry Facility (Harvard Medical School) for identification of target proteins. Excised gel bands were cut into approximately 1mm3 pieces. Gel pieces were then subjected to a modified in-gel trypsin digestion procedure92. Gel pieces were washed and dehydrated with acetonitrile for 10min. followed by removal of acetonitrile. Pieces were then completely dried in a speed-vac. Rehydration of the gel pieces was with 50 mM ammonium bicarbonate solution containing 12.5ng/µl modified sequencing-grade trypsin (Promega, Madison, WI) at 4°C. After 45min., the excess trypsin solution was removed and replaced with 50 mM ammonium bicarbonate solution to just cover the gel pieces. Samples were then placed in a 37°C room overnight. Peptides were later extracted by removing the ammonium bicarbonate solution, followed by one wash with a solution containing 50% acetonitrile and 1% formic acid. The extracts were then dried in a speed-vac (~1h). The samples were then stored at 4°C until analysis. On the day of analysis the samples were reconstituted in 5 - 10 µl of HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A nano-scale reverse-phase HPLC capillary column was created by packing 2.6µm C18 spherical silica beads into a fused silica capillary (100µm inner diameter x ~30cm length) with a flame-drawn tip93. After equilibrating the column each sample was loaded via a Famos autosampler (LC Packings, San Francisco CA) onto the column. A gradient was formed, and peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid). As peptides eluted, they were subjected to electrospray ionization and then entered into a Velos Orbitrap Pro ion-trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program, Sequest (Thermo Fisher Scientific, Waltham, MA)94. All databases include a reversed version of all the sequences, and the data was filtered to between a one and two percent peptide false discovery rate.

Design of custom sgRNA library
The custom sgRNA library was designed using the list of 478 MS-identified genes. For each of these genes, 4 sgRNA sequences were taken from each of two previously published, full-genome CRISPR libraries95,96. (Because sequences for a given gene were sometimes redundant between the two libraries, this approach produced an average of 7 unique sgRNA sequences per gene). Also included were 100 AAVS1-targeting “safe harbor” guides taken from a separate CRISPR library97. Each sequence was appended with the same universal prefix and suffix sequences (Supplementary Table 2) and synthesized commercially in pooled format (GenScript).

Cloning of the CRISPR library
The commercially synthesized oligopool was diluted 1:10 in water and amplified using the Phusion Hot Start Flex polymerase (NEB) according to the manufacturer’s protocol with the Array_F and Array_R primers (Integrated DNA Technologies) (Supplementary Table 2). The lentiCRISPRv2 vector (Addgene #52961) was digested with FastDigest Esp3I (Thermo Fisher) at 37°C for 12h. The digested product was size selected with gel electrophoresis and extracted using the Zymo Gel DNA Extraction Kit. The eluted product was cleaned using the AMPure XP reagent at a 1:1 input-to-reagent ratio, and concentration was measured using the Qubit dsDNA HS Assay Kit (Invitrogen). The digested vector was combined with the amplified inserts in a 5:2 ratio by mass and ligated using a 2X Gibson Assembly Master Mix (NEB) at 50°C for 30min. The ligation product was cleaned using the AMPure XP reagent at a 1:1.4 input-to-reagent ratio, and concentration was again measured using the Qubit dsDNA HS Assay Kit. The final product was transformed into electrocompetent cells (Lucigen), incubated overnight in LB broth at 37°C, and isolated using the Qiagen MaxiPrep kit.

Lentivirus production
Lentivirus for both the pooled CRISPR screen and individual sgRNAs was produced using 293FT cells. For every 1.3µg of plasmid, 1µg of the psPAX2 packaging vector (Addgene #12260) and 0.657µg of the VSVg envelope (Addgene #14888) were diluted into 385µl of Opti-MEM supplemented with 11.6µl of the lipofectamine-2000 transfection reagent and 12.64µl of PLUS reagent (Thermo Fisher). This mixture was added dropwise to 293FT cells, and cells were returned to the incubator for 4h. After the incubation, media were aspirated and replaced with an FBS-rich harvest medium (DMEM high glucose, 30% FBS, 1% PS, 1% NEAA, 1% GlutaMax, 1% sodium pyruvate) for 48h. Viruses were collected, filtered, and either used directly or aliquoted and stored at −80°C. Viral titers were performed as previously described9. Briefly, PC9, GR4, and WZR12 cells were seeded into 6-well plates at a density of 150,000 cells per well and allowed to adhere overnight. The next day, cells were exposed to media containing the polybrene transduction reagent (8µg/ml) and increasing concentrations of library virus before spinfection via centrifugation at 900×g for 1h at room temperature. On Day 2, the media containing library virus and polybrene was removed and replaced with fresh media. On Day 3, the cells were subjected to selection with puromycin (2µg/ml) for 48h. Then, on Day 5, the cells were resuspended and counted to determine the percentage of cells infected per volume of virus and compared with an MOI table to estimate functional viral titer.

Pooled CRISPR screening
On Day 0, PC9, GR4, and WZR12 cells were seeded into 6-well plates at a density of 150,000 cells per well and allowed to adhere overnight. On Day 1, the cells were exposed to media containing library virus and the polybrene transduction reagent (8µg/ml) before spinfection via centrifugation at 900×g for 1h at room temperature. On Day 2, the media containing library virus and polybrene was removed and replaced with fresh media. On Day 3, the cells were subjected to selection with puromycin (2µg/ml) for 48h. Then, on Day 5, the cells were resuspended, counted, divided into triplicates, and carried forward with passage every five days for an estimated 14 population doublings. Throughout the entirety of the screen, each replicate was represented at any given point in time by at least 4×106 cells to guarantee at least 1000x coverage of the custom library. Pellets of at least 4×106 cells were taken at T0, Tf, and at each passage as well. Upon completion of the screen, pellets were lysed and genomic DNA was extracted using the QIAamp Blood Maxi Kit (Qiagen).

CRISPR screen sequencing and analysis
Extracted DNA was amplified with a two-step protocol. First, sgRNA libraries were PCR-amplified from genomic DNA using NEBNext Ultra II Q5 Master Mix according to the manufacturer’s protocol using the PCR 1 Forward and Reverse primers (IDT) (Supplementary Table 2). Following the initial amplification, a second PCR step was undertaken to append the PCR 1 product for each condition with a unique combination of staggered forward and barcoded reverse primers to facilitate pooled sequencing (IDT) (Supplementary Tables 2 and 3). In all cases, the amplified libraries were purified with SPRIselect beads (Beckman Coulter) using right-sided selection. Samples were quantified with the Quant-iT dsDNA Broad Range Assay Kit (Thermo Fisher), pooled, and sequenced on an Illumina NextSeq 500 with 75bp single-end sequencing. Analysis was performed using the Models-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) software analysis pipeline under the default conditions. Data from the GR4 and WZR12 cell lines were collapsed into one condition representing drug resistance, and compared with data from the PC9 parental cell line.

CRISPR screen validation by single gene knockout
To validate screening hits, the top two scoring sgRNA sequences for a given gene were taken from the custom library, commercially synthesized (IDT), cloned into the LCv2 vector, transfected into 293FT cells, and transduced into parental and drug-resistant NSCLC cells as described above (Supplementary Table 2). After 48h of puromycin selection, 350,000 cells per condition were seeded in triplicate into 10-cm dishes and passaged and counted every three days. Total cumulative cell counts over time were estimated using cell counts and doubling time while assuming exponential growth and a negligible death rate. Cumulative counts for a given sgRNA were normalized to cumulative counts for a non-targeting control.

Protein purification
His6-KEAP1 was purified as previously described98. Briefly, One Shot BL21(DE3) chemically competent E. coli cells were transformed with the pET28a-His6-KEAP1 vector (Addgene #62454). Colonies were grown in LB broth containing 50µM ZnCl2 and 50µg/ml kanamycin at 37°C until cultures reached an OD600 of 0.6-0.8, and then were allowed to cool to room temperature. Protein expression was induced with 250µM IPTG and allowed to shake at 19°C for 14h. Bacteria were collected and His6-KEAP1 purified on Ni-NTA agarose beads (Thermo Fisher) using the following buffers: lysis (50mM NaHPO4 (pH 8.0), 10mM Imidazole, 5mM β-mercaptoethanol (BME), 0.01% Triton X-100), washing buffer (50mM NaHPO4 (pH 8.0), 50mM Imidazole, 500mM NaCl, 5mM BME, 0.01% Triton X-100), and elution (50mM NaHPO4 (pH 8.0), 125mM Imidazole, 150mM NaCl, 5mM BME). Following elution, elution fractions were analyzed by gel electrophoresis to select and pool fractions of highest purity. The pooled fractions were then exchanged into storage buffer (50mM Tris (pH 8.0), 10mM DTT, 100mM NaCl, 5% glycerol) using the Amicon Ultra-15 centrifugal filter column with a 10kDa pore size (Millipore). Protein was quantified with the Bradford method before adjusting to a stock concentration of 4mg/ml, aliquoting, flash freezing in liquid nitrogen, and storing at −80°C.

Construction of pLX302 V5-KEAP1
The pLX302 V5-KEAP1 construct was assembled from an empty vector backbone (Addgene #25896) and the pDONR223 KEAP1 WT vector (Addgene #81925) using the Gateway LR Clonase II enzyme according the manufacturer’s instructions.

Thermal shift assay
The thermal shift assay was performed in a 384-well format as previously described in ref. 99. Purified His6-KEAP1 was diluted to a final concentration of 133µg/ml (1.9µM) in assay buffer (50mM Na3PO4, 50mM NaCl, pH 6.0) containing 8x SYPRO Orange diluted from a 5,000x stock (Invitrogen). The mixture was transferred to the wells of a 384-well plate and treated with either vehicle (DMSO) or doses of MCB-613 diluted in assay buffer. All conditions were plated in triplicate. The plate was sealed and subjected to centrifugation at 800×g for 2min to settle the samples before incubating at room temperature for 15min. After the incubation ended, the plate was transferred to a Bio-Rad CFX384 Touch Real-Time PCR Detection System at 25°C. The well temperature was increased in 0.5°C increments with a 10-s equilibration time and fluorescence was measured by the built-in FRET channel until the machine reached 95°C. For each condition, the melting temperature was determined by adjusting the raw values according to a modified Boltzmann Equation and calculating the average of the three replicates.

Cellular thermal shift assay (CETSA)
The cellular thermal shift assay was performed according to previous reports32. First, cells were seeded and allowed to adhere overnight. On the day of the assay, cells were treated in biological triplicate with either vehicle (DMSO) or a 2µM dose of MCB-613 for 1h before trypsinization, room temperature PBS washes, and collection in room temperature PBS supplemented with a protease inhibitor (Roche). For each condition, approximately 1×106 cells were then transferred to each of 12 PCR strip tubes and sealed. A gradient PCR protocol including the following temperatures (°C) was initiated: 42.2, 43.7, 45.3, 46.7, 48.3, 49.8, 50.2, 51.7, 53.3, 54.2, 56.3, and 57.8. Samples were held off of the machine until the target temperature for each well was reached, and then placed on the machine and incubated at the target temperatures for 3min. Each of the samples was then incubated in the thermal cycler at 25°C for an additional 3min, before being allowed to rest for a final 3min at room temperature and on the benchtop. Then, the PCR strip tubes were transferred to pre-chilled racks and snap frozen in liquid nitrogen, before incubating until completely thawed (~3-4min) in a room temperature water bath. The cycle was repeated 3 more times for a total of 4 freeze-thaw cycles to promote cell lysis. After the final freeze-thaw cycle, the total volume of each PCR strip tube (~100µl) was transferred to a 1.5mL Eppendorf tube and centrifuged at 17,000×g for 40min at 4°C. Debris pellets were discarded, and 60µl of supernatant was transferred to a new tube containing 20µl of NuPAGE LDS Sample Buffer (4X) and 4µl of β-mercaptoethanol. Samples were vortexed and allowed to incubate at room temperature for 30–60min before western blotting according to the procedures described below. Targets were detected using primary antibodies anti-KEAP1 clone 144 (MABS514, Sigma) and anti-V5 (#13202, CST) for endogenous and ectopic KEAP1, respectively.

Densitometry analysis
Densitometry analysis of X-ray films from immunoblotting studies was performed using Adobe Photoshop and ImageJ. Images were converted to grayscale in Photoshop, and the intensity of bands was measured in ImageJ. For each blot, measurements were taken using a constant size selection for regions of interest. Background measurements were also collected from the blank regions located just above these bands. Pixel density was inverted by subtracting the measured intensities of both the band and background regions from 255. Net values were determined by subtracting the inverted densities of the background regions from the inverted densities of their corresponding bands of interest. The net value for each band of interest (e.g., KEAP1) was then normalized to the net value of the loading control for that lane (e.g., β-Actin). Finally, these normalized net values were themselves normalized to the 42.2°C band in the CETSA experiment and plotted as a function of temperature.

Western blot
Cells were washed in ice-cold PBS, collected via scraping, and resuspended in ice-cold 1X Cell Lysis Buffer (CST) supplemented with protease and phosphatase inhibitors (Pierce). Cell suspensions were vortexed thoroughly and rotated end-over-end at 4°C for 30min before centrifugation at 21,130×g at 4°C for 20min. The debris pellets were discarded, and the supernatants transferred to fresh Eppendorf tubes before quantification using the Bradford method. Samples were diluted to equal concentrations using lysis buffer and prepared for electrophoresis with NuPAGE LDS Sample Buffer (4X) before boiling at either 70°C for 10min or 95°C for 15min. Prior to electrophoresis, samples were reduced with DTT at a final concentration of 50mM except where otherwise noted. Membranes were probed with primary antibodies anti-KEAP1 clone 144 (MABS514, Sigma), anti-NRF2 (Ab137550, Abcam), anti-ATF4 (#11815, CST), anti-CHOP (#5554, CST), anti-V5-tag (#13202, CST), anti-GFP (#2555, CST), anti-Vinculin (#4650, CST), and anti-β-Actin (#4970, CST), and secondary antibodies anti-rabbit IgG, HRP-linked (#7074, CST) and anti-rat IgG, HRP-linked (#7077, CST). Uncropped and unprocessed scans of all immunoblots are available in the Source Data file.

Co-immunoprecipitation
For co-IP studies, 4–5×106 cells were seeded into two 15-cm dishes per condition and allowed to adhere overnight. The next day, cells were treated with a 2µM dose of MCB-613 for 1h before scraping into ice-cold PBS. Cells were resuspended in IP buffer containing 40mM Tris HCl (pH 7.4), 150mM NaCl, 20mM EDTA, 1mM DTT, and 0.5% NP-40 supplemented with phosphatase and protease inhibitors, thoroughly vortexed, and rotated end-over-end at 4°C for an hour. Samples were then subjected to centrifugation at 21,130×g for 20min at 4°C before harvesting the supernatant and measuring protein concentration using the Bradford method. A small volume of lysate was reserved for input, and the rest (600–800µg) was adjusted so that a constant amount was loaded onto antibody-bound sepharose beads (see below) per sample. Samples were rotated end-over-end at 4°C overnight. The following morning, samples were washed 5 times in ice-cold IP buffer before boiling at 95°C for 5min in NuPAGE LDS Sample Buffer (4X). Lysates were collected from the beads using a 50µl syringe (Hamilton) and analyzed via gel electrophoresis according to the procedures described above.
To prepare beads, 50µl of Recombinant Protein G–Sepharose beads (Invitrogen) per condition were washed 3 times in IP buffer. The beads were then resuspended in IP buffer, and anti-V5-tag(E9H8O) Mouse mAb (#80076) (CST) was added at a 1:50 dilution as instructed by the manufacturer. The bead-antibody combination was then rotated end-over-end at 4°C for at least 5h. After the incubation, the beads were washed 3 times in IP buffer and divided evenly among the experimental conditions.

LC-MS based protein identification from SDS-PAGE gel bands
WZR12 cells were transduced with either pLX302 V5-luciferase (Addgene #47553) or pLX302 V5-KEAP1 and selected with puromycin. After selection, cells bearing V5-KEAP1 were treated with either DMSO or 2 µM MCB-613 for 1h before collection and V5-co-immunoprecipitation, alongside an untreated, V5-luciferase control. Samples were collected, prepared for, and subjected to SDS-PAGE as described above. Coomassie stained SDS-PAGE bands (n=1 per condition) were subjected to standardized in-gel trypsin digestion in which gel bands were subjected to reduction with 10 mM dithiothreitol, alkylated with 20 mM iodoacetamide, and digested with 100ng of sequencing grade modified trypsin (Promega). Extracted peptides were lyophilized to dryness and resuspended in 12μL of 0.2% formic acid/2% acetonitrile. Samples were subjected to chromatographic separation on a Waters NanoAquity UPLC equipped with a 1.7µm BEH130 C18 75µm I.D. X 250mm reversed-phase column. The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. Following a 3µL injection, peptides were trapped for 3min on a 5µm Symmetry C18 180µm I.D. X 20mm column at 5µl/min in 99.9% A. The analytical column was then switched in-line, and a linear elution gradient of 5% B to 40% B was performed over 30min at 400nL/min. The analytical column was connected to a fused silica PicoTip emitter (New Objective, Cambridge, MA) with a 10µm tip orifice and coupled to an Oribtrap Fusion Lumos mass spectrometer (Thermo) through an electrospray interface operating in a data-dependent mode of acquisition. The instrument was set to acquire a precursor MS scan from m/z 200-1500 in the Orbitrap at r=120,000 with MS/MS spectra acquired in the Ion Trap with AGC setting of 1e4 and 100ms. For all experiments, stepped HCD energy settings were 28.5, 30, 31.5v, and a 20s dynamic exclusion was employed for previously fragmented precursor ions.
Raw LC-MS/MS data files were processed in Proteome Discoverer (Thermo Scientific) and then submitted to independent Mascot searches (Matrix Science) against a SwissProt database (Human taxonomy) containing both forward and reverse entries of each protein (20,371 forward entries). Search tolerances were 5ppm for precursor ions and 0.8 Da for productions using trypsin specificity with up to two missed cleavages. Carbamidomethylation (+57.0214 Da on C) was set as a fixed modification, whereas oxidation (+15.9949 Da on M) was considered dynamic mass modifications. All searched spectra were imported into Scaffold (v4.3, Proteome Software), and scoring thresholds were set to achieve a peptide false discovery rate of 1% using the PeptideProphet algorithm.

Iso-TMT (ABPP) sample preparation
Iso-TMT sample preparation was conducted as previously detailed45. WZR12 cells were seeded for a total of six biological replicates and allowed to adhere overnight. The next day, cells were treated with either vehicle (DMSO) (3 samples) or a 2µM dose of MCB-613 (3 samples) for 1h before collection by scraping into ice-cold PBS, centrifugation, and snap freezing in liquid nitrogen. Frozen cell pellets were lysed in DPBS supplemented with Benzonase (Santa Cruz) and protease inhibitors (Roche) using a chilled bath sonicator (Q700, Qsonica) and centrifuged for 3min at 300×g. Protein concentrations were determined by BCA assay (Thermo Fisher Scientific), and 50µg of protein extracts were used per compound treatment. Lysates were treated with vehicle (DMSO) or 500µM of KB02, KB03, or KB05 (Sigma-Aldrich) for 1h, followed by 1mM DBIA treatment for another hour. Following DBIA incubation, lysates were reduced with 5mM 5-tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma-Aldrich) for 2min at room temperature, then alkylated using 20mM chloroacetamide (Sigma-Aldrich) for 30min in the dark at room temperature. Proteins were precipitated using SP3 magnetic beads. Briefly, SP3 magnetic beads (Cytiva) were prewashed with LC-MS grade water (Sigma Aldrich), and 250 µg of combined SP3 beads (1:1, hydrophobic: hydrophilic) and LC-MS grade ethanol (Sigma Aldrich) were added to each sample to achieve a final concentration of 50% ethanol. SP3 incubation was performed for 30min at room temperature, after which the beads were washed three times with 80% HPLC grade ethanol (Sigma Aldrich) and then resuspended with 175µL of Trypsin/Lys-C (1µg, Thermo Fisher Scientific) in 200mM EPPS (Sigma Aldrich), pH 8.4, 5mM CaCl2. Proteins were digested overnight (16h) at 37°C, and digested peptides were enriched with streptavidin magnetic beads (Cytiva) for 1h at room temperature. Beads were then washed three times with DPBS and twice with HPLC-grade water (Sigma Aldrich). Peptides were eluted with 50% acetonitrile (Sigma Aldrich), 0.1% formic acid (Thermo Fisher Scientific), and dried using a Speedvac (Thermo Fisher Scientific).
Cysteine-enriched peptides were reconstituted with 30% acetonitrile, 70% 200mM EPPS pH 8.4, and labeled with 25µg of TMT reagent (Thermo Fisher Scientific) per channel for 75min at room temperature with rotation. Labeling was terminated by the addition of 5% hydroxylamine (Acros Organics) for 15min followed by the addition of 10% formic acid. Peptides from four different cancer cell lines derived from the same lineage, treated with vehicle or three scout fragments, were labeled with 16 TMT tags, pooled, and dried using a Speedvac (Thermo Fisher Scientific). Peptides were then desalted with stagetips using the following procedure: peptides were reconstituted with 5% acetonitrile/0.1% formic acid and loaded onto C18 MicroSpin columns (Nest Group) pre-equilibrated with LC/MS-grade methanol (Fisher Chemical) and LC/MS-grade water containing 0.1% formic acid. C18 spin columns were washed 10 times with LC/MS grade water containing 0.1% formic acid and subsequently eluted with 80% acetonitrile, 0.1% formic acid, and dried using a Speedvac (Thermo Fisher Scientific).
All mass spectrometry (ABPP) samples were analyzed as previously described using an Orbitrap Eclipse™ Tribrid™ Mass Spectrometer coupled with an Easy NanoLC-1200 system (Thermo Fisher Scientific)45. Peptides were separated on a 75-µm capillary column packed with 50 cm of C18 resin (2µm, 100Å; Thermo Fisher Scientific) using a 180-min gradient of 4–35% acetonitrile in 0.1% formic acid, with a flow rate of 300nL/min. The eluted peptides were acquired by data-dependent acquisition and quantified using the synchronous precursor selection (DDA-SPS-MS3) method for TMT quantification. MS1 spectra were acquired in the scan range of 400–1400 m/z at an Orbitrap resolution of 120,000 with a maximum injection time of 50 ms and high-field asymmetric-waveform ion-mobility spectrometry (FAIMS) values set at -40, -50, and -70 compensation voltage (CV). MS2 spectra were acquired by selecting the top twenty most abundant features via collision-induced dissociation in the ion trap with an automatic gain control (AGC) setting of 10K, a quadrupole isolation width of 0.7 m/z, and a maximum ion accumulation time of 50ms. These spectra were relayed in real-time to an external computer for online database searching using Comet real-time searching (RTS) with a database that included cell line mutations (DepMap)100 and human protein databases (release_20210506)101,102. Both the real-time search and the final search utilized the same forward and reverse-sequence human protein databases (Uniprot). Peptides were filtered using simple initial parameters: they did not match a reverse-sequence, contained TMTPro16 isobaric tags, had a maximum PPM error <50, a minimum PPM error >5, and a minimum ΔCorr of 0.10. If peptide spectra met these criteria, an SPS–MS3 scan was performed using up to 20 beta- and gamma-type fragment ions as precursors with an AGC of 250K for a maximum of 250ms, and a normalized HCD collision energy setting of 55 (TMTPro16).
Peptide searches were performed in Proteome Discoverer (2.5, Thermo Fisher Scientific) against the UniProt human protein database (release_20210506) with the following parameters: up to two missed cleavages, 20 ppm precursor tolerance, 0.6 Da fragment ion tolerance, and fully tryptic peptides were allowed with a minimum length of 6. Static modification of TMTPro16 on lysine and peptide N-termini (+304.2071 Da) and carbamidomethylation of cysteine residues (+57.0214 Da), along with oxidation of methionine (+15.9949 Da) and DIBA on cysteine residues (for iso-TMT samples) (+239.1634) as variable modifications were permitted. Results were filtered to a peptide false discovery rate (FDR) of 1%. For the TMT reporter ion quantification, all identified peptide spectral matches (PSMs) from the MS3 scans were extracted by an in-house program, and the reporter ion intensities were adjusted for impurity correction according to the manufacturer's specifications. For quantification of each MS3 spectrum, a total sum signal-to-noise of all reporter ions of 100 (TMT16-plex) was used. Engagement scores were calculated as previously described45.

Generation of HA-Clover KEAP1 constructs
Full-length KEAP1 WT was amplified out of the pET28a-His6-KEAP1 vector using the Phusion Hot Start Flex 2x Master Mix (NEB) according to the manufacturer’s instructions and appended with EcoRV and NotI restriction sites using commercially synthesized primers (IDT) (Supplementary Table 2). Amplified full-length KEAP1 WT and the HA-Clover construct (Addgene #163336) were digested with the EcoRV and NotI enzymes (NEB) at 37°C for 1h before heat inactivation at 65°C for 5min. Digestion fragments were purified by gel electrophoresis, excised, and extracted with the QIAQuick gel extraction kit (Qiagen). The fragments were combined using T4 DNA ligase (NEB) according to the manufacturer’s instructions. To generate KEAP1 truncations, site-directed mutagenesis was performed to introduce nonsense mutations at positions 61, 179, 315, and 598 using commercially synthesized primers (IDT) (Supplementary Table 2). All constructs were validated by Sanger sequencing (Eton Biosciences).

Transient transfection of HA-Clover KEAP1 constructs
293FT cells were transiently transfected with 3µg of each of the HA-Clover KEAP1 constructs diluted into Opti-MEM (Gibco) and supplemented with Lipofectamine 2000 transfection reagent (Invitrogen). After 24h, media was refreshed. After another 24h, cells were treated with vehicle (DMSO) or 2µM MCB-613 for 1h before collection in ice-cold PBS and preparation for western blot.

GFP-Trap assay
293FT cells were transfected with HA Clover KEAP1 WT as described above. After 48h, the cells were washed once in ice-cold PBS and collected in ice-cold RIPA buffer (Sigma). The cells were incubated on ice for 30min with intermittent vortexing, and the resultant lysates were subjected to centrifugation at 9600×g for 10min at 4°C. A fraction of the supernatant was reserved for input and prepared for western blotting as described above. The remaining supernatant was exposed to GFP-Trap agarose beads (Proteintech) and rotated end-over-end at 4°C overnight before washing 5 times with ice-cold RIPA buffer. The beads were then equilibrated in room-temperature binding buffer (25mM Tris (pH 7.5), 100mM NaCl, 0.1% NP-40, 1mM DTT, 5% glycerol). Ten microgram of purified His6-KEAP1 was added, and the samples were rotated end-over-end at room temperature for 15min. MCB-613 (2, 20, or 200µM) was added, and the samples were rotated for another 15min. Samples were washed 3 times in room-temperature binding buffer, re-suspended in 80µl of NuPAGE LDS Sample Buffer (4X), boiled at 95°C for 5min, and analyzed by western blot. Targets were detected using anti-GFP (#2555, CST) and anti-His (#2365, CST) primary antibodies.

Modeling the covalent complex of KEAP1 and MCB-613
Modeling was performed using the RDKit cheminformatics toolkit and the RosettaLigand docking protocol. We used RDKit to generate 100 conformers of MCB-613, modeled in its reacted form with two lysine atoms at each covalent reaction site (SMILES: CCC1CC(C(C2=CN=CC=C2)NC)C(C(C1)C(NC)C3=CN=CC=C3)=O). The conformers were generated using the AllChem.EmbedMolecule function, followed by a minimization step using UFF force field calculations with the Chem.rdForceFieldHelpers module. Conformers with RMSD<0.5 Å to any of the previously sampled conformers were discarded. The lysine Cε atom was then deleted, and the lysine Nζ was replaced with a vanadium atom, which is interpreted as a virtual atom. To parametrize the ligand for use in Rosetta, we used the molfile_to_params.py script provided in the Rosetta release, and modified the resulting params file to allow covalent binding103.
To model the complex, we first generated a dimeric structure of KEAP1 based on the monomer in PDB ID: 4CXT and a generated symmetry mate using PyMol. Each Lys97 residue was replaced with a covalently-linked variant of lysine103, and the dimeric structure was prepacked using the PackRotamersMover and MinMover protocols through the RosettaScripts interface. The ligand was then added to the structure and docked using the RosettaLigand docking XML protocol104, to generate 5,000 models. The center of the two Lys97 NZ atoms in the two different units was used as the starting position of the ligand. To enforce the covalent binding, we defined covalent constraints for each binding site105, using the HARMONIC score function, centered at 0 and with a standard deviation of 0.3. Since each electrophilic warhead can bind to either Lys97, we used two sets of MultiConstraints within an AmbiguousConstraints block to allow both binding options. The selected model received a low atom_pair_constraint score (<4), which indicates that it fits well into the expected covalent bond geometry, and was ranked 4th best by interface score.

Inducible knockdown of KEAP1
Hairpins were prepared and cloned into the pLKO-Tet-On vector (Addgene #21915) as previously described106,107. Briefly, forward and reverse sequences were synthesized commercially (IDT), diluted in 10x annealing buffer (1M NaCl, 100mM Tris-HCl, pH 7.4), heated to 95°C to induce annealing, and allowed to cool to room temperature. Annealed oligos were diluted 1:400 in 0.5x annealing buffer before T4 ligation into gel-purified, AgeI- and EcoRI-digested pLKO-Tet-On, followed by transformation into Stbl3 bacteria and validation by Sanger sequencing for difficult templates (Eton Bioscience). They were then transfected into 293FT cells and transduced into drug-resistant NSCLC cells. After 48h of puromycin selection, puromycin was removed, and cells were cultured in their usual medium without doxycycline for at least five days. 24h prior to seeding, media were supplemented with doxycycline to a concentration of 100ng/μl. The next day, cells were seeded and allowed to adhere overnight. Once cells had adhered, they were treated with either vehicle or MCB-613 to a concentration of 1.25μM for either 2 or 4h before collection, lysis, and western blotting as described above.

GSH reactivity assay
Five microliters of a 20mM DMSO solution of the electrophile was added to a 1000μL solution of 1mM GSH in a 25mM HEPES pH=8 buffer (final electrophile concentration was 200μM). The reaction mixture was left at room temperature. At selected time points, a sample from the reaction mixture was mixed with a formic acid (FA) solution to obtain a final 0.4% of FA in order to quench the reaction. Samples were then injected into the LC/MS. Runs were performed on a Waters ACUITY UPLC BEH instrument in positive ion mode using electrospray ionization. UPLC separation used a C18 column (2.1 mm i.d., 50 mm length). The column was held at 40°C and the autosampler at 10°C. Mobile phase A was 0.1% FA in water, and mobile phase B was 0.1% FA in acetonitrile. The run flow was 0.4 mL/min. The gradient used was 99% A for 1min, then increasing linearly to 95% B for 5.5min, holding at 95% B for 0.75min, changing to 1% B and holding for 1min. Reaction progress quantification was performed by calculating the peak area of the starting material (unlabeled) at each time point, normalized to the starting time point (t=0). Reaction half-life values were derived from a one-phase exponential decay curve fit using Prism.

NAL reactivity assay
NAL (N-Acetyl-L-lysine methyl ester) was dissolved to 100mM in water and diluted to 2mM in a 25mM HEPES, 150mM NaCl, pH=8 buffer. Two microliters of a 10mM electrophile DMSO solution were diluted to 100μL using the prepared NAL solution and let react at room temperature. (Final concentration in the reaction mixture: 5mM NAC and 200μM electrophile.) Reaction was conducted at room temperature. At selected time points, 10μL samples were mixed with 35μL of 0.1% TFA and injected to LC/MS). Reaction progress quantification was performed similarly to described for the GSH assay.

NAC reactivity assay
Two microliters of a 10mM electrophile DMSO solution were diluted to 50μL with reaction buffer (NaPi 25mM, pH=7.5, 50% ACN). Five microliters of this sample and 5μL of reaction buffer were mixed with 35μL of 0.1% TFA in water for the t=0 sample. Then, NAC (N-Acetyl-L-cysteine methyl ester) was dissolved to 100mM in water and diluted to 2mM in the reaction buffer. Forty microliters of NAC solution was added to each sample (final concentration in the reaction mixture: 1mM NAC and 200μM electrophile.) Reaction was conducted at room temperature. At selected time points, 10μL samples were mixed with 35μL of 0.1% TFA and injected into LC/MS. Reaction progress quantification was performed similarly to described for the GSH assay.

DCFDA assay
Cells were seeded into 10-cm dishes and allowed to adhere overnight. The next day, media was aspirated, and cells were washed once with PBS before incubation with 5µM DCFDA for 30min at 37°C. After incubation, DCFDA was removed, and the cells were washed once more with PBS. Cells were then incubated in 1.25µM MCB-613 for 2h at 37°C before fluorescence detection on a Tecan Infinite M1000 plate reader. Background signal from media was subtracted, and treatment wells were normalized to vehicle controls.

Statistics and Reproducibility
Throughout the manuscript, statistical methods, statistical measures, and numbers of replicates and/or repeated experiments have been noted in the figure legends, as well as at times within the Methods. Although exact p-values are not reported in main text figures and/or figure legends due to space constraints, these can be found in the Source Data file. Exact p-values for Supplementary Figs. are reported within the Supplementary Fig. legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors have addressed most of my previous comments appropriately.\nMy only further suggestion is for them to consider/comment on how Keap1 oligomerization consequent to MCB-613 treatment could be causing ROS accumulation. Does Keap1 oligomerized Keap1 acquire a new function and/or lose the ability to interact with another protein? This is important, because in the revised manuscript, they suggest that depletion of Keap1 (and the consequent accumulation of Nrf2) does not cause ROS accumulation, and thus ROS accumulation is independent of loss of functional Keap1 per se. Or, does depletion of Keap1 affect the levels of ROS, which are not further affected by MCB-613 treatment? This is not clear from the way that the data are expressed (Suppl Fig. 7b.", "reviewer_3": "The authors have now addressed my comments satisfactorily and the manuscript should be accepted for publication.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Reviewer #1 (Remarks to the Author):\nThe authors have addressed most of my previous comments appropriately.\nMy only further suggestion is for them to consider/comment on how Keap1 oligomerization consequent to MCB-613 treatment could be causing ROS accumulation. Does oligomerized Keap1 acquire a new function and/or lose the ability to interact with another protein? This is important, because in the revised manuscript, they suggest that depletion of Keap1 (and the consequent accumulation of Nrf2) does not cause ROS accumulation, and thus ROS accumulation is independent of loss of functional Keap1 per se. Or, does depletion of Keap1 affect the levels of ROS, which are not further affected by MCB-613 treatment? This is not clear from the way that the data are expressed (Suppl Fig. 7b.\nWe agree with the reviewer that the exact mechanism between KEAP1 oligomerization and ROS accumulation remains unclear, and that further discussion of this would enhance the manuscript. To the section in lines 485-498, which speculates as to the link between KEAP1 oligomerization and ROS accumulation, we have added the following text: “This raises additional questions. Are there heretofore undescribed substrates of KEAP1 that are involved in this mechanism? Is it possible that MCB-613 binding begets neosubstrates—or even neomorphic functions—for KEAP1?”\nReviewer #2 (Remarks to the Author):\nThe structure of MCB-613 is not druglike and has structural elements that resemble common PAINS. Pleiotropic effects are likely as well. Although the authors mention limited selectivity and non-ideal PK properties of the compound class, it should be pointed out more clearly throughout the manuscript that this is not a druglike molecule. Interestingly, there is a certain structural similarity to curcumin. Are the authors sure that MCB-613 does not have similar PAINS effects in in-vitro and in-vivo assays (see e.g. DOI: 10.1021/acs.jmedchem.6b00975)?\nWhile we agree with the reviewer that MCB-613 is a PAINS-like compound that likely has pleiotropic effects, we believe that a full accounting of these effects is likely beyond the scope of the present manuscript. Thus, we have amended language throughout the manuscript to better reflect the PAINS-like nature of MCB-613. References to the probable PAINS-like nature of the compound are now included in the revised manuscript in lines 33, 102, 138, 173-186, and 429.\nThank you for your clarifications and rephrasing of the manuscript. However, phrasings like “Interestingly, several preliminary studies in other settings also speak to the use of this molecule and its derivatives as treatments for models of cardiovascular and nervous ischemia, studies which suggest that MCB-613 is generally well tolerated and, thus far, free from any obvious toxicities.“ (line 468 ff. in discussion), should be removed due to their potential misleading interpretation as MCB-613 being a drug like molecule. This is not the scope of the presented results/manuscript.\nWe agree with the reviewer and have further amended the language in the discussion. The use of the word “promising” to describe MCB-613 as a probe compound in line 462 has been amended to “instructive.” Additionally, the sentence which the reviewer has highlighted has been amended to read as follows: “Beyond lung cancer alone, several preliminary studies in other settings have also utilized this molecule to draw conclusions about cardiovascular and nervous ischemia.”\nThis study needs to be accompanied by lysine and cysteine reactivity assays. As is common best practice, the reactivity of all relevant compounds i.e. MCB-613, C151-0598 and ARM-143 needs to be quantified in GSH assays vs a common reference compound (see e.g. doi 10.1039/9781839160745-00069 by London and colleagues). Such data would also be essential to support the claim made on page 15 (“…suggesting that this molecule might be less promiscuous and thiol-reactive than initially assumed”). As the compounds are supposed to react with lysine an analogous Lys-reactivity assay (see e.g. doi 10.1039/C7OB02028G) should also be conducted.\nWe appreciate this suggestion by the reviewer, and agree that these studies are important for the evaluation of MCB-613. We have addressed this concern in the following ways: 1) we have tested MCB-613 in a standard GSH reactivity assay, in which the BTK inhibitor ibrutinib was included as a standard control/reference compound. This assay, which is now mentioned in lines 298-301 in the text and included as Supplementary Figure 5a, revealed that MCB-613 is highly cysteine-reactive and rapidly labels GSH. 2) We have also tested MCB-613 in a lysine reactivity assay using N-acetyl-L-lysine methyl ester with a model NHS-ester as a positive control. This assay, which is mentioned in lines 340-345 and included as Supplementary Figure 6a, revealed that MCB-613 shows high lysine reactivity.\nIn both of these studies, we also included ARM-143 and C151-0598. ARM-143, which is included in both figure panels, showed significantly less cysteine and lysine reactivity than MCB-613. This result is expected, as the loss of the dienone structure significantly reduces the conjugation of the molecule and, therefore, its reactivity. Despite this expected decrease in overall activity, however, Supplementary Figure 3h clearly demonstrates that ARM-143 labels MCB-613 and therefore functions as an effective control for MCB-613-induced dimer/oligomerization.\nC151-0598, though tested, is not included in the figure panels. Due to solubility issues with this compound, we were unable to obtain meaningful results from the cysteine and lysine reactivity assays.\nThank you for your clarification and the additional experiments. The authors should interpret these results with respect to the potential/limitations of a pharmacokinetic profile.\nWe agree with the reviewer that such an interpretation is important. We have added the following text to lines 302-303: “The fact that MCB-613, on the other hand, rapidly labels GSH in an in vitro assay further speaks to its tool-like and non-drug-like nature.”\nThe use of N-acetylcystein as ROS Scavenger is interesting in this context. Couldn't NAC quench the covalent warhead?\nWe agree with the reviewer that NAC is theoretically vulnerable to quenching by covalent compounds such as MCB-613. To address this concern, we have performed a NAC reactivity assay with MCB-613, ARM-3-115, positive control RA13 (a reactive chloro-acetamide-based fragment) and negative control ibrutinib. These data, which are included as Supplementary Figure 7a, definitively demonstrate that NAC is not quenched by MCB-613, suggesting that the rescues depicted in Figure 5 are on-target.\nThank you for your clarification and the additional experiments. It is interesting that NAC does not react while GSH does, which is most likely linked to the different assay conditions (pH and co-solvent). The possibility of interference of NAC with the electrophilic moiety should at least be mentioned in the manuscript.\nWe agree with the reviewer. This possibility is now discussed in lines 411-413: “(Although it cannot be definitively ruled out by these studies alone that NAC is working by directly interfering with MCB-613, the data shown here suggest against that possibility.)”\nIt is surprising that very little cysteine modification has been observed in chemoprotomics experiments. Could it be that these compounds react with cysteine in a reversible covalent manner making the interaction difficult to capture? The authors should assess this by experiments with model thiols.\nWe appreciate the reviewer’s point. We believe if target occupancy is sufficiently high, competitive displacement should be detectable even with reversible molecules. For example, non-covalent kinase inhibitors molecules are routinely used in kinome surveying experiments, even in the absence of covalent binding (PMID: 37904048).\nHowever, for ultra-reversible Micheal acceptors (i.e., 2-cyano), it is true that this does result in a diminished signal (PMID: 31222866).\nThe authors should include a corresponding discussion in the manuscript also raising the possibility of reversible covalent cysteine engagement.\nWe agree with the reviewer that the possibility of reversible covalent cysteine engagement should be discussed within the manuscript. We have added the following text to lines 308-314: “One explanation for these findings is that MCB-613 might covalently engage cysteine residues through a reversible mechanism, thus rendering detection difficult in assays such as those described here. Although this cannot be ruled out on the bases of these data alone, the fact that competitive displacement is often detectable even with non-covalent inhibitors led us to speculate instead that MCB-613’s activity might depend upon alternative residues within KEAP1.”\nReviewer #3 (Remarks to the Author):\nThe authors have now addressed my comments satisfactorily and the manuscript should be accepted for publication.\nWe thank the reviewer.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank the reviewer."}]

Decision letter
This is an interesting study showing that EGFR-mutant, EGFR-inhibitor-resistant non-small cell lung cancer cells (NSCLC) are hypersensitive to a small molecule compound, MCB-613. Using an elegant comparison with an inactive structural analog (C151-0598) and click chemistry, combined with proteomic and genomic screens, the authors identify Keap1 as a target for MCB-613, and further show that the double Michael acceptor functionality of MCB-613 acts as a molecular bridge, causing an oligomerization in Keap1, and that this is important for conferring sensitization. Surprisingly, the thiol reactivity of MCB-613 was low. Instead, the compound was found to require K97 in Keap1 for Keap1 oligomerization. They propose that Keap1 oligomerization in increases ROS production, causing ATF4/CHOP-mediated cell death.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author):
This is an interesting study showing that EGFR-mutant, EGFR-inhibitor-resistant non-small
cell lung cancer cells (NSCLC) are hypersensitive to a small molecule compound, MCB-
613. Using an elegant comparison with an inactive structural analog (C151-0598) and click
chemistry, combined with proteomic and genomic screens, the authors identify Keap1 as a
target for MCB-613, and further show that the double Michael acceptor functionality of
MCB-613 acts as a molecular bridge, causing an oligomerization in Keap1, and that this is
important for conferring sensitization. Surprisingly, the thiol reactivity of MCB-613 was low.
Instead, the compound was found to require K97 in Keap1 for Keap1 oligomerization. They
propose that Keap1 oligomerization in increases ROS production, causing ATF4/CHOP-
mediated cell death.
The authors should consider the following points:
• Fig.3b: Why does compound treatment cause an increase in fluorescence at the lower
temperature, and a slight decreases at the higher temperatures?
Although not necessarily intuitive, this pattern is commonly observed in thermal shift assays
and results from fluorescence quenching due to protein aggregation at high temperatures.
In our case, MCB-613 binds purified KEAP1 and destabilizes it, which exposed hydrophobic
residues. The fluorescent probe binds to these residues and fluoresces, and the machine
detects that fluorescence. Once the curve peaks and the protein has fully melted,
disorganized proteins aggregate. This obscures the hydrophobic residues from the probe,
leading to a loss of signal. Since the conditions with the highest concentrations of MCB-613
are the most destabilized, they are the first to quench and lose signal once they cross the
peak.
• Fig 3c and Suppl. Fig. 3b: the V5-tagged Keap1 appears mote thermostable than the
endogenous Keap1. What could be the reason? It will be good to show the corresponding
immunoblot for the endogenous Keap1, which is quantified in Suppl. Fig. 3b. Is the thermal
stability of the dimer affected by the concentration of MCB-613?
At the reviewer’s suggestion, we have included the immunoblot for endogenous KEAP1 as
Supplementary Figure 3c. With respect to the thermostability of endogenous vs. V5-tagged
KEAP1, there are a variety of possible explanations, as there several differences between
these assays. In the first place, the use of an ectopic promoter for V5-KEAP1 expression may
result in different amounts of total KEAP1 protein in these two experiments. The use of
different antibodies (anti-V5 vs anti-KEAP1) may also account for differences. The presence
of a V5 tag, though small, may influence thermostability. With respect to the thermostability
of dimers at varying concentrations, the majority of assays directed against the dimer in
question were run at 2 µM doses of MCB-613. While the question of thermostability and
MCB-613 concentration is interesting, we believe it is ultimately outside of the scope of the
present investigation.
• Fig. 4f: In addition to oligomerization, MCB-613 seems to cause an overall increase of
Keap1. This is in contrast to the endogenous Keap1 monomer, which appears to decrease
(e.g. Fig. 3d). What could be the reason for this difference?
We thank the reviewer for highlighting this difference. Although we cannot be certain about
the exact cause, we agree with the reviewer’s emphasis on the differences in transcriptional
control between these two assays. With respect to the KEAP1 monomer in Fig. 3d, for
example, protein expression is ultimately under control of an endogenous promoter which
is responsive to changing cellular conditions, and it is possible that proteotoxic stress
related to KEAP1 oligomerization may feed back to inhibit further transcription and protein
expression. By contrast, the protein levels in Fig. 4f reflect a GFP-tagged protein which is
driven off of an ectopic promoter which is not responsive to cellular feedback. Additionally,
the blots in Fig. 3d were detected using an anti-KEAP1 antibody, while those in Fig. 4f were
detected using anti-GFP antibody. Differential epitope exposure, especially in the setting of
dimerization and/or oligomerization, may also account for some of these differences.
• Does MCB-613 react with K97 in each monomer of dimeric Keap1? How do the authors
envision formation of the higher molecular weight species? What happens to these species?
We thank the reviewer for the question, and agree that there would be value in investigating
the exact, step-by-step mechanism involved in KEAP1 oligomerization by MCB-613. As now
discussed in the revised version of the manuscript (lines 345-348, 444-446), however, we
cannot definitively claim that MCB-613 interacts directly with KEAP1 at the K97 residues. In
order to support that claim, we would need to demonstrate direct engagement of the K97
residue by MCB-613 via e.g. lysine ABPP and MS, but our attempts to do so were
unsuccessful based on current limitations of the technology. Our data clearly demonstrate,
however, that K97 is required for KEAP1 oligomerization by MCB-613, whether or not it is
directly engaged.
As for the formation of higher molecular weight species, we speculate that, if K97 is directly
engaged by MCB-613, then MCB-613 may even act to cause “daisy chains” of KEAP1
monomers which form higher order oligomers.
• The levels of Nrf2 are similar in cells expressing Keap1-WT and Keap1-K97A mutant. Does
the level of Nrf2 change in Keap1-K97A mutant expressing cells treated with MCB-613?
We thank the reviewer for the question. We have performed this study, and have included
the results in a revised version of Supplementary Figure 6 (Supplementary Fig. 6e). As this
study shows, WZR12 cells which overexpress WT KEAP1 show a greater accumulation of
NRF2 than do cells which overexpress KEAP1 K97A. The residual NRF2 accumulation seen
in the latter setting is presumably due to the presence of endogenous wild-type KEAP1.
• The authors propose that Keap1 oligomerization by MCB-613 leads to ROS induction. How
does this happen? Which is the cellular source(s) of ROS under these conditions? Is the
generation of ROS caused by MCB-613 treatment really due to Keap1 oligomerization? Or, is
it an independent (from Keap1) effect of MCB-613. In Fig. 5j, the authors show nicely that
Keap1 depletion prevents the increase in ATF4 caused by MCB-613. Does Keap1 depletion
also prevent the increase in ROS caused by MCB-613 in the experiment shown in Fig. 5f?
If so, this will provide a better link among Keap1 oligomerization, ROS and ATF4. If not, it
will suggest that the increase in ATF4 is not due to Keap1 oligomerization and the increase in the
levels of ROS may be an independent effect of the compound.
We thank the reviewer for the proposed experiment, and agree that a demonstration of the
KEAP1 dependence of MCB-induced ROS would further enhance the claims presented in the
study. We have performed this study, and included the results in Supplementary Figure 7b,
which clearly shows that the loss of KEAP1 abrogates ROS accumulation after treatment
with MCB-613, demonstrating that this effect is dependent upon KEAP1.
• Why does the sensitization occur only in EGFR inhibitor-resistant cells? Could GCN2 be
involved?
We thank the reviewer for the question. As detailed in lines 389-412, we believe that EGFR-
inhibitor-resistant cells are sensitized to MCB-613 by their high levels of ATF4 and CHOP,
which arise as a consequence of the acquisition of drug resistance. The reviewer is correct
to point out that GCN2 is one of the kinases which phosphorylates eIF2a and drives ATF4
expression. In data which we have not included in the present manuscript (due chiefly to
space constraints), we have knocked out each of the four kinases which feed into the ISR:
GCN2, PERK, PKR, and HRI. None of these knockouts was sufficient to rescue the effect of
MCB-613. This may be because there are overlapping inputs which would require
combinatorial knockouts of some combination of these kinases, or because there is an
alternative mechanism at play. We believe that further investigation of this question, while
certainly interesting, falls outside of the scope of the present manuscript.
• The authors suggest that MCB-613 may cause general proteotoxic stress. Do they see
activation of the heat shock response? Knowing this may help to establish/test for
selectivity.
At the reviewer’s suggestion, we have incorporated data demonstrating that MCB-613 does
not induce activation of the heat shock response. Specifically, we tested PC9, GR4, and
WZR12 cells against standard doses of MCB-613 over multiple timepoints. As expected,
MCB-613 selectively induced ATF4 expression in the drug-resistant derivatives, but not in the
parental cells. However, no increase in HSP60, HSP70, or HSP90 was observed in any of the
cell lines. These data are now included in Supplementary Figure 6g.
• Lines 238- 242: The authors state: “Although prior reports have shown that structurally
diverse electrophiles can oligomerize KEAP1, MCB-613 seems to be unique in that it
induces the formation of a complex which persists even in the face of stringent reducing
conditions, suggesting that it does not arise as a result of non-covalent protein-protein
interactions or disulfide bridges”. This mechanism is not unique to MCB-613. For example,
methylglyoxal has been shown to modify Keap1 by forming a covalent methylimidazole
crosslink between proximal cysteine and arginine residues, which also persists under
reducing conditions (Bollong et al. Nature 2018; 562: 600-604).
We thank the reviewer for this correction, and we have amended the manuscript to reflect
these details (revised manuscript lines 250-251).
• Lines 343- 345: The authors state: “In the presence of oxidative or electrophilic stress,
however, KEAP1 dissociates from substrates and allows for their rapid cellular
accumulation.” This is a common misconception. Neither oxidants, nor electrophiles
cause dissociation of Nrf2 from Keap1; only protein-protein interaction inhibitors do this
(e.g. see Horie et al. Commun Biol. 2021 May 14;4(1):576; Baird et al. Proc Natl Acad Sci U
S A. 2013 Sep 17;110(38):15259-64). The oxidant/electrophile-mediated modification of
Keap1 disables its substrate adaptor function, allowing accumulation of newly synthesized
Nrf2.
We thank the reviewer for this clarification, and have corrected this error in the revised
version of the manuscript.
Reviewer #2 (Remarks to the Author):
In this study Bassil et al. describe their investigations of EGFR-therapy resistant cell lines
with hypersensitivity to compound MCB-613 in a KEAP1-dependent manner. Initially, based
on a compound library of 2,100 molecules they found this molecule acting as a covalent,
lysine-addressing binder of KEAP1, which leads to the dimerization of two KEAP1
monomers and subsequent cellular dysfunctions causing ATF4/CHOP-dependent cell
death. It’s noteworthy that this effect is especially observable in mutated cell lines or
xenografts and apparently to a lesser amount to wildtype EGFR, which is of high interest for
follow-up therapies for current EGFR related cancer treatments.
Their biological examinations and discussion are of high relevance and generally well
structured and conclusive. The discussion of MCB-613 as proof-of-concept molecule for
deeper understanding of the relationship between KEAP1/NFE2L2/CUL3 and EGFR
inhibitor resistance is fair, as long as the compound is not stated as a druglike molecule
due to it’s PAINS-like structure and missing PK data.
Some major and minor issues questions should be addressed (major revision):
1) Is the structure of ARM-143 correctly drawn? (Supp. Fig. 3d.)
We thank the reviewer for the question, and confirm that the structure is drawn correctly.
Although we had originally intended to prepare the strict mono-unsaturated analog of MCB-
613, we discovered during synthesis that the double bond isomerizes from the expected
exocyclic to the unanticipated endocyclic position. We believe that both Supplementary
Figure 3d and the language of the text (“…a closely related structural analogue of MCB-613
that harbors only one α, β-unsaturated bond”) are consistent with this.
2) The structure of MCB-613 is not druglike and has structural elements that resemble
common PAINS. Pleiotropic effects are likely as well. Although the authors mention limited
selectivity and non-ideal PK properties of the compound class, it should be pointed out more
clearly throughout the manuscript that this is not a druglike molecule.
Interestingly, there is a certain structural similarity to curcumin. Are the authors sure that
MCB-613 does not have similar PAINS effects in in-vitro and in-vivo assays (see e.g. DOI:
10.1021/acs.jmedchem.6b00975)?
While we agree with the reviewer that MCB-613 is a PAINS-like compound that likely has
pleiotropic effects, we believe that a full accounting of these effects is likely beyond the
scope of the present manuscript. Thus, we have amended language throughout the
manuscript to better reflect the PAINS-like nature of MCB-613. References to the probable
PAINS-like nature of the compound are now included in the revised manuscript in lines 33,
102, 138, 173-186, and 429.
3) The authors investigate the binding site of MCB-613 within the KEAP1 protein with a HA-
clover system and sequential elongation of the peptides. Activity can is traced back to an
amino acid between residues 90-100, and ultimately K97. Replacement of this lysine with a
non-isostructural alanine (K97A) leads to reduced oligomerization when treated with MCB-
613.
Could the authors perform a mass labeling experiment to proof the covalency of the
investigated interaction? Their applied method sounds plausible, but the final argument is
missing here.
We appreciate the reviewer’s suggestion. To assess target engagement, we optimized and
performed lysine-ABPP; however, detecting engagement at this particular lysine residue
remains technically challenging. In our experiments, the relevant peptide was not observed,
and consequently no definitive engagement signal was detected. Based on our review of the
current state of lysine-ABPP, to our knowledge, no lysine abpp probe has yet been
successfully implemented to detect this lysine. This may be due to a combination of factors
(e.g., poor peptide ionization, limitations in probe design). However, it did shed light on the
low global lysine reactivity MCB-613. We have amended the text in lines 345-348 and 444-446
to reflect this limitation of the current study.
For the reviewer’s convenience, here are references to four other lysine ABPP studies, none
of which were able to detect the peptide in question:
• https://doi.org/10.1038/s41557-021-00765-4
• https://doi.org/10.1021/jacs.4c09073
• https://doi.org/10.1039/d3sc05766f
• https://doi.org/10.1002/anie.202418473
These reports are all now cited in the revised manuscript at lines 345-348.
4) This study needs to be accompanied by lysine and cysteine reactivity assays. As is
common best practice, the reactivity of all relevant compounds i.e. MCB-613, C151-0598
and ARM-143 needs to be quantified in GSH assays vs a common reference compound
(see e.g. doi 10.1039/9781839160745-00069 by London and colleagues). Such data would
also be essential to support the claim made on page 15 (“…suggesting that this molecule might
be less promiscuous and thiol-reactive than initially assumed”). As the compounds are
supposed to react with lysine an analogous Lys-reactivity assay (see e.g. doi
10.1039/C7OB02028G) should also be conducted.
We appreciate this suggestion by the reviewer, and agree that these studies are important
for the evaluation of MCB-613. We have addressed this concern in the following ways: 1) we
have tested MCB-613 in a standard GSH reactivity assay, in which the BTK inhibitor
ibrutinib was included as a standard control/reference compound. This assay, which is
now mentioned in lines 298-301 in the text and included as Supplementary Figure 5a,
revealed that MCB-613 is highly cysteine-reactive and rapidly labels GSH. 2) We have also
tested MCB-613 in a lysine reactivity assay using N-acetyl-L-lysine methyl ester with a
model NHS-ester as a positive control. This assay, which is mentioned in lines 340-345 and
included as Supplementary Figure 6a, revealed that MCB-613 shows high lysine reactivity.
In both of these studies, we also included ARM-143 and C151-0598. ARM-143, which is
included in both figure panels, showed significantly less cysteine and lysine reactivity than
MCB-613. This result is expected, as the loss of the dienone structure significantly reduces
the conjugation of the molecule and, therefore, its reactivity. Despite this expected
decrease in overall activity, however, Supplementary Figure 3h clearly demonstrates that
ARM-143 labels MCB-613 and therefore functions as an effective control for MCB-613-
induced dimer/oligomerization.
C151-0598, though tested, is not included in the figure panels. Due to solubility issues with
this compound, we were unable to obtain meaningful results from the cysteine and lysine
reactivity assays.
5) The use of N-acetylcystein as ROS Scavenger is interesting in this context. Couldn't NAC
quench the covalent warhead?
We agree with the reviewer that NAC is theoretically vulnerable to quenching by covalent
compounds such as MCB-613. To address this concern, we have performed a NAC reactivity
assay with MCB-613, ARM-3-115, positive control RA13 (a reactive chloro-
acetamide-based fragment) and negative control ibrutinib. These data, which are included
as Supplementary Figure 7a, definitively demonstrate that NAC is not quenched by MCB-
613, suggesting that the rescues depicted in Figure 5 are on-target.
6) It is surprising that very little cysteine modification has been observed in chemoprotomics experiments. Could it be that these compounds react with cysteine in a reversible covalent manner making the interaction difficult to capture? The authors should assess this by experiments with model thiols.
We appreciate the reviewer’s point. We believe if target occupancy is sufficiently high,
competitive displacement should be detectable even with reversible molecules. For
example, non-covalent kinase inhibitors molecules are routinely used in kinome surveying
experiments, even in the absence of covalent binding (PMID: 37904048). For
example, non-covalent kinase inhibitors molecules are routinely used in kinome surveying
experiments, even in the absence of covalent binding (PMID: 37904048). However, for
ultra-reversible Micheal acceptors (i.e., 2-cyano), it is true that this does result in a
diminished signal (PMID: 31222866).
7) Is there clear evidence that the presumed reaction with lysine proceeds though a double Michael addition? Reversible covalent imine formation followed by Michael addition may also be a potential mechanism.
Although double Michael addition is a possible reaction mechanism, the reviewer is correct
that clear evidence for this is not provided, and alternative mechanisms, such as reversible
covalent imine formation followed by Michael addition, are also possible. Given this, we
have amended the language throughout the manuscript to avoid favoring one mechanism
over another.
8) Other studies that describe MCB-613 (partially cited here as well) traced back the effect
of EGFR-resistance on TP53 gain-of-function mutations (https://doi.org/10.1038/s41467-
018-03599-w, https://doi.org/10.1038/s41698-024-00557-2) and also described it e.g., for
curcumin with high structural similarities (https://doi.org/10.1016/j.bbrc.2023.08.016).) or
outlined a relationship to the steroid receptor coactivator (10.1016/j.ccell.2015.07.005),
and subsequential the generation of ROS. Can the authors cross check and provide
evidence, that their observed effects in EGFR TKI resistant cell lines do not
(also/partially/completely) stem from other correlations? This would be of high importance
since the KEAP1-dependency phenomenon of EGFR-mutant NSCLCs remains
fragmentated, as mentioned by the authors.
We agree with the reviewer that further investigation of the previously described targets of
MCB-613, including SRC3 and p53 (via USP15) is warranted and would enhance the strength
of the manuscript. We have addressed this comment by including data demonstrating that
knockout of either SRC3 or p53 fails to rescue the effect of MCB-613 in EGFR-mutant, EGFR-
inhibitor resistant NSCLC cells. These data are now included in a revised Supplementary
Figure S2 (S2a-f) and are addressed in the revised manuscript in lines 173-180.
9) The authors should depict the chemical structures of the KEAP1 inhibitors used for
validation in Supplementary Figure 4.
We agree with the reviewer that the manuscript would benefit from the inclusion of these
structures, which are now supplied as Supplementary Figure 4b.
10) The structures included in the 2,100 compound commercial screening library should
be added as supplementary information as SMILES strings
We agree with the reviewer that the manuscript would benefit from the inclusion of these
structures, which are now supplied in Supplementary Table 1.
Reviewer #3 (Remarks to the Author):
The work by Bassil et al. clearly demonstrates that EGFR-driven NSCLC cell lines with
developed resistance to EGFR inhibitors show preferential sensitivity to the compound
MCB-613. They also outline the molecular mechanisms through which this sensitisation
occurs which involves drug-induced dimerisation of KEAP1 and associated IRS. The data
are of very high quality, the authors perform adequate rescue experiements for the
observed phenotypes and their conclusions are adequately supported by the results
presented.
The paper is very well written and clear and the figures very well presented.
The data are of great interest, both from a biological point of view and from a clinical
perspective as the authors propose a novel therapeutic strategy for the treatment of TKI-
resistant EGFR-driven NSCLC. Hence, further translation of their findings could be
transformative.
It is my opinion that the manuscript is acceptable for publication in its current state, but I
would suggest that the authors make the following minor amendments:
- Fig 2g: Statistical assessment of the difference between the curves would be required. If
the differences between consitions are not significant, the authors should clarify this in the
text by stating that they are discussing "non-significant trends". The possible absence of
statistical significance in this precise experiment is not damaging to the rest of the
manuscript, and should therefore not be a concern to recognise if such.
We agree with the reviewer that statistical analysis would be informative. Due to the
statistical challenge inherent in analyzing time series data such as those presented, we
elected to conduct a one-way ANOVA comparison of the final time point (D18) for each of
the four cell lines. This analysis demonstrated statistical significance for each of the three
resistant derivatives (PFR3, GR4, and WZR12) when compared with the parental PC9 cells.
P-values are now included in Figure 2g, and the figure legend has been updated to reflect this
statistical testing.
- Fig 4f: Could the authors suggest an explanation for where the residual dimerisation might
be coming from in the K97A + MCB-613 condition? Mention/explanation of this in the
Discussion section of the manuscript would be welcome.
The reviewer highlights an interesting finding: in certain assays, we observe either residual
dimerization in the setting of KEAP1 K97A expression (Figure 4f), or we observe multiple
oligomer bands (Figure 3h, Figure 4b, Figure 4d). Importantly, these findings always occur
with expression of ectopic KEAP1 against a background of endogenous KEAP1 expression. It
remains unclear whether these bands occur as a result of higher order KEAP1 oligomers
(trimers, et cetera), as a result of differential combinations of wild-type and tagged KEAP1,
or both. These possibilities are now presented in the Discussion (lines 452-456 in the revised
manuscript).
- Line 350-353: Further to the point that the authors are making here and also directly
relevant to TKI resistance in EGFR-driven NSCLC, a manuscript by Roy et al. in Cell Discovery
in 2016 described decreased NRF2 activity and in GSH levels being associated with
resistance to first-generation EGFR TKIs. Citing this paper here would tie up their reasoning
more tightly with the resistance phenotype that they investigate.
We thank the reviewer for highlighting this publication, and agree that its inclusion further
strengthens our findings. This paper is now cited in the location the reviewer recommended
(lines 367-369 in the revised version of the manuscript).
Reviewer #4 (Remarks to the Author):
I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This
is part of the Nature Communications initiative to facilitate training in peer review and to
provide appropriate recognition for Early Career Researchers who co-review manuscripts.
Reviewer #1 (Remarks to the Author):
The authors have addressed most of my previous comments appropriately.
My only further suggestion is for them to consider/comment on how Keap1 oligomerization
consequent to MCB-613 treatment could be causing ROS accumulation. Does
oligomerized Keap1 acquire a new function and/or lose the ability to interact with another
protein? This is important, because in the revised manuscript, they suggest that depletion
of
Keap1 (and the consequent accumulation of Nrf2) does not cause ROS accumulation,
and thus ROS accumulation is independent of loss of functional Keap1 per se. Or, does
depletion of Keap1 affect the levels of ROS, which are not further affected by MCB-613
treatment? This is not clear from the way that the data are expressed (Suppl Fig. 7b.
We agree with the reviewer that the exact mechanism between KEAP1 oligomerization and
ROS accumulation remains unclear, and that further discussion of this would enhance the
manuscript. To the section in lines 485-498, which speculates as to the link between
KEAP1 oligomerization and ROS accumulation, we have added the following text: “This
raises additional questions. Are there heretofore undescribed substrates of KEAP1 that are
involved in this mechanism? Is it possible that MCB-613 binding begets neosubstrates—or
even neomorphic functions—for KEAP1?”
Reviewer #2 (Remarks to the Author):
The structure of MCB-613 is not druglike and has structural elements that resemble
common PAINS. Pleiotropic effects are likely as well. Although the authors mention limited
selectivity and non-ideal PK properties of the compound class, it should be pointed out
more clearly throughout the manuscript that this is not a druglike molecule. Interestingly,
there is a certain structural similarity to curcumin. Are the authors sure that MCB-613
does not have similar PAINS effects in in-vitro and in-vivo assays (see e.g. DOI:
10.1021/acs.jmedchem.6b00975)?
While we agree with the reviewer that MCB-613 is a PAINS-like compound that likely has
pleiotropic effects, we believe that a full accounting of these effects is likely beyond the
scope of the present manuscript. Thus, we have amended language throughout the
manuscript to better reflect the PAINS-like nature of MCB-613. References to the probable
PAINS-like nature of the compound are now included in the revised manuscript in lines 33,
102, 138, 173-186, and 429.
Thank you for your clarifications and rephrasing of the manuscript. However, phrasings like
“Interestingly, several preliminary studies in other settings also speak to the use of this
molecule and its derivatives as treatments for models of cardiovascular and nervous
ischemia, studies which suggest that MCB-613 is generally well tolerated and, thus far, free
from any obvious toxicities.“ (line 468 ff. in discussion), should be removed due to their
potential misleading interpretation as MCB-613 being a drug like molecule. This is not the
scope of the presented results/manuscript.
We agree with the reviewer and have further amended the language in the discussion. The
use of the word “promising” to describe MCB-613 as a probe compound in line 462 has
been amended to “instructive.” Additionally, the sentence which the reviewer has
highlighted has been amended to read as follows: “Beyond lung cancer alone, several
preliminary studies in other settings have also utilized this molecule to draw conclusions
about cardiovascular and nervous ischemia.”
This study needs to be accompanied by lysine and cysteine reactivity assays. As is
common best practice, the reactivity of all relevant compounds i.e. MCB-613, C151-0598
and ARM-143 needs to be quantified in GSH assays vs a common reference compound
(see e.g. doi 10.1039/9781839160745-00069 by London and colleagues). Such data would
also be essential to support the claim made on page 15 (“…suggesting that this molecule
might be less promiscuous and thiol-reactive than initially assumed”). As the compounds
are supposed to react with lysine an analogous Lys-reactivity assay (see e.g. doi
10.1039/C7OB02028G) should also be conducted.
We appreciate this suggestion by the reviewer, and agree that these studies are important
for the evaluation of MCB-613. We have addressed this concern in the following ways: 1)
we have tested MCB-613 in a standard GSH reactivity assay, in which the BTK inhibitor
ibrutinib was included as a standard control/reference compound. This assay, which is
now mentioned in lines 298-301 in the text and included as Supplementary Figure 5a,
revealed that MCB-613 is highly cysteine-reactive and rapidly labels GSH. 2) We have also
tested MCB-613 in a lysine reactivity assay using N-acetyl-L-lysine methyl ester with a
model NHS-ester as a positive control. This assay, which is mentioned in lines 340-345 and
included as Supplementary Figure 6a, revealed that MCB-613 shows high lysine reactivity.
In both of these studies, we also included ARM-143 and C151-0598. ARM-143, which is
included in both figure panels, showed significantly less cysteine and lysine reactivity than
MCB-613. This result is expected, as the loss of the dienone structure significantly reduces
the conjugation of the molecule and, therefore, its reactivity. Despite this expected
decrease in overall activity, however, Supplementary Figure 3h clearly demonstrates that
ARM-143 labels MCB-613 and therefore functions as an effective control for MCB-613-
induced dimer/oligomerization.
C151-0598, though tested, is not included in the figure panels. Due to solubility issues with
this compound, we were unable to obtain meaningful results from the cysteine and lysine
reactivity assays.
Thank you for your clarification and the additional experiments. It is interesting that NAC
does not react while GSH does, which is most likely linked to the different assay conditions
(pH and co-solvent). The possibility of interference of NAC with the electrophilic moiety
should at least be mentioned in the manuscript.
We agree with the reviewer. This possibility is now discussed in lines 411-413: “(Although it
cannot be definitively ruled out by these studies alone that NAC is working by directly
interfering with MCB-613, the data shown here suggest against that possibility.)”
It is surprising that very little cysteine modification has been observed in chemoprotomics experiments. Could it be that these compounds react with cysteine in a reversible covalent manner making the interaction difficult to capture? The authors should assess this by experiments with model thiols.
We appreciate the reviewer’s point. We believe if target occupancy is sufficiently high,
competitive displacement should be detectable even with reversible molecules. For
example, non-covalent kinase inhibitors molecules are routinely used in kinome surveying
experiments, even in the absence of covalent binding (PMID: 37904048).
However, for ultra-reversible Micheal acceptors (i.e., 2-cyano), it is true that this does result
in a diminished signal (PMID: 31222866).
The authors should include a corresponding discussion in the manuscript also raising the
possibility of reversible covalent cysteine engagement.
We agree with the reviewer that the possibility of reversible covalent cysteine engagement
should be discussed within the manuscript. We have added the following text to lines 308-314: “One explanation for these findings is that MCB-613 might covalently engage cysteine residues through a reversible mechanism, thus rendering detection difficult in assays such as those described here. Although this cannot be ruled out on the bases of these data alone, the fact that competitive displacement is often detectable even with non-covalent inhibitors led us to speculate instead that MCB-613’s activity might depend upon alternative residues within KEAP1.”
Reviewer #3 (Remarks to the Author):
The authors have now addressed my comments satisfactorily and the manuscript should
be accepted for publication.
We thank the reviewer.
Reviewer #4 (Remarks to the Author):
I co-reviewed this manuscript with one of the reviewers who provided the listed reports.
This is part of the Nature Communications initiative to facilitate training in peer review and
to provide appropriate recognition for Early Career Researchers who co-review
manuscripts.
We thank the reviewer.