Ternary complex dissociation kinetics contribute to mutant-selective EGFR degradation.

Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life strongly correlates with processive ubiquitination with purified components and mutant-selective degradation in cells. We present cryoelectron microscopy and hydrogen-deuterium exchange mass spectroscopy data on wild-type and mutant EGFR ternary complexes, which demonstrate that potent target degradation can be achieved in the absence of stable compound-induced protein-protein interactions. These results highlight the importance of considering target conformation during degrader development as well as leveraging heterobifunctional ligand binding kinetics to achieve robust target degradation.

CRAF dimerization with ARAF regulates KRAS-driven tumor growth.

KRAS, which is mutated in ∼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulating MAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit.

Structure-based optimization of hydroxylactam as potent, cell-active inhibitors of lactate dehydrogenase.

Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.

Identifying transcriptional programs underlying cancer drug response with TraCe-seq.

Genetic and non-genetic heterogeneity within cancer cell populations represent major challenges to anticancer therapies. We currently lack robust methods to determine how preexisting and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA sequencing (scRNA-seq), we have developed tracking differential clonal response by scRNA-seq (TraCe-seq). TraCe-seq is a method that captures at clonal resolution the origin, fate and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual epidermal growth factor receptor (EGFR) inhibitor-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. We identified a loss of antigrowth activity associated with targeted degradation of EGFR protein and an essential role of the endoplasmic reticulum (ER) protein processing pathway in anti-EGFR therapeutic efficacy. Our results suggest that targeted degradation is not always superior to enzymatic inhibition and establish TraCe-seq as an approach to study how preexisting transcriptional programs affect treatment responses.

RTK-Dependent Inducible Degradation of Mutant PI3Kα Drives GDC-0077 (Inavolisib) Efficacy.

PIK3CA is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer. SIGNIFICANCE: The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in PIK3CA-mutant tumors.See related commentary by Vanhaesebroeck et al., p. 20.This article is highlighted in the In This Issue feature, p. 1.

The promise and peril of KRAS G12C inhibitors.

RAS mutant tumors have been largely refractory to therapies until now. Recent findings published in the New England Journal of Medicine show that sotorasib provides clinical benefit for KRAS p.G12C-mutated non-small-cell lung cancer (NSCLC) and provide mechanistic insights into acquired resistance to KRASG12C-specific inhibition.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.

ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma.

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.

Emerging Trends in Cancer Drug Discovery-From Drugging the "Undruggable" to Overcoming Resistance.

Technology advancement and the courage to challenge dogma have been key elements that have continuously shifted druggability limits. We illustrate this notion with several recent cancer drug-discovery examples, while also giving an outlook on the opportunities offered by newer modalities such as chemically induced proximity and direct targeting of RNA. Treatment resistance is a major impediment to the goal of durable efficacy and cure, but the confluence of new biological insights, novel drug modalities, and drug combinations is predicted to enable transformative progress in this decade and beyond.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

Machine-Learning and Chemicogenomics Approach Defines and Predicts Cross-Talk of Hippo and MAPK Pathways.

Hippo pathway dysregulation occurs in multiple cancers through genetic and nongenetic alterations, resulting in translocation of YAP to the nucleus and activation of the TEAD family of transcription factors. Unlike other oncogenic pathways such as RAS, defining tumors that are Hippo pathway-dependent is far more complex due to the lack of hotspot genetic alterations. Here, we developed a machine-learning framework to identify a robust, cancer type-agnostic gene expression signature to quantitate Hippo pathway activity and cross-talk as well as predict YAP/TEAD dependency across cancers. Further, through chemical genetic interaction screens and multiomics analyses, we discover a direct interaction between MAPK signaling and TEAD stability such that knockdown of YAP combined with MEK inhibition results in robust inhibition of tumor cell growth in Hippo dysregulated tumors. This multifaceted approach underscores how computational models combined with experimental studies can inform precision medicine approaches including predictive diagnostics and combination strategies. SIGNIFICANCE: An integrated chemicogenomics strategy was developed to identify a lineage-independent signature for the Hippo pathway in cancers. Evaluating transcriptional profiles using a machine-learning method led to identification of a relationship between YAP/TAZ dependency and MAPK pathway activity. The results help to nominate potential combination therapies with Hippo pathway inhibition.This article is highlighted in the In This Issue feature, p. 521.

Author Correction: RAS-targeted therapies: is the undruggable drugged?

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dimerization Induced by C-Terminal 14-3-3 Binding Is Sufficient for BRAF Kinase Activation.

The Ras-RAF-MEK-ERK signaling axis, commonly mutated in human cancers, is highly regulated to prevent aberrant signaling in healthy cells. One of the pathway modulators, 14-3-3, a constitutive dimer, induces RAF dimerization and activation by binding to a phosphorylated motif C-terminal to the RAF kinase domain. Recent work has suggested that a C-terminal "DTS" region in BRAF is necessary for this 14-3-3-mediated activation. We show that the catalytic activity and ATP binding affinity of the BRAF:14-3-3 complex is insensitive to the presence or absence of the DTS, while the ATP sites of both BRAF molecules are identical and available for binding. We also present a crystal structure of the apo BRAF:14-3-3 complex showing that the DTS is not required to attain the catalytically active conformation of BRAF. Rather, BRAF dimerization induced by 14-3-3 is the key step in activation, allowing the active BRAF:14-3-3 tetramer to achieve catalytic activity comparable to the constitutively active oncogenic BRAF V600E mutant.

RAS-targeted therapies: is the undruggable drugged?

RAS (KRAS, NRAS and HRAS) is the most frequently mutated gene family in cancers, and, consequently, investigators have sought an effective RAS inhibitor for more than three decades. Even 10 years ago, RAS inhibitors were so elusive that RAS was termed 'undruggable'. Now, with the success of allele-specific covalent inhibitors against the most frequently mutated version of RAS in non-small-cell lung cancer, KRASG12C, we have the opportunity to evaluate the best therapeutic strategies to treat RAS-driven cancers. Mutation-specific biochemical properties, as well as the tissue of origin, are likely to affect the effectiveness of such treatments. Currently, direct inhibition of mutant RAS through allele-specific inhibitors provides the best therapeutic approach. Therapies that target RAS-activating pathways or RAS effector pathways could be combined with these direct RAS inhibitors, immune checkpoint inhibitors or T cell-targeting approaches to treat RAS-mutant tumours. Here we review recent advances in therapies that target mutant RAS proteins and discuss the future challenges of these therapies, including combination strategies.

Negative regulation of RAF kinase activity by ATP is overcome by 14-3-3-induced dimerization.

The RAS-RAF-MEK-ERK signaling axis is frequently activated in human cancers. Physiological concentrations of ATP prevent formation of RAF kinase-domain (RAFKD) dimers that are critical for activity. Here we present a 2.9-Å-resolution crystal structure of human BRAFKD in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that induce an inactive, monomeric conformation of BRAFKD. We also determine how 14-3-3 relieves the negative regulatory effect of ATP through a 2.5-Å-resolution crystal structure of the BRAFKD-14-3-3 complex, in which dimeric 14-3-3 enforces a dimeric BRAFKD assembly to increase BRAF activity. Our data suggest that most oncogenic BRAF mutations alter interactions with ATP and counteract the negative effects of ATP binding by lowering the threshold for RAF dimerization and pathway activation. Our study establishes a framework for rationalizing oncogenic BRAF mutations and provides new avenues for improved RAF-inhibitor discovery.

Partners in Crime: Clandestine Operations among RAS-RAF Accomplices in Promoting Tumorigenesis.

In this issue of Molecular Cell, Terrell et al. (2019) characterize the interactions of RAS-RAF complexes in live cells. Using bioluminescence resonance energy transfer (BRET) technology, they show that individual RAF family members exhibit distinct binding affinities to each RAS family protein, with CRAF exhibiting high binding affinities for all RAS proteins and BRAF exhibiting increased binding affinity for KRAS.

Pharmacological Induction of RAS-GTP Confers RAF Inhibitor Sensitivity in KRAS Mutant Tumors.

Targeting KRAS mutant tumors through inhibition of individual downstream pathways has had limited clinical success. Here we report that RAF inhibitors exhibit little efficacy in KRAS mutant tumors. In combination drug screens, MEK and PI3K inhibitors synergized with pan-RAF inhibitors through an RAS-GTP-dependent mechanism. Broad cell line profiling with RAF/MEK inhibitor combinations revealed synergistic efficacy in KRAS mutant and wild-type tumors, with KRASG13D mutants exhibiting greater synergy versus KRASG12 mutant tumors. Mechanistic studies demonstrate that MEK inhibition induced RAS-GTP levels, RAF dimerization and RAF kinase activity resulting in MEK phosphorylation in synergistic tumor lines regardless of KRAS status. Taken together, our studies uncover a strategy to rewire KRAS mutant tumors to confer sensitivity to RAF kinase inhibition.