Ras proteins are functionally dependent on one or more lipid modifications.1,2 The dynamic palmitoylation of N-Ras by DHHC palmitoyl acyltransferases and depalmitoylation by ABHD17 serine hydrolases is essential for the growth of NRAS-mutant acute myeloid leukemia (AML) cells.3-6 Here we show that ABD778, an in vivo-active ABHD17 inhibitor, selectively reduces the growth of NRAS-mutant AML and melanoma cell lines and is synergistic with the MEK inhibitor PD0325901 (PD901; mirdametinib). Mechanistically, ABD778 and PD901 induce deep and durable suppression of mitogen activated protein kinase (MAPK) pathway activation. Co-treatment extended the survival of mice transplanted with NrasG12D AMLs, which acquired by-pass mutations at relapse that conferred drug resistance and restored MAPK activation. ABD778 augmented the anti-leukemia activity of PI3 kinase, pan-Ras tri-complex, and FLT3 inhibitors, and restored gilteritinib sensitivity in a patient-derived xenograft model of FLT3 inhibitor resistance. These studies validate the palmitoylation cycle as a therapeutic target in NRAS-mutant cancers.

The RASopathies are a group of congenital disorders with overlapping clinical manifestations that are caused by pathogenic germline or early somatic variants that result in the hyperactivation of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway. Given the heterogeneous clinical presentations of these disorders that involve abnormalities across multiple organ systems, multidisciplinary clinical management and progress in scientific research are essential for optimal patient diagnosis and care. The 9th International RASopathies Symposium, a biennial meeting, was organized by the patient advocacy group RASopathies Network and showcased recent discoveries, case studies, and advances in preclinical research. Participants, who included scientists, clinicians, industry representatives, patients, and family advocates, explored knowledge gaps, innovative clinical approaches, and lived experiences of individuals with a RASopathy. Sessions centered around organ systems were introduced with a patient perspective to highlight the burden of disease, continued with presentations from established and early-career investigators. Overall, the RASopathies Symposia serve as a catalyst for sustained community collaboration focused on enhancing patient health and accelerating the translation of discoveries into effective treatments.

Abstract Although KRASG12C-specific inhibitors have been introduced, no approved targeted therapies exist for other clinically significant KRAS mutants, including KRASG12D and KRASG12V. We discovered BBO-11818, a potent, selective, orally bioavailable noncovalent pan-KRAS inhibitor capable of targeting multiple KRAS mutants in both the inactive GDP-bound (OFF) and active GTP-bound (ON) states. BBO-11818 binds in the Switch-II/Helix 3 pocket, inducing conformational changes incompatible with effector binding, and demonstrates high-affinity binding to mutant KRAS with strong selectivity over NRAS and HRAS. BBO-11818 potently inhibited MAPK signaling and cellular viability specifically in KRAS-driven lines and produced tumor regressions in KRAS-mutant xenograft models. Combination studies with anti–PD-1, anti-EGFR antibodies, and a RAS:PI3Kα breaker compound showed enhanced efficacy. BBO-11818 has entered phase I clinical trials for patients with various KRAS mutations in colorectal, pancreatic, and lung cancers (NCT06917079). Significance: We discovered BBO-11818, a potent and selective noncovalent KRAS inhibitor with activity against multiple KRAS mutants in both the active (ON) and inactive (OFF) states. BBO-11818 addresses the need for KRAS inhibitors targeting clinically relevant mutants such as KRASG12D and KRASG12V, either as monotherapy or in combination.

Abstract RAS GTPases are central drivers of proliferation, differentiation, morphology, and apoptosis, yet oncogenic RAS remains largely untreatable: only two KRAS G12C–specific therapies are approved, benefit a minority of patients, and face clinical resistance. A key unresolved problem is how RAS activates its main effector, RAF, inside cells. Bulk affinity measurements show strong RAS–RAF binding but obscure the lifetimes of individual encounters and their spatial context. Here we integrate single-molecule with both in vitro and live-cell approaches to quantify the spatiotemporal dynamics of RAS–RAF binding. To directly visualize one-to-one RAS–RAF and RAF–RAF interactions in a TIRF microscope, we conducted dynamic single-molecule pulldowns: we built artificial 2- or 8-lipid membranes on coverslips (supported lipid biolayers) with tethered recombinant human RAS proteins. Then, BRAF or CRAF proteins from crude human cell lysates were captured by the membrane-bound RAS. This system allowed us to quantitate the biophysical parameters of RAS-RAF complexes diffusing on a lipid bilayer in real time. Strikingly, most individual RAS-RAF encounters only lasted a few tens of milliseconds. These short-lived interactions are consistent with constant interaction turnover detected in conventional co-immunoprecipitation assays. In live cells, we built upon a human HEK cell line devoid of endogenous H/N/KRAS and A/B/CRAF genes (6 KOs), to stably express halo-KRAS4b and snap-CRAF at stoichiometric, low levels. Then, we imaged single-molecule halo-KRAS4b and snap-CRAF using simultaneous two-color TIRF video acquisition, and analyzed the dynamics of the RAS/RAF interaction. Similar to our in vitro results, we observed that CRAF exhibits very short membrane dwell times in live cells, which increased upon MAPK pathway activation with soluble EGF. By bridging millisecond-scale binding kinetics with MAP kinase pathway activation, our work outlines how transient RAS–RAF contacts are regulated in space and time. This could reveal potential vulnerabilities for therapeutic intervention beyond current RAS inhibitors. Citation Format: Rodrigo E. Cáceres-Gutiérrez, Rebika Shreshta, Jean Castillo-Badillo, Vanessa Wall, Scott Eury, Katie Powell, William Burgan, Min Hong, Peter Frank, Dwight V. Nissley, Frank McCormick, Thomas Turbyville. Spatiotemporal control of RAS-RAF signaling at the single-molecule level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5976.

Supplementary Figure S6 shows that BBO-8520 shows anti-tumor activity in NCI-H358 CDX sotorasib-resistant tumors

Supplementary Figure S4 summarizes cysteine profiling, RNA-seq and kinomescan studies demonstrating that BBO-8520 is selective for KRAS G12C and MAPK inhibition.

Supplementary Figure S6 shows densitometry analysis for pERK and pAKT in a western blot experiment in KRASG12D and KRASG12D/A59G cells treated with BBO-11818 or DMSO.

Supplementary Table S2 summarizes the potency of BBO-8520, sotorasib, adagrasib and RMC-6291 on pERK and viability in a panel of KRAS mutant, wild type and BRAF V600E mutant cell lines

Molecular glues, compounds that bind cooperatively at protein-protein interfaces (PPIs), are revolutionizing chemical biology and drug discovery, allowing the modulation of traditional "undruggable" targets. Here, we focus on a native regulatory PPI between the scaffolding protein 14-3-3 and C-RAF, a key component of the MAPK signaling pathway. Extensive drug discovery efforts have focused on the MAPK pathway due to its central role in oncology and developmental disorders (RASopathies). However, the modulation of its protein complexes is underexplored. C-RAF activity is regulated on multiple levels including dimerization, phosphorylation, and complex formation with 14-3-3, which prevents C-RAF activation by binding to a C-RAF sequence centered on phospho-serine 259. We used a fragment-merging approach to design molecular glues that bound to the composite surface of this 14-3-3/C-RAFpS259 complex. Molecular glues stabilized the inhibitory complex up to 300-fold; their glue-based mechanism of action was confirmed by crystallography and biophysical studies. Selectivity among the other RAF isoforms and other RAF phosphorylation sites was evaluated. The best compounds showed excellent selectivity among a broad panel of 80 14-3-3 clients. Cellular assays demonstrated on-target engagement, enhanced phosphorylation levels of C-RAFpS259, and reduced levels of RAF dimerization and ERK phosphorylation. Overall, this approach enabled chemical biology studies for a C-RAF site that was intrinsically disordered prior to 14-3-3 binding and had not been targeted previously. These molecular glues will be useful chemical probes and starting points for drug discovery efforts to modulate native PPI stabilization in the MAPK pathway with applications in oncology and RASopathies.

Supplementary Figure S2 shows the electron density map for BBO-8520 bound to KRAS G12C in the GDP and GppNHp states

Abstract Therapeutically silencing the RAS/MAPK signaling cascade, an oncogenic driver in more than one-third of human cancers, is constrained by a fundamental trade-off: potent pathway inhibition in tumors versus dose-limiting toxicities in normal tissues. BRAF-mutant (BRAF-MUT) cancers are a notable exception, where current clinical RAF inhibitors (RAFis) (Type 1.5 - αC-OUT/DFG-IN) selectively inhibit monomeric BRAF(V600X), while paradoxically activating MAPK signaling pathway in settings where RAF signals as a dimer, including wild-type and RAS-Mutant (RAS-MUT) contexts. While this paradoxical activation limits the broader applicability to BRAF-MUT tumors, it has been therapeutically exploited in vertical MAPK-targeting combinations with MEK or EGFR inhibitors, enhancing antitumor efficacy while restoring physiological MAPK signaling in normal tissues, achieving an improved therapeutic window and enhancing tolerability.To target dimeric RAF-driven tumors, including RAS-MUT tumors, Type 2 (αC-IN/DFG-OUT) RAFis were developed to engage both RAF monomers and dimers. However, as single agents, Type 2 RAFis showed only modest activity. Combining them with MEK inhibitors improved efficacy but also exacerbated toxicities due to MAPK pathway suppression in normal tissues, limiting dosing and ultimately constraining therapeutic benefit.Here, we characterized ELV-3111, a next-generation, highly potent and selective Type 1 RAFi with broad activity across BRAF class I/II/III, CRAF, and RAS-MUT models, including contexts resistant to current MAPK-targeted therapies. Unlike Type 2 RAFis, ELV-3111 induces robust paradoxical MAPK hyperactivation selectively in normal tissues - a phenomenon we successfully modeled in cells. Using complementary biochemical and live-cell assays, alongside molecular dynamics simulations, we demonstrate that this MAPK hyperactivation occurs via a RAS-dependent allosteric mechanism distinct from the paradoxical activation described for Type 1.5 RAFis. This unique property can be therapeutically exploited. Combining ELV-3111 with a MEK inhibitor overcomes the therapeutic ceiling of MAPK pathway targeting by creating a pharmacologically complementary interaction: additive suppression in tumors, where both agents inhibit MAPK signaling, and opposing effects in normal tissues, where MEK inhibition counteracts RAFi-driven hyperactivation. This configuration produced profound and durable regressions across RAS- and BRAF-MUT models, including a RAS-MUT model refractory to current therapies, while maintaining favorable tolerability. This tumor-selective mechanism, previously exploited in BRAF-MUT cancers, can now be extended to RAS-MUT and other dimeric RAF-driven tumors, offering a renewed therapeutic opportunity and the potential to reshape combination strategies across a broader spectrum of MAPK-driven cancers. Citation Format: Mathieu Desaunay, Tara L. Peters, Evangelia Matenoglou, Beau Baars, Bijaya Gaire, Ana Orive-Ramos, Li Ren, Joseph P. Lyssikatos, Michael R. Burkard, Dalton Dacus, Matthew J. Sale, Stuart A. Aaronson, Frank McCormick, Evripidis Gavathiotis, Stefan D. Gross, Poulikos I. Poulikakos. Tumor-selective dimeric and monomeric RAF targeting with a next-generation Type 1 RAF inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2940.

Abstract Aberrant PI3K signaling drives cancer growth, yet tumors activate p110α through distinct mechanisms. In normal cells, the regulatory subunit p85 restrains p110α until activated receptors recruit the complex to membranes. HER2-amplified tumors intensify this route through phosphorylated HER3, which provides multiple phospho-sites that both recruit PI3K and relieve autoinhibition. PIK3CA mutations found in various cancer types, including those with HER2 amplification, can weaken the restraint of p85 and bypass receptor control while preserving the requirement for membrane localization. Given that p110α still requires membrane localization even after autoinhibition is relieved, tumors often use oncogenic KRAS to provide this input. However, most HER2-amplified and many PIK3CA-mutant cancers lack KRAS mutations, suggesting alternative RAS-family proteins may fill this role.To define the contribution of RAS to PI3K signaling in a HER2-amplified context with wild-type p110α, we used KRAS G12C KYSE-410 cells. Through targeted siRNA knockdown, we identified RRAS2, not KRAS G12C, as the dominant PI3K driver, resulting in a ∼60% reduction in pAKT. Replacing endogenous p110α with a RAS-binding-defective mutant produced the same effect, demonstrating that RRAS2 exclusively engages this site. Exogenous expression of oncogenic RRAS2 failed to restore pAKT in the presence of the HER2 kinase inhibitor tucatinib, even though RRAS2 remained GTP-loaded, membrane-localized, and bound to p110α. These results show that RRAS2 alone is insufficient to activate PI3K in the absence of phospho-HER3.To test whether p85 restraint prevents RRAS2 from activating PI3K, we expressed p110α mutants that destabilize the helical (E545K) or C2 (C420R) interface with p85. Both mutants restored RRAS2-driven signaling after tucatinib treatment, demonstrating that RRAS2 activates PI3K only when autoinhibition is relieved. To test the model in a physiological context, we used JIMT1 cells, which endogenously co-amplify HER2 and RRAS2 and carry the C420R mutation in p110α. Tucatininb treatment did not affect pAKT, indicating that RRAS2 can sustain PI3K activity when p110α autoinhibition is relieved. RRAS2 knockdown or re-expression of wild-type p110α restored tucatinib sensitivity, confirming that RRAS2 engagement and p110α–p85 regulation form key regulatory nodes.Further, DepMap analyses reveal increased RRAS2 dependency in cells with destabilizing p110α–p85 mutations in the absence of RTK amplification, supporting a model in which RRAS2-driven PI3K signaling requires both relief of autoinhibition and membrane localization.Together, our findings demonstrate that RRAS2 can drive PI3K signaling in contexts previously seen as RAS-independent and where recruitment mechanisms were unclear. Thus, highlighting an additional regulatory node of PI3K activation in HER2-amplified and PI3K-destabilized contexts. Citation Format: Miranda R. Cabanski-Dunning, Matthew J. Sale, Lucy C. Young, Maria Tarazona-Guzman, Dylan Aguinaldo, Madeleine Sitton, Rony Andre Francois, Frank McCormick. Pi3k autoinhibition dictates rras2 dependency across HER2-amplified and PI3K-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3295.

Current approved KRASG12C inhibitors covalently bind the inactive GDP-bound (OFF) form of KRASG12C. Recently, KRASG12C inhibitors that selectively bind to the GTP-bound (ON) form of both KRASG12C (ON) and (OFF) forms have been reported and entered clinical testing. In principle, KRASG12C (ON) inhibitors may be less susceptible to adaptive mechanisms that promote resistance to (OFF) inhibitors, however the specific mechanisms that differentiate the activity of (ON) versus (OFF) inhibition are not well understood. We profiled the activity of BBO-8520, a covalent dual inhibitor of GTP-bound (ON) and GDP-bound (OFF) KRASG12C, in KRAS G12C -mutant non-small cell lung cancer models. BBO-8520 exerted more potent and sustained inhibition of KRASG12C and anti-tumor activity in vitro and in vivo compared with sotorasib, a KRASG12C (OFF)-only inhibitor. While cells treated with BBO-8520 or sotorasib both exhibited feedback reactivation of MAPK signaling driven by wild-type HRAS/NRAS isoforms, more durable suppression of KRASG12C by BBO-8520 was associated with decreased PI3Kα-AKT activation in vitro. Disruption of the interaction between RAS and PI3Kα using a novel protein:protein interaction inhibitor suppressed PI3Kα-AKT activation and increased the tumor response to sotorasib to a similar level as BBO-8520. Moreover, in some contexts, disruption of RAS-PI3Kα further increased the anti-tumor activity of BBO-8520 monotherapy. These results reveal mechanistic differences between KRAS (ON) and (OFF) inhibitors, highlight the importance of PI3Kα-AKT signaling in driving resistance to KRAS inhibition in lung cancer, and suggest combination strategies that suppress PI3Kα-AKT to improve the response to KRAS inhibitors.

Supplementary Figure S8 shows mean body weight and survival upon rechallenge in a KRASG12D syngeneic model treated with the combination of BBO-11818 and anti–PD-1 antibody. In addition, it shows mean body weight and mean tumor volume in KRASG12V and KRASG12D CDX models treated with BBO-11818 and BBO-10203 or BBO-11818 and cetuximab.

Supplementary Figure S1 shows the Kinact/KI values for BBO-8520, sotorasib and adagrasib in biochemical and cell-based assays

Distinct effector-binding preferences among RAS family GTPases challenge the longstanding view that canonical RAS proteins uniformly bind and activate RAF, PI3Kα, RalGDS, and other downstream effectors. Quantitative binding data, supported by structural insights into effector recognition, instead reveal a division of labor: the canonical RAS subfamily (KRAS, HRAS, NRAS) binds RAF kinases with high affinity, the RRAS subfamily (RRAS2 and MRAS) preferentially engages PI3Kα, and the RAP subfamily (RAP1A and RAP1B) shows the strongest binding to RalGDS. These intrinsic preferences, encoded in the switch regions and further shaped by isoform and effector expression, as well as subcellular localization, establish a hierarchy in which canonical RAS, RRAS2/MRAS, and RAP1A/B primarily activate RAF, PI3Kα, and RalGDS, respectively, in normal cells. Oncogenic mutations at codons G12, G13, or Q61 disrupt this hierarchy by driving sustained accumulation of GTP-bound canonical RAS, enabling engagement of lower-affinity effectors such as PI3Kα and RalGDS. In addition, certain mutations, including KRAS-G12D and -G12V, modestly enhance PI3Kα binding, representing a neomorphic expansion of effector engagement. Together, these effects bypass intrinsic effector selectivity, allowing canonical RAS to co-opt effectors normally associated with other RAS subfamilies and broaden downstream signaling. This framework explains how inherent effector preferences govern normal signaling and how oncogenic mutations override these constraints to expand effector engagement in RAS-driven cancers.

Disulfide tethering is a site-directed method of drug discovery used to identify hits for challenging targets. We applied tethering to target oncogenic KRAS, a small GTPase once considered undruggable due to its high nucleotide affinity and a perceived absence of binding sites. We prepared a library of 2160 disulfide-containing fragments. We screened over 1000 compounds against a panel of 83 engineered cysteine mutants of KRAS G12D in the active conformation and screened the full library for a subset of 30 mutants. For select mutants and hits, we performed 2-mercaptoethanol competition assays (βME-50) to prioritize ligands. Ligandability analysis comparing hit rates across mutant residues enabled the identification of druggable hot spots. Our studies confirmed known binding sites, including the Switch-II / α-helix 3 pocket. In addition, we identified previously undescribed cryptic pockets and validated select hits using computational chemistry and NMR spectroscopy. These pockets represent promising opportunities for future drug discovery campaigns.

Neurofibromin, the protein product of the neurofibromatosis type 1 (NF1) gene, requires the direct binding interaction with SPRED to negatively regulate the RAS-MAPK pathway. Although the region of neurofibromin that stimulates the intrinsic GTPase activity of RAS represents only a small percentage of the entire protein, a large degree of the NF1 structural domains and their correlating mechanistic functions remain elusive. Here, we demonstrate RAS-independent biochemical and signaling functions regulated by the coordinate control of NF1 and SPRED1/2. Utilizing CRISPR-Cas9 methods to ablate NF1 or SPRED1/2 in isogenic "RASless" mouse embryonic fibroblast (MEF) cell lines expressing either the KRAS4b wild-type variant or an oncogenic KRAS-mutation, we show loss of SPRED1/2 phenocopies NF1 loss and their cooperation is required to modulate MAPK-AKT signaling. Moreover, NF1 or SPRED1/2 loss also resulted in a potent suppression of the RAS family GTPases, RRAS and RRAS2, occurring independently of RAS or AKT pathway activation. A transcriptome microarray analysis of the NF1 or SPRED1/2 knockout MEF cells revealed a specific subset of RAS-independent, NF1-SPRED1/2-dependent gene signatures, in which these same genes were also directly regulated by the RAS-GTPase function of neurofibromin. The modulation of these NF1-SPRED1/2-dependent downstream signaling effectors were further corroborated in Schwann cell models derived from Neurofibromatosis type I patients that consisted of either plexiform neurofibroma cells or unaffected nerve cells abrogated of NF1 or neurofibromin RAS-GAP activity. Taken together, this study provides RAS-independent functions that are dependent on the cooperation of NF1 and SPRED1/2 in a manner that is uncoupled from canonical MAPK signaling.

This file contains the supplementary materials and methods for the manuscript.