TEAD transcription factors enable the oncogenic activity of deregulated Hippo signaling and are a promising therapeutic target in oncology. Targeting the TEAD lipid pocket is an established path to inhibit the oncogenic activities of cofactors YAP and TAZ. Here we present two pan-TEAD inhibitors, GNE-8025 and its in vivo brain-penetrant derivative GNE-2181, that covalently bind the lipid pocket at a conserved cysteine. Both small molecules show growth inhibition of YAP-driven tumor cells in vitro and in vivo. Moreover, we show that GNE-8025 increases the activity of a broad range of MAPK pathway inhibitors in vitro as well as the KRASG12C inhibitor Divarasib both in vitro and in vivo. In addition, GNE-2181 inhibits growth of an intracranial tumor model in vivo. Altogether we present a next-generation class of TEAD inhibitors representing a significant advancement towards potent, specific, and effective Hippo-targeting cancer therapies.

Abstract While CDK4/6 inhibitors (CDK4/6i) have improved the treatment of hormone receptor-positive (HR+) breast cancer (BC), resistance remains a major clinical challenge. Resistance to CDK4 inhibition can be driven by higher CDK2 activity, due to overexpression of cyclin E1/E2 or p53 loss of function, and other mechanisms. As such, targeting both CDK4 and CDK2 is hypothesized to achieve more durable cell cycle arrest. This study evaluated the preclinical activity of GDC-4198, a novel CDK4/2 inhibitor currently in early clinical trials, and its potential to overcome CDK2-driven resistance to CDK4 inhibition as a single-agent and in combination with giredestrant, a novel selective estrogen receptor (ER) degrader and full ER antagonist. GDC-4198 has sub-nanomolar potency against CDK4/CycD1 and is a more potent inhibitor of CDK2/CycE or CDK2/CycA than CDK6/CycD3, unlike approved CDK4/6i. Western blots and immunofluorescence data demonstrated that GDC-4198 can directly decrease levels of pNCL, a direct marker of CDK2 activity. Cells engineered with a CDK2-activity reporter further confirmed the difference in inhibition profile of GDC-4198 compared to first generation CDK4/6i. In cell viability assays, we observed that GDC-4198 can maintain more durable growth arrest than CDK4/6 or CDK4-targeted inhibitors. Moreover, the introduction of TP53 knock-out had limited effects on GDC-4198 activity, and its potency was not changed by the overexpression of CCNE1/2, in contrast to other CDK inhibitors. Notably, across these different assays, the single-agent activity of GDC-4198 was comparable to the combination of CDK4- and CDK2-targeting agents. Additional studies in patient-derived cell lines obtained in the setting of metastatic HR+ breast cancer following progression on letrozole/ribociclib, as well as in cell lines with acquired in vitro resistance to palbociclib, confirmed the enhanced anti-proliferation activity of GDC-4198 compared to CDK4/6i. When combined with giredestrant, GDC-4198 showed strong combination benefits across a range of HR+ BC cell lines. In xenograft HR+ BC models, GDC-4198 demonstrated dose-dependent tumor growth inhibition as a single agent and further improved antitumor activity in combination with giredestrant. Pharmacokinetic and pharmacodynamic analyses indicated pRb modulation in tumor samples correlated with drug exposure. Taken together, these preclinical findings demonstrate that GDC-4198 induces more durable cell cycle arrest than first generation CDK4/6i and CDK4-targeting agents by overcoming CDK2-driven adaptive and intrinsic resistance to CDK4 inhibition. These results suggest that GDC-4198 is a promising next generation CDK4/2 inhibitor with the potential to prolong clinical benefit by delaying adaptation in earlier disease settings, as well as to provide benefit to patients who have progressed on approved CDK4/6i. Citation Format: Marc Hafner, Steffan Vartanian, Luca Gerosa, Nont Kosaisawe, Michael S. Hwang, Eva Lin, Yi-Chang Wang, Jason Oeh, Tianyi Chen, Kazi N. Islam, Alice Zheng, Karen Samy, Udi Segal, John G. Moffat, DANIEL ZINGG, Annie Collier, Ioannis Sanidas, Zhi Xie, Seth A. Wander. GDC-4198, a next-generation CDK4/2 inhibitor, induces durable cell cycle arrest and shows combination benefit with giredestrant [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 1910.

KRAS G12C is one of the most prevalent oncogenic mutations in nonsmall cell lung cancer. Herein we describe the discovery and optimization of divarasib (GDC-6036), an orally available, highly potent, and selective covalent KRAS G12C inhibitor. We demonstrate a significant noncovalent binding component of divarasib that contributes to its potency and rapid kinetics. Divarasib has greater potency and kinetics of alkylation compared with other KRAS G12C inhibitors in vitro and shows robust tumor growth inhibition in multiple KRAS G12C-positive cell lines.

TEAD (transcriptional enhanced associate domain) transcription factors (TEAD1-4) serve as the primary effectors of the Hippo signaling pathway in various cancers. Targeted therapy leads to the emergence of resistance and the underlying mechanism of resistance to TEAD inhibition in cancers is less characterized. We uncover that upregulation of the AP-1 (activator protein-1) transcription factors, along with restored YAP (yes-associated protein) and TEAD activity, drives resistance to GNE-7883, a pan-TEAD inhibitor. Acute GNE-7883 treatment abrogates YAP-TEAD binding and attenuates FOSL1 (FOS like 1) activity. TEAD inhibitor resistant cells restore YAP and TEAD chromatin occupancy, acquire additional FOSL1 binding and exhibit increased MAPK (mitogen-activated protein kinase) pathway activity. FOSL1 is required for the chromatin binding of YAP and TEAD. This study describes a clinically relevant interplay between the Hippo and MAPK pathway and highlights the key role of MAPK pathway inhibitors in mitigating resistance to TEAD inhibition in Hippo pathway dependent cancers.

Supplementary Figure S2: Ex vivo RIT1 cell lines have Trp53 genetic knockout and therapeutic vulnerabilities include MAPK/PI3K inhibitors and statins.

The mitogen-activated protein kinase (MAPK) pathway integrates growth factor signaling through extracellular signal-regulated kinase (ERK) to control cell proliferation. To study ERK dynamics, many researchers use an ERK activity kinase translocation reporter (KTR). Our study reveals that this ERK KTR also partially senses cyclin-dependent kinase 2 (CDK2) activity, making it appear as if ERK activity rises as cells progress through the cell cycle. Through single-cell time-lapse imaging, we identified a residual ERK KTR signal that was eliminated by selective CDK2 inhibitors, indicating crosstalk from CDK2 onto the ERK KTR. By contrast, EKAREN5, a FRET-based ERK sensor, showed no CDK2 crosstalk. A related p38 KTR is also partly affected by CDK2 activity. To address this, we developed linear and non-linear computational correction methods that subtract CDK2 signal from the ERK and p38 KTRs. These findings will allow for more accurate quantification of MAPK activities, especially for studies of actively cycling cells.

Supplementary Table S1: Assessment of immunohistochemistry staining intensity on lungs from RIT1 mice. Pathological findings are summarized for all tumors within each animal. Localization of CD3-positive T cells is described. IHC staining intensity is qualitatively scored as +++; strongly positive, +/-; mixed and description is included, -; negative.

Supplementary Figure S1: RIT1 tumors have heterogenous pERK expression.

RIT1 is a RAS-family guanosine triphosphatase that is mutated in 2.4% and amplified in up to 14% of patients with lung adenocarcinoma. Yet the oncogenic potential of RIT1 in the lungs has not been fully established. Consequently, patients with RIT1 alterations are considered "oncogene-negative" and are not eligible for any targeted therapy in the clinic. The role of RIT1 in cancer has been historically understudied due to the lack of in vitro and in vivo models harboring RIT1 alterations. In this study, we generated a murine model of RIT1M90I-mutant lung cancer. RIT1M90I expression induced tumorigenesis in the lungs, and the tumors displayed histopathologic features similar to lung adenocarcinoma in humans. An unbiased chemical compound screen leveraging this model revealed a sensitivity to inhibitors of the MAPK, PI3K, and cholesterol biosynthesis pathways in RIT1-mutant cell lines. The SHP2 inhibitor, migoprotafib, in combination with other MAPK pathway-targeted therapies, effectively suppressed the growth of RIT1-mutant cells ex vivo and in vivo. Finally, RIT1M90I drove resistance to the KRASG12C inhibitor, divarasib, and the combination with migoprotafib reverted this phenotype. Together, these data show that RIT1M90I is a bona fide oncogenic driver of lung cancer and a mediator of targeted therapy resistance as a co-occurring mutation and suggest that patients with RIT1-altered cancer may benefit from combination treatments with an SHP2 inhibitor. SIGNIFICANCE:Development of a mouse model of RIT1M90I-altered non-small cell lung cancer reveals that RIT1M90I is a driver of lung tumorigenesis and that RIT1-mutated tumors are sensitive to MAPK pathway inhibitors. See related commentary by Wu and Vaishnavi, p. 3186 See related article by Mozzarelli et al., p. 3196.

Abstract Acquisition of ARAF mutations upon treatment with the potent and selective RAF dimer (type II) inhibitor belvarafenib has been previously observed, but whether these alterations or others are observed in patients treated with a combination of belvarafenib and the MEK inhibitor cobimetinib is unknown. The mutational profiles from circulating tumor DNA (ctDNA) were determined using FoundationOne liquid ctDNA assay from paired baseline (BL) and end of treatment (EOT) peripheral blood samples from 79 patients with locally advanced or metastatic RAF or RAS-mutant solid tumors treated with belvarafenib and cobimetinib in the HM-RAFI-103 trial (NCT03284502). The proportion of mutations were compared between BL and EOT using a McNemar’s test (unadjusted) to identify mutations significantly enriched or lost following treatment. ARAF mutations were not observed to significantly increase following treatment with belvarafenib + cobimetinib (4% BL vs 4% EOT in all solid tumors, p = 1; 6% BL vs 6% EOT in melanoma), and in patients with pre-existing ARAF mutations, the variant allele frequency did not consistently increase following treatment. The MEK1 gene had the greatest increase in mutations following treatment, present in 5% (4/79) patients at BL vs 16% (13/79) at EOT (p = 0.02) across all solid tumors, which included 44 colorectal cancer (CRC) cases (56%), and 18 melanoma (23%). While CRC comprised the largest fraction of all solid tumors in this study, it did not contribute the greatest gain in MEK1 mutations at EOT, with 7% (3/44) of CRC patients having MEK1 mutations at BL vs 14% (6/44) at EOT (p = 0.2). The indication with the greatest number of patients developing previously undetected MEK1 mutations at EOT was melanoma, where MEK1 mutations were present in 0% (0/18) patients at BL vs 28% (5/18) at EOT (p = 0.07). The MEK1 mutations gained in melanoma, which included Q58_E62del, P124L, and C121S, occurred in the N-terminal or catalytic core domains of MEK1, were known to be of functional impact, and all but one patient developing them had ≥ 1 known to be gain of function via the Clinical Knowledge Database; one patient gained three distinct MEK1 mutations following treatment. The MEK1 C121S mutation was previously observed to appear in a melanoma patient who developed resistance to the BRAF V600 inhibitor vemurafenib, and in vitro studies demonstrated it also conferred resistance to MEK inhibition (Wagle et al., 2011). Melanoma patients in the HM-RAFI-103 trial gaining MEK1 mutations at EOT were more commonly BRAF-mutant(mt) or NRAS non-mt melanoma at BL, but sample size is small and results were non-significant (p = 0.3 and 0.3, for association with BRAFmt and NRASmt, respectively, Fisher’s exact test). All melanoma patients who developed MEK1 mutations post treatment had either a partial response (1/5) or stable disease (4/5) as best overall response. Together, these data suggest the acquisition of gain of function MEK1 mutations following treatment of solid tumors, including melanoma, with combined type II RAF dimer and MEK inhibition. Citation Format: Stephanie Hilz, Marissa Chen, Malgorzata Nowicka, Harini Chakravarthy, Maryam Moshref, Luca Gerosa, Jennifer Eng-Wong, Cassie Chou, Young S. Noh, Yoon-hee Hong, Yibing Yan. Emergence of MEK1 mutations in RAF or RAS-mutant solid tumors following treatment with a combination of selective type II RAF inhibitor belvarafenib and MEK inhibitor cobimetinib [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A012.

Purpose: This study explores the potential of pre-clinical in vitro cell line response data and computational modeling in identifying the optimal dosage requirements of pan-RAF (Belvarafenib) and MEK (Cobimetinib) inhibitors in melanoma treatment. Our research is motivated by the critical role of drug combinations in enhancing anti-cancer responses and the need to close the knowledge gap around selecting effective dosing strategies to maximize their potential. Results: In a drug combination screen of 43 melanoma cell lines, we identified specific dosage landscapes of panRAF and MEK inhibitors for NRAS vs. BRAF mutant melanomas. Both experienced benefits, but with a notably more synergistic and narrow dosage range for NRAS mutant melanoma (mean Bliss score of 0.27 in NRAS vs. 0.1 in BRAF mutants). Computational modeling and follow-up molecular experiments attributed the difference to a mechanism of adaptive resistance by negative feedback. We validated the in vivo translatability of in vitro dose–response maps by predicting tumor growth in xenografts with high accuracy in capturing cytostatic and cytotoxic responses. We analyzed the pharmacokinetic and tumor growth data from Phase 1 clinical trials of Belvarafenib with Cobimetinib to show that the synergy requirement imposes stricter precision dose constraints in NRAS mutant melanoma patients. Conclusion: Leveraging pre-clinical data and computational modeling, our approach proposes dosage strategies that can optimize synergy in drug combinations, while also bringing forth the real-world challenges of staying within a precise dose range. Overall, this work presents a framework to aid dose selection in drug combinations.

Introduction RAS mutations are prevalent in AML, but efforts to target the mitogen activated protein kinase (MAPK) effector pathway have been largely unsuccessful. Belvarafenib is a type II pan-Raf kinase inhibitor that is active against both monomeric and dimeric mutant B-Raf proteins as well as wild-type (WT) Raf homo- and heterodimers (Yen et al. 2021). We observed promising anti-leukemia activity of belvarafenib as a single agent and in combination with the allosteric MEK inhibitor cobimetinib in AML models harboring oncogenic NRAS/Nras or KRAS/Kras mutations (2023 ASH Annual Meeting, Abstract #4172). Belvarafenib treatment unexpectedly had minimal effects on phosphorylated (p) ERK levels and downregulated mTORC1 signaling in multiple RAS-mutant AML cell lines. In this study, we further analyzed belvarafenib's mechanism of action and identified and analyzed candidate resistance mutations that emerged after in vivo treatment. Methods Mouse AMLs that were generated using retroviral insertional mutagenesis were transplanted and treated as previously described (Li et al. 2011, Burgess et al. 2014, 2017). Whole exome sequencing (WES) was performed on DNA extracted from bone marrow cells isolated at euthanasia from recipients of primary Nras- or Kras-mutant AMLs that were treated with either belvarafenib, cobimetinib, belvarafenib + cobimetinib, or vehicle. OCI-AML3 cells were lentivirally transduced with doxycycline-inducible vectors containing constitutively active MEK-DD, a MEK mutation that confers resistance to allosteric MEK inhibitors (L115P), or candidate MEK resistance mutations and cell viability was determined by CellTiter-Glo. Results To investigate whether MAPK pathway inhibition is essential for the inhibitory activity of belvarafenib in AML cells, we expressed doxycycline-inducible MEK-DD and MEKL115P mutant proteins. NRAS-mutant OCI-AML3 cells expressing MEK-DD were resistant to belvarafenib and sensitive to cobimetinib. As expected, MEKL115Pinduced resistance to cobimetinib, but not belvarafenib. To characterize the effects of belvarafenib treatment on mTORC signaling, we treated OCI-AML3 cells with belvarafenib 100nM, cobimetinib 10nM, this combination, or vehicle for 4 or 24 hours (h). While we observed minimal effect of drug treatment at 4h, belvarafenib decreased pAKT, p4EBP1 and p70S6K levels after 24h. At the later time point, cells treated with both drugs displayed profound and synergistic reductions in the levels of all three mTORC target phospho-proteins. Of five independent primary murine AMLs, AML 63A was the most sensitive to belvarafenib and the belvarafenib/cobimetinib combination (median survival of vehicle group, 7d; belvarafenib group, 23d; combination group, 32d; p = 0.003). WES of DNA extracted from the bone marrows of mice with refractory leukemia uncovered distinct Map2k1 mutations (K57T, E203K, N122D) in three independent recipient mice treated with the belvarafenib/cobimetinib combination at variant allele frequencies (VAFs) of 10-33% and a Braf L542H mutation in a belvarafenib-treated recipient at a VAF of 21%. MAP2K1 K57T and E203K mutations and a BRAF mutation (L505H) corresponding to L542H in mouse have been reported in patients who developed clinical resistance after treatment with first generation Raf kinase inhibitors. To functionally interrogate putative resistance mechanisms, we expressed MEKK57T and MEKN122D in NRAS mutant OCI-AML3 cells and analyzed them in parallel with control cells expressing wild-type MEK, MEK-DD, or MEKL115P. Whereas MEKN122D had no effect on sensitivity to belvarafenib or cobimetinib, MEKK57T conferred partial resistance to cobimetinib and more pronounced resistance to belvarafenib. Conclusions Belvarafenib and cobimetinib synergistically inhibit the growth of RAS-mutant AML cell lines in vitro and of primary mouse Nras- and Kras-mutant leukemias mutations in vivo. Although belvarafenib has minimal effects on pERK levels in KRAS/NRAS-mutant AML cell lines at clinically achievable concentrations, genetic analysis of MEK-DD and MEKL115P mutations confirmed MAPK pathway inhibition as a major mechanism of action. We are characterizing candidate resistance mutations in addition to Mapk21 K57T that emerged during belvarafenib treatment. Altogether, our data support further investigation of belvarafenib monotherapy and rational drug combinations in AML.

Introduction Outcomes for children with acute myeloid leukemia (AML) remain poor, with ~40% dying from refractory leukemia or treatment-related toxicity (Gamis et al. 2014). NRAS, KRAS, and NF1 mutations occur in over 40% of pediatric AMLs (Bolouri et al. 2018), but efforts to therapeutically target the RAS/mitogen activated protein kinase (MAPK) pathway have been largely unsuccessful. Belvarafenib, a novel type II pan-RAF kinase inhibitor that inhibits mutant monomeric BRAF proteins and activated RAF homo- and heterodimers, has shown promising safety/efficacy data in adult solid cancers (Yen et al. 2021). Here, we investigate belvarafenib in preclinical models of AML, both alone and in combination with the allosteric MEK inhibitor, cobimetinib. Methods We used a panel of NRAS (OCI-AML3, HL-60, THP-1) or KRAS (NOMO-1, NB4, and SKM-1) mutant human AML cell lines. Viability was determined by CellTiter-Glo. Synergy was assessed by Bliss Independence and Chou Talalay methods. Transcriptome and proteomic profiling were performed as previously described (Pucciarelli et al. 2020). Mouse AMLs were generated using retroviral insertional mutagenesis (Li et al. 2011). Cryopreserved primary AML cells were injected intravenously into sublethally irradiated recipients that were then treated daily with vehicle, belvarafenib, cobimetinib, or the combination until disease progression. Survival curves were generated using Kaplan-Meier analysis. Results Belvarafenib inhibited the growth of AML cells lines with nanomolar potency at IC50 values ranging from 48nM (OCI-AML3) to 310nM (SKM-1). In all cell lines, belvarafenib and cobimetinib were highly synergistic. Western blotting of OCI-AML3 cells treated with either belvarafenib or cobimetinib at their respective IC50 values (50nM belvarafenib, 20nM cobimetinib) for 4 or 24 hours demonstrated discordant effects on downstream MAPK effector proteins (Fig. 1). Whereas cobimetinib potently suppressed phosphorylated ERK (pERK), belvarafenib unexpectedly had no effect. Phosphorylated S6 (pS6) levels were not reduced by either belvarafenib or cobimetinib at their respective IC50 values, but were dramatically reduced by the drug combination. To further investigate these unanticipated biochemical findings, we treated OCI-AML3 cells with belvarafenib, cobimetinib or the combination and performed kinome profiling and transcriptome sequencing (RNAseq) analysis. Exposure to 10nM cobimetinib inhibited several kinases at 4 hours with rebound activation of kinases involved in cell cycle progression at 24 hours. Cells treated with 100nM belvarafenib displayed down-regulation of multiple kinases that persisted at 24 hours. Cells treated with the combination at these doses exhibited the most potent inhibition of many kinases; this effect recapitulated the effect of high dose (500nM) belvarafenib, but not high dose (50nM) cobimetinib. At the transcriptional level, cells treated with the combination showed far greater suppression of negative regulators of MAPK signaling (e.g., DUSPs, SPRY2/4, SPRED1/2) than either agent alone, suggesting potent inhibition of the MAPK pathway by the combination. Intriguingly, OCI-AML3 cells treated with high-dose belvarafenib or low-dose combination unexpectedly exhibited profound down-regulation of mTORC1-regulated genes on GSEA analysis. We extended these in vitro data by performing preclinical trials in mice transplanted with 5 independent primary Nras- or Kras-mutant AMLs that received belvarafenib (15mg/kg), cobimetinib (2mg/kg), the combination or vehicle by oral gavage (Fig. 2). The combination was well tolerated. Belvarafenib prolonged survival in all 5 trials; in 3 of 5, the addition of low-dose cobimetinib further enhanced survival (median survival: vehicle, 9 days; cobimetinib, 13 days; belvarafenib, 22 days; combination, 32 days; p < 0.0001). Conclusions Belvarafenib showed activity in 6 of 6 Ras mutant human AML cell lines and in 5 of 5 primary Ras mutant murine AMLs. Belvarafenib and cobimetinib displayed synergy in all AML cell lines and in 3 of 5 murine AMLs treated in vivo. Mechanistically, we identified distinct biochemical and transcriptional effects of RAF dimer and MEK inhibition in AML cells. We are characterizing these further and pursuing causes of resistance in primary Nras- and Kras-mutant mouse AMLs that relapsed after an initial response to treatment.

The RAS-RAF pathway is one of the most commonly dysregulated in human cancers. The activation of this pathway is tightly regulated with appropriate spatial and temporal signaling cues. The central step in activation of this pathway is the dimerization of RAF kinases. Recent advances in biochemical, enzymatic and structural characterization of various multiprotein complexes in this pathway provide insight into various modes of RAF activity modulation. Kinase RAF and its substrate MEK exist as a pre-formed complex prior to pathway activation. Multiple factors prevent MEK phosphorylation by RAF in this pre-formed complex, including phosphorylation of a Serine residue N-terminal to the kinase domain, which enables 14-3-3 to trap RAF as a monomer. One of our intriguing findings is that cellular ATP exerts a negative regulatory effect on RAF kinase and stabilizes the inactive conformation of RAF monomer. We characterize this negative regulatory effect of ATP of RAF kinase structurally by solving a RAF-ATP complex and enzymology. Upon membrane recruitment of RAF by RAS-GTP, dephosphorylation of the above-mentioned serine residue in RAF occurs by a multiprotein complex composed of phosphatase PP1C, a scaffolding protein SHOC2 and RAS-GTP. We solved the structure of this RAS-SHOC2-PP1C complex and performed a thorough enzymatic analysis of this complex for RAF dephosphorylation. Our results show that RAF specificity is determined by SHOC2 and RAS GTP is responsible for spatial localization both inactive RAF and SHOC2/PP1C to the membrane. This dephosphorylation results in RAF being stabilized as a dimer by 14-3-3, which now binds to phospho-serine residues C-terminal to the kinase domain (S729 in BRAF) in 2 RAF molecules. We solve a structure of the RAF-14-3-3 complex. 14-3-3 induced RAF dimerization increases activity of RAF ~500 fold. Further, oncogenic mutations in RAF and MEK occur basally in tumors, and additional mutations are induced upon pathway inhibitor treatment in the clinic, especially for recent KRAS-G12C treatments. These mutations include RAF mutations that either activate or inactivate the kinase activity of the mutated RAF, but nevertheless result in pathway activation. Our data suggests that these mutations appear to tilt the RAF monomer-dimer equilibrium towards RAF dimer. The molecular mechanism of how the MEK mutations active the pathway in poorly understood. Biochemical and structural characterization of these mutations occurring in MEK kinases suggests that the mechanism of activation is also likely functioning via promoting RAF dimerization. This RAF dimer promotion appears to mechanistically work by relieving the negative regulatory effect of RAF by ATP to result in the “just-right” amount of pathway activation to sustain tumor growth. A comprehensive understanding of the various factors modulating RAF dimerization in the context of normal cell and cancer cells that depend on this pathway can pave the way for more precise interventions for treatment of cancers that depend on the RAS pathway. Citation Format: Jawahar Sudhamsu, Nicholas Liau, Timothy Wendorff, Saeed Izadi, Luca Gerosa. Factors modulating RAF dimerization downstream of RAS – A mechanistic overview [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR02.

The RAS-RAF-MEK-ERK signaling axis is upregulated in many human cancers due to its central role in cell growth, differentiation and survival. Oncogenic transformation frequently arises from RAS, BRAF and MEK mutations that activate the pathway. In the clinic, similar transformations also recur as resistance mutations to KRAS.G12C inhibitor treatment. BRAF mutations at V600 (Class I) activate BRAF by releasing the kinase’s activation segment and rendering BRAF activity dimerization-independent. Yet, it remains unclear how a diverse set of dimerization-dependent BRAF mutations (Class II/III) activate the pathway. MEK mutations are assumed to bypass the RAF node entirely. We previously reported a 2.9-Å-resolution crystal structure of human BRAF kinase domain (BRAFKD) in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that stabilize an inactive, pre-signaling monomeric conformation of BRAFKD and explain how ATP breaks RAF dimers in solution. Surprisingly, the β3-αC loop of MEK provides additional stabilizing interactions to the BRAF-bound AMP-PCP molecule. Utilizing an in vitro RAF dimerization assay, we find that MEK not only modulates RAF-RAF dimerization, it also enhances both the dimer-breaking effect ATP and the dimer-forming effect of paradoxical activator molecules, such as GDC-0879. Structural analysis reveals that all common oncogenic BRAF mutations alter key interactions with ATP that stabilize the inactive monomer conformation. We find ATP is unable to break RAF dimers containing Class I, II or III mutations, confirming these mutants counteract the inhibitory effects of ATP binding by lowering the threshold for RAF dimerization and thus pathway activation. We further find that oncogenic MEK mutants, in particular β3-αC loop deletions, favor RAF-RAF dimerization, even in the presence of ATP. Our study establishes a framework for rationalizing oncogenic BRAF mutations, and suggests that oncogenic MEK mutants can act, at least in part, by promoting RAF dimerization. This model provides a better understanding of activation mechanisms in the context of constantly evolving resistance mutations where oncogenesis continues to depend on the MAPK pathway. Citation Format: Timothy J. Wendorff, Jennifer Kung, Jawahar Sudhamsu. Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A036.

Oncogene-induced senescence is a phenomenon in which aberrant oncogene expression causes non-transformed cells to enter a non-proliferative state. Cells undergoing oncogenic induction display phenotypic heterogeneity, with some cells senescing and others remaining proliferative. The causes of heterogeneity remain unclear. We studied the sources of heterogeneity in the responses of human epithelial cells to oncogenic BRAFV600E expression. We found that a narrow expression range of BRAFV600E generated a wide range of activities of its downstream effector ERK. In population-level and single-cell assays, ERK activity displayed a non-monotonic relationship to proliferation, with intermediate ERK activities leading to maximal proliferation. We profiled gene expression across a range of ERK activities over time and characterized four distinct ERK response classes, which we propose act in concert to generate the ERK-proliferation response. Altogether, our studies map the input-output relationships between ERK activity and proliferation, elucidating how heterogeneity can be generated during oncogene induction.

BRAF is prototypical of oncogenes that can be targeted therapeutically and the treatment of BRAF(V600E) melanomas with RAF and MEK inhibitors results in rapid tumor regression. However, drug-induced rewiring generates a drug adapted state thought to be involved in acquired resistance and disease recurrence. In this article, we study mechanisms of adaptive rewiring in BRAF(V600E) melanoma cells using an energy-based implementation of ordinary differential equation (ODE) modeling in combination with proteomic, transcriptomic and imaging data. We develop a method for causal tracing of ODE models and identify two parallel MAPK reaction channels that are differentially sensitive to RAF and MEK inhibitors due to differences in protein oligomerization and drug binding. We describe how these channels, and timescale separation between immediate-early signaling and transcriptional feedback, create a state in which the RAS-regulated MAPK channel can be activated by growth factors under conditions in which the BRAF(V600E)-driven channel is fully inhibited. Further development of the approaches in this article is expected to yield a unified model of adaptive drug resistance in melanoma.

High-throughput measurement of cells perturbed using libraries of small molecules, gene knockouts, or different microenvironmental factors is a key step in functional genomics and pre-clinical drug discovery. However, it remains difficult to perform accurate single-cell assays in 384-well plates, limiting many studies to well-average measurements (e.g. CellTiter-Glo®). Here we describe a public domain “Dye Drop” method that uses sequential density displacement and microscopy to perform multi-step assays on living cells. We use Dye Drop cell viability and DNA replication assays followed by immunofluorescence imaging to collect single-cell dose-response data for 67 investigational and clinical-grade small molecules in 58 breast cancer cell lines. By separating the cytostatic and cytotoxic effects of drugs computationally, we uncover unexpected relationships between the two. Dye Drop is rapid, reproducible, customizable, and compatible with manual or automated laboratory equipment. Dye Drop improves the tradeoff between data content and cost, enabling the collection of information-rich perturbagen-response datasets.