BRAF Dimer Research Articles

A phase 1, open-label, dose escalation and dose expansion study to evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of PF-07799544 (ARRY-134) as a single agent and in combination with PF-07799933 BRAF dimer inhibitor, in participants 16 years and older with advanced solid tumors.

TPS3180 Background: The MAPK pathway plays a role in several key signaling and phosphorylation events that contribute to tumorigenesis. Inhibition of MEK, along with RAF kinases, has proven to be a successful transformative strategy for melanoma and other MAPK pathway-altered tumors. However, the duration of clinical benefit is limited by a narrow therapeutic index, de novo and acquired resistance and poor brain penetration. PF-07799544 is a next-generation, fully brain penetrant MEK inhibitor. PF-07799933 is an oral selective ATP-competitive small-molecule RAF kinase inhibitor that suppresses BRAF signaling in BRAF V600-mutant and non-V600-BRAF mutant tumors. It displays significantly less paradoxical activation than approved BRAFi and is not “pan RAF” as it spares non-BRAF mutated-containing RAF dimers. We describe here the design of the Phase 1 study of PF-07799544 as Monotherapy or in Combination with a next generation BRAF dimer inhibitor PF-07799933 in participants with BRAF mutated (BRAFm) advanced solid tumors. Methods: The purpose of this first-in-human study is to investigate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and potential clinical benefits of PF-07799544 administered as a single agent in participants with advanced solid tumors (Phase 1a) and in combination with PF-07799933 in participants with BRAF Class 1, 2 and 3 mutated solid tumors with or without brain involvement (Phase 1b). Phase 1a will enroll participants with advanced solid tumors who have progressed on standard of care. The primary objective is to determine monotherapy MTD/RDE of PF-07799544. Once PK measurements indicate the potential for significant WT MEK mutation target coverage, participants with untreated and symptomatic brain metastases (bm) will be allowed to enroll. Phase 1b is comprised of multiple sub-studies, all of which evaluate PF-07799544 in combination with PF-07799933 in participants with BRAFm solid tumors who have progressed on standard of care therapies, including Class 1 BRAFi where indicated. Sub-study B: BRAFm melanoma (V600 and non-V600) will determine the MTDc/RDEc of the combination (Part 1 primary objective), followed by dose expansion cohorts (Part 2) (~80 participants): Cohort 1: BRAF V600 melanoma (asymptomatic and untreated bm permitted), Cohort 2: BRAF V600 melanoma (symptomatic bm), Cohort 3 BRAFm Class 2/3 melanoma (asymptomatic and untreated bm permitted); Sub-study C: BRAFm Class 2 or 3 advanced solid tumor Cohort 1 (asymptomatic and untreated bm permitted and Cohort 2 (symptomatic bm) (~20 each cohort). The main objectives of the dose expansion cohorts in all substudies, will be to evaluate anti-tumor activity, safety, PK and PD. Clinical trial information: NCT05538130 .

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors

Abstract 6623: Ala59 mutants of KRAS cooperate with Nf1 loss to enhance colon tumorigenesis

Abstract Understanding the role of KRAS allele heterogeneity in tumor development, and its influence on therapeutic efficacy, is critical for the improvement of cancer treatment. The Kirsten rat sarcoma (KRAS) gene is the most frequently mutated oncogene in colorectal cancer (CRC) and patients with CRC tumors positive for KRAS mutations have poorer outcomes and limited treatment options. Extensive work over the past four decades has shown that common oncogenic alleles of KRAS are associated with hyperactivation of the MAPK signaling pathway (i.e. RAF/MEK/ERK), and it is for this reason that patients with KRAS mutant tumors are excluded from targeted therapies against EGFR. Recently, we characterized two alleles of KRAS, KRASA59T and KRASA59E, that paradoxically promote KRAS activation but are impaired in their ability to promote BRAF dimerization (Johnson et al. Mol Cell. 2022. PMID: 35202574). Consistent with these biochemical functions, KRASA59T and KRASA59E are enriched in CRC tumors that already have genetic alterations in genes that regulate MAPK signaling (e.g. EGFR, BRAF, NRAS, NF1). Further, unlike other described oncogenic alleles of KRAS, clinical data suggest that these KRASA59T and KRASA59E may not confer intrinsic resistance to anti-EGFR therapies at all. While KRASA59T and KRASA59E may not promote BRAF dimers, here we show that KRASA59T and KRASA59E does promote dimerization between ARAF and CRAF, and using an unpublished conditional mouse mode of K-Ras (i.e. LSL-K-RasA59E) we tested (1) whether Ala59 alleles are oncogenic or (2) require cooperation with additional MAPK pathway activating mutations, and (3) whether Ala59 alleles can promote intrinsic resistance to EGFR inhibition. Here, we show that endogenous K-RasA59E can disturb colon homeostasis and decrease mouse survival in an autochthonous colon tumor model, demonstrating that A59E alone is an oncogenic allele of KRAS in the colon. Breeding of LSL-K-RasA59E mice with mice harboring a conditional knockout of Nf1 (i.e. Nf1fl/fl), a GTPase activating protein (GAP) that promotes activation of wildtype RAS, show that combined expression of K-RasA59E and wildtype RAS activation cooperatively perturb colon homeostasis and decrease mouse survival. Thus, while KRASA59E may not require a second genetic alteration to activate the MAPK signaling pathway, it can cooperate with these alterations to increase the severity of disease. Finally, using mouse colon derived organoids, we show that endogenous K-RasA59E neither promotes EGF independence of organoid growth nor confers on these organoids a resistance to EGFR inhibition. Thus, our data support a unique role for RAF dimer function in in KRAS mutant tumors of the colon in both development and sensitivity to drugs, as well as argue for the inclusion of KRASA59T and KRASA59E bearing CRC tumors in anti-EGFR therapies. Citation Format: Christian William Johnson, Elizabeth M. Terrell, Josh Cook, Bing Shui, Eve O'Donoghue, Shannon Hull, Moon Hee Yang, Shikha Sheth, Rona Yaeger, Scott Kopetz, Deborah K. Morrison, Kevin Haigis. Ala59 mutants of KRAS cooperate with Nf1 loss to enhance colon tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6623.

CTNI-76. EFFICACY OF BRAF INHIBITOR PLIXORAFENIB (FORE8394) IN RECURRENT, PRIMARY CENTRAL NERVOUS SYSTEM TUMORS (PCNST)

Abstract Plixorafenib, a selective potent BRAFi, is uniquely designed to disrupt BRAF dimers and evade paradoxical MAPK pathway activation. Thus, it inhibits both V600 and non-V600 BRAF alterations and may be less prone to toxicities and resistance typical of approved BRAFi. An approved BRAFi, when used with MEKi, has ORR 33% with mDOR 13.6=months and ORR=50% with DOR of 6-29 months in V600+ HGG and LGG, respectively (dabrafenib US Prescribing Information, 2023). Phase 1/2a is a single-arm study (NCT02428712) in patients (n=113) with BRAF-altered advanced tumors to assess safety, PK, and efficacy of oral plixorafenib 900-3600 mg/day alone or with cobicistat (CYP3A4/P-gp inhibitor). Symptomatic TEAEs (rash, pyrexia, myalgia) were low grade and infrequent relative to approved BRAFi. This pre-specified analysis (31Mar2023) was in MAPKi-naïve adults with BRAFV600+ PCNST. 22 patients with PCNST received plixorafenib: 9 LGG, 1 glioneuronal tumor (LGG/GNT; 5 V600+), 12 HGG (10 V600+). Treatment-emergent AEs (TEAEs, ≥ 20%) were limited to LFT changes, G1 increased creatinine, fatigue, headache, and nausea. Only one G3 event (increased ALT) occurred at the RP2D (n=7) showing better tolerability than dabrafenib. 10 BRAFV600+ MAPKi-naïve adults with PCNST (median age 45 yrs, 6 females, 9 non-Hispanic) were efficacy evaluable: 6 HGG (4 glioblastoma, 1 epithelioid glioblastoma, 1 anaplastic astrocytoma); 4 LGG/GNT (1 each: ganglioglioma, pilocytic astrocytoma, xanthoastrocytoma, neuroepithelial tumor). Prior anti-cancer treatments included surgery (8/10), radiotherapy (8/10), and systemic therapy (8/10, including temozolomide [n=8], bevacizumab [n=2], carboplatin [n=1], dendritic cell vaccine [n=1]). 1 was treatment-naïve (unresectable; declined radiation/chemotherapy). ORR=60% (6/10) including HGG=67% (4/6), LGG/GNT=50% (2/4); median time to response=1.94 months; 4/6 responders with DOR > 9 months; mPFS=34.1 months; median follow-up=10.3 months; 4/10 ongoing. Plixorafenib has a benign safety profile and leads to a high ORR and durable response in MAPKi-naïve BRAFV600+ PCNST. A phase 2 study is ongoing to confirm these findings (NCT05503797).

A phase 1, open-label, dose escalation and dose expansion study to evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of PF-07799933 (ARRY-440) as a single agent and in combination therapy in participants 16 years and older with advanced solid tumors with BRAF alterations.

TPS3164 Background: Approved BRAF inhibitors (BRAFi) are inactive against RAF dimers. Accordingly, the acquisition of BRAF dimers ( e.g., through drug-acquired splice variants or BRAF amplification) may lead to therapy resistance. Non-V600 (class II and III) BRAF alterations that occur de novo in diverse tumor types also signal as dimers and therefore have no approved targeted therapeutic options. Additional liabilities of approved BRAFi include toxicity from paradoxical activation of wild-type (WT) RAF and limited brain penetration, a common primary and metastatic site for BRAF-altered cancers. In contrast to approved agents, investigational BRAFi inhibit WT and mutant RAF proteins (A, B, and C-RAF, monomers, dimers), and therefore may be limited by on-target toxicity from “pan-RAF” inhibition. PF-07799933 is an oral selective ATP-competitive small-molecule RAF kinase inhibitor that suppresses BRAF signaling in BRAF V600-mutant and non-V600- BRAF mutant tumors. It displays significantly less paradoxical activation than approved BRAFi and is not “pan RAF” as it spares non-BRAF-containing RAF dimers. We describe here the Phase 1 study of PF-07799933 as monotherapy or in combination with binimetinib or cetuximab in participants with BRAF-altered advanced solid tumors. Methods: The purpose of this first-in-human study is to investigate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and potential clinical benefits of PF-07799933 administered as a single agent and in combination in participants with BRAF Class I, II and III mutated solid tumors with or without brain involvement. Part 1 (monotherapy dose escalation) and Part 2 (combination dose escalation) will enroll participants with BRAF V600 mutation who have progressed on a approved BRAFi or with de novo Class II or III alterations who have progressed on standard of care therapy. The primary objective of Part 1 is to determine monotherapy MTD/RDE of PF-07799933. Participants in Part 1 will be allowed to add on rational combination with either binimetinib or cetuximab at the time of disease progression. Once PK measurements indicate the potential for significant BRAF inhibition in the brain, participants with untreated and symptomatic brain metastases will be allowed to enroll. The primary objective of Part 2 is combination MTDc/RDEc of PF-07799933 with binimetinib or cetuximab. The primary objective of Part 3 is efficacy in defined cohorts (total ~120 participants): Cohort 1-2: BRAF V600 mutant melanoma; Cohort 3: BRAF Class II altered melanoma; Cohorts 4-5: BRAF V600 and Class II altered CRC; Cohort 6: other BRAF V600, Class II/III altered solid tumor not qualifying for Cohorts 1-5. Clinical trial information: NCT05355701 .

Abstract 4045: DCC-3084, a RAF dimer inhibitor, broadly inhibits BRAF class I, II, III, BRAF fusions, and RAS-driven solid tumors leading to tumor regression in preclinical models

Abstract Background: Mutations in the RAS/MAPK pathway are a frequent driver of cancer, with oncogenic RAS or RAF mutations occurring in >30% of all cancers. First generation BRAF inhibitors are approved for use for tumors with Class I BRAF mutations (V600X). However, these drugs are not efficacious in RAF dimer mutant and RAS mutant cancers due to paradoxical activation of RAF dimers. Herein, we describe DCC-3084, a potent and selective investigational Switch Control inhibitor of BRAF and CRAF kinase dimers that targets Class I, II and III BRAF mutations, BRAF fusions, and BRAF/CRAF heterodimers. DCC-3084 combines with inhibitors of additional nodes in the MAPK pathway to potentially target a large unmet medical need in RAS and RAF mutant cancers. Methods: Inhibition of RAF kinases, including off-rate analysis, was measured using recombinant enzymes. X-ray crystallography was used for structure-based drug design. Cellular proliferation was measured using resazurin to monitor cell viability. Synergy in cells was measured using BLISS scores and curve shift analysis. Inhibition of ERK or RSK phosphorylation was measured by AlphaLISA or ELISA. Pharmacokinetics (PK) in the plasma, brain and CSF compartments were measured following oral dosing in Wistar rats. RAF and RAS mutant mouse xenograft models were used to assess PK, pharmacodynamics (PD), and efficacy. Results: DCC-3084 is a potent and selective Switch Control inhibitor of RAF dimers that was designed to target Class I, II, III BRAF mutants, BRAF fusions, and BRAF/CRAF heterodimers. DCC-3084 inhibits BRAF and CRAF, exhibiting slow off-rates (t1/2 >20 hr). Potent single-agent inhibition of MAPK pathway signaling and cellular proliferation was observed in a wide range of Class I, II, III BRAF and BRAF fusion altered cell lines. Synergy was observed in combination with inhibitors of other nodes in the RAS/MAPK pathway in RAS mutant cell lines. DCC-3084 was demonstrated to be CNS penetrable and exhibited dose dependent oral exposure with robust inhibition of the RAS/MAPK pathway in PK/PD models. DCC-3084 accumulated in tumor tissue relative to plasma, further demonstrating a favorable pharmaceutical profile. Oral treatment of DCC-3084 as a single agent resulted in tumor regression in BRAF mutant and KRAS Q61K mutant mouse xenograft models and tumor growth inhibition in KRAS G12C/D mutant models. Additionally, DCC-3084 in combination with a MEKi resulted in tumor regression in KRAS mutant models. Conclusions: The Switch Control inhibitor DCC-3084 broadly inhibits Class I, II and III BRAF mutations, BRAF fusions, and BRAF/CRAF heterodimers leading to tumor regression in preclinical models. The overall preclinical profile of DCC-3084 supports IND-enabling activities towards clinical development in a key area of unmet medical need in RAS and RAF mutant cancers. Citation Format: Stacie L. Bulfer, Bertrand Le Bourdonnec, Jeffery D. Zwicker, Yu Mi Ahn, Gada Al-Ani, Hikmat Al-Hashimi, Chase Crawley, Kristin M. Elliott, Saqib Faisal, Andrew M. Harned, Cale L. Heiniger, Molly M. Hood, Salim Javed, Michael Kennedy, Joshua W. Large, Cynthia B. Leary, Wei-Ping Lu, Kylie Luther, Max D. Petty, Hunter R. Picard, Justin T. Proto, Yeni K. Romero, Forrest A. Stanley, Kristen L. Stoltz, Daniel C. Tanner, Hanumaiah Telikepalli, Mary J. Timson, Lakshminarayana Vogeti, Subha Vogeti, Sihyung Yang, Lexy H. Zhong, Bryan D. Smith, Daniel L. Flynn. DCC-3084, a RAF dimer inhibitor, broadly inhibits BRAF class I, II, III, BRAF fusions, and RAS-driven solid tumors leading to tumor regression in preclinical models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4045.

Structure and RAF family kinase isoform selectivity of type II RAF inhibitors tovorafenib and naporafenib

Upon activation by RAS, RAF family kinases initiate signaling through the MAP kinase cascade to control cell growth, proliferation, and differentiation. Among RAF isoforms (ARAF, BRAF, and CRAF), oncogenic mutations are by far most frequent in BRAF. The BRAFV600E mutation drives more than half of all malignant melanoma and is also found in many other cancers. Selective inhibitors of BRAFV600E (vemurafenib, dabrafenib, encorafenib) are used clinically for these indications, but they are not effective inhibitors in the context of oncogenic RAS, which drives dimerization and activation of RAF, nor for malignancies driven by aberrantly dimerized truncation/fusion variants of BRAF. By contrast, a number of "type II" RAF inhibitors have been developed as potent inhibitors of RAF dimers. Here, we compare potency of type II inhibitors tovorafenib (TAK-580) and naporafenib (LHX254) in biochemical assays against the three RAF isoforms and describe crystal structures of both compounds in complex with BRAF. We find that tovorafenib and naporafenib are most potent against CRAF but markedly less potent against ARAF. Crystal structures of both compounds with BRAFV600E or WT BRAF reveal the details of their molecular interactions, including the expected type II-binding mode, with full occupancy of both subunits of the BRAF dimer. Our findings have important clinical ramifications. Type II RAF inhibitors are generally regarded as pan-RAF inhibitors, but our studies of these two agents, together with recent work with type II inhibitors belvarafenib and naporafenib, indicate that relative sparing of ARAF may be a property of multiple drugs of this class.

239 (PB119) - Preclinical efficacy of BDTX-4933, a brain penetrant MasterKey inhibitor targeting oncogenic BRAF Class I/II/III mutations

Background: FDA-approved BRAF inhibitors target V600 (Class I) mutant monomers and are largely inactive against mutant BRAF dimers. These dimeric mutants found in many solid tumors including primary CNS tumors and brain metastases can drive RAS-independent (Class II) or RAS-dependent (Class III) oncogenic tumor growth. Furthermore, the approved BRAF inhibitors can induce paradoxical RAF activation that limits their activity. Although currently approved BRAF V600 mutation-selective inhibitors demonstrated efficacy in brain tumors and metastases when combined with MEK inhibitors in clinical trials, duration of response tends to be short partly due to limited BBB permeability.

466P Efficacy of BRAF inhibitor FORE8394 in BRAF V600+ patients

FORE8394 is a selective, potent BRAFi (class 1, 2 including fusions). By disrupting BRAF dimers, FORE8394 evades paradoxical MAPK pathway activation and may be less prone to toxicities & resistance typical of 1st generation BRAFi.

Abstract LB083: BRAF dimer-selective inhibitors synergize with BRAF monomer-selective inhibitors to overcome adaptive resistance and retain therapeutic index

Abstract BRAF kinase, a critical component of the RAS/MAPK signaling pathway, requires dimerization for its catalytic activity, whereas the oncoprotein BRAF(V600E) can be catalytically active as a monomer. Current clinical BRAF inhibitors selectively bind and inhibit monomeric over dimeric BRAF (i.e. “BRAF monomer-selective”) providing a high therapeutic index and are now standard practice in combination with MEK inhibitors in the treatment of patients harboring BRAF(V600E) tumors. However adaptive resistance due to RAF dimerization limits their effectiveness. Recently, RAF inhibitors that equipotently inhibit both monomeric and dimeric BRAF have been developed, but they are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues. We identified and characterized a novel class of BRAF inhibitors that preferentially bind and inhibit dimeric over monomeric BRAF(V600E) (i.e. “BRAF dimer-selective inhibitors”). Biochemical analysis using BRAF dimer-selective inhibitors revealed that the two forms of dimeric and monomeric BRAF(V600E) differ in their conformation in their active site and their strength of interaction with MEK. Further, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index, we assessed the triple combination of BRAF monomer- plus BRAF dimer selective inhibitor plus a MEK inhibitor that disrupts the BRAF-MEK complex. The triple combination potently suppressed tumor growth in multiple colorectal and melanoma BRAF(V600E) cell line- and in vivo models that were resistant to the current clinical BRAF and MEK inhibitor combination. Strikingly, while the double combination treatment with the BRAF dimer-selective inhibitor and MEK inhibitor was associated with a significant gradual weight loss in vivo, the triple combination showed no weight loss or other apparent toxicities, indicating a higher Therapeutic Index. Finally, off-label use of the triple combination in patients with advanced BRAF(V600E) cancers that progressed on standard therapies achieved durable tumor control with minimal toxicities. Thus, a rationally designed combinatorial approach of conformation-selective BRAF and MEK inhibitors may be a highly effective and well tolerated therapeutic strategy for patients with BRAF(V600E) tumors. Overall, our work supports the notion that for most tumors a “3-drug” combinatorial strategy will potentially be most effective: one drug targeting MAPK signaling directly, such as a MEK inhibitor (“pathway” inhibitor), one drug targeting mutated BRAF or RAS selectively in the tumor (“therapeutic index” inhibitor, e.g. BRAF monomer-selective inhibitor, RAS(G12C) inhibitors, etc) and one drug targeting components of the feedback loop that is responsible for adaptive resistance (“feedback” inhibitor, e.g. BRAF dimer-selective inhibitors, SHP2 or SOS inhibitors, etc). Our data contribute to the development of a roadmap for the treatment of MAPK-driven tumors using drug combinations tailored to the specific driver oncoprotein. Citation Format: Christos Adamopoulos, Tamer Ahmed, Peter M. Ung, Min Xiao, Stuart Aaronson, Celina Ang, Vito Rebecca, Avner Schlessinger, Meenhard Herlyn, Poulikos I. Poulikakos. BRAF dimer-selective inhibitors synergize with BRAF monomer-selective inhibitors to overcome adaptive resistance and retain therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB083.

Molecular Dynamics Investigation of Oncogenic BRAF Mutations

BRAF is one member of the RAF serine/threonine kinase family and a key component of the RAS‐RAF‐MEK‐ERK (MAPK) signaling pathway, controlling cell growth, proliferation, differentiation, migration and survival. BRAF is the most frequently mutated kinase in human cancers, with over 200 mutations discovered, among which V600E and G469A are the most common BRAF mutations. Current therapies with ATP‐competitive inhibitors are potent against BRAFV600E, yet they display “paradoxical activation” and drug resistance toward BRAFG469Aandother non‐V600 BRAF mutants. Thus, novel BRAF therapies towards non‐V600 variants are urgently needed. However, the biochemical properties of non‐V600 BRAF mutants and the cause of drug response are unclear due to the lack of structures of BRAF mutants. In this study, we applied molecular dynamics (MD) simulations to investigate the structure, dynamics and functions of oncogenic BRAFG469A, in comparison with BRAFWT and BRAFV600E. For each BRAF type (WT or mutant), MD simulations are carried out for its monomer and dimer with or without 1 or 2 bound vemurafenib in explicit aqueous solution. Our work provides atomic information on mechanism of aberrant activation by G469A mutation, “paradoxical activation” and structural dynamics of the nucleotide pocket, activation loop, and dimer and inhibitor/BRAF interfaces. G469A mutation makes hydrophobic contact between methyl of A469 with alkyl chain of K483 in the binding pocket. This stabilizes the K483‐E501 salt‐bridge, which functions to bring αC‐helix towards the active site. Thus, G469A stabilizes the active conformation by positioning αC‐helix close to the P‐loop through increasingly stabilized salt‐bridge relative to WT and V600E. Consequently, our simulations illustrate inability of vemurafenib to disrupt active conformation of BRAFG469A by separating αC‐helix from the nucleotide binding pocket. This decreased structural fluctuation upon inhibitor binding to one protomer explains the ability of “paradoxical activation” of BRAF dimer through allosteric activation.

Thermodynamic and allosteric basis of paradoxical activation in V600E BRAF cancers

The V600E mutation in the protein kinase BRAF is a well-established driver of cancer. The effectiveness of current small-molecule inhibitors designed to target V600E BRAF is severely limited by resistance mechanisms that unavoidably develop only months after treatment. These inhibitors effectively suppress V600E BRAF monomers but do not inhibit V600E BRAF homodimers, and instead elevate their activity through a phenomenon called paradoxical activation (PA). Consequently, molecular lesions promoting the formation of V600E BRAF dimers lead to pharmacological resistance and highlight the inability of current therapeutics to inhibit V600E BRAF dimers as a severe clinical problem.

Abstract P229: Pre-clinical evaluation of next-generation inhibitor targeting a wide spectrum of oncogenic BRAF dimers

Abstract The canonical BRAF V600E (Class I) mutation is a potent oncogene uniquely active as a RAS-independent monomer, successfully targeted by several FDA-approved inhibitors. However, these first-generation BRAF inhibitors are not active against non-canonical BRAF oncogenic mutations, including BRAF-fusions, that drive RAS-independent (Class II) or RAS-dependent (Class III) dimers. As such, developing inhibitors directed against dimeric BRAF oncogenic mutations that avoid paradoxical activation is a major unmet clinical need. We applied proprietary Mutation-Allostery-Pharmacology (MAP) platform technology developed by Black Diamond Therapeutics to identify and validate a group of previously uncharacterized non-canonical oncogenic Class II and Class III BRAF mutation clusters. We further demonstrate that this ensemble of novel and previously validated non-canonical oncogenic BRAF mutants can form the basis of a differentiated drug discovery program aimed at identifying small molecules that potently and selectively target this family of dimeric BRAF mutations. Herein, we describe a small molecule inhibitor, BDTX BRAF-A, with potent anti-proliferative activity directed against tumor cells expressing a wide spectrum of non-canonical Class II/III mutations. This broad activity (“MasterKey” profile) of BDTX BRAF-A is further demonstrated in cell lines that harbor endogenous oncogenic dimer-inducing BRAF mutations and in various solid tumor patient-derived xenograft (PDX) models ex vivo. Importantly, BDTX BRAF-A did not induce paradoxical RAF activation characteristic of Class I BRAF inhibitors. Finally, BDTX BRAF-A achieves robust anti-tumor efficacy and target engagement of dimeric BRAF oncogenes in mouse models. These data support the continued development of rationally designed molecules targeting a broad range of non-canonical BRAF dimer-promoting mutations to extend the prospect of precision medicine in patients. Citation Format: Yoon-Chi Han, Pui Yee Ng, Ryan Schulz, Shao Ning Yang, Alana Lelo, Luisa Shin, Matthew O'Connor, Ivan Jewett, Noboru Ishiyama, Darlene Romashko, Shalabh Thakur, Andrei Salomatov, Sherri Smith, Elizabeth Buck, Christopher Roberts, Matthew Lucas, Tai-An Lin. Pre-clinical evaluation of next-generation inhibitor targeting a wide spectrum of oncogenic BRAF dimers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P229.

Triple MAPK Inhibition Salvaged a Relapsed Post BCMA CAR-T Cell Therapy in Multiple Myeloma Patient with BRAF V600E Dominant Clone

Background:Multiple Myeloma (MM) is a progressive plasma cell neoplasm characterized by heterogeneous clonal expansion. Despite numerous targeted therapy options, failing anti-BCMA CAR-T cell therapy represents a challenging treatment course with dismal prognosis. 53% of relapsed/refractory (rr) MM patients have shown emerging clones harboring mutations within the MAPK signaling pathway including the targetable BRAF V600E in 7% of cases. Allosteric kinase inhibition of MAPK pathway with BRAF monomer selective inhibitors +/- MEK inhibitor have been frequently attempted with rrMM patients with equivocal outcome on both disease progression and survival. This could be ascribed to the feedback activation of the pathway via BRAF dimers induction. In solid tumor contexts, a recent study has revealed that the multi-kinase inhibitor regorafenib is a potent and selective inhibitor of dimeric BRAF. Moreover, the study demonstrated overcoming of this negative feedback by combining two inhibitors targeting BRAF in its monomeric and its dimeric form, respectively, with MEK inhibition leading to more efficacious and tolerable treatment (Adamopoulos C. et al. Cancer Discov. 2021). Here, we show the applicability, effectiveness, and tolerability of the aforementioned triple therapy strategy in hematological malignancy context to salvage post BCMA CAR-T cell therapy in a BRAF V600E rrMM patient.Material and Methods:CD138 + selected cells were collected from bone marrow (BM) and patient's plasmacytoma before and after CAR-T cell therapy relapse, respectively. Samples were processed using the standard GATK 4 workflows and a consensus mutation calling approach was used to call somatic mutations using a matched normal obtained from peripheral blood. PhyloWGS (Deshwar et al, Genome Biol 2015) was then used to perform intra-tumor heterogeneity modeling and phylogeny estimation.CD138 + selected cells from BM of rrMM patient were treated with encorafenib (50nM) and binimetinib (250 nM) +/- regorafinib (1mM) for 48 hours, cells were harvested, lysed and immunoblotted with BRAF V600E and pERK antibodies.In parallel, rrMM patient BM selected CD138 + cells' sensitivity to MAPK inhibitors were assessed by Travera. Cells are incubated with different inhibitors at varying concentrations and combinations for 15 hours before having their single-cell mass distributions measured. Sensitivity is then characterized by the relative statistical difference (Hellinger distance) between conditions and untreated controls.A rrMM patient was started on targeted triple therapy using the following combination: BRAF monomer-inhibitor, dabrafenib (100mg, orally twice daily), a MEK-inhibitor, trametinib (1.5mg, orally for 21/28 days daily), and a BRAF dimer inhibitor, regorafenib (40mg, orally once daily).Results:Whole Exome Sequencing (WES) showed a BRAF V600E mutation was present prior to CAR-T cell therapy and persisted through subsequent treatments. The variant allele frequency of the mutation was 41% post CAR-T cell therapy (Figure 1A).Western blot of CD138 + cells confirmed BRAF V600E mutation with a specific antibody, and showed ERK phosphorylation, which was inhibited with trametinib. Longer exposure to trametinib confirmed a feedback loop activating MAPK which was abrogated with the BRAF dimer inhibitor regorafenib (Figure 1B).Travera analysis showed CD138 + cells' sensitivity to the combination of trametinib at 5mM, and dabrafenib at 1µM. Adding regorafenib at 1µM could slightly improve the response compared to the double combination. (Figure 1C).Clinically, three months of the triple therapy administration has led to prompt reduction in subcutaneous skin lesions as well as 80% reduction in free lambda chain (Figure 1D). Furthermore, the patient exhibited good tolerance to all three medications with minimal side effects (grade 1 fatigue). The treatment regimen allowed the patient to carry out activities of daily living and resume work.Conclusion:We have shown the applicability, effectiveness, and tolerability of the triple MAPK inhibition in a hematological malignancy context to salvage post BCMA CAR-T cell therapy in a rrMM patient with BRAF V600E mutation. Also, we have revealed the power of routine WES in tracking clonal evolution, and identifying targetable mutations which allows tailoring of therapeutics for MM patients. [Display omitted] DisclosuresStevens: Travera: Current Employment. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding.

P-090: BRAF V600E multiple myeloma patient salvaged with triple MAPK inhibition after CAR T relapse

<h3>Background</h3> Despite a growing arsenal of treatment choices, patient relapse post-BCMA-targeted CART therapy remains a challenge and the therapeutic path is still undefined. Among the most frequently observed actionable mutations, BRAF V600E, is present in ~7% of multiple myeloma (MM) patients. The availability of selective inhibitors of BRAF V600E makes this a valuable therapeutic target. However, the clinical efficacy of these inhibitors has been limited and short-lived, frequently due to feedback-induction of BRAF dimers. Novel studies revealed that the multi-kinase inhibitor regorafenib is a potent and selective inhibitor of dimeric BRAF. They have further demonstrated relief of these negative outcomes by inhibiting BRAF in both its monomeric and dimeric form in combination with MEK inhibition leading to more efficacious and tolerable treatment. (Adamopoulos C. et al. Cancer Discov. 21). We present a case of a MM patient with BRAF V600E mutation salvaged with a triple therapy inhibition strategy after relapsing following anti-BCMA CART therapy. <h3>Method</h3> A 61-year-old male with penta-refractory MM (IgA lambda), ISS stage 3 with hyperdiploidy, gain of 1q21 and del13 was treated with anti-BCMA CART therapy, achieving a best response of VGPR, and disease progression after 6 months. He was temporarily salvaged with BCNU/Mel ASCT and achieved a best response of PR until progression with extramedullary disease (subcutaneous skin lesions in lower extremities) and elevated lambda free light chains (FLC, 126.4mg/l) at 6 months. <h3>Results</h3> Targeted sequencing showed a BRAF V600E mutation was present prior to CART therapy in his bone marrow and persisted in his bone marrow and a plasmacytoma with a VAF of 41% at this current relapse. A western blot analysis of bone marrow aspirate confirmed BRAF V600E with a specific antibody, and showed phosphorylation of ERK, which was diminished with trametinib. Longer exposure to trametinib confirmed a feedback loop activating MAPK which was abrogated with the BRAF dimer inhibitor regorafenib. With these findings the patient was started on targeted therapy based on combination of a BRAF monomer-inhibitor, dabrafenib (100mg, orally twice daily), a MEK-inhibitor, trametinib (1.5mg, orally for 21/28 days daily), and a BRAF dimer inhibitor, regorafenib (40mg, orally once daily). Within 3 months of treatment initiation, prompt reduction in subcutaneous skin lesions and 80% reduction in free lambda FLC (27.5 mg/l) was observed. Furthermore, the patient had good tolerance to all three medications with minimal side effects (grade 1 fatigue) and continues treatment. The treatment regimen allowed the patient to carry out activities of daily living and return to work. <h3>Conclusion</h3> Post-CART failure treatment is a challenging and unmet need. NGS may identify targeted therapy that would be able to salvage patients with a tolerable side effect profile.

The next-generation pan-RAF inhibitor, KIN-2787, is active in class II and class III BRAF mutant models.

3116 Background: Oncogenic BRAF gene alterations, leading to aberrantly activated BRAF monomers (Class I mutations) or dimers (Class II and Class III mutations), are observed in approximately 6% of all human cancers. First-generation BRAF inhibitors targeting Class I BRAF mutants, including dabrafenib, encorafenib, and vemurafenib, provide significant clinical benefit to patients with BRAF V600 mutation-driven melanoma and select solid tumors as monotherapies or in combination with other targeted therapies. The currently approved BRAF inhibitors have not, however, proven to be effective in patients with Class II or III BRAF alterations which account for a large proportion (34%) of BRAF mutations. KIN-2787 is an orally available, potent and selective small molecule pan-RAF inhibitor specifically designed to inhibit Class II and III BRAF dimers, in addition to Class I mutants. Methods: The efficacy and tolerability of the pan-RAF inhibitor, KIN-2787, was evaluated in vitro and in vivo in Class I, II, and III BRAF mutation-driven human cancer models. Results: In biochemical assays, KIN-2787 showed low nanomolar to picomolar potency against RAF1, BRAF, and ARAF (IC50 0.06-3.46 nM) with minimal activity towards non-RAF kinases. In cell-based assays, KIN-2787 inhibited RAF activity, as measured by inhibition of downstream ERK phosphorylation (pERK), across multiple BRAF mutant cancer cell lines. Class II and III BRAF mutant cell lines were the most responsive when treated with KIN-2787 (IC50 &lt; 50 nM); 19- and 7-fold more sensitive compared to cells harboring wild-type BRAF, respectively. Dose-dependent inhibition of A-375 (Class I), BxPC-3 (Class II), and WM3629 (Class III) BRAF mutant human xenograft tumor growth was attained with daily KIN-2787 treatment and was well-tolerated. A trend towards greater tumor responses was observed with twice daily (BID) compared to once daily (QD) dosing of KIN-2787; however, the two dosing regimens led to similar tumor growth inhibition (TGI) and regressions (mean TGI up to 101-118%; p ≤0.0001) at equivalent total daily doses. Furthermore, KIN-2787 led to a significant in vivo pharmacodynamic response using either regimen, however, prolonged target coverage, as measured by pERK, was achieved with BID dosing. The impact of KIN-2787 treatment on additional biomarkers, including transcriptional changes and MAPK pathway modulation in cell-based models and patient-derived samples, will be presented at the meeting. Conclusions: KIN-2787 is a next-generation pan-RAF inhibitor with pronounced in vitro and in vivo activity against human cancers driven by Class II and III BRAF mutations. A phase 1 dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 monotherapy in patients with advanced or metastatic solid tumors harboring BRAF gene alterations, including Class II and III mutations, is expected to initiate in 2021.

B-Raf autoinhibition in the presence and absence of 14-3-3

Raf-activating mutations are frequent in cancer. In the basal state, B-Raf is autoinhibited by its upstream Ras-binding domain (RBD) and cysteine-rich domain (RBD-CRD) interacting with its kinase domain (KD) and the 14-3-3 dimer. Our comprehensive molecular dynamics simulations explore two autoinhibition scenarios in the presence and absence of the 14-3-3 dimer. When present, the 14-3-3 interaction with B-Raf stabilizes the RBD-CRD-KD interaction, interfering with the KD dimerization. Raf's pSer365 removal fails to induce large disruption. RBD-CRD release promotes KD fluctuations and reorientation for dimerization, consistent with experimental data. In the absence of 14-3-3, our sampled B-Raf conformations suggest that RBD-CRD can block the KD dimerization surface. Our results suggest a B-Raf activation mechanism, whereby one KD monomer is donated by 14-3-3-free B-Raf KD and the other by 14-3-3-bound KD. This mechanism can lead to homo- and heterodimers. These autoinhibition scenarios can transform autoinhibited B-Raf monomers into active B-Raf dimers.

43P Pre-clinical evaluation of potent and orally bioavailable next-generation inhibitors targeting the family of mutants that drive oncogenic BRAF dimer formation

The canonical BRAF V600E (class I) mutation is a potent oncogene which is uniquely active as a RAS-independent monomer, and which has been successfully targeted by several FDA-approved inhibitors. While active against monomeric BRAF V600E, these first generation BRAF inhibitors induce paradoxical activation of RAS-driven BRAF dimers in cells expressing wild-type RAF, and this can lead to secondary malignancies. More recently, numerous non-canonical BRAF oncogenic mutations including BRAF-fusions have been described as oncogenes that drive RAS-independent (class II) or RAS-dependent (class III) dimers. These non-canonical dimeric BRAF oncogenes are resistant to the first-generation drugs, effective only against the monomeric BRAF V600E mutation. Discovery of an inhibitor directed against the family of dimeric BRAF oncogenic mutations which avoids paradoxical activation is a major unmet need. We applied our proprietary Mutation-Allostery-Pharmacology (MAP) platform technology developed by Black Diamond Therapeutics to identify and validate a group of previously uncharacterized non-canonical oncogenic class II and class III BRAF mutation clusters. We further demonstrate that this ensemble of both novel and previously validated non-canonical oncogenic BRAF mutants can form the basis of a differentiated drug discovery program aimed at identifying small molecules that potently and selectively target this family of dimeric BRAF mutations. Herein, we describe a series of small molecule inhibitors with potent anti-proliferative activity directed against tumor cells harboring dimer-inducing BRAF oncogenic mutations and which are devoid of paradoxical RAF activation. Leading exemplars of BDTX compounds are orally available inhibitors that achieve target engagement of BRAF dimer oncogenes in vivo and robust anti-tumor efficacy in xenograft models in mice. These data support continued development of rationally designed molecules targeting a broad range of non-canonical BRAF dimer-promoting mutations to extend the prospect of precision medicine in patients with BRAF mutant tumors.

Inhibitors of BRAF dimers using an allosteric site

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAFV600E signals as an active monomer in the absence of active RAS, however, in many tumors BRAF dimers mediate ERK signaling. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which leads to tumor resistance. We found that Ponatinib, an FDA-approved drug, is an effective inhibitor of BRAF monomers and dimers. Ponatinib binds the BRAF dimer and stabilizes a distinct αC-helix conformation through interaction with a previously unrevealed allosteric site. Using these structural insights, we developed PHI1, a BRAF inhibitor that fully uncovers the allosteric site. PHI1 exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors. This work shows that Ponatinib and BRAF dimer selective inhibitors will be useful in treating BRAF-dependent tumors.