BRAF Kinase Domain Research Articles

Clinical and Preclinical Activity of EGFR Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Harboring BRAF Class 3 Mutations.

Patients with tumors harboring BRAF class 3 mutations lack targeted therapies. These mutations are characterized by low/absent BRAF kinase domain activation and are believed to amplify already active RAS signaling, potentially triggered by receptor tyrosine kinases like EGFR. Two patients with BRAF class 3-mutated metastatic non-small-cell lung cancer (NSCLC) were treated with erlotinib at our Institution after failure of standard therapies. Two cell lines were established from patients with BRAF class 3-mutated NSCLC, and their sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs) was assessed using EGFR-mutated, BRAF class 1 and 2-mutated, and KRAS-mutated NSCLC cell lines as controls. Patient 1, a 60-year-old male with BRAFD594N-mutated NSCLC, achieved complete response to erlotinib after progression on first- and second-line chemotherapy. Patient 2, a 60-year-old female with BRAFD594G-mutated NSCLC, achieved partial response to erlotinib after progression on first-line chemoimmunotherapy. High baseline phosphorylated EGFR values and reduced EGFR activation following erlotinib were observed in BRAF class 3-mutated and EGFR-mutated cell lines, but not in BRAF class 1-mutated, BRAF class 2-mutated, or KRAS-mutated lines. Erlotinib inhibited 2-dimensional growth in BRAF class 3-mutated cell lines (IC50 6.33 and 7.11 µM) and in the BRAF class 2-mutated cell line (IC50 5.51 µM), albeit at higher concentrations than in EGFR-mutated lines, whereas it showed no effect on BRAF class 1-mutated (IC50, >25 µM) or KRAS-mutated (IC50, >25 µM) lines. These findings were corroborated by 3-dimensional and sphere formation assays. In the Cancer Cell Line Encyclopedia, BRAF class 3-mutated NSCLC cell lines showed greater sensitivity to EGFR-TKIs compared with BRAF class 2-mutated and KRAS-mutated lines. BRAF class 3 mutations in NSCLC may identify a novel targetable population sensitive to EGFR-TKIs.

Tumor-Agnostic Genomic and Clinical Analysis of BRAF Fusions Identifies Actionable Targets.

Even though BRAF fusions are increasingly detected in standard multigene next-generation sequencing panels, few reports have explored their structure and impact on clinical course. We collected data from patients with BRAF fusion-positive cancers identified through a genotyping protocol of 97,024 samples. Fusions were characterized and reviewed for oncogenic potential (in-frame status, non-BRAF partner gene, and intact BRAF kinase domain). We found 241 BRAF fusion-positive tumors from 212 patients with 82 unique 5' fusion partners spanning 52 histologies. Thirty-nine fusion partners were not previously reported, and 61 were identified once. BRAF fusion incidence was enriched in pilocytic astrocytomas, gangliogliomas, low-grade neuroepithelial tumors, and acinar cell carcinoma of the pancreas. Twenty-four patients spanning multiple histologies were treated with MAPK-directed therapies, of which 20 were evaluable for RECIST. Best response was partial response (N = 2), stable disease (N = 11), and progressive disease (N = 7). The median time on therapy was 1 month with MEK plus BRAF inhibitors [(N = 11), range 0-18 months] and 8 months for MEK inhibitors [(N = 14), range 1-26 months]. Nine patients remained on treatment for longer than 6 months [pilocytic astrocytomas (N = 6), Erdheim-Chester disease (N = 1), extraventricular neurocytoma (N = 1), and melanoma (N = 1)]. Fifteen patients had acquired BRAF fusions. BRAF fusions are found across histologies and represent an emerging actionable target. BRAF fusions have a diverse set of fusion partners. Durable responses to MAPK therapies were seen, particularly in pilocytic astrocytomas. Acquired BRAF fusions were identified after targeted therapy, underscoring the importance of postprogression biopsies to optimize treatment at relapse in these patients.

Abstract LB053: A novel BRAF-specific allosteric inhibitor that effectively targets acquired RAFi-resistant BRAF(V600E) cancers and RAS-mutated cancers in vitro and in vivo

Abstract Targeting hyperactive RAS-RAF-MEK-ERK signaling for treating cancers has achieved a promising outcomes, and a number of RAS/RAF inhibitors have been applied to clinical cancer treatment. However, these inhibitors have only a short-term efficacy and their effectiveness is abrogated by acquired drug resistance in less than one year. For those resistant/refractory RAS/RAF-mutated cancers, there’s no effective therapeutics available in clinic. In this study, we developed a novel BRAF-specific inhibitor that docks on an allosteric site on BRAF kinase domain. This inhibitor effectively killed both primary and drug-resistant cancer cell lines with RAS mutation or BRAF(V600E) mutation in vitro and inhibited the growth of xenografted cancers derived from these cell lines in vivo without apparent toxicity. Mechanistically, we found that although this inhibitor blocked the catalytic activity of BRAF in vitro kinase assay, it triggered a super high ERK signaling through improving BRAF/CRAF heterodimerization and hence induced cellular apoptosis of cancer cells with RAS mutation or BRAF(V600E) mutation. Surprisingly, there’s no drug resistance occurring when treating cancer cells with RAS mutation or BRAF(V600E) mutation at a low concentration. To further evaluate the efficacy of this BRAF against RAS/RAF-mutated cancers in vivo, we constructed a series of pancreatic patient-derived xenograft cancer models with Kras G12D or G12V mutations, and found that our BRAF inhibitor exhibited a comparable or much better efficacy than Kras G12D-specific inhibitor on theses PDX models. Altogether, our data indicated that our BRAF-specific allosteric inhibitor may serve as a good therapeutic drug for treating BRAF(V600E)/RAS- cancers, particularly those resistant/refractory cancers, though it still need further evaluations via clinical trials. Citation Format: Jiancheng Hu. A novel BRAF-specific allosteric inhibitor that effectively targets acquired RAFi-resistant BRAF(V600E) cancers and RAS-mutated cancers in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB053.

A novel BRAF::PTPRN2 fusion in meningioma: a case report

Gene fusion events have been linked to oncogenesis in many cancers. However, gene fusions in meningioma are understudied compared to somatic mutations, chromosomal gains/losses, and epigenetic changes. Fusions involving B-raf proto-oncogene, serine/threonine kinase (BRAF) are subtypes of oncogenic BRAF genetic abnormalities that have been reported in certain cases of brain tumors, such as pilocytic astrocytomas. However, BRAF fusions have not been recognized in meningioma. We present the case of an adult female presenting with episodic partial seizures characterized by déjà vu, confusion, and cognitive changes. Brain imaging revealed a cavernous sinus and sphenoid wing mass and she underwent resection. Histopathology revealed a World Health Organization (WHO) grade 1 meningioma. Genetic profiling with next generation sequencing and microarray analysis revealed an in-frame BRAF::PTPRN2 fusion affecting the BRAF kinase domain as well as chromothripsis of chromosome 7q resulting in multiple segmental gains and losses including amplifications of cyclin dependent kinase 6 (CDK6), tyrosine protein-kinase Met (MET), and smoothened (SMO). Elevated pERK staining in tumor cells provided evidence of activated mitogen-activated protein kinase (MAPK) signaling. This report raises the possibility that gene fusion events may be involved in meningioma pathogenesis and warrant further investigation.

Tumor-agnostic genomic and clinical analysis of solid tumors with BRAF fusions.

3146 Background: Approximately 15% of cancers harbor BRAF alterations, of which ~5% are BRAF fusions. BRAF fusions are class II alterations that have worse outcomes than class I V600E alterations. While prior investigational therapies for BRAF fusions had disappointing outcomes, novel therapies are in clinical trials, underscoring the need to better characterize these tumors. Methods: Data from adult and pediatric patients with BRAF fusion+ cancers identified between January 2014 and November 2022 utilizing a center-wide next generation sequencing (NGS) program of >99,000 sequenced samples were analyzed. DNA-based hybrid capture tumor NGS (MSK-IMPACT), ctDNA targeted NGS (MSK-ACCESS), and/or RNA anchored multiplex PCR tumor NGS (MSK-Fusion) were used. Fusions were manually reviewed and considered to have oncogenic potential if they had an in-frame protein fusion involving a non-BRAF partner gene and an intact BRAF kinase domain (exons 11-18). Fusions not previously reported in OncoKB, COSMIC, TCGA, NIH gene, Fusion GDB2, and PubMED were classified as novel. All cases underwent clinical data curation including baseline demographic, tumor characteristics, and treatment histories. Results: 212 patients (0.2%) with BRAF-fusion positive solid tumors were identified. 194 were identified by DNA-based tumor NGS (96 had sufficient tissue for confirmatory RNA-based NGS, all of which were positive). Six were identified by ctDNA NGS and 12 by RNA-based tumor NGS only (5 had insufficient tissue for DNA-based tumor NGS). 83 unique 5’ fusion partners were found, of which 42 were novel. The most frequent tumor types were pilocytic astrocytoma (n=30, 14%), prostate CA (n=28, 13%), melanoma (n=24, 11%), lung CA (n=21, 10%), and colon CA (n=15, 7%). 44% of patients with pilocytic astrocytoma had BRAF fusions, of which 90% were BRAF-KIAA1549. Concomitant alterations (≥10% frequency) included TP53 (24%), TERT (18%), CDKN2A deletions (13%), and CDKN2B deletions (12%). 20% (n=43) were treated with MAPK-pathway directed therapies spanning multiple histologies and lines of therapies. Of the 212 patients, 17 had acquired BRAF fusions after targeted therapy for another oncogene ( EGFR mutation (n=11), BRAF V600E (n=4), FGFR fusion (n=1), NTRK fusion (n=1)). The majority of patients with acquired BRAF fusions had EGFR-mutant lung adenocarcinoma (n=11, 76%); the median time from EGFR targeted therapy initiation to BRAF fusion detection was 25 months (range 16-38 months). Conclusions: A wide variety of adult and pediatric solid tumors harbored de novo BRAF fusions. Complementary RNA sequencing optimized fusion identification in many cases. Multiple novel fusion partners were found. Acquired BRAF fusions were identified after targeted therapy for a variety of distinct oncogenes, the majority of which were EGFR mutations.

Abstract A036: Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation

Abstract 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.

Design, synthesis and characterisation of a novel type II B-RAF paradox breaker inhibitor

The mutation V600E in B-Raf leads to mitogen activated protein kinase (MAPK) pathway activation, uncontrolled cell proliferation, and tumorigenesis. ATP competitive type I B-Raf inhibitors, such as vemurafenib (1) and PLX4720 (4) efficiently block the MAPK pathways in B-Raf mutant cells, however these inhibitors induce conformational changes in the wild type B-Raf (wtB-Raf) kinase domain leading to heterodimerization with C-Raf, causing paradoxical hyperactivation of the MAPK pathway. This unwanted activation may be avoided by another class of inhibitors (type II) which bind the kinase in the DFG-out conformation, such as AZ628 (3) preventing heterodimerization. Here we present a new B-Raf kinase domain inhibitor, based on a phenyl(1H-pyrrolo [2,3-b]pyridin-3-yl)methanone template, that represents a hybrid between 4 and 3. This novel inhibitor borrows the hinge binding region from 4 and the back pocket binding moiety from 3. We determined its binding mode, performed activity/selectivity studies, and molecular dynamics simulations in order to study the conformational effects induced by this inhibitor on wt and V600E mutant B-Raf kinase. We discovered that the inhibitor was active and selective for B-Raf, binds in a DFG-out/αC-helix-in conformation, and did not induce the aforementioned paradoxical hyperactivation in the MAPK pathway. We propose that this merging approach can be used to design a novel class of B-Raf inhibitors for translational studies.

Computational analysis of natural product B-Raf inhibitors

B-Raf protein is a serine-threonine kinase and an important signal transduction molecule of the MAPK signaling pathway that mediates signals from RAS to MEK, ultimately promoting various essential cellular functions. The B-Raf kinase domain is divided into two subdomains: a small N-terminal lobe and a large C-terminal lobe, with a deep catalytic cleft between them. The N-terminal lobe contains a phosphate-binding loop (P-loop) and nucleotide-binding pocket, while the C-terminal lobe binds the protein substrates and contains the catalytic loop. The ligand pharmacophore was generated by using 17 different natural products and the receptor pharmacophore was generated by using protein structures. The reported natural product B-Raf inhibitors were analyzed according to the pharmacophore analysis (HipHop fit), virtual screening tools by Lipinski's rule of five. Thirteen out of seventeen molecules share the best ligand based pharmacophoric model (HipHop_5). The best receptor based pharmacophoric model came as AADHR. The compounds were docked against the B-Raf receptors (PDB ID: 3OG7, 4XV2, 5C9C). The compound DHSilB with cDOCKER interaction energy of −62.7 kcal/mol, −83.3 kcal/mol, −73.6 kcal/mol as well as the compound DHSilA with cDOCKER interaction energy of −63.9 kcal/mol, −63.2 kcal/mol, −74.7 kcal/mol showed satisfactory interaction with the respective receptors. Finally, the MD simulation was run for 100 ns for the top docked compounds DHSilA and DHSilB with the B-Raf proteins (PDB ID: 3OG7, 4XV2 and 5C9C). After the MD simulation run for 100 ns, the ligand 2,3-dehydrosilybin A (DHSilA) was found to be more stable in terms of the trajectories of RMSD, RMSF, Rg and H-bonds.

Abstract 2158: Preclinical evaluation of CFT1946 as a selective degrader of mutant BRAF for the treatment of BRAF driven cancers

Abstract The BRAF kinase is a critical node in the MAPK signaling pathway and is mutated in approximately 8% of human cancers including melanoma (~60%), thyroid (~60%), and lung adenocarcinoma (~10%). The most common mutation in BRAF is V600E (Class I), occurring in half of malignant melanomas. This mutation hyperactivates ERK and signals as a RAF inhibitor-sensitive monomer. BRAF inhibitors including vemurafenib, dabrafenib and encorafenib have produced impressive responses in V600X patients, however resistance usually emerges within a year, including RAS mutation, BRAFV600E amplification, and BRAFV600E intragenic deletion or splice variants. These inhibitors are also ineffective against non-V600 BRAF mutants (Class II & III). To address some of these limitations we have developed CFT1946, a bifunctional degradation activating compound (BiDAC™) degrader comprising a BRAF kinase domain targeting ligand linked to a cereblon ligand. CFT1946 is capable of degrading BRAF V600E (Class I), G469A (Class II), G466V (Class III) mutations, and the p61-BRAFV600E splice variant while maintaining exquisite selectivity against the proteome including WT BRAF and CRAF. In A375 cells, CFT1946 potently degraded BRAFV600E (Emax = 26%; DC50 = 14nM at 24hr) and, inhibited ERK phosphorylation (IC50 = 11nM at 24hr) and cell growth (GI50 = 94nM at 96hr) while having no effect in the mutant KRAS driven cell line HCT116. In A375 xenografts, oral delivery of CFT1946 resulted in deeper tumor regressions when dosed at 10 mg/kg PO BID and compared favorably to a clinically relevant dose of encorafenib. We further evaluated CFT1946 in an engineered A375-BRAFV600E/NRASQ61K double mutant model of BRAF inhibitor resistance. CFT1946 was able to degrade BRAFV600E in these cells and was much more effective than encorafenib at inhibiting viability in vitro. In this model, in vivo dosing of single agent CFT1946 caused robust tumor growth inhibition and combination with the MEK inhibitor, trametinib, resulted in tumor regressions. The combination of encorafenib and trametinib showed no activity in the same model. Next, we demonstrated that CFT1946 was able to degrade additional BRAF mutant proteins including G469A (Class II), G466V (Class III), and the p61-BRAFV600E splice variant using heterologous expression in HEK293T cells. Additionally, we also showed that CFT1946, but not encorafenib, inhibited proliferation of the BRAFG466V heterozygous lung tumor cell line H1666. Based on its activity in preclinical models, including models of BRAF inhibitor resistance, and its drug-like properties we are progressing CFT1946 as a candidate for clinical development in patients with solid tumors bearing BRAF V600X mutations. Further, given CFT1946’s activity on non-V600 BRAF mutations, we are continuing to explore CFT1946 and related BiDAC degraders as therapeutic options for patients bearing Class II or Class III BRAF mutations. Citation Format: Mathew E. Sowa, Bridget Kreger, Joelle Baddour, Yanke Liang, Jeffrey R. Simard, Laura Poling, Ping Li, Robert Yu, Ashley Hart, Roman V. Agafonov, Grace Sarkissian, Joe Sahil Patel, Richard Deibler, Kyle S. Cole, Scott Eron, David Cocozziello, Fazlur Rahman, Moses Moustakim, Christopher G. Nasveschuk, Katrina L. Jackson, Mark Fitzgerald, Victoria Garza, Morgan O’Shea, Gesine Veits, Jeremy L. Yap, Andrew J. Phillips, Elizabeth Norton, Adam S. Crystal, Stewart L. Fisher, Roy M. Pollock. Preclinical evaluation of CFT1946 as a selective degrader of mutant BRAF for the treatment of BRAF driven cancers [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 2158.

Abstract 2674: Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in preclinical models of human RAF/RAS mutant melanoma

Abstract Background: In the US in 2021, invasive melanoma will account for an estimated 106,000 new cases and > 7,000 deaths. Somatic mutations that activate the MAPK signaling pathway are a leading cause of melanoma with 50% harboring oncogenic BRAF alterations and another 20% with activating NRAS mutations. Of note, NRAS mutant melanoma has been shown to be dependent upon RAF signaling via CRAF dimers for downstream activation of MEK/ERK. While targeted therapies are approved for V600 (Class I, monomer-driven) BRAF mutant melanoma, no approved targeted therapy exists for patients with melanoma driven by Class II or Class III dimer-dependent BRAF alterations or NRAS mutations. KIN-2787 is a novel, orally available, potent, and selective pan-RAF inhibitor designed to be effective in RAF-dependent cancers, including all classes of BRAF alterations, by targeting mutant BRAF monomers and RAF dimers, regardless of isoform. Methods: KIN-2787 activity was assessed by suppression of downstream MAPK pathway signaling and subsequent cell growth inhibition in a panel of human melanoma cell lines. In vivo KIN-2787 efficacy was evaluated in BRAF and NRAS mutant melanoma cell- and patient-derived xenograft models. Results: KIN-2787 cellular activity was measured by inhibition of ERK phosphorylation across a panel of melanoma cell lines, including those harboring Class I BRAF alterations, Class II and III BRAF alterations, NRAS mutations, KRAS mutations, and wild type RAF/RAS. In contrast to vemurafenib, an approved BRAF inhibitor with activity limited to Class I BRAF alterations, KIN-2787 was active across all classes of BRAF mutant melanoma cells (EC50 values < 100 nM). NRAS and KRAS mutant cell lines were moderately responsive to KIN-2787 inhibition. Melanoma cells expressing wild type RAS/RAF were the least sensitive to MAPK pathway inhibition by KIN-2787. KIN-2787 also inhibited cell proliferation in BRAF and NRAS mutant melanoma in 2D and 3D cell cultures. Daily KIN-2787 treatment resulted in significant tumor growth inhibition in human melanoma xenograft models bearing Class I, II and III BRAF alterations as well as NRAS mutations and was associated with MAPK pathway suppression. Additionally, KIN-2787 was efficacious in a pre-/post-treatment melanoma PDX pair in which the original tumor was Class I BRAF V600E but acquired a Class II BRAF kinase domain duplication upon progression on dabrafenib + trametinib. Details from the above findings will be presented at the meeting. Conclusions: KIN-2787 is a next-generation, pan-RAF inhibitor with in vitro and in vivo activity against human melanoma driven by BRAF and/or NRAS mutations. Data supports KIN-2787 use in acquired BRAF dimer-dependent resistance to BRAF+MEK inhibitor therapy. A Phase I dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 is ongoing (NCT04913285). Citation Format: Nichol L. G. Miller, Tim S. Wang, Paul Severson, Ping Jiang, Michelle Perez, Noel Timple, Toufike Kanouni, Aleksandra Franovic, Eric S. Martin, Eric Murphy. Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in preclinical models of human RAF/RAS mutant melanoma [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 2674.

Abstract 5775: Landscape of BRAF and RAF1 fusions identified by next-generation sequencing in a Chinese multi-cancer retrospective analysis

Abstract Background: The RAF family includes three members, ARAF, BRAF, and RAF1/CRAF. These kinases function downstream of RAS as part of the MAPK (RAF-MEK-ERK) signaling cascade. The BRAF gene is mutated in ∼7% of cancers, whereas ARAF and RAF1 mutations are rare. Gene fusions in BRAF or RAF1 fusions, which activate the MAPK pathway, can be oncogenic drivers in a variety of cancer types and represent potential targets for targeted therapy. Here we focused on the prevalence of BRAF and RAF1 fusions in a multi-cancer cohort for patients (pts) whom might benefit from MEK/RAF inhibitors in clinical development. Methods: Genomic profiling was performed on samples from 29,102 lung cancer pts, 676 melanoma pts, 392 colorectal cancer pts, 1,463 gastric cancer pts, 1,014 sarcoma pts, 1,079 liver cancer pts, 215 gallbladder carcinoma pts, 297 ovarian cancer pts, 46 tongue cancer pts, 62 thymic carcinoma pts, 6 hemangioma pts, and 890 pancreatic cancer pts using next-generation sequencing (NGS), which covers all exons of BRAF and RAF1. The fusion patterns were analyzed. Results: A total of 5 RAF1 and 114 BRAF fusions were identified in approximately 0.34% (120/35,242) of the patients in this Chinese multi-cancer cohort. The positive rates of RAF1 fusions were 0.0034% (1/29,102) in lung cancer pts, 0.44% (3/676) in melanoma pts, and 0.10% (1/1,014) in sarcoma pts. The fusion partners of RAF1 were LYRM4 in lung cancer, LMCD1 and USP20 in melanoma, and FMR1 in sarcoma. RAF1 fusions involving the intact in-frame RAF1 kinase domain were LYRM4-RAF1, LMCD1-RAF1, and USP20-RAF1, which might be targeted with MEK/RAF inhibitors. The BRAF fusion-positive rates were 0.27% (80/29,102) in lung cancer pts, 1.18% (8/676) in melanoma pts, 2.81% (11/392) in colorectal cancer pts, 0.41% (6/1,463) in gastric cancer pts, 0.20% (2/1,014) in sarcoma pts, 0.19% (2/1,079) in liver cancer pts, 0.47% (1/215) in gallbladder carcinoma pts, 0.34% (1/297) in ovarian cancer pts, 2.17% (1/46) in tongue cancer pts, 1.61% (1/62) in thymic carcinoma pts, 16.67% (1/6) in hemangioma pts, and 0.11% (1/890) in pancreatic cancer pts. The most common 5’partners of BRAF were TRIM24 (4 cases) and SND1 (3 cases). Whereas the most common 3’partners of BRAF were BAIAP2L1 (5 cases) and TRIM24 (4 cases). Furthermore, 57 BRAF fusions contained intact BRAF kinase domain and nearly half of BRAF fusion-positive patients might be sensitive to MEK inhibitor. The most frequent location of the breakpoints occurred in intron 8 of the BRAF gene, followed by intron 7. However, the sensitivity of these fusions to MEK/RAF inhibitors in different cancers needs to be further studied. Conclusions: This study revealed the molecular features of BRAF and RAF1 rearrangements in Chinese cancer patients, which could help to identify therapeutic targets and personalize therapy management, leading to improved patient outcomes. Citation Format: Tao Li, Xin Zhang, Yanling Niu, Tonghui Ma. Landscape of BRAF and RAF1 fusions identified by next-generation sequencing in a Chinese multi-cancer retrospective analysis [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 5775.

Activity and Resistance of a Brain-Permeable Paradox Breaker BRAF Inhibitor in Melanoma Brain Metastasis.

A brain-penetrant BRAF inhibitor demonstrates potent activity in brain metastatic melanoma, even upon relapse following standard BRAF inhibitor therapy, supporting further investigation into its clinical utility.

Neurologic and neurodevelopmental complications in cardiofaciocutaneous syndrome are associated with genotype: A multinational cohort study

PurposeDysregulation of RAS or its major effector pathway is the molecular mechanism of RASopathies, a group of multisystemic congenital disorders. Neurologic complications are especially challenging in the management of the rare RASopathy cardiofaciocutaneous (CFC) syndrome. This study evaluated clinical neurologic and neurodevelopmental features and their associations with CFC syndrome gene variants. MethodsA multinational cohort of 138 individuals with CFC syndrome (BRAF = 90, MAP2K1 = 36, MAP2K2 = 10, KRAS = 2) was recruited. Neurologic presentation was captured via clinician review of medical records and caregiver-completed electronic surveys. Validated measures of seizure severity, adaptive function, and gross motor function were obtained. ResultsThe overall frequency of intellectual disability and seizures was 82% and 55%, respectively. The frequency and severity of seizures was higher among individuals with BRAF or MAP2K1 variants than in those with MAP2K2 variants. A disproportionate incidence of severe, treatment-resistant seizures was observed in patients with variants in the catalytic protein kinase domain of BRAF and at the common p.Y130 site of MAP2K1. Neurodevelopmental outcomes were associated with genotype as well as seizure severity. ConclusionMolecular genetic testing can aid in prediction of epilepsy and neurodevelopmental phenotypes in CFC syndrome. Study results identified potential CFC syndrome-associated variants in the development of relevant animal models for neurologic, neurocognitive, and motor function impairment.

Investigations of BRAF Regulation through Auto‐Inhibition and HRAS Interaction

BRAF is a key member in the MAPK signaling pathway essential for normal cell growth, reproduction, and differentiation. For this reason, BRAF is also the cause of numerous cancers when misregulated or mutated. Upstream of BRAF is the small, compact GTPase, RAS—a protein implicated in nearly a third of all cancers, yet extremely difficult to target due to its shape and shallow binding pocket. Upon interaction with RAS through the RAS binding domain (RBD) and membrane recruitment, BRAF transitions from an inactive, auto‐inhibited monomer to an active dimer and subsequently phosphorylates MEK to propagate the signal. The auto‐inhibited conformation is thought to be stabilized through interactions between the Cysteine Rich Domain (CRD) and the Kinase Domain (KD) of BRAF. Despite its central role in cellular signal transduction, the order of BRAF’s activation steps have yet to be experimentally validated. We propose that by studying the inter‐ and intra‐molecular interactions of BRAF, we will begin to unveil the fine details of RAF regulation, important for developing new strategies towards therapeutic intervention. We performed biophysical, biochemical, and cell‐based assays to further investigate the role of the regulatory regions in BRAF activation and auto‐inhibition. We quantified in vitro binding between the N‐terminal domains of BRAF and the KD as well as binding between the N‐terminal domains of BRAF and HRAS through Surface Plasmon Resonance (SPR). We show that the interaction between RAS and regulatory regions of BRAF disrupt the interaction between the regulatory regions and catalytic region of BRAF, providing direct evidence that RAS binding to BRAF relieves the auto‐inhibitory interactions among BRAF domains. Furthermore, we examined the effect of Raf‐mimetic peptides in disrupting the RAS‐RAF interaction and relief of auto‐inhibition. The studies outlined here are leading to promising new insights into the activation mechanism of BRAF kinase.

Docking, synthesis and biological evaluation of pyridine ring containing Diaryl urea derivatives as anticancer agents

A novel series of pyridine ring containing diaryl urea derivatives (R1-R9) were synthesized in four chemical steps using pyridine-2-carboxylic acid as starting material. The synthesized compounds were design by using Autodock vina in the crystal structure of the Kinase domain of Human B-raf (PDB ID: 4DBN) to get insights into structural requirements for anticancer activity. In vitro anticancer activity against cell line (MCF-7) showed that compounds R3, R6 and R9 were found to be the most potent (Docking score: > -12, IC50 = 17.39 µM) among the synthesized molecules.

Abstract 1106: BRAF and EGFR fusion as a novel mechanism of resistance mechanism to Lazertinib, 3rd- generation EGFR-TKI, in EGFR-mutant NSCLC

Abstract Background: EGFR-TKI is an established first-line therapy for NSCLC with activating EGFR mutations. Osimertinib, third-generation EGFR TKI, was investigated as a first-in-class drug, but lazertinib(YH25448) was also reported as an outstanding drug which had similar efficacy as osimertinib. Nevertheless, acquired resistance is inevitably developed by third-generation EGFR-TKIs in clinic. Even though EGFR C797S mutation has been identified as a major resistant mechanism, the remaining resistance mechanisms to these TKIs are largely unknown. Methods: To explore the mechanism of resistance to lazertinib, we firstly established drug-resistant cells to lazertinib in vitro using four NSCLC cells including the patient-derived cell line (PDC), patient-derived tumor xenograft cell line (PDTC), and ATCC cell lines. To analyze the changes in global genetic alterations and gene expression of established lazertinib-resistant cell lines(YH1R), whole exome sequencing (WES) and RNA sequencing (RNA-seq) were performed. The effect of EGFR/BRAF fusion was investigated in vitro using established YH1R cell line and various patient-derived tumor xenograft samples. Western blot study was used to investigate EGFR and MAPK cascade signaling pathway. We evaluated antitumor activity of MEK inhibitor and lazertinib combination treatment in lazertinib-resistant NSCLC cells with EGFR/BRAF fusion in vitro and in vivo. Results: RNA-seq data revealed a novel EGFR/BRAF fusion transcript (EGFR Exon 19-BRAF Exon 9, in-frame fusion) in lazertinib-resistant PC9GR_YH1R. The fusion gene consisted of EGFR (Exon 1-19) portion and BRAF kinase domain (Exon 9-18). The EGFR/BRAF fusion mRNA and protein were specifically upregulated in PC9GR_YH1R compared to parental cells. Intriguingly, we detected the EGFR/BRAF fusion gene expression in patient-derived xenografts (YHIM-1045 and YHIM-1035) obtained from patients who experienced acquired resistance to lazertinib. Compared with treatment with either single agent, combination treatment of lazertinib and MEK inhibitor, trametinib or selumetinib, was significantly effective at inhibiting cell proliferation as well as EGFR signaling pathways in PC9GR_YH1R in vitro and in vivo. Conclusion: We found a novel EGFR/BRAF fusion gene, as a key driver of acquired resistant mechanism of lazertinib, in NSCLC cell lines and patient-derived xenografts with acquired resistance to lazertinib. Combination treatment of lazertinib and MEK inhibitor showed a strong antitumor effect in lazertinib-acquired resistant NSCLCs, indicating that the combination therapy of EGFR and MEK inhibitors might be a promising therapeutic option for overcoming lazertinib-acquired resistant NSCLC patients in clinic. Citation Format: Seo-Yoon Jeong, Jiyeon Yun, San-Duk Yang, Soo-Hwan Lee, Sangbin Lim, Seok-Young Kim, Min Hee Hong, Sun Min Lim, Hye Ryun Kim, Hye Ryun Kim, Byoung Chul Cho. BRAF and EGFR fusion as a novel mechanism of resistance mechanism to Lazertinib, 3rd- generation EGFR-TKI, in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1106.

Abstract 2036: Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications

Abstract Cancer cells display two modes of focal amplifications (FA): extrachromosomal double minutes (DMs/ecDNAs) and intrachromosomal homogenously staining regions (HSRs). Understanding the plasticity of these two modes is critical for preventing targeted therapy resistance. We developed a combined BRAF plus MEK inhibitor resistance melanoma model that bears high BRAF amplifications through both ecDNA and HSR modes, and investigated FA dynamics in the context of drug resistance plasticity. Cells harboring FAs displayed mode switching between ecDNAs and HSRs, from both de novo genetic changes and selection of pre-existing subpopulations. We found that copy number plasticity is not exclusive to ecDNAs. Single cell-derived clones with HSRs also exhibit BRAF copy number and corresponding HSR length plasticity that allows them to respond to dose reduction and recover from drug addiction. In addition, upon kinase inhibitor escalation, we observed reproducible selection for cells with BRAF kinase domain duplications residing on ecDNAs. Whole genome sequencing of sublines with different FA formats allowed fine reconstructions of BRAF amplicon and revealed conserved structures during the transitions due to drug dose manipulations. The amplification boundaries in our cell line model were found consistent with those in clinically observed cases of combined BRAF/MAPK inhibitor resistance. RNA-seq and CRISPR screening performed on parental, ecDNA and HSR sublines revealed distinct biology and vulnerabilities associated with each kind of FA. Vulnerabilities for genes involved in ecDNA maintenance in DNA repair pathways identified in our model are consistent with previous reports, such as a role for DNA-dependent protein kinase (DNA-PK/PRKDC). Our screen reveals many new targets. In sum, the karyotypic plasticity of FAs allows cancer cells to respond to drug dose challenges through a myriad of mechanisms, including increases or decreases in DMs, shortening of HSRs, and acquisition of secondary resistance mechanisms. The cellular vulnerabilities identified in our study provide roadmaps for therapeutically targeting each kind of FA mode. Citation Format: Kai Song, Jenna Minami, Trevor Ridgley, Arthur Huang, Rachana Jayaraman, William Crosson, Jesus Salazar, Eli Pazol, Senaratne Niroshi, Rao Nagesh, Kim Paraiso, Thomas Graeber. Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2036.

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.

SNP-based detection of allelic imbalance: A novel approach for identifying KIAA1549-BRAF fusion in pilocytic astrocytoma using DNA sequencing

Pilocytic astrocytoma (PA) is the most common glioma subtype found in children, and it is a non-malignant tumor type. The majority of PAs is caused by an approximately 2 Mb tandem duplication within 7q34 which creates an in-frame KIAA1549-BRAF fusion gene. The kinase domain of BRAF is fused to the N-terminal of KIAA1549, whereby BRAF is constitutively activated. We here present a novel approach for identifying KIAA1549-BRAF fusion based on single nucleotide polymorphism (SNP) analysis and next generation sequencing (NGS). Highly polymorphic SNPs in the duplicated area and in adjacent areas were selected and a custom targeted amplicon based NGS panel was designed. The panel was tested on DNA extracted from formalin fixed and paraffin embedded tissue from a retrospective cohort, consisting of biopsies from patients with PA, anaplastic astrocytoma, oligodendroglioma and glioblastoma as well as two non-tumor biopsies. The panel could distinguish chromosome 7 gain from BRAF fusion and correctly identified 8/9 PA samples with KIAA1549-BRAF fusion confirmed by RNA sequencing. The one biopsy where no fusion was detected was fresh frozen and from the RNA sequencing expected to have very low tumor content. No allelic imbalance was detected in either oligodendroglioma or in the non-tumor biopsies.

Abstract A31: Germline RASopathy mutations provide functional insights into the Raf cysteine-rich domain (CRD)

Abstract The three-member RAF kinase family (A-, B- and C-Raf) are direct Ras effectors and key mediators of the proproliferative MAPK pathway. Somatic driver mutations in the B-Raf kinase domain occur frequently in melanoma and cancers of the thyroid and large intestine. These are predominantly the V600E mutation, which allows B-Raf to signal constitutively as a monomer. Germline mutations in B-Raf also occur and are present in ~67% of the childhood developmental RASopathy-cardiofaciocutaneous (CFC) syndrome. Interestingly, many of these CFC mutations cluster around the cysteine-rich domain (CRD) in the B-Raf regulatory region. CRDs are zinc stabilized structures with two flexible loops that are shown to bind phosphatidylserine (PS), DAG and pro-oncogenic phorbol esters in several PKC family members. Although the B-Raf CRD has not been well characterized, prior studies examining the C-Raf CRD show that it binds to PS through its first flexible loop but does not bind DAG or phorbol esters, likely due to its truncated second loop, which is found in all Raf members. In addition to PS binding, C-Raf CRD may also interact directly with Ras, suggesting a second Ras binding site outside of its Ras binding domain (RBD). Interestingly, B-Raf RASopathy mutations occur in both CRD loops and commonly introduce a positively charged residue suited to interacting with negatively charged lipids in the plasma membrane (PM). Of note, Q257R, which is the most common mutation in CFC, maps to the truncated second loop. Our in-cell studies with full-length B-Raf have shown that RASopathy mutations in both loops enhance biologic activity, PM localization and Ras interaction. However, the biologic activity of loop 1 mutations requires an intact RBD, which is not the case for the loop 2 mutant, Q257R. These data suggest that the Q257R mutant either has increased binding to Ras or allows B-Raf to signal independently of Ras. To further investigate these findings, we are examining differences in localization, Ras interaction and lipid binding using the isolated CRDs of C-Raf, B-Raf and the B-Raf RASopathy mutants. As may be predicted, the RASopathy mutations increased membrane localization over WT B-Raf, with Q257R being the most PM localized. Somewhat surprisingly, expression of the wild-type B-Raf CRD in cells showed greatly enhanced PM localization compared to that of wild-type C-Raf, suggesting functional differences between their CRDs, which we are currently investigating. Citation Format: Russell Spencer-Smith, Elizabeth M. Terrell, Constance Agamasu, Daniel A. Ritt, Alyson K. Freeman, Andrew G. Stephen, Deborah K. Morrison. Germline RASopathy mutations provide functional insights into the Raf cysteine-rich domain (CRD) [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A31.