Potent BRAF Inhibitor Research Articles

Into the Groove: A Multitechnique Insight into the DNA–Vemurafenib Interaction

This study explores the interaction between Vemurafenib (VEM), a potent BRAF inhibitor, and calf thymus DNA (ctDNA) using a comprehensive array of biophysical and computational techniques. The primary objective is to understand the potential off-target effects of VEM on DNA, given its established role in melanoma therapy targeting the BRAF V600E mutation. The investigation employed methods such as ultraviolet–visible absorption spectroscopy, steady-state fluorescence, circular dichroism, isothermal titration calorimetry, and advanced molecular dynamics simulations. The results indicate that VEM interacts with DNA primarily through a minor groove-binding mechanism, causing minimal structural disruption to the DNA double helix. Viscosity measurements and melting temperature analyses further confirmed this non-intercalative mode of binding. Calorimetry data revealed an exothermic, thermodynamically favorable interaction between VEM and ctDNA, driven by both enthalpic and entropic factors. Finally, computer simulations identified the most probable binding site and mode of VEM within the minor groove of the nucleic acid, providing a molecular basis for the experimental findings.

Novel gene therapy for drug-resistant melanoma: Synergistic combination of PTEN plasmid and BRD4 PROTAC-loaded lipid nanocarriers

Patients suffering from BRAF mutant melanoma have tumor recurrence within merely 7 months of treatment with a potent BRAF inhibitor (BRAFi) like vemurafenib (VEM). It has been proven that diverse molecular pathways driving BRAFi resistance converge to activation of c-Myc in melanoma. Therefore, we identified a novel combinatorial therapeutic strategy by targeting loss of PTEN tumor suppressor gene and upregulated BRD4 oncoprotein as Myc-dependent vulnerabilities of drug-resistant melanoma. Being promising therapeutic targets, we decided to concomitantly deliver PTEN plasmid and BRD4 targeted PROteolysis-TArgeting Chimera (ARV) to drug the “undruggable” c-Myc in BRAFi-resistant melanoma. Since PTEN plasmid and ARV are distinct in their physicochemical properties, we fabricated PTEN-plasmid loaded LNPs (PL-NANO) and ARV-825 loaded nanoliposomes (AL-NANO) to yield mean particle size of <100 nm and >99% encapsulation efficiency for each therapeutic payload. Combination of PL-NANO and AL-NANO displayed synergistic tumor growth inhibition and substantial apoptosis in in vitro 2D and 3D models. Importantly, simultaneous delivery of PL-NANO and AL-NANO achieved significant upregulation of PTEN expression levels and degradation of BRD4 protein to ultimately downregulate c-Myc levels in BRAFi-resistant melanoma cells. Altogether, lipid nanocarriers delivering this novel lethal cocktail stands as “one-of-a-kind” gene therapy to target “undruggable” c-Myc oncogene in BRAFi-resistant melanoma.

Identification of the Clinical Candidate PF-07284890 (ARRY-461), a Highly Potent and Brain Penetrant BRAF Inhibitor for the Treatment of Cancer.

Mutant BRAFV600E is one of the most common oncogenic drivers in metastatic melanoma. While first generation BRAFV600E inhibitors are capable of controlling tumors systemically, they are unable to adequately treat tumors that have metastasized to the brain due to insufficient penetration across the blood-brain barrier (BBB). Through a combination of structure-based drug design (SBDD) and the optimization of physiochemical properties to enhance BBB penetration, we herein report the discovery of the brain-penetrant BRAFV600E inhibitor PF-07284890 (ARRY-461). In mice studies, ARRY-461 proved to be highly brain-penetrant and was able to drive regressions of A375 BRAFV600E tumors implanted both subcutaneously and intracranially. Based on compelling preclinical safety and efficacy studies, ARRY-461 was progressed into a Phase 1 A/B clinical trial (NCT04543188).

A Practical Review of Encorafenib and Binimetinib Therapy Management in Patients with BRAF V600E-Mutant Metastatic Non-Small Cell Lung Cancer.

According to current guidelines, targeted therapy with a combination of BRAF plus MEK inhibitors is the preferred first-line treatment for patients with BRAF V600E-mutant metastatic non-small cell lung cancer (NSCLC). In the open-label, single-arm, phase2 PHAROS trial (NCT03915951), the combination of encorafenib, a potent BRAF inhibitor, and binimetinib, a potent MEK inhibitor, demonstrated durable antitumor activity with a manageable safety profile in this patient population. On the basis of the results of this study, the combination of encorafenib plus binimetinib was approved by the US Food and Drug Administration on October 11, 2023, for patients with BRAF V600E-mutant metastatic NSCLC. In this review, we summarize the efficacy and safety of encorafenib plus binimetinib from the PHAROS study. In addition, we discuss strategies to manage adverse reactions with this combination therapy with the intent of minimizing unnecessary treatment discontinuations in these patients.

Abstract 593: DCC-3084, a brain penetrant RAF dimer inhibitor, broadly inhibits BRAF class I, II, and III alterations leading to growth inhibition of intracranially implanted tumors 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 &amp;gt;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 brain penetrant, potent and selective investigational switch-control inhibitor of BRAF and CRAF kinases, shown preclinically to target all relevant aberrant signaling mechanisms (monomers, heterodimers and homodimers) and inhibits growth of subcutaneously and intracranially implanted tumors in preclinical models. Methods: Inhibition of RAF kinases was measured using recombinant enzymes. Cellular proliferation was measured using resazurin to monitor cell viability. 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. Subcutaneous or intracranial BRAF mutant mouse xenograft models were used to assess PK, pharmacodynamics (PD), and efficacy. Results: DCC-3084 treatment resulted in potent single-agent inhibition of MAPK pathway signaling, and cellular proliferation in a wide range of Class I, II, III BRAF and BRAF fusion altered cell lines. DCC-3084 was demonstrated to be CNS penetrable and exhibited dose dependent oral exposure with robust inhibition of the MAPK pathway in PK/PD models. Oral treatment of DCC-3084 as a single agent resulted in tumor regression in subcutaneously implanted BRAF Class I and BRAF fusion xenograft models. Additionally, DCC-3084 resulted in tumor regression or growth inhibition in intracranially implanted BRAF Class I and BRAF fusion tumor 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. DCC-3084 CNS penetration enables potential for further research in brain metastases or primary brain cancer. Citation Format: Gada Al-Ani, Stacie L. Bulfer, Jefferey D. Zwicker, Chase K. Crawley, Kristin M. Elliot, Salim Javed, Qi Groer, Molly M. Hood, Joshua W. Large, Kylie Luther, Yeni K. Romero, Forrest A. Stanley, Daniel C. Tanner, Hanumaiah Telikepalli, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DCC-3084, a brain penetrant RAF dimer inhibitor, broadly inhibits BRAF class I, II, and III alterations leading to growth inhibition of intracranially implanted tumors in preclinical models [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 593.

Network pharmacology and molecular docking analysis on potential molecular targets and mechanism of action of BRAF inhibitors for application in wound healing.

Topical application of BRAF inhibitors has been shown to accelerate wound healing in murine models, which can be extrapolated into clinical applications. The aim of the study was to identify suitable pharmacological targets of BRAF inhibitors and elucidate their mechanisms of action for therapeutic applicability in wound healing, by employing bioinformatics tools including network pharmacology and molecular docking. The potential targets for BRAF inhibitors were obtained from SwissTargetPrediction, DrugBank, CTD, Therapeutic Target Database, and Binding Database. Targets of wound healing were obtained using online databases DisGeNET and OMIM (Online Mendelian Inheritance in Man). Common targets were found by using the online GeneVenn tool. Common targets were then imported to STRING to construct interaction networks. Topological parameters were assessed using Cytoscape and core targets were identified. FunRich was employed to uncover the signaling pathways, cellular components, molecular functions, and biological processes in which the core targets participate. Finally, molecular docking was performed using MOE software. Key targets for the therapeutic application of BRAF inhibitors for wound healing are peroxisome proliferator-activated receptor γ, matrix metalloproteinase 9, AKT serine/threonine kinase 1, mammalian target of rapamycin, and Ki-ras2 Kirsten rat sarcoma viral oncogene homolog. The most potent BRAF inhibitors that can be exploited for their paradoxical activity for wound healing applications are Encorafenib and Dabrafenib. By using network pharmacology and molecular docking, it can be predicted that the paradoxical activity of BRAF inhibitors can be used for their potential application in wound healing.

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 &amp;gt;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 &amp;gt;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.

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.

Abstract 1473: Nonclinical development of PF-07284890 (ARRY-461), a potent, brain-penetrant, small molecule inhibitor of BRAF V600-mutation-driven tumors in vitro and in vivo

Abstract Although approved BRAF inhibitors have transformed the treatment of patients with certain BRAF V600 mutant cancers, their long-term efficacy is thought to be limited by poor brain penetration. As a result, disease progression in the brain is a significant cause of morbidity and mortality. PF-07284890 (ARRY-461) is an orally bioavailable, brain penetrant, potent, small molecule inhibitor targeting BRAF V600 mutant tumors and is in clinical development in patients with BRAF V600 metastatic melanoma with progression to the brain. In biochemical in vitro studies, PF-07284890 inhibits BRAF and CRAF with IC50's of 5.8 and 4.1 nM, respectively. Additionally, PF-07284890 inhibits the clinically relevant kinase domain BRAF V600 mutants (V600E and V600K) (IC50 = 24-25 nM). In cell-based systems, PF-07284890 potently inhibits phosphorylation of ERK, a downstream marker of BRAF inhibition, and potently inhibits proliferation of BRAF V600E/K mutant melanoma cell lines (IC50 18-38 nM). PF-07284890 was designed to distribute to the brain and, as such, in vitro experiments indicate that PF-07284890 has high cellular membrane permeability and is not a substrate for human P glycoprotein (P-gp). After oral administration of PF-07284890 to mice and rats, PF-07284890 distributes to the brain where the free fraction adjusted exposure in the brain was approximately proportional to the free fraction adjusted exposure in plasma (i.e., Cbrain,u/Cplasma,u approaches 1.0). In vivo, PF-07284890 inhibits phosphorylation of ERK in A375 BRAF V600E tumors, achieving maximal target inhibitions at a dose of 10 mg/kg. PF-07284890 has been evaluated for its ability to control BRAF V600E cell line and patient-derived melanoma xenograft tumor growth in nude mice when implanted both subcutaneously and intracranially. Dose-related tumor growth inhibition was demonstrated at dose levels ranging from 1 to 30 mg/kg, BID in both subcutaneous and intracranial xenograft models. In all models, regardless of tumor location, maximal efficacy was observed in dose ranges of 10-30 mg/kg BID. In the intracranial A375-luc BRAF V600E melanoma xenograft model, significant and durable tumor regressions were seen at doses of 10 and 30 mg/kg/day which translated to a profound survival benefit in animals receiving PF-07284890 (median survival &amp;gt; 55 days post-implantation) compared to control animals (median survival = 15 days). GLP safety studies demonstrated a good safety profile for PF-07284890. PF-07284890 is fully brain penetrant, with the potential to address this key unmet medical need and thereby defines a new class of brain penetrant, potent and selective BRAF inhibitors. Citation Format: Karyn Bouhana, Deborah Anderson, Walter DeWolf, Suzy Brown, Lance Williams, Li Ren, David Moreno, Ross Wallace, Jay Brad Fell, Dylan Hartley, Patrice Lee. Nonclinical development of PF-07284890 (ARRY-461), a potent, brain-penetrant, small molecule inhibitor of BRAF V600-mutation-driven tumors in vitro and in vivo [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 1473.

A Fluoro Derivative of Embelin, as Potent B-RAF Inhibitor in Melanoma

Melanoma is one of the most common forms of skin cancer, and B-RAF is a mutated protein found in most melanomas. The important function of B-RAF is normal cell growth and survival. Most of the known B-RAF mutations are V600E mutations. Vemurafenib is the fluorine-based drug currently used for V600E mutations. However, this drug has side effects, therefore, more potent drugs with fewer side effects are required. This study aims to develop a more effective lead compound as a B-RAF inhibitor from hydroxyquinone by structural modification of embelin, a naturally occurring hydroxybenzoquinone. It has the potency of detoxifying blood and is hence useful in a wide range of skin diseases. Thus, a fluorine substituted semisynthetic derivative of embelin, 5-(3-chloro-4-trifluoromethoxy phenyl amino)-2-hydroxy-3-undecyl- [1, 4] benzoquinone to fight against skin cancer was prepared. Fluoro derivative of embelin was synthesized by the direct condensation of embelin with 3-chloro-4- trifluoromethoxy aniline. The structure of the product was characterized using various spectral data obtained from IR, 1H NMR, 19F NMR, 12C NMR, and mass spectrum. Various in vitro studies like antiproliferative study in A375 Cell Lines (B-RAF Elisa), western blotting analysis, gene expression study by reverse transcriptase PCR, caspase assay, flow cytometry analysis, clonogenic assay, and transwell migration assay were carried out to find its biological activity. A semisynthetic derivative of Embelin 5-(3-Chloro-4-trifluoromethoxy phenyl amino)-2-hydroxy-3- undecyl- [1, 4] benzoquinone (EOCF) was prepared, and the structure of the derivative was confirmed by spectral analysis. The MTT assay proves that the fluoro derivative of embelin exhibited better anti-cancer activity in melanoma cell lines than the parent compound, embelin. Western blot analysis showed that B-RAF expression level was reduced by the addition of derivative than the parent compound embelin. The Caspase ELISA analysis indicated that the derivative was found to be a good apoptotic marker. From the flow cytometry analysis, it was observed that cell arrest occurs at the G0/G1 phase. Its antimetastatic activity was determined using clonogenic assay. It indicated that the derivative EOCF inhibits the metastatic effects in melanoma cell lines. The migratory potential of melanoma cells was significantly reduced in the presence of EOCF when the transwell migration assay was conducted. This work established that the potency of the synthesized compound was more than the parent compound, embelin, when it was structurally modified with 3-chloro-4-trifluoromethoxy aniline. The derivative can be used as a lead molecule for further drug discovery.

LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors.

Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254. LXH254 was profiled in biochemical, in vitro, and in vivo assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF. We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive. LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.

Design, synthesis and molecular modeling of phenyl dihydropyridazinone derivatives as B-Raf inhibitors with anticancer activity

Three new series of phenyl dihydropyridazinone derivatives 4b–8i have been designed, synthesized and evaluated for their anticancer activity against different cancer cell lines. Nine compounds showed strong inhibitory activity, among which compound 8b exhibited potent activity against PC-3 cell line with IC50 value of 7.83 µM in comparison to sorafenib (IC50 11.53 µM). Compounds 6a, 6c, 7f-h and 8a-d were further screened for their B-Raf inhibitory activity where seven compounds 7f-h and 8a-d showed high B-Raf inhibition with ranges of IC50 values 70.65–84.14 nM and 24.97–44.60 nM, respectively when compared to sorafenib (IC50 44.05 nM). Among the tested compounds, 8b was the most potent B-Raf inhibitor with IC50 value of 24.79 nM. Cell cycle analysis of MCF-7 cells treated with 8b showed cell cycle arrest at G2-M phase with significant apoptotic effect. Molecular modeling study was performed to understand the binding mode of the most active synthesized compounds with B-Raf enzyme.

Abstract LB-B10: Targeting a new allosteric site modulates selectivity of BRAF dimer inhibition

Abstract BRAF mutants are present in ~50% of melanoma patients as well as in colorectal (~10%), thyroid carcinomas (~40%) and other malignancies. FDA-approved RAF inhibitors Vemurafenib, Dabrafenib and Encorafenib show remarkable responses in melanoma patients with BRAFV600E tumors. However, these drugs ultimately become ineffective due to adaptive and acquired resistance mechanisms, many of which depend on signaling by poorly inhibited BRAF homo- or hetero-dimers. In about 20-30% of melanoma tumors, intrinsic resistance is driven by a constitutive BRAF dimer splice variant, p61-BRAFV600E, while in resistant colorectal and thyroid tumors RAF dimerization is maintained by frequent RAS activation. An adaptive resistance mechanism depends on inhibitor-induced relief of ERK-mediated feedback pathway inhibition, which stimulates RAS-GTP and increases RAF dimers. Finally, oncogenic non-V600E BRAF species in some tumors signal as obligated dimers. Challenged by the clinical relevance of BRAF dimerization, we initiated a systematic quest for novel inhibitors that specifically recognize BRAFV600E dimers. We developed a phenotypic cellular assay to screen for inhibitors of ERK-signaling, using VEM-resistant melanoma cells that express p61-BRAFV600E dimers. We discovered new potent BRAF inhibitors, including a FDA approved kinase inhibitor. We obtained novel crystal structures of this drug with BRAFV600E and BRAFWT. We used these structural insights and scaffold-based drug-design methods to develop PHI1, a first-in-class αC-IN BRAF inhibitor. Remarkably, the crystal structure of BRAFV600E/PHI1 complex revealed that this compound targets a new BRAF allosteric site, which uniquely modulates the αC-helix conformation. In vitro and cellular studies showed that PHI1 is potent and selective against resistant cells with p61-BRAFV600E dimers. In addition, SAR analysis indicated that selectivity for dimer inhibition is lost upon modification of PHI1’s moiety targeting the allosteric pocket, suggesting that this site modulates specificity for BRAF dimers. Co-treatment experiments revealed that PHI1 inhibits the second site within the dimers with positive cooperativity driven from first site occupancy by αC-OUT inhibitors, in contrast to current RAF inhibitors that exhibit no, or negative, cooperativity. Based on these data, co-treatment of PHI1 with various clinical RAF and MEK inhibitors was explored in BRAF dimer-dependent tumors resulting in promising synergistic effects. This work provides a new class of allosteric BRAF inhibitors that may broadly diminish clinically untargeted oncogenic signaling dependent on BRAF dimers. Citation Format: Paul (Bogos) Agianian, Xiomaris M Cotto-Rios, Nadege Gitego, Emmanouil Zachariouadakis, Evripidis Gavathiotis. Targeting a new allosteric site modulates selectivity of BRAF dimer inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B10. doi:10.1158/1535-7163.TARG-19-LB-B10

Determining Resistance Mechanisms in BRAF-mutated Multiple Myeloma

Introduction: Constant clonal evolution and outgrowth of clones that harbor resistance mutations are likely explanations for the emergence of drug-resistant disease in multiple myeloma (MM). Activating mutations in BRAF, KRAS and NRAS have been suggested as potential therapeutic targets. In this study, we investigate resistance to BRAF inhibition in the context of BRAF-mutated MM which accounts for about 5-12% of all patients with relapsed/ refractory MM. Methods: Resistance to dabrafenib was modeled in vitro in the BRAF-mutated MM cell lines (MMCL) U266 (K601Nmut) and DP6 (BRAFV600Emut). Low-pass whole genome sequencing (LPWGS), RNA sequencing, ChIP sequencing and immunoblotting were performed for genomic, transcriptomic, epigenomic and molecular characterization. Functional validation was performed by genome editing using CRISPR/Cas9 technology. Results: Modeling of dabrafenib resistance in vitro revealed an initial decline of cell numbers, followed by a plateau phase and a gradual outgrowth of resistant cells after ~80 days of treatment. As expected, exposure of BRAFmut MMCL to dabrafenib led to initial downregulation of pERK and pMEK. At later timepoints, upregulation of pERK and pMEK was observed, suggesting that re-activation of the ERK/MEK pathway ultimately overcomes BRAF inhibition. This outgrowth was associated with highly distinct copy number profiles in each resistant clone, implying clonal selection with outgrowth of genetically resistant clones as one mechanism of drug resistance in MM. Additionally, we found that BRAF inhibition of BRAFmut MMCL promotes changes of the transcriptional circuitry that appears independent of clonal outgrowth of genetically resistant clones. These transcriptional changes were highly homogenous, occurred as early as 7-14 days after starting treatment and were associated with de-differentiation of MMCL into a more immature B lymphocytic phenotype. This phenotype was associated with greater mRNA expression of CD19 and CD81, as well as upregulation of the B-lymphocyte activation antigen B7-2 (CD86) and PI3K pathway genes. We next investigated if targeting the PI3K pathway and B7.2 can be exploited for effective killing of dabrafenib-resistant BRAFmut MM cells. Studies for the PI3Kδ inhibitor idelalisib in dabrafenib-persistent MMCL revealed higher sensitivity as compared to dabrafenib-naïve controls. Genome editing suggests a survival advantage for CD86WT as compared to CD86KO MMCL. Conclusions: Our data suggest that resistance to BRAF inhibition in vitro is mediated by two distinct mechanisms: 1) clonal outgrowth of genetically distinct resistant clones, and 2) transcriptional rewiring that leads to activation of alternative signaling pathways. The latter is characterized by changes in cellular differentiation and upregulation of PI3K and CD28/CD86 signaling. These concepts may provide a framework for revealing therapeutic vulnerabilities and to overcome drug resistance mediated by genetic heterogeneity in MM. Disclosures Munshi: Oncopep: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy; Amgen: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Yee:Karyopharm: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Adaptive: Consultancy. Raje:Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Merck: Consultancy. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria. OffLabel Disclosure: Dabrafenib is a potent inhibitor of BRAF mutated at codon 600 (BRAFV600). Here we explored the efficacy of dabrafenib a preclinical model of multiple myeloma cell lines with BRAFV600E and BRAFK601N mutations.

BRAF Oncogenic Activation Induces Radioresistance Through Non-homologous End-Joining Repair Which is Abrogated by Targeted Inhibition of BRAF Mutational Activity

BRAF activating mutations, most commonly BRAFV600E/K, are driver mutations commonly found in solid tumors for which ionizing radiation (IR) is used, including thyroid, melanoma, colorectal, and lung cancer. Whether BRAFV600E mutation fosters radioresistance through nuclear DNA repair mechanisms is unclear. We hypothesize that BRAFV600E promotes radioresistance through heightened double-strand break repair mechanisms, which can be attenuated by targeted inhibition of the BRAF oncogene. We tested the effects of a BRAFV600E mutation using papillary and anaplastic thyroid cancer cell lines that possessed either wild-type or mutant BRAFV600E. We evaluated the role of BRAF and downstream targets in radiation clonogenic, neutral comet, nuclear foci, immunofluorescence, immunoblotting, RT-PCR, functional DNA repair reporter, and in vivo tumor growth assays. Radiation clonogenic assay showed BRAFV600E thyroid cancer cell lines were resistant to IR, and introduction of BRAFV600E into wild-type BRAF thyroid cancer TPC-1 cells led to significant IR resistance (dose enhancement factor, DEF 0.70). Selectively targeting BRAFV600E with a potent mutant BRAF inhibitor (vemurafenib, Vem) significantly sensitized BRAFV600E to IR, but not wild-type BRAF thyroid cancer cells (DEFs 1.37-1.78). Immunoblotting demonstrates that vemurafenib potently inhibited ERK signaling in BRAFV600E mutants but not BRAF wild-type cells. Vem delayed DNA repair in BRAFV600E cells after IR as evidenced by comet tail assays at 15 min, 1, 6, and 24 hrs (p<0.005) and γ-H2AX nuclear foci assays at 6 and 24 hrs (p<0.01). Moreover, Vem altered the kinetics of 53BP and RAD51 nuclear foci in BRAFV600E cells at 6 and 24 hrs after IR (p<0.05). Assessment of double-strand DNA repair using functional reporter assays revealed that BRAFV600E mutant was associated with enhanced non-homologous end-joining (NHEJ) repair activity, while Vem significantly reduced NHEJ repair activity by ∼40% (p<0.01). BRAFV600E mutation led to upregulation of XLF, a nuclear protein involved in NHEJ-mediated DNA repair, in thyroid cancer cell lines and in the publicly-available TCGA dataset (p<2.4e-14). Additionally, Vem treatment resulted in decreased XLF expression and genetic down-regulation of XLF sensitized BRAFV600E cells to IR (p<0.05). Importantly, Vem in combination with IR resulted in sustained tumor regression of BRAFV600E thyroid tumor xenografts, with minimal evidence of tumor regrowth after 60 days. BRAFV600E mutation promotes NHEJ activity leading to radioresistance, and Vem selectively radiosensitizes BRAFV600E thyroid cancer cells by inhibiting NHEJ repair. Our findings suggest that combining BRAF inhibition and IR may improve the therapeutic outcome for patients with BRAFV600E mutant thyroid cancer.

Abstract CT019: ASN003, a novel highly selective BRAF and PI3K dual inhibitor: Phase I PK/PD results in patients with advanced solid tumors

Abstract Background RAS-RAF-MEK and PI3K-AKT-mTOR are two major pathways involved in tumor cell signaling and growth. Components of these pathways are frequently mutated in a broad range of tumors. ASN003 is a highly selective and potent (low nM IC50) inhibitor of BRAF and PI3K-α and -δ. ASN003 shows strong antitumor activity in tumor models harboring BRAF and PIK3CA mutations, and also in patient-derived xenograft (PDX) models that are resistant to selective BRAF and MEK inhibitors. Patients and Methods: Oral once-daily ASN003 is being evaluated for safety/tolerability and preliminary efficacy in eligible patients with advanced solid tumors using an accelerated dose titration design (Part A) and enrolling cohorts of patients diagnosed with melanoma, CRC, NSCLC and other solid tumors with a BRAF, PIK3CA mutations at MTD (Part B). Pharmacokinetic (PK) profile and the pharmacodynamic (PD) effects of ASN003 on tumor tissue biomarkers such as pERK and pS6 are being investigated in both parts of the study. Results: Patient accrual in Part A is ongoing. As of January 2018, fifteen eligible patients were enrolled in dose levels ranging from 10 - 400 mg QD and 400 mg BID. ASN003 has been well tolerated. Treatment-related adverse events (TRAEs) were mostly mild (G1) to moderate (G2). TRAEs include, rash (G3), hypersensitivity (G3) (n=1), diarrhea (G2) (n=1), hyperglycemia (G2) (n=1), rash maculo-papular (G2) (n=1), vomiting (G1) (n=3), nausea (G1) (n=2), hyperglycemia (G1) (n=2), and dry mouth/lips/skin, transient blurred vision, rash acneiform, ALT increase, cognitive disturbance and hyperkalemia. The PK profile showed dose-dependent increase in systemic exposure at steady state (Cmax up to 5,990 ng/mL, AUC0-T up to 110,000 ng.h/mL) and a half-life of 14-25 hours in general. Early evidence of biomarker changes in repeat biopsies, and clinical disease control have been observed. Conclusions: ASN003 is a novel small molecule, with uniquely selective and potent inhibition of BRAF, PI3K-α and -δ kinases. To date, ASN003 was well tolerated at doses up to 400 mg QD/BID and achieved good systemic exposure. Updated and detailed clinical safety/efficacy and PK/PD results will be presented. Citation Format: Drew Rasco, Nehal Lakhani, Ryan Sullivan, Monica Mita, Jaimini Shah, Helen Usansky, Sanjeeva Reddy, Niranjan S. Rao, Sarper Toker, Louis Denis, Keith Flaherty, Anthony Tolcher. ASN003, a novel highly selective BRAF and PI3K dual inhibitor: Phase I PK/PD results in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT019.

Design and biological evaluation of novel triaryl pyrazoline derivatives with dioxane moiety for selective BRAFV600E inhibition

A series of novel selective BRAFV600E inhibitory agents (Compound 1–16) 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-N,3-diaryl-4,5-dihydro-1H-pyrazole-1-carbothioamides have been designed and synthesized. Their anti-proliferation and BRAF inhibitory activities were evaluated. Though 15, 4 and 12 all displayed comparable activity with the positive control Vemurafenib, only 12 indicated fine selectivity on BRAFV600E (IC50 = 0.06 μM for BRAFV600E; GI50 = 0.52 μM for A375) over BRAFWT at both kinase and cell levels. This result satisfied the designing concept of improving activity and introducing selectivity. Flow cytometry analysis and western blot convinced the apoptosis induction and kinase inhibitory activity. Docking simulation inferred the differences in binding patterns of BRAFV600E and BRAFWT, pointing out that the future orientation might be seeking for outer space binding of BRAFV600E and avoiding interactions with HIS573 of BRAFWT. These results brought potent BRAF inhibitors one step further to selective agents, enhancing the potential for safe medication.

Abstract B147: A phase 1 PK/PD study of ASN003, a novel, highly selective BRAF and PI3K dual inhibitor, in patients with advanced solid tumors

Abstract Background: RAS-RAF-MEK and PI3K-AKT-mTOR are two major pathways involved in tumor cell signaling and growth. Components of these pathways are frequently mutated in a broad range of tumors. ASN003 is a highly selective and potent (low nM IC50) inhibitor of BRAF and PI3K-α and -δ (low affinity for PI3K-β). ASN003 shows strong antitumor activity in tumor models harboring BRAF and PIK3CA or PTEN mutations, and also in patient-derived xenograft (PDX) models that are resistant to selective BRAF and MEK inhibitors. Patients and Methods: Oral once-daily ASN003 is being evaluated for safety/tolerability and preliminary efficacy in eligible patients with advanced solid tumors using an accelerated dose titration design (Part A) and enrolling cohorts of patients diagnosed with melanoma, CRC, NSCLC, and other solid tumors with a BRAF, PIK3CA, or PTEN mutation at MTD (Part B). Pharmacokinetic (PK) profile and the pharmacodynamic (PD) effects of ASN003 on tumor tissue biomarkers such as pERK and pS6 are investigated in both parts of the study. Results: Patient accrual in Part A is ongoing. As of June 2017, nine eligible patients are enrolled in dose levels ranging from 10 - 240 mg QD. ASN003 has been well tolerated. Treatment-related adverse events (TRAEs) were mild (G1) to moderate (G2). TRAEs include diarrhea (G2) (n=1), nausea/vomiting (G1), blurred vision (G1), and dry mouth/lips/skin (G1). Transient G1 elevation of glucose and insulin c-peptide levels has been noted in 1 pt. No G3/4 or serious TRAEs have been observed to date. The PK profile showed dose-dependent, systemic exposure with Cmax ranging from 74 to 1055 ng/mL, AUCT from 912 to 20100 ng.h/mL. Conclusions: ASN003 is a novel small molecule, with uniquely selective and potent inhibition of BRAF, PI3K-α and -δ kinases. To date, ASN003 was well tolerated at doses up to 240 mg QD and achieved good systemic exposure. Updated and detailed clinical safety/efficacy and PK/PD results will be presented. Citation Format: Drew Rasco, Nehal Lakhani, Ryan Sullivan, Monica Mita, Jaimini Shah, Helena Usansky, Sanjeeva Reddy, Niranjan Rao, Louis J. Denis, Anthony Tolcher, Keith Flaherty. A phase 1 PK/PD study of ASN003, a novel, highly selective BRAF and PI3K dual inhibitor, in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B147.

Dabrafenib Therapy in 30 Patients with Melanoma Metastatic to the Brain: a Single-centre Controlled Retrospective Study in Hungary.

Dabrafenib is a potent BRAF inhibitor, which showed intracranial tumor activity. The purpose of our retrospective analysis was to evaluate the efficacy of dabrafenib for patients with melanoma brain metastasis (BM). We studied 30 BRAF mutant melanoma patients with BM, who received dabrafenib after local control of the brain between 2014 and 2017. Eastern Cooperative Oncology Group Performance Status (ECOG) was 0-2. The control arm consisted of 204 melanoma patients from our institutional melanoma database with BM and ECOG 0-2 treated with local therapies and/or chemotherapy, between 2003 and 2015. We found the intracranial disease control rate (DCR) was 83% including four (13%) complete remissions (CR), nine (30%) partial remissions (PR) and twelve (40%) stable diseases (SD) in contrast to five (17%) progressive diseases (PD). With a median follow-up of 14months, median progression-free survival (PFS) and overall survival (OS) were 5.5months, and 8.8months, respectively. If calculated from BM onset, the OS turned to be 11.8months on the dabrafenib arm, while it was only 6.0months in the control arm (HR=0.45, p=0.0014). Higher risk of progression was observed with increasing ECOG (HR =4.06, p=0.00027) and if more than 2 extracranial organs were involved (HR=3.4, p=0.0077). Elevated lactate dehydrogenase (LDH) was non-significantly associated with worse clinical outcome. Remarkable intracranial activity of dabrafenib in real practice was confirmed by our analysis.