Potent BRAF Inhibitor Research Articles

Abstract 967: Vemurafenib regulates miRNA-211 expression in melanoma - effects on extracellular vesicle RNA cargo and function

Abstract In melanoma, more than 50% of the patients harbor BRAF somatic missense mutations, most affecting the V600 region. Vemurafenib, a potent BRAF inhibitor used for the treatment of late stage melanoma, has shown promising results by causing programmed cell death in melanoma cells. Recently, extracellular vesicles including apoptotic bodies, microvesicles and exosomes has been shown to contain genetic information especially RNA with distinct repertoire of RNA, which could be transferred from one cell to another. Here we investigate the effects of Vemurafenib on melanoma cells and the RNA content in their subsets of extracellular vesicles. Upon vemurafenib treatment, we found that the melanoma cells harboring the BRAF mutation significantly increases the RNA and protein cargo in the different subsets of extracellular vesicles. The RNA profile also changes substantially in apoptotic bodies and microvesicles with significant changes in the ribosomal RNA ratio. Further, using small RNA sequencing, we found that extracellular vesicles from treated cells harbor unique miRNAs. The significantly different miRNA detected with sequencing analysis was validated in vitro with PCR and confirmed that miR-211 is upregulated in cells as well as subsets of extracellular vesicles after treatment. Furthermore, miR-211 was validated to be significantly upregulated in tumors tissues harvested from patient derived xenograft (PDX) as well as cell line derived xenograft and its vesicular subsets. This shows that vemurafenib induces miR-211 upregulation in BRAF mutant cells and PDX especially in the subsets of extracellular vesicles. miR-211 induction upon oncogene treatment could be potentially used as a biomarker in melanoma patients with BRAF mutation. Citation Format: Taral R. Lunavat, Lesley Cheng, Berglind Einarsdottir, Cecilia Lässer, Jonas A. Nilsson, Andrew F. Hill, Jan Lötvall. Vemurafenib regulates miRNA-211 expression in melanoma - effects on extracellular vesicle RNA cargo and function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 967.

Abstract 3568: Delta-tocotrienol and simvastatin induce cytotoxicity and synergy in BRAF mutant SK-MEL-28 but not in wild type BRAF SK-MEL-2 melanoma cancer cells

Abstract Targeting the mutant BRAF protein is an accepted approach to the treatment of metastatic melanoma. Potent and specific BRAF inhibitors like vemurafenib and dabrafenib are superior to chemotherapy in treatment of BRAF mutant melanomas which represent nearly 50% of all melanomas. Previous studies have shown that certain isoforms of vitamin E and statins can have synergistic anti-cancer activity. We determined whether a combination of delta-tocotrienol (DT3), an unsaturated vitamin E isoform, and simvastatin, an HMG-CoA reductase inhibitor, can exert an anti-neoplastic activity on BRAF-mutated SK-MEL-28 and BRAF-wild type SK-MEL-2 melanoma cell lines and whether a differential effect would be evident. MTS assays were used to analyze cytotoxicity. SK-MEL-28 and SK-MEL-2 cells were cultured in MEM media containing 10% serum and plated in 96-well culture plates for 24 hours then treated with DT3 (0-40 μM), simvastatin (0-5 μM), or a combination and dosed again at 48 hours. SK-MEL-28 and SK-MEL-2 cells grown in 60 mm plates and were treated with DT3 at concentrations of 40, 30, 20 μM, simvastatin at a concentrations of 20, 10, 5 μM or dissolution vehicle as a control for 6 h. Protein concentration of cell lysates was measured spectrophotometrically (GLO Max Multi+, Promega), using a BCA protein assay kit. The samples were run in SDS PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with antibodies against Hsp 70 (Enzo Life Sciences, Farmingdale, NY), Hsp 90 (Santa Cruz, Dallas, TX), pS6 and pBAD (Cell Signaling, Danvers, MA). Using MTS assay, we found that DT3 (IC50 38.8 μM) and simvastatin (IC50 22.7μM) have cytotoxic effects on melanoma cell line SK-MEL-28, but on the SK-MEL-2 cells DT3 does not have an effect at the concentrations studied (10-40 μM DT3) yet simvastatin (IC50 16.9 μM) does have cytotoxicity. Further studies determined that combinations of these drugs display a synergistic effect on SK-MEL-28 by inhibition of pS6 and pBAD and subsequent apoptosis. However, these effects are not observed in SK-MEL-2 cells; treated SK-MEL-2 cells show over-expression of Hsp70 and Hsp90 suggestive of a rescue effect leading to lesser cytotoxic activity. The selective cytotoxicity observed in BRAF-mutated cells and not in wild type BRAF melanoma cell lines by both DT3 and simvastatin warrants further research into the potential therapeutic use of these combinations. This observation has added importance in the light of recent findings that show the acquisition of BRAF mutation is an early event in melanogenesis and hence these compounds may have a key role in chemoprevention approaches to melanoma. Citation Format: Kelley Cross, Victoria Palau, Marianne Brannon, Janet Lightner, Megan Dycus, William Stone, Koyamangalath Krishnan. Delta-tocotrienol and simvastatin induce cytotoxicity and synergy in BRAF mutant SK-MEL-28 but not in wild type BRAF SK-MEL-2 melanoma cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3568.

Design, synthesis, anticancer evaluation and molecular docking of new V600EBRAF inhibitors derived from pyridopyrazinone

Design and synthesis of some new pyridopyrazinone derivatives as anti-proliferative agents is described. The cytotoxic activities of the synthesized compounds against melanoma cell line (LOXIMVI), ovarian cell line (OVCAR3), thyroid cell lines (CAL62, FTC133, BCPAP and ML1) and colon cell lines (HT29 and HCT116) were investigated. Results revealed that most compounds were active and compound 3d was the most active one. It exhibited promising activity against all tested cell lines. In addition, in vitro kinase assay against both WT BRAF and V600E BRAF was performed for all synthesized compounds. Furthermore, molecular docking of tested compounds was established with active site of V600E BRAF kinase domain. Results of kinase inhibition assay and molecular docking revealed that, compounds 1, 3d, e, h, i, 5d, e and 6b were potent inhibitors for V600E BRAF kinase enzyme involved in number of cancer types as melanoma, ovarian and thyroid cancer. The newly synthesized pyridopyrazinones substituted with different substituents at C-3 or fused with triazine heterocycle at C-3 and C-4 afforded potent V600E BRAF inhibitors and exhibited promising cytotoxic activities against different cancer types such as melanoma, ovarian, thyroid and colon cancer.

EBI-907, a novel BRAFV600E inhibitor, has potent oral anti-tumor activity and a broad kinase selectivity profile

abstractThe oncogenic mutation of BRAFV600E has been found in approximately 8% of all human cancers, including more than 60% of melanoma and 10% of colorectal cancers. The clinical proof of concept in treating BRAFV600E-driving melanoma patients with the BRAF inhibitors has been well established. We have sought to identify and develop novel BRAFV600E inhibitors with more favorable profiles. Our chemistry effort has led to the discovery of EBI-907 as a novel BRAFV600E inhibitor with potent anti-tumor activity in vitro and in vivo. In a LanthaScreen BRAFV600E kinase assay, EBI-907 showed an IC50 of 4.8 nM, which is >10 -fold more potent than Vemurafenib (IC50 = 58.5 nM). In addition, EBI-907 showed a broader kinase selectivity profile, with potent activity against a number of important oncogenic kinases including FGFR1-3, RET, c-Kit, and PDGFRb. Concomitant with such properties, EBI-907 exhibits potent and selective cytotoxicity against a broader range of BRAFV600E-dependent cell lines including certain colorectal cancer cell lines with innate resistance to Vemurafenib. In BRAFV600E-dependent human Colo-205 and A375 tumor xenograft mouse models, EBI-907 caused a marked tumor regression in a dose-dependent manner, with superior efficacy to Vemurafenib. Our results also showed that combination with EGFR or MEK inhibitor enhanced the potency of EBI-907 in cell lines with innate or acquired resistance to BRAF inhibition alone. Our findings present EBI-907 as a potent and promising BRAF inhibitor, which might be useful in broader indications.

Discovery of EBI-907: A highly potent and orally active B-Raf(V600E) inhibitor for the treatment of melanoma and associated cancers.

A novel series of pyrazolo[3,4-c]isoquinoline derivatives was discovered as B-Raf(V600E) inhibitors through scaffold hopping based on a literature lead PLX4720. Further SAR exploration and optimization led to the discovery of potent B-Raf(V600E) inhibitors with good oral bioavailability in rats and dogs. One of the compounds EBI-907 (13g) demonstrated excellent in vivo efficacy in B-Raf(V600E) dependent Colo-205 tumor xenograft models in mouse and is under preclinical studies for the treatment of melanoma and B-Raf(V600E) associated cancers.

Vemurafenib (VEM) in Relapsed Refractory Multiple Myeloma Harboring BRAFV600 Mutations (V600m): A Cohort of the Histology-Independent VE-Basket Study

Background: Whole-genome sequencing has identified a 4-6% incidence of BRAF mutations in multiple myeloma (MM). We undertook a histology-independent, "basket" study of VEM in BRAF V600m-positive cancers (NCT01524978). Six disease cohorts were prespecified; remaining tumors were classified in a 7th "all-comers" cohort. Here we present preliminary efficacy and safety data for the MM cohort.Methods: A multicenter, Simon, 2-stage adaptive design in patients with relapsed refractory BRAF V600m-positive MM who were receiving VEM (960 mg bid) until disease progression (PD) or unacceptable toxicity was used. Primary end point is investigator-assessed response rate (RR) at week 8 by International Myeloma Working Group criteria. Secondary objectives include overall RR, clinical benefit rate, duration of response, progression-free survival, overall survival, and safety. Stage 1 was complete after the 7th patient received a minimum of 8 weeks of treatment, died, or withdrew early from the study.Results: Eight patients had been enrolled in the MM cohort at the time of the data cutoff (December 3, 2014). Data are presented on patients in stage 1. Twelve patients were screened, of which 4 patients did not meet eligibility criteria. Of the 8 patients enrolled in the study, 6 were men and 2 were women, with a median age of 64 years (range, 55-68). High-risk features were seen in 3 patients by cytogenetics and fluorescence in situ hybridization (FISH). Prior treatment included immunomodulators (IMiDs) in 100% of patients, proteasome inhibitors in 75%, and chemotherapy (melphalan, bendamustine, cytoxan, doxorubicin, etoposide, and cisplatin) in 87.5%. Patients had received between 2 and 7 lines of treatment before enrolling in the BASKET trial, and 5 were refractory to IMiDs or proteasome inhibitors, or both. Median duration of treatment was 3.3 months (range, 1-5) at the time of data cutoff; 3 patients continue to be treated and 5 patients discontinued study drug. Response data were available for 7 patients at the end of 2 cycles. One patient achieved partial response (PR); 4 patients had stable disease; 1 patient had progressive disease; and 1 patient response was reported as not evaluable (objective response rate [ORR] week 8, 14%; 95% confidence interval [CI], 0.4-57.9). Of the patients enrolled, 71% (95% CI, 29.0-96.3) had clinical benefit with single-agent VEM. Responses occurred beyond 2 cycles: 1 patient went on to achieve very good PR (after cutoff date, January 2015). Three patients experienced disease progression between study days 57 and 85, and 1 of these patients died as a result of progressive disease. Single-agent VEM was well tolerated, with a safety profile similar to that observed in melanoma patients. Seven patients (88%) had at least 1 adverse event (AE) of grade 3 or 4, and 3 patients (38%) had at least 1 serious AE, including sepsis and lower respiratory tract infection, that was attributable to the underlying disease. Toxicity was manageable, and 1 patient discontinued treatment because of lower respiratory tract infection and skin lesions. Dose modification was necessary in 5 of 8 patients because of toxicity.Conclusions: This is the first mutation-specific clinical trial in MM. VEM has promising activity in patients with BRAF V600m-positive MM despite these patients being heavily pretreated. Because obvious clinical benefit for patients has been observed, the decision to recruit additional patients was made, and recruitment is ongoing. Updated efficacy results from all patients currently participating in the study will be presented. DisclosuresRaje:Amgen: Consultancy; Celgene: Consultancy; Astra Zeneca: Research Funding; Eli Lilly: Research Funding; BMS: Consultancy; Roche: Consultancy; Takeda: Consultancy; Onyx: Consultancy. Off Label Use: Vemurafenib is a potent inhibitor of BRAF mutated at codon 600 (BRAFV600). Here we explored the efficacy of vemurafenib in multiple myeloma patients with BRAFV600 mutations.. Chau:Roche: Research Funding. Hyman:Chugai Pharma: Consultancy; Biotherapeutics: Consultancy; Atara: Consultancy, Honoraria. Ribrag:Gilead: Membership on an entity's Board of Directors or advisory committees; Esai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmamar: Honoraria, Membership on an entity's Board of Directors or advisory committees. Blay:roche: Research Funding. Tabernero:Taiho: Consultancy; Millennium: Consultancy; Merck: Consultancy; Amgen: Consultancy; Imclone: Consultancy; Chugai: Consultancy; Merck Serono: Consultancy; Boehringer Ingelheim: Consultancy; Eli Lilly: Consultancy; Celgene: Consultancy; Sanofi: Consultancy; Roche: Consultancy; Novartis: Consultancy; Symphgen: Consultancy. Wolf:Roche: Consultancy, Equity Ownership, Honoraria, Research Funding. Sirzen:F. Hoffmann-La Roche: Employment, Equity Ownership. Faris:Merrimack Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding; N-of-One-Therapeutics: Consultancy. Kaiser:Bristol-Myers Squibb: Consultancy; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Chugai: Consultancy. Veronese:F. Hoffmann-La Roche: Employment. Makrutzki:F. Hoffmann-La Roche: Employment. Lasserre:F. Hoffmann-La Roche: Employment, Other: Unspecified, Patents & Royalties. Puzanov:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Baselga:Roche: Consultancy.

Encorafenib (LGX818), a potent BRAF inhibitor, induces senescence accompanied by autophagy in BRAFV600E melanoma cells

Encorafenib (LGX818) is a new-generation BRAF inhibitor that is under evaluation in clinical trials. However, the underlying mechanism remains to be elucidated. Here we show that LGX818 potently decreased ERK phosphorylation and inhibited proliferation in BRAFV600E melanoma cell lines. Moreover, LGX818 downregulated CyclinD1 in a glycogen synthase kinase 3β-independent manner and induced cell cycle arrest in the G1 phase, Surprisingly, LGX818 triggered cellular senescence in BRAFV600E melanoma cells, as evidenced by increased β-galactosidase staining, while no appreciable induction of apoptosis was detected, as determined by Annexin V and propidium iodide staining and immunoblot analysis of caspase-3 processing and poly (ADP-ribose) polymerase cleavage. Increased p27KIP1 expression and retinoblastoma protein activation were detected during LGX818-induced senescence. Additionally, inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 1B by AZ191 reversed LGX818-induced CyclinD1 turnover and senescence. Interestingly, autophagy is triggered through inhibition of the mTOR/70S6K pathway during LGX818-induced senescence. Moreover, autophagy inhibition by pharmacological and genetic regulation attenuates LGX818-induced senescence. Notably, combining LGX818 with autophagy modulators has anti-proliferative effect in LGX818-resistant BRAF mutant melanoma cells. Altogether, we uncovered a mechanism by which LGX818 exerts its anti-tumor activity in BRAFV600E melanoma cells.

Discovery of RAF265: A Potent mut-B-RAF Inhibitor for the Treatment of Metastatic Melanoma.

Abrogation of errant signaling along the MAPK pathway through the inhibition of B-RAF kinase is a validated approach for the treatment of pathway-dependent cancers. We report the development of imidazo-benzimidazoles as potent B-RAF inhibitors. Robust in vivo efficacy coupled with correlating pharmacokinetic/pharmacodynamic (PKPD) and PD-efficacy relationships led to the identification of RAF265, 1, which has advanced into clinical trials.

A Combined Pharmacophore Modeling, 3D QSAR and Virtual Screening Studies on Imidazopyridines as B-Raf Inhibitors.

B-Raf kinase is an important target in treatment of cancers. In order to design and find potent B-Raf inhibitors (BRIs), 3D pharmacophore models were created using the Genetic Algorithm with Linear Assignment of Hypermolecular Alignment of Database (GALAHAD). The best pharmacophore model obtained which was used in effective alignment of the data set contains two acceptor atoms, three donor atoms and three hydrophobes. In succession, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on 39 imidazopyridine BRIs to build three dimensional quantitative structure-activity relationship (3D QSAR) models based on both pharmacophore and docking alignments. The CoMSIA model based on the pharmacophore alignment shows the best result (q2 = 0.621, r2pred = 0.885). This 3D QSAR approach provides significant insights that are useful for designing potent BRIs. In addition, the obtained best pharmacophore model was used for virtual screening against the NCI2000 database. The hit compounds were further filtered with molecular docking, and their biological activities were predicted using the CoMSIA model, and three potential BRIs with new skeletons were obtained.

Evaluation of Safety, Tolerability, Pharmacokinetics and Efficacy of Dabrafenib, a Braf Inhibitor, in Japanese Patients with Braf V600 Mutation-Positive Advanced Solid Tumors: a Phase I Study

ABSTRACT Aim: Dabrafenib, a potent and selective BRAF inhibitor, has demonstrated clinical significance for BRAF V600 mutation-positive metastatic melanoma and has been approved in the US and EU. However, there is less clinical experience of treatment with dabrafenib in the Japanese population. Therefore, this study has been designed and conducted to evaluate safety, tolerability, pharmacokinetics (PK) and efficacy of dabrafenib in Japanese patients with BRAF V600 mutation-positive advanced solid tumors as the first clinical trial. Methods: Patients with BRAF V600 mutation-positive advanced solid tumors refractory to standard therapies were enrolled. Dose escalation was performed according to a standard 3 + 3 design. Dabrafenib was given twice a day (BID), which started 7 days after a single dose administration for a single-dose PK analysis. Antitumor activity was evaluated based on RECIST version 1.1. This study was registered at www.clinicaltrials.gov as NCT01582997. Results: Twelve patients (11 melanoma and 1 papillary thyroid cancer) with BRAF V600E mutation received dabrafenib at doses of 75 mg (n = 3), 100 mg (n = 3), or 150 mg (n = 6). The median duration of treatment was approximately 39 weeks. No dose-limiting toxicities were observed in these three dose cohorts. The most common adverse events (AEs) included alopecia, leucopenia, pyrexia and arthralgia. Most AEs were reported as grade 1 or 2. Squamous-cell carcinoma or keratoacanthoma, which is a well-documented BRAF inhibitor class toxicity, was not observed in this study. Preliminary PK results showed no obvious ethnic difference. Partial responses were confirmed in 7 patients (58.3%), 6 melanoma patients and 1 papillary thyroid cancer patient. The data also suggested dabrafenib had an antitumor activity in Japanese patients with BRAF V600 mutation-positive melanoma as well as papillary thyroid cancer. Conclusions: Compared with the data published for the caucasian population, dabrafenib exhibited comparable tolerability, safety and PK profiles in Japanese patients at the dose of 150 mg BID. This dose also gave a suggestive outcome for the potential efficacy to the Japanese population. Disclosure: A. Suzuki: A. Suzuki is an employee of GlaxoSmithKline K.K; A. Mukaiyama: A. Mukaiyama is an employee of GlaxoSmithKline K.K. All other authors have declared no conflicts of interest.

535P - Encorafenib (Lgx818), an Oral Braf Inhibitor, in Patients (Pts) with Braf V600E Metastatic Colorectal Cancer (Mcrc): Results of Dose Expansion in an Open-Label, Phase 1 Study

ABSTRACT Aim: Few treatment options exist for pts with BRAF-mutant mCRC. Encorafenib, a potent and selective oral BRAF inhibitor, showed signs of efficacy in pts with BRAF-mutant advanced melanoma in a phase 1 dose-escalation study. Here we present results from pts with BRAF-mutant mCRC enrolled in the dose-expansion phase of the same trial. Methods: Adult pts with nonresectable, advanced mCRC with a BRAF V600 mutation, for whom there was no existing effective therapy, received encorafenib 300 mg (n = 6) or 450 mg (n = 12) once daily. Responses were assessed by investigator per RECIST 1.1. Results: All 18 pts had a BRAF V600E mutation. Median age was 62 y (range, 34-73). Ten pts (55.6%) were female. Median number of prior treatment regimens was 3 (range, 1-8); 1 pt received prior therapy with a BRAF inhibitor (vemurafenib). At the data cutoff (7 Jan 2014), median treatment duration was 11 wks; 4 pts (22.2%) remained on treatment, and 14 (77.8%) discontinued due to progressive disease (PD; n = 10), adverse events (AEs; n = 3), and death (n = 1, not related to study drug). Twelve pts (66.7%) achieved a best response of stable disease (SD). No pt achieved confirmed complete response (CR) or confirmed partial response (PR) by the data cutoff (Table). Progression-free survival (PFS) events occurred in 13 pts (PD, n = 10; death, n = 3). Median PFS was 4.0 mo. The most common AEs of any grade were palmar-plantar erythrodysaesthesia syndrome (66.7%), myalgia (44.4%), and dry skin (44.4%). Three pts had dose-limiting toxicities (arthralgia and myalgia [n = 1]; insomnia and myalgia [n = 1]; bone pain and vomiting [n = 1]; all grade 3), all on encorafenib 450 mg. Encorafenib 300 mg n = 6 Encorafenib 450 mg n = 12 All pts N = 18 Best Overall Response, n (%) _CRa 0 0 0 _PRa 0 0 0 _SD 4 (66.7) 8 (66.7) 12 (66.7) __With unconfirmed CR/PR 1 (16.7) 2 (16.7) 3 (16.7) _PD 1 (16.7) 3 (25.0) 4 (22.2) __With unconfirmed CR/PR 0 2 (16.7) 2 (11.1) _Unknownb 1 (16.7) 1 (8.3) 2 (11.1) Median PFS, mo (95% CI) 2.3 (1.7-7.2) 4.0 (1.8-5.5) 4.0 (1.8-5.6) a CR and PR required confirmation in a second assessment at least 4 wks after the first. b Best response was unknown for 2 pts who achieved SD before 6 wks of treatment. Conclusions: Encorafenib showed modest activity in this difficult-to-treat population, with SD observed in two-thirds of pts. Studies in BRAF-mutant mCRC are ongoing in combination with epidermal growth factor receptor and phosphoinositide 3-kinase inhibitors. Disclosure: M. Hidalgo: has been on a board of directors and/or advisory board for and received research funding from Novartis; R. von Moos: has consulted for Novartis, Roche, GSK, BMS, MSD; received research funding from Amgen, Merck, Roche; received honoraria from Novartis, Roche, GSK, BMS, Sanofi; A. Arance: has received research funding from Roche; has consulted with Roche, BMS, GSK D. Michel, A. Seroutou and T. Demuth: is an employee of Novartis Pharma AG; J. Tabernero: has consulted for Amgen, Imclone, Lilly, Merck KGaA, Millenium, Novartis, Roche, Sanofi, Celgene, Chugai, Taiho and has received honoraria from Amgen, Merck KGaA, Novartis, Roche, Sanofi. All other authors have declared no conflicts of interest.

Vemurafenib and aPTT prolonging: A case report.

e20021 Background: Metastatic melanoma (MM) is a disease with poor prognosis; Vemurafenib (V) is a potent inhibitor of BRAF with V600E mutation with well established antitumor activity in this sett...

Abstract C254: Discovery of a novel and potent BRAF (V600E) inhibitor with a unique kinase targeting profile, EBI-907: Pharmacological characterization and potential to overcome drug resistance and paradoxical activation.

Abstract The RAS/RAF/MEK/ERK pathway plays a central role in regulating cell proliferation, differentiation, and growth. The oncogenic mutation of BRAF (V600E) has been found in 8% of all human cancers, including more than 60% of melanoma, 45% of papillary thyroid cancer, 10% of colorectal cancers, and a small subset of ovarian, breast, lung cancers, and leukemia. Although BRAF-specific inhibitors such as Vemurafenib (PLX-4032) and Dabrafenib (GSK2118436) have been approved by FDA and several other BRAF inhibitors are at different stages of clinical development, they carry some liabilities and drug resistance rapidly developed. We have sought to identify and develop more potent BRAF (V600E) inhibitors with favorable pharmacological and safety profiles. Our chemistry effort has led to the discovery of EBI-907 as a novel BRAF (V600E) inhibitor with potent anti-tumor activity in vitro and in vivo. In a LanthaScreen BRAF (V600E) kinase assay, EBI-907 is a highly potent inhibitor displaying a low single-digit nanomolar activity (IC50 = 4.9 nM), which is >10-fold more potent than Vemurafenib (IC50 = 59 nM). EBI-907 also exhibits high potency in selectively inhibiting the proliferation of BRAF (V600E)-dependent cell lines (A375 and Colo205) and cellular Erk phosporylation, with superior activity to Vemurafenib. In multiple preclinical species (mice, rats, and dogs), EBI-907 exhibited an excellent oral bioavailability. In a BRAF (V600E)-dependent human Colo-205 tumor xenograft mouse model, EBI-907 caused a partial or complete tumor regression in a dose-dependent manner, with superior efficacy than Vemurafenib. In addition, we have also observed a broader kinase selectivity profile for EBI-907, displaying potent activity against a number of important oncogenic kinases including BRK, FGFR1, c-Kit, and PDGFRb. Preliminary toxicity studies in rodent models showed that EBI-907 was well tolerated and has a high safety margin. More importantly, EBI-907 showed a favorable pattern of drug-induced paradoxical activation of MAPK pathway, pointing to a possibility to “break” the inhibitor paradox. Finally, our results showed that combined use of EBI-907 and a MEK inhibitor completely abolished the paradoxical activation and overcame the drug resistance induced by BRAF inhibitor alone. Our findings not only present EBI-907 as a potent and promising BRAF inhibitor, but also point out a possibility to develop next generation targeted therapies to treat BRAF mutated cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C254. Citation Format: Jiayin Zhang, Dong Liu, Ru Shen, Yinfa Yan, Liuqing Yang, Minsheng Zhang, Guoqing Cao, Hu Cao, Beibei Fu, Aishen Gong, Biao Lu, Qiming Sun, Hong Wan, Pangke Yan, Lei Zhang, Lianshan Zhang, Jingsong Cao. Discovery of a novel and potent BRAF (V600E) inhibitor with a unique kinase targeting profile, EBI-907: Pharmacological characterization and potential to overcome drug resistance and paradoxical activation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C254.

Concomitant Oral and Intravenous Pharmacokinetics of Dabrafenib, a BRAF Inhibitor, in Patients with BRAF V600 Mutation‐Positive Solid Tumors

Dabrafenib is an orally bioavailable, potent, and selective inhibitor of human wild-type BRAF and CRAF kinases as well as mutant forms of BRAF kinase. The aim of this phase 1, single-center, open-label study in four patients with BRAF mutation-positive solid tumors was to determine the absolute bioavailability of a 150 mg oral dose of dabrafenib. A microtracer study approach, in which a 50 µg radiolabeled intravenous (IV) microdose of dabrafenib was given concomitantly with a 150 mg oral dose, was used to simultaneously recover IV and oral pharmacokinetic parameters. The least squares mean (90% CI) absolute bioavailability of dabrafenib (HPMC capsules) was 94.5% (81.3%, 109.7%). Median T(max) after oral administration was 2.0 hours and the geometric mean terminal half-life was 4.8 hours. The geometric mean clearance and volume of distribution after IV administration were 12.0 L/h and 45.5 L, respectively. Human clearance and volume of distribution at steady state were in agreement with predictions made using allometric scaling of pharmacokinetic parameters from four preclinical species. In conclusion, dabrafenib absolute bioavailability was high, whereas first-pass metabolism was low. Furthermore, the microtracer approach provided an innovative and efficient method for assessing the absolute bioavailability of dabrafenib in patients with advanced cancer.

Dabrafenib; Preclinical Characterization, Increased Efficacy when Combined with Trametinib, while BRAF/MEK Tool Combination Reduced Skin Lesions

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAFV600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G1 cell cycle arrest, followed by cell death. In a BRAFV600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.

Vitiligo in 2 patients receiving vemurafenib for metastatic melanoma

To the Editor: Vemurafenib is a potent inhibitor of BRAF approved for the treatment of metastatic melanoma with the V600E mutation. 1 Sosman J.A. Kim K.B. Schuchter L. et al. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med. 2012; 366: 707-714 Crossref PubMed Scopus (1749) Google Scholar This new therapy is associated with a high rate of cutaneous adverse events. 2 Sinha R. Edmonds K. Newton-Bishop J.A. Gore M.E. Larkin J. Fearfield L. Cutaneous adverse events associated with vemurafenib in patients with metastatic melanoma: practical advice on diagnosis, prevention and management of the main treatment-related skin toxicities. Br J Dermatol. 2012; 167: 987-994 Crossref PubMed Scopus (106) Google Scholar , 3 Huang V. Hepper D. Anadkat M. Cornelius L. Cutaneous toxic effects associated with vemurafenib and inhibition of the BRAF pathway. Arch Dermatol. 2012; 148: 628-633 Crossref PubMed Scopus (18) Google Scholar We present two patients treated with vemurafenib who developed vitiligo. To our knowledge, no cases of vitiligo have been previously described after treatment with vemurafenib.

BKM120 combined with vemurafenib in vemurafenib-refractory BRAF mutant metastatic melanoma: Two cases.

e20010 Background: PTEN loss and PI3K activation are common in BRAF mutant metastatic melanoma, and PI3K activation has been implicated as a cause of acquired resistance to BRAF inhibitors. Two vemurafenib refractory were treated with the potent BRAF inhibitor, vemurafenib, and the pan-class I PI3K inhibitor BKM120. Methods: The study enrolled BRAF-V600E/K mutant metastatic melanoma patients with an ECOG PS ≥ 2 and adequate organ function. Vemurafenib refractory BRAF-V600E/K mutant melanoma patients started both vemurafenib twice daily and BKM120 daily on cycle 1, day 1 after a vemurafenib washout of at least 14 days. Serial biopsies prior to treatment, on cycle 1 day 15, and at progression were obtained for pharmacodynamics analysis in patients with visible or palpable tumors. Results: 3 BRAF inhibitor refractory patients were treated on study with vemurafenib 720 mg PO bid and BKM120 60 mg PO daily. One patient was inevaluable due to non-compliance and had minimal exposure to study drug. Pre-treatment biopsy specimens were available in the remaining 2 vemurafenib-refractory patients. Both patient expressed PTEN at baseline and had demonstrable pAKT and pS6 staining. One patient had a mixed response to treatment with a 35.9% reduction in target lesions and two new small subcutaneous lesions. This patient also developed dose limiting febrile neutropenia on trial. The second patient tolerated treatment well but had widespread disease progression at 8 weeks. Conclusions: Combination therapy with vemurafenib and BKM120 in BRAF-V600E/K mutant melanoma led to substantial regression of several tumors in a PTEN+ patient with prior disease progression on vemurafenib alone. A phase I dose escalation trial in ongoing. Clinical trial information: NCT01512251.

Initial results from a phase I, open-label, dose escalation study of the oral BRAF inhibitor LGX818 in patients with BRAF V600 mutant advanced or metastatic melanoma.

9028 Background: LGX818, a potent and selective BRAF inhibitor (BRAFi) being investigated in BRAF V600 mutant melanoma, has unique biochemical properties with a dissociation half-time > 10 times longer than other BRAF inhibitors. Methods: A phase I trial of LGX818 administered orally once (qd) or twice (bid) daily in BRAF V600 tumors was initiated to define the maximum tolerated dose (MTD)/recommended phase II dose (RP2D) and to assess pharmacokinetics and clinical activity in BRAFi–naive or pretreated patients with BRAF V600 mutant advanced melanoma. Baseline assessment of biomarkers from MAPK/PI3K pathways and pharmacodynamics were also evaluated. Results: Fifty-four patients have been enrolled in the dose-escalation phase (dose levels [DLs], 50-700 mg qd [n=42] and 75-150 mg bid [n=12]). LGX818 plasma concentrations increased proportionally by dose with a mean t1/2 of 4 hours and steady state in ≈ 15 days. The MTD/RP2D (450 mg qd) was well tolerated. Seven patients had a dose limiting toxicity (DLT): 5 at qd (1 each with hand-foot skin reaction [HFSR], foot pain, fatigue, diarrhea/rash, insomnia/asthenia) and 2 at bid (1 facial paresis/confusion, 1 musculoskeletal pain/neuralgia). All DLTs were grade 3 and reversible. The most common adverse events (≥ 20%) suspected to be treatment related were cutaneous (rash, dry skin, HFSR, pruritus, keratosis pilaris, alopecia), pain in extremity, arthralgia, and fatigue. Squamous cell carcinoma was observed in 2 patients (1 naive and 1 pretreated). As of 30 Sept 2012, the preliminary efficacy (all DLs) in patients with at least 1 postbaseline tumor assessment was 16 partial responses [PRs] (67%; 12 confirmed) out of 24 BRAFi–naive patients and 2 PRs (8.3%; 1 confirmed) among 24 BRAFi–pretreated patients. Responses were seen at all DLs from 50 to 550 mg qd. Updated safety and efficacy including time to event endpoints will be reported. Conclusions: Initial results from this study identified the MTD/RP2D as 450 mg/day and provided an early sign of promising activity in advanced melanoma. Expansion cohorts are ongoing in BRAFi–naive and BRAFi–pretreated melanoma and colorectal cancer. Clinical trial information: NCT01436656.

RASopathic Skin Eruptions during Vemurafenib Therapy

PurposeVemurafenib is a potent inhibitor of V600 mutant BRAF with significant impact on progression-free and overall survival in advanced melanoma. Cutaneous side effects are frequent. This single-center observational study investigates clinical and histological features of these class-specific cutaneous adverse reactions.Patients and MethodsPatients were all treated with Vemurafenib 960 mg b.i.d. within local ethic committees approved clinical trials. All skin reactions were collected and documented prospectively. Cutaneous reactions were classified by reaction pattern as phototoxic and inflammatory, hair and nail changes, keratinocytic proliferations and melanocytic disorders.ResultsVemurafenib was well tolerated, only in two patients the dose had to be reduced to 720 mg due to arthralgia. 26/28 patients (93%) experienced cutaneous side effects. Observed side effects included UVA dependent photosensitivity (n = 16), maculopapular exanthema (n = 14), pruritus (n = 8), folliculitis (n = 5), burning feet (n = 3), hair thinning (mild alopecia) (n = 8), curly hair (n = 2) and nail changes (n = 2). Keratosis pilaris and acanthopapilloma were common skin reactions (n = 12/n = 13), as well as plantar hyperkeratosis (n = 4), keratoacanthoma (n = 5) and invasive squamous cell carcinoma (n = 4). One patient developed a second primary melanoma after more than 4 months of therapy (BRAF and RAS wild type).ConclusionVemurafenib has a broad and peculiar cutaneous side effect profile involving epidermis and adnexa overlapping with the cutaneous manifestations of genetic diseases characterized by activating germ line mutations of RAS (RASopathy). They must be distinguished from allergic drug reaction. Regular skin examination and management by experienced dermatologists as well as continuous prophylactic photo protection including an UVA optimized sun screen is mandatory.

Potent BRAF kinase inhibitors based on 2,4,5-trisubstituted imidazole with naphthyl and benzothiophene 4-substituents

The RAS–RAF–MEK–ERK pathway is hyperactivated in 30% of human cancers. BRAF is a serine–threonine kinase, belonging to this pathway that is mutated with high frequency in human melanoma and other cancers thus BRAF is an important therapeutic target in melanoma. We have designed inhibitors of BRAF based on 2,4,5-trisubstituted imidazoles with naphthyl and benzothiophene-4-substituents. Two compounds were discovered to be potent BRAF inhibitors: 1-(6-{2-[4-(2-dimethylamino-ethoxy)phenyl]-5-(pyridin-4-yl)-1H-imidazol-4-yl} benzo[b]thiophen-3-yl)-2,2,2-trifluoroethanol (1i) with BRAF IC50=190nM and with cellular GI50=2100nM, and 6-{2-[4-(2-dimethylamino-ethoxy)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-naphthalen-1-ol (1q) with IC50=9nM and GI50=220nM.