Small Molecule Inhibitors Of BRAF Research Articles

Beyond BRAF: where next for melanoma therapy?

In recent years, melanoma has become a poster-child for the development of oncogene-directed targeted therapies. This approach, which has been exemplified by the development of small-molecule BRAF inhibitors and the BRAF/MEK inhibitor combination for BRAF-mutant melanoma, has brought new hope to patients. Despite these successes, treatment failure seems near inevitable in the majority of cases—even in individuals treated with the BRAF/MEK inhibitor doublet. In the current review, we discuss the future of combination strategies for patients with BRAF-mutant melanoma as well as the emerging therapeutic options for patients with NRAS-mutant and BRAF/NRAS-wild-type melanoma. We also outline some of the newest developments in the in-depth personalisation of therapy that should allow melanoma treatment to continue shaping the field precision cancer medicine.

MAPK Pathway Inhibition Enhances the Efficacy of an Anti-Endothelin B Receptor Drug Conjugate by Inducing Target Expression in Melanoma

Therapies targeting the mitogen-activated protein (MAP) kinase pathway in melanoma have produced significant clinical responses; however, duration of response is limited by acquisition of drug resistance. Rational drug combinations may improve outcomes in this setting. We assessed the therapeutic combination of an antibody-drug conjugate (ADC) targeting the endothelin B receptor (EDNRB) with small-molecule inhibitors of the MAP kinase signaling pathway in melanoma. Cell lines and tumor models containing either mutant BRAF or NRAS, or wild-type for both, were exposed to small-molecule inhibitors of BRAF and MEK. Expression of EDNRB was analyzed and the therapeutic impact of combining the anti-EDNRB ADC with the BRAF and MEK inhibitors was assessed. Increased expression of EDNRB in response to inhibition of BRAF and/or MEK was observed and augmented the antitumor activity of the ADC. Enhanced target expression and ADC antitumor activity were realized irrespective of the response of the tumor model to the BRAF or MEK inhibitors alone and could be achieved in melanoma with mutant NRAS, BRAF, or neither mutation. Cells that acquired resistance to BRAF inhibition through long-term culture retained drug-induced elevated levels of EDNRB expression. Expression of EDNRB was not enhanced in normal human melanocytes by inhibition of BRAF and the combination of the ADC with MAPK inhibitors was well-tolerated in mice. The anti-EDNRB ADC combines well with BRAF and MEK inhibitors and could have therapeutic use in the majority of human melanoma cases.

Emerging insights into resistance to BRAF inhibitors in melanoma

Melanoma is the most aggressive form of skin cancer. The treatment of patients with advanced melanoma is rapidly evolving due to an improved understanding of molecular drivers of this disease. Somatic mutations in BRAF are the most common genetic alteration found in these tumors. Recently, two different mutant-selective small molecule inhibitors of BRAF, vemurafenib and dabrafenib, have gained regulatory approval based on positive results in randomized phase III trials. While the development of these agents represents a landmark in the treatment of melanoma, the benefit of these agents is limited by the frequent and rapid onset of resistance. The identification of several molecular mechanisms of resistance to BRAF inhibitors is rapidly leading to the clinical testing of combinatorial strategies to improve the clinical benefit of these agents. These mechanisms, and the lessons learned from the initial testing of the BRAF inhibitors, provide multiple insights that may facilitate the development of targeted therapies against other oncogenic mutations in melanoma, as well as in other cancers.

Targeting The BRAF-MEK-ERK Pathway In Hairy Cell Leukemia

BackgroundHairy cell leukemia (HCL) is a peripheral B-cell neoplasm with indolent clinical course and peculiar morphology and phenotype. Recently, we identified the BRAF-V600E kinase mutation as the genetic lesion underlying HCL and distinguishing it from other leukemias and lymphomas, including HCL-mimics such as splenic marginal zone lymphoma and HCL-variant. The BRAF-V600E mutation is known to be an oncogenic driver in melanoma and other solid tumors thorough constitutive phosphorylation of the kinase MEK (the immediate downstream target of BRAF), which in turns phosphorylates the kinase ERK. Thus, the BRAF-MEK-ERK pathway appears an ideal therapeutic target in HCL to be attacked by small-molecule BRAF inhibitors (such as PLX4720, Vemurafenib and Dabrafenib) and MEK inhibitors (such as Trametinib), which have already proven effective in clinical trials of BRAF-V600E-positive melanoma patients and (for Vemurafenib) in anecdotal HCL patients. However, a thorough analysis of the effects of these drugs in primary HCL cells has not been conducted so far. AimTo study in vitro the effects of BRAF and MEK inhibitors on the specific biochemical, morphological and anti-apoptotic features of HCL, using primary leukemic cells purified from HCL patients (as we previously showed that the putative “HCL” cell lines described in the literature do not carry the BRAF-V600E mutation and are, thus, unlikely to be of true HCL origin). MethodsUsing CD19-MACS, we purified primary hairy cells from the peripheral blood of 20 HCL patients and 12 HCL-like patients and treated these cells in vitro with different BRAF inhibitors (Vemurafenib, PLX4720 and Dabrafenib) or the MEK inhibitor Trametinib, at various concentrations (up to 1 µM) for different time periods (30 minutes to 72 hours), and monitored the phosphorylation status of MEK and ERK by Western blotting. Primary hairy cells were also followed, up to 5 days, for: i) the potential loss of their typical hairy projections (rich in F-actin), through phalloidin staining and confocal microscopy analysis (in 6 HCL and 5 HCL-like patients); ii) the potential reduction of viability, through the MTT or WST metabolic assays (in 12 HCL and 6 HCL-like patients); and iii) the potential induction of apoptosis, through double staining with AnnexinV/Propidium-Iodide and flow cytometry analysis (in 12 HCL and 4 HCL-like patients). ResultsTreatment with any BRAF inhibitors resulted in consistent, rapid, sustained and dose-dependent MEK and ERK dephosphorylation at all time points in all HCL cases, as opposed to vehicle-treated HCL cells and to inhibitor-treated HCL-like cells. Trametinib also produced a strong ERK dephosphorylation in HCL cells. These early biochemical events were followed, in a time frame of 2-3 days, by a consistent and statistically significant reduction of the hairy projections in still viable leukemic cells (AnnexinV-negative). After additional 1-2 days, we observed loss of viability (20% to 60% decrease relative to the drug vehichle – p value <0.05 in all instances) and induction of apoptosis (20% to 59% relative increase – p value <0.05 in all instances) specifically in leukemic cells of the vast majority of HCL patients as opposed to none of the HCL-like patients. ConclusionThese results represent a comprehensive pre-clinical evidence of the significant anti-leukemic activity obtained through inhibition of the BRAF-MEK-ERK pathway in HCL.V.P. and A.S. equally contributed to this work.E.T. and B.F. equally contributed to this work. Disclosures:No relevant conflicts of interest to declare.

The intersection of immune-directed and molecularly targeted therapy in advanced melanoma: where we have been, are, and will be.

In three years, four drugs have gained regulatory approval for the treatment of metastatic and unresectable melanoma, with at least seven other drugs having recently completed, currently in, or soon to be in phase III clinical testing. This amazing achievement has been made following a remarkable increase of knowledge in molecular biology and immunology that led to the identification of high-valued therapeutic targets and the clinical development of agents that effectively engage and inhibit these targets. The discovery of either effective molecularly targeted therapies or immunotherapies would have led to dramatic improvements to the standard-of-care treatment of melanoma. However, through parallel efforts that have showcased the efficacy of small-molecule BRAF and MAP-ERK kinase (MEK) inhibitors, as well as the immune checkpoint inhibitors, namely ipilimumab and the anti-PD1/PDL1 antibodies (lambrolizumab, nivolumab, MPDL3280), an opportunity exists to transform the treatment of melanoma specifically and cancer generally by exploring rational combinations of molecularly targeted therapies, immunotherapies, and molecular targeted therapies with immunotherapies. This overview presents the historical context to this therapeutic revolution, reviews the benefits and limitations of current therapies, and provides a look ahead at where the field is headed.

Immunotherapy at Large: Balancing tumor immunity and inflammatory pathology

Immunotherapy at Large: Balancing tumor immunity and inflammatory pathology

Discovery and Optimization of a Novel Series of Potent Mutant B-RafV600E Selective Kinase Inhibitors

B-Raf represents an attractive target for anticancer therapy and the development of small molecule B-Raf inhibitors has delivered new therapies for metastatic melanoma patients. We have discovered a novel class of small molecules that inhibit mutant B-Raf(V600E) kinase activity both in vitro and in vivo. Investigations into the structure-activity relationships of the series are presented along with efforts to improve upon the cellular potency, solubility, and pharmacokinetic profile. Compounds selectively inhibited B-Raf(V600E) in vitro and showed preferential antiproliferative activity in mutant B-Raf(V600E) cell lines and exhibited selectivity in a kinase panel against other kinases. Examples from this series inhibit growth of a B-Raf(V600E) A375 xenograft in vivo at a well-tolerated dose. In addition, aminoquinazolines described herein were shown to display pERK elevation in nonmutant B-Raf cell lines in vitro.

3′-Deoxy-3′-18F-Fluorothymidine PET Predicts Response to V600EBRAF-Targeted Therapy in Preclinical Models of Colorectal Cancer

Selective inhibition of oncogenic targets and associated signaling pathways forms the basis of personalized cancer medicine. The clinical success of (V600E)BRAF inhibition in melanoma, coupled with the emergence of acquired resistance, underscores the importance of rigorously validating quantitative biomarkers of treatment response in this and similar settings. Because constitutive activation of BRAF leads to proliferation in tumors, we explored 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET to noninvasively quantify changes in tumor proliferation that are associated with pharmacologic inhibition of (V600E)BRAF downstream effectors and that precede changes in tumor volume. Human colorectal cancer (CRC) cell lines expressing (V600E)BRAF were used to explore relationships between upregulation of p27 and phosphorylation of BRAF downstream effectors on small-molecule (V600E)BRAF inhibitor exposure. Athymic nude mice bearing (V600E)BRAF-expressing human CRC cell line xenografts were treated with a small-molecule (V600E)BRAF inhibitor (or vehicle) daily for 10 d. Predictive (18)F-FLT PET was conducted before changes in tumor volume occurred. Correlations were evaluated among PET, inhibition of phosphorylated MEK (p-MEK) and phosphorylated-ERK (p-ERK) by Western blot, tumor proliferation by histology, and small-molecule exposure by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Treatment of CRC cell lines with PLX4720 reduced proliferation associated with target inhibition and upregulation of p27. In vivo, PLX4720 treatment reduced (18)F-FLT uptake, but not (18)F-FDG uptake, in Lim2405 xenografts before quantifiable differences in xenograft volume. Reduced (18)F-FLT PET reflected a modest, yet significant, reduction of Ki67 immunoreactivity, inhibition of p-MEK and p-ERK, and elevated tumor cell p27 protein levels. Both (18)F-FLT PET and (18)F-FDG PET accurately reflected a lack of response in HT-29 xenografts, which MALDI imaging mass spectrometry suggested may have stemmed from limited PLX4720 exposure. We used preclinical models of CRC to demonstrate (18)F-FLT PET as a sensitive predictor of response to (V600E)BRAF inhibitors. Because (18)F-FLT PET predicted reduced proliferation associated with attenuation of BRAF downstream effectors, yet (18)F-FDG PET did not, these data suggest that (18)F-FLT PET may represent an alternative to (18)F-FDG PET for quantifying clinical responses to BRAF inhibitors.

Docking Studies on a Series of Novel Potent BRAF Inhibitors

BRAF, a serine/threonine specific protein kinase that is part of the MAPK pathway and acts as a downstream effector of RAS, is a potential therapeutic target in melanoma. We have studied a series of small-molecule BRAF inhibitors based on a 1H-imidazo[4,5-b]pyridine-2(3H)-one scaffold (ring A) as the hinge binding moiety and a number of substituted phenyl rings C that interact with the allosteric binding site. Based on molecular docking obtained by using GOLD, the interaction models on the receptor site of BRAF are guiding the design of potential inhibitory structures directed against BRAF activity.

Targeting Oncogenic Drivers and the Immune System in Melanoma

Melanoma is one of the most common cancers in Western countries but has defied the trend of reductions in age-adjusted mortality observed in most other cancers in recent years. Biologically, melanoma is characterized by a high propensity to metastasize at low tumor volumes necessitating the need for effective drug therapies to support efforts in prevention and early detection for reducing mortality. Efforts to study the clinical biology of melanoma have led to a new understanding of the disease, with genomic studies identifying several targetable oncogenes, in particular the protein kinases BRAF and KIT. Biologic studies have also identified a variety of immunologic targets, including the programmed death 1 (PD-1) and cytotoxic T-cell lymphocyte-associated antigen 4 (CTLA-4) inhibitory molecules expressed on T lymphocytes. After several decades of clinical trials that failed to demonstrate improvement in overall survival in patients with advanced melanoma, small molecule inhibitors of BRAF or MEK and inhibition of CTLA-4 can improve survival in patients with advanced disease. These early clinical studies have provided a great opportunity to improve mortality in melanoma with the significant potential of combinations of signaling inhibitors or signaling inhibitors combined with immunologic agents, particularly when used in the adjuvant setting, and to transform the care of patients with this most challenging of cancers.

Vemurafenib

Oral vemurafenib (Zelboraf(®)) is a first-in-class, small molecule BRAF(V600E) inhibitor indicated for the treatment of unresectable or metastatic melanoma in BRAF(V600) mutation-positive patients (EU) or BRAF(V600E) mutation-positive patients (USA). Compared with intravenous dacarbazine, vemurafenib significantly improved overall survival and progression-free survival in patients with unresectable, previously untreated, BRAF(V600E) mutation-positive, stage IIIC or IV melanoma. Oral vemurafenib was generally well tolerated, with cutaneous adverse events among the most commonly occurring adverse events.

Ipilimumab in melanoma

Ipilimumab is a fully human monoclonal antibody directed against the cytotoxic T-lymphocyte antigen-4 receptor. Blocking cytotoxic T-lymphocyte antigen-4 signaling has been shown to enhance T-cell activation and to amplify T-cell-mediated immunity. Ipilimumab, either as a single agent or in combination with gp100 vaccination, significantly prolonged overall survival in a randomized Phase III trial in patients with disease progression after prior treatment when compared with gp100 alone. In previously untreated patients, the addition of ipilimumab to dacarbazine also significantly prolonged overall survival. The most common adverse events are immune related. Adherence to established treatment algorithms in patients with immune-related adverse events is advocated. Predictive factors for the activity of ipilimumab have not been identified but would be of great value in the selection of patients who are most likely to benefit from this innovative immunotherapy. Outstanding issues include the role of ipilimumab in the adjuvant treatment of patients who are at high risk for relapse, and the optimal treatment sequence for patients with BRAFV600 mutant melanoma, as small-molecule BRAF inhibitors have also been shown to improve the survival for this subgroup of patients.

Impact of P-Glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2) on the Brain Distribution of a Novel BRAF Inhibitor: Vemurafenib (PLX4032)

Vemurafenib [N-(3-{[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]carbonyl}-2,4-difluorophenyl)propane-1-sulfonamide(PLX4032)] is a novel small-molecule BRAF inhibitor, recently approved by the Food and Drug Administration for the treatment of patients with metastatic melanoma with a BRAF(V600E) mutation. The objective of this study was to investigate the role of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in the distribution of vemurafenib to the central nervous system. In vitro studies conducted in transfected Madin-Darby canine kidney II cells show that the intracellular accumulation of vemurafenib is significantly restricted because of active efflux by P-gp and BCRP. Bidirectional flux studies indicated greater transport in the basolateral-to-apical direction than the apical-to-basolateral direction because of active efflux by P-gp and BCRP. The selective P-gp and BCRP inhibitors zosuquidar and (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino(1',2':1,6)pyrido(3,4-b)indole-3-propanoic acid-1,1-dimethylethyl ester (Ko143) were able to restore the intracellular accumulation and bidirectional net flux of vemurafenib. The in vivo studies revealed that the brain distribution coefficient (area under the concentration time profile of brain/area under the concentration time profile of plasma) of vemurafenib was 0.004 in wild-type mice. The steady-state brain-to-plasma ratio of vemurafenib was 0.035 ± 0.009 in Mdr1a/b(-/-) mice, 0.009 ± 0.006 in Bcrp1(-/-) mice, and 1.00 ± 0.19 in Mdr1a/b(-/-)Bcrp1(-/-) mice compared with 0.012 ± 0.004 in wild-type mice. These data indicate that the brain distribution of vemurafenib is severely restricted at the blood-brain barrier because of active efflux by both P-gp and BCRP. This finding has important clinical significance given the ongoing trials examining the efficacy of vemurafenib in brain metastases of melanoma.

Targeting mutant BRAF in melanoma: current status and future development of combination therapy strategies.

The discovery of activating BRAF mutations in ∼50% of all melanomas has proved to be a turning point in the therapeutic management of the disseminated disease. In this commentary, we review the latest research delineating the role of mutant BRAF in melanoma initiation and progression and discuss the remarkable 10-year journey leading up to the recent U.S. Food and Drug Administration approval of the small-molecule BRAF inhibitor vemurafenib. We further outline the most recent findings on the mechanisms that underlie intrinsic and acquired BRAF inhibitor resistance and describe ongoing preclinical and clinical studies designed to delay or abrogate the onset of therapeutic escape. It is hoped that our evolving understanding of melanoma genetics and intracellular signaling coupled with a growing armamentarium of signal transduction inhibitors will lead to significant improvements in the level and durability of therapeutic response in metastatic melanoma.

Role of BRAF in Thyroid Oncogenesis

BRAF, a cytoplasmic serine-threonine protein kinase, plays a critical role in cell signaling as an activator within the mitogen-activated protein kinase (MAPK) pathway. The most common BRAF mutation is the V600E transversion, which causes constitutive kinase activity. This mutation has been found in a multitude of human cancers, including both papillary thyroid cancer (PTC) and papillary-derived anaplastic thyroid cancer (ATC), in which it initiates follicular cell transformation. With such a high frequency of BRAF mutations in PTC (44%) and PTC-derived ATC (24%), research in BRAF(V600E) detection for diagnostic purposes has shown high sensitivity and specificity for tumor cell presence. BRAF(V600E) in PTC has also provided valuable prognostic information, as its presence has been correlated with more aggressive and iodine-resistant phenotypes. Such findings have initiated research in targeting oncogenic BRAF in cancer therapeutics. Although multiple phase II clinical trials in patients with iodine-refractory metastatic PTC have shown significant efficacy for sorafenib, a first-generation BRAF inhibitor, the mechanism by which it mediates its effect remains unclear because of multiple additional kinase targets of sorafenib. Additionally, preclinical and clinical studies investigating combination therapy with agents such as selective (PLX 4032) and potent (BAY 73-4506 and ARQ 736) small-molecule BRAF inhibitors and MAP/extracellular signal-regulated kinase (ERK) kinase inhibitors (AZD6244) hold great promise in the treatment of BRAF(V600E) cancers and may eventually play a powerful role in changing the clinical course of PTC and ATC.

Small molecule inhibitors of BRAF in clinical trials

Over the last few years, BRAF has emerged as a validated target in melanoma. This review summarises recent advances in the development of BRAF inhibitors, focussing on agents that have been assessed clinically.

Hyperspectral imaging: A non-invasive method of imaging melanoma lesions in a patient with stage IV melanoma, being treated with a RAF inhibitor

Utilizing a macroscopic Prism and Reflectance Imaging Spectroscopy System (MACRO-PARISS), spectral data are taken from human skin using a fiber optic light probe. The subject was an oncology patient at the University of Pennsylvania, with biopsy proven melanoma, who underwent chemotherapy with a novel small molecule BRAF inhibitor as part of a Phase I clinical trial. The BRAF mutation had been previously identified in this patient. Cutaneous lesions were identified by a clinical oncologist, and lesions were imaged periodically over a 1 month period of time. Spectra are classified using a linearity-independent algorithm over a wavelength range of 450-920 nm. Spectral signatures, or characteristic features, of generated spectra correlate with gross features of lesions that are readily observable. Using PARISS we demonstrated differences between spectral signatures of treated lesions over a treatment course, indicating spectral data correlation with changes in disease. PARISS may be useful to better establish a noninvasive means of following disease progression, and ultimately establish characteristic spectral signatures to define disease presence in the future.

Acquired and intrinsic BRAF inhibitor resistance in BRAF V600E mutant melanoma

The discovery of activating BRAF V600E mutations in 50% of all cutaneous melanomas has revolutionized the understanding of melanoma biology and provided new strategies for the therapeutic management of this deadly disease. Highly potent small molecule inhibitors of BRAF are now showing great promise as a novel therapeutic strategy for melanomas harboring activating BRAF V600E mutations and are associated with high levels of response. This commentary article discusses the latest data on the role of mutated BRAF in the development and progression of melanoma as the basis for understanding the mechanism of action of BRAF inhibitors in the preclinical and clinical settings. We further address the issue of BRAF inhibitor resistance and outline the latest insights into the mechanisms of therapeutic escape as well as describing approaches to prevent and abrogate the onset of both intrinsic and acquired drug resistance. It is likely that our evolving understanding of melanoma genetics and signaling will allow for the further personalization of melanoma therapy with the goal of improving clinical responses.

Using quantitative proteomic analysis to understand genotype specific intrinsic drug resistance in melanoma

The discovery of activating BRAF V600E mutations in 50% of all melanoma patients and the development of small molecule BRAF inhibitors looks set to revolutionize the therapy of disseminated melanoma. However, in the recent clinical trial of the BRAF inhibitor, vemurafenib (PLX4032), a significant percentage of BRAF V600E mutant melanoma patients did not meet the RECIST criteria for a response. Recent work from our lab identified loss of the tumor suppressor phosphatase and tensin homolog (PTEN) as being a possible mediator of intrinsic BRAF inhibitor resistance. In this commentary, we describe the development of a novel mass spectrometry based proteomic screen of Bcl-2 family proteins that was used to delineate the PTEN-dependent differences in apoptosis signaling observed when BRAF was inhibited. We further discuss how use of these sensitive quantitative proteomic methods gives unique insights into the signaling of cancer cells that are not captured through routine biochemical techniques and how this may lead to the development of combination therapy strategies for overcoming intrinsic BRAF inhibitor resistance.

Discovery and optimization of thieno[2,3-d]pyrimidines as B-Raf inhibitors

The serine/threonine specific protein kinase B-Raf is part of the MAPK pathway and is an interesting oncology target. We have identified thieno[2,3-d]pyrimidines as a core scaffold of small molecule B-Raf inhibitors. The SAR of analogs in this series will be described.