Selective BRAF Inhibitors Research Articles

Selectively Targeting Mutant BRAF in Thyroid Cancer

Selectively Targeting Mutant BRAF in Thyroid Cancer

Abstract B87: Genomic complexity and BRAF/MEK-dependence in V600E BRAF mutant melanoma

Abstract Constitutive ERK activation, often the result of BRAF mutation, is a common finding in human cancer. BRAF mutations occur in approximately 8% of human tumors, with the highest frequency observed in melanoma (40–70%). Supporting its classification as an oncogene, V600E BRAF stimulates ERK signaling, induces proliferation and is capable in model systems of promoting transformation. BRAF mutations are, however, common in nevi suggesting that BRAF mutation alone is insufficient for tumorigenesis. These observations suggest that additional alterations cooperate with BRAF in promoting melanoma formation and that inhibition of oncogenic BRAF may not be sufficient in many tumors to induce apoptosis in some BRAF mutant melanomas. Prior studies have demonstrated that loss of PTEN is a common event in melanoma and PTEN has been shown to cooperate with BRAF mutation in accelerating invasive melanoma formation in genetic murine models. With the goal of identifying model systems in which to study the the role played by PTEN in promoting tumorigenesis, we performed MALDI-TOF mass spectrometry genotyping on a panel of 116 melanoma cell lines. Seventy-two (62%) harbored BRAF mutations (66 V600E, 3 V600K, 3 in exon 11). Proteomic profiling of the class of V600E BRAF mutant cell lines demonstrated significant variability in the expression of PTEN and pAKT. Nine cells lines harboring V600E BRAF mutations were found to express no PTEN protein and high pAKT by immunoblotting. The majority of these cell lines had small (1–2) base-pair insertions and deletions leading to a frame shift and early truncation. Array CGH confirmed focal homozygous deletion of the PTEN gene in three of the models. These deletions were small and in one case detected by the Agilent 1M but not the 244K platform. We have previously reported that mutations of BRAF are associated with enhanced and selective sensitivity to MEK inhibition when compared to either receptor tyrosine kinase-driven cells or cells harboring a RAS mutation. To determine the MAPK pathway dependence of V600E BRAF mutant cell lines as a function of PTEN expression, we used PD0325901, a selective allosteric inhibitor of MEK1/2. A subset of the V600E BRAF mutant, PTEN null or mutant cells were dependent upon MEK for proliferation with IC50 values ranging from 1.1 to >500nM. These data suggest that PTEN loss is insufficient to confer absolute resistance to MAPK-pathway inhibition. MEK inhibition was, however, cytostatic in all V600E BRAF, PTEN null cell lines whereas MEK inhibition induced significant apoptosis in a subset of V600E BRAF, PTEN wild-type models. PTEN null, BRAF mutant cell lines were sensitive to two selective PI3 kinase inhibitors and the combination of MEK and PI3 kinase inhibition was more effective than either alone in a subset of models. In summary, our data suggest that PTEN is one of several concurrent genetic/epigenetic alterations that condition MEK-dependence in melanoma cell lines harboring V600E BRAF mutations. Given the promising preliminary data with BRAF and MEK selective inhibitors in early stage clinical trials, these data may aid in the identification of those patients most likely to benefit from selective inhibitors of the MAPK pathway. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B87.

Anticipating drug resistance in the MAP kinase pathway

Anticipating drug resistance in the MAP kinase pathway

Identification of direct transcriptional targets of V600EBRAF/MEK signalling in melanoma

Oncogenic mutations in BRAF are common in melanoma and drive constitutive activation of the MEK/ERK pathway. To elucidate the transcriptional events downstream of (V600E)BRAF/MEK signalling we performed gene expression profiling of A375 melanoma cells treated with potent and selective inhibitors of (V600E)BRAF and MEK (PLX4720 and PD184352 respectively). Using a stringent Bayesian approach, we identified 69 transcripts that appear to be direct transcriptional targets of this pathway and whose expression changed after 6 h of pathway inhibition. We also identified several additional genes whose expression changed after 24 h of pathway inhibition and which are likely to be indirect transcriptional targets of the pathway. Several of these were confirmed by demonstrating their expression to be similarly regulated when BRAF was depleted using RNA interference, and by using qRT-PCR in other BRAF mutated melanoma lines. Many of these genes are transcription factors and feedback inhibitors of the ERK pathway and are also regulated by MEK signalling in NRAS mutant cells. This study provides a basis for understanding the molecular processes that are regulated by (V600E)BRAF/MEK signalling in melanoma cells.

Discovery of highly potent and selective type I B-Raf kinase inhibitors

A series of pyrazolo[1,5-α]pyrimidine analogs has been prepared and found to be potent and selective B-Raf inhibitors. Molecular modeling suggests they bind to the active conformation of the enzyme.

6BA Early efficacy signal demonstrated in advanced melanoma in a phase I trial of the oncogenic BRAF-selective inhibitor PLX4032

6BA Early efficacy signal demonstrated in advanced melanoma in a phase I trial of the oncogenic BRAF-selective inhibitor PLX4032

Development of a Novel Chemical Class of BRAF Inhibitors Offers New Hope for Melanoma Treatment

Evaluation of: Niculescu-Duvaz D, Gaulon C, Dijkstra HP et al.: Pyridoimidazolones as novel potent inhibitors of v-Raf murine sarcoma viral oncogene homologue B1 (BRAF). J. Med. Chem. 52, 2255-2264 (2009). There are currently no therapies known to alter the clinical course of disseminated melanoma. Mutational profiling studies have shown that the majority of melanomas harbor activating mutations in the serine/threonine kinase BRAF at the V600E position. Preclinical work has validated mutated BRAF as a therapeutic target in melanoma, and a number of BRAF-selective small-molecule inhibitors are now undergoing clinical evaluation. The only BRAF inhibitor to be investigated extensively in clinical trials of melanoma at this time is sorafenib--a compound with very limited single-agent activity. As sorafenib has poor cellular activity against the BRAF V600E mutation, the conclusion that many researchers have arrived at is that sorafenib did not provide a test of the therapeutic value of BRAF inhibition. Thus, more highly selective BRAF inhibitors have been sought. The current paper describes the identification of a new class of BRAF inhibitors that contain pyridoimidazolone as the ATP hinge-binding domain and a rigid imidazolone group. Building upon this novel scaffold, the authors derived a series of compounds with low nanomolar potency against mutated BRAF in isolated kinase assays, and low micromolar potency in cellular assays. These new chemical leads represent a significant step forward in the search for new, potent BRAF-selective small-molecule inhibitors.

Selecting subjects for a therapeutic target in colorectal cancer (CRC): Using a clinical database to enrich for patients harboring the BRAFV600E mutation

11003 Introduction: The BRAFV600E mutation (BRAF) causes constitutive activation of the RAS-induced MAPK pathway and is found in 10% of colon cancers. B-RAF inhibitors are in early clinical development, but their development in CRC will be challenging unless subsets of patients (pts) with higher rates of BRAF can be defined. The mutation rate in rectal tumors, the concordance between primary and metastases, and the prognostic/predictive significance of BRAF are current gaps in knowledge. Methods: 481 primary tumors and 80 matched primary-metastasis (prim-met) pairs were analysed from a pre-defined cohort of pts with CRC based on age (≥ 70 vs < 70 years), gender, tumor site (right-R, left-L and rectum), stage (A to C vs D) and ≥ 2 years follow-up. BRAF was assessed by routine sequencing of exon 15 and by a mutant-specific PCR assay. KRAS (KRAS-codon 12 and 13) and MSI (Bethesda markers) status were also examined. Results: Overall prevalence of BRAF was 11%. BRAF (see table ) was independently associated with increasing age, female gender and R-sided cancer, but not with stage. Mutations were rare in rectal cancers. BRAF was associated with inferior overall survival in stage D pts (log-rank, p = 0.0003; HR 0.38, 95% CI, 0.10–0.51). Survival analysis will be further stratified by treatment received. No difference in outcome was seen in preliminary analysis of earlier stage cancers. Mutation frequencies in the prim-met pairs were 38% (30/80) and 1.3% (1/80) for KRAS and BRAF, respectively. Overall concordance was 88% (70/80) for KRAS and 100% (80/80) for BRAF status. Conclusions: The development of selective B-Raf inhibitors in CRC is potentially more attractive due to the ability to define patient subsets with a higher prevalence of BRAF mutations. Analysis of the primary tumor reliably predicts the status of metastatic disease in the same patient. The association between BRAFand poor outcome will need to be considered when interpreting the result of studies targeting this mutation. [Table: see text] [Table: see text]

The Crystal Structure of BRAF in Complex with an Organoruthenium Inhibitor Reveals a Mechanism for Inhibition of an Active Form of BRAF Kinase

Substitution mutations in the BRAF serine/threonine kinase are found in a variety of human cancers. Such mutations occur in approximately 70% of human malignant melanomas, and a single hyperactivating V600E mutation is found in the activation segment of the kinase domain and accounts for more than 90% of these mutations. Given this correlation, the molecular mechanism for BRAF regulation as well as oncogenic activation has attracted considerable interest, and activated forms of BRAF, such as BRAF(V600E), have become attractive targets for small molecule inhibition. Here we report on the identification and subsequent optimization of a potent BRAF inhibitor, CS292, based on an organometallic kinase inhibitor scaffold. A cocrystal structure of CS292 in complex with the BRAF kinase domain reveals that CS292 binds to the ATP binding pocket of the kinase and is an ATP competitive inhibitor. The structure of the kinase-inhibitor complex also demonstrates that CS292 binds to BRAF in an active conformation and suggests a mechanism for regulation of BRAF by phosphorylation and BRAF(V600E) oncogene-induced activation. The structure of CS292 bound to the active form of the BRAF kinase also provides a novel scaffold for the design of BRAF(V600E) oncogene selective BRAF inhibitors for therapeutic application.

Pharmacodynamics of 2-{4-[(1E)-1-(Hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridine-4-yl)-1H-pyrazol-1-yl}ethan-1-ol (GDC-0879), a Potent and Selective B-Raf Kinase Inhibitor: Understanding Relationships between Systemic Concentrations, Phosphorylated Mitogen-Activated Protein Kinase Kinase 1 Inhibition, and Efficacy

The Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase signaling pathway is involved in cellular responses relevant to tumorigenesis, including cell proliferation, invasion, survival, and angiogenesis. 2-[4-[(1E)-1-(Hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridine-4-yl)-1H-pyrazol-1-yl]ethan-1-ol (GDC-0879) is a novel, potent, and selective B-Raf inhibitor. The objective of this study was to characterize the relationship between GDC-0879 plasma concentrations and tumor growth inhibition in A375 melanoma and Colo205 colon cancer xenografts and to understand the pharmacodynamic (PD) marker response requirements [phosphorylated (p)MEK1 inhibition] associated with tumor growth inhibition in A375 xenografts. Estimates of GDC-0879 plasma concentrations required for tumor stasis obtained from fitting tumor data to indirect response models were comparable, at 4.48 and 3.27 microM for A375 and Colo205 xenografts, respectively. This was consistent with comparable in vitro potency of GDC-0879 in both cell lines. The relationship between GDC-0879 plasma concentrations and pMEK1 inhibition in the tumor was characterized in A375 xenografts after oral doses of 35, 50, and 100 mg/kg. Fitting pMEK1 inhibition to an indirect response model provided an IC(50) estimate of 3.06 microM. pMEK1 inhibition was further linked to A375 tumor volume data from nine different GDC-0879 dosing regimens using an integrated pharmacokinetic-PD model. A simulated PD marker response curve plot of K (rate constant describing tumor growth inhibition) versus pMEK1 inhibition generated using pharmacodynamic parameters estimated from this model, showed a steep pMEK1 inhibition-response curve consistent with an estimated Hill coefficient of approximately equal 8. A threshold of >40% pMEK1 inhibition is required for tumor growth inhibition, and a minimum of approximately 60% pMEK1 inhibition is required for stasis in A375 xenografts treated with GDC-0879.

Tumour cell survival signalling by the ERK1/2 pathway

Several advances in recent years have focused increasing attention on the role of the RAF-MEK-ERK1/2 pathway in promoting cell survival. The demonstration that BRAF is a human oncogene mutated at high frequency in melanoma, thyroid and colon cancer has provided a pathophysiological context, whilst the description of potent and highly selective inhibitors of BRAF or MEK has allowed a more informed and rational intervention in both normal and tumour cells. In addition, separate studies have uncovered new mechanisms by which the ERK1/2 pathway can control the activity or abundance of members of the BCL-2 protein family to promote cell survival. It is now apparent that various oncogenes co-opt ERK1/2 signalling to de-regulate these BCL-2 proteins and this contributes to, and even underpins, survival signalling in some tumours. New oncogene-targeted therapies allow direct or indirect inhibition of ERK1/2 signalling and can cause quite striking tumour cell death. In other cases, inhibition of the ERK1/2 pathway may be more effective in combination with other conventional and novel therapeutics. Here, we review recent advances in our understanding of how the ERK1/2 pathway regulates BCL-2 proteins to promote survival, how this is de-regulated in tumour cells and the opportunities this might afford with the use of new targeted therapies.

Knowledge-based design of 7-azaindoles as selective B-Raf inhibitors

The synthesis of a 7-azaindole series of novel, potent B-Raf kinase inhibitors using knowledge-based design was carried out. Compound 6h exhibits not only excellent potency in both the enzyme assay (IC 50 = 2.5 nM) and the cellular assay (IC 50 = 63 nM), but also has an outstanding selectivity profile against other kinases.

The identification of potent, selective and CNS penetrant furan-based inhibitors of B-Raf kinase

Modification of the potent imidazole-based B-Raf inhibitor SB-590885 resulted in the identification of a series of furan-based derivatives with enhanced CNS penetration. One such compound, SB-699393 ( 17), was examined in vivo to challenge the hypothesis that selective B-Raf inhibitors may be of value in the treatment of stroke.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity

BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.

Demonstration of a Genetic Therapeutic Index for Tumors Expressing Oncogenic BRAF by the Kinase Inhibitor SB-590885

Oncogenic BRAF alleles are both necessary and sufficient for cellular transformation, suggesting that chemical inhibition of the activated mutant protein kinase may reverse the tumor phenotype. Here, we report the characterization of SB-590885, a novel triarylimidazole that selectively inhibits Raf kinases with more potency towards B-Raf than c-Raf. Crystallographic analysis revealed that SB-590885 stabilizes the oncogenic B-Raf kinase domain in an active configuration, which is distinct from the previously reported mechanism of action of the multi-kinase inhibitor, BAY43-9006. Malignant cells expressing oncogenic B-Raf show selective inhibition of mitogen-activated protein kinase activation, proliferation, transformation, and tumorigenicity when exposed to SB-590885, whereas other cancer cell lines and normal cells display variable sensitivities or resistance to similar treatment. These studies support the validation of oncogenic B-Raf as a target for cancer therapy and provide the first evidence of a correlation between the expression of oncogenic BRAF alleles and a positive response to a selective B-Raf inhibitor.