Harboring BRAF V600E Mutation Research Articles

Abstract 4326: Responses of a direct in vivo xenograft model of melanoma to MKK1/2 inhibitor

Abstract Metastatic melanoma is the most aggressive form of skin cancer, accounting for 80% of skin cancer deaths due to its high resistance to conventional chemotherapy. Recent studies have shown that metastatic melanomas are susceptible to targeted therapeutics using inhibitors of BRAF or MKK1/2 protein kinases. Here, we study the effect of the MKK1/2 inhibitor, AZD6244, on a direct in vivo xenograft model of melanoma, as well as the effect of MKK1/2 inhibitors, AZD6244 and CI1040 and the BRAF-V600E inhibitor, PLX4720, on established cell lines and cells from direct in vivo xenografts grown in 2D culture. Treatment with CI1040 elicited strong cell death responses in six of nine established melanoma cell lines, reaching 65-80% apoptosis after 48 h. Xenografts of established melanoma cell lines were also responsive to MKK1/2 inhibitor, showing suppressed tumor growth in animals treated with CI1040 in the presence or absence of temozolomide. Likewise, MKK1/2 inhibitor suppressed tumor growth in a direct in vivo xenograft model of melanoma, where tumors harboring BRAF-V600E mutations showed complete growth inhibition in response to AZD6244. One tumor harboring BRAF-WT showed partial growth suppression with AZD6244 alone, but complete suppression was achieved when AZD6244 was combined with temozolomide. Unexpectedly, cells derived from direct in vivo xenografts and grown in 2D culture showed relatively low apoptotic responses when treated with MKK1/2 or BRAF inhibitors. The results suggest that tumor and cell growth conditions might influence mechanisms underlying the oncogene addiction response to constitutive activation of the BRAF/MKK/ERK pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4326. doi:10.1158/1538-7445.AM2011-4326

PLX4032: does it keep its promise for metastatic melanoma treatment?

Importance of the field: Activating mutations in the BRAF kinase gene have been identified in 50% of all melanomas. PLX4032, a selective and potent inhibitor of BRAF V600E mutant tumor cells, has shown inhibition of tumor growth in cell lines harboring BRAF V600E mutations. Data from early clinical trials showed promising results in the treatment of patients with metastatic melanoma.Areas covered in this review: An extensive literature search was conducted that included published articles and abstracts on PLX4032 to evaluate the existing data in both preclinical and Phase I–II studies.What the reader will gain: The review comprises the rationale for choosing a selective BRAF inhibitor for certain types of melanoma, its mode of action, associated toxicities and potential pitfalls.Take home message: Despite the convincing response rates in Phase I trials, duration of tumor response is limited in some patients, and a cure cannot be expected. Intrinsic and acquired PLX4032 resistance still has to be investigated; signaling pathway switching is probably the most important factor for development of resistance. Combination therapy with simultaneous inhibition of different pathways might be more effective and warrants further investigation. The toxicity profile of PLX4032 is considerably low, and special attention is needed to address the development of keratoacanthomas and cutaneous squamous cell carcinomas.

BRAFV600E mutation influences hypoxia-inducible factor-1α expression levels in papillary thyroid cancer

Hypoxia-inducible factor-1α is found frequently overexpressed in solid tumors cells, exerting an important role in angiogenesis, glucose metabolism, cell proliferation, survival and invasion. In thyroid carcinomas, hypoxia-inducible factor-1α expression was found increased in differentiated, poorly differentiated, medullary and anaplastic variants. Hypoxia represents the principal stimulus responsible for hypoxia-inducible factor-1α induction. Other nonhypoxic stimuli increase hypoxia-inducible factor-1α synthesis through the activation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways in a cell-type-specific manner. We have previously shown the role of BRAFV600E mutation in papillary thyroid cancer cells as a factor that facilitates tumor cell growth and progression. In this study, we tested the hypothesis that BRAFV600E mutation influences hypoxia-inducible factor-1α expression in papillary thyroid carcinoma cells. We analyzed 27 papillary thyroid carcinomas, 13 of which presented BRAFV600E mutation. In tumor tissues, immunoreactivity for hypoxia-inducible factor-1α was detected in the majority of analyzed BRAFV600E mutated cases. Transcriptional analyses revealed elevated hypoxia-inducible factor-1α levels with significant differences between wild-type and mutated group. A BRAF wild-type papillary thyroid carcinoma cell line and a BRAFV600E mutated papillary thyroid carcinoma cell line were selected to study the effects of BRAF mutation on hypoxia-inducible factor-1α expression in vitro. Knockdown of mutant BRAFV600E or both the wild type and the BRAFV600E by RNA interference induced a significant reduction of hypoxia-inducible factor-1α expression at mRNA and protein levels. Pharmacological inhibition of BRAF significantly reduces hypoxia-inducible factor-1α expression levels in papillary thyroid carcinoma cell line harboring BRAFV600E mutation. Our results suggest that hypoxia-inducible factor-1α is expressed in papillary thyroid carcinomas and is regulated not only by hypoxia but also by BRAFV600E-mediated signaling pathway.

Preclinical In vivo Evaluation of Efficacy, Pharmacokinetics, and Pharmacodynamics of a Novel MEK1/2 Kinase Inhibitor RO5068760 in Multiple Tumor Models

Targeting the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway represents a promising anticancer strategy. Recently, we have reported a novel class of potent and selective non-ATP-competitive MEK1/2 inhibitors with a unique structure and mechanism of action. RO5068760 is a representative of this class showing significant efficacy in a broad spectrum of tumors with aberrant mitogen-activated protein kinase pathway activation. To understand the relationship between systemic exposures and target (MEK1/2) inhibition as well as tumor growth inhibition, the current study presents a detailed in vivo characterization of efficacy, pharmacokinetics, and pharmacodynamics of RO5068760 in multiple xenograft tumor models. For inhibition of MEK1/2 as measured by the phosphorylated ERK levels, the estimated EC(50)s in plasma were 1.36 micromol/L (880 ng/mL) and 3.35 micromol/L (2168 ng/mL) in LOX melanoma and HT-29 colorectal cancer models, respectively. A similar EC(50) (1.41 micromol/L or 915 ng/mL) was observed in monkey peripheral blood lymphocytes. To achieve tumor growth inhibition (>or=90%), an average plasma drug concentration of 0.65 or 5.23 micromol/L was required in B-RafV600E or K-Ras mutant tumor models, respectively, which were remarkably similar to the IC(90) values (0.64 or 4.1 micromol/L) determined in vitro for cellular growth inhibition. With equivalent in vivo systemic exposures, RO5068760 showed superior efficacy in tumors harboring B-RafV600E mutation. The plasma concentration time profiles indicate that constant p-ERK suppression (>50%) may not be required for optimal efficacy, especially in highly responsive tumors. This study may facilitate future clinical trial design in using biochemical markers for early proof of mechanism and in selecting the right patients and optimal dose regimen.

A phase I study of XL281, a selective oral RAF kinase inhibitor, in patients (Pts) with advanced solid tumors

3513 Background: XL281 is a potent and selective inhibitor of wild type and mutant RAF kinases showing anti-tumor activity in multiple xenograft models. Mutations in KRAS or BRAF can activate the RAF/MEK/ERK pathway in human tumors and may promote sensitivity to RAF kinase inhibitors. Methods: Pts were enrolled in successive cohorts of XL281 orally once daily on a 28-day cycle. Tumor response was assessed per RECIST every 8 wks. Plasma pharmacokinetic and pharmacodynamic samples were collected. The maximum tolerated dose (MTD) was expanded to 10 pts each with colorectal (CRC), melanoma, papillary thyroid (PTC) and NSCLC. Pre- and post-dose tumor and surrogate tissues were obtained. Biomarker and genotype analyses of pathway genes were performed. Results: The dose escalation phase is complete; 30 pts were treated with XL281. DLTs of fatigue, nausea, vomiting, and diarrhea were observed at the MAD (225 mg). The MTD is 150 mg. The most common related AEs included Grade 1/2, fatigue (48%), diarrhea (35%), nausea (35%), vomiting (35%) and anorexia (30%). Three pts had related AEs ≥G3: hypokalemia, nausea, and vomiting. One pt with an ocular melanoma demonstrated a cPR of 4 mos duration. Twelve pts had SD (3 -17+ mos), including 2 with I131-refractory PTC harboring BRAF V600E mutations (15+ and 17+ mos). Nine of these pts had decreases in target lesions (5–29%), including a pt with KRAS mutant CRC on study for 20 wks with marked symptomatic improvement. At the MTD, paired biopsies from 4 pts (3 melanoma, 1 NSCLC) show an average 72 % decrease in pMEK, 68 % decrease in pERK, 24 % decrease in Ki67 (proliferation) and 64 % increase in TUNEL (apoptosis). Three of 6 evaluable pts in the MTD cohort show SD at first assessment, including 1 melanoma pt with a NRAS Q61R mutation who showed a 20% decrease in target lesions. Conclusions: XL281 was generally well tolerated and the MTD was established at 150 mg. One cPR occurred in an ocular melanoma subject, and clinical benefit (PR or SD) occurred in 43% (13/30) of pts in the dose-escalation phase. XL281 demonstrates biological activity by modulation of the RAF pathway in tumor and surrogate tissue, with decreases in cell proliferation and increases in apoptosis. [Table: see text]

Genetic Subgrouping of Melanoma Reveals New Opportunities for Targeted Therapy

The discovery of activating oncogenic BRAF V600E mutations in the majority of melanomas has not yet been translated into more effective therapy. The failure of agents may be due to lack of sufficiently targeted therapeutics, but is more likely based on the activation of multiple oncogenic pathways in melanomas in addition to the mitogen-activated protein kinase signaling pathway. In contrast, there are groups of melanomas that instead rely on either c-KIT or CRAF signaling that may be amenable to single-agent targeted therapy. In the current review, we discuss how knowledge about these new melanoma subgroups may lead to improved strategies for treating melanomas harboring BRAF V600E mutations.

BRAFV600E mutations in malignant melanoma are associated with increased expressions of BAALC

BachgroundActivating BRAF mutations are present in approximately 50% of melanomas. Although different downstream target genes of the most common mutant V600E have been identified, the contribution of activating BRAF mutations to malignant transformation needs further clarification.MethodsMicroarray gene analysis was performed for human melanoma cell lines harboring BRAFV600E mutations in comparison to cell lines without this mutation.ResultsThis analysis revealed a more than two fold down-regulation of 43 and an increase of 39 gene products. BAALC (Brain and acute Leukaemia, cytoplasmatic) was most prominently regulated, since it was up-regulated in mutated cell lines by a mean of 11.45. Real time PCR analyses with RNA from melanoma cell lines (n = 30) confirmed the BRAF-activation dependent up-regulation of BAALC.ConclusionBAALC, which has been associated with cell dedifferentiation and migration, may function as a downstream effector of activating BRAF mutations during melanomagenesis.

Mutant V600E BRAF Increases Hypoxia Inducible Factor-1α Expression in Melanoma

Mutations in the BRAF serine/threonine kinase gene are frequently found in cutaneous melanomas. Activation of hypoxia inducible factor-1alpha (HIF-1alpha) in response to both hypoxic stress and oncogenic signals has important implications in cancer development and progression. Here, we report that mutant BRAF(V600E) increases HIF-1alpha expression in melanoma cells. Our microarray profiling data in 35 melanoma and melanocyte cell lines showed that HIF-1alpha gene expression was significantly increased in melanomas harboring BRAF(V600E) mutation. Stable suppression of mutant BRAF(V600E) or both wild-type and mutant BRAF(V600E) by RNA interference in melanoma cells resulted in significantly decreased HIF-1alpha expression. Knockdown of mutant BRAF(V600E) induced significant reduction of cell survival and proliferation under hypoxic conditions, whereas knockdown of both wild-type and mutant BRAF(V600E) resulted in further reduction. The effects of BRAF knockdown can be rescued by reintroducing BRAF(V600E) into tumor cells. Transfection of BRAF(V600E) into melanoma cells with wild-type BRAF induced significantly more hypoxic tolerance. Knockdown of HIF-1alpha in melanoma cells resulted in decreased cell survival under hypoxic conditions. Pharmacologic inhibition of BRAF by BAY 43-9006 also resulted in decreased HIF-1alpha expression. Although HIF-1alpha translational rate was not changed, the protein was less stable in BRAF knockdown cells. In additional, von Hippel-Lindau protein expression was significantly increased in BRAF knockdown cells. Our data show for the first time that BRAF(V600E) mutation increases HIF-1alpha expression and melanoma cell survival under hypoxic conditions and suggest that effects of the oncogenic V600E BRAF mutation may be partially mediated through the HIF-1alpha pathway.