An in silico study of the molecular basis of B-RAF activation and conformational stability

<p>Abstract</p> <p>Background</p> <p>B-RAF kinase plays an important role both in tumour induction and maintenance in several cancers and it is an attractive new drug target. However, the structural basis of the B-RAF activation is still not well understood.</p> <p>Results</p> <p>In this study we suggest a novel molecular basis of B-RAF activation based on molecular dynamics (MD) simulations of B-RAF^WTand the B-RAF^V600E, B-RAF^K601Eand B-RAF^D594Vmutants. A strong hydrogen bond network was identified in B-RAF^WTin which the interactions between Lys601 and the well known catalytic residues Lys483, Glu501 and Asp594 play an important role. It was found that several mutations, which directly or indirectly destabilized the interactions between these residues within this network, contributed to the changes in B-RAF activity.</p> <p>Conclusion</p> <p>Our results showed that the above mechanisms lead to the disruption of the electrostatic interactions between the A-loop and the αC-helix in the activating mutants, which presumably contribute to the flipping of the activation segment to an active form. Conversely, in the B-RAF^D594Vmutant that has impaired kinase activity, and in B-RAF^WTthese interactions were strong and stabilized the kinase inactive form.</p

Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles

B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase

Adaptive upregulation of FOXD3 and resistance to PLX4032/4720-induced cell death in mutant B-RAF melanoma cells.

Melanoma cells driven by mutant v-raf murine sarcoma viral oncogene homolog B1 (B-RAF) are highly resistant to chemotherapeutic treatments. Recent phase 1 results with PLX4032/RG7204/vemurafenib, which selectively inhibits B-RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)1/2 signaling in mutant B-RAF cells, has given encouragement to this struggling field. Nearly all patients in the phase 1-3 studies saw at least some response and the overall response rates ranged from 48 and 81%. However, despite initial tumor shrinkage, most responders in the trial experienced tumor relapse over time. These findings indicate that both intrinsic and acquired resistance may affect the clinical efficacy of PLX4032. It is critical to optimize PLX4032 activity to improve response rates and understand why some patients with the B-RAF mutation do not respond. We have previously shown that the stemness factor, Forkhead box D3 (FOXD3), is upregulated following inhibition of B-RAF-MEK signaling in mutant B-RAF melanoma cells. Here, we show that upregulation of FOXD3 following treatment with PLX4032 and PLX4720 (the non-clinical tool compound for PLX4032) confers resistance to cell death. Small interfering RNA-mediated knockdown of FOXD3 significantly enhanced the cell death response after PLX4032/4720 treatment in mutant B-RAF melanoma cell lines. Additionally, upregulation of FOXD3 after PLX4720 treatment was attenuated in non-adherent conditions and correlated with enhanced cell death. Ectopic expression of FOXD3 in non-adherent cells significantly reduced cell death in response to PLX4720 treatment. Together, these data indicate that upregulation of FOXD3 is an adaptive response to RAF inhibitors that promotes a state of drug resistance

What links BRAF to the heart function? New insights from the cardiotoxicity of BRAF inhibitors in cancer treatment

The RAS-related signalling cascade has a fundamental role in cell. It activates differentiation and survival. It is particularly important one of its molecules, B-RAF. B-RAF has been a central point for research, especially in melanoma. Indeed, it lacked effective therapeutic weapons since the early years of its study. Molecules targeting B-RAF have been developed. Nowadays, two classes of molecules are approved by FDA. Multi-target molecules, such as Sorafenib and Regorafenib, and selective molecules, such as Vemurafenib and Dabrafenib. Many other molecules are still under investigation. Most of them are studied in phase 1 trials. Clinical studies correlate B-RAF inhibitors and QT prolongation. Though this cardiovascular side effect is not common using these drugs, it must be noticed early and recognize its signals. Indeed, Oncologists and Cardiologists should work in cooperation to prevent lethal events, such as fatal arrhythmias or sudden cardiac death. These events could originate from an uncontrolled QT prolongation

Activity of the Heat Shock Protein 90 Inhibitor Ganetespib in Melanoma

Heat shock protein 90 (HSP90) is involved in the regulation of diverse biological processes such as cell signaling, proliferation and survival, and has been recently recognized as a potential target for cancer therapy. Ganetespib is a potent ATP competitive inhibitor of HSP90. Ganetespib downregulated the expression of multiple signal transducing molecules including EGFR, IGF-1R, c-Met, Akt, B-RAF and C-RAF, resulting in pronounced decrease in phosphorylation of Akt and Erk1/2 in a panel of five cutaneous melanoma cell lines including those harboring B-RAF and N-RAS mutations. Ganetespib exhibited potent antiproliferative activity on all five of these cell lines, with IC50 values between 37.5 and 84 nM. Importantly, Ganetespib is active on B-RAF mutated melanoma cells that have acquired resistance to B-RAF inhibition. Ganetespib induced apoptosis and cell cycle arrest at G1 and/or G2/M phase. Ganetespib induced cell cycle arrest was accompanied by altered expression of cyclin-dependent kinase inhibitor (CDKI) p21Cip1 and p27Kip1, cyclins B1, D1 and E, and/or cyclin-dependent kinases 1, 2 and 4. HSP90 is functionally important for melanoma cells and HSP90 inhibitors such as ganetespib could potentially be effective therapeutics for melanoma with various genetic mutations and acquired resistance to B-RAF inhibition

The Effect of Gefitinib on B-RAF Mutant Non-small Cell Lung Cancer and Transfectants

Abstract:We previously reported one patient with squamous cell carcinoma of the lung that showed the long-term effect to gefitinib with complete response. In the present report, we examine the epidermal growth factor receptor (EGFR) and K-RAS, HER2, and B-RAF mutations in this patient to find a B-RAF exon11 mutation, resulting in a substitution of valine by phenylalanine at codon 470 (V470F) as a novel type of B-RAF mutation in human cancers. In addition, the fluorescence in situ hybridization analysis for EGFR showed the high polysomy status. B-RAF is a nonreceptor serine/threonine kinase whose kinase domain has a structure similar to other protein kinases, including EGFR members. Of interest, the B-RAF V470F mutation corresponds to a position similar to the EGFR G719X mutation located on the phosphate binding (P)-loop of EGFR that clamps ATP into the catalytic cleft. This observation suggests that gefitinib may have an anti-cancer effect on B-RAF mutant tumors. Indeed, previous reports demonstrated that H1666 cells harboring B-RAF G465V mutations showed sensitivity to gefitinib, inhibiting phosphorylation of ERK1/2. We examined the effect of gefitinib on transient transfectants of the B-RAF mutant, but no drastic inhibition of ERK1/2 phosphorylation that was one of the downstream molecules of B-RAF was induced by gefitinib.In summary, we found a novel B-RAF V470F mutation in lung squamous cell carcinoma that showed response to gefitinib. However, our in vitro investigation did not explain the response observed in this particular patient. Further investigation is necessary to elucidate the mechanism of tumor sensitivity to EGFR tyrosine kinase inhibitors

Significato prognostico della mutazione di B-RAF nel carcinoma differenziato della tiroide

Recentemente è stata individuata una mutazione somatica puntiforme del gene di B-RAF(V600E);questa si ritrova esclusivamente nei PTC e nei cancri anaplastici derivati da PTC.La prevalenza della mutazione varia dal 23 al 62%.Scopo del presente lavoro è individuare il significato prognostico della mutazione di B-RAF attraverso lo studio retrospettivo dei dati clinici dei pazienti afferiti al Dipartimento di Endocrinologia di Pisa dal 1987

Mutation analysis of B-RAF gene in human gliomas

The RAS/RAF/MEK/ERK kinase pathway is pivotal in the transduction of mitogenic stimuli from activated growth factor receptors, which regulates cell proliferation, survival, and differentiation. Up-regulation of this pathway due to RAS mutations is found in approximately 30% of human tumors. Recently, activating mutations of B-RAF were identified in a large proportion of human cancers. Gliomas are the most frequent primary central nervous system tumors and the molecular mechanisms that underlie the development and progression of these tumors are far from being completely understood. The purpose of this study was to clarify the incidence of B-RAF mutations and their possible relation with tumor progression in a series of 82 human gliomas, including 49 astrocytic and 33 oligodendroglial tumors. The analysis of B-RAF hotspot regions, exons 11 and 15, showed presence of B-RAF mutations in only 2 out of 34 (6%) glioblastomas, and absence in the remaining histological types. Both mutations were located in the hotspot residue 600 (V600E) at exon 15, which leads to constitutive B-RAF kinase activity. These data suggest that activating mutations of B-RAF are not a frequent event in gliomas; nevertheless, when present they are associated with highgrade malignant lesions

Novel mechanisms of resistance to vemurafenib in melanoma – V600E B-Raf reversion and switching VEGF-A splice isoform expression

Targeting activating mutations in the proto-oncogene B-Raf, in melanoma, has led to increases in progression free survival. Treatment with vemurafenib, which inhibits the most common activating-mutated form of B-Raf (B-RafV600E), eventually results in resistance to therapy. VEGF-A is the principal driver of angiogenesis in primary and metastatic lesions. The bioactivity of VEGF-A is dependent upon alternative RNA splicing and pro-angiogenic isoforms of VEGF-A are upregulated in many disease states dependent upon angiogenesis, including cancers. Using techniques including RT-PCR, Western blotting, ELISA and luciferase reporter assays, the effect of vemurafenib on proliferation, ERK1/2 phosphorylation and the levels of pro- and anti-angiogenic VEGF-A isoforms was investigated in melanoma cell types expressing either wild-type B-Raf or B-RafV600E, including a primary melanoma culture derived from a highly vascularised and active nodule taken from a patient with a V600E mutant melanoma. The primary melanoma culture was characterised and found to have reverted to wild-type B-Raf. In B-RafV600E A375 cells ERK1/2 phosphorylation, pro-angiogenic VEGF-A mRNA, total VEGF-A protein expression and VEGF-A 3’UTR activity were all decreased in a concentration-dependent manner by vemurafenib. Conversely vemurafenib treatment of wild-type B-Raf cells significantly increased ERK1/2 phosphorylation, pro-angiogenic VEGF-A mRNA and total VEGF-A expression in a concentration-dependent manner. A switch to pro-angiogenic VEGF-A isoforms, with a concomitant upregulation of expression by increasing VEGF-A mRNA stability, may be an additional oncogenic and pathological mechanism in B-RafV600E melanomas, which promotes tumor-associated angiogenesis and melanoma-genesis. We have also identified the genetic reversal of B-RafV600E to wild-type in an active melanoma nodule taken from a V600E-positive patient and continued vemurafenib treatment for this patient is likely to have had a detrimental effect by promoting B-RafWT activity