Raf Kinase Family Research Articles

Comparative interactome analysis reveals distinct and overlapping properties of Raf family kinases

The three mammalian Raf proteins (A-Raf, B-Raf, and C-Raf) are key components of the MAPK pathway. Although diverse functions have been proposed for Raf kinases, it is still not clear how interacting proteins contribute to differences in the signaling functions of the three Raf kinases. Here, we report the comparative interactomes of the three Raf kinases under serum-starved and EGF-stimulated conditions. We identified nearly 400 novel interacting proteins; some interacted with all three isoforms while others interacted exclusively with one or two. Comparing the interactomes of the three Raf kinases under different conditions revealed Raf proteins perform distinct functions through specific interactions. Our interactome data help define the differences between the three Raf kinases and may uncover new functions or regulatory mechanisms. Knowledge of Raf kinase protein-protein interactions will help us to investigate the function of specific pathways in the future.

Small molecule inhibitor agerafenib effectively suppresses neuroblastoma tumor growth in mouse models via inhibiting ERK MAPK signaling.

Neuroblastoma (NB) is the most common extracranial solid tumor in early childhood. Despite intensive multimodal therapy, nearly half of children with high-risk disease will relapse with therapy-resistant tumors. Dysregulation of MAPK pathway has been implicated in the pathogenesis of relapsed and refractory NB patients, which underscores the possibility of targeting MAPK signaling cascade as a novel therapeutic strategy. In this study, we found that high expressions of RAF family kinases correlated with advanced tumor stage, high-risk disease, tumor progression, and poor overall survival. Targeted inhibition of RAF family kinases with the novel small molecule inhibitor agerafenib abrogated the activation of ERK MAPK pathway in NB cells. Agerafenib significantly inhibited the cell proliferation and colony formation ability of NB cells in vitro, and its combination with traditional chemotherapy showed a synergistic pro-apoptotic effect. More importantly, agerafenib exhibited a favorable toxicity profile, potently suppressed tumor growth, and prolonged survival in NB mouse models. In conclusion, our preclinical data suggest that agerafenib might be an effective therapeutic agent for NB treatment, both as a single-agent and in combination with chemotherapy.

The dimer-dependent catalytic activity of RAF family kinases is revealed through characterizing their oncogenic mutants

Although extensively studied for three decades, the molecular mechanisms that regulate the RAF/MEK/ERK kinase cascade remain ambiguous. Recent studies identified the dimerization of RAF as a key event in the activation of this cascade. Here, we show that in-frame deletions in the β3-αC loop activate ARAF as well as BRAF and other oncogenic kinases by enforcing homodimerization. By characterizing these RAF mutants, we find that ARAF has less allosteric and catalytic activity than the other two RAF isoforms, which arises from its non-canonical APE motif. Further, these RAF mutants exhibit a strong oncogenic potential, and a differential inhibitor resistance that correlates with their dimer affinity. Using these unique mutants, we demonstrate that active RAFs, including the BRAF(V600E) mutant, phosphorylate MEK in a dimer-dependent manner. This study characterizes a special category of oncogenic kinase mutations, and elucidates the molecular basis that underlies the differential ability of RAF isoforms to stimulate MEK-ERK pathway. Further, this study reveals a unique catalytic feature of RAF family kinases that can be exploited to control their activities for cancer therapies.

Revealing A-Raf functions through its interactome

A-Raf is a member of the Raf kinase family. Unlike B-Raf and C-Raf, the functions of A-Raf remain obscure. To gain more insight into the biological functions of A-Raf, we investigated the A-Raf interactome using proteomics. We found 132 proteins that interact with A-Raf and confirmed the interaction of 12 of these proteins with A-Raf by western blotting. Our data suggested that A-Raf regulates apoptosis, RNA catabolism, GTPase activity, and cell adhesion by interacting with proteins located in different cellular compartments. We identified all ten hallmarks of cancer in these interacting proteins, suggesting that A-Raf is involved in carcinogenesis. Our results also indicated that A-Raf may play a role in different diseases and signaling pathways. These findings have identified potential regulators of A-Raf and provide a systemic insight into its biological functions.

C-RAF function at the genome-wide transcriptome level: A systematic view

C-RAF was the first member of the RAF kinase family to be discovered. Since its discovery, C-RAF has been found to regulate many fundamental cell processes, such as cell proliferation, cell death, and metabolism. However, the majority of these functions are achieved through interactions with different proteins; the genes regulated by C-RAF in its active or inactive state remain unclear. In the work, we used RNA-seq analysis to study the global transcriptomes of C-RAF bearing or C-RAF knockout cells in quiescent or EGF activated states. We identified 3353 genes that are promoted or suppressed by C-RAF. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these genes are involved in drug addiction, cardiomyopathy, autoimmunity, and regulation of cell metabolism. Our results provide a panoramic view of C-RAF function, including known and novel functions, and have revealed potential targets for elucidating the role of C-RAF.

MEK drives BRAF activation through allosteric control of KSR proteins.

RAF family kinases have prominent roles in cancer. Their activation is dependent on dimerization of their kinase domains, which has emerged as a hindrance for drug development. In mammals, RAF family kinases include three catalytically competent enzymes (ARAF, BRAF and CRAF) and two pseudokinases (KSR1 and KSR2) that have been described as scaffolds owing to their apparent ability to bridge RAF isoforms and their substrate, mitogen-activated protein kinase kinase (MEK). Kinase suppressor of Ras (KSR) pseudokinases were also shown to dimerize with kinase-competent RAFs to stimulate catalysis allosterically. Although GTP-bound RAS can modulate the dimerization of RAF isoforms by engaging their RAS-binding domains, KSR1 and KSR2 lack an RAS-binding domain and therefore the regulatory principles underlying their dimerization with other RAF family members remain unknown. Here we show that the selective heterodimerization of BRAF with KSR1 is specified by direct contacts between the amino-terminal regulatory regions of each protein, comprising in part a novel domain called BRS in BRAF and the coiled-coil-sterile α motif (CC-SAM) domain in KSR1. We also discovered that MEK binding to the kinase domain of KSR1 asymmetrically drives BRAF-KSR1 heterodimerization, resulting in the concomitant stimulation of BRAF catalytic activity towards free MEK molecules. These findings demonstrate that KSR-MEK complexes allosterically activate BRAF through the action of N-terminal regulatory region and kinase domain contacts and challenge the accepted role of KSR as a scaffold for MEK recruitment to RAF.

Towards Piecing Together the RAS-RAF Puzzle: Dynamics of Membrane-Associated KRAS4b-RAF RBD/CRD Ternary Complex

The RAS family of small guanine nucleotide-binding proteins are critical for various cellular signaling pathways and are among the most common culprits in human cancer, with KRAS4b mutations occurring at high rates in pancreatic, lung, and colorectal cancer. Membrane-anchored RAS binds to effectors from the RAF kinase family at two domains: (i) the RAS-binding domain (RBD) whose complex with RAS has been solved experimentally, and (ii) the cysteine-rich domain (CRD) that mutational assays show can bind RAS independently. However, the structure of the RAS/RBD/CRD ternary complex still remains unknown, including its membrane orientation and interactions. We have used a combination of all-atom and coarse-grained molecular dynamics (MD) simulations to investigate these questions, focusing on the complex between KRAS4b and the RBD/CRD domains of BRAF that experiments show is the initially-activated RAF isoform. First, single-domain simulations of CRD or RBD alone indicate their associations with the membrane. Next, binary complex simulations based on the RAS/RBD crystal structure show multiple dominant membrane orientations, where RBD could either be away from the membrane or making direct membrane interactions. Finally, extensive simulations based on computational docking of CRD to the RAS/RBD crystal structure suggest a viable ternary complex model where CRD embeds into the membrane via both of its hydrophobic loops, RAS interacts with the membrane at the surface covering helices 4 and 5 of the G domain, and RBD away from the membrane. We are currently investigating if interactions of KRAS4b with the RBD/CRD domains of the CRAF isoform lead to changes in the ternary complex structure and to differential membrane interactions. These studies should provide insights that will contribute to our understanding of both normal and cancer-activated signaling pathways.

Ras-Mediated Activation of the Raf Family Kinases.

The extracellular signal-regulated kinase (ERK) cascade comprised of the Raf, MEK, and ERK protein kinases constitutes a key effector cascade used by the Ras GTPases to relay signals regulating cell growth, survival, proliferation, and differentiation. Of the ERK cascade components, the regulation of the Raf kinases is by far the most complex, involving changes in subcellular localization, protein and lipid interactions, as well as alterations in the Raf phosphorylation state. The Raf kinases interact directly with active, membrane-localized Ras, and this interaction is often the first step in the Raf activation process, which ultimately results in ERK activation and the downstream phosphorylation of cellular targets that will specify a particular biological response. Here, we will examine our current understanding of how Ras promotes Raf activation, focusing on the molecular mechanisms that contribute to the Raf activation/inactivation cycle.

Separase Inhibitor Sepin-1 Inhibits Foxm1 Expression and Breast Cancer Cell Growth.

Sepin-1, a potent non-competitive inhibitor of separase, inhibits cancer cell growth, but the mechanisms of Sepin-1-mediated growth inhibition are not fully understood. Here we report that Sepin-1 hinders growth of breast cancer cells, cell migration, and wound healing. Inhibition of cell growth induced by Sepin-1 in vitro doesn’t appear to be through apoptosis but rather due to growth inhibition. Following Sepin-1 treatment caspases 3 and 7 are not activated and Poly (ADP-ribose) polymerase (Parp) is not cleaved. The expression of Forkhead box protein M1 (FoxM1), a transcription factor, and its target genes in the cell cycle, including Plk1, Cdk1, Aurora A, and Lamin B1, are reduced in a Sepin-1-dependent manner. Expressions of Raf kinase family members A-Raf, B-Raf, and C-Raf also are inhibited following treatment with Sepin-1. Raf is an intermediator in the Raf-Mek-Erk signaling pathway that phosphorylates FoxM1. Activated FoxM1 can promote its own transcription via a positive feedback loop. Sepin-1-induced downregulation of Raf and FoxM1 may inhibit expression of cell cycle-driving genes, resulting in inhibition of cell growth.

Predictive Simulation Modeling in JAK2-Mutant MPNs Identified Ruxolitinib and Sorafenib As a Synergistic Drug Combination for Disease Modifying Therapy

Abstract Background: Despite the identification of a number of genes that underlie the genetic complexity of the myelofibrosis (MF) phenotype and even with the subsequent FDA approval of a targeted therapy for MF that is ruxolitinib, the median overall survival time of MF patients is still only 5-6 years. This unchanged survival may be explained, in part, via the complex mutation patterns that contribute to MF. Consequently, treating MF patients remains a significant challenge in the hematology clinic. Aim: To identify new drugs for MF patients, we used predictive simulation modeling of JAK2-V617F expressing MPN cells that could readily be re-purposed for MF. Methods: : To identify combinations of other drugs that can enhance the effect of Ruxolitinib, we (1) employed predictive simulation to model a JAK2-V617F bearing cell line (2) identified the key molecular disease characteristics of the JAK2-driven disease network, and (3) experimentally validated the predictions. The predictive simulation approach from Cellworks provides a representation of disease physiology incorporating signaling and metabolic networks with an integrated phenotype view. We modelled the JAK2-V617F expressing SET2 cell line based on its genomic aberration data. We then screened a library of drugs on the computational model in combination with Ruxolotinib, by assessing the impact on disease specific biomarkers and phenotypes of proliferation, viability, and apoptosis. Results: The modeling and digital drug screening identified a short list of drugs that were predicted to inhibit Jak2 signaling. Validation studies were conducted with the representative JAK2 driven SET2 cell line which harbors the JAK2-V617F mutation and was predicted to lead to subsequent activation of STAT3, STAT5, ERK via SHC1, AKT via IRS1-PIK3CA and STAT1 (Fig 1). JAK2 is known to phosphorylate STAT3 on Y705. Predictive modeling also discovered activation of parallel signaling via mutations of ALK, IRS2 and RPTOR. ALK is also known to activate STAT3 via Y705 phosphorylation and ERK via SHC1. IRS2 leads to activation of PIK3CA-AKT-MTOR. SET2 cells also harbor a RPTOR mutation which also activates MTOR pathway further. With higher ERK and MTOR signaling, there was S727 phosphorylation of STAT3. Using digital drug screening on the SET2 simulation model, sorafenib mitigated the effect of JAK2 mutation by inhibiting pathways downstream of JAK2, rather than decreasing the JAK2 mutation burden itself. Sorafenib is a small molecule kinase inhibitor that targets multiple proteins including VEGFR, PDGFR, and several Raf family kinases. It is approved for use in renal, liver, and thyroid cancers and is under clinical investigation for a number of other neoplasias. A potential role for sorafenib in MF has not been fully investigated. Sorafenib is known to inhibit ERK via inhibition of RAF. Sorafenib was also predicted to inhibit STAT3, STAT5, STAT1 and AKT1 via activation of PTPN6 in the SET2 model. With down-regulation of ERK and AKT1-MTOR pathway, Sorafenib was predicted to inhibit STAT3 S727 phosphorylation. Hence, with sorafenib and ruxolitinib, not only was JAK2 burden reduced, but also parallel pathway signaling via ALK, IRS2 and RPTOR. Using in vitro validation, we found that sorafenib was highly efficacious in reducing Jak2-V617F cell viability in a dose-dependent manner (GI50 = 6 uM) . Moreover, when Jak2-V617F cells were treated with both ruxolitinib and sorafenib, there was synergy, suggesting that the compounds were acting on multiple, cooperating signaling pathways. A prior report also showed synergistic MPN regression with ruxolitinib and sorafenib in BaF3/Jak2-V617F and HEL cell lines and primary specimens (PMID: 23560534), thereby providing independent evidence for the rationale use of these compounds in MF patients harboring mutation of PDGFRA/B, NRAS, KRAS in addition to JAK2 mutations. Conclusion: Thus, in addition to identifying a novel drug regimen for MF, we demonstrate that successful treatment of MF likely requires targeting multiple, non-redundant, constitutively active, and cooperative signaling pathways, rather than a one-biomarker/one-drug approach as is the current clinical standard. Further, we demonstrate how predictive simulation modeling can be used for precision medicine purposes in MF to inform clinical treatment decision-making of FDA approved drugs, with the intent of providing significant health and cost benefits. Download : Download high-res image (220KB) Download : Download full-size image Figure 1 . Disclosures Singh: Cellworks Research India Pvt. Ltd: Employment. Pampana: CellWorks: Employment. Usmani: Cellworks: Employment. Sauban: Cellworks: Employment. Kushwaha: CellWorks: Employment. Ramakrishnan: CellWorks: Employment. Abbasi: Cellworks Group Inc.: Employment. Vali: Cellworks Group Inc.: Employment. Cogle: Celgene: Other: Membership on Steering Committee for Connect MDS/AML Registry.

Tolerability of oral sorafenib in pet dogs with a diagnosis of cancer.

Sorafenib is a multi-target small molecule inhibitor of the RAF kinase family and VEGFR-2/PDGFR. The US Food and Drug Administration approved sorafenib in human patients with liver, thyroid, or renal carcinoma. The aim of this study was to help guide future pharmacokinetic (PK) studies of sorafenib in dogs with a cancer diagnosis. Client-owned dogs were eligible if they had a cytologic or histologic diagnosis of cancer. Patients were enrolled at escalating doses of sorafenib. Patients were evaluable for the study if they received at least one dose of sorafenib and presented 1 week later for a follow-up examination, blood work, and assessment of drug tolerability. The goal of this study was not to define a maximum tolerated dose as may be reasonable in conventional cytotoxic chemotherapy drugs, but rather to describe the tolerability of this drug in dogs with a cancer diagnosis, as a prequel to future sorafenib PK studies. No patients in the study had any evidence of adverse events that were attributable to sorafenib. Doses of 3 mg/kg were well tolerated and associated with a suggestion of clinical activity, supportive of future PK, and pharmacodynamic analysis. Such future studies are recommended at this dose to define the associated exposure achieved and determine a reasonable schedule for sorafenib administration.

Decoding the full picture of Raf1 function based on its interacting proteins.

Raf1 is a member of the Raf kinase family and regulates many fundamental cell processes, including proliferation, differentiation, apoptosis, motility, and metabolism. However, the functions of Raf1 have not been completely elucidated. To better understand Raf1 function, we investigated the proteins that interacted with Raf1. We identified 198 Raf1 interacting proteins and our data suggested that Raf1 may regulate cell processes through these interactions. These interaction partners were involved in all ten hallmarks of cancer, suggesting that Raf1 is involved in different aspects of carcinogenesis. In addition, we showed that Raf1 interacting proteins were enriched in six signaling pathways and many human diseases. The interaction partners identified in this study may represent oncological candidates for future investigations into Raf1 function. Our findings have provided an overview of Raf1 function from a systems biology perspective.

Abstract CT002: A Phase IB study of RAF dimer inhibitor BGB-283 in patients with B-RAF or K-RAS/N-RAS mutated solid tumors

Abstract Background: BGB-283 is a novel inhibitor of the RAF dimer with potent and reversible inhibitory activities against RAF family kinases including wild type A-RAF, B-RAF, C-RAF and B-RAF V600E, as well as EGFR. BGB-283 demonstrated acceptable safety and early clinical activity in its Phase 1A study. In this Phase IB dose expansion study, we further evaluated safety and tolerability of BGB-283 and investigated efficacy in pre-selected patients (pts) with B-RAF or K-RAS/N-RAS-mutated solid tumors. Methods: This Phase 1B study was a multicenter, open-label, multiple-arm, dose expansion study. Pts were treated at the RP2D of BGB-283 at 30 mg/day. There were 10 different expansion arms for: B-RAF V600-mutated melanoma (not previously treated with a B-RAF or MEK inhibitor, and B-RAF and/or MEK inhibitor-resistant pts), colorectal cancer (CRC), non-small cell lung cancer (NSCLC), papillary thyroid cancer (PTC) and other solid tumors with B-RAF mutations; K-RAS/N-RAS-mutated endometrial cancer, NSCLC, CRC, and other solid tumors with K-RAS/N-RAS or NF-1 mutations. The primary end point was the response rate based on RECIST Version 1.1. Adverse events (AEs) are reported per CTCAE V4.03. Results: As of 19th Sep 2016, 96 pts were enrolled: median age was 63 years (range: 20 to 82 years) with all pts having baseline ECOG PS of 0 or 1. Forty-seven (49.0%) subjects had received ≥3 prior lines of treatment, 22 (22.9%) had received 2 prior lines of treatment, and 18 (18.8%) had received 1 prior line of treatment. BGB-283 was generally well-tolerated. Drug-related AEs were mostly Grade 1/2 in severity; the most common were fatigue in 37 (38.5%) subjects, nausea in 16 (16.7%), decreased appetite in 21 (21.9%), and diarrhea in 17 (17.7%). A total of 34 (35.4%) subjects experienced study drug-related Grade 3/4 AEs, notably drug-related thrombocytopenia (6.3%), palmar-plantar erythrodysesthesia syndrome (1.0%) and hypertension (8.3%). In the cohorts with previously-untreated B-RAF V600-mutated melanoma (n=7), PTC with B-RAF mutation (n=3) and NSCLC with K-RAS mutation (n=6), the response rates were 42.9% (95% confidence interval [CI], 9.9, 81.6), 33.3% (95% CI, 0.8, 90.6) and 16.7% (95% [0.4, 64.1]), respectively. Confirmed PR was seen in the single case of ovarian cancer with B-RAF mutation enrolled and also in one of 4 melanoma subjects with a confirmed B-RAF V600 mutation, who had not responded to B-RAF and/or MEK inhibitor(s). One unconfirmed PR was found in two NSCLC subjects with a B-RAF mutation. Conclusions: BGB-283 was generally well-tolerated during the Phase 1B stage of the study. Antitumor activity was not only observed in subjects with B-RAF V600-mutated solid tumors including melanoma, PTC, and ovarian cancer, but also in subjects with K-RAS-mutated NSCLC. When added to the efficacy data in the Phase IA portion of this study, with objective responses noted in these tumor subtypes as well as in K-RAS-mutated endometrial carcinoma, BGB-283 demonstrated a desirable risk-benefit profile for further efficacy and safety investigation. Citation Format: Jayesh Desai, Hui Gan, Catherine Barrow, Michael B. Jameson, Ben Solomon, Victoria Atkinson, Andrew Haydon, Michael Millward, Stephen Begbie, Michael Brown, Benjamin Markman, William Patterson, Andrew Hill, Lisa Horvath, Adnan Nagrial, Gary Richardson, Christopher Jackson, Michael Friedlander, David Gibbs, Phillip Parente, Jason Yang, Lai Wang, Yunxin Chen, Lusong Luo. A Phase IB study of RAF dimer inhibitor BGB-283 in patients with B-RAF or K-RAS/N-RAS mutated solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT002. doi:10.1158/1538-7445.AM2017-CT002

Abstract 4260: 3D colorectal cancer organoids as preclinical models for assessment of activity of RAF inhibitor BGB-283

Abstract Tumor organoids are three-dimensional (3D) cultures of cancer cells. They can be derived from the tumor cell lines, patient-derived xenografts and tumor tissues from cancer patients, thereby providing an attractive in vitro assay to optimize anticancer treatment. Oncogenic B-RAF, which drives cell transformation and proliferation, has been detected in 5-15% of colorectal cancers (CRC). Despite the remarkable clinical activities achieved by vemurafenib in treating B-RAF V600E metastatic melanoma, their clinical efficacy in B-RAF V600E CRC is far less impressive. BGB-283, currently under clinical development for RAF and RAS mutated solid tumors, is a novel inhibitor of the RAF dimer with inhibitory activities against RAF family kinases. In this study, we evaluated the anti-proliferative effect of BGB-283 and compared it to BRAF inhibitor vemurafenib in CRC patient tumor derived organoids models. BGB-283 exhibited therapeutic potentials in tumors with mutations in the MAPK pathway. Herein, we reported the establishment of tumor organoid models from 19 CRC patients’ tumor tissues with a success rate of 83%. Organoids maintained many characteristics of their original tumors based on their histology. Our studies using primary CRC tumor organoids showed that in contrast to vemurafenib, which only inhibited B-RAF V600E CRC tumors, BGB-283 had inhibition effect on both B-RAF V600E and KRAS mutated tumors. These findings support BGB-283 as a promising clinical stage antitumor agent in treating CRC harboring B-RAF V600E and KRAS mutations. In addition, we showed that patient-derived organoids can be a valuable tool to test drug sensitivity in a personalized treatment and help to fill the gap between tumor genetics and patient trials. Citation Format: Xiaoran Wu, Chunyan Fu, Xi Yuan, Lianhai Zhang, Xiaohong Wang, Jiafu Ji, Lusong Luo. 3D colorectal cancer organoids as preclinical models for assessment of activity of RAF inhibitor BGB-283. [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 4260.

Abstract CT005: Phase I study of RAF dimer inhibitor BGB-283 in patients with B-RAF or K-RAS/N-RAS mutated solid tumors

Abstract Background: BGB-283 is a novel inhibitor of the RAF dimer with potent and reversible inhibitory activities against RAF family kinases including wild type ARAF, BRAF, CRAF and BRAF V600E, as well as EGFR. We report here the initial findings of the dose escalation component of an ongoing phase I first-in-human, dose escalation and expansion trial of BGB-283. Methods: Safety, tolerability, pharmacokinetics, recommended phase 2 dose and antitumor activity of BGB-283 were evaluated in patients (pts) with advanced BRAF, N- or KRAS mutant solid tumors. The phase Ia dose escalation study utilized a modified 3+3 dose escalation design. Adverse events (AEs) are reported per CTCAE V4.03 and responses per RECIST V1.1. Results: As of 31st Oct 2015, 31 pts (mutations: 18 KRAS; 3 NRAS; 7 BRAF V600E; 2 BRAF non-V600 and 1 NRAS/BRAF non-V600) enrolled in 7 cohorts received BGB-283 from 5 mg QD to 60 mg QD. BGB-283 was well tolerated. MTD was determined to be 40 mg/QD and dose-limiting toxicity (DLT) was thrombocytopenia. Most frequent treatment-related AEs were fatigue (68%), anorexia (48%), constipation (42%), thrombocytopenia (39%), nausea (39%), vomiting (39%), dermatitis acneiform (39%), hand-foot syndrome (35%), hypertension (35%) and dysphonia (32%). Frequent treatment-related Grade 3/4 AEs included thrombocytopenia (13%), fatigue (10%) and ALT increase (10%). Cutaneous malignancies associated with the approved first-generation BRAF inhibitors have not been observed in pts treated with BGB-283, with a mean follow-up of 6 months. The plasma concentrations of BGB-283 increased proportionally from 5 mg QD through 50 mg QD. The mean t½ of BGB-283 was around 110 hours. Among 29 evaluable pts, one of the only two melanoma pts with a BRAF V600E mutation had a partial response (PR) (on treatment over 249 days). In addition, there were two confirmed PRs including 1 pt with KRAS mutated endometrial cancer (on treatment over 456 days) and 1 thyroid cancer pt with BRAF V600E mutation (on treatment over 481 days); and one unconfirmed PR for 1 pt with KRAS mutated NSCLC (stable disease with transient PR and on treatment for over 572 days). Except for the K-RAS mutated endometrial cancer patient, all other three responders are still on study treatment at the cutoff date. Furthermore, 14 of the 29 evaluable pts achieved prolonged stable disease (3 pts have been on treatment for over 300 days), with the disease control rate at 62%. Finally, 18F-FDG uptake was measured at baseline and at the end of cycle 1 to evaluate BGB-283 pharmacodynamic activity. Partial metabolic responses were observed in 13/30 (43%) pts. Conclusions: BGB-283 was well tolerated and the MTD on a continuous once daily dosing was determined at 40 mg/day. Promising clinical activity was observed at multiple dose levels in pts with BRAF or KRAS mutated tumors. BGB-283 activity and safety are being further investigated in a number of molecularly-defined tumors in an ongoing phase Ib expansion study. Citation Format: Jayesh Desai, Hui Gan, Catherine Barrow, Michael B. Jameson, Grant Mcarthur, Ben Tran, Michael Lam, Laird Cameron, Andrew Weickhardt, Jason Yang, Lai Wang, Zhen Qin, Lusong Luo, Ben Solomon. Phase I study of RAF dimer inhibitor BGB-283 in patients with B-RAF or K-RAS/N-RAS mutated solid tumors. [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 CT005.

Stabilization of C-RAF:KSR1 complex by DiRas3 reduces availability of C-RAF for dimerization with B-RAF

RAF family kinases are central components of the Ras-RAF-MEK-ERK cascade. Dimerization is a key mechanism of RAF activation in response to physiological, pathological and pharmacological signals. It is mediated by a dimer interface region in the RAF kinase domain that is also conserved in KSR, a scaffolding protein that binds RAF, MEK and ERK. The regulation of RAF dimerization is incompletely understood. Especially little is known about the molecular mechanism involved in the selection of the dimerization partner. Previously, we reported that Ras-dependent binding of the tumour suppressor DiRas3 to C-RAF inhibits the C-RAF:B-RAF heterodimerization. Here we show that DiRas3 binds to KSR1 independently of its interaction with activated Ras and RAF. Our data also suggest that depending on the local stoichiometry between DiRas3 and oncogenic Ras, DiRas3 can either enhance homodimerization of KSR1 or recruit KSR1 to the Ras:C-RAF complex and thereby reduce the availability of C-RAF for binding to B-RAF. This mechanism, which is shared between A-RAF and C-RAF, may be involved in the regulation of Ras12V-induced cell transformation by DiRas3.

Autophosphorylation on S614 inhibits the activity and the transforming potential of BRAF

The BRAF proto-oncogene serine/threonine-protein kinase, known as BRAF, belongs to the RAF kinase family. It regulates the MAPK/ERK signalling pathway affecting several cellular processes such as growth, survival, differentiation, and cellular transformation. BRAF is mutated in ~8% of all human cancers with the V600E mutation constituting ~90% of mutations. Here, we have used quantitative mass spectrometry to map and compare phosphorylation site patterns between BRAF and BRAF V600E. We identified sites that are shared as well as several quantitative differences in phosphorylation abundance. The highest difference is phosphorylation of S614 in the activation loop which is ~5fold enhanced in BRAF V600E. Mutation of S614 increases the kinase activity of both BRAF and BRAF V600E and the transforming ability of BRAF V600E. The phosphorylation of S614 is mitogen inducible and the result of autophosphorylation. These data suggest that phosphorylation at this site is inhibitory, and part of the physiological shut-down mechanism of BRAF signalling.

Abstract A91: BGB-283 exhibits potentials in enhancing tumor immune sensitization and lymphocyte function

Abstract Oncogenic B-RAF, which drives cell transformation and proliferation, has been detected in a variety of human malignancies. Prior studies suggested that B-RAF-targeting therapies could increase antigen expression, decrease immune suppressive factors in tumor microenvironment, and improve homing of T effector cells to the tumors. Thus, selective BRAF inhibitors were suggested to possess immunosensitization properties and could be used in combination with immunotherapies to achieve more durable disease control/response in treating BRAF V600E melanomas. BGB-283 is a novel inhibitor of the RAF dimer with potent and reversible inhibitory activities against RAF family kinases including wild type A-RAF, B-RAF, C-RAF and B-RAF V600E. In addition, BGB-283 has potent inhibitory activity against EGFR (Mol. Can. Ther. onlinefirst 2015; doi: 10.1158/1535-7163). BGB-283 is currently under Phase 1 clinical investigations. In the present study, BGB-283 showed significant stimulation of melanocyte-differentiation antigens and HLA-A expression in BRAF V600E melanoma cell lines. Intermediate level of BGB-283 was found to stimulate proliferation of human peripheral blood mononuclear cell (PBMC) and isolated human CD4+ and CD8+ T cells. In addition, BGB-283 potentiated IFNγ releasing from PBMCs at concentration range of 30∼300 nM. In newly developed the 3D tumor spheroid/PBMC co-culture system, BGB-283 improved PBMC function as measured by cell proliferation and IFNγ production. The presence of PBMC also enhanced the anti-proliferation activity of BGB-283 on A375 BRAF V600E mutant melanoma cells in the co-culture system. Further studies have shed light on the mechanistic underpinning of the immunomodulation effect of BGB-283 and its potential differentiation from vemurafenib. These findings support BGB-283 as a promising clinical stage antitumor drug that could potentially combine with immunotherapies by enhancing immune sensitization and T cell function. Citation Format: Xi Yuan, Mi Lian, Xiaoran Wu, Lusong Luo. BGB-283 exhibits potentials in enhancing tumor immune sensitization and lymphocyte function. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A91.

Abstract SY14-02: Regulation of kinase signaling complexes with small molecule inhibitors

Abstract Kinases catalyse the phosphorylation of target substrates on hydroxyl-containing residues as a means to nucleate multi-component complexes or to stabilize unique conformational states. Pseudokinases constitute a subclass of these enzymes that were originally predicted as inactive based on mutations of key conserved active site residues. Like the active kinases, pseudokinases appear to play important functional roles in signal transduction pathways and several pseudokinases have been shown to be important mediators of diseases, including cancer and diabetes. Many pseudokinases share homology inside and outside of the kinase domain with catalytically active kinases. Examples include KSR1 and KSR2, the pseudokinase relatives within the RAF-family of kinases. And Her3, the pseudokinase member of the EGFR family. In both examples, the pseudokinase has been demonstrated to allosterically stimulate the activity of their active ancestral partners through the formation of higher-order complexes. The ability to allosterically modulate active signaling kinases appears to be a dedicated feature of several pseudokinases that overlaps or appears redundant with the ability of active kinases to modulate themselves. For example, KSR forms a high affinity dimer with RAF related kinases and this drives RAF catalytic activation. However, RAF may dimerize with itself to drive its own activation. Clinical inhibitors that bind to RAF are thought to exploit the ability of RAF to regulate itself, leading to activation, rather than inhibition, of RAF when dimeric. Similarly, Her3 dimerizes with EGFR through the formation of an asymmetric dimer, but EGFR appears able to form this dimer in the absence of Her3 as well. In this case, the clinical kinase inhibitor bosutinib, has been shown to function as an agonist through direct modulation of Her3-EGFR complexes. Because pseudokinase function can be redundant with the scaffolding abilities of active kinases, it has been difficult to dissect their precise roles in biology using conventional genetic or biochemical techniques. Small molecules that could antagonize pseudokinase dependent activities would be valuable tools that could be used to functionally annotate the biology and therefore pharmacology of this class of proteins. In particular, direct small molecule antagonists would allow one to disable pseudokinase structure and function on very short timescales and could also provide new leads for therapeutic development. I will describe my laboratories work on developing small molecule modulators of KSR. In particular, I will describe biochemical and cell based approaches used to identify KSR antagonists and furthermore X-ray crystallographic analysis of a lead compound bound to a KSR associated complex. Citation Format: Arvin Dar. Regulation of kinase signaling complexes with small molecule inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY14-02. doi:10.1158/1538-7445.AM2015-SY14-02

Regulation of RAF protein kinases in ERK signalling

RAF family kinases were among the first oncoproteins to be described more than 30 years ago. They primarily act as signalling relays downstream of RAS, and their close ties to cancer have fuelled a large number of studies. However, we still lack a systems-level understanding of their regulation and mode of action. The recent discovery that the catalytic activity of RAF depends on an allosteric mechanism driven by kinase domain dimerization is providing a vital new piece of information towards a comprehensive model of RAF function. The fact that current RAF inhibitors unexpectedly induce ERK signalling by stimulating RAF dimerization also calls for a deeper structural characterization of this family of kinases.