Small Molecule BRAF Research Articles

Structure-Based De Novo Design for the Discovery of Miniprotein Inhibitors Targeting Oncogenic Mutant BRAF.

Being a component of the Ras/Raf/MEK/ERK signaling pathway crucial for cellular responses, the VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has emerged as a promising target for anticancer drug discovery due to oncogenic mutations that lead to pathway hyperactivation. Despite the discovery of several small-molecule BRAF kinase inhibitors targeting oncogenic mutants, their clinical utility has been limited by challenges such as off-target effects and suboptimal pharmacological properties. This study focuses on identifying miniprotein inhibitors for the oncogenic V600E mutant BRAF, leveraging their potential as versatile drug candidates. Using a structure-based de novo design approach based on binding affinity to V600E mutant BRAF and hydration energy, 39 candidate miniprotein inhibitors comprising three helices and 69 amino acids were generated from the substructure of the endogenous ligand protein (14-3-3). Through in vitro binding and kinase inhibition assays, two miniproteins (63 and 76) were discovered as novel inhibitors of V600E mutant BRAF with low-micromolar activity, with miniprotein 76 demonstrating a specific impediment to MEK1 phosphorylation in mammalian cells. These findings highlight miniprotein 76 as a potential lead compound for developing new cancer therapeutics, and the structural features contributing to its biochemical potency against V600E mutant BRAF are discussed in detail.

Abstract 1995: DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant colorectal cancer

Abstract The 5-year survival rate for metastatic colorectal cancer (CRC) is less than 14%. Hyperactivation of the ERK/MAPK pathway occurs at a high frequency in CRC, due to mutations (RAS, BRAF) or overexpression/amplification (EGFR) of its signalling components. Aberrant activation of this pathway drives tumour growth and survival and several inhibitors of this pathway are now clinically utilized. The BRAF V600E activating mutation occurs in ~10% of CRC patients and is correlated with an extremely poor prognosis. Use of the small molecule BRAF inhibitor encorafenib in combination with the EGFR inhibitor cetuximab is now approved for BRAF V600E mutant patients, however, the objective response rate is only ~20% and the treatment is rarely curative. Therefore, there is an urgent need to identify predictive biomarkers of response to encorafenib+cetuximab treatment. Dual-specificity phosphatase 4 (DUSP4) is rapidly induced upon activation of ERK/MAPK signaling upon which it acts as a critical negative regulator of signaling output by directly de-phosphorylating ERK. We have observed that DUSP4 is highly expressed in BRAF-mutant CRC cell lines and primary tumours, although there is a range of expression. Whether DUSP4 expression status impacts on response to encorafenib+cetuximab therapy is unknown. The aim of this study therefore was to determine the role of DUSP4 on sensitivity of BRAF-mutant CRC cells to encorafenib+cetuximab treatment in vitro. To examine this, we determined the impact of DUSP4 knock-down, knock-out and re-expression on sensitivity of BRAF-mutant CRC cancer cell lines to encorafenib+cetuximab treatment. Screening a panel of BRAF mutant CRC cell lines for sensitivity to encorafenib+cetuximab revealed a subset of resistant cell lines had either low or undetectable DUSP4 protein expression, while all sensitive cell lines had high DUSP4 expression. siRNA-mediated knockdown of DUSP4 in DUSP4 expressing BRAF-mutant CRC cell lines increased pERK and pFRA1, suggesting activation of ERK/MAPK signalling. DUSP4 knockdown also reduced sensitivity of BRAF mutant CRC cell lines to encorafenib+cetuximab compared to control. These findings were further validated using DUSP4 knock-out with CRISPR-Cas9. Conversely, DUSP4 re-expression in BRAF-mutant CRC cell lines lacking DUSP4 expression, decreased pERK and increased sensitivity to encorafenib+cetuximab treatment. These results demonstrate that DUSP4 regulates ERK/MAPK signalling in BRAF mutant CRC cell lines and that loss of DUSP4 promotes resistance to encorafenib plus cetuximab treatment. These findings warrant investigation of DUSP4 expression status as a predictive biomarker of encorafenib+cetuximab response in patient samples. Citation Format: Kristen Needham, Fiona Chionh, Stan Kaczmarczyk, Connor Kearney, Laura Jenkins, Ian Luk, John M. Mariadason. DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1995.

A phase 1, multicenter, open-label study of a new BRAF inhibitor ABM-1310 in adult patients (pts) with BRAFv600-mutated solid tumors.

3098 Background: ABM-1310 is a novel small-molecule BRAF inhibitor with high water solubility, cell permeability, and blood-brain barrier penetration as demonstrated in preclinical studies. Here we report preliminary findings from a first-in-patient phase 1 study of ABM-1310. Methods: This US multicenter, open-label, dose-escalation with cohort expansion trial includes adult pts with advanced BRAFv600-mutated solid tumors, including those with active brain metastasis from solid tumors or primary malignant brain tumors. Previous BRAF/MEK inhibitor treatment is permitted. In dose escalation Part A, ABM-1310 monotherapy is given orally twice a day (25–250 mg bid). In dose escalation Part B, ABM-1310 (100 and 200 mg bid) is given in combination with the MEK inhibitor cobimetinib (60 mg QD d1–21 q4w). Primary objectives: determine maximum tolerated dose and recommended phase 2 dose of ABM-1310 ± cobimetinib. Secondary objectives: safety/tolerability; pharmacokinetics; preliminary efficacy. Dose-limiting toxicities (DLTs) are assessed during cycle 1 by a 3+3 design. Results: As of November 28, 2022, 20 pts (65% male; median age 57.5 years) have been treated with ABM-1310 monotherapy across six dose levels in Part A: 25 mg bid (n=3); 50 mg bid (n=3); 100 mg bid (n=4); 150 mg bid (n=4); 200 mg bid (n=4); 250 mg bid (n=2). All pts experienced adverse events (AEs), most frequently skin rash (40%) and QT prolongation (20%). Three pts had drug-related grade ≥3 AEs (nausea/vomiting, QT prolongation and rash) and two had drug-related serious AEs (SAEs) (nausea/vomiting and creatinine increase). Among 16 efficacy evaluable pts, two (pleomorphic xanthoastrocytoma and glioblastoma) had a partial response (PR) and eight had stable disease (SD) as their best response (per RECIST or RANO). In Part B, two ABM-1310 dose levels were assessed in combination with cobimetinib: 100 mg bid (n=3) and 200 mg bid (n=3). Four pts had AEs and drug-related AEs (rash and QT prolongation). There were no SAEs. Among three evaluable pts, one (melanoma) had a PR and one had SD as their best response. There were no confirmed DLTs in either dose-escalation Part A or B, although a few pts had ABM-1310 treatment interruption and subsequent dose reduction. No pts discontinued prematurely for any safety or tolerability reason and there were no drug-related deaths. Preliminary assessment of ABM-1310 drug exposure vs dosage showed a linear dose-proportional relationship. This trial is ongoing. Conclusions: ABM-1310 at the currently tested doses up to 200 mg bid, either alone or in combination with cobimetinib, was generally well tolerated, with no new safety signals. Preliminary efficacy data demonstrate favorable activity of ABM-1310 in pts with BRAFv600-mutated solid tumors, including primary CNS tumors, and pts previously refractory to other BRAF/MEK inhibitors and I/O therapies. Clinical trial information: NCT04190628 .

ERK1/2 inhibitors act as monovalent degraders inducing ubiquitylation and proteasome-dependent turnover of ERK2, but not ERK1.

Innate or acquired resistance to small molecule BRAF or MEK1/2 inhibitors (BRAFi or MEKi) typically arises through mechanisms that sustain or reinstate ERK1/2 activation. This has led to the development of a range of ERK1/2 inhibitors (ERKi) that either inhibit kinase catalytic activity (catERKi) or additionally prevent the activating pT-E-pY dual phosphorylation of ERK1/2 by MEK1/2 (dual-mechanism or dmERKi). Here, we show that eight different ERKi (both catERKi or dmERKi) drive the turnover of ERK2, the most abundant ERK isoform, with little or no effect on ERK1. Thermal stability assays show that ERKi do not destabilise ERK2 (or ERK1) in vitro, suggesting that ERK2 turnover is a cellular consequence of ERKi binding. ERK2 turnover is not observed upon treatment with MEKi alone, suggesting it is ERKi binding to ERK2 that drives ERK2 turnover. However, MEKi pre-treatment, which blocks ERK2 pT-E-pY phosphorylation and dissociation from MEK1/2, prevents ERK2 turnover. ERKi treatment of cells drives the poly-ubiquitylation and proteasome-dependent turnover of ERK2 and pharmacological or genetic inhibition of Cullin-RING E3 ligases prevents this. Our results suggest that ERKi, including current clinical candidates, act as 'kinase degraders', driving the proteasome-dependent turnover of their major target, ERK2. This may be relevant to the suggestion of kinase-independent effects of ERK1/2 and the therapeutic use of ERKi.

Sustained release hydrogel for durable locoregional chemoimmunotherapy for BRAF-mutated melanoma

The wide prevalence of BRAF mutations in diagnosed melanomas drove the clinical advancement of BRAF inhibitors in combination with immune checkpoint blockade for treatment of advanced disease. However, deficits in therapeutic potencies and safety profiles motivate the development of more effective strategies that improve the combination therapy's therapeutic index. Herein, we demonstrate the benefits of a locoregional chemoimmunotherapy delivery system, a novel thermosensitive hydrogel comprised of gelatin and Pluronic® F127 components already widely used in humans in both commercial and clinical products, for the co-delivery of a small molecule BRAF inhibitor with immune checkpoint blockade antibody for the treatment of BRAF-mutated melanoma. In vivo evaluation of administration route and immune checkpoint target effects revealed intratumoral administration of antagonistic programmed cell death protein 1 antibody (aPD-1) lead to potent antitumor therapy in combination with BRAF inhibitor vemurafenib. The thermosensitive F127-g-Gelatin hydrogel that was evaluated in multiple murine models of BRAF-mutated melanoma that facilitated prolonged local drug release within the tumor (>1 week) substantially improved local immunomodulation, tumor control, rates of tumor response, and animal survival. Thermosensitive F127-g-Gelatin hydrogels thus improve upon the clinical benefits of vemurafenib and aPD-1 in a locoregional chemoimmunotherapy approach for the treatment of BRAF-mutated melanoma.

Clinical efficacy of T-cell therapy after short-term BRAF-inhibitor priming in patients with checkpoint inhibitor-resistant metastatic melanoma

PurposeDespite impressive response rates following adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, improvement is needed to increase the efficacy and broaden the applicability of this...

Encorafenib: A Review in Metastatic Colorectal Cancer with a BRAF V600E Mutation.

Encorafenib (Braftovi®) is an oral small molecule BRAF inhibitor used in combination with cetuximab for the treatment of adult patients with metastatic colorectal cancer (mCRC) with a BRAF V600E mutation, who have received prior systemic therapy. In a clinical trial in adults with BRAF V600E-mutated mCRC who had disease progression after one or two previous regimens (BEACON CRC), encorafenib plus cetuximab was associated with a significantly longer median overall survival (OS), a higher objective response rate (ORR) and longer median progression-free survival (PFS), compared with standard therapy. Encorafenib plus cetuximab had a manageable tolerability profile in BEACON CRC. Current evidence suggests that encorafenib plus cetuximab combination therapy is an important targeted regimen for patients with mCRC and a BRAF V600E mutation who have received prior therapy.

Abstract 4038: ABM-1310, A novel BRAF Inhibitor, combined with EGFR and MEK inhibitors, inhibits colorectal tumor growth and increases overall survival in vivo

Abstract Colorectal cancer (CRC) is a cancer that starts in the colon or the rectum, and the current treatment mainly relies on chemotherapies. For example, ERBITUX® /cetuximab is an FDA approved targetedtherapy drug to treat CRC with wild-type KRAS, the 11% low response rate limits its clinical application. BRAF inhibitors have been widely used clinically for melanoma and lung cancer, however, they have dismally low response rate at only approximately 5% of colorectal cancer patients with BRAFv600 mutation. Here, we report that ABM-1310, a novel small molecule inhibitor of BRAF (specifically inhibit BRAFv600 mutant), has shown good potency in colon cancer xenograft models, when combined with a MEK inhibitor and EGFR antibody. In the HT-29 xenograft subcutaneous model, 10 to 25mg/kg/day oral dose of ABM-1310 combined with cetuximab (0.1mg/dose, twice a week) and 3 mg/kg/day of Binimetinib (MEK inhibitor) significantly inhibited HT-29 tumor growth with TGI of 85% to 92% after 34-days treatment. As a monotherapy or combined with cetuximab alone, ABM-1310 also brought the TGI of 74% and 82% at 25mg/kg/day, which indicates ABM-1310 played a key role in tumor growth inhibition in the combinations. Beside with Binimetinib, we also tested combination of ABM-1310 with other different MEK inhibitors in this model. ABM-1310 is highly water-soluble small molecule with high cell permeability and blood brain barrier penetration. ABM-1310 shown good efficacy in melanoma A375 (luciferase labelled cells) in the subcutaneous/intracranial/intracardiac xenograft models (brain metastasis models). In the HT-29 colon intracranial model, ABM-1310 also demonstrated significant tumor inhibition effect and increased animal overall survival. Compared with vehicle or marketed BRAF inhibitor vemurafenib, the median survival time of ABM-1310 group was >90 days vs 51 days (vemurafenib) and 38 days (vehicle), respectively. In Summary, ABM-1310, as a novel small molecule BRAF inhibitor, combined with EGFR and MEK inhibitors, has shown strong anti-tumor effect in preclinical in vivo models of colon cancer with BRAFv600 mutation, especially with brain metastasis. Citation Format: Yong Hu, Chen Chen, Charles Huang, Min Xu, Youqin Chen, Xiyuan Zhao, Li Zhao. ABM-1310, A novel BRAF Inhibitor, combined with EGFR and MEK inhibitors, inhibits colorectal tumor growth and increases overall survival in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4038.

Abstract 4177: Preclinical development of ABM-1310, a novel BRAF Inhibitor to treat cancer with brain metastasis

Abstract BRAF is a key molecule activated by RAS in the MAPK pathway that mainly controls cell proliferation. BRAF mutation is detected in about 8% of all cancer population, and more than 50% is BRAFv600e, which causes constitutive MAPK activation. BRAFv600 mutation is found in various cancers such as melanoma, colorectal cancer, glioma, lung cancer, thyroid cancer, pancreatic cancer, ovary cancer etc. ABM-1310 is a novel small molecule BRAF inhibitor that specific inhibits BRAFv600 mutant form with high water solubility, cell permeability, and blood brain barrier (BBB) penetration. Although current target or immune-therapies are reported with some variable effects, melanoma with brain metastasis is still a significant challenge in clinic. With high BBB penetration and high exposure in brain, ABM-1310 has good potential to treat tumors bearing BRAFv600 mutation with brain metastasis. In-vitro data shown similar on-target enzyme activity of ABM-1310 to marketed BRAF inhibitors on BRAFv600e, as well as high anti-proliferation activity in multiple cancer cell lines with BRAF V600E mutation like A375, Colo-829, and HT-29 etc. In vivo pharmacology studies demonstrated that ABM-1310 had good potency of tumor growth inhibition with oral dose as low as 1mg/kg/day. ABM-1310 treatment also improved animal overall survival in several cell-line derived xenograft (CDX) models, including subcutaneous, intracranial and intracardiac melanoma models. Non-clinical pharmacokinetics studies also shown ABM-1310 had a favorable ADME profile both in vitro and in vivo in animals. ABM-1310 demonstrated excellent brain penetration in animals. Single-dose, seven-day repeat dose non-GLP studies and four-week GLP toxicity studies in SD rats and beagle dogs all revealed that ABM-1310 had a good safety profile with NOAEL of 100mg/kg/day in SD rats and 30mg/kg/day in beagle dogs. Based on these supportive preclinical study results, IND of ABM-1310 was submitted in 2019 Q4 to investigate its safety in humans as signal reagent, which will be followed by further clinic development of ABM-1310, or combination with other molecules to treat cancers with BRAFv600 mutation, especially with brain metastasis. Citation Format: Yong Hu, Chen Chen, Charles Huang, Min Xu, Youqin Chen, Xiyuan Chen, Li Zhao. Preclinical development of ABM-1310, a novel BRAF Inhibitor to treat cancer with brain metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4177.

Systemic review on B-RafV600E mutation as potential therapeutic target for the treatment of cancer

Cancer is one of the major public catastrophes worldwide and as per WHO, cancer is the leading cause of death universally after CVS disorders accounting for 9.6 million deaths in 2018. WHO statistics revealed five dangerous types of cancer viz. lung, breast, colorectal, prostate and skin. In male, lung cancer causes highest death, while in female, breast cancer causes the most. Alteration in MAPK signalling pathway plays a significant role in majority of cancer cases. Raf protein is activated by phosphorylation via downstream regulation of the MAPK pathway. Raf composed of 3 subtypes, viz. A-Raf, B-Raf, and C-Raf. B-Raf kinase plays a significant role in healthy cell growth in the MAPK pathway and the problem associated with B-Raf mutation leads to the development of cancer and other diseases. The progression of mutant B-Raf (B-RafV600E) protein is higher in cancer as compare to other diseases. In 2002, B-RafV600E mutation was identified for the first time in the development of cancer. The frequency of B-RafV600E mutation is higher in melanoma, thyroid, colorectal and ovarian cancer. We have covered small molecule B-RafV600E inhibitors reported in various literatures; from 2002 to 2020 and also covered clinical trial data. To widen the scope of readers, we compiled details of small molecules, specifically inhibiting B-RafV600E mutant and showing anti-proliferative activity against various cancer cell lines along with in-vivo data. We believe that the information covered here will be important in signifying the potentials of B-RafV600E mutation and its inhibitors as potent anticancer agents.

A phase II study evaluating atezolizumab (A), cobimetinib (C), and vemurafenib (V) in patients (pts) with BRAF-mutant melanoma and central nervous system (CNS) metastases (mets).

TPS10081 Background: CNS mets, a common complication of melanoma, are associated with poor survival prognosis (median of 4-5 months). The rationale for combining PD-L1 pathway blockade using atezolizumab (A) with the small molecule BRAF pathway–targeted inhibitors cobimetinib (C) and vemurafenib (V) for the treatment of BRAFV600 mutation–positive melanoma is based on preclinical and translational evidence supporting the synergistic antitumoral effects of these approaches. Recently, the phase 3 IMspire150 study (NCT02908672) demonstrated improved progression-free survival outcomes with A + C + V vs C + V. Methods: This phase 2 study (NCT03625141) is currently evaluating A + C + V in pts with BRAFV600 mutation–positive advanced melanoma and CNS mets. The study originally included a parallel cohort evaluating A + C in pts with BRAFV600wild-type advanced melanoma and CNS mets. This cohort was closed at an enrollment of 15 pts after the primary analysis of the phase 3 IMspire170 study (NCT03273153), which showed no added benefit with A + C vs pembrolizumab in pts with BRAFV600 wild-type disease. Eligible pts are aged ≥18 years with histologically confirmed melanoma and magnetic resonance imaging–confirmed brain mets ≥5 mm in at least 1 dimension. In addition, pts should not have received prior systemic treatment for metastatic disease; they were required to have ECOG performance status ≤2 and adequate hematologic and end organ function. Prior stereotactic or surgical therapy of ≤10 brain mets is allowed. The primary endpoint is intracranial objective response rate (ORR) with A + C + V in BRAFV600mutation–positive melanoma as assessed by an independent review committee. Key secondary endpoints include investigator-assessed intracranial ORR, extracranial ORR, overall ORR, safety, and quality of life. Exploratory biomarker analyses are planned. The sample size for the cohort will be approximately 60 pts. No formal statistical hypothesis is being tested in this study. The primary study analysis and the analyses of all efficacy endpoints and safety summaries will be based on data collected ≤6 months after the last pt is enrolled. Clinical trial information: NCT03625141.

Discovery of Selective Small Molecule Degraders of BRAF-V600E.

BRAF is among the most frequently mutated oncogenes in human cancers. Multiple small molecule BRAF kinase inhibitors have been approved for treating melanoma carrying BRAF-V600 mutations. However, the benefits of BRAF kinase inhibitors are generally short-lived. Small molecule-mediated targeted protein degradation has recently emerged as a novel pharmaceutical strategy to remove disease proteins through hijacking the cellular ubiquitin proteasome system (UPS). In this study, we developed thalidomide-based heterobifunctional compounds that induced selective degradation of BRAF-V600E, but not the wild-type BRAF. Downregulation of BRAF-V600E suppressed the MEK/ERK kinase cascade in melanoma cells and impaired cell growth in culture. Abolishing the interaction between degraders and cereblon or blocking the UPS significantly impaired the activities of these degraders, validating a mechanistic role of UPS in mediating targeted degradation of BRAF-V600E. These findings highlight a new approach to modulate the functions of oncogenic BRAF mutants and provide a framework to treat BRAF-dependent human cancers.

Quantitative Interrogation of the Human Kinome Perturbed by Two BRAF Inhibitors.

Oncogenic BRAF mutations contribute to the development of a number of cancers, and small-molecule BRAF inhibitors have been approved by the Food and Drug Administration (FDA) for anticancer therapy. In this study, we employed two targeted quantitative proteomics approaches for monitoring separately the alterations in protein expression and ATP binding affinities of kinases in cultured human melanoma cells elicited by two FDA-approved small-molecule BRAF inhibitors, dabrafenib and vemurafenib. Our results showed that treatment with the two inhibitors led to markedly different reprograming of the human kinome. Furthermore, we confirmed that vemurafenib could compromise the ATP binding capacity of MAP2K5 in vitro and inhibit its kinase activity in cells. Together, our targeted quantitative proteomic methods revealed profound changes in expression levels of kinase proteins in cultured melanoma cells upon treatment with clinically used BRAF inhibitors and led to the discovery of novel putative target kinases for these inhibitors.

Mechanism of BRAF Activation through Biochemical Characterization of the Recombinant Full-Length Protein.

BRAF kinase plays an important role in mitogen-activated protein kinase (MAPK) signaling and harbors activating mutations in about half of melanomas and in a smaller percentage in many other cancers. Despite its importance, few in vitro studies have been performed to characterize the biochemical properties of full-length BRAF. Herein, a strategy to generate an active, intact form of BRAF protein suitable for in vitro enzyme kinetics is described. It is shown that purified, intact BRAF protein autophosphorylates the kinase activation loop and this can be enhanced by binding the MEK protein substrate through an allosteric mechanism. These studies provide in vitro evidence that BRAF selectively binds to active RAS and that the BRAF/CRAF heterodimer is the most active form, relative to their respective homodimers. Full-length BRAF analysis with small-molecule BRAF inhibitors shows that two drugs, dabrafenib and vemurafenib, can modestly enhance kinase activity of BRAF at low concentration. Taken together, this characterization of intact BRAF contributes to a framework for understanding its role in cell signaling.

Metastatic Melanoma: Insights Into the Evolution of the Treatments and Future Challenges.

Melanoma is the deadliest form of skin cancer. While associated survival prognosis is good when diagnosed early, it dramatically drops when melanoma progresses into its metastatic form. Prior to 2011, the favored therapies include interleukin-2 and chemotherapies, regardless of their low efficiency and their toxicity. Following key biological findings, two new types of therapy have been approved. First, there are the targeted therapies, which rely on small molecule B-Raf and MEK inhibitors and allow the treatment of patients with B-Raf mutated melanoma. Second, there are the immunotherapies, with anti-CTLA-4 and anti-PD-1 antibodies that are used for patients harboring a B-Raf wild-type status. Both approaches have significantly improved patient survival, compared with alkylating agents, in the treatment of unresectable melanoma. Herein, we review the evolution of the treatment of melanoma starting from early discoveries to current therapies. A focus will be provided on drug discovery, synthesis, and mode of action of relevant drugs and the future directions of the domain to overcome the emergence of the resistance events.

Inhibition of the CRAF/prohibitin interaction reverses CRAF-dependent resistance to vemurafenib.

Activating BRAF mutations promote constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway and are common in a variety of human malignancies, including melanoma and colon cancer. Several small molecule BRAF inhibitors such as vemurafenib have been developed and demonstrate remarkable clinical efficacy. However, resistance typically emerges in most melanoma patients. Studies have demonstrated that reactivation of MAPK signaling via CRAF overexpression and dysregulation is a mechanism for vemurafenib resistance in melanoma. Prohibitins (PHBs) are highly conserved proteins that are thought to control the cell cycle, senescence and tumor suppression. PHB1 is essential for CRAF-mediated ERK1/2 activation through direct binding to CRAF. We developed a CRAF-mediated model of vemurafenib resistance in melanoma cells to assess the importance of the interaction between CRAF and PHB1 in resistance to BRAF-targeting agents. We demonstrate that CRAF overexpression renders melanoma cells resistant to BRAF-targeting agents. Moreover, treatment with the natural compound rocaglamide A disrupts the interaction between PHB and CRAF in melanoma cells, thus reducing MEK1/2 and ERK1/2 signaling, inhibiting melanoma cell growth and inducing apoptosis. The efficacy of these compounds was also demonstrated in a human melanoma xenograft model. Taken together, these data suggest that PHB1 may serve as a novel, druggable target in CRAF-mediated vemurafenib resistance.

Radiotherapy with BRAF inhibitor therapy for melanoma: progress and possibilities.

The introduction of small molecule BRAF(V600) kinase inhibitors represents a milestone in the targeted therapy of patients with metastatic melanoma by a significant increase in therapeutic efficacy in terms of overall and progression-free survival compared with conventional chemotherapy. Beside BRAF(V600) inhibitor treatment, radiotherapy is a further mainstay for the therapy of metastatic melanoma and thus a concomitant or sequential application of BRAF(V600) inhibitors and radiotherapy is inevitable. Recent reports show a significant radiosensitization of the irradiated healthy tissue in patients with melanoma after the combination of radiotherapy and BRAF(V600) inhibitors, evoking concern in clinical practice. We review interactions of BRAF(V600) inhibitors and radiation with regard to antitumor effects and an increased radiotoxicity in the healthy tissue.

ATCT-11RECURRENT GANGLIOGLIOMA TREATED WITH BRAF INHIBITORS

BACKGROUND: A mutation in the BRAF oncogene, BRAF V600E has been characterized as commonly present in melanoma as well as a variety of WHO Grade I gliomas including gangliogliomas wherein the mutation is present in approximately 30%. The presence of the BRAF V600E mutation in gangliogliomas suggests the possibility that use of small molecule BRAF inhibitors may have efficacy in the recurrent setting. PATIENTS AND METHODS: Thirteen ganglioglioma tumor specimens were screened by real-time polymerase-chain reaction assay (Cobas 4800 BRAF V600 Mutation test, Roche Molecular Systems) for the presence of the V600E mutation, amongst which 3 (23%) were positive. Three patients with recurrent ganglioglioma and positive for the V600E mutation were treated with dabrafenib, a second generation BRAF inhibitor, following disease progression after previous therapy that included surgery, radiotherapy and temozolomide chemotherapy. Dabrafenib was administered orally at a dose of 150mg twice per day. RESULTS: Dabrafenib was well tolerated and there were no CTC Grade 3 or higher toxicities. The median number of cycles (defined as 1-month of dabrafenib) was 7 with a range of 4-10. Best radiographic response following 2 cycles of dabrafenib was stable disease in 2 patients and partial response in 1 patient. Two patients following progression on dabrafenib elected another salvage therapy and one patient proceeded to hospice. Median progression free survival was 7 months (range 4-10 months). Median overall survival from onset of dabrafenib was 12 months (range 6-16 months). CONCLUSIONS: A small case series such as the present is hypothesis generating only but suggests inhibition of BRAF in gangliogliomas with V600E mutations may if confirmed in a larger and prospective trial constitute a novel treatment paradigm.

Abstract 2924: MAPK pathway inhibition enhances the efficacy of an anti-endothelin B receptor drug conjugate by inducing target expression in melanoma

Abstract Therapies targeting the MAP kinase pathway in melanoma have produced significant clinical responses; however, duration of response is limited by acquisition of drug resistance. Rationale 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 wildtype for both, were exposed to small molecule inhibitors or 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 anti-tumor 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 utility in the majority of human melanoma cases. Citation Format: Jyoti Asundi, Paul Polakis, Jennifer A. Lacap, Michelle Nannini. MAPK pathway inhibition enhances the efficacy of an anti-endothelin B receptor drug conjugate by inducing target expression in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2924. doi:10.1158/1538-7445.AM2014-2924

Abstract 2611: Function blocking ERBB3 antibody inhibits adaptive response to RAF inhibitor

Abstract ERBB3/HER3 is a member of the EGFR family of receptor tyrosine kinase. Our lab recently showed that treating melanoma cells with the FDAapproved small molecule BRAF inhibitor vemurafenib leads to an increase in ERBB3 signaling when grown in the presence of its ligand neuregulin (NRG1). Here, we show that targeting ERBB3 with a monoclonal antibody inhibits its activation and downstream signaling in the presence of vemurafenib and NRG1. This inhibition of ERBB3 led to a decrease in proliferation and increase in apoptosis in vitro as measured by EdU and AnnexinV/PI staining. Targeting ERBB3 in combination with mutant BRAF in vivo resulted in a delay of tumor re-growth in immune deficient mice compared to targeting mutant BRAF alone. Taken together, these results indicate that targeting ERBB3 in combination with mutant BRAF in melanoma may help enhance the initial effects of vemurafenib in the clinic. Citation Format: Curtis Harrison Kugel, Edward J. Hartsough, Michael A. Davies, Andrew E. Aplin. Function blocking ERBB3 antibody inhibits adaptive response to RAF inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2611. doi:10.1158/1538-7445.AM2014-2611