BRAF Inhibitor Vemurafenib Research Articles

Cell-free DNA BRAF V600E measurements during BRAF inhibitor therapy of metastatic melanoma: long-term analysis.

We assessed the status of the BRAF V600E mutation in cell-free circulating tumor DNA (cfDNA) isolated from the plasma of patients with metastatic melanoma treated with the BRAF inhibitor vemurafenib, collected at different time points during therapy to evaluate the sensitivity and specificity of quantitative polymerase chain reaction and droplet digital polymerase chain reaction (ddPCR) and the correlation between the level of plasma cfDNA p.V600E and the long-term clinical outcome. cfDNA in patients with BRAF-mutated melanoma (n = 62) was analyzed at baseline and at 4-8 weeks from the start of vemurafenib therapy. BRAF mutations were assessed using tumor tissue-derived DNA and circulating cfDNA from plasma samples. Quantification of BRAF V600E was performed in cfDNA using ddPCR. cfDNA V600E was detected in the plasma of 48/62 (77%) patients at baseline and in 18/62 (29%) patients after 4-8 weeks of treatment. Patients positive for BRAF mutations in cfDNA at baseline had shorter progression-free survival (PFS) and overall survival (OS) compared with patients with undetectable cfDNA BRAF mutations. Undetectable cfDNA p.V600E at baseline and after 4-8 weeks of therapy was associated with the best prognosis. When treated as a continuous variable, the log-transformed concentration of baseline cfDNA p.V600E was significantly associated with both PFS and OS. This effect was retained in the multivariate OS Cox model adjusted for Eastern Cooperative Oncology Group performance status, the presence of brain metastases, patient age, and previous systemic treatment. Monitoring of plasma BRAF p.V600E cfDNA concentrations in patients with metastatic melanoma on targeted therapy may have prognostic value. Undetectable cfDNA p.V600E before and during treatment was associated with a favorable prognosis.

Identifying drug targets in tissues and whole blood with thermal-shift profiling.

Monitoring drug-target interactions with methods such as the cellular thermal-shift assay (CETSA) is well established for simple cell systems but remains challenging in vivo. Here we introduce tissue thermal proteome profiling (tissue-TPP), which measures binding of small-molecule drugs to proteins in tissue samples from drug-treated animals by detecting changes in protein thermal stability using quantitative mass spectrometry. We report organ-specific, proteome-wide thermal stability maps and derive target profiles of the non-covalent histone deacetylase inhibitor panobinostat in rat liver, lung, kidney and spleen and of the B-Raf inhibitor vemurafenib in mouse testis. In addition, we devised blood-CETSA and blood-TPP and applied it to measure target and off-target engagement of panobinostat and the BET family inhibitor JQ1 directly in whole blood. Blood-TPP analysis of panobinostat confirmed its binding to known targets and also revealed thermal stabilization of the zinc-finger transcription factor ZNF512. These methods will help to elucidate the mechanisms of drug action in vivo.

Substantial intrinsic variability in chemoradiosensitivity of newly established anaplastic thyroid cancer cell-lines

Background: Well characterized human cell lines are needed for preclinical treatment studies of anaplastic thyroid cancer (ATC).Aims/Objectives: The aim was to establish, verify and characterize a panel of ATC cell lines.Material and methods: Cell lines were established from ATC fine-needle aspiration biopsies and characterized genetically and functionally regarding treatment sensitivities.Results: Eight cell lines were established in vitro and the anaplastic thyroid origin was verified. Seven of the cell lines were also grown as xenografts. The cell lines harboured complex karyotypes with modal numbers in hyperdiploid to near-pentaploid range. Five were TP53 mutated and three carried the BRAFV600E mutation. None had rearrangements of RET. For doxorubicin, IC50 ranged from 0.42 to 46 nmol/L and for paclitaxel from 1.6 to 196 nmol/L. Radiation sensitivity varied between 2.6 and 6.3 Gy. Two of the BRAF mutated cell lines displayed high sensitivity to vemurafenib, while the third was similar to the wild-type ones.Conclusions and significance: We describe a series of new ATC cell lines demonstrating large heterogeneity in the response to cytostatic drugs and the BRAF inhibitor vemurafenib. The observations are relevant to future attempts to optimize treatment combinations for ATC.

Connecting Cancer Pathways to Tumor Engines: A Stratification Tool for Colorectal Cancer Combining Human In Vitro Tissue Models with Boolean In Silico Models.

To improve and focus preclinical testing, we combine tumor models based on a decellularized tissue matrix with bioinformatics to stratify tumors according to stage-specific mutations that are linked to central cancer pathways. We generated tissue models with BRAF-mutant colorectal cancer (CRC) cells (HROC24 and HROC87) and compared treatment responses to two-dimensional (2D) cultures and xenografts. As the BRAF inhibitor vemurafenib is—in contrast to melanoma—not effective in CRC, we combined it with the EGFR inhibitor gefitinib. In general, our 3D models showed higher chemoresistance and in contrast to 2D a more active HGFR after gefitinib and combination-therapy. In xenograft models murine HGF could not activate the human HGFR, stressing the importance of the human microenvironment. In order to stratify patient groups for targeted treatment options in CRC, an in silico topology with different stages including mutations and changes in common signaling pathways was developed. We applied the established topology for in silico simulations to predict new therapeutic options for BRAF-mutated CRC patients in advanced stages. Our in silico tool connects genome information with a deeper understanding of tumor engines in clinically relevant signaling networks which goes beyond the consideration of single drivers to improve CRC patient stratification.

Role of VEGFR-1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib.

The vascular endothelial growth factor receptor‐1 (VEGFR‐1) is a tyrosine kinase receptor frequently expressed in melanoma. Its activation by VEGF‐A or placental growth factor (PlGF) promotes tumour cell survival, migration and invasiveness. Moreover, VEGFR‐1 stimulation contributes to pathological angiogenesis and induces recruitment of tumour‐associated macrophages. Since melanoma acquired resistance to BRAF inhibitors (BRAFi) has been associated with activation of pro‐angiogenic pathways, we have investigated VEGFR‐1 involvement in vemurafenib resistance. Results indicate that human melanoma cells rendered resistant to vemurafenib secrete greater amounts of VEGF‐A and express higher VEGFR‐1 levels compared with their BRAFi‐sensitive counterparts. Transient VEGFR‐1 silencing in susceptible melanoma cells delays resistance development, whereas in resistant cells it increases sensitivity to the BRAFi. Consistently, enforced VEGFR‐1 expression, by stable gene transfection in receptor‐negative melanoma cells, markedly reduces sensitivity to vemurafenib. Moreover, melanoma cells expressing VEGFR‐1 are more invasive than VEGFR‐1 deficient cells and receptor blockade by a specific monoclonal antibody (D16F7 mAb) reduces extracellular matrix invasion triggered by VEGF‐A and PlGF. These data suggest that VEGFR‐1 up‐regulation might contribute to melanoma progression and spreading after acquisition of a drug‐resistant phenotype. Thus, VEGFR‐1 inhibition with D16F7 mAb might be a suitable adjunct therapy for VEGFR‐1 positive tumours with acquired resistance to vemurafenib.

Real-World Toxicity Experience with BRAF/MEK Inhibitors in Patients with Erdheim-Chester Disease.

BackgroundErdheim‐Chester disease (ECD) is a rare non‐Langerhans cell histiocytosis. The BRAF inhibitor vemurafenib is approved by the U.S. Food and Drug Administration (FDA) for patients with ECD harboring a BRAF V600E mutation. Successful treatment has also been reported with MEK‐targeted therapies, likely because of the fact that BRAF mutant–negative patients harbor MEK pathway alterations. In our Rare Tumor Clinic, we noted that these patients have frequent drug‐related toxicity, consistent with previous reports indicating the need to markedly lower doses of interferon‐alpha when that agent is used in these patients.Patients and MethodsWe performed a review of ten patients with ECD seen at the Rare Tumor Clinic at University of California San Diego receiving 16 regimens of targeted BRAF, MEK, or combined therapies.ResultsThe median age of the ten patients with ECD was 53 years (range, 29–77); seven were men. The median dose percentage (percent of FDA‐approved dose) tolerated was 25% (range, 25%–50%). The most common clinically significant adverse effects resulting in dose adjustments of targeted therapies were rash, arthralgias, and uveitis. Renal toxicity and congestive heart failure were seen in one patient each. In spite of these issues, eight of ten patients (80%) achieved a partial remission on therapy.DiscussionPatients with ECD appear to require substantially reduced doses of BRAF and MEK inhibitors but are responsive to these lower doses.

Classification and Prognosis of Central Nervous System Involvement in Erdheim-Chester Disease

Background: Erdheim-Chester disease (ECD) is a non-Langerhans cell histiocytosis, characterized by the infiltration of tissues by foamy CD68+CD1a- histiocytes, with 1500 known-cases since 90 years. Mutations activating the MAPK pathway are found in more than 80% of ECD patients, mainly the BRAFV600E in 57 to 70%. Central nervous system (CNS) involvement during ECD lead to significant morbidity and mortality. We assessed CNS manifestations in a French cohort of 253 ECD patients. Since 2012, close to 200 patients worldwide with multi-systemic and refractory ECD (e.g. heart and/or CNS involvements) have benefited from BRAF and MEK inhibitors, which have proven highly beneficial. Methods: CNS manifestations were determined by clinical examination and brain and/or spine magnetic resonance imaging (MRI). Targeted therapy efficacy was assessed using physician and radiologist global assessment. Results: Ninety-seven (38%) among the 253 of the whole cohort of ECD patients had CNS involvement. CNS involvement was significantly associated with a younger age at diagnosis (mean 55.5 years) and at symptoms onset (mean 50.5 years), and also with the presence of the BRAFV600E mutation (in 77% of cases), xanthelasma (34%), and diabetes insipidus (36%). Median survival among patients with CNS involvement was significantly lower than in ECD patients without CNS involvement (124 months versus 146 months, p=0.03). Seventy-four CNS MRI were centrally reviewed, corresponding to 3 patterns: tumoral in 66%, degenerative in 50%, and vascular in 18%. Targeted therapy (BRAF and/or MEK inhibitors) was associated with improvement of symptoms in 43% of patients, and MRI improvement in 45%. Conclusions: CNS manifestations are typically associated with a poor prognosis in ECD. Three distinct patterns may be recognized: tumoral, degenerative, and vascular. Targeted therapy leads to clinical and/or imaging improvement in almost 50% of patients. OffLabel Disclosure: Targeted therapies such as BRAF inhibitor (vemurafenib) and MEK inhibitor (cobimetinib) are used in severe histiocytosis in EU but are off label

First Line Chemo-Free Therapy with the BRAF Inhibitor Vemurafenib Combined with Obinutuzumab Is Effective in Patients with Hcl

First Line Chemo-Free Therapy with the BRAF Inhibitor Vemurafenib Combined with Obinutuzumab Is Effective in Patients with Hcl

EXTH-65. PLASMA AND CEREBROSPINAL FLUID PHARMACOKINETICS COMPARISON OF BRAF AND MEK INHIBITORS FOLLOWING SINGLE AND EXTENDED ADMINISTRATION IN A PRE-CLINICAL NON-HUMAN PRIMATE MODEL

Abstract BACKGROUND Aberrations in the MAPK signaling pathway are present in adult and pediatric CNS tumors. The BRAF and MEK inhibitors Dabrafenib, Vemurafenib, Selumetinib and Trametinib, alone or in combination, have demonstrated clinical efficacy. However, minimal CSF penetration has been demonstrated in pre-clinical models with single dose administration. CSF penetration of these agents was compared following single and extended oral administration in a nonhuman CSF Lateral Reservoir model to determine if CSF penetration increases at steady state. METHODS Agents were administered orally, to each subject (n=9), in single or multiple doses, as serial single agent studies, with washout periods between studies. The following dosages (Human equivalent dose HED) were utilized in the studies: Single Dosage-Selumetinib 2.5 mg/kg (50.0 mg/m2), Dabrafenib 8.0 mg/kg (160 mg/m2), Vemurafenib 26 mg/kg (520 mg/m2), Trametinib 0.06 and 0.12 mg/kg (1.2 and 2.4 mg/m2). Extended dosages-Selumetinib 2.5 mg/kg BID x 3 days (50.0 mg/m2), Dabrafenib 4.0 mg/kg BID x 2 days (160 mg/m2/day), Vemurafenib 26 mg/kg BID x 3 days (520 mg/m2), Trametinib 0.05 mg/kg x 10 days BID (1.0mg/m2). Paired serial plasma and CSF samples were collected. Agents were quantified by uHPLC-MS/MS. Pharmacokinetic parameters were calculated via noncompartmental methods. Plasma exposure reported as Area Under the Curve (AUC) and CSF penetration as ratio of CSF:plasma AUC. Statistical significance determined by t-test. RESULTS Single administration AUCinf/D (hr*ng/ml/mg) vs. extended administration AUCtau/D (hr*ng/ml/mg) for each agent: Vemurafenib-Plasma 30.9–351.4 vs.29.2–379.7, CSF-non-quantifiable; Dabrafenib-Plasma 29.3–102.6 vs.3.4–58.2, CSF-0.12–0.54 vs 0.04–0.19, % CSF:Plasma-0.3–0.84 vs.0.32–1.3; Trametinib-Plasma AUC indeterminable, CSF-non-quantifiable; Selumetinib-Plasma 69.8–133.0 vs.26.5–81.2; CSF-AUC indeterminable vs. 0.37–0.86, % CSF:Plasma-Indeterminable vs. 0.78–1.4. T-test: A downward trend in Dabrafenib and Selumetinib plasma AUC and a marked upward trend in Selumetinib CSF AUC. CONCLUSIONS With the exception of Selumetinib, the CSF exposure of BRAF and MEK inhibitors studied did not markedly increase following extended dosing.

Effects of Glutathione Transferase-Targeting Nitrobenzoxadiazole Compounds in Relation to PD-L1 Status in Human Melanoma Cells

Background: PD-L1 is a membrane protein with inhibitory effects on immune responses, whose expression has been correlated with high aggressiveness and the propensity of melanoma to metastasize. The nitrobenzoxadiazole (NBD) NBDHEX and its analog MC3181 are endowed with strong antitumor activity towards melanoma and a significant ability to reduce its adhesion and invasiveness. Therefore, we investigated whether PD-L1 status could affect cell sensitivity to the cytotoxic effects of NBDs. We then evaluated the effects of NBDHEX on PD-L1 expression and autophagy in melanoma cells. We used the BRAF-mutated A375 melanoma cell line and an A375 variant population enriched for PD-L1+ cells as a model. The cytotoxic effects of NBDs were evaluated in comparison to those of the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine. Methods: The effect of NBDHEX on autophagy was determined by measuring LC3-II and p62 protein levels by Western blot. The cytotoxic activity of the compounds was evaluated by sulforhodamine B assay. PD-L1 expression and plasma membrane localization were analyzed by FACS and Western blot analysis. Results: NBDHEX behaves as a late-autophagy inhibitor in A375 melanoma cells, as previously found in other tumor cell lines. NBDHEX and MC3181 showed strong and comparable cytotoxic activity in both parental and PD-L1+ A375 cells, with IC₅₀ values in the sub-micromolar range. Conversely, cells sorted for high PD-L1 expression had lower sensitivity to both the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine. NBDHEX treatment did not change the total expression and cell surface localization of PD-L1 in both parental and PD-L1+ A375 cells. Conclusions: Our data suggest that NBDs may represent a promising treatment strategy for melanoma with elevated PD-L1 expression.

Signaling pathways in melanoma biology and new targeted therapeutic approaches

Despite the broad prevention programs and early detection and therapy progress, melanoma of skin is still responsible for 0.6% of deaths caused by tumour disease. Every year 300,000 patients are diagnosed and 60,000 die from the most malignant of skin cancer. Generally, melanoma is formed as a result of mutation of growth pathways responsible for proliferation and apoptosis. One of the most investigated pathway, mutated in 90% of melanomas, is RAS > RAF > MEK > ERK also known as mitogen-activated protein kinase (MAPK) pathway. The second one is phosphoinositide-3-OH kinase (PI3K) pathway. The better understanding of melanoma biology resulted in research of inhibitors, which can affect presented pathways and prevent uncontrolled proliferation of melanoma. The BRAF inhibitors vemurafenib and dabrafenib and MEK inhibitor trametinib seem to be the most successful ones. Recent advances in biology of melanoma provided new interesting therapeutic targets. One of the most inquiring is microphthalmia associated transcription factor (MITF), the principal regulator of melanocyte lineage. MITF perform the role of so-called ‘survival’ or ‘addiction’ oncogene. Moreover, the interleukin-1 receptor-associated kinases (IRAKs) might clarify the connection between the inflammatory environment and melanoma carcinogenesis. IRAKs play a key role as mediators of toll-like receptor (TLR) and interleukin-1 receptor (IL1R) in inflammation signalling processes. Moreover, it was observed that metformin cause cell cycle arrest in melanoma cells, secondly leading to activation of autophagy and apoptosis. Although due to targeted and immunotherapy the prognosis of patients with metastatic melanoma is incomparably better, melanoma in its advanced stadium is still predominantly lethal. Therefore, the most present research concentrates on acquired resistance against targeted therapy.

Vemurafenib acts as an aryl hydrocarbon receptor antagonist: Implications for inflammatory cutaneous adverse events.

In recent years, the BRAF inhibitor vemurafenib has been successfully established in the therapy of advanced melanoma. Despite its superior efficacy, the use of vemurafenib is limited by frequent inflammatory cutaneous adverse events that affect patients' quality of life and may lead to dose reduction or even cessation of anti-tumor therapy. To date, the molecular and cellular mechanisms of vemurafenib-induced rashes have remained largely elusive. In this study, we deployed immunohistochemistry, RT-qPCR, flow cytometry, lymphocyte activation tests, and different cell-free protein-interaction assays. We here demonstrate that vemurafenib inhibits the downstream signaling of the canonical pathway of aryl hydrocarbon receptor (AhR) in vitro, thereby inducing the expression of proinflammatory cytokines (eg, TNF) and chemokines (eg, CCL5). In line with these results, we observed an impaired expression of AhR-regulated genes (eg, CYP1A1) and an upregulation of the corresponding proinflammatory genes in vivo. Moreover, results of lymphocyte activation tests showed the absence of drug-specific T cells in respective patients. Taken together, we obtained no hint of an underlying sensitization against vemurafenib but found evidence suggesting that vemurafenib enhances proinflammatory responses by inhibition of canonical AhR signaling. Our findings contribute to our understanding of the central role of the AhR in skin inflammation and may point toward a potential role for topical AhR agonists in supportive cancer care.

MEK inhibitor cobimetinib rescues a dRaf mutant lethal phenotype in Drosophila melanogaster.

Since Drosophila melanogaster has proven to be a useful model system to study phenotypes of oncogenic mutations and to identify new anti-cancer drugs, we generated human BRAFV600E homologous dRaf mutant (dRafA572E ) Drosophila melanogaster strains to use these for characterisation of mutant phenotypes and exploit these phenotypes for drug testing. For mutant gene expression, the GAL4/UAS expression system was used. dRafA572E was expressed tissue-specific in the eye, epidermis, heart, wings, secretory glands and in the whole animal. Expression of dRaf A572E under the control of an eye-specific driver led to semi-lethality and a rough eye phenotype. The vast majority of other tissue-specific and ubiquitous drivers led to a lethal phenotype only. The rough eye phenotype was used to test BRAF inhibitor vemurafenib and MEK1/2 inhibitor cobimetinib. There was no phenotype rescue by this treatment. However, a significant rescue of the lethal phenotype was observed under a gut-specific driver. Here, MEK1/2 inhibitor cobimetinib rescued Drosophila larvae to reach pupal stage in 37% of cases as compared to 1% in control experiments. Taken together, the BRAFV600E homolog dRaf A572E exerts mostly lethal effects in Drosophila. Gut-specific dRaf A572E expression might in future be developed further for drug testing.

Abstract 4864: Targeted melanoma therapies as radiosensitizers

Abstract Introduction: In BRAFV600 mutant melanoma combination of BRAF and MEK inhibitors is associated with a rapid response in approximately 70% of patients, and an improvement in survival. Radiotherapy is used in the treatment of melanoma in multiple clinical settings including stereotactic radiosurgery for brain metastasis, oligo-metastatic extracranial disease and palliative radiotherapy to symptomatic lesions. Preclinical studies, and a small number of clinical case reports suggest the BRAF inhibitor vemurafenib and the MEK inhibitor trametinib leads to increased radiosensitivity. Aims: To determine if BRAF Inhibitors dabrafenib, vemurafenib and encorafenib, alone or in combination with their corresponding MEK Inhibitors, are radiation-sensitising in BRAF-mutant treatment naive melanoma cells. Methods: Three melanoma cell lines harbouring a BRAF V600E mutation were studied: A375, SkMel28 and MM200. The half-maximal inhibitory concentration (IC50) for each cell line was determined by MTS assays for vemurafenib, dabrafenib, encorafenib, and the MEK inhibitors trametinib, binimetinib and cobimetinib. Clonogenic assays were conducted on each cell line. The optimal dose of BRAF and MEK inhibitors was determined from MTS assay and single drug clonogenic assays. Cells were treated with BRAF or MEK inhibitor alone and in combination. Cells were also treated with increasing doses of radiation (X-RAD 320 irradiator, PXI). The mode of cell death and changes in cell morphology were determined by live cell imaging (Wide Field Live Cell Observer, Ziess) for five days following the above treatments. Results:Using Clonogenic assays, we confirmed that radiation sensitivity varies across different melanoma cell lines, with MM200 being significantly more sensitive to radiation then SKMel28. Clonogenic assays demonstrated that vemurafenib, dabrafenib, encorafenib, trametinib and binimetinib increased radiosensitivity across all three-cell lines. Further, the level of radiosensitsation was greater in combination across all three sets, than with either BRAF or MEK inhibitor alone. Using live cell imaging, we were able to confirm this increase in cell death, and visualise differences in the mode of cell death and morphology with these therapies. Conclusions: Vemurafenib, dabrafenib, encorafenib, trametinib and binimetinib were shown in this study to be radiosensitisers in BRAFV600Emutant melanoma cell lines. Radiosensitisation was greatest with combined BRAF and MEK inhibition. Further studies are being conducted to determine if this sensitisation continues after BRAF resistance develops. Citation Format: Jessica L. Smith, Cecilia Chang, Tim Wang, Helen Rizos, Han Shen, Matteo Carlino, Eric Hau. Targeted melanoma therapies as radiosensitizers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4864.

Abstract 3550: microRNA-211 promotes aggressive melanoma growth in vivo by epigenetic modification, and contributes to BRAFV600E inhibitor resistance via ERK5 signaling

Abstract The microRNA miR-211 is an established participant in melanomagenesis, but controversy exists as to whether it acts as a bone fide tumor suppressor or oncogene. Here we ectopically expressed miR-211 in the BRAF v600E-mutant A375 melanoma cell line and examined its effect in xenografts in vivo. The miR-211 ectopic expression promoted aggressive tumor xenograft growth with extensive cell proliferation, and angiogenesis. ChIP-seq and single cell sequencing analysis of xenograft tissues demonstrated that aggressive tumor formation is partly associated with H3K27me3 and H3K4me3, and migration of cells from mouse tissues to tumor locus. Interrogation of xenograft transcriptomics data revealed activation of the ERK5 pathway, itself negatively regulated by miR-211 target genes, BIRC2 and DUSP6, further confirmed as direct miR-211 target genes by RNA immunopurification with RNA-seq (RIP-seq) and site-directed mutagenesis. miR-211 conferred resistance to the BRAF inhibitor vemurafenib, and MEK inhibitor cobimetinib with corresponding increases in ERK5 phosphorylation. The miR-211-ERK5 axis may represent a novel therapeutic target, but however, miR-211 is exquisitely pleiotropic in the complex in vivo tumor environment and its context must be considered carefully in diagnostic and therapeutic development. Citation Format: Bongyong Lee, Anupama Sahoo, Junko Sawada, Dimitrios G. Zisoulis, John Marchica, Sanjay Sahoo, Fabiana I Alves De Lima Layng, Darren Finlay, Joseph Mazar, Piyush Joshi, Masanobu Komatsu, Kristiina Vuori, Garth Powis, Petrus R. de Jong, Animesh Ray, Ranjan J. Perera. microRNA-211 promotes aggressive melanoma growth in vivo by epigenetic modification, and contributes to BRAFV600E inhibitor resistance via ERK5 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3550.

Conjunctival Melanoma Targeted Therapy: MAPK and PI3K/mTOR Pathways Inhibition.

To analyze the activity of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases/mechanistic target of rapamycin (PI3K/mTOR) pathways in benign and malignant conjunctival melanocytic proliferations and explore whether specific inhibitors can suppress growth of conjunctival melanoma (CJM) cells. The presence of a BRAF V600E mutation and activation of ERK, MEK, S6, and AKT were assessed with immunohistochemistry in 35 conjunctival nevi and 31 melanomas. Three CJM cell lines were used: CRMM1, carrying the BRAF V600E mutation; CRMM2, harboring the NRAS Q61L mutation; and T1527A, with a BRAF G466E mutation. WST-1 assays were performed with a BRAF inhibitor (vemurafenib), two MEK inhibitors (trametinib, selumetinib), a PI3K inhibitor (pictilisib), and a dual PI3K/mTOR inhibitor (dactolisib). The phosphorylation of ERK, MEK, and S6 were tested with western blots and apoptosis with cleaved caspase-3 immunostaining. A BRAF V600E mutation was detected in 42.6% of nevi and in 35.5% of CJM. MEK and ERK activation were higher in CJM, occurring in 62.9% and 45.7% of the nevi and 90.3% and 96.8% of the CJM, respectively. There was also a significant increase in S6 activation in CJM (90.3%) compared with the nevi (20%). CRMM1 was sensitive to trametinib and the PI3K inhibitors but only marginally to vemurafenib. CRMM2 was moderately sensitive to pictilisib, whereas T1527A was resistant to all drugs tested. The MAPK pathway activity in CJM is increased, not only as a consequence of the BRAF V600E mutation. Targeted therapy may be useful for patients with CJM, especially those with activating BRAF mutations, whereas NRAS-mutated melanomas are relatively resistant.

Efficacy of Vemurafenib in Patients With Non-Small-Cell Lung Cancer With BRAF V600 Mutation: An Open-Label, Single-Arm Cohort of the Histology-Independent VE-BASKET Study.

PURPOSETo study whether BRAF V600 mutations in non–small-cell lung cancer (NSCLC) may indicate sensitivity to the BRAF inhibitor vemurafenib, we included a cohort of patients with NSCLC in the vemurafenib basket (VE-BASKET) study. On the basis of observed early clinical activity, we expanded the cohort of patients with NSCLC. We present results from this cohort.METHODSThis open-label, histology-independent, phase II study included six prespecified cohorts, including patients with NSCLC, and a seventh all-comers cohort. Patients received vemurafenib (960 mg two times per day) until disease progression or unacceptable toxicity. The primary end point of the final analysis was objective response rate (Response Evaluation Criteria in Solid Tumors, version 1.1). Secondary end points included progression-free survival, overall survival, and safety. Because the prespecified clinical benefit endpoint was met in the initial NSCLC cohort, the cohort was expanded.RESULTSSixty-two patients with BRAF V600–mutant NSCLC were enrolled and treated: 13% (n = 8) had received no prior systemic therapy, and 87% (n = 54) had received prior therapies. The objective response rate was 37.1% (95% CI, 25.2% to 50.3%) overall, 37.5% (95% CI, 8.5% to 75.5%) in previously untreated patients, and 37.0% (24.3% to 51.3%) in previously treated patients. Median progression-free survival was 6.5 months (95% CI, 5.2 to 9.0 months), and median overall survival was 15.4 months (95% CI, 9.6 to 22.8 months). The most common all-grade adverse event was nausea (40%). The safety profile of vemurafenib was similar to that observed in melanoma studies.CONCLUSIONVemurafenib showed promising activity in patients with NSCLC harboring BRAF V600 mutations. The safety profile of vemurafenib was similar to previous observations in patients with melanoma. Our results suggest a role for single-agent BRAF inhibition in patients with NSCLC and BRAF V600 mutations.

Essential Oil of Mentha aquatica var. Kenting Water Mint Suppresses Two-Stage Skin Carcinogenesis Accelerated by BRAF Inhibitor Vemurafenib.

The v-raf murine sarcoma viral homolog B1 (BRAF) inhibitor drug vemurafenib (PLX4032) is used to treat melanoma; however, epidemiological evidence reveals that it could cause cutaneous keratoacanthomas and squamous cell carcinoma in cancer patients with the most prevalent HRASQ61L mutation. In a two-stage skin carcinogenesis mouse model, the skin papillomas induced by 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) (DT) resemble the lesions in BRAF inhibitor-treated patients. In this study, we investigated the bioactivity of Mentha aquatica var. Kenting Water Mint essential oil (KWM-EO) against PDV cells, mouse keratinocytes bearing HRASQ61L mutation, and its effect on inhibiting papilloma formation in a two-stage skin carcinogenesis mouse model with or without PLX4032 co-treatment. Our results revealed that KWM-EO effectively attenuated cell viability, colony formation, and the invasive and migratory abilities of PDV cells. Induction of G2/M cell-cycle arrest and apoptosis in PDV cells was also observed. KWM-EO treatment significantly decreased the formation of cutaneous papilloma further induced by PLX4032 in DT mice (DTP). Immunohistochemistry analyses showed overexpression of keratin14 and COX-2 in DT and DTP skin were profoundly suppressed by KWM-EO treatment. This study demonstrates that KWM-EO has chemopreventive effects against PLX4032-induced cutaneous side-effects in a DMBA/TPA-induced two-stage carcinogenesis model and will be worth further exploration for possible application in melanoma patients.

Plasma lncRNA expression profile as a prognostic tool in BRAF-mutant metastatic melanoma patients treated with BRAF inhibitor.

Long non-coding RNAs (lncRNA) are dysregulated in many cancer types. Abnormal baseline levels of these lncRNAs display diagnostic and prognostic potential in cancer patients. The aim of this study was to evaluate the prognostic value of plasma lncRNAs in BRAF-mutant advanced melanoma patients treated with a BRAF inhibitor. Total RNA was isolated from plasma samples collected from 58 advanced BRAF-mutant melanoma patients and 15 healthy donors. The expression levels of 90 lncRNAs were estimated using the LncProfiler qPCR Array Kit (SBI) and LightCycler 96 (Roche). LncRNA expression levels correlated with responses to the BRAF inhibitor (vemurafenib) treatment. The patients were stratified into three groups based on their lncRNA levels with various lncRNA expressions (low, medium, and high). A Cox proportional hazards regression model was used to determine the lncRNAs that were significantly associated with both progression-free and overall survivals (PFS and OS, respectively) in patients receiving vemurafenib. The expression level of 12 lncRNAs was down-regulated, while five lncRNAs were up-regulated in melanoma patients compared to healthy donors. Kaplan-Meier analysis showed that upregulation or downregulation of 11 and 16 different lncRNAs were associated with longer median PFS and OS, respectively. Further analysis demonstrated that the baseline lncRNAs for IGF2AS, anti-Peg11, MEG3, Zeb2NAT are independent prognostic factors in BRAF-mutant advanced melanoma patients treated with vemurafenib. Evaluation of plasma lncRNAs expression level for advanced melanoma diagnosis and prognosis evaluation appears to be a safe and valuable method; however, this method requires further validation in larger cohorts and randomized trials.

Highlights of This Issue

Treatments combining chemotherapeutic drugs with BCL-2/XL inhibitors are precluded due to dose-limiting toxicities in patients. Here, Zoeller and colleagues describe a novel drug treatment strategy that avoids the systemic toxicities associated with such combined treatments by selectively targeting the chemotherapeutic agent to the tumor cells using an antibody-drug conjugate (ADC). This therapeutic strategy combines T-DM1, the HER2-targeted ADC, with inhibitors of the pro-survival proteins BCL-2/XL. The authors provide in vivo evidence that BCL-2/XL inhibition significantly enhanced the effectiveness of T-DM1. These pre-clinical results provide rationale for additional investigation and clinical translation of treatment strategies that combine antibody-drug conjugates and apoptotic inhibitors.Human papillomavirus negative (HPV[-]) head and neck squamous cell carcinoma (HNSCC) represents a distinct genetic alteration pattern associated with a worse prognosis and increased radioresistance. To address this, Yang and colleagues focused on Inhibitor of Apoptosis Proteins (IAPs). IAP overexpression was associated with poor overall survival and with HPV[-] HNSCC. Therefore, they administered a SMAC mimetic (LCL161) that degrades cIAP1 to radiosensitize HPV[-] HNSCC mouse xenografts. Chemoradiotherapy with LCL161 was well tolerated and led to a significant reduction in tumor size by initiating apoptosis. Taken together, the results demonstrate the potency of SMAC mimetics in HPV[-] HNSCC and support the initiation of clinical trials with radiation and IAP inhibitors in this population of patients.BRD4 inhibitors were recently shown to suppress expression of the immune checkpoint ligand PD-L1 and to restore immune-active environments by reducing myeloid-derived suppressor cells and increasing anti-tumor immunity. Despite the crucial role they play in tumor immunity, macrophages have not been characterized in the context of BET inhibitors. Assuming a connection to macrophage programming and informed by previous results implicating PI3K signaling, Joshi and colleagues demonstrated a dual PI3K/BRD4 inhibitor (SF2523) reduced tumor growth, immunosuppression, and metastasis. Their results link BRD4 to the polarization of immunosuppressive macrophages. Furthermore, they demonstrate BRD4 inhibitors (JQ1) and/or PI3K/BRD4 dual inhibitors (SF2523) stimulate anti-tumor immune responses and are a therapeutic strategy for cancers driven by macrophage-dependent immunosuppression.The response to therapies for non-Langerhans cell histiocytosis (non-LCH), such as Erdheim-Chester disease (ECD) and Rosai-Dorfman disease (RDD), is dependent on the mutational landscape of the disease. For example, BRAFV600E-positive ECD respond to BRAF inhibitors dabrafenib and vemurafenib. Similarly, RDD patients with MAPK pathway alterations potentially respond to MEK inhibitors. Therefore, Janku and colleagues sought to define the molecular profile in 39 non-LCH patients by sequencing tumor samples and plasma-derived cfDNA. They demonstrate 50% of evaluable patients had BRAFV600E and that 58% contained at least one alteration in the MAPK pathway. Their results highlight the practicality of cfDNA for non-LCH patients and suggest MAPK activation is a hallmark of non-LCH.