Pseudoprogression,radionecrosis, inflammation or true tumor progression? challenges associated with glioblastoma response assessment in an evolving therapeutic landscape

BRAF^V600E is a common finding in glioma (about 10–60% depending on histopathologic subclassification). BRAF^V600E monotherapy shows modest preclinical efficacy against BRAF^V600E gliomas and also induces adverse secondary skin malignancies. Here, we examine the molecular mechanism of intrinsic resistance to BRAF^V600E inhibition in glioma. Furthermore, we investigate BRAF^V600E/MEK combination therapy that overcomes intrinsic resistance to BRAF^V600E inhibitor and also prevents BRAF^V600E inhibitor induced secondary malignancies. Immunoblotting and Human Phospho-Receptor Tyrosine Kinase Array assays were used to interrogate MAPK pathway activation. The cellular effect of BRAF^V600E and MEK inhibition was determined by WST-1 viability assay and cell cycle analysis. Flanked and orthotopic GBM mouse models were used to investigate the in vivo efficacy of BRAF^V600E/MEK combination therapy and the effect on secondary malignancies. BRAF^V600E inhibition leads to recovery of ERK phosphorylation. Combined BRAF^V600E and MEK inhibition prevents reactivation of the MAPK signaling, which correlates with decreased cell viability and augmented cell cycle arrest. Similarly, mice bearing BRAF^V600E glioma showed reduced tumor growth when treated with a combination of BRAF^V600E and MEK inhibitor compared to BRAF^V600E inhibition alone. Additional benefit of BRAF^V600E/MEK inhibition was reflected by reduced cutaneous squamous-cell carcinoma (cSCC) growth (a surrogate for RAS-driven secondary maligancies). In glioma, recovery of MAPK signaling upon BRAF inhibition accounts for intrinsic resistance to BRAF^V600E inhibitor. Combined BRAF^V600E and MEK inhibition prevents rebound of MAPK activation, resulting in enhanced antitumor efficacy and also reduces the risk of secondary malignancy development.     

BRAFV600E mutation: A promising target in colorectal neuroendocrine carcinoma

To determine the role of BRAF^V600E mutation and MAPK signaling as well as the effects of BRAF and MEK directed therapy in gastroenteropancreatic neuroendocrine neoplasia (GEP-NEN), with a focus on highly aggressive gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC). Using Sanger sequencing of BRAF exon 15 we determined the frequency of BRAF^V600E mutations in 71 primary GEP-NENs. MEK phosphorylation was examined by immunohistochemistry in corresponding tissue samples. To evaluate the biological relevance of BRAF^V600E mutation and MAPK signaling in GEP-NECs, effects of a pharmacological BRAF and MEK inhibition were analyzed in NEC cell lines both in vitro and in vivo. BRAF^V600E mutation was detected in 9.9% of all GEP-NENs. Interestingly, only NECs of the colon harbored BRAF^V600E mutations, leading to a mutation frequency of 46.7% in this subgroup of patients. In addition, a BRAF^V600E mutation was significantly associated with high levels of MEK phosphorylation (pMEK) and advanced tumor stages. Pharmacological inhibition of BRAF and MEK abrogated NEC cell growth, inducing G1 cell cycle arrest and apoptosis only in BRAF^V600E mutated cells. BRAF inhibitor dabrafenib and MEK inhibitor trametinib prevented growth of BRAF^V600E positive NEC xenografts. High frequencies of BRAF^V600E mutation and elevated expression levels of pMEK were detected in biologically aggressive and highly proliferative colorectal NECs. We provide evidence that targeting BRAF oncogene may represent a therapeutic strategy for patients with BRAF mutant colorectal NECs.     

Development of a potent small‐molecule degrader against oncogenic BRAFV600E protein that evades paradoxical MAPK activation

BRAF mutations are frequently observed in melanoma and hairy‐cell leukemia. Currently approved rapidly accelerated fibrosarcoma (RAF) kinase inhibitors targeting oncogenic BRAF V600 mutations have shown remarkable efficacy in the clinic, but their therapeutic benefits are occasionally hampered by acquired resistance due to RAF dimerization–dependent reactivation of the downstream MAPK pathway, which is known as paradoxical activation. There is also a concern that paradoxical activation of the MAPK pathway may trigger secondary cancer progression. In this study, we developed chimeric compounds, proteolysis targeting chimeras (PROTACs), that target BRAF^V600E protein for degradation. CRBN(BRAF)‐24, the most effective chimera, potently degraded BRAF^V600E in a ubiquitin‐proteasome system (UPS)‐dependent manner and inhibited the proliferation of BRAF^V600E‐driven cancer cells. In BRAF wild‐type cells, CRBN(BRAF)‐24 induced neither BRAF^WT degradation nor paradoxical activation of the MAPK pathway. Biochemical analysis revealed that CRBN(BRAF)‐24 showed more potent and sustained suppression of MAPK signaling than a BRAF^V600E inhibitor, PLX‐8394, in BRAF^V600E‐driven cancer cells. Targeted degradation of BRAF^V600E by CRBN(BRAF)‐24 could be a promising strategy to evade paradoxical activation of the RAF‐MAPK pathway.     

Pluripotency markers are differentially induced by MEK inhibition in thyroid and melanoma BRAFV600E cell lines

Oncogenic mutations in BRAF are common in melanoma and thyroid carcinoma and drive constitutive activation of the MAPK pathway. Molecularly targeted therapies of this pathway improves survival compared to chemotherapy; however, responses tend to be short-lived as resistance invariably occursCell line models of melanoma and thyroid carcinoma, +/? BRAF^V600E activating mutation, were treated with the MEK inhibitor PD0325901. Treated and naive samples were assayed for expression of key members of the MAPK pathway. Global microRNA expression profiling of naive and resistant cells was performed via next generation sequencingand indicated pluripotency pathways in resistance. Parental cell lines were progressed to holoclones to confirm the miRNA stemness profileMembers of the MIR302/373/374/520 family of embryonic stem cell specific cell cycle regulating (ESCC) microRNAs were identified as differentially expressed between resistant BRAF^V600E melanoma and thyroid cell lines. Upregulated expression of gene and protein stemness markers, upregulated expression of MAPK pathway genes and downregulation of the ESCC MIR302 cluster in BRAF^V600E melanoma indicated an increased stem-like phenotype in resistant BRAF^V600E melanoma. Conversely, downregulated expression of gene and protein stemness markers, downregulated expression of MAPK pathway genes, upregulation of the ESCC MIR520 cluster, reeexpression of cell surface receptors, and induced differentiation-associated morphology in resistant BRAF^V600E indicate a differentiated phenotype associated with MEK inhibitor resistance in BRAF^V600E thyroid cellsThe differential patterns of resistance observed between BRAF^V600E melanoma and thyroid cell lines may reflect tissue type or de novo differentiation, but could have significant impact on the response of primary and metastatic cells to MEK inhibitor treatment. This study provides a basis for the investigation of the cellular differentiation/self-renewal access and its role in resistance to MEK inhibition.     

EBI-907, a novel BRAFV600E inhibitor,has potent oral anti-tumor activity and a broad kinase selectivity profile

The oncogenic mutation of BRAF^V600E has been found in approximately 8% of all human cancers, including more than 60% of melanoma and 10% of colorectal cancers. The clinical proof of concept in treating BRAF^V600E-driving melanoma patients with the BRAF inhibitors has been well established. We have sought to identify and develop novel BRAF^V600E inhibitors with more favorable profiles. Our chemistry effort has led to the discovery of EBI-907 as a novel BRAF^V600E inhibitor with potent anti-tumor activity in vitro and in vivo. In a LanthaScreen BRAF^V600E kinase assay, EBI-907 showed an IC50 of 4.8 nM, which is >10 -fold more potent than Vemurafenib (IC50 = 58.5 nM). In addition, EBI-907 showed a broader kinase selectivity profile, with potent activity against a number of important oncogenic kinases including FGFR1-3, RET, c-Kit, and PDGFRb. Concomitant with such properties, EBI-907 exhibits potent and selective cytotoxicity against a broader range of BRAF^V600E-dependent cell lines including certain colorectal cancer cell lines with innate resistance to Vemurafenib. In BRAF^V600E-dependent human Colo-205 and A375 tumor xenograft mouse models, EBI-907 caused a marked tumor regression in a dose-dependent manner, with superior efficacy to Vemurafenib. Our results also showed that combination with EGFR or MEK inhibitor enhanced the potency of EBI-907 in cell lines with innate or acquired resistance to BRAF inhibition alone. Our findings present EBI-907 as a potent and promising BRAF inhibitor, which might be useful in broader indications.     

Sema6A and Mical1 control cell growth and survival of BRAFV600E human melanoma cells

We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAF^V600E vs NRAS^Q61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAF^V600E and NRAS^Q61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAF^V600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRAS^Q61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAF^V600E melanoma.     

Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAFV600E patient-derived papillary thyroid carcinoma preclinical model

BRAF^V600E mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAF^V600E inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAF^V600E selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAF^V600E-PTC, intrathyroidal BRAF^V600E-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAF^V600E-PTC orthotopic mouse. We find that metastatic BRAF^V600E-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAF^WT-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAF^V600E-PTC cells but lesser in metastatic BRAF^V600E-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment–associated pro-metastatic molecules, with no effects in BRAF^WT-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAF^WT/V600E-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAF^V600E-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAF^V600E-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAF^V600E-positive PTC.     

Mitochondrial complex I inhibitor deguelin induces metabolic reprogramming and sensitizes vemurafenib‐resistant BRAFV600E mutation bearing metastatic melanoma cells

Treatment with vemurafenib, a potent and selective inhibitor of mitogen‐activated protein kinase signaling downstream of the BRAF^V600E oncogene, elicits dramatic clinical responses in patients with metastatic melanoma. Unfortunately, the clinical utility of this drug is limited by a high incidence of drug resistance. Thus, there is an unmet need for alternative therapeutic strategies to treat vemurafenib‐resistant metastatic melanomas. We have conducted high‐throughput screening of two bioactive compound libraries (Siga and Spectrum libraries) against a metastatic melanoma cell line (A2058) and identified two structurally analogous compounds, deguelin and rotenone, from a cell viability assay. Vemurafenib‐resistant melanoma cell lines, A2058R and A375R (containing the BRAF^V600E mutation), also showed reduced proliferation when treated with these two compounds. Deguelin, a mitochondrial complex I inhibitor, was noted to significantly inhibit oxygen consumption in cellular metabolism assays. Mechanistically, deguelin treatment rapidly activates AMPK signaling, which results in inhibition of mTORC1 signaling and differential phosphorylation of mTORC1's downstream effectors, 4E‐BP1 and p70S6 kinase. Deguelin also significantly inhibited ERK activation and Ki67 expression without altering Akt activation in the same timeframe in the vemurafenib‐resistant melanoma cells. These data posit that treatment with metabolic regulators, such as deguelin, can lead to energy starvation, thereby modulating the intracellular metabolic environment and reducing survival of drug‐resistant melanomas harboring BRAF ^V600E mutations.     

Nuclear interaction of Arp2/3 complex and BRAFV600E promotes aggressive behavior and vemurafenib resistance of thyroid cancer

The presence of mutant BRAF ^V600E correlates with the risk of recurrence in papillary thyroid cancer (PTC) patients. However, not all PTC patients with BRAF ^V600E are associated with poor prognosis. Thus, understanding the mechanisms by which certain PTC patients with nuclear BRAF ^V600E become aggressive and develop resistance to a selective BRAF inhibitor, PLX-4032, is urgently needed. The effect of nuclear localization of BRAF^V600E using in vitro studies, xenograft mouse-model and human tissues was evaluated. PTC cells harboring a nuclear localization signal (NLS) of BRAF^V600E were established and examined in nude mice implanted with TPC1-NLS-BRAF^V600E cells followed by PLX-4032 treatment. Immunohistochemical (IHC) analysis was performed on 100 PTC specimens previously confirmed that they have BRAF^V600E mutations. Our results demonstrate that 21 of 100 (21%) PTC tissues stained with specific BRAF^V600E antibody had nuclear staining with more aggressive features compared to their cytosolic counterparts. In vitro studies show that BRAF^V600E is transported between the nucleus and the cytosol through CRM1 and importin (α/β) system. Sequestration of BRAF^V600E in the cytosol sensitized resistant cells to PLX-4032, whereas nuclear BRAF^V600E was associated with aggressive phenotypes and developed drug resistance. Proteomic analysis revealed Arp2/3 complex members, actin-related protein 2 (ACTR2 aliases ARP2) and actin-related protein 3 (ACTR3 aliases ARP3), as the most enriched nuclear BRAF^V600E partners. ACTR3 was highly correlated to lymph node stage and extrathyroidal extension and was validated with different functional assays. Our findings provide new insights into the clinical utility of the nuclear BRAF^V600E as a prognostic marker for PTC aggressiveness and determine the efficacy of selective BRAF^V600E inhibitor treatment which opens new avenues for future treatment options.     

Functional characterization of the novel BRAF complex mutation,BRAFV600delinsYM,identified in papillary thyroid carcinoma

An activating mutation in the BRAF gene is the most common genetic alteration in papillary thyroid carcinomas (PTCs). The mutation in PTCs is almost a c.1799T>A transversion, resulting in a p.V600E amino acid substitution (BRAF^V600E). Here, we report a novel complex BRAF mutation identified in 4/492 Japanese PTC cases (0.81%). The mutation was comprised of one nucleotide substitution at position 1798, followed by an in‐frame insertion of three nucleotides, c.1798delinsTACA in Exon 15, resulting in p.V600delinsYM. In silico three‐dimensional protein structure prediction implied altered kinase activity of this mutant. In vitro kinase assay and western blotting revealed that this mutation conferred high kinase activity on the BRAF protein, leading to constitutive activation of the MAPK signaling pathway. The mutation also showed high transforming ability in focus formation assay using NIH3T3 cells. The degree of all the functional characteristics was comparable to that of BRAF^V600E, and treatment with a BRAF inhibitor Sorafenib was also equally effective in this mutant. These findings suggest that the novel BRAF mutation, BRAF^V600delinsYM, is a gain‐of‐function mutation and plays an important role in PTC development.     

Melanoma patient derived xenografts acquire distinct Vemurafenib resistance mechanisms

Variable clinical responses, tumor heterogeneity, and drug resistance reduce long-term survival outcomes for metastatic melanoma patients. To guide and accelerate drug development, we characterized tumor responses for five melanoma patient derived xenograft models treated with Vemurafenib. Three BRAF^V600E models showed acquired drug resistance, one BRAF^V600E model had a complete and durable response, and a BRAF^V600V model was expectedly unresponsive. In progressing tumors, a variety of resistance mechanisms to BRAF inhibition were uncovered, including mutant BRAF alternative splicing, NRAS mutation, COT (MAP3K8) overexpression, and increased mutant BRAF gene amplification and copy number. The resistance mechanisms among the patient derived xenograft models were similar to the resistance pathways identified in clinical specimens from patients progressing on BRAF inhibitor therapy. In addition, there was both inter- and intra-patient heterogeneity in resistance mechanisms, accompanied by heterogeneous pERK expression immunostaining profiles. MEK monotherapy of Vemurafenib-resistant tumors caused toxicity and acquired drug resistance. However, tumors were eradicated when Vemurafenib was combined the MEK inhibitor. The diversity of drug responses among the xenograft models; the distinct mechanisms of resistance; and the ability to overcome resistance by the addition of a MEK inhibitor provide a scheduling rationale for clinical trials of next-generation drug combinations.     

BRAFV600E melanoma cells secrete factors that activate stromal fibroblasts and enhance tumourigenicity

Background:

Melanoma, the most lethal form of skin cancer, is responsible for over 80% of all skin cancer deaths and is highly metastatic, readily spreading to the lymph nodes or metastasising to other organs. The frequent genetic mutation found in metastatic melanoma, BRAF^V600E, results in constitutive activation of the mitogen-activated protein kinase pathway.

Methods:

In this study, we utilised genetically engineered melanoma cell lines and xenograft mouse models to investigate how BRAF^V600E affected cytokine (IL-1β, IL-6, and IL-8) and matrix metalloproteinase-1 (MMP-1) expression in tumour cells and in human dermal fibroblasts.

Results:

We found that BRAF^V600E melanoma cells expressed higher levels of these cytokines and of MMP-1 than wild-type counterparts. Further, conditioned medium from the BRAF^V600E melanoma cells promoted the activation of stromal fibroblasts, inducing expression of SDF-1 and its receptor CXCR4. This increase was mitigated when the conditioned medium was taken from melanoma cells treated with the BRAF^V600E specific inhibitor, vemurafenib.

Conclusions:

Our findings highlight the role of BRAF^V600E in activating the stroma and suggest a mechanistic link between BRAF^V600E and MMP-1 in mediating melanoma progression and in activating adjacent fibroblasts in the tumour microenvironment.     

Non-V600E BRAF mutations and EGFR signaling pathway in colorectal cancer

The Raf murine sarcoma viral oncogene homolog B (BRAF^V600E) mutation (MT) in metastatic colorectal cancer (CRC) is a well-known prognostic indicator and a negative predictive biomarker for antiepidermal growth factor receptor (EGFR) treatment. However, the clinical characteristics and significance of BRAF^non-V600E MTs remain unclear. Here, we evaluated the clinical characteristics of BRAF^non-V600E MTs vs. those of other MTs in the EGFR signaling pathway, including BRAF^V600E. Consecutive CRC patients in our institute from June 2012 to November 2013 were enrolled in our study. Multiplex genotyping of the EGFR pathway was performed with archival samples using a Luminex Assay for BRAF^V600E/BRAF^non-V600E, KRAS/NRAS exons 2–4, and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA). We analyzed correlations among the MT profiles, clinical data and primary tumor locations in CRC. All statistical analyses were performed using R software. CRC samples (824) from 374 (45.4%) male and 450 (54.6%) female patients were analyzed, of which 154 (18.7%), 202 (24.5%), 270 (32.8%) or 198 (24.0%) had Stages I, II, III or IV or recurrent CRC, respectively. The frequencies of BRAF^V600E/BRAF^non-V600E, KRAS (including exons 2–4), NRAS and PIK3CA MTs were 5.3/1.7, 41.4, 3.3 and 9.6%, respectively. The characteristics of patients with the BRAF^V600E MT were an age of ≥65 years old, a right-sided primary tumor location, poorly differentiated histology and an advanced disease stage. In contrast, the characteristics of patients with BRAF^non-V600E MTs were a left-sided primary tumor location and well-differentiated histology. BRAF^non-V600E MTs were relatively rare and showed different characteristics compared to the BRAF^V600E MT. These results may contribute to future precision medicine.     

Targeting glutamine transport to suppress melanoma cell growth

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAF^WT (C8161 and WM852) and BRAF^V600E mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2‐mediated glutamine transport is a potential therapeutic target for both BRAF^WT and BRAF^V600E melanoma.     

Advances in the therapy of BRAFV600E metastatic colorectal cancer

ABSTRACT

Introduction: BRAF^V600E metastatic colorectal cancer (CRC) is an aggressive tumor subset with an approximate 8% incidence. In these patients, standard chemotherapy has limited efficacy, and the recent development of novel-targeted treatment regimens may significantly improve clinical outcome.

Area covered: This review provides an overview of available data regarding advances in the first–line treatment of BRAF^V600E metastatic CRC including patient tumors with microsatellite instability. The implications of BRAF^V600E in earlier stage CRC are also discussed.

Expert opinion: Recently, significant progress has been achieved in improving tumor response rates using a novel-targeted regimen in patients with BRAF^V600E metastatic CRC. The implications of BRAF^V600E in non-metastatic CRC are also becoming more evident and remains an area of ongoing investigation. The majority of CRCs with microsatellite instability high are sporadic and frequently harbor BRAF^V600E. All patients with microsatellite instability high metastatic CRCs, irrespective of BRAF^V600E, are candidates for immune checkpoint inhibitors. The optimal sequencing of treatment regimens for patients with BRAF^V600E metastatic CRCs is an important area for future research.     

Dysfunctional oxidative phosphorylation makes malignant melanoma cells addicted to glycolysis driven by the V600EBRAF oncogene

Oncogene addiction describes how cancer cells exhibit dependence on single oncogenes to escape apoptosis and senescence. While oncogene addiction constitutes the basis for new cancer treatment strategies targeting individual kinases and pathways activated by oncogenic mutations, the biochemical basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to ^V600EBRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS). Notably, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that ^V600EBRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes an addiction to ^V600EBRAF. Finally, the senescence response associated with inhibition of ^V600EBRAF is rescued by overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing direct evidence that oncogene addiction rests on a metabolic foundation.     

Identification of MET and SRC Activation in Melanoma Cell Lines Showing Primary Resistance to PLX4032

PLX4032/vemurafenib is a first-in-class small-molecule BRAF^V600E inhibitor with clinical activity in patients with BRAF mutant melanoma. Nevertheless, drug resistance develops in treated patients, and strategies to overcome primary and acquired resistance are required. To explore the molecular mechanisms involved in primary resistance to PLX4032, we investigated its effects on cell proliferation and signaling in a panel of 27 genetically characterized patient-derived melanoma cell lines. Cell sensitivity to PLX4032 was dependent on BRAF^V600E and independent from other gene alterations that commonly occur in melanoma such as PTEN loss, BRAF, and MITF gene amplification. Two cell lines lacking sensitivity to PLX4032 and harboring a different set of genetic alterations were studied as models of primary resistance. Treatment with the MEK inhibitor UO126 but not with PLX4032 inhibited cell growth and ERK activation. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA indicating alternative activation of MEK-ERK signaling. Genetic characterization by multiplex ligation-dependent probe amplification and analysis of phosphotyrosine signaling by MALDI-TOF mass spectrometry analysis revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. The combination of PLX4032 with drugs or siRNA targeting MET was effective in inhibiting cell growth and reducing cell invasion and migration in melanoma cells with MET amplification; similar effects were observed after targeting SRC in the other cell line, indicating a role for MET and SRC signaling in primary resistance to PLX4032. Our results support the development of classification of melanoma in molecular subtypes for more effective therapies.     

Lung Cancer Driven by BRAFG469V Mutation Is Targetable by EGFR Kinase Inhibitors

IntroductionMutations in BRAF occur in 2% to 4% of patients with lung adenocarcinoma. Combination dabrafenib and trametinib, or single-agent vemurafenib is approved only for patients with cancers driven by the V600E BRAF mutation. Targeted therapy is not currently available for patients harboring non-V600 BRAF mutations.MethodsA lung adenocarcinoma patient-derived xenograft model (PHLC12) with wild-type and nonamplified EGFR was tested for response to EGFR tyrosine kinase inhibitors (TKIs). A cell line derived from this model (X12CL) was also used to evaluate drug sensitivity and to identify potential drivers by small interfering RNA knockdown. Kinase assays were used to test direct targeting of the candidate driver by the EGFR TKIs. Structural modeling including, molecular dynamics simulations, and binding assays were conducted to explore the mechanism of off-target inhibition by EGFR TKIs on the model 12 driver.ResultsBoth patient-derived xenograft PHLC12 and the X12CL cell line were sensitive to multiple EGFR TKIs. The BRAF^G469V mutation was found to be the only known oncogenic mutation in this model. Small interfering RNA knockdown of BRAF, but not the EGFR, killed X12CL, confirming BRAF^G469V as the oncogenic driver. Kinase activity of the BRAF protein isolated from X12CL was inhibited by treatment with the EGFR TKIs gefitinib and osimertinib, and expression of BRAF^G469V in non–EGFR–expressing NR6 cells promoted growth in low serum condition, which was also sensitive to EGFR TKIs. Structural modeling, molecular dynamic simulations, and in vitro binding assays support BRAF^G469V being a direct target of the TKIs.ConclusionsClinically approved EGFR TKIs can be repurposed to treat patients with non-small cell lung cancer harboring the BRAF^G469V mutation.     

New Therapeutic Opportunities Based on DNA Mismatch Repair and <Emphasis Type="Italic">BRAF</Emphasis> Status in Metastatic Colorectal Cancer

Recently, colorectal cancer (CRC) subtyping consortium identified four consensus molecular subtypes (CMS1–4). CMS1 is enriched for deficient mismatch repair (dMMR) and BRAF ^V600E tumors. Intriguingly, this subtype has better relapse-free survival but worse overall survival after relapse compared with the other subtypes. Growing evidence is accumulating on the benefit of specific therapeutic strategies such as immune checkpoint inhibition therapy in dMMR tumors and mitogen-activated protein kinase (MAPK) pathway targeted therapy in tumors harboring BRAF ^V600E mutation. After reviewing dMMR prognostic value, immune checkpoints as major targets for dMMR carcinomas will be highlighted. Following, BRAF ^V600E prognostic impact will be reviewed and therapeutic strategies with the combination of cytotoxic agents and especially the combinations of BRAF and MAPK inhibitors will be discussed.     

EGFR tyrosine kinase inhibition radiosensitizes and induces apoptosis in malignant glioma and childhood ependymoma xenografts

Malignant gliomas and childhood ependymomas have a high rate of treatment failure. Epidermal growth factor receptor (EGFR) activation has been implicated in the tumorigenesis and radioresistance of many cancers, including brain tumors. Therefore, combining EGFR targeting with irradiation is a potentially attractive therapeutic option. We evaluated the tyrosine kinase inhibitor gefitinib for its antitumor activity and potential to radio-sensitize in vivo in two xenograft models: an EGFR amplified glioma and an EGFR expressing ependymoma, both derived from primary tumors. When administered at 100 mg/kg for 5 consecutive days, gefitinib-induced partial tumor regression in all treated EGFR amplified IGRG88 glioma xenografts. The addition of 1 Gy of irradiation prior to gefitinib administration resulted in 5 complete and 4 partial regressions for the 9 treated tumors as well as a significant tumor growth delay of 33 days for the combined treatment compared to 19 days for each therapy alone, suggesting additive antitumor activity. Tumor regression was associated with inhibition of AKT and MAPK pathways by gefitinib. In contrast, the ependymoma IGREP83 was sensitive to irradiation, but remained resistant to gefitinib. Combined treatment was associated with inhibition of radiation-induced MAPK phosphorylation and significant induction of apoptotic cell death though radiation-induced AKT phosphorylation was maintained. Depending on the scheduling of both therapies, a trend towards superior antitumor activity was observed with combined treatment. Thus, EGFR targeting through tyrosine kinase inhibition appears to be a promising new approach in the treatment of EGFR-driven glioma, particularly in combination with radiation therapy.