Oncogenic BRAF V600E Mutation Research Articles

Presyncope and Ophthalmoparesis Due to Intracranial Erdheim-Chester Disease.

An 82-year-old female presented to the emergency department with presyncope and was found to be bradycardic with proptosis and ophthalmoparesis. MRI revealed an extra-axial enhancing mass compressing the medulla and bilateral enhancing retro-orbital masses. Imaging, including nuclear medicine bone scan, PET CT, and cardiac MRI raised the suspicion for a histiocytic neoplasm. These findings, along with a fibrohistiocytic infiltrate on bone biopsy and a BRAF V600E oncogenic mutation on plasma cell-free DNA confirmed a diagnosis of Erdheim-Chester disease. These enhancing masses invoke a broad differential, including a histiocytic or granulomatous process, fungal infection, amyloidosis, IgG4 disease, and lymphoma. Systematic laboratory, radiologic, pathology, and genetic testing yielded a diagnosis of this rare histiocytic disorder with frequent neurologic involvement.

Abstract 1159: BRAF- mediated PHGDH induction establishes a metabolic vulnerability in melanoma

Abstract Phosphoglycerate Dehydrogenase (PHGDH) gene, encoding the rate limiting enzyme of the serine synthesis pathway, is located at chromosome locus 1p12 and frequently amplified in melanoma. PHGDH is thought to be required for the proliferation of melanoma cells having chromosome 1p12 amplification. However, the role of PHGDH in chromosome 1p12 non-amplified melanoma is poorly understood. We observed that PHGDH is overexpressed in melanoma cell lines that do not harbor chromosome 1p12 amplification compared to melanocytes. Additionally, PHGDH is upregulated in melanomas compared to nevi. Hyperactivation of the MAPK pathway through activating mutations in BRAF or NRAS occurs in nearly all melanomas. Since PHGDH is universally upregulated in melanoma cells, we hypothesized that oncogenic BRAF mutations upregulate PHGDH expression, thereby generating a metabolic vulnerability in melanoma. Pharmacologic inactivation of the MAPK pathway using inhibitors of mutant BRAF/MEK/ERK resulted in the downregulation of PHGDH expression both in vitro and in xenograft tumors in vivo. On the other hand, acute expression of oncogenic BRAF (BRAFV600E) in melanocytes resulted in upregulation of PHGDH expression. Downregulation of PHGDH in 1p12 non-amplified melanoma cell lines decreased their cell proliferation and colony formation ability in vitro. We also observed that genetic knockdown of PHGDH in a BrafV600E/WT; Pten−/- (BPP) melanoma mouse model significantly increased survival. In addition to de-novo synthesis, serine can be taken up from extracellular compartments. Depletion of extracellular serine from the media resulted in upregulation of PHGDH expression and reduced proliferation of melanoma cells in vitro. By contrast, serine-glycine starvation in BPP mice did not affect tumor development or survival. These results implicate the dependency of melanoma tumors on de-novo serine synthesis through PHGDH. To investigate the therapeutic applicability of the PHGDH dependency, we inhibited the MAPK pathway with BRAF inhibitor along with extracellular serine-glycine depletion. We hypothesized that inhibiting the BRAF-mediated PHGDH expression along with extracellular serine-glycine starvation effectively depletes serine specifically in melanoma cells. Notably, cell viability was significantly reduced in vitro when BRAF inhibition was combined with serine-glycine depletion. Furthermore, our preliminary studies suggests that serine-glycine depletion potentiates the effect of mutant BRAF inhibition on melanoma growth in BPP mouse models. Overall, these results suggest the importance of PHGDH for melanoma cell proliferation and tumor growth irrespective of its amplification status. Combined MAPK inhibition and serine starvation may therefore exploit a metabolic vulnerability of melanoma cells and could be explored for melanoma therapy. Citation Format: Neel Jasani, Benjamin Posorske, Florian Karreth. BRAF- mediated PHGDH induction establishes a metabolic vulnerability in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1159.

The Histone Deacetylase Inhibitor ITF2357 (Givinostat) Targets Oncogenic BRAF in Melanoma Cells and Promotes a Switch from Pro-Survival Autophagy to Apoptosis.

Histone deacetylase inhibitors (HDACI) are epigenetic compounds that have been widely considered very promising antitumor agents. Here, we focus on the effects of the pan-HDAC inhibitor ITF2357 (Givinostat) in comparison with SAHA (Vorinostat) in melanoma cells bearing BRAF V600E oncogenic mutation. Our results indicate both ITF2357 and SAHA dose-dependently reduce the viability of BRAF-mutated SK-MEL-28 and A375 melanoma cells. The comparison of IC50 values revealed that ITF2357 was much more effective than SAHA. Interestingly, both inhibitors markedly decreased oncogenic BRAF protein expression levels, ITF2357 being the most effective compound. Moreover, the BRAF decrease induced by ITF2357 was accompanied by a decrease in the level of phospho-ERK1/2. The inhibitor of upstream MEK activity, U0126, reduced ERK1/2 phosphorylation and dramatically potentiated the antitumor effect of ITF2357, exacerbating the reduction in the BRAF level. ITF2357 stimulated an early pro-survival autophagic response, which was followed by apoptosis, as indicated by apoptotic markers evaluation and the protective effects exerted by the pan-caspase inhibitor z-VADfmk. Overall, our data indicate for the first time that ITF2357 targets oncogenic BRAF in melanoma cells and induces a switch from autophagy to classic apoptosis, thus representing a possible candidate in melanoma targeted therapy.

Oncogene-induced maladaptive activation of trained immunity in the pathogenesis and treatment of Erdheim-Chester disease

AbstractTrained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation.

NRF2 DLG Domain Mutations Identified in Japanese Liver Cancer Patients Affect the Transcriptional Activity in HCC Cell Lines.

Geographically, East Asia had the highest liver cancer burden in 2017. Besides this, liver cancer-related deaths were high in Japan, accounting for 3.90% of total deaths. The development of liver cancer is influenced by several factors, and genetic alteration is one of the critical factors among them. Therefore, the detailed mechanism driving the oncogenic transformation of liver cells needs to be elucidated. Recently, many researchers have focused on investigating the liver cancer genome and identified somatic mutations (MTs) of several transcription factors. In this line, next-generation sequencing of the cancer genome identified that oxidative stress-related transcription factor NRF2 (NFE2L2) is mutated in different cancers, including hepatocellular carcinoma (HCC). Here, we demonstrated that NRF2 DLG motif mutations (NRF2 D29A and L30F), found in Japanese liver cancer patients, upregulate the transcriptional activity of NRF2 in HCC cell lines. Moreover, the transcriptional activity of NRF2 mutations is not suppressed by KEAP1, presumably because NRF2 MTs disturb proper NRF2-KEAP1 binding and block KEAP1-mediated degradation of NRF2. Additionally, we showed that both MTs upregulate the transcriptional activity of NRF2 on the MMP9 promoter in Hepa1-6 and Huh7 cells, suggesting that MT derived gain-of-function of NRF2 may be important for liver tumor progression. We also found that ectopic overexpression of oncogenic BRAF WT and V600E increases the transcriptional activity of NRF2 WT on both the 3xARE reporter and MMP9 promoter. Interestingly, NRF2 D29A and L30F MTs with oncogenic BRAF V600E MT synergistically upregulate the transcription activity of NRF2 on the 3xARE reporter and MMP9 promoter in Hepa1-6 and Huh7 cells. In summary, our findings suggest that MTs in NRF2 have pathogenic effects, and that NRF2 MTs together with oncogenic BRAF V600E MT synergistically cause more aberrant transcriptional activity. The high activity of NRF2 MTs in HCC with BRAF MT warrants further exploration of the potential diagnostic, prognostic, and therapeutic utility of this pathway in HCC.

BRAF inhibition curtails IFN-gamma-inducible PD-L1 expression and upregulates the immunoregulatory protein galectin-1 in melanoma cells.

Although melanoma is considered one of the most immunogenic malignancies, spontaneous T‐cell responses to melanoma antigens are ineffective due to tumor cell‐intrinsic or microenvironment‐driven immune evasion mechanisms. For example, oncogenic BRAF V600E mutation in melanoma cells fosters tumor immune escape by modulating cell immunogenicity and microenvironment composition. BRAF inhibition has been shown to increase melanoma cell immunogenicity, but these effects are transient and long‐term responses are uncommon. For these reasons, we aimed to further characterize the role of BRAF‐V600E mutation in the modulation of PD‐L1, a known immunoregulatory molecule, and galectin‐1 (Gal‐1), a potent immunoregulatory lectin involved in melanoma immune privilege. We report herein that vemurafenib downregulates IFN‐γ‐induced PD‐L1 expression by interfering with STAT1 activity and by decreasing PD‐L1 protein translation. Surprisingly, melanoma cells exposed to vemurafenib expressed higher levels of Gal‐1. In coculture experiments, A375 melanoma cells pretreated with vemurafenib induced apoptosis of interacting Jurkat T cells, whereas genetic inhibition of Gal‐1 in these cells restored the viability of cocultured T lymphocytes, indicating that Gal‐1 contributes to tumor immune escape. Importantly, Gal‐1 plasma concentration increased in patients progressing on BRAF/MEK inhibitor treatment, but remained stable in responding patients. Taken together, these results suggest a two‐faceted nature of BRAF inhibition‐associated immunomodulatory effects: an early immunostimulatory activity, mediated at least in part by decreased PD‐L1 expression, and a delayed immunosuppressive effect associated with Gal‐1 induction. Importantly, our observations suggest that Gal‐1 might be utilized as a potential biomarker and a putative therapeutic target in melanoma patients.

Comprehensive genomic profiling aids in understanding the lesion origins of a patient with six synchronous invasive lung adenocarcinomas: a case study

BackgroundSynchronous multiple primary lung cancers (sMPLC) are rare forms of lung cancer, and their diagnosis remains as a significant challenge. Distinguishing sMPLC from advanced disease is important as their prognoses and therapeutic management vary dramatically.Case presentationThe patient was a 56-year-old Chinese male who exhibited six synchronous invasive adenocarcinomas at diagnosis [T2(6)N0M0], and who achieved durable clinical benefit under adjuvant chemotherapy for 41 months following wedge resection and lobectomy. Whole-exome sequencing revealed that two lesions (L4 and L6) in the left upper lobe of the patient’s lung shared 28 nonsynonymous mutations; thus, suggesting that the lesions may have arisen from a common ancestor at the early stages of tumorigenesis, and spread into distinct histologic subtypes. Moreover, while L5 was in the same lobe as L4 and L6, it represented a distinct lineage as it did not share any mutations with other lesions. Notably, the BRAF V600E oncogenic mutation was exclusive to L5. In addition, the KRAS G12C mutation was identified in three lesions (L1-L3) located in the right lung, which may have resulted from convergent evolution.ConclusionWe report a patient with six synchronous invasive adenocarcinomas who demonstrated durable clinical benefits under adjuvant chemotherapy following surgical treatment. While cancer staging is one of the many challenges associated with sMPLC, the data generated through next-generation sequencing can provide information on lesion origins, and thus, advance the era of precision medicine.

Carotenoids from Rhodomonas salina Induce Apoptosis and Sensitize A2058 Melanoma Cells to Chemotherapy

Melanoma is an aggressive tumor with invasive and metastatic potential, frequently exhibiting multidrug resistance mechanisms. In our continuous search for antimelanoma molecules, we have identified some effective marine compounds capable of not only inducing cell death, but also of sensitizing chemoresistant tumor cells to clinically used anticancer drugs. In this report, the cryptophyte Rhodomonas salina (Wislouch) D.R.A.Hill & R.Wetherbee, Pyrenomonadaceae, was chemically investigated in order to identify pigments efficiently inhibiting melanoma cells proliferation. All pharmacological tests were performed on A2058 cells expressing the oncogenic BRAF V600E mutation and resistant to dacarbazine treatment. Flash chromatography of R. salina ethanol extract led to purification of alloxanthin and crocoxanthin, which showed significant antiproliferative activity against A2058 cells, exhibiting IC50 = 29 and 50 μM, respectively. These carotenoids promoted growth inhibition, decreased cell migration, and induced apoptosis and sub-G1 cells accumulation after 72 h of treatment. In addition, alloxanthin potentiated the cytotoxic activity of vemurafenib (a BRAF inhibitor) and restored the sensitivity of A2058 cells to dacarbazine treatment. Graphical Abstract

Abstract 3589: Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy

Invasive melanoma is an aggressive form of skin cancer with poor clinical outcomes. Nearly 50% of melanoma patients exhibit oncogenic BRAFV600E mutations. Existing BRAF kinase inhibitor drugs provide a short-term benefit prior to the rapid onset of drug resistance. Emerging results suggest that BRAF and other kinase inhibitors induce metabolic reprogramming from glycolytic energy production to oxidative phosphorylation (OXPHOS) in a subset of malignant melanomas. Metabolic reprogramming to OXPHOS is emerging as an important mechanism of resistance to BRAF kinase inhibitors and is associated with worse prognosis. Consequently, there is a significant opportunity for multimodal therapeutic approaches that selectively target the metabolic reprogramming associated with resistance to BRAF kinase inhibitors and promote immune-targeted therapy. We have previously developed mitochondria-targeted drugs by exploiting the enhanced negative mitochondrial membrane potential of cancer cells to selectively target and disrupt tumor growth. We hypothesized that metabolic reprogramming from glycolysis to OXPHOS in BRAF inhibitor-resistant melanoma cells would make them susceptible to OXPHOS inhibitors. In this study, we have modified the structure of a naturally occurring polyphenol, magnolol into a mitochondria-targeted-magnolol (Mito-MGN). We tested the effects of Mito-MGN on mitochondrial respiration and bioenergetic function, cellular redox status, and mitophagy in wild type (WT) and BRAF-resistant (metabolically reprogrammed) melanoma cells. Our data show that Mito-MGN inhibits mitochondrial respiration, melanoma cell proliferation and invasion at concentrations more than 200-fold lower than that of magnolol. Furthermore, Mito-MGN increases ROS formation and oxidation of mitochondrial peroxiredoxin. Preliminary results also indicate that BRAF inhibitor resistant melanoma cells exhibit enhanced OXPHOS as a consequence of metabolic reprogramming and that Mito-MGN effectively inhibits proliferation and mitochondrial respiration in the resistant cells. Mito-MGN inhibited mitochondrial complex I activity in both WT and resistant cells. The IC 50 values to inhibit complex I-mediated respiration was consistent with the values calculated for inhibition of cell respiration. We conclude that inhibition of complex I is a key initial event that is responsible for the anti-proliferative effects observed in the presence of Mito-MGN against wild-type as well as drug-resistant melanoma cells. Citation Format: Gang Cheng, Jacek Zielonka, Micael Hardy, Michael B. Dwinell, Balaraman Kalyanaraman. Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy [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 3589.

Abstract 3589: Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy

Abstract Invasive melanoma is an aggressive form of skin cancer with poor clinical outcomes. Nearly 50% of melanoma patients exhibit oncogenic BRAFV600E mutations. Existing BRAF kinase inhibitor drugs provide a short-term benefit prior to the rapid onset of drug resistance. Emerging results suggest that BRAF and other kinase inhibitors induce metabolic reprogramming from glycolytic energy production to oxidative phosphorylation (OXPHOS) in a subset of malignant melanomas. Metabolic reprogramming to OXPHOS is emerging as an important mechanism of resistance to BRAF kinase inhibitors and is associated with worse prognosis. Consequently, there is a significant opportunity for multimodal therapeutic approaches that selectively target the metabolic reprogramming associated with resistance to BRAF kinase inhibitors and promote immune-targeted therapy. We have previously developed mitochondria-targeted drugs by exploiting the enhanced negative mitochondrial membrane potential of cancer cells to selectively target and disrupt tumor growth. We hypothesized that metabolic reprogramming from glycolysis to OXPHOS in BRAF inhibitor-resistant melanoma cells would make them susceptible to OXPHOS inhibitors. In this study, we have modified the structure of a naturally occurring polyphenol, magnolol into a mitochondria-targeted-magnolol (Mito-MGN). We tested the effects of Mito-MGN on mitochondrial respiration and bioenergetic function, cellular redox status, and mitophagy in wild type (WT) and BRAF-resistant (metabolically reprogrammed) melanoma cells. Our data show that Mito-MGN inhibits mitochondrial respiration, melanoma cell proliferation and invasion at concentrations more than 200-fold lower than that of magnolol. Furthermore, Mito-MGN increases ROS formation and oxidation of mitochondrial peroxiredoxin. Preliminary results also indicate that BRAF inhibitor resistant melanoma cells exhibit enhanced OXPHOS as a consequence of metabolic reprogramming and that Mito-MGN effectively inhibits proliferation and mitochondrial respiration in the resistant cells. Mito-MGN inhibited mitochondrial complex I activity in both WT and resistant cells. The IC50 values to inhibit complex I-mediated respiration was consistent with the values calculated for inhibition of cell respiration. We conclude that inhibition of complex I is a key initial event that is responsible for the anti-proliferative effects observed in the presence of Mito-MGN against wild-type as well as drug-resistant melanoma cells. Citation Format: Gang Cheng, Jacek Zielonka, Micael Hardy, Michael B. Dwinell, Balaraman Kalyanaraman. Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy [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 3589.

Zeaxanthin from Porphyridium purpureum induces apoptosis in human melanoma cells expressing the oncogenic BRAF V600E mutation and sensitizes them to the BRAF inhibitor vemurafenib

Zeaxanthin, an abundant carotenoid present in fruits, vegetables and algae was reported to exert antiproliferative activity and induce apoptosis in human uveal melanoma cells. It also inhibited uveal melanoma tumor growth and cell migration in nude mice xenograft models. Here we report that zeaxanthin purified from the rhodophyte Porphyridium purpureum (Bory) K.M.Drew & R.Ross, Porphyridiaceae, promotes apoptosis in the A2058 human melanoma cell line expressing the oncogenic BRAF V600E mutation. Zeaxanthin 40μM (IC50) induced chromatin condensation, nuclear blebbing, hypodiploidy, accumulation of cells in sub-G1 phase, DNA internucleosomal fragmentation and activation of caspase-3. Western blot analysis revealed that zeaxanthin induced up-regulation of the pro-apoptotic factors Bim and Bid and inhibition of NF-κB transactivation. Additionally, zeaxanthin sensitized A2058 melanoma cells in vitro to the cytotoxic activity of vemurafenib, a BRAF inhibitor widely used for the clinical management of melanoma, suggesting its potential interest as dietary adjuvant increasing melanoma cells sensitivity to chemotherapy.

Molecular insight into mutation-induced conformational change in metastasic bowel cancer BRAF kinase domain and its implications for selective inhibitor design

Oncogenic BRAF V600E mutation confers constitutive activation for the kinase and is closely related to the pathogenesis of metastasic bowel cancer (MBC). Here, the V600E-induced conformational change in MBC BRAF kinase domain is characterized systematically at structural, energetic and dynamic levels. The mutation is observed to cause a conformational conversion of the kinase’s activation loop from DFG-out to DFG-in, thus activating the kinase. Electrostatic force is primarily responsible for the conformational conversion and stabilization of DFG-in associated with the mutation. Molecular docking calculations are employed to analyze the binding mode difference of mutant-selective inhibitors between the DFG-out and DFG-in conformations of BRAF kinase. It is revealed that the mutation can reshape inhibitor selectivity profile by altering kinase loop conformation. Several compounds are determined to have a high or moderate selectivity for mutant over wild-type kinase. The selectivity is primarily originated from hydrogen bond interactions of inhibitor ligands with mutant rather than wild type due to the conformational difference in kinase domain.

Robust Multiplexed Clustering and Denoising of Digital PCR Assays by Data Gridding.

Digital PCR (dPCR) relies on the analysis of individual partitions to accurately quantify nucleic acid species. The most widely used analysis method requires manual clustering through individual visual inspection. Some automated analysis methods have emerged but do not robustly account for multiplexed targets, low target concentration, and assay noise. In this study, we describe an open source analysis software called Calico that uses "data gridding" to increase the sensitivity of clustering toward small clusters. Our workflow also generates quality score metrics in order to gauge and filter individual assay partitions by how well they were classified. We applied our analysis algorithm to multiplexed droplet-based digital PCR data sets in both EvaGreen and probes-based schemes, and targeted the oncogenic BRAF V600E and KRAS G12D mutations. We demonstrate an automated clustering sensitivity of down to 0.1% mutant fraction and filtering of artifactual assay partitions from low quality DNA samples. Overall, we demonstrate a vastly improved approach to analyzing ddPCR data that can be applied to clinical use, where automation and reproducibility are critical.

Copper chelation as targeted therapy in a genetic mouse model of oncogenic BRAF-driven thyroid cancer.

e23148 Background: Over half of papillary thyroid cancers (PTC) contain the MAPK pathway activating, oncogenic BRAF V600E mutation. Early clinical trials using inhibitors to this mutant protein or its substrates, the MEK1/2 kinases, prolonged progression-free survival or stabilized disease in patients with advanced PTC. However, the toxicities of these inhibitors are uniquely highlighted in an indolent disease like PTC, which would require patients to endure toxicities long-term. Our goal was to determine if tetrathimoybdate (TM), a well-tolerated copper chelator we have previously shown to inhibit BRAF-mutated melanoma via MEK inhibition, can inhibit BRAF-driven PTC growth. Methods: We assessed TM in comparison to current standard of care (SOC), Lenvatinib and Sorafenib, and mutant BRAF inhibitor, Vemurafenib. Anchorage independent growth assays were used to test the inhibitory effect of these drugs on human BRAF-mutated PTC cell lines. We then confirmed these findings by treating a genetically engineered mouse model (GEMM) of aggressive BRAF-driven PTC. Results: TM inhibited 57.5% of colony growth in vitro, which was not significantly different from the 42.4% and 32.2% inhibition by Sorafenib and Lenvatinib, respectively. TM inhibition was less effective than the 70.3% inhibition by Vemurafenib ( p =0.04). We confirmed these results in vivo, where mice on the TM arm, on average, were observed to have 14.8% of their thyroid glands occupied by tumor, a statistically significant reduction from the mice in the control arm, whose tumor load averaged 23.6% ( p= 0.008). This 37.4% reduction in tumor burden was not statistically different from the 35.2% reduction measured in the Vemurafenib arm, where mice on average had 15.3% of their thyroid glands replaced by tumor. Conclusions: The copper chelator, TM, was as effective as the SOCs, Lenvatinib and Sorafenib, at inhibiting the growth of human PTC in vitro. Although TM was slightly less effective than Vemurafenib in vitro, TM was as effective as Vemurafenib at reducing tumor load in a GEMM of BRAF-driven PTC. Success of TTM in these PTC models may next inform a Phase I trial assessing TM in patients with advanced PTC.

Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer.

603 Background: Oncogenic BRAFV600E mutations activate MAP kinase signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer (CRC). In BRAFV600E mutant CRCs, treatment failure may be related to BRAFV600E-mediated apoptosis resistance that occurs by an as yet undefined mechanism. Methods: BRAF isogenic RKO CRC cells and RKO, HT29, WiDr cell lines were treated with cobimetinib ± the small molecule MCL-1 inhibitor, A-1210477. Apoptosis was measured by Annexin V staining and caspase cleavage. Gene knockdown or overexpression was achieved by lentivirus; ERK siRNA was utilized. Competitive RT-PCR was performed for MCL-1 mRNA expression. HT-29 cells with control or MCL-1 shRNA were xenografted into SCID mice, treated with cobimetinib or vehicle, and tumor volume was measured. Results: We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using CRC cell lines isogenic for BRAF. BRAFV600E-induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by ERK siRNA that suppressed MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively.MEK /ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced pro-apoptotic BIM to a greater extent than did vemurafenib in BRAFV600E cell lines. MCL-1 knockdown vs control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. A-1210477 also enhanced cobimetinib-induced apoptosis in vitrothat was due to disruption of the interaction of MCL-1 with pro-apoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. Conclusions: BRAFV600E-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism.

Chemotherapeutic drug selectivity between wild-type and mutant BRaf kinases in colon cancer.

Oncogenic BRaf V600E mutation is involved in the development, invasion and metastasis of colon cancer. Selective inhibition of BRafV600E mutant has been recognized as a therapeutic strategy for the cancer. Here, we carried out atomistic molecular dynamics (MD) simulations to characterize the structural basis, energetic property, and dynamics behavior of conformational change in BRaf activation loop upon the mutation. It is found that V600E mutation destabilizes inactive DFG-out conformation of activation loop and promotes its conversion to the active DFG-in conformation, thus conferring constitutive activity for BRaf kinase. A further analysis revealed that the conformational change is induced by electrostatic effect of the negatively charged mutant residue Glu600, which can form a potent salt bridge with the positively charged residue Lys570; this is naturally consistent with phosphorylation of activation loop to activate the kinase. Both of them introduce a negative charge to activation loop and, consequently, the DFG-out is destabilized and conversed to DFG-in. Energetic analysis unraveled that small-molecule kinase inhibitor PLX4720 has a similar selectivity profile for mutant over wild-type kinases and for phosphorylated and dephosphorylated kinases. This can be substantiated in part by in vitro kinase assay that the inhibitor exhibits 12.6 and 10.4-fold higher potencies against mutant than wild type and against phosphorylated than dephosphorylated, respectively. It is suggested that the activation loop conformation, but neither V600E mutation nor phosphorylation, directly determines inhibitor affinity; the mutation and phosphorylation can only indirectly influence inhibitor binding via regulation of activation loop conformation. Graphical Abstract Chemotherapeutic drug selectivity between wild-type and mutant BRaf kinases in colon cancerᅟ.

BRAF V600E Targetable Mutation in Relapsed/Refractory Multiple Myeloma (R/R MM) Patients: A High Incidence in R/R MM Detected Using Cell Sorting Screening

Background:The oncogenic BRAF V600E mutation activates the MAPK signaling pathway resulting in cell growth and survival of tumor cells carrying the mutation. Targeting BRAF has shown its efficacy in metastatic melanomas. BRAF V600E mutation has been described in multiple myeloma (MM), with an incidence of 4-10% in previous reports. We report here the incidence of BRAF mutations in a series of MM patients treated at our center.Methods:From February 2012 to March 2016, 94 MM patients were tested for BRAF mutations. Eighty-one samples came from patients with relapsed/refractory MM (R/R MM) and 13 samples from newly diagnosed patients. Eighty-eight samples were collected from bone marrow aspirates, 3 from extramedullary plasmacytoma biopsies, 1 from pleural effusion, and 2 from peripheral blood. Targeted sequencing for BRAF mutations using Sanger direct sequencing or targeted next generation sequencing were performed on all samples after CD138 cell (after 2015). In addition, NGS was performed for 2 patients. From 2012 to 2015, we used material obtained from scraping bone marrow smears with > 20% plasma cells. Since 2015, we began using immunomagnetic cell-sorting based on the CD138 cell surface marker (Stem Cells®).Results:BRAF mutations were detected in 8 samples out of the 84 that could have been screened (9.5%). When considering only the relapsed/refractory population, the incidence of BRAF mutations was 10.8%. One sample had the inactivating D594N BRAF mutation and the other 7 had the V600E BRAF mutation. The 8 mutated patients had relapsed/refractory MM at the time of analysis. Of note, 5 out of the 8 mutated patients (62.5%) had an IgA MM. Seventy-six samples had no BRAF mutation detectable. Ten samples were not tested because of the small percentage of MM cells on the bone marrow smears. The failure rate was 16% with the scraping technique and 3.4% with the sorting cell technique. Five out of 7 patients harboring the V600E BRAF mutation were treated with a BRAF inhibitor in different clinical trials. One mutated patient could not be treated due to exclusion criteria related to an underlying condition (terminal renal failure requiring chronic hemodialysis) and one patient received allogeneic stem-cell transplantation at relapse. No BRAF mutation was detected in the 13 newly diagnosed patients.Conclusion:Our results show that BRAF screening is feasible in routine practice and can help orienting therapy in relapsed/refractory MM patients. The incidence of 9.5% that we found in our series compares favorably with previously published results, and we observed a slightly increased incidence of 10.8% in the R/R population. The cell sorting technique seems to be more effective than the scraping technique for MM sample DNA acquisition and BRAF mutation screening. Results of BRAF inhibitors therapy in MM are eagerly awaited. DisclosuresLacroix:sanofi: Research Funding; qiagen: Membership on an entity's Board of Directors or advisory committees; roche: Membership on an entity's Board of Directors or advisory committees; astrazeneca: Membership on an entity's Board of Directors or advisory committees. Michot:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Ribrag:ArgenX: Research Funding; Esai: Membership on an entity's Board of Directors or advisory committees; Infinity: Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; NanoString: Membership on an entity's Board of Directors or advisory committees.

A new NFIA:RAF1 fusion activating the MAPK pathway in pilocytic astrocytoma

Pilocytic astrocytoma (PA) is one of the most common brain cancers among children and activation of the Mitogen-Activated Protein Kinase (MAPK) pathway is considered the hallmark. In the majority of cases, oncogenic BRAF fusions or BRAF V600E mutations are observed, while RAF1 or NF1 alterations are more rarely found. However, in some cases, no apparent cancer driver events can be identified. Here, we describe a novel fusion between the transcription factor nuclear factor 1A (NFIA) and Raf-1 proto-oncogene (RAF1) in a 5-year old boy with PA. The novel fusion was identified as part of a comprehensive genomic tumor profiling. We show that the NFIA:RAF1 fusion results in constitutive Raf1 kinase activity, leading to activation of downstream MEK1/2 cascade and increased proliferation of cancer cells. The NFIA:RAF1 fusion displayed distinct subcellular localization towards the plasma membrane indicative of Raf-1 activation, in contrast to both wild type NFIA and Raf-1, which were localized in the nucleus and cytoplasm, respectively. In conclusion, our data support the existence of rare oncogenic RAF1 fusions with constitutive Raf-1 activity. This highlights the need for broad genetic testing in order to refine diagnostics of PA and to unravel potential treatment options, e.g. with MEK inhibitors.

Abstract 2115: MEK/ERK and MCL-1 inhibition synergistically reverse apoptosis resistance in colon cancer cells with BRAF(V600E)-mediated MCL-1-upregulation

Abstract Background. Oncogenic BRAFV600E mutations in colorectal cancer (CRC) are associated with poor prognosis and treatment resistance. BRAF mutations activate MAP kinase signaling and may confer apoptosis resistance by upregulating anti-apoptotic BCL-2/BCL-XL/MCL-1 proteins. Results. Ectopic expression of mutant BRAFV600E was shown to potently activate MEK/ ERK that mediated the phosphorylation (T163) and upregulation of MCL-1, but not BCL-2 or BCL-XL. MEK/ERK-mediated phosphorylation of MCL-1 (T163) increased its protein stability, shown by an MCL-1 phospho-mimic mutant. In human colon cancers, mutant vs wild-type BRAF was associated with increased MCL-1 expression by IHC. MEK/ERK inhibition by cobimetinib induced pro-apoptotic BIM in BRAF mutant CRC cell lines (HT-29, RKO), and suppressed phospho-MCL-1/MCL-1 proteins as did ERK siRNA. Suppression of MCL-1 by shRNA or using a small molecule MCL-1 inhibitor (A-1210477) synergistically enhanced cobimetinib-induced apoptosis (Table) that was associated with BAK/BAX activation. A-1210477 antagonized MCL-1 by disrupting its interaction with BAK and BIM proteins, shown by immunoprecipitation. Conclusion. BRAF/MEK/ERK induces upregulation of MCL-1 via phosphorylation/stabilization to confer apoptosis resistance that can be overcome by combined ERK and MCL-1 inhibition, suggesting a novel therapeutic strategy against BRAF mutant CRCs. Drug-induced apoptosis (annexin V) in CRC cell linesDrugsHT-29 % apoptosisRKO % apoptosisDMSO16.842.11A-1210477 (2.5 μM)29.5222.88Cobimetinib(5 μM)28.0421.05Combination53.2250.93 Citation Format: Hisato Kawakami, Shengbing Huang, Frank A. Sinicrope. MEK/ERK and MCL-1 inhibition synergistically reverse apoptosis resistance in colon cancer cells with BRAF(V600E)-mediated MCL-1-upregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2115.

ENDOCRINE TUMORS: BRAF V600E mutations in papillary craniopharyngioma.

Papillary craniopharyngioma (PCP) is an intracranial tumor that results in high levels of morbidity. We recently demonstrated that the vast majority of these tumors harbor the oncogenic BRAF V600E mutation. The pathologic diagnosis of PCP can now be confirmed using mutation specific immunohistochemistry and targeted genetic testing. Treatment with targeted agents is now also a possibility in select situations. We recently reported a patient with a multiply recurrent PCP in whom targeting both BRAF and MEK resulted in a dramatic therapeutic response with a marked anti-tumor immune response. This work shows that activation of the MAPK pathway is the likely principal oncogenic driver of these tumors. We will now investigate the efficacy of this approach in a multicenter phase II clinical trial. Post-treatment resection samples will be monitored for the emergence of resistance mechanisms. Further advances in the non-invasive diagnosis of PCP by radiologic criteria and by cell-free DNA testing could someday allow neo-adjuvant therapy for this disease in select patient populations.