BRAFV600E Cells Research Articles

BRAF V600E in a preclinical model of pleomorphic Xanthoastrocytoma: Analysis of the tumor microenvironment and immune cell infiltration dynamics in vivo

Low-grade glioma is the most common brain tumor affecting pediatric patients (pLGG) and BRAF mutations constitute the most frequent genetic alterations. Within the spectrum of pLGGs, approximately 70-80% of pediatric patients diagnosed with transforming Pleomorphic Xanthoastrocytoma (PXA) harbor the BRAF V600E mutation. However, the impact of glioma BRAF V600E cells regulation of tumor-infiltrating immune cells and their contribution to tumor progression remains unclear. Moreover, the efficacy of BRAF inhibitors in treating pLGGs is limited compared to their impact on BRAF-mutated melanoma. Here we report a novel immunocompetent RCAS-BRAF V600E murine glioma model. Pathological assessment indicates this model appears to be consistent with diffuse gliomas and morphological features of PXA. Our investigations revealed distinct immune cells signatures associated with increased trafficking and activation within the tumor microenvironment (TME). Intriguingly, immune system activation within the TME also generated a pronounced inflammatory response associated with dysfunctional CD8+ T cells, increased presence of immunosuppressive myeloid cells and regulatory T cells (Tregs). Further, our data suggests tumor-induced inflammatory processes such as cytokine storm. These findings suggest a complex interplay between tumor progression and the robust inflammatory response within the TME in preclinical BRAF V600E LGGs, which may significantly influence animal survival.

Abstract 4760: Inhibition of FASN postpones development of resistance to BRAF inhibitors in colorectal cancer

Abstract Introduction: Mutations in proto-oncogene BRAF occur in about 10-15% of CRC patients and BRAFV600E is the most common. For patients who undergo BRAF-targeted therapy, resistance develops 4-6 months after treatment initiation and results in more aggressive disease. We found that development of resistance to BRAF inhibitors (BRAFi) is associated with an increase in lipid metabolism and expression of fatty acid synthase (FASN). FASN is a key enzyme of lipid synthesis overexpressed in CRC. Therefore, our hypothesis is that inhibition lipid metabolism via FASN will postpone development of resistance to BRAFi. Methods: We established CRC cells resistant to PLX8394, a novel BRAFi. To evaluate differences in parental and resistance cells, CellTiterGlo 2.0, PrestoBlue, CytoSelectTM Invasion Assay, Triglyceride Assay, Seahorse XF, and confocal microscopy were used. Combination of PLX8394 and TVB3664 or C75 (FASN inhibitors) was tested on cell viability, colony formation, and synergy studies in parental and BRAFi resistant cells. Results: The development of resistance to BRAFi promotes cellular proliferation and increases cyclin D and survivin expression in vitro and in vivo. BRAFi resistance is also associated with an increase in invasive properties and loss of E-cadherin expression. Metabolic changes include an increase in lipid metabolism, oxidative phosphorylation, and triglycerides storage. RNAseq and western blot analysis show significant upregulation of FASN in BRAFi resistant cells. Using cell viability and soft agar colony formation assays, we show that combined PLX8394 and TVB3664 treatment leads to a significantly higher decrease in cell viability and colony formation as compared to each drug alone in parental cells but not in BRAFi resistant cells. The calculation of a Bliss synergy score confirms that combination treatment with C75 and PLX8394 has synergetic effect in BRAFV600E cells. To further confirm that FASN contributes to resistance to BRAFi, we show that HT29 FASN shRNA cells are more susceptible to PLX8394 treatment as compared to control cells. Importantly, our data show that the long-term treatment with PLX8394 in combination with TVB3664 postpones development of resistance to BRAFi as compared to cells treated with PLX8394 alone. Conclusion: Our study demonstrates that resistance to BRAFi is associated with a significant increase in proliferation, metastasis, and lipid metabolism. We demonstrate that combination of FASN inhibitors and BRAFi postpones development of resistance in BRAFV600E cells. However, FASN inhibition does not sensitize cells to BRAFi in already resistance cells, suggesting that this approach cannot be used to overcome acquired resistance to BRAFi. In summary our data suggest that an addition of TVB3664 at the beginning of BRAF treatment regimen could be an efficacious treatment strategy for BRAFV600E CRC patients. Citation Format: Mariah Geisen, Josiane weber-Tessmann, Courtney Kelson, Daheng He, Chi Wang, Abu Saleh Mosa Faisal, Jill Kolesar, Yekaterina Zaytseva. Inhibition of FASN postpones development of resistance to BRAF inhibitors in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4760.

Regulation of MEK inhibitor selumetinib sensitivity by AKT phosphorylation in the novel BRAF L525R mutant

BackgroundOncogenic mutations in BRAF genes are found in approximately 5–10% of colorectal cancers. The majority of BRAF mutations are located within exons 11–15 of the catalytic kinase domains, with BRAF V600E accounting for more than 80% of the observed BRAF mutations. Sensitivity to BRAF- and mitogen-activated protein kinase (MEK) inhibitors varies depending on BRAF mutations and tumor cell types. Previously, we newly identified, BRAF L525R-mutation, in the activation segment of the kinase in colorectal cancer patient. Here, we characterized the function of the BRAF L525R mutation.MethodsHEK293 cells harboring a BRAF mutation (V600E or L525R) were first characterized and then treated with cetuximab, dabrafenib, and selumetinib. Cell viability was measured using WST-1 assay and the expression of proteins involved in the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways was evaluated using western blot analysis.ResultsThe MEK inhibitor selumetinib effectively inhibited cell proliferation and ERK phosphorylation in BRAF L525R cells but not in BRAF V600E cells. Further studies revealed that AKT phosphorylation was reduced by selumetinib in BRAF L525R cells but not in BRAF V600E cells or selumetinib-resistant BRAF L525R cells. Moreover, the AKT inhibitor overcame the selumetinib resistance.ConclusionsWe established a model system harboring BRAF L525R using HEK293 cells. BRAF L525R constitutively activated ERK. AKT phosphorylation caused sensitivity and resistance to selumetinib. Our results suggest that a comprehensive network analysis may provide insights to identify effective therapies.

Abstract IA009: Tumor organoids of multi-metastatic colorectal cancer: From research tools to treatment decision tools

Abstract Most patients with colorectal cancer (CRC) do not have an “actionable” target based on molecular tumor profiling. Ex vivo drug sensitivity testing of tumor organoids holds promise as a complementary approach in precision oncology. We have established a pre-clinical pharmacogenomics platform for CRC at Oslo University Hospital. A living biobank of 208 organoids of distinct liver lesions from 100 patients treated by hepatic resection for metastatic CRC was generated in the observational phase of the project. Sensitivity testing of custom drug libraries (n=40-47 drugs) and molecular profiling (targeted DNA sequencing, RNA sequencing, multiplex immunohistochemistry) indicated that the organoids recapitulate well-known pharmacogenomic associations, phenotypic tumor features, and clinical treatment responses (n=35 patients published (ref 1,2)). Evaluation of multiple lesions (n=2-6) per patient suggested diversity among patients in the level of inter-metastatic heterogeneity of drug sensitivities, supporting the need for multi-tumor evaluations in a clinical setting. A collection of CRC cell lines (n=107) was screened for sensitivity to more than 500 drugs and this suggested a molecular basis for response to selected targeted agents, including to PARP inhibition in a small subset of the cell lines (ref 3). Combination screens suggested possibility to overcome acquired resistance to dual BRAF and EGFR blockade in BRAFV600E cells (unpublished data). The living biobank serves as a reference for drug nominations in an ongoing phase II umbrella trial of pharmacogenomics-guided treatment in patients with metastatic CRC (EVIDENT; EU Clinical Trials Register no. 2020-003395-41). Several ad hoc patient cases have been screened as a pilot for the trial, and data of clinical benefit from this functional precision oncology strategy will be presented.

Abstract 355: Molecular characterization of BRAF V600E CRC in response to Src-targeted therapy

Abstract Oncogenic mutations in B-type Raf Kinase (BRAF) (particularly V600E) are present in 7-10% of metastatic colorectal cancer (mCRC), conferring resistance to conventional therapies and a poor prognosis. Src Family Kinases (SFKs) play a relevant role in CRC, where Src activation is associated with an aggressive phenotype and a worse prognosis; and could be involved in chemoresistance mechanisms. Dasatinib is a potent Src inhibitor with demonstrated anti-proliferative activity, although deeper knowledge is needed. Thus, we aimed to explore the role of Src in BRAF V600E CRC and to investigate this tumor subgroup in response to Dasatinib. SRC activation was evaluated by Western Blot. We generated Knock-Out (KO) Src cell lines by CRISPR/Cas9 system and stable Src overexpression (OE) by lentivirus. Cell viability was analysed by crystal violet and MTS assay. Phospho-kinase proteins screening was performed using the Proteome Profiler Human Phospho-Kinase Array kit. Expression levels of Src and phospho-Src (p-Src) displayed an inverse correlation with IC50 values for Dasatinib in a panel of 7 CRC cell lines including BRAF V600E (HT29, COLO201, RKO, LS411N) and BRAF WT (DIFI, HCA7 and LIM1215), and these doses were able to inhibit Src activation in all cell lines. Focusing on HT29 cells, inhibition of p-Src occurs in a dose-dependent manner and in shortened periods of treatment. Dasatinib showed a significant inhibition of proliferation and migration on HT29. Moreover, stable Src-OE HT29 cells showed higher clonogenicity, migration and proliferation capacities, as well as higher resistance to Dasatinib. Conversely, HT29 Src KO cells were more sensitive. In line with those results, silencing Src in other BRAF V600E cells (RKO and LS411N) also resulted in a remarkable reduction in clonogenicity and migration abilities. Xenograft mouse model of SRC depletion showed that the growth of SRC KO tumors was significantly slower than WT tumors. Phospho-kinase array quantification in basal and Src-OE HT29 cells showed up- and downregulated proteins related to classical tumorigenic signaling pathways such as MAPK (JNK1/2, MSK), PI3K/Akt (Akt, TOR, GSK3), and others. Further studies to explore resistance mechanisms mediated by Src/SFKs are required. To conclude, lower p-Src expression at baseline is related to a higher resistance to Dasatinib in all CRC cell lines. Dasatinib showed a significant anti-proliferative activity in a subset of CRC cell lines and regulates the activity of Src in a time and dose-dependent manner. Modified expression of Src (OE and KO) in BRAF V600E CRC cells significantly regulates clonogenicity, proliferation and migration abilities in vitro. Src overexpression is responsible of the acquired resistance to Dasatinib and regulates several pathways that could be involved in the tumorigenesis of BRAF V600E CRC, although further studies to explore resistance mechanisms mediated by Src/SFKs are required. Citation Format: Beatriz Rubio Cuesta, Beatriz Gil Calderón, Patricia LLamas Granda, Jacinto Sarmentero, Carlos Carretero Puche, María del Carmen Riesco, Rocío García Carbonero, Beatriz Soldevilla. Molecular characterization of BRAF V600E CRC in response to Src-targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 355.

Abstract 2422: Migration of V600E-expressing cells is suppressed by ERK-dependent feedback inhibition of RAC1 and rescued in melanoma by secondary mutations

Abstract Oncogenic activation of ERK occurs in as many as 50% of human tumors and in the majority of melanomas. Activated BRAF V600E hyperactivates ERK signaling and causes ERK dependent dysregulation of proliferation. However, BRAF V600E alone is insufficient to drive melanoma formation in genetically engineered mice models. Moreover, 82% of benign nevi harbor BRAF V600E mutations. In contrast, in BRAF V600E/PTEN-/- GEMM models, rapid onset of aggressive and disseminated melanomas occurs. We show here that cell migration and invasion of melanocytes expressing BRAFV600E is depressed in vitro and in vivo and significantly accelerated by pharmacologic inhibition of ERK signaling or by deletion of PTEN. Upon ERK inhibition, cells lose their cell-cell junctions and become single cells with actin rich protrusions and a mesenchymal morphology. ERK pathway activation affects each step of the migration cycle, impeding cell spreading, assembly of stress fibers, and focal contact formation, all of which are associated with decreased mobility and invasion. Hyperactivation of ERK signaling by BRAF V600E has been shown to cause potent feedback inhibition of RTK signaling and wild type RAS. We now show that this is associated with feedback inhibition of RAC1, a RAS-like small GTPase required for cell migration. Moreover, ERK pathway inhibition reactivates RAC in an RTK-dependent manner. Consistent with these findings, expression of RAC1 P29S, an activating mutant, and overexpression of PREX2, an activator of RAC1, both rescue migration in BRAF V600E cells, as does loss of PTEN expression, which converges with RAC1 activation downstream. Taken together, these data suggest that whereas hyperactivation of ERK signaling by BRAFV600E dysregulates proliferation, ERK dependent feedback inhibition of RAC1 inhibits cell mobility and invasiveness and prevents full transformation. Selection of secondary genetic events that overcome or bypass RAC1 feedback is thus required for full transformation. In support of this view, RAC1 mutation, PTEN loss, or PREX2 amplification occurs in 38% of human melanomas. Thus, oncogene induced feedback may be deleterious and directs tumor evolution by causing selection of mutations that restore functions necessary for transformation Citation Format: Sunyana Gadal, Jacob A. Boyer, Hongyan Li, Ning Fan, Eric Chan, Yevgeniy Romin, Elisa De Stanchina, Rona Yeager, Marcus Bosenberg, Katia M. Todorova, Neal Rosen. Migration of V600E-expressing cells is suppressed by ERK-dependent feedback inhibition of RAC1 and rescued in melanoma by secondary mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2422.

Abstract 748: Role of LINC01614 in papillary thyroid cancer

Abstract Papillary thyroid cancer (PTC) accounts for the vast majority of thyroid cancer cases (~80%), with a significantly higher prevalence in females, but much more aggressive and with poorer outcomes in males. Despite a high survival rate, disease recurrence and metastasis remain significant issues. Therefore, there is an unmet need for the elucidation and identification of molecular markers associated with PTC, to aid in identifying early-stage disease, as well as patient prognosis and therapeutic approaches. Long intergenic non-coding RNA (lincRNA) molecules comprise the largest subclass of RNA molecules within the human genome, yet the majority of their functions are yet to be identified. Evidence supports that these non-coding RNA molecules play a role in protein and RNA localization and scaffolding - implying a critical role in the fine tuning of gene expression. Recently, lincRNAs have been identified as major players in cancer development and progression. This deems lincRNAs as attractive, specific biomarkers and/or therapeutic targets, making them important aspects for further study. Data from our lab identified LINC01614 as being significantly upregulated (~10-fold increase) in PTC, when compared to normal thyroid tissue. There was also ~30-fold increase in LINC01614 in male PTC when compared to females, highlighting potential sex-related correlation for study. Thus, we are studying LINC01614 as a potential biomarker for diagnosis and prognosis, as well as a target for therapeutic intervention. LINC01614 was found to be upregulated in various thyroid cancer cell lines, with significant upregulation in the TPC1 (~5-fold; RET/PTC rearrangement) and K1 (~2-fold; BRAFV600E) cell lines when compared to the “normal”, immortalized thyroid cancer cell line (Nthy-ori-3-1). To decipher a phenotype for LINC01614 upregulation in PTC, we utilized the CRISPRi technique to decrease its expression in TPC1. The knockdown of LINC01614 resulted in a ~75% reduction in proliferation in TPC1 cells. Studies are underway to investigate the role of LINC01614 in migration, invasion, morphology, and tumorigenicity in TPC1 cells, as well as in the BRAFV600E cells, K1. Exploring the role that lincRNAs have in cancer phenotypes can lead to a plethora of different molecular markers for therapeutic intervention. The mechanisms by which these non-coding molecules regulate the expression of genes and other elements of the cellular milieu can serve as an open revenue for understanding how they impact PTC development and progression. Citation Format: Danielle Quaranto, Michelle Carnazza, Nicole DeSouza, Sina Dadafarin, Augustine Moscatello, Raj K. Tiwari, Jan Geliebter. Role of LINC01614 in papillary thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 748.

Analysis of the Role of FRMD5 in the Biology of Papillary Thyroid Carcinoma.

Background: Thyroid carcinoma (TC) is the most common endocrine system malignancy, and papillary thyroid carcinoma (PTC) accounts for >80% of all TC cases. Nevertheless, PTC pathogenesis is still not fully understood. The aim of the study was to elucidate the role of the FRMD5 protein in the regulation of biological pathways associated with the development of PTC. We imply that the presence of certain genetic aberrations (e.g., BRAF V600E mutation) is associated with the activity of FRMD5. Methods: The studies were conducted on TPC1 and BCPAP (BRAF V600E) model PTC-derived cells. Transfection with siRNA was used to deplete the expression of FRMD5. The mRNA expression and protein yield were evaluated using RT-qPCR and Western blot techniques. Proliferation, migration, invasiveness, adhesion, spheroid formation, and survival tests were performed. RNA sequencing and phospho-kinase proteome profiling were used to assess signaling pathways associated with the FRMD5 expressional status. Results: The obtained data indicate that the expression of FRMD5 is significantly enhanced in BRAF V600E tumor specimens and cells. It was observed that a drop in intracellular yield of FRMD5 results in significant alternations in the migration, invasiveness, adhesion, and spheroid formation potential of PTC-derived cells. Importantly, significant divergences in the effect of FRMD5 depletion in both BRAF-wt and BRAF-mutated PTC cells were observed. It was also found that knockdown of FRMD5 significantly alters the expression of multidrug resistant genes. Conclusions: This is the first report highlighting the importance of the FRMD5 protein in the biology of PTCs. The results suggest that the FRMD5 protein can play an important role in controlling the metastatic potential and multidrug resistance of thyroid tumor cells.

Multi-sample measurement of hyperpolarized pyruvate-to-lactate flux in melanoma cells.

Hyperpolarized [1-13 C] pyruvate can be used to examine the metabolic state of cancer cells, highlighting a key metabolic characteristic of cancer: the upregulated metabolic flux to lactate, even in the presence of oxygen (Warburg effect). Thus, the rate constant of 13 C exchange of pyruvate to lactate, kPL , can serve as a metabolic biomarker of cancer presence, aggressiveness and therapy response. Established in vitro hyperpolarized experiments dissolve the probe for each cell sample independently, an inefficient process that consumes excessive time and resources. Expanding on our previous development of a microcoil with greatly increased detection sensitivity (103 -fold) compared with traditional in vitro methods, we present a novel microcoil equipped with a 10-μL vertical reservoir and an experimental protocol utilizing deuterated dissolution buffer to measure metabolic flux in multiple mass-limited cell suspension samples using a single dissolution. This method increases efficiency and potentially reduces the methodological variability associated with hyperpolarized experiments. This technique was used to measure pyruvate-to-lactate flux in melanoma cells to assess BRAF-inhibition treatment response. There was a significant reduction of kPL in BRAFV600E cells following 24 and 48 hours of treatment with 2 μM vemurafenib (P ≤ .05). This agrees with significant changes observed in the pool sizes of extracellular lactate (P ≤ .05) and glucose (P ≤ .001) following 6 and 48 hours of treatment, respectively, and a significant reduction in cell proliferation following 72 hours of treatment (P ≤ .01). BRAF inhibition had no significant effect on the metabolic flux of BRAFWT cells. These data demonstrate a 6-8-fold increase in efficiency for the measurement of kPL in cell suspension samples compared with traditional hyperpolarized in vitro methods.

CSF1R Is Required for Differentiation and Migration of Langerhans Cells and Langerhans Cell Histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare disorder characterized by tissue accumulation of CD1a+CD207+ LCH cells. In LCH, somatic mutations of the BRAF V600E gene have been detected in tissue LCH cells, bone marrow CD34+ hematopoietic stem cells, circulating CD14+ monocytes, and BDCA1+ myeloid dendritic cells (DC). Targeting BRAF V600E in clonal Langerhans cells (LC) and their precursors is a potential treatment option for patients whose tumors have the mutation. The development of mouse macrophages and LCs is regulated by the CSF1 receptor (CSF1R). In patients with diffuse-type tenosynovial giant cell tumors, CSF1R inhibition depletes tumor-associated macrophages (TAM) with therapeutic efficacy; however, CSF1R signaling in LCs and LCH has not been investigated. We found through IHC and flow cytometry that CSF1R is normally expressed on human CD1a+CD207+ LCs in the epidermis and stratified epithelia. LCs that were differentiated from CD14+ monocytes, BDCA1+ DCs, and CD34+ cord blood progenitors expressed CSF1R that was downregulated upon maturation. Immature LCs migrated toward CSF1, but not IL34. Administration of the c-FMS/CSF1R kinase inhibitors GW2580 and BLZ945 significantly reduced human LC migration. In LCH clinical samples, LCH cells (including BRAF V600E cells) and TAMs retained high expression of CSF1R. We also detected the presence of transcripts for its ligand, CSF1, but not IL34, in all tested LCH cases. CSF1R and CSF1 expression in LCH, and their role in LC migration and differentiation, suggests CSF1R signaling blockade as a candidate rational approach for treatment of LCH, including the BRAF V600E and wild-type forms of the disease.

Abstract B18: Clostridium perfringens lethal toxin specifically targets RAS and disrupts RAS signaling pathway

Abstract Clostridium perfringens lethal toxin (TpeL) belongs to the family of large clostridial glucosylating cytotoxins. Clostridial toxins are glucosyltransferases that modify and deactivate small GTPases of the RHO and RAS subfamily. TpeL mono-glucosylates in the switch I domain (Thr35) of RAC and RAS small GTPases. RAS family proteins function as key regulators of cell proliferation, differentiation, survival and gene expression. Moreover, mutations in RAS proteins are highly prevalent in human cancers. Considering the specificity of TpeL for RAS, we decided to investigate the biochemical interaction between TpeL and RAS, and potentially develop TpeL as a tool to disrupt the RAS signaling pathway in vivo. First, we used RAS-dependent MEFs expressing different KRAS mutations and BRAF V600E to assess the specificity of TpeL in vivo. We expected that proliferation should be inhibited in MEF cells expressing RAS isoforms, but MEF cells expressing BRAF V600E should not be affected. We found that TpeL treatment did not affect the viability of the BRAF V600E cells and only induced toxicity in RAS expressing cells. Consistent with this result, TpeL treatment inhibited MAPK signal transduction in the RAS expressing cells, but not in the BRAF V600E cells. Surprisingly, both cell viability as well as pERK levels remained unaffected in the KRAS Q61R MEF cells, suggesting that KRAS Q61R is resistant to TpeL. Furthermore, TpeL treatment of cancer cell lines shows significant differences in potency, with KRAS G12C cell lines being more sensitive to the toxin. Our cellular data suggest a correlation between TpeL sensitivity and intrinsic hydrolysis rate of the KRAS alleles. Biochemical assays corroborate our hypothesis that TpeL preferentially glucosylates KRAS GDP loaded in vitro. Moreover, TpeL glucosylation inhibits GEF-mediated nucleotide exchange, effectively blocking KRAS on the GDP inactive state. To further follow up the correlation between KRAS GDP levels and TpeL sensitivity, we used a combination treatment of TpeL and EGFR/FGFR inhibitors. Combination screen shows a synergistic effect; we observed increased TpeL sensitivity in combination with the inhibitors. By elucidating the mechanism of substrate binding and substrate specificity, we have gained insight into the TpeL-RAS interaction and we are working on developing the toxin as a therapeutic tool. Citation Format: Maria Abreu-Blanco, Ming Yi, Jean-Paul Denson, Matthew Holderfield. Clostridium perfringens lethal toxin specifically targets RAS and disrupts RAS signaling pathway [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B18.

A Combinatorial Strategy for Targeting BRAF V600E-Mutant Cancers with BRAFV600E Inhibitor (PLX4720) and Tyrosine Kinase Inhibitor (Ponatinib).

Most aggressive thyroid cancers are commonly associated with a BRAF V600E mutation. Preclinical and clinical data in BRAF V600E cancers suggest that combined BRAF and MEK inhibitor treatment results in a response, but resistance is common. One mechanism of acquired resistance is through persistent activation of tyrosine kinase (TK) signaling by alternate pathways. We hypothesized that combination therapy with BRAF and multitargeting TK inhibitors (MTKI) might be more effective in BRAF V600E thyroid cancer than in single-agent or BRAF and MEK inhibitors. The combined drug activity was analyzed to predict any synergistic effect using high-throughput screening (HTS) of active drugs. We performed follow-up in vitro and in vivo studies to validate and determine the mechanism of action of synergistic drugs. The MTKI ponatinib and the BRAF inhibitor PLX4720 showed synergistic activity by HTS. This combination significantly inhibited proliferation, colony formation, invasion, and migration in BRAF V600E thyroid cancer cell lines and downregulated pERK/MEK and c-JUN signaling pathways, and increased apoptosis. PLX4720-resistant BRAF V600E cells became sensitized to the combination treatment, with decreased proliferation at lower PLX4720 concentrations. In an orthotopic thyroid cancer mouse model, combination therapy significantly reduced tumor growth (P < 0.05), decreased the number of metastases (P < 0.05), and increased survival (P < 0.05) compared with monotherapy and vehicle control. Combination treatment with ponatinib and PLX4720 exhibited significant synergistic anticancer activity in preclinical models of BRAF V600E thyroid cancer, in addition to overcoming PLX4720 resistance. Our results suggest this combination should be tested in clinical trials.

Inhibiting BRAF Oncogene–Mediated Radioresistance Effectively Radiosensitizes BRAFV600E-Mutant Thyroid Cancer Cells by Constraining DNA Double-Strand Break Repair

Activating BRAF mutations, most commonly BRAFV600E, are a major oncogenic driver of many cancers. We explored whether BRAFV600E promotes radiation resistance and whether selectively targeting BRAFV600E with a BRAF inhibitor (vemurafenib, BRAFi) sensitizes BRAFV600E thyroid cancer cells to radiotherapy. Immunoblotting, neutral comet, immunocytochemistry, functional reporter, and clonogenic assays were used to analyze the outcome and molecular characteristics following radiotherapy with or without BRAFV600E or vemurafenib in thyroid cancer cells. BRAFV600E thyroid cancer cell lines were associated with resistance to ionizing radiation (IR), and expression of BRAFV600E into wild-type BRAF thyroid cancer cells led to IR resistance. BRAFi inhibited ERK signaling in BRAFV600E mutants, but not BRAF wild-type thyroid cancer cell lines. BRAFi selectively radiosensitized and delayed resolution of IR-induced γH2AX nuclear foci in BRAFV600E cells. Moreover, BRAFi impaired global DNA repair and altered the resolution of 53BP1 and RAD51 nuclear foci in BRAFV600E cells following IR. BRAFV600E mutants displayed enhanced nonhomologous end-joining (NHEJ) repair activity, which was abolished by BRAFi. Intriguingly, BRAFV600E mutation led to upregulation of XLF, a component of NHEJ, which was prevented by BRAFi. Importantly, BRAFi in combination with radiotherapy resulted in marked and sustained tumor regression of BRAFV600E thyroid tumor xenografts. BRAFV600E mutation promotes NHEJ activity leading to radioresistance and BRAFi selectively radiosensitizes BRAFV600E thyroid cancer cells through inhibiting NHEJ. Our findings suggest that combining BRAFi and radiation may improve the therapeutic outcome of patients with BRAFV600E-mutant thyroid cancer.

Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma.

Melanocortin 1 receptor (MC1R) is under investigation as a target for drug delivery for metastatic melanoma therapy and imaging. The purpose of this study was to determine the potential of using BRAF inhibitors (BRAFi) and histone deacetylase inhibitors (HDACi) to enhance the delivery of MC1R-targeted radiolabeled peptide ([212Pb]DOTA-MC1L) by pharmacologically upregulating the MC1R expression in metastatic melanoma cells and tumors. MC1R expression was analyzed in de-identified melanoma biopsies by immunohistochemical staining. Upregulation of MC1R expression was determined in BRAFV600E cells (A2058) and BRAF wild-type melanoma cells (MEWO) by quantitative real-time polymerase chain reaction, flow cytometry, and receptor-ligand binding assays. The role of microphthalmia-associated transcription factor (MITF) in the upregulation of MC1R was also examined in A2058 and MEWO cells. The effectiveness of [212Pb]DOTA-MC1L α-particle radiotherapy in combination with BRAFi and/or HDACi was determined in athymic nu/nu mice bearing A2058 and MEWO human melanoma xenografts. High expression of MC1R was observed in situ in clinical melanoma biopsies. BRAFi and HDACi significantly increased the MC1R expression (up to 10-fold in mRNA and 4-fold in protein levels) via MITF-dependent pathways, and this increase led to enhanced ligand binding on the cell surface. Inhibition of MITF expression antagonized the upregulation of MC1R in both BRAFV600E and BRAFWT cells. Combining [212Pb]DOTA-MC1L with BRAFi and/or HDACi improved the tumor response by increasing the delivery of 212Pb α-particle emissions to melanoma tumors via augmented MC1R expression. These data suggest that FDA-approved HDACi and BRAFi could improve the effectiveness of MC1R-targeted therapies by enhancing drug delivery via upregulated MC1R.

Contextual role of E2F1 in suppression of melanoma cell motility and invasiveness.

The general transcription factor E2F1 reportedly functions in a protumorigenic manner in several cancer models. We show that the genetic context of cancer cells influence E2F1's role to impede the protumorigenic role. Thirty to fifty percent of melanoma patients carry mutant BRAF with about 90% of mutant BRAF melanomas being V600E mutation. Tissue microarrays from melanoma patients were used to establish an association between E2F1 and BRAFV600E . We show for the first time that low E2F1 levels in BRAFV600E melanomas are associated with lymph node metastasis. Genetic manipulation of E2F1 in BRAFV600E and BRAFwt cells were used to determine its role in malignant melanoma progression by examining effects on migration and invasion. E2F1-mediated negative regulation of myosin light chain kinase (MYLK) increased migration and invasion in BRAFV600E cells by phosphorylating myosin light chain and increased stress fiber formation. We show that E2F1 inhibits extracellular signal-regulated kinase (ERK) activation in BRAFV600E cells and provide evidence for a negative feedback loop between E2F1 and ERK in these cells. This study shows for the first time that E2F1 has a cancer protective role in oncogenic BRAF-activated melanoma cells and that loss of E2F1 can allow disease progression through a novel mechanism of E2F1-mediated MYLK regulation. This study has implications for oncogenic BRAF-activated tumors and resistance to targeted oncogenic BRAF therapy.

Utx Insufficiency Cooperates with Braf V600E in the Induction of Myeloma in Mice

Introduction:Dysfunction of epigenetic pathways has been frequently implicated in hematological malignancies. In multiple myeloma (MM), EZH2, a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3), acts as an oncogene as evidenced by its overexpression, which was found to be positively correlated with disease progression (Kalushkova et al. PloS One. 2010). We have shown that inhibition of both EZH2 and its homolog EZH1 is effective in eradicating MM cells in vitro and in vivo (Rizq et al. Clin Cancer Res. 2017). In addition, inactivating somatic mutations in UTX/KDM6A, an X-linked histone demethylase that removes di- and tri-methyl groups from H3K27, are found in 3 - 10% of MM patients (van Haaften et al. Nat Genet. 2009 and Pawlyn et al. Clin Cancer Res. 2016), indicating a tumor suppressive role for UTX, which has yet to be delineated. Up till now, no mouse model has been generated to test Utx insufficiency in post germinal center (GC) B cells and plasma cells. On the other hand, an activating mutation V600E in the BRAF kinase gene is closely associated with aggressive disease features such as extramedullary disease and shorter overall survival in MM patients (Andrulis et al. Cancer Discov. 2013) and could accelerate induction of myeloma in mice.Methods:To investigate whether loss of Utx cooperates with Braf V600E in myelomagenesis in mice, we generated and analyzed mice with conditional knock-out allele of Utx and/or knock-in allele of Braf V600E combined with Cγ1-Cre allele, in which Cre is activated by immunization in post GC B cells.Results:Loss of Utx and Braf V600E synergistically induced post GC B-cell lymphoma and plasma cell neoplasms in mice and significantly shortened the survival of mice compared with control mice and either allele alone. Utx-/-Braf V600E females succumbed to death earlier than Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales. Of note, plasma cell neoplasms developed at a high frequency in Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales and, less frequently, in Utx-/-Braf V600E females. Mice with plasma cell neoplasms showed expansion of CD138+ plasma cells in bone marrow as well as spleen and/or lymph nodes, exhibiting extramedullary disease. Loss of Utx alleles and expression of Braf V600E were confirmed by genomic PCR of plasma cells. Importantly, the clonality of plasma cells was demonstrated by genomic PCR detecting rearrangements of immunoglobulin heavy and light chain genes. In addition, M protein was detected by serum protein electrophoresis (SPEP) at a high frequency. Notably, we were able to establish murine myeloma cell lines from moribund compound mice. These cells readily engrafted in the bone marrow of NOG mice after transplantation and caused myeloma-associated phenotypes including paraplegia in recipient mice. Interestingly, Utx-/-Braf V600E cells were sensitive to dual inhibition of EZH2 and EZH1 but not to specific inhibition of EZH2 in culture. They also showed decreased susceptibility to proteasome inhibitors when compared with human MM cell lines. To gain insight into the changes in the transcriptional landscape following Utx loss, we performed RNA sequencing (RNA seq) and then gene set enrichment analysis (GSEA). We found positive enrichment of gene sets related to Myc, implying that Myc is one of the main drivers of myelomagenesis in our mouse model. In addition, gene sets related to MM were significantly enriched following Utx loss. We are now working on ChIP sequencing (ChIP seq) of UTX-related histone modifications to evaluate the epigenetic impact of Utx loss on myelomagenesis.Conclusion:Utx insufficiency cooperates with Braf V600E in the induction of myeloma in mice. Our mouse model is a promising tool for understanding the role of epigenetic dysregulation in the pathogenesis of MM and evaluating novel anti-myeloma agents. DisclosuresOkuno:Celgene: Research Funding. Tamaru:Nichirei Bioscience INC.: Research Funding; Takeda Pharmaceutical Company Limited: Speakers Bureau.

BRAF Oncogenic Activation Induces Radioresistance Through Non-homologous End-Joining Repair Which is Abrogated by Targeted Inhibition of BRAF Mutational Activity

BRAF activating mutations, most commonly BRAFV600E/K, are driver mutations commonly found in solid tumors for which ionizing radiation (IR) is used, including thyroid, melanoma, colorectal, and lung cancer. Whether BRAFV600E mutation fosters radioresistance through nuclear DNA repair mechanisms is unclear. We hypothesize that BRAFV600E promotes radioresistance through heightened double-strand break repair mechanisms, which can be attenuated by targeted inhibition of the BRAF oncogene. We tested the effects of a BRAFV600E mutation using papillary and anaplastic thyroid cancer cell lines that possessed either wild-type or mutant BRAFV600E. We evaluated the role of BRAF and downstream targets in radiation clonogenic, neutral comet, nuclear foci, immunofluorescence, immunoblotting, RT-PCR, functional DNA repair reporter, and in vivo tumor growth assays. Radiation clonogenic assay showed BRAFV600E thyroid cancer cell lines were resistant to IR, and introduction of BRAFV600E into wild-type BRAF thyroid cancer TPC-1 cells led to significant IR resistance (dose enhancement factor, DEF 0.70). Selectively targeting BRAFV600E with a potent mutant BRAF inhibitor (vemurafenib, Vem) significantly sensitized BRAFV600E to IR, but not wild-type BRAF thyroid cancer cells (DEFs 1.37-1.78). Immunoblotting demonstrates that vemurafenib potently inhibited ERK signaling in BRAFV600E mutants but not BRAF wild-type cells. Vem delayed DNA repair in BRAFV600E cells after IR as evidenced by comet tail assays at 15 min, 1, 6, and 24 hrs (p<0.005) and γ-H2AX nuclear foci assays at 6 and 24 hrs (p<0.01). Moreover, Vem altered the kinetics of 53BP and RAD51 nuclear foci in BRAFV600E cells at 6 and 24 hrs after IR (p<0.05). Assessment of double-strand DNA repair using functional reporter assays revealed that BRAFV600E mutant was associated with enhanced non-homologous end-joining (NHEJ) repair activity, while Vem significantly reduced NHEJ repair activity by ∼40% (p<0.01). BRAFV600E mutation led to upregulation of XLF, a nuclear protein involved in NHEJ-mediated DNA repair, in thyroid cancer cell lines and in the publicly-available TCGA dataset (p<2.4e-14). Additionally, Vem treatment resulted in decreased XLF expression and genetic down-regulation of XLF sensitized BRAFV600E cells to IR (p<0.05). Importantly, Vem in combination with IR resulted in sustained tumor regression of BRAFV600E thyroid tumor xenografts, with minimal evidence of tumor regrowth after 60 days. BRAFV600E mutation promotes NHEJ activity leading to radioresistance, and Vem selectively radiosensitizes BRAFV600E thyroid cancer cells by inhibiting NHEJ repair. Our findings suggest that combining BRAF inhibition and IR may improve the therapeutic outcome for patients with BRAFV600E mutant thyroid cancer.

Dissecting RAF Inhibitor Resistance by Structure-based Modeling Reveals Ways to Overcome Oncogenic RAS Signaling

Clinically used RAF inhibitors are ineffective in RAS mutant tumors because they enhance homo- and heterodimerization of RAF kinases, leading to paradoxical activation of ERK signaling. Overcoming enhanced RAF dimerization and the resulting resistance is a challenge for drug design. Combining multiple inhibitors could be more effective, but it is unclear how the best combinations can be chosen. We built a next-generation mechanistic dynamic model to analyze combinations of structurally different RAF inhibitors, which can efficiently suppress MEK/ERK signaling. This rule-based model of the RAS/ERK pathway integrates thermodynamics and kinetics of drug-protein interactions, structural elements, posttranslational modifications, and cell mutational status as model rules to predict RAF inhibitor combinations for inhibiting ERK activity in oncogenic RAS and/or BRAFV600E backgrounds. Predicted synergistic inhibition of ERK signaling was corroborated by experiments in mutant NRAS, HRAS, and BRAFV600E cells, and inhibition of oncogenic RAS signaling was associated with reduced cell proliferation and colony formation.

IL6/STAT3 axis mediates resistance to BRAF inhibitors in thyroid carcinoma cells

Thyroid carcinomas (TCs) bearing BRAF mutations represent approximately 26–53% of human thyroid malignancies and, differently from melanomas, are poorly sensitive to BRAF inhibitors (BRAFi), and develop acquired resistance through activation of alternative signaling pathways. A whole-genome gene expression analysis of TC BRAF V600E cells exposed to PLX4032 identified JAK/STAT among the most significantly modulated signaling pathways. Interestingly, both transient exposure and chronic adaptation to PLX4032 resulted in upregulation of IL6/STAT3 axis and this impaired the cytostatic activity of PLX4032. Mechanistically, exposure to PLX4032 enhanced IL6 secretion and this, in turn, was responsible for STAT3 upregulation, activation of ERK signaling and poor sensitivity to BRAF inhibition. Consistently, the dual blockade of STAT3 (by siRNA or pharmacological inhibition) or IL6 signaling (by the humanized anti-human IL6 receptor antibody, tocilizumab) and BRAF (by PLX4032) improved the inhibition of cell cycle progression compared to PLX4032 single agent. These data support the role of IL6/STAT3 signaling pathway in modulating TC cell response to PLX4032 and candidate IL6 targeting as a strategy to improve the activity of PLX4032 in BRAF V600E TC cells.

Abstract 1361: Characterization of TpeL, a RAS specific clostridial toxin

Abstract Clostridium perfringes lethal toxin (TpeL) belongs to the family of large clostridial glycosylating cytotoxins. Clostridial toxins are glycosyltransferases that modify and deactivate small GTPases of the RHO and RAS subfamily. TpeL mono-glycosylates in the switch I domain (Thr35) of RAC and RAS small GTPases. Glycosylation of RAS at Thr35 prevents binding to the primary effector, RAF kinase and results in a blockade RAS signal transduction. RAS family proteins function as key regulators of cell proliferation, differentiation, survival and gene expression. Moreover, mutations in RAS proteins are highly prevalent in human cancers. Considering the specificity of TpeL for RAS, we decided to investigate the biochemical interaction between TpeL and RAS, and potentially develop tools to disrupt the RAS signaling pathway. In order to assess the effect of TpeL in vivo, we use RAS-dependent MEFs expressing different KRAS mutations and BRAF V600E. We expect that proliferation should be inhibited in MEF cells expressing RAS isoforms, but MEF cells expressing BRAF V600E should not be affected by any toxin. We found that TpeL treatment did not affect the viability of the BRAF V600E cells and only induced toxicity in RAS expressing cells. Consistent with this result, TpeL treatment inhibited MAPK signal transduction in the RAS expressing cells, but not in the BRAF V600E cells. Surprisingly, both cell viability as well as pERK levels remained unaffected in the KRAS Q61R MEF cells treated with TpeL, suggesting that KRAS Q61R is resistant to glycosylation. This may be due in part to the relatively slow intrinsic rate of GTP hydrolysis and very high levels of KRAS-GTP in KRAS Q61R cells. We also developed and adapted biochemical assays to study TpeL-KRAS interaction in vitro. Based on Alpha Assay and UDP Glow, TpeL activity does not appear to be nucleotide specific, in contrast to previously reported findings. We are currently testing if TpeL RAS glycosylation is competitive with or is inhibited by other molecules that bind to switch I, such as the RBD domain of RAF1 or GEF/GAP proteins. These data may indicate that the lack of activity in KRAS Q61R expressing MEF cells is due not to the high levels of RAS-GTP, but to the constitutive binding to effector molecules, which prevent TpeL from accessing KRAS. We also hope to identify the critical residues required for TpeL binding and catalysis. By elucidating the mechanism of substrate binding and substrate specificity we may gain insight into novel binding pockets that could be exploited therapeutically. Citation Format: Maria T. Abreu-Blanco. Characterization of TpeL, a RAS specific clostridial toxin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1361. doi:10.1158/1538-7445.AM2017-1361