BRAF-mutant Cells Research Articles

Lobeglitazone, A Peroxisome Proliferator-Activated Receptor-Gamma Agonist, Inhibits Papillary Thyroid Cancer Cell Migration and Invasion by Suppressing p38 MAPK Signaling Pathway.

BackgroundPeroxisome proliferator-activated receptor-gamma (PPAR-γ) ligands have been widely shown to correlate with epithelial-mesenchymal transition (EMT) and cancer progression. Lobeglitazone (LGZ) is a novel ligand of PPAR-γ; and its role in EMT and metastasis in papillary thyroid carcinoma (PTC) is poorly understood. We aimed to investigate the role of LGZ in metastatic behavior of PTC cells.MethodsHalf maximal inhibitory concentration (IC50) values of LGZ in BRAF-mutated PTC cell lines (BCPAP and K1) were determined using MTT assay. Rosiglitazone (RGZ), the PPAR-γ ligand was used as a positive control. The protein expression of PPAR-γ, cell-surface proteins (E-cadherin, N-cadherin), cytoskeletal protein (Vimentin), transcription factor (Snail), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2 pathway, and matrix metalloproteinase (MMP)-2 expression were measured using Western blotting. Changes in E-cadherin expression were also determined using immunocytochemistry. Cell migration and invasion were analyzed using wound healing and Matrigel invasion assays.ResultsTreatment with LGZ or RGZ significantly inhibited transforming growth factor-beta1 (TGF-β1)-induced EMT-associated processes such as fibroblast-like morphological changes, EMT-related protein expression, and increased cell migration and invasion in BCPAP and K1 cells. LGZ restored TGF-β1-induced loss of E-cadherin, as observed using immunocytochemistry. Furthermore, LGZ and RGZ suppressed TGF-β1-induced MMP-2 expression and phosphorylation of p38 MAPK, but not ERK1/2. Although there was no change in PPAR-γ expression after treatment with LGZ or RGZ, the effect of downstream processes mediated by LGZ was hampered by GW9662, a PPAR-γ antagonist.ConclusionLGZ inhibits TGF-β1-induced EMT, migration, and invasion through the p38 MAPK signaling pathway in a PPAR-γ-dependent manner in PTC cells.

Tissue architecture delineates field cancerization in BRAFV600E-induced tumor development.

ABSTRACTCancer cells hijack developmental growth mechanisms but whether tissue morphogenesis and architecture modify tumorigenesis is unknown. Here, we characterized a new mouse model of sporadic thyroid carcinogenesis based on inducible expression of BRAF carrying a Val600 Glu (V600E) point mutation (BRAFV600E) from the thyroglobulin promoter (TgCreERT2). Spontaneous activation of this Braf-mutant allele due to leaky activity of the Cre recombinase revealed that intrinsic properties of thyroid follicles determined BRAF-mutant cell fate. Papillary thyroid carcinomas developed multicentrically within a normal microenvironment. Each tumor originated from a single follicle that provided a confined space for growth of a distinct tumor phenotype. Lineage tracing revealed oligoclonal tumor development in infancy and early selection of BRAFV600E kinase inhibitor-resistant clones. Somatic mutations were few, non-recurrent and limited to advanced tumors. Female mice developed larger tumors than males, reproducing the gender difference of human thyroid cancer. These data indicate that BRAFV600E-induced tumorigenesis is spatiotemporally regulated depending on the maturity and heterogeneity of follicles. Moreover, thyroid tissue organization seems to determine whether a BRAF-mutant lineage becomes a cancerized lineage. The TgCreERT2;BrafCA/+ sporadic thyroid cancer mouse model provides a new tool to evaluate drug therapy at different stages of tumor evolution.

EIF4B enhances ATF4 expression and contributes to cellular adaptation to asparagine limitation in BRAF-mutated A375 melanoma

ATF4 is a crucial transcription factor in the integrated stress response, a major adaptive signaling pathway activated by tumor microenvironment and therapeutic stresses. BRAF inhibitors, such as vemurafenib, induce ATF4 in BRAF-mutated melanoma cells, but the mechanisms of ATF4 induction are not fully elucidated. Here, we show that ATF4 expression can be upregulated by eukaryotic initiation factor 4B (eIF4B) in BRAF-mutated A375 cells. Indeed, eIF4B knockout (KO) prevented ATF4 induction and activation of the uORF-mediated ATF4 translation mechanism during vemurafenib treatment, which were effectively recovered by the rescue of eIF4B. Transcriptome analysis revealed that eIF4B KO selectively influenced ATF4-target gene expression among the overall gene expression changed by vemurafenib. Interestingly, eIF4B supported cellular proliferation under asparagine-limited conditions, possibly through the eIF4B-ATF4 pathway. Our findings indicate that eIF4B can regulate ATF4 expression, thereby contributing to cellular stress adaptation, which could be targeted as a therapeutic approach against malignancies, including melanoma.

Abstract 2361: The functional roles of miRNA-193a-3p in colorectal cancer

Abstract Background: Patients with BRAF-mutated colorectal cancer (CRC) have a poor prognosis despite recent therapeutic advances such as combination therapy with BRAF, MEK, and EGFR inhibitors. Aim: Our aim was to clarify the functions of the microRNAs (miRNAs) involved in oncogenesis and sensitivity to BRAF inhibitors and MEK inhibitors in BRAF-mutated CRC. Methods and Results: To identify miRNAs that sensitize a BRAF inhibitor dabrafenib (DAB) and a MEK inhibitor trametinib (TRA), we screened 240 miRNAs in BRAF-mutated CRC cell line RKO. Among the screened miRNAs, we tried to elucidate the novel functional roles of miR-193a-3p in CRC cell lines. We found that miR-193a-3p overexpression inhibited cell proliferation in CRC cell lines. The protein array analysis revealed that protein phosphorylation levels were altered by miR-193a-3p overexpression in various pathways such as cell-cycle-related pathways and PI3K-AKT pathways. The enhanced cytotoxicity by miR-193a-3p in addition to DAB and TRA was validated in multiple BRAF-mutated cell lines. Furthermore, the enhanced cytotoxicity by miR-193a-3p in addition to DAB and TRA was shown to be through inhibiting reactivation of MAPK signaling. This enhanced cytotoxicity by miR-193a-3p was even observed under treatment of DAB, TRA, and an anti-EGFR antibody. Conclusions: Our study shows that miR-193a-3p regulates multiple proteins involved in oncogenesis, and proposes that the combination therapy of miR-193a-3p and BRAF/MEK inhibitors could be a promising treatment strategy in BRAF-mutated CRC. Citation Format: Sakura Hiraide, Masanobu Takahashi, Yuya Yoshida, Keigo Komine, Chikashi Ishioka. The functional roles of miRNA-193a-3p in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2361.

Abstract 598: Resistance mechanisms to BRAF inhibition identified by single circulating tumor cell and cell-free tumor DNA molecular profiling in BRAF-mutant non-small cell lung cancer

Abstract Background: Combination therapy with dabrafenib + trametinib demonstrated robust activity in patients (pts) with BRAFV600E-mutant advanced non-small cell lung cancer (NSCLC), but its resistance mechanisms are poorly known. Liquid biopsy components such as circulating tumor cells (CTCs) and cell-free (cf) tumor DNA can provide a comprehensive genomic picture of tumor content. Molecular profiling of single CTCs from pts with BRAF-V600Emutant NSCLC was performed to carry out a pilot study to identify resistance mutations at failure to dabrafenib + trametinib and to compare the mutations detected on CTCs to the mutations found on cfDNA and tumor biopsies. Patients and Methods: Eight pts with advanced BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib were prospectively enrolled between Jul 2018 and Mar 2019 at Gustave Roussy (IDRCB2008-A00585-50). Bloods samples were collected. Matched tissue-cfDNA and CTCs were available in 3 pts and matched tissue-CTCs for 4 pts. Single CTC isolation strategy included RosetteSep enrichment, immunofluorescent staining (Hoechst/CD45/cytokeratins) and fluorescence activated cell-sorting. The process to identify CTC mutations included Ampli1 whole-genome amplification, quality controls, multiplex targeted PCR with the Ampli1 CHPCustomBeta cancer panel developed by (Menarini Silicon Biosystems) and next-generation sequencing (NGS). The cfDNA was analyzed using InVisionFirst-Lung. Tissue samples were analyzed using targeted NGS in the MATCH-R trial (Recondo G; NPJ Precis Oncol 2020). Results: Single CTCs were isolated from 7 pts. As baseline characteristics, the median age was 66 years, 5 (71%) were smoker; all the pts with adenocarcinoma histology. Most of the pts received dabrafenib + trametinib as 2nd line (86%). The median of CTCs isolated by patient was 20 (8-28). A wide spectrum of mutations in CTCs was observed at treatment failure that were involved in the main cancer pathways, including MAPK (n=1; NRAS), tyrosine kinase receptors (n=5; EGFR, ALK, FLT3, HER2,…), signal transduction (n=4; IDH1, EZH2,⋯), and DNA repair (n=2; AKT1, ATM,⋯). In the same CTC, several mutations were observed in 5/7 patients, commonly involving more than one cancer pathways. A higher degree of mutational diversity was observed in CTCs compared to tumor tissue biopsies and cfDNA. In the 3 patients with an available tumor/liquid biopsy, only 1 shared mutations between CTCs and matched tumor and cfDNA. Conclusion: Single CTC profiling reveals a wide spectrum of therapeutic resistance mutations not detected by other analyses in pts with BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib. Integration of single CTC sequencing to tumor and cfDNA analysis, provides important perspectives to assess heterogeneous resistance mechanisms and to guide precision medicine in BRAFV600E- NSCLC. Citation Format: Laura Mezquita, Marianne Oulhen, Agathe Aberlenc, Marc Deloger, Aurélie Honoré, Marianna Garonzi, Genny Buson, Claudio Forcato, Yann Lecluse, Mihaela Aldea, Maud NgoCamus, Claudio Nicotra, Karen Howarth, Ludovic Lacroix, Luc Friboulet, Benjamin Besse, Nicolò Manaresi, David Planchard, Françoise Farace. Resistance mechanisms to BRAF inhibition identified by single circulating tumor cell and cell-free tumor DNA molecular profiling in BRAF-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 598.

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.

Proteomic Profiling of BRAFV600E Mutant Colon Cancer Cells Reveals the Involvement of Nucleophosmin/c-Myc Axis in Modulating the Response and Resistance to BRAF Inhibition by Vemurafenib.

BRAFV600E mutations are found in approximately 10% of colorectal cancer patients and are associated with worse prognosis and poor outcomes with systemic therapies. The aim of this study was to identify novel druggable features of BRAFV600E-mutated colon cancer (CC) cells associated with the response and resistance to BRAFV600E inhibitor vemurafenib. Towards this aim, we carried out global proteomic profiling of BRAFV600E mutant vs. KRAS mutant/BRAF wild-type and double wild-type KRAS/BRAF CC cells followed by bioinformatics analyses. Validation of selected proteomic features was performed by immunohistochemistry and in silico using the TCGA database. We reveal an increased abundance and activity of nucleophosmin (NPM1) in BRAFV600E-mutated CC in vitro, in silico and in tumor tissues from colon adenocarcinoma patients and demonstrate the roles of NPM1 and its interaction partner c-Myc in conveying the resistance to vemurafenib. Pharmacological inhibition of NPM1 effectively restored the sensitivity of vemurafenib-resistant BRAF-mutated CC cells by down-regulating c-Myc expression and activity and consequently suppressing its transcriptional targets RanBP1 and phosphoserine phosphatase that regulate centrosome duplication and serine biosynthesis, respectively. Altogether, findings from this study suggest that the NPM1/c-Myc axis could represent a promising therapeutic target to thwart resistance to vemurafenib in BRAF-mutated CC.

LGG-05. GENERATION OF NOVEL MOUSE MODELS FOR BRAF V600E MUTANT GLIOMAGENESIS TO GAIN MECHANISTIC INSIGHTS INTO TUMOR FORMATION AND PROGRESSION

BackgroundThe BRAF V600E mutation occurs in ~ twenty percent of histologically diverse pediatric gliomas and is the second most common mutation in pediatric low-grade gliomas (LGG). BRAF V600E expression in LGG with balanced CDKN2A is associated with a higher rate for progression than for BRAF V600E wildtype tumors, and despite adjuvant therapy, consisting of resection, radiation and chemotherapy. Progression invariably occurs in BRAF V600E mutant CDKN2A deleted gliomas, marking a high-risk group. Here, we aim to overcome the lack BRAF V600E mutant glioma models that allow for studies of stem and progenitor cells and the immune system ability to understand progression. MethodsWe develop novel immunocompetent, stem and progenitor cell-based mouse models for BRAF mutant gliomas, including genetically engineered mouse models (GEMMs), orthotopic glioma models derived from gliomas in GEMMs as well as in vitro models of those tumors. BRAF mutant mouse brains and cells were analyzed by immunofluorescence staining, flow cytometry, mass cytometry and RNA sequencing. ResultsOngoing model development studies indicate that BRAF V600E mutant gliomas in murine brain exhibit very similar neuroanatomical preferences to human gliomas. The BRAF V600E mutation exacerbates the heterogenous cell cycling pattern of normal neural stem and progenitors and expands a symmetrically dividing progenitor population. Cellular plasticity rather than cellular lineage hierarchy drives the generation of a therapy resistant stem cell pool. Transcriptomic analyses of neuroglial stem cells with induced BRAF V600E expression provide insights into mechanisms for neoplastic transformation and progression. ConclusionAnalyses of two independent BRAF V600E mutant mouse models provide novel insights into the role for tumor intrinsic factors, such as plasticity and stemness, and the tumor microenvironment in progression.

BIOL-05. MAPK PATHWAY INHIBITION SENSITIZES TO IMMUNOTHERAPY IN BRAF-MUTANT GLIOMAS

BackgroundBRAF alterations frequently occur in pediatric low-grade gliomas. Previously, we showed that dabrafenib and trametinib (D+T) that target MAPK pathway can mediate the antitumor effect in a preclinical model of BRAF-mutant glioma (PMC5342782). Here, we further investigate the effect of MAPK pathway inhibitors on cancer cells and tumor-infiltrating immune cells to maximize the therapeutic efficacy in malignant gliomas. MethodsDrug concentrations in tumor, brain and plasma were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RNA sequencing and Gene Set Enrichment Analysis were performed using patient-derived BRAF-mutant glioma lines upon D+T treatment. Molecular profiles of drug-resistant clones were assessed for understanding of glioma heterogeneity and exploring new therapeutic targets. Results. BRAF-mutant stem-like glioma cells were particularly resistant to BRAF or MAPK inhibitor, along with aggressive phenotype in mice. LC-MS/MS showed effective D+T drug delivery in tumor regions. The transcriptome analysis demonstrated that D+T upregulate HLA molecules and downregulate immunosuppressive factors in patient-derived BRAF-mutant glioma lines. Consistent with these molecular changes, D+T led to changes in the proportions of tumor-infiltrating immune cells, including CD8+ cytotoxic T lymphocytes and FOXP3+ regulatory T cells. Furthermore, the therapeutic effect of D+T was further enhanced in combination with immune checkpoint inhibition. ConclusionsThe present study highlights the immunomodulatory activity of MAPK pathway inhibitors in BRAF-mutant gliomas.

LSD1 and Aberrant DNA Methylation Mediate Persistence of Enteroendocrine Progenitors That Support BRAF-Mutant Colorectal Cancer.

Despite the connection of secretory cells, including goblet and enteroendocrine (EEC) cells, to distinct mucus-containing colorectal cancer histologic subtypes, their role in colorectal cancer progression has been underexplored. Here, our analysis of The Cancer Genome Atlas (TCGA) and single-cell RNA-sequencing data demonstrates that EEC progenitor cells are enriched in BRAF-mutant colorectal cancer patient tumors, cell lines, and patient-derived organoids. In BRAF-mutant colorectal cancer, EEC progenitors were blocked from differentiating further by DNA methylation and silencing of NEUROD1, a key gene required for differentiation of intermediate EECs. Mechanistically, secretory cells and the factors they secrete, such as trefoil factor 3, promoted colony formation and activation of cell survival pathways in the entire cell population. Lysine-specific demethylase 1 (LSD1) was identified as a critical regulator of secretory cell specification in vitro and in a colon orthotopic xenograft model, where LSD1 loss blocks formation of EEC progenitors and reduces tumor growth and metastasis. These findings reveal an important role for EEC progenitors in supporting colorectal cancer. SIGNIFICANCE: This study establishes enteroendocrine progenitors as a targetable population that promotes BRAF-mutant colorectal cancer and can be blocked by LSD1 inhibition to suppress tumor growth.

The next-generation pan-RAF inhibitor, KIN-2787, is active in class II and class III BRAF mutant models.

3116 Background: Oncogenic BRAF gene alterations, leading to aberrantly activated BRAF monomers (Class I mutations) or dimers (Class II and Class III mutations), are observed in approximately 6% of all human cancers. First-generation BRAF inhibitors targeting Class I BRAF mutants, including dabrafenib, encorafenib, and vemurafenib, provide significant clinical benefit to patients with BRAF V600 mutation-driven melanoma and select solid tumors as monotherapies or in combination with other targeted therapies. The currently approved BRAF inhibitors have not, however, proven to be effective in patients with Class II or III BRAF alterations which account for a large proportion (34%) of BRAF mutations. KIN-2787 is an orally available, potent and selective small molecule pan-RAF inhibitor specifically designed to inhibit Class II and III BRAF dimers, in addition to Class I mutants. Methods: The efficacy and tolerability of the pan-RAF inhibitor, KIN-2787, was evaluated in vitro and in vivo in Class I, II, and III BRAF mutation-driven human cancer models. Results: In biochemical assays, KIN-2787 showed low nanomolar to picomolar potency against RAF1, BRAF, and ARAF (IC50 0.06-3.46 nM) with minimal activity towards non-RAF kinases. In cell-based assays, KIN-2787 inhibited RAF activity, as measured by inhibition of downstream ERK phosphorylation (pERK), across multiple BRAF mutant cancer cell lines. Class II and III BRAF mutant cell lines were the most responsive when treated with KIN-2787 (IC50 < 50 nM); 19- and 7-fold more sensitive compared to cells harboring wild-type BRAF, respectively. Dose-dependent inhibition of A-375 (Class I), BxPC-3 (Class II), and WM3629 (Class III) BRAF mutant human xenograft tumor growth was attained with daily KIN-2787 treatment and was well-tolerated. A trend towards greater tumor responses was observed with twice daily (BID) compared to once daily (QD) dosing of KIN-2787; however, the two dosing regimens led to similar tumor growth inhibition (TGI) and regressions (mean TGI up to 101-118%; p ≤0.0001) at equivalent total daily doses. Furthermore, KIN-2787 led to a significant in vivo pharmacodynamic response using either regimen, however, prolonged target coverage, as measured by pERK, was achieved with BID dosing. The impact of KIN-2787 treatment on additional biomarkers, including transcriptional changes and MAPK pathway modulation in cell-based models and patient-derived samples, will be presented at the meeting. Conclusions: KIN-2787 is a next-generation pan-RAF inhibitor with pronounced in vitro and in vivo activity against human cancers driven by Class II and III BRAF mutations. A phase 1 dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 monotherapy in patients with advanced or metastatic solid tumors harboring BRAF gene alterations, including Class II and III mutations, is expected to initiate in 2021.

High BRAF variant allele frequencies are associated with distinct pathological features and responsiveness to target therapy in melanoma patients

BackgroundBRAF mutant melanoma patients are commonly treated with anti-BRAF therapeutic strategies. However, many factors, including the percentage of BRAF-mutated cells, may contribute to the great variability in patient outcomes.Patients and methodsThe BRAF variant allele frequency (VAF; defined as the percentage of mutated alleles) of primary and secondary melanoma lesions, obtained from 327 patients with different disease stages, was assessed by pyrosequencing. The BRAF mutation rate and VAF were then correlated with melanoma pathological features and patients’ clinical characteristics. Kaplan–Meier curves were used to study the correlations between BRAF VAF, overall survival (OS), and progression-free survival (PFS) in a subset of 62 patients treated by anti-BRAF/anti-MEK therapy after metastatic progression.ResultsA highly heterogeneous BRAF VAF was identified (3%-90%). Besides being correlated with age, a higher BRAF VAF level was related to moderate lymphocytic infiltration (P = 0.017), to melanoma thickness according to Clark levels, (level V versus III, P = 0.004; level V versus IV, P = 0.04), to lymph node metastases rather than cutaneous (P = 0.04) or visceral (P = 0.03) secondary lesions. In particular, a BRAF VAF >25% was significantly associated with a favorable outcome in patients treated with the combination of anti-BRAF/anti-MEK drug (OS P = 0.04; PFS P = 0.019), retaining a significant value as an independent factor for the OS and the PFS in the multivariate analysis (P = 0.014 and P = 0.003, respectively).ConclusionThese results definitively support the role of the BRAF VAF as a potential prognostic and predictive biomarker in melanoma patients in the context of BRAF inhibition.

Deregulated FASN Expression in BRAF Inhibitor-Resistant Melanoma Cells Unveils New Targets for Drug Combinations.

Simple SummaryStrategies to overcome resistance to targeted therapy represent a clinical need for metastatic melanoma. Altered lipid metabolism has been identified in the metabolic reprogramming associated with melanoma progression. Lipid metabolism genes such as fatty acid synthase (FASN) and 24-dehydrocholesterol reductase (DHCR24) have been proposed to contribute to tumor aggressiveness, but the therapeutic value of lipid metabolism inhibitors, particularly in drug-resistant melanoma, is unknown. Here, we found that molecular targeting of FASN in melanoma cells resistant to the BRAF inhibitor PLX4032 increased the sensitivity to the drug. Up-regulation of DHCR24 upon FASN targeting revealed the activation of druggable compensatory pathways sustaining the growth of resistant cells.Metabolic changes promoting cell survival are involved in metastatic melanoma progression and in the development of drug resistance. In BRAF-inhibitor resistant melanoma cells, we explored the role of FASN, an enzyme involved in lipogenesis overexpressed in metastatic melanoma. Resistant melanoma cells displaying enhanced migratory and pro-invasive abilities increased sensitivity to the BRAF inhibitor PLX4032 upon the molecular targeting of FASN and upon treatment with the FASN inhibitor orlistat. This behavior was associated with a marked apoptosis and caspase 3/7 activation observed for the drug combination. The expression of FASN was found to be inversely associated with drug resistance in BRAF-mutant cell lines, both in a set of six resistant/sensitive matched lines and in the Cancer Cell Line Encyclopedia. A favorable drug interaction in resistant cells was also observed with U18666 A inhibiting DHCR24, which increased upon FASN targeting. The simultaneous combination of the two inhibitors showed a synergistic interaction with PLX4032 in resistant cells. In conclusion, FASN plays a role in BRAF-mutated melanoma progression, thereby creating novel therapeutic opportunities for the treatment of melanoma.

AIM2 Inhibits BRAF-Mutant Colorectal Cancer Growth in a Caspase-1-Dependent Manner.

Absent in melanoma 2 (AIM2), a DNA sensor that plays an important role in natural immunity system, has been reported to participate in colorectal cancer (CRC) development. However, the functional role of AIM2 in BRAF-mutant CRC remains unclear. In this study, we first investigated AIM2 expression level in BRAF-mutant CRC tumor tissues. Overexpression of AIM2 in CRC cells was performed to investigate the effect of AIM2 on CRC cell viability, and cell death detection and caspase activity assay were performed to explore the mechanism that AIM2 impacts the growth of BRAF-mutant CRC cells. Moreover, we confirmed the antitumor effect of AIM2 in BRAF-mutant CRC cell-derived tumor xenograft (CDX) models as well as patient-derived organoids (PDOs). Herein, we reported that AIM2 expression was lower in BRAF-mutant than that in BRAF wild-type CRC tumor tissues. Restoring the expression of AIM2 in BRAF-mutant CRC cells greatly inhibits the tumor cell growth by inducing necrotic cell death. Mechanism studies revealed that AIM2-induced cell death is in a caspase-1-dependent manner. Additionally, overexpression of AIM2 significantly inhibits tumor growth and metastasis in BRAF-mutant CRC in vivo, which was further confirmed in BRAF-mutant CRC PDOs. Taken together, our data suggested that AIM2 inhibits BRAF-mutant colon cancer growth in a caspase-1-dependent manner, which may provide evidence to understand the pathogenesis of CRC with BRAF-mutant, as well as new strategies for manipulation of CRC.

Combined Toxicity of Gas Plasma Treatment and Nanoparticles Exposure in Melanoma Cells In Vitro.

Despite continuous advances in therapy, cancer remains a deadly disease. Over the past years, gas plasma technology emerged as a novel tool to target tumors, especially skin. Another promising anticancer approach are nanoparticles. Since combination therapies are becoming increasingly relevant in oncology, both gas plasma treatment and nanoparticle exposure were combined. A series of nanoparticles were investigated in parallel, namely, silica, silver, iron oxide, cerium oxide, titanium oxide, and iron-doped titanium oxide. For gas plasma treatment, the atmospheric pressure argon plasma jet kINPen was utilized. Using three melanoma cell lines, the two murine non-metastatic B16F0 and metastatic B16F10 cells and the human metastatic B-Raf mutant cell line SK-MEL-28, the combined cytotoxicity of both approaches was identified. The combined cytotoxicity of gas plasma treatment and nanoparticle exposure was consistent across all three cell lines for silica, silver, iron oxide, and cerium oxide. In contrast, for titanium oxide and iron-doped titanium oxide, significantly combined cytotoxicity was only observed in B16F10 cells.

Inhibition of BRAF and ERK1/2 has synergistic effects on thyroid cancer growth in vitro and in vivo.

Mutations in the BRAF gene are highly prevalent in thyroid cancer. However, the response rate of thyroid tumors to BRAF-directed therapies has been mixed. Increasingly, combination therapies inhibiting the MAPK pathway at multiple nodes have shown promise. Recently developed ERK1/2 inhibitors are of interest for use in combination therapies as they have the advantage of inhibiting the most downstream node of the MAPK pathway, therefore preventing pathway reactivation. Here, we examined the effect of combined BRAF inhibition (dabrafenib) and ERK1/2 inhibition (SCH772984) on the growth and survival of a panel of BRAF-mutant thyroid cancer cell lines using in vitro and in vivo approaches. We found that resistance due to MAPK pathway reactivation occurs quickly with single-agent BRAF inhibition, but can be prevented with combined BRAF and ERK1/2 inhibition. Combined inhibition also results in synergistic growth inhibition, decreased clonogenic survival, and enhanced induction of apoptosis in a subset of BRAF-mutant thyroid cancer cells. Finally, combined inhibition of BRAF and ERK1/2 results in enhanced inhibition of tumor growth in an anaplastic thyroid cancer in vivo model. These results provide key rationale to pursue combined BRAF and ERK1/2 inhibition as an alternative therapeutic strategy for BRAF-mutant advanced thyroid cancer patients.

Differential Sensitivity of Wild-Type and BRAF-Mutated Cells to Combined BRAF and Autophagy Inhibition.

BRAF inhibitors are insufficient monotherapies for BRAF-mutated cancer; therefore, we investigated which inhibitory pathway would yield the most effective therapeutic approach when targeted in combination with BRAF inhibition. The oncogenic BRAF inhibitor, PLX4720, increased basal autophagic flux in BRAF-mutated cells compared to wild-type (WT) BRAF cells. Interestingly, early autophagy inhibition improved the effectiveness of PLX4720 regardless of BRAF mutation, whereas late autophagy inhibition did not. Although ATG5 knockout led to PLX4720 resistance in both WT and BRAF-mutated cells, the MEK inhibitor trametinib exhibited a synergistic effect on PLX4720 sensitivity in WT BRAF cells but not in BRAF-mutated cells. Conversely, the prolonged inhibition of endoplasmic reticulum (ER) stress reduced basal autophagy in BRAF-mutated cells, thereby increasing PLX4720 sensitivity. Taken together, our results suggest that the combined inhibition of ER stress and BRAF may simultaneously suppress both pro-survival ER stress and autophagy, and may therefore be suitable for treatment of BRAF-mutated tumors whose autophagy is increased by chronic ER stress. Similarly, for WT BRAF tumors, therapies targeting MEK signaling may be a more effective treatment strategy. Together, this study presents a rational combination treatment strategy to improve the efficacy of BRAF inhibitors depending on BRAF mutation status.

PD-L1 Is an Independent Prognostic Marker in Middle Eastern PTC and Its Expression Is Upregulated by BRAFV600E Mutation

Simple SummaryThis study was conducted to investigate the prognostic significance of programmed death-ligand 1 (PD-L1) expression in a large cohort of Middle Eastern papillary thyroid carcinoma (PTC) patients and to explore the correlation of PD-L1 and BRAFV600E mutations in PTC tumors and cell lines. We found PD-L1 over-expression in PTC patients and it was significantly associated with aggressive clinico-pathological parameters and BRAF mutation. PTC patients with co-existing PD-L1 over-expression and BRAF mutation had a poor disease-free survival. In vitro studies showed that BRAF inhibition induces PD-L1 expression in BRAF-mutated PTC cell lines via mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase (MEK/ERK) pathway activation. Silencing of PD-L1 in BRAF-mutated cell lines significantly attenuated cell growth. Our data suggest that PD-L1 could represent a useful prognostic marker for risk stratification in Middle Eastern PTC and that a programmed cell death protein 1 (PD-1)/PD-L1 inhibitor could be a potential therapeutic option for aggressive PTC cancers, such as the tall cell variant, BRAF mutation-positive patients that are unresponsive to standard PTC treatment.PD-L1 inhibition is a promising therapeutic target whose efficacy has been demonstrated in several cancers. Immunohistochemistry was performed to assess PD-L1 protein expression in PTC. We further conducted in vitro analysis to investigate the role of PD-L1 in regulating BRAFV600E in PTC cell lines. PD-L1 over-expression was noted in 32.4% (473/1458) of cases and significantly associated with aggressive clinico-pathological parameters. Importantly, PD-L1 was found to be an independent poorer prognostic marker. We also found PD-L1 to be significantly associated with BRAF mutation and patients with co-existing PD-L1 over-expression and BRAF mutation had a poor disease-free survival compared to patients with BRAF mutation alone. In vitro analysis showed high expression of PD-L1 in BRAF-mutated PTC cell lines compared to a BRAF wild-type cell line. Inhibition of BRAF using vemurafenib induced PD-L1 expression in BRAF-mutated cell lines without affecting cell growth. Knockdown of PD-L1 in BRAF-mutated cell lines significantly decreased the cell growth and induced apoptosis. Our data suggest that PD-L1 might represent a useful prognostic marker in Middle Eastern PTC and PD-L1 inhibition could be a potential therapeutic option for aggressive PTC cancers, such as the tall cell variant, BRAF mutation-positive patients that are unresponsive to standard treatment.

AZD0364 Is a Potent and Selective ERK1/2 Inhibitor That Enhances Antitumor Activity in KRAS-Mutant Tumor Models when Combined with the MEK Inhibitor, Selumetinib.

The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.

NAMPT Over-Expression Recapitulates the BRAF Inhibitor Resistant Phenotype Plasticity in Melanoma.

Simple SummaryMalignant melanoma (MM) is the most fatal skin cancer due to its high metastatic potential. Treatment strategies are dramatically changing due to the introduction of BRAF/MEK inhibitors (i) and immunotherapy; however, multiple resistant mechanisms rapidly occur including metabolic rewiring. This study aimed to establish the driver role of the nicotinamide adenine dinucleotide (NAD)-biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in BRAFi resistance development. We defined that NAMPT over-expressing MM cells were strikingly similar to cells that acquired resistance to BRAFi in terms of growth, invasion, and phenotype plasticity. These findings confirmed NAMPT as a key factor in melanoma progression and in the onset of BRAFi resistance in melanoma patients, opening new therapeutic possibilities for this subset of patients.Serine–threonine protein kinase B-RAF (BRAF)-mutated metastatic melanoma (MM) is a highly aggressive type of skin cancer. Treatment of MM patients using BRAF/MEK inhibitors (BRAFi/MEKi) eventually leads to drug resistance, limiting any clinical benefit. Herein, we demonstrated that the nicotinamide adenine dinucleotide (NAD)-biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) is a driving factor in BRAFi resistance development. Using stable and inducible NAMPT over-expression systems, we showed that forced NAMPT expression in MM BRAF-mutated cell lines led to increased energy production, MAPK activation, colony-formation capacity, and enhance tumorigenicity in vivo. Moreover, NAMPT over-expressing cells switched toward an invasive/mesenchymal phenotype, up-regulating expression of ZEB1 and TWIST, two transcription factors driving the epithelial to mesenchymal transition (EMT) process. Consistently, within the NAMPT-overexpressing cell line variants, we observed an increased percentage of a rare, drug-effluxing stem cell-like side population (SP) of cells, paralleled by up-regulation of ABCC1/MRP1 expression and CD133-positive cells. The direct correlation between NAMPT expression and gene set enrichments involving metastasis, invasiveness and mesenchymal/stemness properties were verified also in melanoma patients by analyzing The Cancer Genome Atlas (TCGA) datasets. On the other hand, CRISPR/Cas9 full knock-out NAMPT BRAFi-resistant MM cells are not viable, while inducible partial silencing drastically reduces tumor growth and aggressiveness. Overall, this work revealed that NAMPT over-expression is both necessary and sufficient to recapitulate the BRAFi-resistant phenotype plasticity.