Frequent Activating Mutations Of JAK-STAT Pathway Genes In Natural Killer Cell Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 812-812
Author(s):  
Can Kucuk ◽  
Bei Jiang ◽  
Xiaozhou Hu ◽  
Philippe Gaulard ◽  
Wenyan Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Sanger Sequencing ◽  
Nk Cell ◽  
Killer Cell ◽  
Negative Regulator ◽  
Rna Seq ◽  
Activating Mutations ◽  
Oncogenic Mutations ◽  
Stat3 Mutations ◽  
Stat Pathway

Abstract Background Natural killer cell lymphomas (NKCLs) are rare diseases with poor prognosis. There are few studies that reported oncogenic mutations in this disease. Identifying mutations critical to the neoplastic transformation of NK cells is crucial for the development of targeted therapies. Methods We performed RNA sequencing (RNA-Seq) on NKCL cases (n=17), malignant NK cell lines (n=3) and resting or activated normal NK cells (n=3) to analyze the genome-wide mutation profile in NKCLs. All SNVs detected by RNA-Seq were validated by Sanger sequencing using the corresponding genomic DNA (gDNA). We expanded our analysis to include specific sequencing of the SH2 domains of STAT3 and STAT5B using 37 additional NKCL cases and 6 NK cell lines. Retroviral or lentiviral transduction was performed to express SOCS6 or STAT3 shRNA in NK cell lines, respectively. After transduction, cells were tracked by quantifying the % of GFP+ cells. Apoptosis was assessed by quantifying AnnexinV-PE stained cells. Western blot was performed on 6 NK cell lines using p-STAT3 (Tyr705) antibody. q-MSP and q-RT-PCR were used to detect SOCS6 promoter methylation and mRNA expression in NK cell lines, respectively. Results RNA-Seq showed frequent oncogenic mutations in JAK/STAT pathway members STAT3 (18%), STAT5B (6%), BRAF (6%) and MAP2K1 (6%). Targeted Sanger sequencing of 37 additional NKCL cases showed one patient with activating STAT3 and two patients with activating STAT5B mutations leading to 7.4% (4 of 54) and 6% (3 of 50) total mutation frequency, respectively. STAT3 and STAT5B mutations were located in the SH2 domain and three of four STAT3 mutations and all STAT5B mutations were previously observed in NK- or T-LGL leukemia cases. Oncogenic activities of two other JAK-STAT pathway genes, BRAF (G469A), MAP2K1 (K57N) have been reported in solid tumors and leukemias. The JAK/STAT pathway mutations were present in a total of 53% of the NKCL cases with RNA-Seq data available (n=17). Intriguingly, targeted sequencing revealed oncogenic STAT3 mutations in 50% of malignant NK cell lines (n=6), which were associated with p-STAT3 expression detected by western blot.STAT3 knock-down resulted in reduced growth in a NK cell line with STAT3 mutation. In agreement with Kimura et. al. Leuk Lymphoma 2013, we were not able to detect JAK3 mutations as reported previously. In an accompanying DNA methylation analysis, we observed epigenetic silencing of SOCS6, a negative regulator of JAK-STAT3 signaling, in NKCL samples. Reintroduction of SOCS6 showed negative selection pressure associated with increased apoptosis in limiting concentrations of IL2 in two SOCS6-null NK cell lines with activating STAT3 mutations suggesting a possible cooperation of oncogenic JAK-STAT pathway mutations and the epigenetic silencing of a negative regulator of this pathway. Conclusions We have identified a high incidence of activating mutations of STAT3, STAT5B and other oncogenic JAK-STAT pathway genes in NKCLs. SOCS6 was frequently methylated in NKCLs with corresponding low gene expression. There was evidence suggestive of cooperation of genetic and epigenetic mechanisms in the activation of the JAK-STAT pathway in NKCLs. This study suggests that JAK-STAT pathway inhibition may be a therapeutic option in NKCLs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mutational Landscape of Aggressive Natural Killer Cell Leukemia and Drug Sensitivity Profiling Reveal Therapeutic Options in Natural Killer Cell Malignancies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2921-2921
Author(s):  
Olli Dufva ◽  
Matti Kankainen ◽  
Tiina Kelkka ◽  
Shady Adnan Awad ◽  
Nodoka Sekiguchi ◽  
...  
Keyword(s):  
Natural Killer ◽  
Cell Lines ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Somatic Mutations ◽  
Nk Cell ◽  
Killer Cell ◽  
Epigenetic Modifiers ◽  
Whole Exome ◽  
Stat Pathway

Abstract INTRODUCTION Natural killer (NK) cell malignancies are rare aggressive neoplasms that are classified by the WHO as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK-cell leukemia (ANKL). Recently, genome and exome level studies in NKTCL have shed light on the mutational spectrum of the disease. However, somatic mutations in ANKL have not been characterized. Here, we identified somatic mutations in 14 cases of ANKL to further clarify the genetic landscape underlying malignant NK cell proliferation. We compared the discovered variants to those detected in NKTCL to understand whether the two diseases harbor common molecular alterations. Moreover, we used high-throughput drug screening and RNA sequencing on NK cell lines derived from ANKL, NKTCL and other malignant NK cell proliferations to identify therapeutically actionable drivers of malignant NK cell growth. METHODS We performed whole-exome sequencing on genomic DNA extracted from peripheral blood or bone marrow samples of 14 ANKL patients. To compare the mutational profiles in ANKL and NTKCL, we re-analyzed the published whole-exome NKTCL data from Jiang et al. (Nat Genet 2015) using our somatic variant calling pipeline. For profiling of drug responses, we used a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs to screen the ANKL cell lines IMC-1, KHYG-1 and NK-92, NKTCL cell lines NK-YS and SNK-6 as well as DERL-7, KAI3, NKL, YT and IL-2-stimulated NK cells from healthy donors. All drugs were tested in 384-well format in 5 concentrations over a 10,000-fold concentration range for 72 h, cell viability was measured and normalized dose response curve values were used to calculate a drug sensitivity score (DSS) for each drug. Finally, we performed amplicon sequencing of known cancer driver genes and RNA sequencing on the cell lines and healthy NK cells to identify driver mutations and integrate gene expression profiles with drug sensitivity patterns. RESULTS We identified recurrent somatic activating mutations in STAT3 in 21% (3 of 14) of ANKL patients. Other mutated genes included RAS-MAPK pathway molecules (BRAF, NRAS, KRAS), protein phosphatases regulating JAK-STAT and PI3K-AKT-mTOR pathways (PTPRT, PTPRK, INPP5D) as well as several epigenetic modifiers (TET2, ARID2, KDM2B, SETD7, SETD2) and the tumor suppressor TP53. Interestingly, we detected mutations in genes recurrently mutated in NKTCL, such as the RNA helicase DDX3X and the cell surface receptor FAS. Re-analysis of the published NKTCL data revealed a high frequency of missense mutations in receptor type and non-receptor type protein phosphatases (e.g. PTPRC, PTPRR, PTPRT, PTPN1, PTPN2, PTPN3), many of which have established roles as negative regulators of JAK-STAT signaling. These findings potentially expand the subset of NK cell tumors where the JAK-STAT pathway is somatically activated and implicate deregulated JAK-STAT signaling as a major driver in these diseases. The malignant NK cell lines used in drug sensitivity profiling frequently harbored mutations in same genes and pathways, including STAT3 (N=3), STAT5B (N=1), DDX3X (N=2), KRAS (N=1), FAS (N=2) and several epigenetic modifiers, thus validating these cell lines as relevant disease models. The drug sensitivities in NK cell lines showed a high correlation and the cell lines formed a distinct group from other lymphoid and myeloid leukemia cell lines in unsupervised hierarchical clustering, suggesting an NK-cell specific drug response pattern. The most effective targeted drugs across all NK cell lines included HDAC inhibitors, inhibitors of Aurora and Polo-like kinases, JAK inhibitors, HSP inhibitors and CDK inhibitors as well as the Bcl-2 inhibitor navitoclax. Compared to other leukemia and lymphoma cell lines, JAK inhibitors, navitoclax and methotrexate emerged as the most NK-cell specific compounds. CONCLUSIONS Our genetic data demonstrate extensive heterogeneity in the mutational spectrum of ANKL and implicate JAK-STAT and RAS-MAPK signaling as well as disruption of epigenetic modifiers in the disease pathogenesis. Integrated drug sensitivity and gene expression profiling corroborates the JAK-STAT pathway as a major therapeutically actionable driver of malignant NK cell proliferation and identifies other potential novel targeted therapy options such as Bcl-2 inhibition in NK cell malignancies. Disclosures Suzumiya: Chugai: Honoraria, Research Funding; Toyama Chemical: Research Funding; Kyowa Hakko kirin: Research Funding; Astellas: Research Funding; Eisai: Honoraria, Research Funding; Takeda: Honoraria. Ohshima:Chugai: Research Funding, Speakers Bureau; Kyowa Kirin: Research Funding, Speakers Bureau. Mustjoki:Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

scholarly journals Differential responsiveness to BRAF inhibitors of melanoma cell lines BRAF V600E-mutated

2020 ◽  
Author(s):  
Muna Al Hashmi ◽  
Seetharama S Konduru ◽  
Lee Silcock ◽  
Lotfi Chouchane ◽  
Valentina Mattei ◽  
...  
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Sanger Sequencing ◽  
Braf V600e ◽  
Biological Behavior ◽  
Rna Expression ◽  
Rna Seq ◽  
Braf Inhibitors ◽  
Melanoma Cell Lines ◽  
Specific Inhibitors

Abstract Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation. DNA and RNA mutation status were assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non -specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. The results obtained were also confirmed in three additional commercial cell lines. Cell lines carrying V600E mutations at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

HLA Influence on NK Cell Expansion

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4924-4924
Author(s):  
Jennifer Schellekens ◽  
Anna Stserbakova ◽  
Madis Tõns ◽  
Hele Everaus ◽  
Marcel GJ Tilanus ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Cell Expansion ◽  
Nk Cell ◽  
Killer Cell ◽  
Hla Class I ◽  
Class I ◽  
Haematopoietic Stem Cell ◽  
Specific Priming

Abstract Natural Killer (NK) cells are effector cells in the innate immune system. The anti-leukaemic capacities of NK cells in haematopoietic stem cell transplantation make these cells a potential treatment modality to improve clinical outcome. Immunotherapy with NK cells requires transfusion of large quantities, which obviates the need for an in vitro culture system for NK cells. The killer cell immunoglobulin-like receptors (KIR) on NK cells recognise defined groups of HLA class I alleles. To elucidate the influence of these interactions on proliferation, the peripheral blood mononuclear cells (PBMCs) of 29 patients and donors were cultured in CellGro SCGM with IL-2 and OKT3 antibody to expand the NK cell fraction. The killer cell immunoglobulin-like receptor (KIR) and HLA repertoire were determined by sequence specific priming and sequence based typing respectively. The percentage of NK cell expansion from the total PBMC fraction varied between 5.4% and 71.6%. A significantly better NK cell expansion was observed for individuals homozygous for HLA-C epitope group 2 (p<0.05). For evaluation of cytolytic competence of the cultured NK cells, specific killing of an HLA class I expression deficient LCL 721.221 cell line and three 721.221 cell lines transfected with different HLA-C alleles was determined. A significantly better NK cell-induced specific cytotoxicity was observed towards the untransfected 721.221 cells compared to the HLA-C transfected 721.221 cells. No significant differences were observed between killing of the three HLA-C transfected 721.221 cell lines. We have shown that cytolytic capacities of the cultured NK cells are maintained and in vitro expansion of NK cells is dependant on the presence of HLA-C alleles.

Exome Sequencing of Aggressive Natural Killer Cell Leukemia and Drug Profiling Highlight Candidate Driver Pathways in Malignant Natural Killer Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 700-700
Author(s):  
Olli Dufva ◽  
Tiina Kelkka ◽  
Shady Awad ◽  
Nodoka Sekiguchi ◽  
Heikki Kuusanmäki ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Exome Sequencing ◽  
Cell Lines ◽  
Hierarchical Clustering ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Somatic Mutations ◽  
Nk Cell ◽  
Negative Regulator

Abstract Background Natural killer (NK) cell malignancies are rare lymphoid neoplasms characterized by aggressive clinical behavior and poor treatment outcomes. Clinically they are classified as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK cell leukemia (ANKL). Both subtypes are almost invariably associated with Epstein-Barr virus (EBV). Recently, genomic studies in NKTCL have identified recurrent somatic mutations in JAK-STAT pathway molecules STAT3 and STAT5b as well as in the RNA helicase gene DDX3X in addition to previously detected chromosomal aberrations. Here, we identified somatic mutations in 4 cases of ANKL in order to understand whether these entities share common alterations at the molecular level. To further establish common patterns of deregulated oncogenic signaling pathways operating in malignant NK cells, we performed drug sensitivity profiling using NK cell lines representing ANKL, NKTCL and other malignant NK cell proliferations. We aimed to identify sensitivities to agents that selectively target components of pathways required for survival of malignant NK cells in an unbiased manner. Methods Exome sequencing was performed on peripheral blood or bone marrow of ANKL patients using the NK cell negative fraction or other healthy tissue as control. Profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs. The NK cell lines KAI3, KHYG-1, NKL, NK-YS, NK-92, SNK-6 and YT and IL-2-stimulated and resting NK cells from healthy donors were used as sample material. All drugs were tested on a 384-well format in 5 different concentrations over a 10,000-fold concentration range for 72 h and cell viability was measured. A Drug Sensitivity Score (DSS) was calculated for each drug using normalized dose response curve values. Results The ANKL patients displayed mutations in genes reported as recurrently mutated in NKTCL, such as FAS, TP53, NRAS, STAT3 and DDX3X. Additionally, novel alterations in genes previously implicated in the pathogenesis of NKTCL were detected. These included an inactivating mutation in INPP5D (SHIP), a negative regulator of the PI3K/mTOR pathway and a missense mutation in PTPRK, a negative regulator of STAT3 activation. Interestingly, the total number of nonsilent somatic mutations in 3 out of 4 ANKL patients (97, 82 and 45) was remarkably high compared to other hematological malignancies analyzed in our variant calling pipeline. Analysis of drug sensitivities in NK cell lines showed a close correlation between all cell lines and a markedly higher correlation with those of IL-2 stimulated than resting healthy NK cells, suggesting that malignant NK cells may share a common drug response pattern. Furthermore, in an unsupervised hierarchical clustering the NK cell lines formed a distinct group from other leukemia cell lines tested (Fig. A). Among pathway-selective compounds (namely, kinase inhibitors and rapalogs), the drugs most selective for malignant NK cells fell into two major categories: PI3K/mTOR inhibitors (e.g. temsirolimus, buparlisib) and inhibitors of aurora and polo-like kinases such as rigosertib and GSK-461364 (Fig. B). JAK inhibitors (e.g. ruxolitinib, gandotinib) and CDK inhibitors (e.g. dinaciclib) showed strong efficacy in both malignant NK cells and IL-2 activated healthy NK cells. Conclusions Our exome sequencing results suggest that candidate driver alterations affecting similar signaling pathways underlie the pathogenesis of ANKL as has been reported in NKTCL. Drug sensitivity profiling highlights the PI3K/mTOR pathway as a potential major driver of malignant NK cell proliferation, whereas JAK-STAT signaling appears to be essential in both healthy and malignant NK cells. Components of these pathways harbored mutations in our small cohort of ANKL patients and have been shown to be deregulated by mutations or other mechanisms in previous studies, underlining their importance as putative drivers. The systematic large-scale characterization of drug responses also identified these pathways as potential targets for novel therapy strategies in NK cell malignancies. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

Genomic Analyses Reveal Global Functional Alterations That Promote Tumor Growth and Novel Tumor Suppressor Genes in Natural Killer-Cell Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3792-3792
Author(s):  
Can Kucuk ◽  
Javeed Iqbal ◽  
Ronald J. deLeeuw ◽  
Gopesh Srivastava ◽  
Wayne Tam ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Natural Killer ◽  
Candidate Genes ◽  
Cell Lines ◽  
Promoter Methylation ◽  
Cell Activation ◽  
Array Cgh ◽  
Nk Cell ◽  
Killer Cell ◽  
Amino Acid Sequences

Abstract Background: Natural Killer (NK)-cell lymphomas/leukemias (NKL) account for 1–2 % of all non-Hodgkin lymphomas. Although the incidence of NKL is relatively low, the clinical course of these lymphomas is highly aggressive. To elucidate the recurrent genomic abnormalities and the associated changes in the gene expression profile, we performed GEP and array-CGH studies on seven clinically well defined cases and eight well characterized cell lines derived from NKL patients. Methods: Array-CGH was performed on a tiling BAC array and GEP on an Affymetrix 133 plus2 array. The two data sets were correlated to identify functional alterations associated with the genetic abnormalities. Candidate genes on del 6q21 were identified and further studied for mutations and promoter methylation. Results: Our aCGH study identified frequent recurrent gains (> 25 %) in 1q, 2p, 7q, 13q, 17q and 20pter-qter. Regions of loss with a frequency (> 25%) included 1p, 6q, 7p, 9p, 9q, 1011q, 12q, 13q, 15q, 16p, 17p, 18p, and 18q consistent with other studies. Only 30%-50% of the genes residing in the gained or deleted regions showed corresponding increased or decreased expression. Many of the genes with increased expression were involved in cell proliferation, growth and energy metabolic processes important for the neoplastic cells. In deleted regions, genes showing decreased expression included transcription factors or repressors (e.g. SP4, PRDM1, NCOR1 and ZNF10), tumor suppressors or negative regulators of the cell cycle (e.g. CDKN2C, CDKN2A, CDKN2B, CHFR and FOXO1A). 6q21 is a frequent deleted region and we found a minimal region of involvement including only three known genes (PRDM1, ATG5 and AIM1), that had consistent low expression. Sequence analysis of these genes revealed mutations in PRDM1 and AIM1 in two out of eight cell lines resulting in truncated proteins of PRDM1 and changes in conserved amino acid sequences of AIM1. Only one cell line with a point mutation in a non-conserved region in ATG5 was detected. DNA methylation analysis showed that the promoter of PRDM1 was highly methylated in NK cell lines having low PRDM1 expression. Reversal of promoter methylation with decitabine treatment resulted in increased PRDM1 transcription consistent with down-regulation of PRDM1 expression on promoter methylation. Finally, we observed progressive up-regulation of PRDM1 expression in primary NK cells in response to IL2 treatment suggestive of a role of PRDM1 on the regulation of NK-cell activation. Conclusion: Combination of high resolution genomic and transcriptional profiling in NK-cell malignancies has provided evidence of a general tumor promoting effect of genomic copy number alterations as well as the identification of candidate genes in a commonly deleted region in 6q. Specifically, we propose PRDM1 as a tumor suppressor gene in NK cell malignancies and loss-offunction of PRDM1 in combination with the haplo-insufficiency of ATG5 might have significant roles in the pathogenesis of NK-cell malignancies.

Jagged2 promotes the development of natural killer cells and the establishment of functional natural killer cell lines

Blood ◽  
2005 ◽  
Vol 105 (9) ◽  
pp. 3521-3527 ◽  
Author(s):  
Sarah L. DeHart ◽  
Marc J. Heikens ◽  
Schickwann Tsai
Keyword(s):  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Lines ◽  
Nk Cell ◽  
Killer Cell ◽  
Interleukin 2 ◽  
Lymphoid Cells ◽  
Killer Cells ◽  
Hematopoietic Stem

AbstractEmerging evidence indicates that Notch receptors and their ligands play important roles in the development of T cells and B cells. However, little is known about their possible roles in the development of other lymphoid cells. Here we demonstrate that Jagged2, a Notch ligand, stimulates the development of natural killer (NK) cells from Lin- Sca-1+ c-kit+ hematopoietic stem cells. Our culture system supports NK cell development for 2 to 3 months, often leading to the establishment of continuous NK cell lines. The prototype of such cell lines is designated as KIL. KIL depends on interleukin-7 for survival and proliferation and is NK1.1+ CD3- TCRαβ- TCRδγ- CD4- CD8- CD19- CD25+ CD43+ CD45+ CD49b- CD51+ CD94+ NKG2D+ Mac-1-/low B220- c-kit+ perforin I+ granzyme B+ Notch-1+, and cytotoxic. Like normal natural killer cells, the T-cell receptor-β loci of KIL remain in the germ-line configuration. In response to interleukin-2, KIL proliferates extensively (increasing cell number by approximately 1010-fold) and terminally differentiates into adherent, hypergranular NK cells. Our findings indicate that Jagged2 stimulates the development of natural killer cells and the KIL cell line preserves most properties of the normal NK precursors. As such, KIL provides a valuable model system for NK cell research.

scholarly journals Differential responsiveness to BRAF inhibitors of melanoma cell lines BRAF V600E-mutated

2020 ◽  
Author(s):  
Muna Al Hashmi ◽  
Seetharama S Konduru ◽  
Lee Silcock ◽  
Lotfi Chouchane ◽  
Valentina Mattei ◽  
...  
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Sanger Sequencing ◽  
Control Cell ◽  
Braf V600e ◽  
Rna Expression ◽  
Rna Seq ◽  
Braf Inhibitors ◽  
Melanoma Cell Lines ◽  
Specific Inhibitors

Abstract BACKGROUND. Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation. METHODS. DNA and RNA mutation status were assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non -specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation.RESULTS. Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele.We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line.The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional commercial cell lines.CONCLUSION. Cell lines carrying V600E mutations at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

scholarly journals Dysregulated Translational Control Is a Central Feature of MM Mediated By 14-3-3ε Interactome Promoting Multiple Myeloma Cell Growth and Viability

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3176-3176
Author(s):  
Yan Xu ◽  
Mariateresa Fulciniti ◽  
Matthew Ho ◽  
Mehmet Kemal Samur ◽  
Michael A Lopez ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cell Viability ◽  
Cell Lines ◽  
Translation Initiation ◽  
Translational Control ◽  
Plasma Cells ◽  
Negative Regulator ◽  
Central Feature ◽  
Rna Seq ◽  
Equity Ownership

Abstract 14-3-3 proteins are chaperone and scaffold proteins that exert a widespread influence on cellular processes through binding to serine/threonine-phosphorylated residues on target proteins, forcing conformational changes or influencing their interactions with other molecules. Altered 14-3-3 expression is associated with development and progression of cancer. We therefore evaluated the status of all 14-3-3 isoforms in plasma cells disorders in publically available gene expression profiling (GEP) data. Using independent patient datasets, we observed a consistent higher expression of YWHAE (coding gene for the isoform 14-3-3ε) in MM and plasma cell leukemia (PCL) patients, while no consistent differences were observed with the other isoforms. Moreover, we also confirmed higher expression of YWHAE in our RNA-seq data from 420 newly-diagnosed MM patients, with relatively low expression in normal plasma cells. Finally, 14-3-3ε was also found to be constitutively expressed at protein level in primary patient MM cells and in a large panel of MM cell lines, with significantly lower expression in healthy donor B cells. To evaluate if 14-3-3ε represents a functional dependency in MM, we performed genetic perturbation of YWHAE in a panel of MM cell lines. Depletion of YWHAE using 3 different shRNA inhibited cell proliferation and induced cell apoptosis across 5 different cell lines, independently of their genetic background. We next performed CRISPR-Cas9-mediated YWHAE knock out (KO) in H929 and JJN3 cells and observed a significant decrease in cell viability and a robust apoptotic response. H929 YWHAE KO cells infected with FLAG-YWHAE addback lentiviral construct completely rescued this phenotype, confirming that loss of YWHAE is responsible for the defective cell viability and apoptotic phenotype. These observations were corroborated by ectopic overexpression of YWHAE in H929 WT cells that significantly promoted MM cell viability. To elucidate the underlying molecular mechanisms, proteins immunocomplexed co-precipitated with FLAG in H929 KO cells with 14-3-3ε-FLAG addback were analyzed by mass spectrometry. Protein analysis revealed interaction of 14-3-3ε with a large number of proteins, enriched in mTORC1, PI3K-AKT-mTOR and unfolded protein response (UPR) pathway-related genes. Among these, TSC2 and mTORC1 proteins were further studied. WB analysis confirmed interaction of 14-3-3ε with p-mTOR (S2448) and its upstream negative regulator p-TSC2 (S939), while mTORC1 downstream targets, p-p70 S6k and p-4E-BP1, did not interact with 14-3-3ε. WB analysis also revealed activation of TSC2 and consequent inhibition of mTORC1 (via decrease of p-mTOR S2448 levels) in YWHAE KD cells. YWHAE-FLAG addback reversed these effects. Additionally, GEP data in KD cells confirmed a significant impact on mTORC1 pathways. Importantly, YWHAE expression highly correlated (R> 0.8) with genes involved in the mTORC1 pathway, including PSMC4, COPS5, EIF2S2, in our RNA-seq dataset, demonstrating a clinical significance of 14-3-3ε and mTORC1 cooperation in the context of myeloma. One of the most conserved functions of mTORC1 is to promote translation. We therefore assessed the impact of YWHAE on global translational efficiencies in MM cells, and observed significant impact on nascent protein synthesis by YWHAE modulation. 14-3-3ε KD induced 4EBP1 de-phosphorylation through inhibited mTORC1, and concomitantly induced EIF2α phosphorylation. Both effects inhibited translation initiation complex formation, mechanistically supporting a strong protein synthesis arrest. These data show the modulation of several hubs of the signaling apparatus controlling translation initiation in response to YWHAE modulation, ultimately producing a marked protein synthesis inhibition. Deregulated translational control is a central feature of MM. Our findings highlight a unique function for YWHAE as promoter of MM cell survival through regulation of mTOR-dependent protein synthesis and apoptosis. Pharmacological inhibition of YWHAE/14-3-3ε is therefore a possibility to specifically target malignancies with deregulated translational control such as MM. Disclosures Anderson: C4 Therapeutics: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

Sensitivity of human glioma and brain cells to natural killer cell lysis

1992 ◽  
Vol 76 (6) ◽  
pp. 986-990 ◽  
Author(s):  
Rene L. Myers ◽  
Ronald L. Whisler ◽  
Ralph E. Stephens ◽  
Craig A. Sponseller ◽  
Kimberly Livingston ◽  
...  
Keyword(s):  
Natural Killer ◽  
Cell Lines ◽  
Nk Cell ◽  
Killer Cell ◽  
Target Population ◽  
Growth Fraction ◽  
Target Cells ◽  
Cell Sensitivity ◽  
Content Type

✓ Using an in vitro monolayer natural killer (NK) cytolysis assay, the authors examined the effects of serum concentration and epidermal growth factor (EGF) on sensitivity to NK cytolysis. It was found that target cells cultured in high concentrations of serum (10% fetal bovine serum (FBS)) had higher cytotoxicity levels than those in low serum concentrations (0% to 0.5% FBS). Exposure of target cells to EGF had no effect on their sensitivity to NK cytolysis. Both glioma cell lines showed decreased NK cell sensitivity with longer times in culture. The results of cytofluorometric studies on these cell lines indicate that the differences in NK cell sensitivity may reflect the growth fraction of the target population and that a population with a higher proportion of cycling cells is more susceptible to lysis by NK cells. Whether it is possible to separate the proliferative rate of these cells from their NK cell sensitivity is unknown, but worthy of consideration.

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