Homozygous Cell Lines Research Articles

Abstract 183: Inhibition of BRAF kinase alone in BRAF V600E-mutated undifferentiated thyroid carcinoma results in no growth arrest

Abstract Background: Undifferentiated thyroid carcinoma is the most aggressive type of thyroid carcinoma. It is refractory to most of the conventional therapies and its median overall survival is less than six months. Mutation in BRAF gene was found around 25% of undifferentiated thyroid carcinoma. Mutated BRAF particularly the V600E mutated BRAF auto-activates the BRAF-MEK proliferation-survival cell signalling pathway. The objectives of our current study were to inhibit the BRAF kinase and investigate its effect on undifferentiated thyroid carcinoma proliferation. Materials and methods: 8050C, a homozygous BRAF V600E mutate undifferentiated thyroid carcinoma cell line, was used to develop anti-BRAF shRNA stable cell line. Quantitative real time polymerase chain reaction (qPCR) and western blot were performed for ratifying the BRAF inhibition efficiency of stable cell line. Proliferation and colony formation assay were done to see the effect of BRAF inhibition on undifferentiated thyroid carcinoma proliferation. In addition, impact of BRAF inhibition on proliferation cell signalling pathways were checked through western blot analysis. Results: Efficiency of BRAF knockdown was around 87% which resulted in approximately 83% inhibition of BRAF kinase (p < 0.05). About 54% proliferation inhibition (p < 0.05) was observed in the BRAF inhibited group compare to control group in the first week. But the proliferation rate in the BRAF inhibited group increased over the subsequent weeks and we did not observed any sign of cell growth arrest. In addition, colony formation assay revealed approximately 13% reduction of colony formation ability (p < 0.05) in BRAF inhibited group compare to control group. Western blot analysis of signalling kinases discovered reduction of around 72% MEK kinase activity after BRAF kinase inhibition. As we observed increasing trend of proliferation after an initial sharp drop in cell proliferation after BRAF kinase inhibition, we hypothesized and analysed the alternative PI3K-AKT proliferation pathway. Surprisingly we found about 63% increase of AKT kinase activity in the BRAF inhibited group compare to control group (p < 0.05). Conclusions: Inhibition of BRAF kinase reduced cell proliferation initially, but this proliferation inhibition did not lead to cell growth arrest at the end. Increased AKT kinase activity counteracted the effect of BRAF kinase inhibition as AKT kinase has the ability to contribute in overall cell proliferation. Therefore, both kinases need to be inhibited simultaneously for cell growth arrest in undifferentiated thyroid carcinoma. Citation Format: Md. Atiqur Rahman, Ali Salajegheh, Haleh Vosgha, Hamidreza Maroof, Robert A. Smith, Alfred KY Lam. Inhibition of BRAF kinase alone in BRAF V600E-mutated undifferentiated thyroid carcinoma results in no growth arrest. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 183.

Establishment of a quenching probe method for detection of NPM1 mutations in acute myeloid leukemia cells.

Nucleophosmin (NPM1) mutations, generally consisting of a four base-pair insertion, are present in ~60% of all cytogenetically normal acute myeloid leukemia (AML) cases. The mutation is clinically significant as an important prognostic factor. Direct sequencing is the current standard method of mutation detection, however, it is quite costly and time consuming. The present study aimed to establish a highly sensitive quenching probe (QP) method to detect NPM1 mutations efficiently. Melting curve analysis was performed using a QP, following polymerase chain reaction for amplification of the involved region of the gene. The curve derived from the fluorescent intensity with respect to the temperature of OCI/AML3, a heterozygous NPM1 mutant AML cell line, was W-shaped with melting peaks at 61°C and 68°C. That of M-07e, the homozygous wild type cell line, was V-shaped with a melting peak at 68°C. Thus, the curve derived from the mutant allele was easily discriminated from that of the wild-type allele. The mutant allele was detected in concentrations as low as 3% as determined by a subsequent sensitivity study. With a short testing time and a high sensitivity, this assay was applicable for NPM1-mutated AML patient samples and is appropriate for screening NPM1 mutations. It does require further examination as to whether it would be useful as a detection method for other mutant alleles since NPM1 mutations may consist of 61 known types of mutant sequences. To the best of our knowledge, this is the first report describing the QP method for the detection of NPM1 mutations.

Tyrosin Kinase Inhibition Restores the Membrane Localization of FLT3-ITD

The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regards to oncogenic alterations that do not only serve as prognostic markers but also as targets for therapy in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Due to sequence-dependent protein conformation changes, FLT3-ITD tends to auto-phosphorylate and displays a constitutive intracellular localization. As tyrosine kinase inhibitors (TKIs) efficiently block FLT3 auto-phosphorylation, we set out to study the effect of TKI on the subcellular localization of the FLT3 receptor and its mutants.We visualized FLT3 WT and its mutants (N676K, D835Y, W51 (ITD), NPOS (ITD), K644R, W51-K644R) in transiently transfected U2OS cells, using anti-FLT3 immunofluorescence in combination with confocal microscopy. Treatment with 50nM of the TKI AC220 for 6 hours restored the membrane localization of both ITD (Figure 1 A) and D835Y. The TKI-induced surface expression of FLT3-ITD was confirmed by flow cytometry (mean fluorescence intensity, MFI) of BaF3 cells stably expressing FLT3 WT or mutants and in the homozygous (ITD/ITD) AML cell line (MV4-11). Similar results were obtained in cells from an AML patient derived xenograft (pdx) with FLT3-ITD and loss of heterozygosity (LOH) (Figure 1 B). Western blot analysis showed increased glycosylation of FLT3-ITD after TKI-treatment (Figure 1 C) suggesting that auto-phosphorylation prevents physiological processing of the receptor, which is required for maturation and surface expression. To monitor the subcellular localization of FLT3 and its mutants in vivo we have generated a FLT3-GFP fusion. Proliferation assays in BaF3 cells confirmed that the C-terminal GFP-tag does not alter the function of FLT3. Upon FLT3-ligand (FL) stimulation of FLT3-GFP we observed FLT3-internalization by life cell imaging in U2OS cells. Conversely, TKI treatment increased cell surface expression of FLT3-GFP.Our findings have translational implications when considering FLT3 as a target for immunotherapy in AML, since FLT3 surface expression might facilitate antigen recognition. Especially FLT3-ITD positive patients with LOH may benefit from a combined TKI-immunotherapy approach. Therefore, we currently study the effect of AC220 on FLT3 antibody-dependent cellular cytotoxicity (ADCC), mediated by natural killer (NK) cells. Taken together, we discovered that TKI alters the subcellular localization and protein processing of specific FLT3 mutants. However, the complex interplay between the individual posttranslational modifications and their role in FLT3 traffic remains elusive. [Display omitted] DisclosuresKrupka:AMGEN Research (Munich): Research Funding. Subklewe:AMGEN Research (Munich): Research Funding.

The MICA-129Met/Val dimorphism affects plasma membrane expression and shedding of the NKG2D ligand MICA.

The MHC class I chain-related molecule A (MICA) is a ligand for the activating natural killer (NK) cell receptor NKG2D. A polymorphism causing a valine to methionine exchange at position 129 affects binding to NKG2D, cytotoxicity, interferon-γ release by NK cells and activation of CD8+ T cells. It is known that tumors can escape NKG2D-mediated immune surveillance by proteolytic shedding of MICA. Therefore, we investigated whether this polymorphism affects plasma membrane expression (pmMICA) and shedding of MICA. Expression of pmMICA was higher in a panel of tumor (n = 16, P = 0.0699) and melanoma cell lines (n = 13, P = 0.0429) carrying the MICA-129Val/Val genotype. MICA-129Val homozygous melanoma cell lines released more soluble MICA (sMICA) by shedding (P = 0.0015). MICA-129Met or MICA-129Val isoforms differing only in this amino acid were expressed in the MICA-negative melanoma cell line Malme, and clones with similar pmMICA expression intensity were selected. The MICA-129Met clones released more sMICA (P = 0.0006), and a higher proportion of the MICA-129Met than the MICA-129Val variant was retained in intracellular compartments (P = 0.0199). The MICA-129Met clones also expressed more MICA messenger RNA (P = 0.0047). The latter phenotype was also observed in mouse L cells transfected with the MICA expression constructs (P = 0.0212). In conclusion, the MICA-129Met/Val dimorphism affects the expression density of MICA on the plasma membrane. More of the MICA-129Met variants were retained intracellularly. If expressed at the cell surface, the MICA-129Met isoform was more susceptible to shedding. Both processes appear to limit the cell surface expression of MICA-129Met variants that have a high binding avidity to NKG2D.

Generation of Hypertension-Associated STK39 Polymorphism Knockin Cell Lines With the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System.

Previous genome-wide association studies identified serine threonine kinase 39 (STK39), encoding STE20/SPS1-related proline/alanine-rich kinase, as one of a limited number of hypertension susceptibility genes. A recent meta-analysis confirmed the association of STK39 intronic polymorphism rs3754777 with essential hypertension, among previously reported hypertension-associated STK39 polymorphisms. However, the biochemical function of this polymorphism in the mechanism responsible for hypertension is yet to be clarified. We generated rs3754777G>A knockin human cell lines with clustered regularly interspaced short palindromic repeats-mediated genome engineering. Homozygous (A/A) and heterozygous (G/A) knockin human embryonic kidney cell lines were generated using a double nickase, single-guide RNAs targeting STK39 intron 5 around single-nucleotide polymorphism, and a 100-bp donor single-stranded DNA oligonucleotide. Reverse transcription polymerase chain reaction with sequencing analyses revealed the identical STK39 transcripts among the wild-type and both knockin cell lines. Quantitative reverse transcription polymerase chain reaction showed increased STK39 mRNA expression, and immunoblot analysis revealed increases in total and phosphorylated STE20/SPS1-related proline/alanine-rich kinase with increased phosphorylated Na-K-Cl cotransporter isoform 1 in both knockin cell lines. The largest increases in these molecules were observed in the homozygous cell line. These findings indicated that this intronic polymorphism increases STK39 transcription, leading to activation of the STE20/SPS1-related proline/alanine-rich kinase-solute carrier family 12A signaling cascade. Increased interactions between STE20/SPS1-related proline/alanine-rich kinase and the target cation-chloride cotransporters may be responsible for hypertension susceptibility in individuals with this polymorphism.

OR39 De novo assembly of the major histocompatibility complex using single-molecule real-time sequencing of large contiguous DNA fragments captured by targeted region specific extraction

Aim Utilize our region-specific extraction (RSE), targeted DNA capture methodology to generate large, contiguous DNA fragments (5–60 Kbp) from the MHC for sequencing on the PacBio RSII single molecule real-time sequencing (SMRT) platform to produce long sequenced reads (10–15 Kbp) for de novo assembly and characterization of the MHC. This unique combination of technologies produces long sequenced reads (up to 60 Kbp) that may eventually enable the construction of large, phased haplotype blocks and haplotype resolved de novo assembly of the MHC. Methods Genomic DNA from the homozygous cell line, COX (which has a fully characterized MHC haplotype) was enriched for 4 Mbp of the MHC (chr6: 29618227–33618227) using the RSE DNA capture methodology [1] . DNA fragment lengths were calculated using a BioAnalyzer prior to sequencing. SMRTbell DNA libraries were constructed according to the Pacbio standard protocol “20 kb Template Preparation Using BluePippin Size-selection system”. Libraries were sequenced on the PacBio RS II instrument (P6-C4 chemistry). Computational analysis was carried out using the PacBio SMRT portal HGAP 2 de novo assembly algorithm. Assembled contigs were evaluated using QUAST with the COX haplotype sequence as reference. Results Captured DNA fragments from the MHC were calculated to be ∼12 Kbp on average (ranging from ∼5 to 60 Kbp). The observed read length distribution following PacBio RSII sequencing reveals an average read length of ∼3.5 Kbp, with some reads as long as 60 Kbp. We are able to de novo assemble 91% of the targeted region, with 99.99% accuracy. The N50 and NG50 for the assembly were calculated to be 33,234 bp and 92,824 bp, respectively. The largest contig aligned to the COX reference was found to be ∼ 200Kbp. Conclusions Our targeted resequencing and de novo assembly approach represents a comprehensive method to characterize 4 Mbp of the Human MHC. We demonstrate the unique ability to de novo assemble and fully characterize 91% of the targeted MHC for the homozygous cell line COX with 99.9% accuracy as compared to the annotated COX haplotype reference sequence.

A catalog of HLA region SNPs with functional annotations, disease associations and correlations with HLA types

The HLA complex is the most gene dense and polymorphic region in the human genome, and not surprisingly, shows a large number of disease associations. The latest genome assembly contains > 250 K SNPs in the HLA region (chr6:28.5 to 34.5 Mb). The emphasis in disease association studies has been on the HLA gene polymorphisms, despite the fact that non-HLA genes vastly outnumber HLA genes. We adopted a comprehensive approach to systematically analyze all HLA region SNPs by functionally annotating, and mapping them to conserved extended haplotypes by genotyping 95 reference cell lines using the Illumina ImmunoChip. We also measured expression levels of classical HLA genes in eight HLA homozygous reference cell lines at baseline and after three treatments (heat shock, LPS, and IFN-gamma). We screened the GWAS catalog, dbGAP and GRASP database for disease associations of SNPs, and assigned CADD, RegulomeDB, and PolyPhen/SIFT scores to all SNPs. SNPs were assessed for alterations they cause in transcription factor binding sites, or microRNA/microRNA binding site sequences. The overall data revealed SNPs representing known HLA associations, suggested mechanisms for some HLA associations via linkage disequilibrium between HLA alleles and functional SNPs, explained the source of the extra amount of DNA specific to the DRB4 family of haplotypes as copy number variation, and unraveled areas of the HLA region with insufficient coverage in GWAS arrays. The SNPs with the most deleterious effects were mainly in non-HLA genes of the region, and most have not been included in GWAS arrays. We also noted that there are more disease associations in the HLA region than listed in the GWAS catalog. Given the insufficient coverage in GWAS arrays due to difficulties in assay design and structural variation, sequencing-based studies are needed to unravel even more associations missed by GWAS. The catalog produced in this work combines HLA and SNP data with functional effects and induced expression levels, and should be of great value in the design and interpretation of HLA and disease association studies.

Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

Abstract 2442: XPO1 is selinexor prime target: validation by mutating cysteine 528 on both XPO1 alleles using CRISPR/Cas9 genome editing

Abstract Human Exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports a variety of different cargo proteins out of the cell's nucleus. Inhibition of XPO1-mediated protein export results in nuclear retention of cargo proteins including tumour suppressor proteins and is a novel therapeutic strategy for cancer. Selinexor, an orally bioavailable XPO1 inhibitor, effectively demonstrates potent activity against multiple types of cancer. Here we directly demonstrate the high selectivity of selinexor for XPO1 inside living cells. Selinexor binds to the cargo-binding pocket of XPO1 and inhibits the formation of XPO1-cargo complex. The binding of selinexor to XPO1 involves a slowly reversible Michael addition type interaction of its acrylate moiety with cysteine 528 residue of XPO1. Using CRISPR/Cas9 genome editing we have now created a homozygous Jurkat T-ALL cell line carrying a Cys528Ser mutation in XPO1. This cell line was used to study the specificity of selinexor for XPO1 inside the cancer cell. Selinexor was highly toxic to wild-type Jurkat cells in the low nanomolar range while the mutant cells were resistant up to micromolar concentrations. These results were confirmed by resistance to induction of caspase dependent apoptosis as well as cell cycle arrest in mutant cells. The single Cys528Ser mutation thus confers resistance to selinexor. In addition, selinexor failed to accumulate cargo proteins in the nucleus of mutant cells. Moreover, the Cys528Ser substitution conferred resistance to the direct interaction of this drug to XPO1 in cells. In conclusion, these results validate XPO1 as the selinexor's prime target and pinpoint its unique selectivity to the Cys528 residue of XPO1. Our findings also demonstrate the general utility of CRISPR/Cas9 genome editing for introducing point mutations in the genome of cancer cells for drug-target specificity validation. Citation Format: Jasper E. Neggers, Thomas Vercruysse, Maarten Jacquemyn, Els Vanstreels, Erkan Baloglu, Sharon Shacham, Marsha Crochiere, William Senapedis, Yosef Landesman, Dirk Daelemans. XPO1 is selinexor prime target: validation by mutating cysteine 528 on both XPO1 alleles using CRISPR/Cas9 genome editing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2442. doi:10.1158/1538-7445.AM2015-2442

Very long haplotype tracts characterized at high resolution from HLA homozygous cell lines.

The HLA region of chromosome 6 contains the most polymorphic genes in humans. Spanning ~5Mbp the densely packed region encompasses approximately 175 expressed genes including the highly polymorphic HLA class I and II loci. Most of the other genes and functional elements are also polymorphic, and many of them are directly implicated in immune function or immune-related disease. For these reasons, this complex genomic region is subject to intense scrutiny by researchers with the common goal of aiding further understanding and diagnoses of multiple immune-related diseases and syndromes. To aid assay development and characterization of the classical loci, a panel of cell lines partially or fully homozygous for HLA class I and II was assembled over time by the International Histocompatibility Working Group (IHWG). Containing a minimum of 88 unique HLA haplotypes, we show that this panel represents a significant proportion of European HLA allelic and haplotype diversity (60-95%). Using a high-density whole genome array that includes 13,331 HLA region SNPs, we analyzed 99 IHWG cells to map the coordinates of the homozygous tracts at a fine scale. The mean homozygous tract length within chromosome 6 from these individuals is 21Mbp. Within HLA, the mean haplotype length is 4.3Mbp, and 65% of the cell lines were shown to be homozygous throughout the entire region. In addition, four cell lines are homozygous throughout the complex KIR region of chromosome 19 (~250kbp). The data we describe will provide a valuable resource for characterizing haplotypes, designing and refining imputation algorithms and developing assay controls.

CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells.

Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein. Here, we establish CRISPR/Cas9-mediated generation of heterozygous and homozygous human knockin cell lines expressing fluorescently tagged proteins from their respective native genomic loci at close to endogenous levels. We tagged three different proteins, exhibiting various localizations and expression levels, with the reversibly switchable fluorescent protein rsEGFP2. We demonstrate the benefit of endogenous expression levels compared to overexpression and show that typical overexpression-induced artefacts were avoided in genome-edited cells. Fluorescence activated cell sorting analysis revealed a narrow distribution of fusion protein expression levels in genome-edited cells, compared to a pronounced variability in transiently transfected cells. Using low light intensity RESOLFT (reversible saturable optical fluorescence transitions) nanoscopy we show sub-diffraction resolution imaging of living human knockin cells. Our strategy to generate human cell lines expressing fluorescent fusion proteins at endogenous levels for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution approaches.

Epigenetic Regulation of RGS2 in Cystic Fibrosis

Cystic fibrosis (CF) is a recessive autosomal disease that affects the respiratory, gastrointestinal and reproductive systems. At pulmonary level, the absence of CFTR function (cystic fibrosis transmembrane conductance regulator encoding an ion channel chlorine) leads to a decrease in mucociliary clearance, resulting in mucus accumulation. A new approach based on epigenetic regulation of gene expression has been investigated and we identified a list of hypermethylated genes in dF508 cell lines, the most frequently observed mutation in the CF population. Among the CF hypermethylated genes, RGS2 (Regulator of G protein signaling 2) gene demonstrated either transcript or protein downregulation. RGS2 is a key modulator of bronchial hyperresponsiveness, plays a critical role in fibrosis‐related diseases and inflammation.We hypothesized that variation in RGS2 protein expression could be the result of modulation of promoter methylation and has an impact on cytokine regulation. The first objective was to verify that methylation observed on RGS2 promoter in dF508 homozygous cell lines induces a modulation of protein expression. Then, to assess the consequences of this downregulation of RGS2, we performed an shRNA‐mediated RGS2 knockdown followed by DMNQ treatment in non‐CF cells and monitored the expression of inflammatory mediators.We confirmed that methylation of RGS2 promoter has an impact on RGS2 gene expression using azacythidine and oxidative stress treatments and that RGS2 is downregulated at the protein level in CF cells and in dF508 homozygous patients. We observed a relation between RGS2 downregulation by shRNA and the upregulation of 3 modulators of early inflammation: CHI3L1, S100A12 and TNFa.

Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function.

The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection.

Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering.

The ability to rapidly and specifically modify the genome of mammalian cells has been a long-term goal of biomedical researchers. Recently, the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system from bacteria has been exploited for genome engineering in human cells. The CRISPR system directs the RNA-guided Cas9 nuclease to a specific genomic locus to induce a DNA double-strand break that may be subsequently repaired by homology-directed repair using an exogenous DNA repair template. Here we describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of a point mutation in human cells. Using this method, homozygous clonal cell lines can be constructed in 5-6 weeks. This method can also be adapted to insert larger DNA elements, such as fluorescent proteins and degrons, at defined genomic locations. CRISPR/Cas9 genome engineering offers exciting applications in both basic science and translational research.

Abstract 5602: BRAF V600 mutated allelic specific imbalance (MASI) impact on sensitivity to vemurafenib

Abstract V600 mutations of BRAF are present in 40% of cutaneous melanoma and are currently targeted for therapy by specific inhibitors (vemurafenib, dabrafenib). While BRAFV600 mutations are mostly heterozygous, the impact of BRAF mutated allelic specific imbalance (MASI) on the sensitivity to BRAF inhibitors and on clinical outcome has not been evaluated so far. Here we compared cell proliferation, tumor growth and vemurafenib sensitivity of heterozygous versus homozygous BRAF V600E mutated melanoma cell lines. We also performed a retrospective study evaluating the impact on overall survival and drug response of BRAF V600E mutated melanoma patients with or without MASI. Methods: We conducted proliferation and cytotoxity assays to compare cell growth and sensitivity to vemurafenib and trametinib in heterozygous, versus homozygous BRAF V600 mutated human melanoma cell lines. Their in vivo tumorigenesis and sensitivity to vemurafenib was also evaluated upon engraftment in SCID mice. A retrospective series of 179 patients with metastatic melanoma harboring a BRAF V600 mutations were analyzed and early first line treatment response was compared between patients with or without MASI. Results: Our results showed that heterozygous BRAF V600 mutated melanoma cell lines proliferate less rapidly than homozygous cell lines in vitro (cell proliferation counts after 7 days range from 3.6 x106 to 7.7x106 cells for homozygote cells, and from 4.8x104 to 5.5x105 for heterozygous cell lines) and in vivo (palpable tumors appeared on average 7 days after inoculation for homozygous cells but after 39 days for heterozygous cells). Heterozygous cell lines are more resistant to BRAF inhibitors either in vitro or in vivo (IC50 of vemurafenib ranged from 0,1 to 0,198 µM for homozygous cells and from 8,62 to 22,65 µM for heterozygous cell lines). We have also shown that patients with BRAF V600 mutated tumors with MASI may have initially a poorer prognosis (median overall survival 733 days IC95% [381; 1762] versus 1011 days IC95%[879;2969] p=0.236) but tend to have better responses to systemic drugs than patients without MASI (percentage of primary resistance 23% versus 34%; p=0.56). In conclusion: This translational study shows that a MASI of BRAF in melanoma is associated with a higher cell proliferation and tumorigenic potential in vitro and in vivo. This mutational status is also associated with a better response to BRAF inhibitor in vitro and in mice xenografts. Clinical relevance of these data should be more thoroughly evaluated on larger and more homogeneous series of patients however our preliminary data show a trend toward a clinical-experimental correlation with similar yet still statistically insignificant results. If confirmed, our data suggest that study of the MASI of BRAF mutation could serve as a predictive marker of patient response to vemurafenib. The role of paradoxical activation of the MAPK signaling pathway in cells without MASI of BRAF needs to be further explored. Citation Format: Amelie Boespflug, Luc Thomas, Pascal Rousset, Charles Dumontet, Julie Caramel, Carole Ferraro-Peyret, Pierre Paul Bringuier, Brigitte Balme, Stephane Dalle. BRAF V600 mutated allelic specific imbalance (MASI) impact on sensitivity to vemurafenib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5602. doi:10.1158/1538-7445.AM2014-5602

48-OR

Aim Capture and sequence by NGS the 4Mb of the MHC and derive genotypes of 33 MHC genes (HLA and non-HLAs) using Omixon Target HLA Typing software. Methods We applied our Region Specific Extraction method to target and capture 4Mb of the MHC region with custom designed capture oligos on 7 samples having known HLA genotypes: 5 homozygous cell lines (COX, DBB, JVM, PGF, and QBL) and 2 heterozygous samples (mixture of PGF and COX DNA, and a standard lab control). Each sample was whole-genome amplified, prepared for sequencing using Illumina TruSeq DNA sample prep kit and size selected for DNA fragments ranging from 300-400 bp. Paired-end 2x100bp sequencing was performed on the Illumina HiSeq. Each sample was genotyped for 33 genes within the MHC by Omixon Target using all paired-end reads. Derived genotypes were compared to known genotypes from the IMGT/HLA database for the cell lines and Sanger sequencing for the heterozygous control sample. Results We sequenced 7 samples throughout the 4Mb of the MHC region. On average, each sample had 284 million reads which were analyzed with Omixon Target. The 33 genes examined were Class I HLA-A, B, C, E, F, G, H, J, K, L, and V, Class II genes HLA-DMA, DMB, DOA, DOB, DPA1, DPB1, DQA1, DQB1, DRA, DRB1, DRB3, DRB4, DRB5, Class II pseudogenes HLA-DRB2, DRB6, DRB7, DRB8, DRB9, and MHC related genes MICA, MICB, TAP1, and TAP2. Omixon Target provided genotypes for 90% of the possible alleles; the remaining 10% of alleles (no genotype) were Class II pseudogenes. The average depth across the alleles ranged from 50–3000, a small percentage of the overall reads per sample. In the cases where the true HLA genotype was known, we determined the correct allele with 93% accuracy. Conclusions Our method of capturing and sequencing the MHC coupled with Omixon Target has yielded highly accurate genotypes for 33 HLA and non-HLA, MHC genes. This work sets the stage for further detailed characterization of the MHC and derivation of clinically relevant HLA typing from MHC NGS data. Major: Omixon, Inc.: Other: Contractor. Hague: Omixon, Inc.: Other: Contractor. Berces: Omixon, Inc.: Employee; Stockholder.

34-OR

Expressed by NK cells and T cells, KIR are critical for immune responses to infection, cancer and tissue transplants. KIR also contribute to autoimmunity and pregnancy syndromes. The human KIR locus is polygenic and polymorphic, with over 300 protein variants described, yet remains poorly characterized worldwide. We have developed a method to sequence complete KIR haplotypes, in parallel with HLA haplotypes, from genomic DNA. After capture of fragments with oligonucleotide probes the entire KIR locus is sequenced to high depth (>500x) using Illumina paired-end technology. In this way we analyzed 104 HLA homozygous cell lines from the 12th IHWC, for which there was limited prior KIR knowledge. To acquire these data we created a pipeline that automatically analyses the KIR -specific reads; it attributes sequencing-reads to specific KIR genes, determines presence/absence genotype by read depth, and then calls high-resolution genotypes for each of the 13 KIR and two pseudogenes that are present. The genotype calls were independently verified on eight of the IHWC cell lines using our high-resolution pyrosequencing method, also targeted to all KIR genes and alleles. The pipeline was further validated by comparison with 25 KIR-specific SNPs present in a whole-genome array (Omni1Quad). Only four IHWC cell lines are completely homozygous for all KIR genes and alleles, but ∼25% cells are homozygous for KIR gene content, due to high KIR A haplotype frequency. Accordingly, we are using population genetics to determine the composition of the 204 distinct KIR haplotypes represented in the 104 IHWC cells. All these methods were validated using the COX cell line, for which both KIR haplotypes were sequenced previously using standard techniques. Both methods gave 100% coverage and complete concordance. This versatile technique and the data so far obtained provide valuable contributions to future immunogenetic studies in transplantation and of other clinical diagnostics.

Heterozygous and Homozygous JAK2V617F States Modeled by Induced Pluripotent Stem Cells from Myeloproliferative Neoplasm Patients

JAK2V617F is the predominant mutation in myeloproliferative neoplasms (MPN). Modeling MPN in a human context might be helpful for the screening of molecules targeting JAK2 and its intracellular signaling. We describe here the derivation of induced pluripotent stem (iPS) cell lines from 2 polycythemia vera patients carrying a heterozygous and a homozygous mutated JAK2V617F, respectively. In the patient with homozygous JAK2V617F, additional ASXL1 mutation and chromosome 20 allowed partial delineation of the clonal architecture and assignation of the cellular origin of the derived iPS cell lines. The marked difference in the response to erythropoietin (EPO) between homozygous and heterozygous cell lines correlated with the constitutive activation level of signaling pathways. Strikingly, heterozygous iPS cells showed thrombopoietin (TPO)-independent formation of megakaryocytic colonies, but not EPO-independent erythroid colony formation. JAK2, PI3K and HSP90 inhibitors were able to block spontaneous and EPO-induced growth of erythroid colonies from GPA+CD41+ cells derived from iPS cells. Altogether, this study brings the proof of concept that iPS can be used for studying MPN pathogenesis, clonal architecture, and drug efficacy.

An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2α as an Endogenous Clock Regulator

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.

Abstract 1988: Divergent control of Cav-1 expression in non-cancerous Li-Fraumeni Syndrome and human cancer cell lines.

Abstract Li-Fraumeni Syndrome (LFS) is primarily characterized by development of tumors exhibiting germ-line mutations in the p53 gene. Cell lines developed from patients of a LFS family have decreased p53 activity as evidenced by the absence of apoptosis upon etoposide treatment. To test our hypothesis that changes in gene expression beyond p53 per se are contributing to the development of tumors, we compared gene expression in non-cancerous skin fibroblasts of LFS-affected (p53 heterozygous) versus non-affected (p53 wild-type homozygous) family members. Expression analysis showed that several genes were differentially regulated in the p53 homozygous and heterozygous cell lines. We were particularly intrigued by the decreased expression (∼88%) of a putative tumor suppressor protein, caveolin-1 (Cav-1), in the p53-mutant cells. Decreased expression of Cav-1 was also seen in both p53-knockout and p21-knockout HTC116 cells suggesting that p53 controls Cav-1 expression through p21 and leading to the speculation that p53, Cav-1 and p21may be part of a positive auto-regulatory feedback loop. The direct relationship between p53 and Cav-1 was also tested with HeLa cells (containing inactive p53), which expressed a significantly lower Cav-1 protein. A panel of nonfunctional and p53-deficient colon and epithelial breast cancer cell lines showed undetectable expression of Cav-1 supporting the role of p53 in the control of Cav-1. However, in two aggressively metastasizing breast cancer cell lines, Cav-1 was strongly expressed suggesting a possible role in tumor metastasis. Thus, there is a divergent control of Cav-1 expression as evidenced in non-cancerous Li-Fraumeni Syndrome and some aggressive human cancer cell lines. Citation Format: Zaki A. Sherif, Ahmed S. Sultan. Divergent control of Cav-1 expression in non-cancerous Li-Fraumeni Syndrome and human cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1988. doi:10.1158/1538-7445.AM2013-1988