Journal: Cancers

Article Title: TGFβ1-Induced EMT in the MCF10A Mammary Epithelial Cell Line Model Is Executed Independently of SNAIL1 and ZEB1 but Relies on JUNB-Coordinated Transcriptional Regulation

doi: 10.3390/cancers15020558

Figure Lengend Snippet: Analyses of TFBM activity suggests a prominent function for AP-1 family members JUN and JUNB in TGFβ1-induced EMT. ( a ) De novo motif analysis of DARs from the TGFβ1 time course experiment using HOMER. For each time point of the TGFβ1 treatment, all significantly identified TF motifs are shown. Bar plots display the percentage of DARs and random background genomics regions in which the motifs were enriched. Statistical significance is shown next to the corresponding bar as −log 10 of the adj. p -value. ( b ) Number of TFBMs enriched in proximal and distal DARs or both according to IMAGE. Examples for TFBMs specifically enriched in proximal, distal, or proximal and distal DARs are given. ( c ) DARs with predicted motif activities of JUN and JUNB are more numerous and have broader regulatory scopes compared to DARs with SNAIL1 and ZEB1 motif activities. For each time point of TGFβ1 treatment, the DARs with predicted activities of SNAIL1, ZEB1, JUN, and JUNB motifs were identified by IMAGE, and their numbers were plotted. The heatmaps below represent, for each time point, the average values of log 2 FC of accessibility calculated for all DARs harboring a given TF motif and which are associated with annotated genes. The annotation bars to the left of each of the heatmaps specify the location of the DARs (proximal, distal, or both) in which the TF motifs are enriched. The annotation bars to the right of each of the heatmaps show whether the genes annotated to the DARs were previously identified as epithelial markers (Epi), mesenchymal markers (Mes), or have no known connection to EMT (unknown).

Article Snippet: After digestion with BamHI, the JUNB PCR fragment was inserted in sense and antisense orientations into the retroviral vector pWZL-Blast-SNAIL1-ER (addgene plasmid #18798; http://n2t.net/addgene:18798 (accessed on 28 August 2022); RRID: addgene_18798) [ ], whereby the SNAIL1 coding sequences were deleted from the vector.

Techniques: Activity Assay

Journal: Cancers

Article Title: TGFβ1-Induced EMT in the MCF10A Mammary Epithelial Cell Line Model Is Executed Independently of SNAIL1 and ZEB1 but Relies on JUNB-Coordinated Transcriptional Regulation

doi: 10.3390/cancers15020558

Figure Lengend Snippet: SNAIL1 and ZEB1 are not required for TGFβ1-induced EMT. ( a , b ) Schematic representation of the human SNAI1 and ZEB1 genes and proteins. Exons are depicted as boxes with protein-coding parts colored in black and UTRs in white. Relevant functional domains of the proteins are indicated by color and linked to the exonic region by which they are encoded. DB: destruction box; NES: nuclear export signal; SID: Smad interaction domain; HB: homeobox; CID: CtBP interaction domain. The locations of sgRNA targets used for inactivation of SNAI1 and ZEB1 are marked by red arrows. ( c , d ) Detection of SNAIL1 and ZEB1 proteins by Western blot using nuclear extracts from MCF10A SNAI1 and ZEB1 wild type (WT) and knock-out (KO) cell clones. Glycogen synthase kinase-3 beta (GSK-3 beta) was used as loading control. Molecular weights are given in kilodaltons (kDa). One representative result from three independent biological replicates is presented. ( e , f ) Representative phase-contrast microscopy pictures from one of three independent biological replicates showing the indicated MCF10A SNAI1 and ZEB1 WT and KO clones after 72 h of TGFβ1 treatment. The scale bar represents 200 µm. ( g , h ) Gene expression analysis of epithelial and mesenchymal marker genes after 72 h of TGFβ1 treatment. RNA levels were measured by qRT-PCR and are shown as relative expression compared to the RNA levels of GAPDH . In the box plots each dot represents the result of a single qRT-PCR measurement. Dot color identifies MCF10A SNAI1 and ZEB1 WT and KO cell clones. Stars indicate p -values corrected for multiple testing by the false discovery rate (FDR) method. *: FDR < 0.05, ns: not significant; Mann-Whitney U test. ( i , j ) Detection of epithelial and mesenchymal markers after 72 h of TGFβ1 treatment by Western blot using the cytoplasmic fractions of protein lysates from MCF10A SNAI1 and ZEB1 WT and KO cell clones. Beta-actin was used as loading control. Molecular weights are given in kilodaltons (kDa). Representative results from one of three independent biological replicates are shown. Uncropped versions of immunoblots including densitometry readings can be found in .

Article Snippet: After digestion with BamHI, the JUNB PCR fragment was inserted in sense and antisense orientations into the retroviral vector pWZL-Blast-SNAIL1-ER (addgene plasmid #18798; http://n2t.net/addgene:18798 (accessed on 28 August 2022); RRID: addgene_18798) [ ], whereby the SNAIL1 coding sequences were deleted from the vector.

Techniques: Functional Assay, Western Blot, Knock-Out, Clone Assay, Microscopy, Expressing, Marker, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cancers

Article Title: TGFβ1-Induced EMT in the MCF10A Mammary Epithelial Cell Line Model Is Executed Independently of SNAIL1 and ZEB1 but Relies on JUNB-Coordinated Transcriptional Regulation

doi: 10.3390/cancers15020558

Figure Lengend Snippet: Increased JUNB activity is sufficient to induce EMT in MCF10A cells. ( a ) Schematic representation of the gene expression cassette for a fusion protein consisting of the human JUNB coding region (JUNB sense) and a mutant estrogen receptor hormone binding domain (ER). A control construct (CTRL) harbored the JUNB coding region in opposite orientation (JUNB antisense). The angled arrow indicates the TSS; the white box represents the 5′-untranslated region. ( b ) Simultaneous detection of endogenous JUNB und ectopic JUNB-ER expression by Western blot using nuclear extracts from MCF10A cells that had been stably transduced with a retroviral vector for expression of JUNB-ER or the control construct. Glycogen synthase kinase-3 beta (GSK-3 beta) was used as loading control. Molecular weights are given in kilodaltons (kDa). One representative result from three independent biological replicates is presented. ( c ) Representative phase-contrast microscopy pictures from one of three independent biological replicates showing the indicated MCF10A JUNB-ER and CTRL cells. The scale bar represents 200 µm. ( d ) Gene expression analysis of epithelial and mesenchymal marker genes in MCF10A JUNB-ER and CTRL cells. RNA levels were measured by qRT-PCR and are shown as relative expression compared to those of GAPDH . ( e ) Detection of epithelial and mesenchymal markers in the cytoplasmic (Fibronectin, N-cadherin, EpCAM, RBM47) and nuclear fractions (SNAIL1, SNAIL2, ZEB1, JUN) of protein lysates from MCF10A JUNB-ER and CTRL cells. Beta-actin and GSK-3beta were used as loading controls. Molecular weights are given in kilodaltons (kDa). Representative results from one of three independent biological replicates are shown. ( f ) Results from transwell migration assays performed with MCF10A JUNB-ER or CTRL cells. Depicted is the area covered by cells on the bottom surface of transwell inserts relative to the value of EtOH treated cells. ( g ) Spheroid invasion assays performed with cellular aggregates embedded in a collagen I matrix. For the quantification single cells and small aggregates (exemplarily marked by arrow heads) that had detached from the bulk of the cell aggregates were counted in two fields of view per cell line and condition. Pictures of representative spheroids from one out of three independent biological replicates are shown on the right. The scale bars represent 100 μm. ( b , e ) Uncropped versions of immunoblots including densitometry readings can be found in . ( b – g ) For all experiments, cells were treated with 100 nM 4-OHT or a corresponding volume of ethanol (EtOH) for 72 h prior to harvest. ( d , f , g ) Bars represent the mean values from at least three independent biological replicates. Error bars depict the standard error of the mean. Stars indicate p -values corrected for multiple testing by the FDR method. *: FDR < 0.05, **: FDR < 0.01, ***: FDR < 0.001, ns: not significant; one-way ANOVA.

Article Snippet: After digestion with BamHI, the JUNB PCR fragment was inserted in sense and antisense orientations into the retroviral vector pWZL-Blast-SNAIL1-ER (addgene plasmid #18798; http://n2t.net/addgene:18798 (accessed on 28 August 2022); RRID: addgene_18798) [ ], whereby the SNAIL1 coding sequences were deleted from the vector.

Techniques: Activity Assay, Expressing, Mutagenesis, Binding Assay, Construct, Western Blot, Stable Transfection, Transduction, Plasmid Preparation, Microscopy, Marker, Quantitative RT-PCR, Migration

Journal: Cancer cell

Article Title: Vitamin B6 addiction in acute myeloid leukemia

doi: 10.1016/j.ccell.2019.12.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse AML cell lines, human AML cell lines, iMEF cell line, 3T3 cell line and human sarcoma cell line were infected with virus encoding Cas9 (Addgene 52962) followed with blastcitidin selection to generate Cas9 cell lines.

Techniques: Plasmid Preparation, Recombinant, Mutagenesis, RNA Extraction, Gel Extraction, Clone Assay, Purification, Flow Cytometry, Sequencing, CRISPR, Functional Assay, shRNA, Software

Journal: PLoS Pathogens

Article Title: Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants

doi: 10.1371/journal.ppat.1009996

Figure Lengend Snippet: (A) Flow cytometry analysis of Dystroglycan-1 (IIH6; red) and Heparan sulfate (10E4; blue) expression in 293T WT, Δ DAG1 , Δ EXTL3 , and Δ DAG1 EXTL3 cells generated by CRISPR Cas9-mediated knockout. (B) Infection assay using a lentiviral (LV) reporter system encoding eGFP. LVs were pseudotyped with VSV-G, as a control, and low (HPI WT Y155H or Arm 53b L260F) or high (HPI high H155Y or Arm Cl13 260L) affinity LCMV GP variants. Cells were transduced with an MOI of 1 (determined for 293T WT) and eGFP signal was measured 72 h later by flow cytometry. (C) Infection assay with low (WE HPI or Arm 53b) and high (Arm Cl13) affinity variants of LCMV in different knockout variants of 293T cells. The cells were infected with an MOI of 1 (determined by semi-functional quantitative flow cytometry assay for 293T WT cells) for 2 h at 37°C and 16 h p.i. quantified by flow cytometry via LCMV N-staining. Shown are the means ± SD of three replicates.

Article Snippet: Human EXT1 – 2 , SDC1 – 4 and GPC1 sequences were amplified from 293T cDNA and subcloned into a lentiviral vector (#119863; pLenti CMVie-IRES-BlastR; Addgene, Watertown, USA).

Techniques: Flow Cytometry, Expressing, Generated, CRISPR, Knock-Out, Infection, Transduction, Functional Assay, Staining

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) NetMHCPan 4.1 summary of the top 10 peptide sequences that are most likely to be presented by HLA-A2 from the first 30 amino acids of KRAS G12V . Peptides containing the G12V mutation (including mutations in red text) are in black text; others are in gray. (b) Log 10 (EC 50 (M)) comparison of recognition of synthetic KRAS G12V 5-14 peptide (KLVVVGAVGV) presented by HLA-A2 by an initially generated set of MHC class I-restricted TCRs isolated to be specific for this epitope. Lower values indicate higher functional avidity. (c) % IFNγ + T cells, transduced with TCR 2 , after exposure to B-LCL cell lines expressing different Class I HLA alleles found on CFPAC1 cells without KRAS G12V 5-14 peptide loaded. Cell lines are color coded by allele. (d) MS results of RAS family peptide fragments detected after tryptic digestion of immunoprecipitated whole KRAS protein from cell lysate of DAN-G and CFPAC1 pancreatic adenocarcinoma cell lines. Lysine side chain trimethylation is indicated in red with an asterisk. (e) Reconstitution of properly folded HLA-A2 surface expression on T2 cells after exogeneous loading by wild type or G12V/D mutant KRAS 5-14 peptides with or without lysine-5 side chain methylations. (f) Additional views of structural models of HLA-A2-presented KRAS G12V 5-14 peptide including possible lysine-5 side chain methylation, supplementing by highlighting the protrusion of the lysine-5 side chain (circled in red) out of the HLA-A2 groove. (g) Overview of structural prediction of a TCR-pMHC complex using a combined Alphafold-Rosetta pipeline. Alphafold allows for deep learning-based prediction of proteins and protein complexes, which we applied to predict TCR-pMHC complex structure. However, this does not yet contain PTMs, which Alphafold cannot incorporate reliably. Thus, we used the resulting structure as input into Rosetta, which used physics-based energy functions to introduce PTMs and reoptimize the structure to accommodate them.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Mutagenesis, Comparison, Generated, Isolation, Functional Assay, Transduction, Expressing, Immunoprecipitation, Methylation, Structural Proteomics, Introduce

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Log 10 (EC 50 (M)) comparison of recognition of different concentrations of a synthetic KRAS G12V 5-14 peptide (KLVVVGAVGV) presented by HLA-A2 by different MHC class I-restricted TCRs specific for the epitope. Lower values indicate higher functional avidity. (b) Killing of live CFPAC1 and DAN-G HLA-A2 + KRAS G12V+ pancreatic adenocarcinoma cells cocultured with CD8 + T cells expressing the TCRs shown in (a) using a 4:1 T cell:tumor cell ratio. Cocultures were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein; NR: red fluorescent protein; NG: green fluorescent protein. (c) ARTEMIS MS data of peptides eluted from HLA-A2. Data is from the 293F cell line, as commonly used for proteomics assays requiring high protein expression. All cell lines were transduced with an HLA-A2 single chain secreted dimer as well as a KRAS G12V constitutive expression construct to increase presentation and the likelihood of detecting a presented peptide by MS, of KRAS G12V -related epitopes. The first 100 amino acids of KRAS G12V are shown, with the G12V mutation highlighted in yellow. Epitopes predicted by NetMHCPan 4.1 are shown in purple; epitopes detected from ARTEMIS are shown in blue. ARTEMIS detection of KRAS G12V 5-14 is outlined in red. (d) Heatmap of – log 10 (EC 50 (μg/ml)) of CD8 + T cells expressing TCR 2 or TCR 19 against candidate KRAS G12V epitopes, with and without methylation of the lysine-5 side chain of the epitope, presented by HLA-A2. Higher values indicate higher functional avidity. EC 50 values were calculated from T cell exposure to peptide concentrations ranging from 1 µg/ml to 10 - µg/ml. Gray squares indicate EC 50 calculations that lacked a stable fit, e.g., due to lack of response to peptide even at high doses. (e) Rosetta structural modeling of HLA-A2 presenting KRAS G12V 5-14 and its methylated variants. Red circles indicate the amine group of the lysine-5 side chain; arrows indicate its movement with different methylation states. A reference point is drawn at the same position in each image to help illustrate positional changes. (f) Alanine scan of the KRAS G12V epitope and resulting response by CD8 + T cells expressing TCR 2 and TCR 19 . The epitope with each individual residue substituted with alanine (or, for alanine-11, substituted with either glycine or threonine) was presented by antigen-presenting cells to stimulate primary human CD8 + T cells expressing a TCR, and subsequent IFNγ expression was measured. Higher IFNγ induction indicates greater tolerance for amino acid substitution at that position. X-axis numbers indicate the residue number on the KRAS protein, and letters indicate the amino acid substitution. A: alanine; G: glycine; T: threonine. Last column is unmodified KRAS G12V 5-14.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Comparison, Functional Assay, Expressing, Imaging, Transduction, Construct, Mutagenesis, Methylation, Residue

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Structural model, as predicted by a combined Alphafold-Rosetta pipeline, of a ‘core’ CDR3α sequence (sequence EDNT) of an existing TCR recognizing KRAS G12V 5-14 and its relation to the lysine-5 side chain (circled in red) of the epitope as presented by HLA-A2. (b) Generation of TCR mutagenesis libraries by mutagenesis of the core CDR3α sequence followed by transduction of the TCRs into Nur77-GFP reporter Jurkat cells and subsequent clonal expansions. Tetramer binding patterns of HLA-A2-KRAS G12V 5-14 peptide with unmodified or methylated lysine-5 side chain at the end of cell stimulation are shown. Each circle indicates a population with a specific pattern of unmethylated vs. methylated peptide tetramer binding that was sorted and sequenced. (c) Examples of mutated CDR3α ‘core’ sequences resulting from sorted clones of the mutagenesis library that exhibit the noted different patterns of PTM peptide recognition. (d) Heatmap of – log 10 (EC 50 (μg/ml)) of TCRs derived from the mutagenesis library and the original TCR 2 responding to different degrees of lysine-5 methylation of KRAS G12V 5-14 presented by HLA-A2, as calculated from dose response assays. Gray squares indicate EC 50 values that lacked a stable fit, e.g., due to limited response to peptide even at high doses. TCR 4UM is the 4 th TCR clonotype identified from an initial sequencing dataset from sorted Jurkat cells binding to both unmethylated (“U”) and methylated (“M”) tetramers; other TCRs are labeled by changes in the ‘core’ CDR3α sequence (e.g., EDST). (e) Killing of HLA-A2 + KRAS + CFPAC1 and DAN-G pancreatic adenocarcinoma cells by CD8 + T cells expressing selected mutagenized TCRs. Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein. NR: red fluorescent protein; NG: green fluorescent protein.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Sequencing, Mutagenesis, Transduction, Binding Assay, Methylation, Cell Stimulation, Clone Assay, Derivative Assay, Labeling, Expressing, Imaging

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: Structural predictions of unmethylated and methylated variants of KRAS G12V 5-14, as well as unmethylated wild type or G12D peptide, presented by HLA-A2 to interface with (a) TCR 2 and (b) mutagenized TCR EDST TCRs. The CDR3α ‘core’ sequence is shown as individual amino acids within the TCRs. In (b), the TCR 2 -pMHC interfaces from (a) are overlaid with transparency to facilitate visual comparison. With TCR EDST , residues in the core sequence of CDR3α and the lysine-5 side chain of each variant of KRAS G12V epitope appear closer to each other than with TCR 2 .

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Methylation, Sequencing, Comparison, Variant Assay

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Dose response curves of CD8 + T cells expressing selected TCRs generated from the HLA-A2 KRAS G12V TCR mutagenesis library after exposure to wild type or G12D KRAS 5-14 peptides presented by HLA-A2. (b) Failure of CD8 + T cell killing and progressive growth of human HLA-A2 + tumor cell lines that express wild type KRAS (HeLa cervical carcinoma cell line; left) or KRAS G12D (Panc1 pancreatic adenocarcinoma; right) and no KRAS G12V . Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein. NR: red fluorescent protein; NG: green fluorescent protein. (c) Alanine scan of KRAS G12V 5-14 epitope tested against TCRs selected from the mutagenesis library. Each cell indicates the resulting expression of Nur77-GFP upon replacement of a KRAS G12V residue with alanine (or in the case of alanine-11, replacement with either glycine or threonine) and with or without lysine-5 side chain dimethylation. (d) IFNγ secretion by CD8 + T cells expressing mutagenized TCRs after exposure to synthetic peptides from the human proteome fitting the K-x-x-V-V-x-A-x-x-x tolerance pattern identified from the alanine scan shown in (c), with a concentration of 0.1 μg/ml for each peptide. (e) Dose response curves of CD8 + T cells expressing mutagenized TCRs after exposure to the potential human off-target peptides demonstrated above to generate the three largest responses at 0.1 μg/ml: CFA61 23-32/607-616/667-676 , RSLBB 35-44 , and TRXR1 342-351 , compared to the KRAS G12V epitope.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Expressing, Generated, Mutagenesis, Imaging, Residue, Concentration Assay

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Experimental workflow to enrich and characterize T cell clones with TCRs recognizing a defined peptide epitope. (b) Tetramer binding patterns of primary human T cells stimulated with unmethylated and methylated KRAS G12V peptide presented by HLA-A2. Plots also indicate gates used to sort cells with different affinities for unmethylated and methylated epitopes. (c) Dose response curves of TCRs against wild type and KRAS G12D 5-14 peptides presented by HLA-A2. (d) Tumor cell growth during coculture of CD8 + T cells expressing selected TCRs with HLA-A2 + , wild type KRAS HeLa and KRAS G12D Panc1 pancreatic adenocarcinoma cell lines. Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein. NR: red fluorescent protein; NG: green fluorescent protein. (e) Alanine scan of KRAS G12V 5-14 epitope tested against TCR A2UoM1-1 . Each box indicates the resulting expression of Nur77-GFP upon replacement of a KRAS G12V residue with alanine (or in the case of alanine-11, replacement with either glycine or threonine). (f) Dose response curve of CD8 + Nur77-GFP Jurkat cells expressing TCR A2UoM1-1 after exposure to EPIPL 1948-1957 , the potential human off-target peptide as determined from alanine scan results.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Clone Assay, Binding Assay, Methylation, Expressing, Imaging, Residue

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Heatmap of –log 10 (EC 50 (μg/ml)) of selected TCRs responding to the unmethylated and methylated epitopes presented by HLA-A2, as calculated from dose response assays. Higher values indicate higher functional avidity. Gray squares indicate EC 50 values that lacked a stable fit, e.g., due to limited response to peptide even at high doses. (b) Killing of HLA-A2 + KRAS G12V+ CFPAC1 and DAN-G pancreatic adenocarcinoma cells by CD8 + T cells expressing selected TCRs. Cocultures of fluorescently labeled tumor cells and TCR-T cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein; NR: red fluorescent protein; NG: green fluorescent protein. (c) Alphafold-Rosetta visualizations of TCR:pMHC complexes for TCR A2UoM1-1 , isolated from expansion of responding primary human CD8 + T cells from normal repertoires. Structures were generated for unmethylated and mono/di/trimethylated versions of the epitope, presented by HLA-A2. The four CDR3α amino acids in closest proximity to the lysine-5 side chain of KRAS G12V are shown with individual molecular bonds.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Methylation, Functional Assay, Expressing, Labeling, Imaging, Isolation, Generated

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Overall workflow: DAN-G pancreatic adenocarcinoma cells were transduced with a CRISPR knockout library targeting methylation-related genes, and then cocultured with CD8 + T cells expressing either TCR 2 or mutagenized TCR EDST , which respectively have weak or strong recognition of methylated KRAS G12V epitopes. Tumor cells surviving at the end of coculture were sequenced to identify gRNA enrichment or depletion associated with immune evasion. (b) Volcano plot summarizing log 2 (fold change) of gRNA read frequencies targeting individual genes recovered after coculture of DAN-G cells with T cells expressing TCR EDST versus TCR 2 . The threshold of significance was set at log 10 ( p ) < 1.2 ( p < 0.05) and is denoted by the horizontal dashed line. Mean counts of all gRNAs per gene were used to calculate fold change values. The data point for gRNAs targeting SUPT6H is specifically labeled. The knockouts depleted or enriched after cocultures using TCR EDST versus TCR 2 are colored blue and red, respectively. (c) Killing of DAN-G cells with or without SUPT6H knockout in coculture with selected TCR-T cells (untransduced or expressing TCR 2 , TCR EDST , or TCR A2UoM1-1 ). Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed green nuclear fluorescent protein (“NG”). (d) Kaplan-Meier survival plots for individuals who have tumors with KRAS mutations, using public TCGA data. Left: only tumors with the KRAS G12V mutant, with or without concurrent SUPT6H mutation. Right: tumors with any KRAS mutant, with or without a concurrent SUPT6H mutation.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Transduction, CRISPR, Knock-Out, Methylation, Expressing, Labeling, Imaging, Mutagenesis