primary antibodies targeting nsd2  (Danaher Inc)

Journal: bioRxiv

Article Title: Targeting high-risk multiple myeloma genotypes with optimized anti-CD70 CAR-T cells

doi: 10.1101/2024.02.24.581875

Figure Lengend Snippet: A. Cell surface capture proteomics of myeloma (AMO-1 and MM.1S) cell lines in comparison to acute myeloid leukemia (AML) cell lines indicated surface expression of CD70. Re-analysis of primary patient sample surface proteomic datasets (NatComm = Ferguson et al, Nature Communications (2022); Blood = Anderson et al, Blood (2022)) also indicates detectable CD70 in some samples. Plot shows normalized abundance based on mass spectrometric intensity. B. Example mass spectra from MM.1S cell line demonstrating peptides from CD70 identified by cell surface proteomics. C. CoMMpass transcript data ( n = 776) demonstrating CD70 expression as a function of Revised International Staging System (R-ISS) stage (1, 2, 3) at diagnosis. D. Box plot of NSD2 (left) and CD70 (right) expression grouped by gene expression subtype in newly diagnosed multiple myeloma (NDMM) CoMMpass samples ( n = 764). Gene expression subtypes are from Zhan et al. (CD-1: Cyclin D1; CD-2: Cyclin D1 and CD20; HP: Hyperdiploid; LB: Low Bone Disease; MF: MAF; MS: MMSET; PR: Proliferation) E. Volcano plot of genes associated with CD70 expression in CoMMpass NDMM samples ( n = 764). Genes significantly associated with CD70 (FDR <0.01) are shown in red (positive) and blue (negative) with the numbers listed above and select genes labelled. F. Top Gene Set Enrichment Analysis (GSEA) Hallmark pathways associated with CD70 expression. Top 5 pathways positively (red) and negatively (blue) associated with CD70 expression are shown, and only pathways with an FDR < 0.01 are included. G. Top GSEA positional gene sets associated with CD70 expression. Only the top 5 pathways positively and negatively associated with CD70 expression are shown.

Article Snippet: Primary antibodies targeting NSD2 (Abcam Cat# ab75359), HDAC2 (Millipore Cat# 05-814), H3 (Cell Signaling Technology Cat# 14269) and H3K36me2 (Abcam Cat# ab9049) were diluted in blocking buffer at 1:1000.

Techniques: Comparison, Expressing

Journal: bioRxiv

Article Title: Targeting high-risk multiple myeloma genotypes with optimized anti-CD70 CAR-T cells

doi: 10.1101/2024.02.24.581875

Figure Lengend Snippet: A. Knockout of the driver oncogene NSD2 in t(4;14) myeloma cell lines KMS-11 and KMS-34 led to decreased surface CD70 expression by flow cytometry. n = 3 biological replicates. p -value by t -test. *p<0.05, **p<0.01. B. MM.1S or AMO.1 myeloma cells were treated with the indicated dose of azacytidine or 0.1% DMSO for 4 days, and surface CD70 assessed by flow cytometry 3 days later. p -value by 2-way ANOVA. * p <0.05, ** p <0.01. n = 3 technical replicates. C. An eXtreme Gradient Boosting (XGB) model (see Methods) extracts features of transcription factor gene expression from CoMMpass ( n = 776) that best-model CD70 expression in patient tumors. 80% of data was used as a test set with 20% left out as a training set, with subsequent 5-fold cross validation (see Methods). n = 776 total samples included in the analysis. R 2 value corresponds to Pearson correlation between model-predicted CD70 expression in the test set and measured tumor CD70 expression in CoMMpass. D. Shapley Additive Explanations (SHAP) analysis suggest expression of transcription factors most strongly impacting CD70 expression levels in CoMMpass tumors. E. Correlation of CD70 with TFAP2A and PAX5 in the CoMMpass dataset. F. Surface CD70 by flow cytometry on LP-1 cells measured after Cas9 RNP nucleofection with sgRNA targeting TFAP2A , CD70 , or scramble control. Flow cytometry mean fluorescence intensity shown as fold-change versus isotype control. p -value by t -test. *** p <0.005, ****p<0.001. n = 3 technical replicates, representative of 2 biological replicates. Mean +/− S.D. shown.

Article Snippet: Primary antibodies targeting NSD2 (Abcam Cat# ab75359), HDAC2 (Millipore Cat# 05-814), H3 (Cell Signaling Technology Cat# 14269) and H3K36me2 (Abcam Cat# ab9049) were diluted in blocking buffer at 1:1000.

Techniques: Knock-Out, Expressing, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Targeting high-risk multiple myeloma genotypes with optimized anti-CD70 CAR-T cells

doi: 10.1101/2024.02.24.581875

Figure Lengend Snippet: A. Immunoblots confirming NSD2 knockout in KMS11 and KMS34 cell lines. H3K36me2 signal decrease confirms functional loss of histone methylation in the context of NSD2 loss. Total HDAC2 and Histone H3 levels are unchanged. B. Called peaks from ATAC-seq (data from Jin et al (2018) at the CD70 locus, across normal memory B-cells (MB), plasmablasts, normal plasma cells, and malignant plasma cells derived from multiple myeloma (MM) patients (ten illustrated here; 24 total included in study). Dashed red lines highlight major ATAC-seq peak present only in myeloma patient samples. Transcription factor motifs (listed in ) extracted from this peak.

Article Snippet: Primary antibodies targeting NSD2 (Abcam Cat# ab75359), HDAC2 (Millipore Cat# 05-814), H3 (Cell Signaling Technology Cat# 14269) and H3K36me2 (Abcam Cat# ab9049) were diluted in blocking buffer at 1:1000.

Techniques: Western Blot, Knock-Out, Functional Assay, Methylation, Derivative Assay