sem b-all cell line  (DSMZ)

Journal: Cancer Gene Therapy

Article Title: Functional characterization of the PI3K/AKT/MTOR signaling pathway for targeted therapy in B-precursor acute lymphoblastic leukemia

doi: 10.1038/s41417-022-00491-0

Figure Lengend Snippet: A Schematic representation of the shRNA library screen. RCH-ACV leukemia cells (E2A-PBX1 + /pre-BCR + ) were transduced with shRNA sublibraries. After puromycin selection, the frequency of individual shRNAs was quantified at day 0 and at day 12 by deep sequencing. The experiment was performed in duplicate. B Dot plot shows Mann–Whitney U test p values for enrichment (increase proliferation) and depletion (decrease proliferation) of targeted genes by shRNA knockdown and analyzed by deep-sequencing. Each dot represents a gene. C Small drug screen. RCH-ACV cells were treated with several small molecule inhibitors and viable cells were enumerated after four days. Graph shows half inhibitory growth concentration (IC50). At least three independent experiments were performed per treatment.

Article Snippet: E2A-PBX1 + B-ALL cell lines RCH-ACV and 697, as well as E2A-PBX1 - B-ALL cell lines REH and SEM were obtained from DSMZ (Braunschweig, Germany) in 2013, and were authenticated in 2020 in DSMZ.

Techniques: shRNA, Transduction, Selection, Sequencing, MANN-WHITNEY, Concentration Assay

Journal: Cancer Gene Therapy

Article Title: Functional characterization of the PI3K/AKT/MTOR signaling pathway for targeted therapy in B-precursor acute lymphoblastic leukemia

doi: 10.1038/s41417-022-00491-0

Figure Lengend Snippet: Titration curves for human leukemia cells cultured with increasing concentrations of ( A ) torin-1, ( B ) capivasertib and ( C ) everolimus. Viable cells were counted with trypan blue exclusion assay after four days. Data represent mean ± SEM of three independent experiments. D Graph shows means of half inhibitory growth concentration (IC50) of three independent experiments. E Titration curves of torin-1 for colony forming assay using murine E2A-PBX1 + /PreBCR + leukemia cells. Data represent mean ± SEM of three independent experiments. CFU, colony forming units. F Kaplan–meier curve represent disease-free survival of recipient mice transplanted with murine E2A-PBX1 + /PreBCR + leukemia cells after sublethal irradiation and treatment with vehicle or torin-1 (20 mg/kg b.w./d) starting at day 8 after transplantation. Each cohort contains 5 mice. Statistical analysis was performed by log-rank test.

Article Snippet: E2A-PBX1 + B-ALL cell lines RCH-ACV and 697, as well as E2A-PBX1 - B-ALL cell lines REH and SEM were obtained from DSMZ (Braunschweig, Germany) in 2013, and were authenticated in 2020 in DSMZ.

Techniques: Titration, Cell Culture, Trypan Blue Exclusion Assay, Concentration Assay, Irradiation, Transplantation Assay

Journal: Cell Death Discovery

Article Title: Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A -rearranged acute B-lymphoblastic leukemia

doi: 10.1038/s41420-022-01093-3

Figure Lengend Snippet: A Dose-response curves (nonlinear regression, curve fit) of cell lines SEM, RS4;11, REH, and NALM-6 were incubated with increasing concentrations of VEN for 72 h. Proliferation and metabolic activity were assessed by trypan blue staining and WST-1 assay, respectively. Mean ± SD of 1–3 biological replicates. B Erythrocyte and PBMC cytotoxicity were evaluated by hemolysis and calcein-AM assay, respectively. The blood of five healthy donors was used. Mean ± SD of three technical replicates. Hemolytic activity was assessed by hemoglobin release after 120 min incubation with 10 nM VEN or 1% SDS (positive control). For viability testing, PBMCs were incubated with 10 nM VEN or DMSO (control) for 24 h. C Cells were treated with 2.5 nM (RS4;11) or 10 nM (SEM, PBMCs) VEN for 48 h and subsequently spun onto microscopic slides and Pappenheim stained. Representative images of three independent biological replicates at 100x magnification.

Article Snippet: Human B—ALL cell lines SEM, RS4;11, REH, and NALM-6 were purchased from DSMZ (Braunschweig, Germany) and maintained at 37 °C and 5% CO 2 in IMDM medium (SEM), Alpha MEM medium (RS4;11) or RPMI 1640 medium (REH, NALM-6), all supplemented with 10% heat-inactivated fetal calf serum and 100 μg/ml penicillin/streptomycin (all PAN—biotech, Aidenbach, Germany).

Techniques: Incubation, Activity Assay, Staining, WST-1 Assay, Calcein AM Assay, Positive Control, Control

Journal: Cell Death Discovery

Article Title: Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A -rearranged acute B-lymphoblastic leukemia

doi: 10.1038/s41420-022-01093-3

Figure Lengend Snippet: A Cells were incubated with increasing concentrations of VEN for 72 h before staining with Annexin V-FITC and propidium iodide and subsequent flow cytometry. Mean ± SD of 1–4 biological replicates; two-way ANOVA with post hoc Dunnett’s multiple comparisons test. Asterisks indicate significance compared to the respective DMSO control. B Dose-response curves (nonlinear regression, curve fit) of cell lines incubated with increasing concentrations of VEN for 72 h. Apoptotic cells were assessed by AnnV/PI staining and flow cytometry and early and late apoptotic/necrotic) cells were added to calculate the amount of apoptotic cells. Mean ± SD of 1–4 biological replicates. C Protein expression of cleaved caspase-3 was analyzed by intracellular flow cytometry after 48 h incubation with 2.5 nM (RS4;11) or 10 nM (SEM) VEN or DMSO (control). Mean ± SD of three biological replicates; paired t -test.

Article Snippet: Human B—ALL cell lines SEM, RS4;11, REH, and NALM-6 were purchased from DSMZ (Braunschweig, Germany) and maintained at 37 °C and 5% CO 2 in IMDM medium (SEM), Alpha MEM medium (RS4;11) or RPMI 1640 medium (REH, NALM-6), all supplemented with 10% heat-inactivated fetal calf serum and 100 μg/ml penicillin/streptomycin (all PAN—biotech, Aidenbach, Germany).

Techniques: Incubation, Staining, Flow Cytometry, Control, Expressing

Journal: Cell Death Discovery

Article Title: Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A -rearranged acute B-lymphoblastic leukemia

doi: 10.1038/s41420-022-01093-3

Figure Lengend Snippet: A Expression of BCL-2 pathway proteins was assessed by immunoblot. Two to three individual biological replicates (gray) and expression mean (red); multiple t -tests, asterisks indicate significance vs time-matched DMSO control. B Total BCL-2 pathway member protein expression was analyzed by intracellular flow cytometry after 48 h incubation. Mean ± SD of three to five biological replicates; ratio paired t -test. C The time-dependent influence of VEN on BCL-2 phosphorylation was measured by immunoblot. BCL-2 and p-BCL-2 bands of three (SEM) or two (RS4;11) individual biological replicates were quantified. Relative BCL-2 phosphorylation values of all replicates (gray), as well as the mean of those experiments (red), are indicated in the graphs. Ratio paired t -test. D Protein expression of phosphorylated and total BCL-2 was assessed by intracellular flow cytometry after 48 h incubation with VEN or DMSO (control). Absolute expression values of controls was set to 100% and the relative change in protein expression following VEN incubation is indicated in the figure. Mean ± SD of five (SEM) or four (RS4;11) biological replicates; paired t -test of each protein vs. respective control. E Functional assessment of Bax-mediated apoptosis induction was performed by Bax translocation assay after 48 h VEN incubation. Four representative images of four biological replicates per cell line and treatment group, 40-fold magnification.

Article Snippet: Human B—ALL cell lines SEM, RS4;11, REH, and NALM-6 were purchased from DSMZ (Braunschweig, Germany) and maintained at 37 °C and 5% CO 2 in IMDM medium (SEM), Alpha MEM medium (RS4;11) or RPMI 1640 medium (REH, NALM-6), all supplemented with 10% heat-inactivated fetal calf serum and 100 μg/ml penicillin/streptomycin (all PAN—biotech, Aidenbach, Germany).

Techniques: Expressing, Western Blot, Control, Flow Cytometry, Incubation, Phospho-proteomics, Functional Assay, Translocation Assay

Journal: Cell Death Discovery

Article Title: Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A -rearranged acute B-lymphoblastic leukemia

doi: 10.1038/s41420-022-01093-3

Figure Lengend Snippet: A Weight progression of ten animals per study group. The dotted lines indicate the treatment period. Multiple t -tests. B , C Tumor cell proliferation was monitored by peripheral blood (PB) blast frequency measurement via flow cytometry (GFP + cells, B ) and in vivo bioluminescence imaging (BLI, C ). Each line represents an individual animal. BLI imaging was discontinued when technical saturation was reached. Mean ± SD of ten animals per group where four animals were sacrificed following therapy finalization at d25 (dotted line); Mann–Whitney test vs. time-matched controls. D Representative BLI images of three individual mice per group in ventral position. E Kaplan–Meier survival analysis. The dotted lines indicate the treatment period. Six animals per group; log-rank test. F The growth rate of leukemic blasts was calculated based on PB blast frequency values measured by flow cytometry. Values of d14 and d21 were used to assess the doubling time during treatment while values of d28 and d35 (SEM) or d35 and d42 (RS4;11) were used for posttreatment calculation. Mean ± SD of three to ten animals per group and time point; Mann–Whitney test. G Pharmacokinetic analyses were conducted one and 2 h after VEN p.o. application to investigate VEN concentrations in PB by liquid coupled mass spectrometry. Each dot represents an individual animal. Mean ± SD of six VEN-treated animals; Wilcoxon matched-pairs signed-rank test. H Analysis of VEN concentration in peripheral blood 24 h after application and the tumor cell doubling time of the respective animal during VEN treatment. Cumulative analysis of SEM and RS4;11-derived xenograft models. Linear regression where each dot represents an individual animal. Pearson’s correlation. I , J Determination of blast frequency in PB, bone marrow (BM), and spleen by flow cytometry ( I ) and spleen parameters ( J ) were assessed when the mice reached humane endpoints (30% blasts in PB or weak performance status). Mean ± SD of five to six animals per group; Mann–Whitney test. K Isolated BM or spleen cells were spun onto microscopic slides and Pappenheim stained. Four representative images of five to six mice per group at 100x magnification.

Article Snippet: Human B—ALL cell lines SEM, RS4;11, REH, and NALM-6 were purchased from DSMZ (Braunschweig, Germany) and maintained at 37 °C and 5% CO 2 in IMDM medium (SEM), Alpha MEM medium (RS4;11) or RPMI 1640 medium (REH, NALM-6), all supplemented with 10% heat-inactivated fetal calf serum and 100 μg/ml penicillin/streptomycin (all PAN—biotech, Aidenbach, Germany).

Techniques: Flow Cytometry, In Vivo, Imaging, MANN-WHITNEY, Mass Spectrometry, Concentration Assay, Derivative Assay, Isolation, Staining

Journal: Genome Research

Article Title: A KMT2A-AFF1 gene regulatory network highlights the role of core transcription factors and reveals the regulatory logic of key downstream target genes

doi: 10.1101/gr.268490.120

Figure Lengend Snippet: Developing a GRN model to assess regulatory impact of KMT2A-AFF1. (A) Schematic illustrating the concept of KMT2A-AFF1-targeted TFs, leading to indirect downstream regulation. (B) Proportion of KMT2A-AFF1-bound genes in SEM cells (nearest annotated promoter in ChIP-seq) that overlap with KMT2A-AFF1 targets from patient samples. (C) DEGs from nascent RNA-seq after 96 h KMT2A-AFF1 KD. DEGs are defined as FDR < 0.05 (n = 3). Shaded area represents KMT2A-AFF1-bound genes. (D) GRN creation workflow using nascent RNA-seq and ChIP-seq data (Methods). (E) Visualization of the whole network. Node color represents down-regulation (red) and up-regulation (blue) upon KMT2A-AFF1 KD. (F) The top 20 genes of the KMT2A-AFF1 GRN by degree centrality. Lines indicate predicted interaction from protein to gene locus, with arrowheads pointing downstream. (G) KMT2A-AFF1 KD DEGs that are unbound by KMT2A-AFF1, as highlighted in C. Shaded areas represent MAZ, ELF1, or RUNX1-bound genes.

Article Snippet: SEM cells, a KMT2A-AFF1 B cell ALL line ( Greil et al. 1994 ), were purchased from DSMZ ( https://www.dsmz.de ).

Techniques: ChIP-sequencing, RNA Sequencing

Journal: Genome Research

Article Title: A KMT2A-AFF1 gene regulatory network highlights the role of core transcription factors and reveals the regulatory logic of key downstream target genes

doi: 10.1101/gr.268490.120

Figure Lengend Snippet: KMT2A-AFF1 and intermediate TFs cooperate to regulate cascade and FFL motif targets. (A) MAGeCK analysis of CRISPR screen comparing T0 (baseline) and T18 (venetoclax), plotting −log10 FDR against log2 gRNA FC. (Left) Key genes; (right) core GRN TFs. (B) Select cascade motifs in the KMT2A-AFF1 GRN that explain interactions between KMT2A-AFF1 and CASP9. Node color represents logFC response to KMT2A-AFF1 KD. (C,E) Western blot in SEM cells showing RUNX1 protein levels after 48 h KMT2A-AFF1 KD (C) or 48 h RUNX1 KD (E), with GAPDH as a loading control. (D,F) qRT-PCR assaying KMT2A-AFF1, RUNX1 and CASP9 expression following 48 h KMT2A-AFF1 KD (D) or 48 h RUNX1 KD (F) in SEM cells (n = 3). Expression normalized to GAPDH and shown relative to NT control. (G,H) Subnetworks illustrating interactions from KMT2A-AFF1 and cooperative TFs that feed into MYC (G) and BCL2 (H). (I,J) qRT-PCR analysis probing mature MYC mRNA (I) and BCL2 pre-mRNA (J) after 96 h KD targeting genes as indicated (n = 3, n = 5 for NT and RUNX1 KD). Expression normalized to GAPDH mature mRNA levels and shown relative to NT control. (K) Colony assay counts after 96 h KD targeting genes as indicated (n = 3, n = 5 for NT and RUNX1 KD). Colony counts shown relative to NT control. Error bars represent standard error of the mean; (#) P < 0.1; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

Article Snippet: SEM cells, a KMT2A-AFF1 B cell ALL line ( Greil et al. 1994 ), were purchased from DSMZ ( https://www.dsmz.de ).

Techniques: CRISPR, Western Blot, Control, Quantitative RT-PCR, Expressing, Colony Assay