Journal: Journal of Translational Medicine

Article Title: Aberrant SPOP-CHAF1A ubiquitination axis triggers tumor autophagy that endows a therapeutical vulnerability in diffuse large B cell lymphoma

doi: 10.1186/s12967-022-03476-7

Figure Lengend Snippet: High CHAF1A exerts tumor-promoting biological effects in DLBCL. A The RT-qPCR analysis indicated the down-regulated CHAF1A mRNA expressions shCHAF1A DLBCL cell lines (DB, U2932 and FARAGE) and shCtrl cells. B The RT-qPCR analysis indicated the elevated CHAF1A expressions in EV and OE OCI-Ly7 cells. C MTT assay showed suppressed cell viability when CHAF1A was knocked down by shCHAF1A in three independent DLBCL cell lines as compared to control (shCtrl) cells. D In contrast, CHAF1A overexpression could cellular proliferative activity of OCI-Ly7. E The colony generation of DLBCL cells by colony formation assays showed CHAF1A KD could attenuate colony formation ability, but CHAF1A OE could rescue the suppressed capacity. F Besides, CHAF1A KD could also attenuate migration ability of cells by Transwell assay, but CHAF1A OE could also rescue the impaired migration capacity. G Animal assays on nude mice with OCI-Ly7 cells showed enhanced tumor growth rate in the CHAF1A OE group compared with the control group (EV). Representative tumor graphs were shown on the left, while H&E and IHC staining of Ki-67 were exhibited on the right. Scale bar = 50 μm H Record of tumor volumes at the indicated timepoints and generation of tumor growth curves in two mice groups. I After the animal sacrifice, tumor weight of mice derived from the two groups were compared. Experiments were performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Human lymphoblastoid B cell (GM12878), human renal epithelial cells (293 T) and DLBCL cells (OCI-LY7, DB, U2932, and FARAGE) were purchased from American Type Culture Collection (ATCC, USA).

Techniques: Quantitative RT-PCR, MTT Assay, Control, Over Expression, Activity Assay, Migration, Transwell Assay, Immunohistochemistry, Derivative Assay

Journal: Journal of Translational Medicine

Article Title: Aberrant SPOP-CHAF1A ubiquitination axis triggers tumor autophagy that endows a therapeutical vulnerability in diffuse large B cell lymphoma

doi: 10.1186/s12967-022-03476-7

Figure Lengend Snippet: Aberrant SPOP-CHAF1A ubiquitination axis contributes to accumulated CHAF1A expressions in DLBCL. A Co-Immunoprecipitation (IP) analysis based on U2932 cells with IgG or anti-SPOP antibody indicated the endogeous interactions between SPOP and CHAF1A, as shown by western blotting assay. B The schematic representation of SPOP-WT and deletion mutants. Binding capacity of SPOP to CHAF1A is indicated with the representative symbol. C Western blotting assay showed the altered CHAF1A proteins in whole cell lysate (WCLs) from U2932 cells which were co-transfected with plasmids of FLAG-CHAF1A and WT SPOP or mutants. D Western blotting analysis showed the elevated CHAF1A proteins in CHAF1A KD cells (U2932, FARAGE) relative to control cells. Besides, the corresponding mRNA levels of CHAF1A in indicated groups were quantified and shown on the right, and no altereations were observed. E Quantification of CHAF1A proteins in WCLs of U2932 cells (parental, sgSPOP) for 12 h and then treated with 50 μg/ml cycloheximide (CHX) and harvested at different time points. At each time point, the intensity of CHAF1A was normalized to the intensity of actin and then to the data at 0 h. F Quantification of CHAF1A proteins in WCLs of FARAGE cells (EV, myc-SPOP) for 12 h and then treated with 50 μg/ml cycloheximide (CHX) and harvested at different time points. G After the 293 T cells were transfected with the indicated plasmids and treated with 20 μM MG132 for 8 h, the western blotting assay showed the in vivo CHAF1A ubiquitination levels by WT SPOP or mutants. H Illustration of tumor-associated mutations across SPOP gene in lymphoid malignancies. DLBCL: diffuse large B Cell lymphoma; PCM: plasma cell myeloma; ALL: acute lymphoid leukemia; NKTL: NK-T cell lymphoma; CLL: chronic lymphocytic leukemia; unclassified LNs unclassified lymphoid malignancies. I Western blotting assay showed the differential in vivo ubiquitination levels of CHAF1A in 293 T cells transfected with plasmids of FLAG-CHAF1A and Myc-SPOP or relevant mutations. J Comparison of putative SPOP binding sites in CHAF1A with the SPOP-binding consensus motif defined in the known SPOP substrates, where amino acid sequence alignment of the SBC motif (Φ-π-S-S/T-S/T; Φ: nonpolar residues, π: polar residues) was identified. K Western blotting assay showed the altered protein levels of WT-CHAF1A and mutants with deleted SBC motif when U2932 cells were transfected with Myc-SPOP, respectively. Experiments were performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Human lymphoblastoid B cell (GM12878), human renal epithelial cells (293 T) and DLBCL cells (OCI-LY7, DB, U2932, and FARAGE) were purchased from American Type Culture Collection (ATCC, USA).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Binding Assay, Transfection, Control, In Vivo, Clinical Proteomics, Comparison, Sequencing

Journal: Journal of Translational Medicine

Article Title: Aberrant SPOP-CHAF1A ubiquitination axis triggers tumor autophagy that endows a therapeutical vulnerability in diffuse large B cell lymphoma

doi: 10.1186/s12967-022-03476-7

Figure Lengend Snippet: Down-regulated or mutated SPOP promotes DLBCL maliganant aggressiveness depending on CHAF1A. A The violin plot exhibited the differential expression levels of SPOP between DLBCL and normal tissues based on the GSE83632 data set. B Kaplan–Meier (K–M) survival curves analysis also showed that low SPOP levels may appear to correlate with shorter OS months of DLBCL patients based on the GSE83632 data set. C The CCK8 assays in three DLBCL cell lines (DB, U2932, FARAGE) revealed that WT SPOP, but not the δBTB mutant, could suppress cell proliferation, but simultaneous overexpression of CHAF1A could partially rescue the impaired cell growth ability. D Proliferation ability of OCI-Ly7 cells was enhanced by SPOP depletion, which could be partailly impaired by CHAF1A KD. E Similarly, SPOP depletion could enhance migration of OCI-Ly7 cells, which could be partially suppressed by CHAF1A KD. F Tumor volumes curves of FARAGE-derived tumor models showed that WT SPOP, but not the δBTB mutant, could suppress in vivo tumor growth, whereas tumor-associated SPOP F102I mutant could enhance in vivo tumor growth. G The OCI-Ly7-derived tumor models showed that SPOP depletion could enhance tumor in vivo growth, which could be partially suppressed by CHAF1A KD. Tumor graph was shown on the left; Quantification of tumor volumes was shown on the middle panel; Tumor weight was compared and shown on the right panel. Experiments were performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Human lymphoblastoid B cell (GM12878), human renal epithelial cells (293 T) and DLBCL cells (OCI-LY7, DB, U2932, and FARAGE) were purchased from American Type Culture Collection (ATCC, USA).

Techniques: Quantitative Proteomics, Mutagenesis, Over Expression, Migration, Derivative Assay, In Vivo

Journal: Journal of Translational Medicine

Article Title: Aberrant SPOP-CHAF1A ubiquitination axis triggers tumor autophagy that endows a therapeutical vulnerability in diffuse large B cell lymphoma

doi: 10.1186/s12967-022-03476-7

Figure Lengend Snippet: SPOP-CHAF1A axis controls tumor autophagy of DLBCL in a TFEB-dependent manner. A Positive associations between CHAF1A and TFEB expressions based on TCGA-DLBCL samples. B The RT-qPCR assay showed that CHAF1A OE could promote TFEB expression (left), while CHAF1A KD reduced TFEB mRNA levels (right panel). C The ChIP-qPCR of H3K27ac markers and CHAF1A in promoter of TFEB gene in U2932 cells, as indicated (N = 4). D The RT-qPCR assay showed that CHAF1A KD could suppress TFEB transcriptional activity in U2932 cells (left panel) and FARAGE cells (middle panel). Relative luciferase activities were normalized versus control. The OCI-Ly7 cells were transfected with TFEB reporters together with TFEB vectors alone or TFEB + CHAF1A plasmids to show the regulation of TFEB reporter activity by CHAF1A (right panel). E Western blotting assay showed that CHAF1A-KD decreased the levels of autophagy-related markers (p62, and Beclin-1) in CHAF1A-silenced U2932 and FARAGE cells relative to control cells. F-G The luciferase activity ( F ) and mRNA levels ( G ) of lysosomal genes were measured in control and SPOP-OE OCI-Ly7 cells transfected with siCtrl or siTFEB, individually. H The tumor growth curves were obtained from OCI-Ly7-derived tumor models to show that targeting TFEB significantly suppressed the enhanced tumor growth induced by SPOP deficiency. I The tumor weight of mice from indicated groups was shown and compared. Experiments were performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Human lymphoblastoid B cell (GM12878), human renal epithelial cells (293 T) and DLBCL cells (OCI-LY7, DB, U2932, and FARAGE) were purchased from American Type Culture Collection (ATCC, USA).

Techniques: Quantitative RT-PCR, Expressing, ChIP-qPCR, Activity Assay, Luciferase, Control, Transfection, Western Blot, Derivative Assay

Journal: Journal of Translational Medicine

Article Title: Aberrant SPOP-CHAF1A ubiquitination axis triggers tumor autophagy that endows a therapeutical vulnerability in diffuse large B cell lymphoma

doi: 10.1186/s12967-022-03476-7

Figure Lengend Snippet: CHAF1A knockdown reversed the promotive effects of SPOP deficiency on DLBCL cell autophagy. A , B The confocal microscopy showed the autophagy flux in the indicated groups of DLBCL cells. C The TEM analysis showed the number of autophagic vacuoles in the indicated groups of DLBCL cells

Article Snippet: Human lymphoblastoid B cell (GM12878), human renal epithelial cells (293 T) and DLBCL cells (OCI-LY7, DB, U2932, and FARAGE) were purchased from American Type Culture Collection (ATCC, USA).

Techniques: Knockdown, Confocal Microscopy

Journal: Nature medicine

Article Title: TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma

doi: 10.1038/s41591-020-0757-z

Figure Lengend Snippet: a, Effects of vincristine and doxorubicin, both components of the R-CHOP regimen, on PS exposure in Karpas422 TMEM30A−/− and wild-type control. The top panel (i) compares wild-type (red) and TMEM30A−/− (blue) events counted by flow cytometry. The lower panel (ii) compares TMEM30A−/− expressing recombinant TMEM30A (rhTMEM30A, red) and a transformation empty-vector control (Empty, blue). b, Increased apoptotic signaling following incubation with doxorubicin as measurable by caspase-3 activation by western blot analysis (i), and vincristine, as measurable by a G2/M block through flow cytometry (ii), was compared in Karpas422 TMEM30A−/−, wild type and TMEM30A transformants. Cropped images from the same western blot are shown. β-actin is used as a loading control. See Source Data Fig. 5. Experiments were repeated independently three times with similar results. c, Doxorubicin uptake in Karpas422 TMEM30A−/−, wild type and TMEM30A transformants over time was detected by flow cytometry. Graphs represent mean values ± s.d. (n = 4). All P values are based on an unpaired two-tailed Student’s t-test. d, Externalized PS following increasing doxorubicin incubation times was measured in gated B-cells from DLBCL patient tumor biopsies expressing normal or TMEM30A-null (summary bar plot, left; movement of each sample, right). Graphs represent mean values ± s.d. (n = 3). P value is based on a two-way ANOVA test. e, Doxorubicin uptake in gated B-cells from DLBCL patient tumor biopsies was measured over time by flow cytometry. Graphs represent mean values ± s.d. (n = 3). P value is based on a two-way ANOVA test. f, Alterations in PS externalization (i) and doxorubicin (ii) uptake including XKR8 knockout in NU-DUL-1 wild type (WT) and TMEM30A−/− cells over time.

Article Snippet: Human DLBCL cell lines DOHH-2 and Karpas422 were purchased from DSMZ and ATCC, respectively, and maintained in RPMI-1640 (Life Technologies) containing 10% or 20% FBS (Life Technologies), respectively.

Techniques: Control, Flow Cytometry, Expressing, Recombinant, Transformation Assay, Plasmid Preparation, Incubation, Activation Assay, Western Blot, Blocking Assay, Two Tailed Test, Knock-Out

Journal: Nature medicine

Article Title: TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma

doi: 10.1038/s41591-020-0757-z

Figure Lengend Snippet: Human ATP8A2 was expressed with or without human TMEM30A or missense mutants in HEK293T cells. a, The expressed complex was purified on an ATP8A2 immunoaffinity column and the input and eluted samples were subjected to SDS gel electrophoresis and western blots were labeled for ATP8A2 with the Atp6C11 and Cdc50-7F4 antibodies. Cropped images from the same Western blot are shown. Tubulin was detected as a loading control for the input lanes. Independent experiments were repeated twice with similar results. Source Data Extended Fig. 3. b, ATPase activity is measured as nmols of ATP hydrolyzed per 30 min at 37 °C. ATPase activities of the isolated complexes were measured in either 100% dioleylphosphatidylcholine (PC) or a mixture of 80% dioleoylphosphatidylcholine: 20% dioleylphosphatidylserine (PS). Samples are: ATP8A2 - expression in the absence of expressed TMEM30A; ATP8A2/WT - co-expression of ATP8A2 with WT- TMEM30A; and ATP8A2/ W41L, ATP8A2/C94R, ATP8A2/D181Y. Graphs represent mean values ± s.d. (n = 3). c, Distributions of genetic alterations of TNFAIP3, PRDM1 and EPHA7 in the patients with TMEM30A mutation and deletion. The header includes status of TMEM30A mutation and heterozygous deletion. MT; mutation, CN; copy number. d, Distributions of recurrent mutation, COO subtypes and IPI groups in TMEM30A mutated and wild type DLBCL patients. The header includes the status of TMEM30A mutation and CNAs.

Article Snippet: Human DLBCL cell lines DOHH-2 and Karpas422 were purchased from DSMZ and ATCC, respectively, and maintained in RPMI-1640 (Life Technologies) containing 10% or 20% FBS (Life Technologies), respectively.

Techniques: Purification, SDS-Gel, Electrophoresis, Western Blot, Labeling, Control, Activity Assay, Isolation, Expressing, Mutagenesis

Journal: Nature medicine

Article Title: TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma

doi: 10.1038/s41591-020-0757-z

Figure Lengend Snippet: a, Single-state diffusion coefficients and b, confinement-diameters are shown for both cell lines, and c, BCR tracks and d, transition rates are measured for DOHH-2. The dots indicate median values; lines indicate 95% confidence intervals ((n=3, DOHH-2) (n = 2, NU-DUL-1). (E-F) TMEM30A mutation does not alter CD19 diffusion and confinement diameters. Cells were labelled with anti-CD19 Fab-Cy3, settled onto poly-l-lysine coated coverslips and imaged for 10 s at 33 Hz. e, Single-state diffusion coefficients were calculated for all tracks and cumulative frequency curves are shown. The dots on the curves indicate the median values and are reported in brackets. Total number of tracks analyzed in each condition is shown above the plot. (f) The cumulative frequency curves of the confinement-diameters with the median values (dots on the curves) indicated in brackets. g, BCR activation of DLBCL cell lines following incubation with F(ab)2 fragments does not lead to an increase in apoptotic signaling through caspase 3 activation, measurable by western blot analysis. Cells (Karpas422) pretreated with vincristine were included as a positive control. Cropped images from the same Western blot are shown. B-actin is used as a loading control. Experiments were repeated independently three times with similar results. Source Data Extended Fig. 6. h, Ca2+ mobilization was measured in wild type (WT) and TMEM30A−/− NU-DUL-1, DOHH-2 and Karpas422 cells, following HBSS, F(ab)2 addition (10 and 40 μg/ml) and ionomycin. Graphs represent mean values ± s.d. (n = 3). Significance is evaluated using one-way ANOVA.

Article Snippet: Human DLBCL cell lines DOHH-2 and Karpas422 were purchased from DSMZ and ATCC, respectively, and maintained in RPMI-1640 (Life Technologies) containing 10% or 20% FBS (Life Technologies), respectively.

Techniques: Diffusion-based Assay, Mutagenesis, Activation Assay, Incubation, Western Blot, Positive Control, Control

Journal: Nature medicine

Article Title: TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma

doi: 10.1038/s41591-020-0757-z

Figure Lengend Snippet: a, Frequencies and type of mutations identified by deep targeted sequencing in 57 recurrently mutated genes in 347 DLBCL tumors. Genes significantly enriched in ABC-DLBCL and GCB-DLBCL are highlighted in blue and orange, respectively. Colored asterisks represent the pathways to which mutations belong. b, Significant (q-value<0.25) focal amplifications (red) and deletions (blue) identified by GISTIC 2.0 analysis (n = 338). Representative genes (functionally known in DLBCL, as well as other cancers) within significant GISTIC regions are annotated. Genes significantly enriched in ABC-DLBCL and GCB-DLBCL are highlighted in blue and orange, respectively. Colored asterisks represent the pathways to which CNAs belong.

Article Snippet: Human DLBCL cell lines DOHH-2 and Karpas422 were purchased from DSMZ and ATCC, respectively, and maintained in RPMI-1640 (Life Technologies) containing 10% or 20% FBS (Life Technologies), respectively.

Techniques: Mutagenesis, Sequencing

Journal: Nature medicine

Article Title: TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma

doi: 10.1038/s41591-020-0757-z

Figure Lengend Snippet: Mutated genes (frequency > 5%) and representative CNAs within GISTIC regions were evaluated. Frequencies of genetic alterations are depicted according to COO subtype. Genes significantly enriched (adjusted p < 0.10, two-sided fisher’s exact test) in ABC-DLBCL (blue) and GCB-DLBCL (orange) are highlighted (left panel). ⋆P < 0.05, ⋆⋆P < 0.01, ⋆⋆⋆P < 0.001 (adjusted by multiple test). Forest plot summarizes the results of univariate analyses (time-to progression (TTP)) for each genomic alteration, in all DLBCL (grey), in ABC-DLBCL (light blue) and in GCB-DLBCL (light-orange). Hazard ratios and 95% confidence intervals are shown. Yellow circles represent significant hazard ratios (p < 0.05) (right panel). HETD, heterozygous deletion; HOMD, homozygous deletion; AMP, amplification.

Article Snippet: Human DLBCL cell lines DOHH-2 and Karpas422 were purchased from DSMZ and ATCC, respectively, and maintained in RPMI-1640 (Life Technologies) containing 10% or 20% FBS (Life Technologies), respectively.

Techniques: Amplification

Journal: Nature medicine

Article Title: TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma

doi: 10.1038/s41591-020-0757-z

Figure Lengend Snippet: a,b,c, Forest plots summarize the results of univariate analyses (TTP and OS) for mutated genes and representative copy-number alterations (CNAs) within GISTIC regions, which are significantly associated with prognosis (unadjusted P value < 0.05), in all-DLBCL (n = 347) (a), in ABC-DLBCL (n = 104) (b) and GCB-DLBCL (n = 183) (c). Hazard ratios and 95% confidence intervals are shown. Colored circles represent significant hazard ratios, and colored genes are significantly associated with both TTP and OS (blue, favorably prognostic; red, unfavorably prognostic). d, Frequency of selection of the genetic feature by the Cox–lasso predictive models generated in 238 iterations of leave-one-out cross validation. COO and IPI are included as control variables in all models. The bar plot (blue, favorable outcome; red, unfavorable outcome) shows the genes repeatedly selected in the 238 models. e, xseq-derived cis and trans effects of loss-of-function and hotspot mutations in all DLBCL tumors and each COO subtype (left). P(D) represents the probability that a recurrently mutated gene influences gene expression across the population of patients. A larger P(D) value represents a stronger inferred effect. Histogram (right) shows the number of each mutation in COO subtypes. f, Frequencies of occurrence of hemizygous deletion or copy-neutral loss of heterozygosity (cnLOH) in the loss-of-function mutated tumors. g, Proportions of mutation type in recurrently mutated genes. Mutated genes with frequency >10% are depicted.

Article Snippet: Human DLBCL cell lines DOHH-2 and Karpas422 were purchased from DSMZ and ATCC, respectively, and maintained in RPMI-1640 (Life Technologies) containing 10% or 20% FBS (Life Technologies), respectively.

Techniques: Selection, Generated, Biomarker Discovery, Control, Derivative Assay, Gene Expression, Mutagenesis

Journal: Nature medicine

Article Title: TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma

doi: 10.1038/s41591-020-0757-z

Figure Lengend Snippet: a, TMEM30A protein domain and the locations of 42 mutations identified in this study, affecting 38 DLBCL tumors, as well as 5 mutations found in transformed folllicular lymphoma (FL) samples in our previous study20. Multiple mutations affecting the same position are annotated. b, Schematic structure of the P4-ATPase and TMEM30A subunits consisting of two membrane-spanning domains with a large extracellular loop; 95% of variants are localized to extracellular domains, which are required for assembly with P4-ATPase. Flippase activities are annotated with colored stars. c, Distribution of DLBCL tumors harboring TMEM30A mutations, heterozygous deletions (Het-Del), and cnLOH, TMEM30A mutation without CNAs and heterozygous deletion without mutations. d, Heterozygous deletions were validated using fluorescence in situ hybridization (FISH) assays in all nine analyzed samples. Two-color FISH assays use a red probe interrogating TMEM30A and a green probe interrogating PIM1 on the chromosomal 6p arm as a reference. e, SNP6.0 copy-number heat map illustrating broad deletions affecting TMEM30A, EPHA7, PRDM1 and TNFAIP3 locus (dense blue line and arrow) on chromosome 6q11-24 in representative DLBCL tumors. f, Distribution of recurrent mutations, COO subtypes and IPI groups in 38 TMEM30A-mutated DLBCL tumors (left). The header includes the status of TMEM30A mutation and CNAs. Histograms (right) show the frequency of each alteration within TMEM30A mutated (dark gray) and non-mutated tumors (light gray). Two-sided Fisher’s exact test was used to compare these frequencies according to the two groups. *P < 0.05, ***P < 0.001. g, Evaluating the evolutionary timing of TMEM30A mutations and 6q deletions. Each column depicts an evolutionary timing scenario for TMEM30A mutations and 6q deletions.

Article Snippet: Human DLBCL cell lines DOHH-2 and Karpas422 were purchased from DSMZ and ATCC, respectively, and maintained in RPMI-1640 (Life Technologies) containing 10% or 20% FBS (Life Technologies), respectively.

Techniques: Transformation Assay, Membrane, Mutagenesis, Fluorescence, In Situ Hybridization

Journal: Nature medicine

Article Title: TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma

doi: 10.1038/s41591-020-0757-z

Figure Lengend Snippet: a, Bar plot showing the frequencies of TMEM30A mutation in lymphomas and other hematological malignancies, based on the previous literature and publicly available datasets. b, Deep targeted sequence data of a representative transformed FL case (FL1004) for the affected position of the TMEM30A visualized in integrative genomics viewer (left) and variant allelic frequency visualized in pie chart (right), demonstrating the proportion of variant reads (variation indicated in green) at the time of diagnosis (FL-Grade 1), transformation (DLBCL) and relapse after transformation (FL-Grade 2). The time between initial diagnosis (FL-Grade1) and first relapse (FL-transformed) and between first relapse and second relapse (FL-Grade 2) is shown in years (y). c–e, Kaplan–Meier curves represent TTP (top) and OS (bottom) according to TMEM30A mutation in all DLBCL (c), in IPI-high/high-intermediate group (d) and according to TMEM30A genetic status in all DLBCL (e). P values were derived from two-sided log-rank test (c,d) and Cox proportional-hazards model (e).

Article Snippet: Human DLBCL cell lines DOHH-2 and Karpas422 were purchased from DSMZ and ATCC, respectively, and maintained in RPMI-1640 (Life Technologies) containing 10% or 20% FBS (Life Technologies), respectively.

Techniques: Mutagenesis, Sequencing, Transformation Assay, Variant Assay, Biomarker Discovery, Derivative Assay

Journal: Nature medicine

Article Title: TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma

doi: 10.1038/s41591-020-0757-z

Figure Lengend Snippet: a, b, Deep targeted sequencing data of transformed FL samples with TMEM30A mutation detected at the time of diagnosis or transformation (A). The affected positions of the TMEM30A are visualized in an integrative genomic viewer. Variant allelic frequencies are visualized in the pie charts per case (B). Those data demonstrate a fraction of variants at the time of diagnosis (upper row) and transformation (lower row). The number in the pie charts represents the coverage of sequencing. FL1179 exhibited the opposite pattern (T1-positive/T2-negative), however the variant was a missense mutation supported by relatively low coverage (five reads out of 81 (VAF=6%). (c–e) Subtype analysis of survival according to TMEM30A genetic alteration. Kaplan Meier curves represent TTP (upper) and OS (bottom) according to TMEM30A mutation in ABC-DLBCL (C), in GCB-DLBCL (D) and in IPI-low/low-intermediate group (E). P values were derived from two-sided log-rank test.

Article Snippet: Human DLBCL cell lines DOHH-2 and Karpas422 were purchased from DSMZ and ATCC, respectively, and maintained in RPMI-1640 (Life Technologies) containing 10% or 20% FBS (Life Technologies), respectively.

Techniques: Mutagenesis, Sequencing, Transformation Assay, Biomarker Discovery, Variant Assay, Derivative Assay

Journal: OncoTargets and therapy

Article Title: ALDH1A1 induces resistance to CHOP in diffuse large B-cell lymphoma through activation of the JAK2/STAT3 pathway

doi: 10.2147/OTT.S107957

Figure Lengend Snippet: Correlation between ALDH1A1, STAT3, or p-STAT3 expression and clinicopathological parameters in 88 DLBCL patients

Article Snippet: The human DLBCL cell lines Pfeiffer (CRL 2632) and Farage (CRL 2630) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: ALDH1A1 induces resistance to CHOP in diffuse large B-cell lymphoma through activation of the JAK2/STAT3 pathway

doi: 10.2147/OTT.S107957

Figure Lengend Snippet: ALDH1A1 expression in DLBCL cell lines and clinical specimens. Notes: ( A ) The levels of ALDH1A1 protein were quantified using Western blot in two DLBCL cell lines (Farage and Pfeiffer). GAPDH blotting was used as a loading control. Bar graphs are derived from densitometric scanning of the blots. The density of the ALDH1A1 blot was normalized against that of GAPDH to obtain a relative blot density. Bars are mean ± SD from three independent experiments. ( B ) Representative images of ALDH1A1, STAT3, and p-STAT3 immunohistochemical staining in low sensitivity group and high sensitivity group DLBCL patients (magnification: 200×). ( C ) Mean immunohistochemiscal staining scores for ALDH1A1, STAT3, and p-STAT3 in low-sensitivity group and high-sensitivity group DLBCL patients. * P <0.05. ( D ) The comparison of overall survival between patients with high ALDH1A1 expression and low ALDH1A1 expression. Patients with low expression of ALDH1A1 had a longer survival than those with high expression (log-rank test, P =0.0033). Abbreviations: ALDH1A1, aldehyde dehydrogenase 1A1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation; DLBCL, diffuse large B-cell lymphoma; p-STAT3, phosphorylated-STAT3.

Article Snippet: The human DLBCL cell lines Pfeiffer (CRL 2632) and Farage (CRL 2630) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Western Blot, Control, Derivative Assay, Immunohistochemical staining, Staining, Comparison, Standard Deviation

Journal: OncoTargets and therapy

Article Title: ALDH1A1 induces resistance to CHOP in diffuse large B-cell lymphoma through activation of the JAK2/STAT3 pathway

doi: 10.2147/OTT.S107957

Figure Lengend Snippet: Correlation between ALDH1A1 and STAT3, p-STAT3 in DLBCL

Article Snippet: The human DLBCL cell lines Pfeiffer (CRL 2632) and Farage (CRL 2630) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: ALDH1A1 induces resistance to CHOP in diffuse large B-cell lymphoma through activation of the JAK2/STAT3 pathway

doi: 10.2147/OTT.S107957

Figure Lengend Snippet: Multivariate analysis of factors contributing to overall survival in DLBCL patients

Article Snippet: The human DLBCL cell lines Pfeiffer (CRL 2632) and Farage (CRL 2630) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques:

Journal: OncoTargets and therapy

Article Title: ALDH1A1 induces resistance to CHOP in diffuse large B-cell lymphoma through activation of the JAK2/STAT3 pathway

doi: 10.2147/OTT.S107957

Figure Lengend Snippet: The influence of ALDH1A1 on chemoresistance in Pfeiffer cells. Notes: Cells were transfected with empty vector (“Pfeiffer-NC”), ALDH1A1-shRNA vector (“Pfeiffer-shRNA”), and ALDH1A1 expression vector (“Pfeiffer-ALDH1A1”). The blank cells were not transfected (“Pfeiffer”). ( A ) Expression levels of ALDH1A1 and GAPDH proteins were confirmed by Western blotting. ( B ) The IC50 values of CHOP were determined by CCK-8 assay. Cells were treated with increasing concentrations of CHOP for 48 hours. ( C ) The long-term effect of ALDH1A1 on chemoresistance was studied by colony formation assay. ( D ) Flow cytometric analysis of apoptosis in cells combined with CHOP (0 and 640 ng/mL). Data are expressed as the mean ± SD of three individual experiments. * P <0.05. Abbreviations: ALDH1A1, aldehyde dehydrogenase 1A1; CHOP, combinatorial cyclophosphamide, doxorubicin, vincristine, and prednisone; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation; CCK-8, Cell Counting Kit-8; shRNA, short hairpin RNA; CI, confidence interval; IC50, half maximal inhibitory concentration.

Article Snippet: The human DLBCL cell lines Pfeiffer (CRL 2632) and Farage (CRL 2630) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transfection, Plasmid Preparation, shRNA, Expressing, Western Blot, CCK-8 Assay, Colony Assay, Standard Deviation, Cell Counting, Concentration Assay

Journal: OncoTargets and therapy

Article Title: ALDH1A1 induces resistance to CHOP in diffuse large B-cell lymphoma through activation of the JAK2/STAT3 pathway

doi: 10.2147/OTT.S107957

Figure Lengend Snippet: ALDH1A1 regulates the JAK2/STAT3 pathway in Pfeiffer cells. Notes: ( A ) Effects of ALDH1A1 on the expressions of JAK2 and p-JAK2 (Y570, Y931, and Y1007) were analyzed by Western blotting. ( B ) Expression levels of STAT3 and p-STAT3 (Y705, S727) were analyzed by Western blotting. GAPDH blotting was used as a loading control. Bar graphs are derived from densitometric scanning of the blots. The density of the ALDH1A1, JAK2/p-JAK2, and STAT3/p-STAT3 blots was normalized against that of GAPDH to obtain a relative blot density. Bars are mean ± SD from three independent experiments. * P <0.05. Abbreviations: ALDH1A1, aldehyde dehydrogenase 1A1; SD, standard deviation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; shRNA, short hairpin RNA; Pfeiffer-NC, cells transfected with empty vector; Pfeiffer-shRNA, ALDH1A1-shRNA vector; Pfeiffer-ALDH1A1, ALDH1A1 expression vector; p-STAT3, phosphorylated-STAT3; p-JAK2, phosphorylated-JAK2.

Article Snippet: The human DLBCL cell lines Pfeiffer (CRL 2632) and Farage (CRL 2630) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Expressing, Control, Derivative Assay, Standard Deviation, shRNA, Transfection, Plasmid Preparation

Journal: OncoTargets and therapy

Article Title: ALDH1A1 induces resistance to CHOP in diffuse large B-cell lymphoma through activation of the JAK2/STAT3 pathway

doi: 10.2147/OTT.S107957

Figure Lengend Snippet: Effects of WP1066 on the JAK2/STAT3 pathway and CHOP resistance induced by ALDH1A1 overexpression. Notes: ( A ) The protein levels of ALDH1A1, JAK2/p-JAK2, STAT3/p-STAT3, and pathway targets (Bcl-2, cyclin D1) were assayed by Western blotting. GAPDH was used as an internal control. ( B ) The IC50 values of CHOP were determined by CCK8 assay. ( C ) Colony formation assay was performed to determine the long-term effects of WP1066. ( D ) Flow cytometric analysis of apoptosis in cells combined with CHOP (0 and 640 ng/mL). Data are expressed as the mean ± SD of three individual experiments. * P <0.05. Abbreviations: ALDH1A1, aldehyde dehydrogenase 1A1; SD, standard deviation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CCK-8, Cell Counting Kit-8; CHOP, combinatorial cyclophosphamide, doxorubicin, vincristine, and prednisone; shRNA, short hairpin RNA; Pfeiffer-NC, cells transfected with empty vector; Pfeiffer-shRNA, ALDH1A1-shRNA vector; Pfeiffer-ALDH1A1, ALDH1A1 expression vector; Pfeiffer-ALDH1A1+WP1066, Pfeiffer-ALDH1A1 cells treated with WP1066; p-STAT3, phosphorylated-STAT3; p-JAK2, phosphorylated-JAK2; IC50, half maximal inhibitory concentration.

Article Snippet: The human DLBCL cell lines Pfeiffer (CRL 2632) and Farage (CRL 2630) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Over Expression, Western Blot, Control, CCK-8 Assay, Colony Assay, Standard Deviation, Cell Counting, shRNA, Transfection, Plasmid Preparation, Expressing, Concentration Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Chimeric antigen receptor T cells targeting CD79b show efficacy in lymphoma with or without co-targeting CD19

doi: 10.1158/1078-0432.CCR-19-1337

Figure Lengend Snippet: (A) Bar graph showing the percent CD79b and CD19 expression on human B cell tumor cell lines determined by flow cytometry (B) Percent CD79b and CD19 expression on MCL-patient-derived xenografts determined by flow cytometry (mean±SEM shown, *<0.05, t test). (C) Percent CD79b and CD19 expression on circulating tumor cells obtained from MCL patients, gated as CD3-CD20+CD5+ (n=6 patients, mean±SEM shown, ****<0.0001, t test). MM: multiple myeloma, BL: Burkitt’s lymphoma, DLBCL: diffuse large B cell lymphoma, MCL: mantle cell lymphoma. (D) Two second-generation chimeric antigen receptors against CD79b were constructed, utilizing a humanized antibody-derived single-chain variable fragment with a light-heavy (CAR79b (L/H), middle) or heavy-light (CAR79b (H/L), bottom) chain configuration. A second-generation CAR against CD19 was included as a control (CAR19, top). (E) Cytotoxicity of CAR T cells after overnight co-culture with target cells at different effector-target ratios (n=2 healthy donors, mean±SEM shown, **<0.01, ***<0.001, ANOVA). (F) Effector cytokine production measured in collected cell culture supernatants after overnight co-culture of CAR T cells and target cells at a 1:1 ratio. Cytokine analysis was performed using a Luminex array (n=2 healthy donors, mean+SEM shown).

Article Snippet: Cell lines and culture The human cell lines Jeko-1, JVM-2 and Granta-519 (MCL), SuDHL-4 and SuDHL-6 (DLBCL), Raji and Daudi (Burkitt’s lymphoma), and MM.1s (Multiple Myeloma) were obtained from American Type Culture Collection (ATCC) and cultured in accordance with the supplier’s recommendations.

Techniques: Expressing, Flow Cytometry, Derivative Assay, Construct, Control, Co-Culture Assay, Cell Culture, Luminex

Journal: Experimental Hematology & Oncology

Article Title: VISTA drives macrophages towards a pro-tumoral phenotype that promotes cancer cell phagocytosis yet down-regulates T cell responses

doi: 10.1186/s40164-024-00501-x

Figure Lengend Snippet: Ectopic expression of VISTA augmented phagocytosis of cancer cells in two manners. ( A ) Phagocytosis of B cell lymphoma cell lines by THP-1 derived M2c macrophages expressing the VISTA extracellular domain (ECD) or empty vector (EV) cells. (B) Phagocytosis of B cell lymphoma cell lines by THP-1 derived M2c macrophages expressing VISTA or EV cells. (C) Increase of phagocytosis by VISTA and VISTA ECD compared to EV-transduced THP-1 derived M2c macrophages in a panel of DLBCL cell lines. (D) Binding of recombinant (His tagged) human VISTA to B cell lymphoma cell lines determined using anti-His tag antibody staining (APC). (E) Percentage of phagocytosis of SU-DHL-2 (left panel) and Daudi (right panel) by VISTA-, ECD-, or EV-transduced THP-1 derived macrophages. F-G. Percent change in phagocytosis by VISTA- or ECD-transduced THP-1 derived macrophages compared to EV transduced THP-1 derived M0-like, M2-like, and M1-like macrophages in Daudi (F) and SU-DHL-2 (G) cell lines. H-I. Percentage of phagocytosis by VISTA-, ECD-, or EV-transduced CD34 + CB cell derived M2-like macrophages in Daudi (H) and SU-DHL-2 (I) cell lines. n.s., not significant. * P < 0.05, ** P < 0.01, and *** P < 0.001 by student’s t-test

Article Snippet: Human monocytic cell line THP-1, promyeloid cell line HL60 and DLBCL cell lines Daudi, SU-DHL-2, SU-DHL-4, SU-DHL-6, SU-DHL-10, U2932, SC1, OCI-LY3, RI1 were obtained from Deutsche Sammlung from Microorganism und Zellkulturen, (Braunschweig, Germany) or ATCC.

Techniques: Expressing, Derivative Assay, Plasmid Preparation, Binding Assay, Recombinant, Staining

Journal: Experimental Hematology & Oncology

Article Title: VISTA drives macrophages towards a pro-tumoral phenotype that promotes cancer cell phagocytosis yet down-regulates T cell responses

doi: 10.1186/s40164-024-00501-x

Figure Lengend Snippet: VISTA triggered changes of cytokine secretion during phagocytosis. A . Measurement of IL-1β in supernatants from VISTA-, ECD-, or EV-transduced THP-1 derived macrophages. B-C. IL-1β concentration in the supernatant of VISTA-, ECD-, or EV-transduced THP-1 derived macrophages after 3 h phagocytosis experiments with Daudi (B) and SU-DHL-2 (C) cells. D-E. IL-1β (D) or IL-10 (E) concentration in the supernatant of VISTA-, ECD-, or EV-transduced CD34 + CB-derived macrophages after phagocytosis experiment with Daudi cells. F-G. IL-1β (F) and IL-10 (G) concentration in the supernatant of VISTA-, ECD-, or EV-transduced CD34 + CB-derived macrophages after phagocytosis experiment with SU-DHL-2 cells. * P < 0.05, ** P < 0.01, and *** P < 0.001 by one-way ANOVA with post hoc intergroup comparisons using Dunnett’s test

Article Snippet: Human monocytic cell line THP-1, promyeloid cell line HL60 and DLBCL cell lines Daudi, SU-DHL-2, SU-DHL-4, SU-DHL-6, SU-DHL-10, U2932, SC1, OCI-LY3, RI1 were obtained from Deutsche Sammlung from Microorganism und Zellkulturen, (Braunschweig, Germany) or ATCC.

Techniques: Derivative Assay, Concentration Assay