EMBO/soda-nel huggingface dataset:
A preclinical model of peripheral T-cell lymphoma GATA3 reveals DNA damage response pathway vulnerability (2022)
Figure 2

Figure 2. The lymphoma originated from a single positive T-cell clone in a Cγ1-Cre Blimp1fl/fl mouse.

A. 5 x 105 P3 splenocytes were implanted IV into congenic CD45.1 mice and resultant lymphomas that developed were evaluated for expression of B and T-cell lineage markers by flow cytometry. Top plots are of a control C57bl/6J (CD45.2+) spleen. Bottom plots show immunophenotype of splenocytes from a CD45.1 mouse with an expanded CD45.2+ lymphoma. The data is representative of five animals.

B. Flow cytometry analysis of CD4 expression on lymphomas cells from four mice at different passages (P3-P6) . In top panel, CD8-TCRβ+ cells are gated. IHC for CD4 in P3 and P5 spleens. Each IHC image is representative of 3 mice.

C. Flow cytometric TCRvβ repertoire analysis of whole spleens from two lymphoma passages. Each color represents the relative usage of a single TCRvβ on total Thy1+CD3+ T-cells containing a mix of lymphoma cells and host T-cells. The percent of positive stained cells was normalized to the sum of total TCRvβ+ cells. Data is representative of two experiments and all passages.

D. T-cell transcript complementary determining region 3 (CDR3) sequencing of C57bl/6J (top) and lymphoma-bearing spleen. The chord diagram shows numbers of distinct TRBV and TRBJ clones. The dominant CDR3 sequence in the lymphoma is CASGDTQANSDYTF. Note that Trbv13-2 gene corresponds to TCRvβ8.2 protein.

E. CD8-TCRβ8mid T-cell quantification across lymphoid and non-lymphoid tissues. Samples were collected from three lymphoma-bearing mice with low, medium and high tumor burden. Data are compared to that of a lymphoma-free mouse (tumor burden = 'none'). The CD4 and CD8 expression pattern of gated TCRvβ8+ cells is shown in the dot plot at right. Sizes of tumor dissemination was confirmed in three independent experiments.

F, G (F) PCR for Cγ1-Cre and wildtype Cγ1 alleles and (G), wildtype, floxed or excised Blimp1 alleles in DNA from wildtype (wt) control C57bl/6J cells and purified T-cells or CD45.2+ cells from mouse with lymphoma (T1, T2). PCR data were confirmed in at least 3 independent tests. Lad. = ladder.
A preclinical model of peripheral T-cell lymphoma GATA3 reveals DNA damage response pathway vulnerability (2022)
Figure 5

Figure 5. mPTCL has an oncogenic β-catenin mutation and overactive MYC.

A. Allele frequencies of predicted deleterious mutations detected by exome sequencing of mPTCL T-cells (n=4). Deleterious variants have a SIFT (sorting intolerant from tolerant) score ≤0.05. In red are OncoKB-listed oncogenes or tumor suppressors. Each oncogene in red has a SIFT score=0, indicating high likelihood to be deleterious. Bars represent SEM. The dotted line represents the lower allele frequency cutoff for clonal heterozygous variants.

B. IHC for β-catenin in spleens (left) and magnified regions of mesenteric lymph node (right) in a normal C57bl/6J mouse and a mouse with mPTCL. Images are representative of six mice.

C. Ingenuity Pathway Analysis predicted upstream regulators in purified TCRvβ8+ mPTCL vs. CD4+ T-cells (n=6). A selection of upstream regulators is shown. The full set can be found in Dataset EV4. Z-score ≥2 or ≤2 represents predicted activation or inhibition of a regulator, respectively.

D. Log2-transformed expression levels of Myc transcript in TCRvβ8+ vs. wildtype CD4+ T-cells (n=6). Adjusted p=5.78E-08. P-values were calculated by ROSALIND.

E. Immunostain for c-MYC in mPTCL vs. C57bl/6J spleens (representative of three mice).

F. Western blot for c-MYC in whole spleen lysates from control C57bl/6J and mPTCL mice. Quantification of c-MYC expression normalized to vinculin housekeeper gene levels (n=6). T-test p=0.0007. Bars represent SEM.

G. GSEA for the top 12 (of 16) enriched MYC-regulated gene signatures in purified TCRvβ8+ lymphoma cells. Red line indicates FDR q=0.05. FDR Q-values can be found in Dataset EV3. *** p<0.001.
A preclinical model of peripheral T-cell lymphoma GATA3 reveals DNA damage response pathway vulnerability (2022)
Figure 6

Figure 6. Interrogation of potential targets for therapeutic intervention in mPTCL.

A. GSEA of HALLMARK gene signature was performed on whole mPTCL-infiltrated vs. wildtype spleens and purified TCRvβ8+ mPTCL vs. wildtype CD4+ T-cells (n=6). 18 of the top 20 positive enriched signatures that were identified in whole spleen and purified cells are shown. Red circles depict significant enrichment (FDR q-value <0.05). NES: normalized enrichment score. FDR Q-values can be found in Dataset EV5.

B. Western blot for phosphorylated and total ZAP70, AKT, S6 and GSK3β in lysates from three control (C) C57bl/6J and three mPTCL (T) spleens. Murine DLBCL cell line A20 is a positive control for constitutively active AKT signaling.

C, D (C) Kaplan-Meyer curve showing time to welfare endpoint (survival) in mice implanted IV with 104 mPTCL cells and dosed with PI3Kδ inhibitor PI-3065 at 75 mg/kg PO QD (n=10) (log-rank test p>0.9999) or (D), the pan-AKT inhibitor capivasertib (AZD5363) dosed at 130 mg/kg PO BID 4 days on/3 days off (4/3; n=9) vs. vehicle control (n=10) (log-rank test p=0.0498 in favor of the control arm). Results in C and D are representative of three independent studies of PI3K pathway inhibitors.

E. GSEA of REACTOME terms in the comparison of transcriptome from whole spleens and purified cells as in (A). A selection of 18 of the top 35 pathways are shown (all with FDR p-value <0.05). FDR Q-values can be found in Dataset EV5.

F. Heatmap of log2 normalized expression of significantly upregulated genes (p-adjusted <0.05) in 'REACTOME Activation of ATR in response to replication stress' gene signature from panel E.

G. Immunostain for DNA damage marker γH2AX in wildtype and mPTCL spleens. mPTCL data is representative of six mice. Arrowhead indicates positive nuclear foci.

H. Western blot for γH2AX, total H2AX, CHK1 and RPA32 in control C57bl/6J and mPTCL spleens. GAPDH is a loading control. Three representative samples per group are shown in the blot (left) and quantification was performed on n=6 at right. T-test p=0.0798 (CHK1; not statistically significant, ns), p=8.69E-05 (RPA32), p=2.11E-03 (γH2AX). ***p<0.001.
A preclinical model of peripheral T-cell lymphoma GATA3 reveals DNA damage response pathway vulnerability (2022)
Figure 7

Figure 7. Sensitivity of PTCL to ATR pathway inhibition. A. Sanger screen IC50 drug sensitivity data (IC50 values) for DDR inhibitors targeting ATR, CHK1/2 and Wee1 in T-ALL, PTCL (DEL, KARPAS-299, SUP-M2) and pan-cancer cell lines. ANOVA p=0.413 (AZD6738, ATR inhibitor), p=0.750 (AZD772, CHK1/2 inhibitor), p=0.119 (MK-8776, ATR inhibitor) and 0.282 (AZD1775, Wee1 inhibitor). Bars represent SEM of biological replicates.

B. PTCL (red) and T-ALL (yellow) human cell lines were dosed in vitro with AZD6738 for 72h and viable cell numbers assayed by luminescence readout using Cell TiterGlo. Average IC50 values from three experiments are plotted. Bars represent SEM.

C. Representative dose-response curve of AZD6738. PC9 and NCI-H197 lung cancer cell lines are DDR inhibitor sensitive and resistant, respectively, and included as drug sensitivity controls. Dotted line represents 50% normalized luminescence levels.

D. Kaplan-Meyer curve of mice implanted IV with mPTCL splenocytes and dosed with ATR inhibitor ceralasertib (AZD6738) 50 mg/kg PO QD (n=9) or vehicle (n=10). On day 38, mice on AZD6738 therapy were randomized to discontinue therapy (n=4, group A) or continue therapy for a further 14 days (n=5, group B). The dosing period is indicated by grey arrows. Log-rank test p=0.0004 (ATR group A, stop day 38) and p=0.0001 (ATR, stop day 52) vs. the vehicle control arm. Group A vs. B: p=0.0040. Median survival times: days 23 (vehicle), 47 (A) and 54 (B).

E. Flow cytometry dot plots of TCRvβ8mid ICOShi infiltrating TCRβ+ T-cells from spleens. Mice were implanted IV with mPTCL splenocytes and administered intermittent AZD6738 (50 mg/kg PO QD) for 71 days-post implant (see Figure S6D). Tumor burden representative of 6 out of 8 mice on prolonged ATR inhibition therapy (day 71 post-implant) is compared to endpoint spleens from a vehicle-treated mouse and an ATR-treated mouse that relapsed off-therapy. A wildtype C57bl/6J spleen is shown in blue. Percentages of total viability marker-negative cells are gated.

F. Western blot for γH2AX relative to total H2AX levels of spleen lysates from mice dosed for 71 days with AZD6738 (ATR; n=4) relative to untreated mPTCL spleens taken at endpoint (n=6). T-test p=2.70E-06. PC9 cells dosed in vitro 24h with 1 µM aphidicolin (Aph; an inhibitor of DNA replication) or with AZD6738 (ATR) are positive controls for DNA damage. Bars represent SEM. ***p<0.001

Expanded View Figures
Innate, translation-dependent silencing of an invasive transposon in Arabidopsis (2021)
Figure 1

Figure 1. EVD shGAG is both a trigger and a target of RDR6-dependent but miRNA-independent siRNAs.

(A) EVD flGAG-POL and spliced shGAG mRNAs are distinguishable using specific PCR primer sets (arrows) for quantification and northern analysis. (35S) Cauliflower Mosaic Virus 35S promoter, (Pr) protease, (IN) integrase, (RT-RNase) reverse-transcriptase-RNase; red squares: stop codons.

(B) sRNA-seq reads profile of EVD expressed from 35S:EVDWT in WT (black) or rdr6 (red). (RPM) reads per million. Positions are indicated in nucleotides (nt) from the start of the 35S sequence. Dashed vertical lines: shGAG and GAG-POL 3' ends.

(C) Northern analysis of EVD RNA isoforms using a probe for the GAG region or for ACTIN2 (ACT2) as a loading control.

(D) qPCR quantification of shGAG and flGAG-POL normalized to ACT2 and to GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C SUBUNIT (GAPC) levels. qPCR was performed on n=3 biological replicates; bars: standard error. (**) = p-value < 0.01 (two-sided t-test between indicated values).

(E) Western analysis of GAG and RDR6 with Coomassie (coom.) staining as a loading control. Arrow indicates cognate RDR6 protein band.

(F-G) sRNA-seq profiles from EVD de-repressed in the ddm1 (F) or ddm1 dcl1 (G) backgrounds. Different siRNA size categories are stacked. Nomenclature as in (B).
Innate, translation-dependent silencing of an invasive transposon in Arabidopsis (2021)
Figure 2

Figure 2. The EVD intron and terminator suffice to initiate PTGS.

(A) The 35S:GFP-EVDint/ter-GUS fusion was made by introducing the EVD intron and proximal shGAG terminator (including the premature cleavage and polyadenylation site; PCAP) between the GFP and GUS coding sequence. Like EVD, it spawns full length unspliced and short spliced mRNAs. Red squares: stop codons.

(B) Expression levels of shGFP (spliced) and GFP-EVDint/ter-GUS (unspliced) transcripts, relative to ACT2 and AT4G26410 (RHIP1), in the WT or rdr6 background. qPCR was performed on three biological replicates and error bars represent the standard error on. (*) = p-value < 0.05 (two-sided t-test against corresponding controls).

(C) sRNA-seq profile mapped on the genomic 35S:GFP-EVDint/ter-GUS locus. (RPM) Reads per million. Positions indicated in nucleotides (nt) from the start of the 35S sequence. Dashed vertical lines: shGFP and GFP-GUS 3' ends.

(D) Low molecular weight RNA analysis of the GFP- and GUS-spanning regions. tasiRNA255 is a control for the rdr6 mutation and miR159 provides a loading control.
Innate, translation-dependent silencing of an invasive transposon in Arabidopsis (2021)
Figure 3

Figure 3. Impact of splicing and premature termination on EVD silencing.

(A-C) Constructs and isoforms transcribed from 35S:EVDwt (A), 35S:EVDΔintron (B) and 35S:EVDmU1 (C). Probes for northern analysis of GAG exon 1 (GAG-ex1), intron (GAG-in), exon2 (GAG-ex2) and the POL region are depicted with black lines. Red squares: stop codons.

(D) Relative expression levels of spliced and unspliced transcripts in the three EVD constructs relative to ACT2. qPCR was performed on three biological replicates and error bars represent the standard error. (ns.) = non-significant, (*) = p-value < 0.05, (**) = p-value < 0.01, (two-sided t-test between indicated samples/targets).

(E) High and low molecular-weight RNA analysis of EVD GAG (GAG-ex1) and EVD intron (GAG-in) in two independent T1 bulks from each indicated line. The filled arrows on the right hand-side or with an asterisk on the blots correspond to the transcripts depicted in (A-C). ACT2: loading control for mRNAs; tasiR255, miR173 and U6: loading controls for sRNAs. Hybridizations for GAG-ex2 and POL probes are found in Supp. Fig.5A.

(F) Western blot analysis of the GAG protein with Coomassie (coom.) staining as a loading control.
Innate, translation-dependent silencing of an invasive transposon in Arabidopsis (2021)
Figure 5

Figure 5. Splicing promotes translation and siRNA biogenesis from short-spliced mRNAs by influencing nucleocytoplasmic distribution of RNA isoforms. (A) Comparison of RNA isoforms and sRNA patterns generated by 35S:GFP-GUS and 35S:GFP-EVDint/ter-GUS. High and low molecular-weight RNA analysis using a GFP or GUS probe in two independent transgenic lines from each construct in the WT or rdr6 background. mRNA isoforms are indicated with arrows and correspond to the transcripts depicted in Fig.2A. EtBr staining of the agarose gel and miR171 probe serve as loading control for mRNAs and sRNAs, respectively.

(B). Western blot analysis of the translation products from GFP and GFP-GUS transcripts. Coomassie (coom.) staining as a loading control. Black arrow: GFP-GUS fusion protein; white arrow: GFP protein.

(C) Nucleo-cytosolic distribution of 35S:EVD and 35S:GFP-EVDint/ter-GUS RNA isoforms in rdr6 relative to that of ACT2 analyzed by qPCR. RNA extracted from Total, nuclear (Nucl) and cytoplasmic (Cyto) fractions was reverse transcribed with random hexamers and oligo(dT). snoRNA U5 is shown as a nuclear-only RNA control.

(D) Same as in (C) but using exclusively oligo(dT) to reverse transcribe poly(A)+ RNAs.

Data information: Both in (C) and (D), qPCR was performed on n=3 biological replicates; bars: standard error. (*) = p-value < 0.05, (**) = p-value < 0.01 (two-sided t-test between indicated samples).
Innate, translation-dependent silencing of an invasive transposon in Arabidopsis (2021)
Figure 6

Figure 6. Intense, discrete ribosome stalling on shGAG correlates with RDR6-dependent siRNA accumulation.

(A) EVD, ACT2 and TAS1c capped and uncapped 5'ends from nanoPARE and Smart-seq2 libraries along 20-21 nt siRNA in WT and ddm1.

(B) RIBO-seq coverage profiles from 35S:EVD in WT or rdr6. RPM: Reads per million. (C) Ribosomal footprints on shGAG in rdr6 displaying codon occupancy at P-sites to calculate codon coverage. The coverage observed at each codon position was divided by the expected mean coverage along the entire GAG coding sequence.

(D) Maximal individual codon coverage over the expected coverage for all translated transcripts of Arabidopsis. Vertical lines indicate the strength of stalling sites of shGAG in the WT or rdr6 background. Percentages specify the proportion of transcripts with more pronounced stalling events than shGAG.

(E) Overlay between 35S:EVD siRNAs (red) and RIBO-seq profiles (black) in the WT background.

(F) Schematic representation of putative ribosome stalling-linked mRNA breakage generating 5'OH ends. Lack of 5'PO4 prevents XRN 5'->3' exonucleolytic activity (see Fig.6A), granting the RNA to be used as template by RDR6.

(G) Overlap between ribosomal footprints and mapping of 5'OH ends from 35S:EVD in rdr6 cloned through RtbC ligation. Regions investigated are highlighted in grey. 5'OH ends were only successfully cloned from region #1. Alignment of sequenced clones to EVD is displayed in Appendix Fig. S9.
Loss of AhR potentiates FoxM1 signaling to enhance self-renewal of colonic stem/progenitor cells (2020)
Figure 1

Figure 1. AhR signaling modulates the percentage of stem cells in colonic crypts.

(A-B) Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (A) Induced Cyp1a1 mRNA expression in response to different treatments (n=3 per group). (B) Assessment of GFP+ stem cell percentages (n=3 replicates per treatment). Bars with different letters are significantly different (p<0.05).

(C) Scheme of the generation of transgenic Lgr5-GFPCreERT2 X Tomatof/f mice. eGFP and CreERT2 expression are controlled by the intestinal stem cell specific marker Lgr5 promoter, and the tdTomato reporter cassette is knocked into the Rosa26 locus. A floxed STOP cassette is upstream of the tdTomato gene, thus preventing tdTomato expression in the absence of tamoxifen. Upon tamoxifen exposure, CreERT2 recombinase will be translocated into the nucleus, and the STOP sequence between the loxP sites is excised and the tdTomato reporter is permanently and constitutively expressed in intestinal stem cells and their progeny.

(D) Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type (Lgr5-GFPCreERT2 X Tomatof/f, GT) and AhR knock out (AhRf/f X Lgr5-GFPCreERT2 X Tomatof/f, HGT) mice. Top left panel shows representative cells from WT negative control animal (no GFP or Tomato expression). Top right panel shows Tomato only control (CDX2P-CreERT2 X Tomatof/f). Middle panels represent DMSO or TCDD treatment in AhR WT mice, respectively. Mice were gavaged every other day, 4 times total, with vehicle or TCDD (25 µg/kg bw) and terminated 1 d later. Bottom left panel is from a representative AhR WT mouse and bottom right panel is from a representative AhR KO mouse.

(E) Induced Cyp1a1 mRNA expression in stem cells after oral gavage with TCDD (n=4).

(F) Percentage of Lgr5+ stem cells (n=4 mice per group) in (D).

(G) Quantitative analysis of the percentage of Lgr5+ stem cells in (D), n=7-10 mice per group.

(H-I). Representative immunohistochemistry and violin plots of GFP+ stem cells per crypt, n=60~70 crypts from n=3 mice per group. Scale 50 µm. Solid line indicates median, and dotted lines indicate 25th/75th quartiles.

Data information: Data were shown as mean ± SD in (A)(B) and (E-G). One-way ANOVA with Turkey test was determined in (A) and (B). Two-tailed Student's t-test was determined in (E-G) and (I).
Loss of AhR potentiates FoxM1 signaling to enhance self-renewal of colonic stem/progenitor cells (2020)
Figure 2

Figure 2. Functional characterization of AhR signaling in mouse colonic stem and progenitor cells.

(A) Representative flow cytometry gating and fluorescence microscopy images of sorted GFPhigh stem cells and GFPlow progenitor cells from Lgr5-EGFP-IRES-CreERT2 mice.

(B) Representative brightfield images of organoids generated from GFPhigh stem cells isolated from tamoxifen treated AhR WT mice (Lgr5-GFPCreERT2 X CDX2P-CreERT2 [GC]) and KO mice (AhRf/f X Lgr5-GFPCreERT2 X CDX2P-CreERT2 [HGC]), treated with DMSO (control) or TCDD for 5 d. Data are from ≥4 mice, scale bar 200 µm.

(C, D) The organoid forming efficiency (C) and diameter (D) from sorted GFPhigh stem cells in AhR WT or KO mice treated for 5 d with DMSO or TCDD (10 nM). n=4-5 mice per group in (C). Each dot represents an individual organoid and grey line indicates mean in (D). Bars with different letters are significantly different (p<0.05).

(E-G) Organoid growth from GFPlow progenitor cells. (E) Representative brightfield images of organoids. (F) Organoid forming efficiency and (G) diameter. n=4-8 mice per group in (F). Each dot represents an individual organoid and grey line indicates mean in (G). Scale bar 200 μm. Bars with different letters are significantly different (p<0.05).

(H, I) (H) Lgr5 expression in organoids and (I) sorted stem cells in the absence or presence of TCDD treatment (n≥3 independent samples per group).

(J) Representative brightfield images for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d. Scale 200 µm.

Data information: Data were shown as mean ± SD in (C)(F)(H) and (I). A 2-way ANOVA with Tukey's multiple comparisons test was carried out in (D)(G)(H) and (I). Paired student t-test was performed within genotype in (C) and (F).
Loss of AhR potentiates FoxM1 signaling to enhance self-renewal of colonic stem/progenitor cells (2020)
Figure 3

Figure 3. FoxM1 pathway affected by AhR in mouse colonic stem and progenitor cells.

(A) Venn diagrams of differentially expressed genes following comparison of AhR KO (KO/WT) and TCDD effects (TCDD/vehicle in WT mice) in sorted stem and progenitor cells; KO (HGC mice); WT (GC mice). An FDR<0.1 cutoff was used.

(B) Heatmap comparison of the top 25 enriched Diseases and Biological Functions in AhR KO (KO/WT) and TCDD (TCDD/vehicle in WT mice) treated cells as identified by IPA.

(C) Heatmap comparison of the top 25 upstream regulators between AhR KO (KO/WT) and TCDD (TCDD/vehicle in WT mice) treated cells.

(D-G) Gene Set Enrichment Analysis (GSEA) was performed in sorted stem and progenitor cells from AhR WT (n=3) and KO (n=4) mice (D&E) and in WT mice in response to vehicle and TCDD treatment (F&G). Normalized enrichment scores (NES) and FDRs were calculated using the Public Interaction Database (PID) for the FoxM1 Pathway gene set.
Loss of AhR potentiates FoxM1 signaling to enhance self-renewal of colonic stem/progenitor cells (2020)
Figure 4

Figure 4. FoxM1 is a direct target of AhR.

(A) FoxM1, Plk1, and Cdc25B mRNA expression in stem and progenitor cells with and without AhR, n=4 mice per group.

(B) FoxM1, Plk1, and Cdc25B mRNA expression in WT stem and progenitor cells with and without TCDD, n=3-4 mice per group.

(C) Schematic representation of the FoxM1 promoter region containing a putative dioxin response element.

(D-E) Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT colonic organoids (D) or WT and KO crypts (E) treated with DMSO or TCDD, n=3 or 4 per group. A DNA fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control.

(F) FoxM1 luciferase activity determination for young adult mouse colonocytes (YAMCs) treated with DMSO or 10 nM TCDD for 1 d, n=3 per group.

(G, H) Expression of FoxM1 and PLK1 at mRNA and protein level, respectively, n>=3 independent organoids per treatment.

(I) Representative brightfield images of mouse colonic organoids generated from WT Lgr5+-GFPhi stem cells treated with DMSO or 5µM FDI-6. Scale bar 200 µm.

(J, K) Quantification of organoid forming efficiency (J) and organoid area (K) from WT colonic stem cells treated with DMSO or 5µM FDI-6. Data are represented as mean ± SEM in (K).

(L) Organoid expression of Lgr5 in response to FDI-6. Lgr5 expression level was dramatically suppressed by FDI-6 in a dose-dependent manner, n>=3 per treatment.

(M) Representative brightfield images for secondary organoid formation following treatment with DMSO or 5 µM FDI-6 for 2 d. Scale 200 µm.

Data information: Data were shown as mean ± SD in (A)(B)(D-F)(J) and (L) or mean ± SEM in (K). Two-tailed Student's t-test was performed in (A)(B)(D) and (F). A 2-way ANOVA with Tukey's multiple comparisons test was carried out in (E)(J)(K) and (L). Paired student t-test was performed within genotype in (G).