Journal: bioRxiv

Article Title: Targeting Monoallelic CREBBP / EP300 Mutations in Germinal Center-Derived B-Cell Lymphoma with a First-in-Class Histone Acetyltransferase Activator

doi: 10.1101/2025.03.13.642871

Figure Lengend Snippet: a , Half-maximal inhibitory concentrations (IC 50 ) of novel histone acetyltransferase (HAT)-activating compounds in B-cell lymphoma (BCL) cell lines harboring various CREBBP / EP300 mutations. Data are presented as mean IC 50 ( n = 3). b , Mean IC 50 of each HAT-activating compound across wild-type (WT), CREBBP mut , and EP300 mut BCL cell lines. Cytotoxicity is compared using the WT IC 50 /mutated IC 50 ratio, with a larger value indicating greater selective cytotoxicity. c , Chemical structure of YF2, the compound that induces the greatest selective cytotoxicity in both CREBBP - and EP300 -mutated cell lines compared to WT cells. d , Cell viability following 6-day exposure to HAT activators, either CTPB (black) or YF2 (red), in BCL cell lines. Cell viability is calculated as a percentage of respective vehicle-treated controls and presented as means ± standard error of the mean (s.e.m.). Treatment conditions from the same cell line are compared using a linear regression analysis ( n = 3). e , Cytotoxicity of peripheral blood mononuclear cells (PBMCs) from healthy donors or patients diagnosed with leukemia/lymphoma treated with YF2 for six days in vitro . Cell viability is expressed as a percentage of the total cell population and presented as means ± s.e.m. All leukemia/lymphoma subtypes are compared to healthy donors using unpaired two-sided t-tests ( n = 2–5). PTCL, peripheral T-cell lymphoma; MCL, mantle cell lymphoma; FL, follicular lymphoma; CLL, chronic lymphocytic leukemia; MZL, marginal zone lymphoma; DLBCL, diffuse large B-cell lymphoma; ATLL, adult T-cell leukemia/lymphoma. f , YF2 IC 50 in T-cell lymphoma (TCL) and BCL cell lines harboring various CREBBP / EP300 mutations. Data are presented as means ± s.e.m ( n = 2–5). g , Combined YF2 IC 50 across the cell types shown in . Data are presented as means and box plots display the first, second, and third quartiles. Mean IC 50 of each cell type are compared using a one-way analysis of variance (ANOVA) followed by a Tukey’s Honestly Significant Difference (HSD) post-hoc test ( n = 5–7). h , Transient knockout of p300 in CREBBP / EP300 WT OCI-Ly7 cells increased sensitivity to YF2 as measured through flow cytometry. Drug resistance returned when the cells regained baseline p300 expression ( n = 1). i , Apoptosis and cell death in BCL cell lines following 6-day YF2 treatment. Data are presented as means ± s.e.m. Treatment conditions from the same cell line are compared using a Kruskal-Wallis test followed by a Dunn’s post-hoc test ( n = 3). j , Parental and histone deacetylase inhibitor (HDACi)-resistant cell lines treated with increasing concentrations of YF2 over six days and assessed for changes in cell viability. SU-DHL-6 cells possess both CREBBP and EP300 mutations, whereas OCI-Ly7 cells are CREBBP / EP300 WT. Cell viability is calculated as a percentage of respective vehicle-treated controls and is presented as means ± s.e.m. YF2 IC 50 of parental and HDACi-resistant cells are compared using a Mann-Whitney U test ( n = 3). Asterisks (*) denote statistical significance: * = P < 0.05, ** = P < 0.01, and *** = P < 0.001. n.s. = not significant.

Article Snippet: For all subsequent experiments, YF2 was purchased commercially from MedChemExpress (#HY-16531A).

Techniques: In Vitro, Knock-Out, Flow Cytometry, Expressing, Histone Deacetylase Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Targeting Monoallelic CREBBP / EP300 Mutations in Germinal Center-Derived B-Cell Lymphoma with a First-in-Class Histone Acetyltransferase Activator

doi: 10.1101/2025.03.13.642871

Figure Lengend Snippet: a , Ribbon diagram of p300 depicting predicted YF2 binding sites including the bromo (circled in yellow), RING (blue), and HAT (red) domains. The p300 protein is colored dark blue and the predicted binding sites are highlighted in warmer colors. b , Ribbon and surface diagram of the p300 RING domain showing predicted YF2 interactions (in cyan and circled in blue). c , Schematic representation of the domain architecture of CREBBP/p300 and proposed YF2 binding to CREBBP as assessed through thermal shift assays. The catalytic domain comprises the bromodomain (Bd; yellow), RING domain (blue), plant homeodomain (PHD; gray), and HAT domain (red). JQ1 is a known bromodomain inhibitor used as a positive control. Data are presented as mean protein melting temperatures (T m ) and box plots display the first, second, and third quartiles. Treatment conditions are compared using a one-way ANOVA followed by a Tukey’s HSD post-hoc test ( n = 3–4). d , Proposed mechanism of YF2 binding and HAT activation. YF2 binding to the bromo/RING domain causes a conformational change that reveals the previously blocked substrate-binding pocket and lysine-rich autoregulatory loop (AL), promoting CREBBP/p300 auto-acetylation that increases HAT activity. Created with BioRender.com . e , Auto-acetylation of CREBBP and p300 incubated with 50 nM YF2 for 1, 5, or 10 min and visualized via immunoblotting ( n = 3–6). f , Quantification of CREBBP auto-acetylation shown in . Data are calculated as normalized fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treated and control samples from the same time point are compared using a Mann-Whitney U test ( n = 5–6). g , Quantification of p300 auto-acetylation shown in . Data are calculated as normalized fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treated and control samples from the same time point are compared using a Mann-Whitney U test ( n = 3–4). h , Cell-free acetylation of histone H3 following exposure to increasing concentrations of YF2 over 30 min ( n = 3). i , Quantification of histone H3 cell-free acetylation shown in . Model fitting to calculate the half maximal effective concentration (EC 50 ) is performed using the drc package ( n = 3). j , Histone H3K14 and H3K27 acetylation in SU-DHL-6 cells treated with 6 µM YF2 for two days as measured by mass spectrometry. Data are calculated as fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treated and control samples from the same histone target are compared using an unpaired two-sided t-test ( n = 3). k , Protein histone acetylation of BCL cell lines and PBMCs isolated from a patient with DLBCL treated with YF2 in vitro over three days and assessed via immunoblotting. Data are calculated as normalized fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treatment conditions from the same cell line are compared using a Kruskal-Wallis test followed by a Dunn’s post-hoc test ( n = 1–3). Asterisks (*) denote statistical significance: * = P < 0.05, ** = P < 0.01, and *** = P < 0.001. n.s. = not significant.

Article Snippet: For all subsequent experiments, YF2 was purchased commercially from MedChemExpress (#HY-16531A).

Techniques: Binding Assay, Positive Control, Activation Assay, Activity Assay, Incubation, Western Blot, Control, MANN-WHITNEY, Concentration Assay, Mass Spectrometry, Isolation, In Vitro

Journal: bioRxiv

Article Title: Targeting Monoallelic CREBBP / EP300 Mutations in Germinal Center-Derived B-Cell Lymphoma with a First-in-Class Histone Acetyltransferase Activator

doi: 10.1101/2025.03.13.642871

Figure Lengend Snippet: a , Cell-free acetylation of p53 in the presence of CREBBP and increasing concentrations of YF2 over 30 min ( n = 3). b , Cell-free acetylation of p53 in the presence of p300 and increasing concentrations of YF2 over 30 min ( n = 3). c , Activity of YF2-induced p53 acetylation in the presence of either CREBBP or p300 shown in and , respectively. Data are calculated as percentage change from respective vehicle-treated controls and presented as means ± s.e.m. CREBBP and p300 activity are compared using a linear regression analysis ( n = 3). d , Protein expression of BCL cell lines treated with YF2 for 24 h as evaluated via immunoblotting. Data are calculated as normalized fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treatment conditions from the same cell line are compared using a Kruskal-Wallis test followed by a Dunn’s post-hoc test ( n = 3). e , Differential expression of p53-associated genes in three BCL cell lines treated with 10 µM YF2 for 24 h as assessed by a p53 signaling array. Data are calculated as fold changes relative to respective vehicle-treated controls. Fold changes greater than 1.5 are highlighted in red ( n = 3). f , Enrichment of p21 expression following treatment with 10 µM YF2 for 24 h as measured through chromatin immunoprecipitation. Data are calculated as fold enrichment relative to respective IgG controls and presented as means ± s.e.m. Treated and control samples from the same cell line are compared using an unpaired two-sided t-test ( n = 3). g , Cell-free acetylation of BCL6 in the presence of p300 and increasing concentrations of YF2 over 30 min ( n = 3). h , Activity of YF2-induced BCL6 acetylation shown in . Data are calculated as percentage change from respective vehicle-treated controls and presented as means ± s.e.m ( n = 3). Asterisks (*) denote statistical significance: * = P < 0.05, ** = P < 0.01, and *** = P < 0.001.

Article Snippet: For all subsequent experiments, YF2 was purchased commercially from MedChemExpress (#HY-16531A).

Techniques: Activity Assay, Expressing, Western Blot, Chromatin Immunoprecipitation, Control

Journal: bioRxiv

Article Title: Targeting Monoallelic CREBBP / EP300 Mutations in Germinal Center-Derived B-Cell Lymphoma with a First-in-Class Histone Acetyltransferase Activator

doi: 10.1101/2025.03.13.642871

Figure Lengend Snippet: a , Schematic depicting CREBBP / EP300 double-mutated SU-DHL-6 cells treated with 12 µM YF2 for 7 days, after which samples are collected for subsequent analyses. Created with BioRender.com . b , Principal component analysis plot depicting variance between YF2-treated and control samples ( n = 3–5). c , Heat map depicting YF2-induced differential expression of genes contributing to the EZB (EZH2 mutations and BCL2 translocations) DLBCL subtype identified by Wright et al. d , Heat map depicting YF2-induced differential expression of gene-expression signatures contributing to the EZB DLBCL subtype identified by Schmitz et al. e , Gene ontology (GO) over-representation analysis on significantly differentially expressed genes following YF2 treatment. Select significant (adjusted P value < 0.05) pathways are shown. f , Kyoto Encyclopedia of Genes and Genomes (KEGG) over-representation analysis on significantly differentially expressed genes following YF2 treatment. Select significant (adjusted P value < 0.05) pathways are shown. g , Gene set enrichment analyses (GSEA) of various antigen processing and presentation pathways following YF2 exposure. h , Differential expression of various antigen presentation genes following YF2 treatment. i , UpSet plot depicting human gene-disease association enrichment analysis on significantly differentially expressed genes following YF2 treatment.

Article Snippet: For all subsequent experiments, YF2 was purchased commercially from MedChemExpress (#HY-16531A).

Techniques: Control, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Targeting Monoallelic CREBBP / EP300 Mutations in Germinal Center-Derived B-Cell Lymphoma with a First-in-Class Histone Acetyltransferase Activator

doi: 10.1101/2025.03.13.642871

Figure Lengend Snippet: a , Schematic depicting mouse serum and tumor collection following YF2 treatment: SCID/beige mice are engrafted with CREBBP / EP300 double-mutated SU-DHL-6 lymphoma cells and treated with 40 or 60 mg/kg YF2 for 5 days, after which serum and tumor samples are collected over 24 h for subsequent analyses. Created with BioRender.com . b , Differential YF2 serum concentrations over 24 h following treatment. Data are presented as means ± s.e.m. Treatment conditions of the same time point are compared using an unpaired two-sided t-test ( n = 3). c , Differential YF2 tumor concentrations over 24 h following treatment. Data are presented as means ± s.e.m. Treatment conditions of the same time point are compared using an unpaired two-sided t-test ( n = 2–3). d , YF2 maximum plasma concentration (C max ) and time to maximum plasma concentration (T max ) in the murine samples from and . e , Differential tumor volumes in engrafted mice following treatment. Data are presented as means ± s.e.m. Treated and control samples are compared using a linear regression analysis ( n = 4–8). f , Histone acetylation in murine tumors treated with 40 mg/kg YF2 for five days as measured by mass spectrometry. Data are calculated as fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treated and control samples of the same histone target were compared using an unpaired two-sided t-test ( n = 3). g , Schematic depicting SU-DHL-6-engrafted mouse model: SCID/beige mice are engrafted with SU-DHL-6 lymphoma cells and treated with 40 mg/kg YF2 or vehicle over 28 days. Created with BioRender.com . h , Differential tumor volumes in SU-DHL-6-engrafted mice following treatment. Data are presented as individual values. Treated and control samples are compared using a linear regression analysis ( n = 6–7). i , Survival rates of SU-DHL-6-engrafted mice following treatment. Data are presented as the overall survival rate on a given day following treatment. Survival rates for each treatment are compared using a log-rank test ( n = 7). j , Schematic depicting patient-derived xenograft (PDX) mouse model: SCID/beige mice are engrafted with a CREBBP / EP300 double-mutated patient-derived DLBCL cell line and treated with 40 mg/kg YF2 or vehicle over 56 days. Created with BioRender.com . h , Differential tumor volumes in PDX mice following treatment. Data are presented as individual values. Treated and control samples are compared using a linear regression analysis ( n = 12). i , Survival rates of PDX mice following treatment. Data are presented as the overall survival rate on a given day following treatment. Survival rates for each treatment are compared using a log-rank test ( n = 12). Asterisks (*) denote statistical significance: * = P < 0.05, ** = P < 0.01, and *** = P < 0.001.

Article Snippet: For all subsequent experiments, YF2 was purchased commercially from MedChemExpress (#HY-16531A).

Techniques: Concentration Assay, Control, Mass Spectrometry, Derivative Assay

Journal: bioRxiv

Article Title: Targeting Monoallelic CREBBP / EP300 Mutations in Germinal Center-Derived B-Cell Lymphoma with a First-in-Class Histone Acetyltransferase Activator

doi: 10.1101/2025.03.13.642871

Figure Lengend Snippet: a , Differential expression of antigen presentation genes in SU-DHL-6 cells treated with 10 µM YF2 for 24 h and assessed by a gene expression array. Data are calculated as fold changes relative to respective vehicle-treated controls. Genes with a log 2 (fold change) > 0.5 and adjusted P value < 0.05 are highlighted in red ( n = 3). b , Cell surface expression of major histocompatibility complex class I (MHC-I) in BCL cell lines exposed to YF2 over six days as measured via flow cytometry. Data are calculated as fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treatment conditions from the same cell line are compared using a Kruskal-Wallis test followed by a Dunn’s post-hoc test ( n = 3). c , Cell surface expression of MHC class II (MHC-II) in BCL cell lines exposed to YF2 over six days as measured via flow cytometry. Data are calculated as fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treatment conditions from the same cell line are compared using a Kruskal-Wallis test followed by a Dunn’s post-hoc test ( n = 3). d , Protein expression of BCL cell lines treated with 15 µM YF2 for six days as evaluated via immunoblotting ( n = 3). B2M, beta-2-microglobulin. e , Enrichment of HLA-A and HLA-C expression following treatment with 10 µM YF2 for 24 h as measured via chromatin immunoprecipitation. Data are calculated as fold enrichment relative to respective IgG controls and presented as means ± s.e.m. Treated and control samples from the same cell line are compared using an unpaired two-sided t-test ( n = 3). f , Schematic depicting BALB/c mice treated with 50 mg/kg YF2 for 14 days, after which blood and spleen samples are collected for subsequent analyses. Created with BioRender.com . g , Cell-surface expression of MHC-I and MHC-II in murine blood B cells following treatment as assessed via flow cytometry. Data are measured as mean fluorescence intensity (MFI) and presented as means ± s.e.m. Treated and control samples are compared using unpaired two-sided t-tests ( n = 9–10). h , Differential cellular differentiation in murine splenic B cells following treatment as assessed via flow cytometry. Data are measured as a percentage of total B cells and presented as means ± s.e.m. Treated and control samples of the same molecular targets are compared using unpaired two-sided t-tests ( n = 11–12). i , Variable dark and light zone populations in murine splenic B cells following treatment as assessed via flow cytometry. Data are calculated as a percentage of total germinal center (GC) B cells and presented as means ± s.e.m. Treated and control samples of the same molecular targets are compared using unpaired two-sided t-tests ( n = 11–12). j , Total B cell counts in YF2-treated and control murine spleens. Data are measured as B cell counts and presented as means ± s.e.m. Treated and control samples were compared using an unpaired two-sided t-test ( n = 10–11). k , Plasma cell populations in murine spleens following treatment as assessed via flow cytometry. Data are calculated as a percentage of the total cell population and presented as means ± s.e.m. Treated and control samples are compared using an unpaired two-sided t-test ( n = 3). l , Spatial transcriptomic profiling in murine spleens following treatment. Spatial feature expression is normalized across all treatments for each target ( n = 1). Asterisks (*) denote statistical significance: * = P < 0.05, ** = P < 0.01, and *** = P < 0.001.

Article Snippet: For all subsequent experiments, YF2 was purchased commercially from MedChemExpress (#HY-16531A).

Techniques: Expressing, Gene Expression, Flow Cytometry, Western Blot, Chromatin Immunoprecipitation, Control, Fluorescence, Cell Differentiation

Journal: bioRxiv

Article Title: Targeting Monoallelic CREBBP / EP300 Mutations in Germinal Center-Derived B-Cell Lymphoma with a First-in-Class Histone Acetyltransferase Activator

doi: 10.1101/2025.03.13.642871

Figure Lengend Snippet: a , Schematic depicting mouse combination treatment study: BALB/c mice are engrafted with murine CREBBP / EP300 -mutated A20 lymphoma cells, treated with 50 mg/kg YF2 and 200 µg/mouse anti-PD-L1 antibody, and monitored for changes in mouse weight, tumor volume, and overall survival. Select mice are euthanized and their tumors collected on day 14. Created with BioRender.com . b , Weight changes in mice following combination treatment. Data are presented as means ± s.e.m. Treatment conditions are compared using a linear regression analysis ( n = 9–10). c , Differential tumor volumes in mice following combination treatment. Data are presented as individual values. Treatment conditions are compared using a linear regression analysis ( n = 9–10). d , Survival rates of mice following combination treatment. Data are presented as the overall survival rate on a given day following treatment. Survival rates for each treatment are compared using a log-rank test ( n = 9–10). e , Variable expression of immunogenic markers in murine tumors following combination treatment as assessed via flow cytometry. Data are measured as MFI and presented as means ± s.e.m. Treatment conditions of the same marker are compared using a Kruskal-Wallis test followed by a Dunn’s post-hoc test ( n = 3–5). f , Spatial transcriptomic profiling of immunogenic markers in murine tumors following combination treatment. Spatial feature expression is normalized across all treatments for each target ( n = 1). g , Spatial organization of cell types, denoted as “topics,” in the YF2-treated tumor sample. The number of designated cell types is optimized using the latent Dirichlet allocation model. h , Spatial expression of topic 3, characterized as putative T cells, showing T-cell infiltration in the YF2-treated tumor sample. i , GSEA of the immune response pathway using topic 3 gene expression. Asterisks (*) denote statistical significance: * = P < 0.05, ** = P < 0.01, and *** = P < 0.001. n.s. = not significant.

Article Snippet: For all subsequent experiments, YF2 was purchased commercially from MedChemExpress (#HY-16531A).

Techniques: Expressing, Flow Cytometry, Marker, Gene Expression

Journal: Cell

Article Title: Quorum-sensing- and type VI secretion-mediated spatiotemporal cell death drives genetic diversity in Vibrio cholera

doi: 10.1016/j.cell.2022.09.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Saccharomyces cerevisiae ;YF2 , Belden Lab Collection , AAM-25.

Techniques: Virus, Variant Assay, Recombinant, Plasmid Preparation, Gene Expression, Over Expression, Luciferase, Activity Assay, Software