Journal: Nature Communications

Article Title: Targeting dependency on a paralog pair of CBP/p300 against de-repression of KREMEN2 in SMARCB1-deficient cancers

doi: 10.1038/s41467-024-49063-w

Figure Lengend Snippet: a Immunoblot analysis of SMARCB1 and β-actin expression in JMU-RTK-2 + SMARCB1 and JMU-RTK-2 -SMARCB1 cells. The experiments were repeated twice independently with similar results. b Heatmap showing the viability of HEK293T, JMU-RTK-2 + SMARCB1, and JMU-RTK-2 -SMARCB1 cells transfected with siRNAs targeting 30 paralog pairs. Cells were transfected for 48 h with the indicated siRNAs. The cells were then reseeded and transfected repeatedly with the indicated siRNAs for 48 h. The cells were then reseeded and incubated for 7 days. Cell viability is shown as a heatmap. c Heatmap showing the viability of SMARCB1-proficient (786-O and H460) and SMARCB1-deficient (G402 and HS-ES-2R) cell lines transfected with the indicated paralog pairs of siRNAs. Cells were transfected for 48 h with the indicated siRNAs. The cells were then reseeded and transfected repeatedly with the indicated siRNAs for 48 h. The cells were then reseeded and incubated for 7 days. Cell viability is shown as a heatmap. d Immunoblot analysis of SMARCB1, CBP, p300, β-actin, and histone H3 and H3K27ac expression in SMARCB1-proficient and SMARCB1-deficient cell lines. e Viability of SMARCB1-proficient (786-O, VMRC-RCZ, 786-O, ES2, H460, and H2228) and SMARCB1-deficient (NEPS, G402, JMU-RKT-2, HS-ES-2R, G401, and HS-ES-1) cell lines transfected with siRNAs specific for CREBBP + EP300 , or with NT (non-targeting) siRNA. Cells were transfected for 48 h with the indicated siRNAs. The cells were then reseeded and transfected repeatedly with the indicated siRNAs for 48 h. The cells were then reseeded and incubated for 7 days. Data are presented as the mean ± SEM (standard error of the mean), n = 3 independent experiments. f Viability of JMU-RTK-2 +SMARCB1 and JMU-RTK-2 -SMARCB1 cells transfected with siRNAs specific for CREBBP and/or EP300 , or with NT siRNA. Cells were transfected for 48 h with the indicated siRNAs. The cells were then reseeded and transfected repeatedly with the indicated siRNAs for 48 h. The cells were then reseeded and incubated for 7 days. Data are presented as the mean ± SEM, n = 3 independent experiments. g Viability of SMARCB1-proficient (786-O and VMRC-RCZ) and SMARCB1-deficient (HS-ES-1, NEPS, G402, and HS-ES-2R) cell lines transfected with siRNAs specific for CREBBP and/or EP300 , or with NT siRNA. Cells were transfected for 48 h with the indicated siRNAs. The cells were then reseeded and transfected repeatedly with the indicated siRNAs for 48 h. The cells were then reseeded and incubated for 7 days. Data are presented as the mean ± SEM, n = 3 independent experiments. h Chemical structures of CBP/p300 dual inhibitors A-485, inobrodib, and CP-C27. i Histone acetylation (HAT) activity of CBP and p300 in vitro. The IC 50 (50% inhibitory concentration) values denoting the inhibitory effects of CP-C27 are shown. Data are presented as the mean ± SD (standard deviation), n = 2 independent experiments. j IC 50 values of the CBP/p300 inhibitors CP-C27, A-485, and inobrodib, the EZH2 inhibitor tazemetostat, and the EZH1/EZH2 inhibitor valemetostat in HEK293T, JMU-RTK-2 +SMARCB1, and JMU-RTK-2 -SMARCB1 cells. Cells were treated with inhibitors for 6 days and IC 50 values were calculated based on cell viability. Data are presented as the mean ± SEM, n = 3 independent experiments. k – m IC 50 values for CBP/p300 inhibitors CP-C27 ( k ), A-485 ( l ), and inobrodib ( m ) in SMARCB1-proficient (H460, H1048, H2009, H2228, 786-O, H358, Caki-1, HEK293T, VMRC-RCZ, and ES2) and SMARCB1-deficient (HS-ES-2M, HS-ES-2R, A-204, NEPS, G401, G402, HS-ES-1, and JMU-RTK-2) cells (corresponding to Supplementary Fig. ). Cells were treated with inhibitors for 6 days and IC 50 values were calculated based on cell viability. Data are presented as the mean ± SEM, (SMARCB1+ ; n = 10 biological independent cell lines, SMARCB1-; n = 8 biological independent cell lines). For all experiments, p- values were determined by an unpaired two-tailed Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The ability of CP-C27 to inhibit p300 HAT activity was evaluated using a SensoLyte HAT (p300) Assay Kit (ANASPEC, AS-72172).

Techniques: Western Blot, Expressing, Transfection, Incubation, Activity Assay, In Vitro, Concentration Assay, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting dependency on a paralog pair of CBP/p300 against de-repression of KREMEN2 in SMARCB1-deficient cancers

doi: 10.1038/s41467-024-49063-w

Figure Lengend Snippet: a Schematic flow illustrating the method used to identify KREMEN2 as a determinant for synthetic lethality in SMARCB1-deficient cells treated with a CBP/p300 inhibitor. KREMEN2 was identified and selected as a gene that is upregulated in SMARCB1-deficient cells, and downregulated in A-485 treated SMARCB1-deficient cells but not in SMARCB1-proficient cells. A set of 471 genes concordantly upregulated in SMARCB1-deficient cells (JMU-RTK-2, HS-ES-2R), but not SMARCB1-proficient cells (JMU-RTK-2 + SMARCB1, 786-O), and a set of 50 genes that were concordantly downregulated in SMARCB1-deficient cells (JMU-RTK-2, HS-ES-2R) treated with A-485, but not in SMARCB1-proficient cells (JMU-RTK-2 + SMARCB1, 786-O), were isolated. Next, a set of 22 genes that overlapped these two gene sets was isolated. In addition, a set of 54 genes that was concordantly downregulated in two other SMARCB1-deficient cells (G402, NEPS) after treatment with A-485 was identified. The KREMEN2 gene was identified from the 22 and 54 overlapping genes. b Viability of JMU-RTK-2 +SMARCB1 and JMU-RTK-2 -SMARCB1 cells transfected with the indicated siRNAs. Cells were transfected for 48 h with the indicated siRNAs. The cells were reseeded and transfected repeatedly with the indicated siRNAs for 48 h. The cells were then reseeded and incubated for 7 days. Data are presented as the mean ± SEM (standard error of the mean), n = 3 independent experiments. c Viability of SMARCB1-proficient (VMRC-RCZ, HEK293T, H460, H2228, and 786-O) and SMARCB1-deficient (HS-ES-1, NEPS, HS-ES-2R, G402, and JMU-RTK-2) cell lines transfected with the indicated siRNAs. Cells were transfected for 48 h with the indicated siRNAs. The cells were reseeded and transfected repeatedly with the indicated siRNAs for 48 h. The cells were then reseeded and incubated for 7 days. Data are presented as the mean ± SEM, n = 3 independent experiments. d Viability of HS-ES-2R mock cells and HS-ES-2R +KREMEN2 cells transfected with the indicated siRNAs. Cells were transfected for 48 h with the indicated siRNAs. The cells were reseeded and transfected repeatedly with the indicated siRNAs for 48 h. The cells were then reseeded and incubated for 7 days. Data are presented as the mean ± SEM, n = 3 independent experiments. e Expression of KREMEN2 mRNA in JMU-RTK-2 +SMARCB1 and JMU-RTK-2 -SMARCB1 cells (relative to that in JMU-RTK-2 -SMARCB1 cells). KREMEN2 mRNA was not detected (ND) in JMU-RTK-2 + SMARCB1. Data are presented as the mean ± SD (standard deviation), n = 3 independent experiments. f Expression of KREMEN2 mRNA in SMARCB1-proficient (HEK293T, H460, 786-O, VMRC-RCZ, and H2228) and SMARCB1-deficient (JMU-RTK-2, G402, NEPS, HS-ES-1, and HS-ES-2R) cell lines (relative to that in JMU-RTK-2 cells). Data are presented as the mean ± SD, n = 3 independent experiments. g Localization signals generated by H3K4me3, H3K4me1, H3K27ac, H3K27me3, CUT&RUN-seq, p300 ChIP-seq, ATAC-seq, and RNA-seq around the KREMEN2 locus in JMU-RTK-2 +SMARCB1 and JMU-RTK-2 -SMARCB1 cells. H3K4me3-localized regions denote the promotor regions of the KREMEN2 locus. H3K4me1-localized regions denote the enhancer regions of the KREMEN2 locus. h – n Enrichment of CUT&RUN signals for H3K4me3 ( h ), H3K4me1 ( i ), H3K27ac ( j ), H3K27me3 ( k ), CBP ( l ), p300 ( m ), and EZH2 ( n ) (calculated relative to the CUT&RUN signal for normal IgG) at the indicated regions distant from the transcription start site (TSS) of the KREMEN2 gene in JMU-RTK-2 + SMARCB1 and JMU-RTK-2 -SMARCB1 cells. Data are presented as the mean ± SD, n = 3 independent experiments. For all experiments, p -values were determined by an unpaired two-tailed Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The ability of CP-C27 to inhibit p300 HAT activity was evaluated using a SensoLyte HAT (p300) Assay Kit (ANASPEC, AS-72172).

Techniques: Isolation, Transfection, Incubation, Expressing, Standard Deviation, Generated, ChIP-sequencing, RNA Sequencing, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting dependency on a paralog pair of CBP/p300 against de-repression of KREMEN2 in SMARCB1-deficient cancers

doi: 10.1038/s41467-024-49063-w

Figure Lengend Snippet: a Percentage of Annexin V-positive cells within the JMU-RTK-2 +SMARCB1 and JMU-RTK-2 -SMARCB1 cell populations treated with 2 μM A-485 for 96 h. Data are presented as the mean ± SD (standard error), n = 3 independent experiments. b Percentage of Annexin V-positive cells within the SMARCB1-proficient (786-O) and SMARCB1-deficient (HS-ES-2R) cell populations treated with the indicated concentrations of A-485 for 6 days. Data are presented as the mean ± SD, n = 3 independent experiments. c Schematic flow diagram showing identification of apoptotic markers induced by simultaneous inhibition of CBP/p300 specifically in SMARCB1-deficient cells. A set of 3,135 genes upregulated or downregulated by > 2-fold in JMU-RTK-2 -SMARCB1, but not in JMU-RTK-2 +SMARCB1 cells, was identified. In addition, a set of 1,163 genes upregulated or downregulated > 2-fold in SMARCB1-deficient cells (HS-ES-2R) but not in SMARCB1-proficient cells (786-O), was identified. Then, a set of 332 genes that overlapped between these gene sets was isolated. Wikipathway analysis identified 112 molecular pathways that were significantly associated with the 332 genes. To identify apoptotic markers induced by simultaneous inhibition of CBP/p300 specifically in SMARCB1-deficient cells, we focused on two apoptosis pathways. Among the genes related to these apoptosis pathways, CASP6 and CASP9 were identified as genes concordantly upregulated specifically in SMARCB1-deficient cells (see also Fig. 5d). d Heatmap showing changes (relative to non-treatment) in mRNA levels in apoptosis pathways (Hs-Apoptosis-WP254-106302 and Hs-Apoptosis-Modulation-and-Signaling-WP1772-107525) induced by treatment with 2 μM A-485 for 24 h. The pro-apoptotic marker genes CASP6 and CASP9 were identified as genes upregulated specifically in SMARCB1-deficient cells treated with CBP/p300 inhibitor. CASP6 and CASP9 are denoted by red arrows. e Expression of CASP6 mRNA (relative to that in NT (non-treated) cells) in SMARCB1-proficient (786-O) and SMARCB1-deficient (JMU-RTK-2, HS-ES-2R, and G402) cell lines treated with 0.2 or 0.4 μM CP-C27 for 24 h. Data are presented as the mean ± SD, n = 3 independent experiments. f Viability of JMU-RTK-2 +SMARCB1 and JMU-RTK-2 -SMARCB1 cells transfected with or without siRNAs targeting EP300 , KREMEN2 , and/or KREMEN1 . Cells were transfected with indicated siRNAs for 48 h. The cells were reseeded and incubated for 7 days. Data are presented as the mean ± SEM (standard error of the mean), n = 3 independent experiments. g Viability of SMARCB1-proficient (786-O) and SMARCB1-deficient (HS-ES-2R) cell lines transfected with or without siRNAs targeting EP300 , KREMEN2 , and/or KREMEN1 . Cells were transfected with indicated siRNAs for 48 h. Cells were reseeded and transfected with indicated siRNAs for 48 h. The cells were reseeded and incubated for 7 days. Data are presented as the mean ± SEM, n = 3 independent experiments. h , i Expression of CASP6 mRNA (relative to that in siNT (non-targeting)-transfected cells) in SMARCB1-deficient JMU-RTK-2 ( h ) and HS-ES-2R ( i ) cells transfected with or without siRNAs targeting EP300 or KREMEN1 , for 96 h. Data are presented as the mean ± SD, n = 3 independent experiments. j , k Expression of CASP6 mRNA (relative to that in siNT-transfected cells) in SMARCB1-deficient JMU-RTK-2 ( j ) and HS-ES-2R ( k ) cells transfected with siNT, or with siRNAs targeting KREMEN2 or KREMEN1 , for 24 h. Data are presented as the mean ± SD, n = 3 independent experiments. l , m Expression of CASP6 mRNA (relative to that in siNT-transfected HS-ES-2R mock cells) in HS-ES-2R mock and HS-ES-2R +KREMEN2 cells transfected with siNT, or with siRNAs targeting KREMEN2 ( l ) or CREBBP + EP300 ( m ), for 96 h. Data are presented as the mean ± SD, n = 3 independent experiments. n Expression of CASP6 mRNA (relative to that in HS-ES-2R mock cells) in HS-ES-2R mock and HS-ES-2R +KREMEN2 cells treated without or with 0.2 or 0.4 μM CP-C27 for 24 h. Data are presented as the mean ± SD, n = 3 independent experiments. o NanoBiT activity of KREMEN1 (relative to that in non-treated cells) in HS-ES-2R NanoBiT cells (HS-ES-2R +KREMEN1-SmBiT +KREMEN1-LgBiT) treated with 0.2 or 0.4 μM CP-C27 for 24 h. Data are presented as the mean ± SEM, n = 3 independent experiments. p , NanoBiT activity of KREMEN1 (relative to that in siNT-transfected cells) in HS-ES-2R NanoBiT cells (HS-ES-2R +KREMEN1-SmBiT +KREMEN1-LgBiT) transfected with siNT (−), or with siRNAs targeting KREMEN2 (+), for 96 h. Data are presented as the mean ± SEM, n = 3 independent experiments. q NanoBiT activity of KREMEN1 (relative to that in cells without KREMEN2 cDNA) in HS-ES-2R NanoBiT cells (HS-ES-2R +KREMEN1-SmBiT +KREMEN1-LgBiT) transduced without (−) or with the KREMEN2 cDNA vector (+). Data are presented as the mean ± SEM, n = 3 independent experiments. For all experiments, p -values were determined by an unpaired two-tailed Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The ability of CP-C27 to inhibit p300 HAT activity was evaluated using a SensoLyte HAT (p300) Assay Kit (ANASPEC, AS-72172).

Techniques: Inhibition, Isolation, Marker, Expressing, Transfection, Incubation, Activity Assay, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting dependency on a paralog pair of CBP/p300 against de-repression of KREMEN2 in SMARCB1-deficient cancers

doi: 10.1038/s41467-024-49063-w

Figure Lengend Snippet: a , b , c Normalized enrichment scores (NES) of significantly enriched biological signaling signatures identified among each of the gene sets downregulated upon treatment with 2 μM CBP/p300 inhibitor A-485 for 24 h ( a ) upon depletion of CREBBP + EP300 for 96 h ( b ) or upon depletion of KREMEN2 for 96 h ( c ) in SMARCB1-deficient cell lines (JMU-RTK-2 and HS-ES-2R), as determined by the Molecular Signatures Database Hallmark Gene Set collection in Gene Set Enrichment Analysis (GSEA). Gene sets with an FDR (False Discovery Rate) q -value < 0.25 and a Normal p -value < 0.05 were considered to be enriched significantly. p -values were determined by an unpaired two-tailed Student’s t -test. d Identification of five significantly enriched biological signaling signatures that overlapped among gene sets downregulated upon treatment with 2 μM CBP/p300 inhibitor A-485 for 24 h, upon depletion of CREBBP + EP300 for 96 h, or upon depletion of KREMEN2 for 96 h. e Identification of core enrichment genes among the gene sets comprising TNFα/NF-kB- and IL-6/JAK2/STAT3-related signatures. Core enrichment genes are the subset of genes that contributes most to the enrichment result. Core enrichment genes in each TNFA-SIGNALING-VIA-NFKB or IL6-JAK-STAT3-SIGNALING signature overlapped with 11 genes and three genes, respectively, among core enrichment genes upon treatment with 2 μM CBP/p300 inhibitor A-485 for 24 h, upon depletion of CREBBP + EP300 for 96 h, or upon depletion of KREMEN2 for 96 h. f Immunoblot analysis of phosphorylated proteins in protein microarrays derived from SMARCB1-deficient JMU-RTK-2 cells treated without or with 2 μM A-485 for 16 h. g Heatmap showing the signal intensities of phosphorylated proteins (relative to that in non-treated cells) in SMARCB1-deficient JMU-RTK-2 cells treated without or with 2 μM A-485 for 16 h. h Immunoblot analysis of AKT1, AKT pS473, PRAS40, PRAS40 pT246, histone H3, H3K27ac, and β-actin expression in SMARCB1-deficient cell lines (JMU-RTK-2, G402, and HS-ES-2R) treated without or with 2 μM or 4 μM A-485 for 16 h. The experiments were repeated twice independently with similar results. i Immunoblot analysis of AKT1, AKT pS473, PRAS40, PRAS40 pT246, histone H3, H3K27ac, CBP, p300, and β-actin expression in SMARCB1-deficient cell lines (JMU-RTK-2, G402, and HS-ES-2R) transfected with or without siRNAs for CREBBP + EP300 for 96 h. The experiments were repeated twice independently with similar results. j Immunoblot analysis of AKT1, AKT pS473, PRAS40, PRAS40 pT246, and β-actin expression in SMARCB1-deficient cell lines (JMU-RTK-2, G402, and HS-ES-2R) transfected with or without siRNAs for KREMEN2 for 96 h. The experiments were repeated twice independently with similar results. k Immunoblot analysis of AKT1, AKT pS473, PRAS40, PRAS40 pT246, histone H3, H3K27ac, and β-actin levels in SMARCB1-deficient cell lines (JMU-RTK-2) treated without or with CP-C27 after transfection with or without siRNAs for KREMEN1 . Cells were transfected for 48 h with the indicated siRNAs. The cells were then reseeded and incubated for 24 h. The cells were then treated with 0.2 μM CP-C27 for 16 h. The experiments were repeated twice independently with similar results. l Immunoblot analysis of AKT1, AKT pS473, PRAS40, PRAS40 pT246, and β-actin expression in SMARCB1-deficient cell lines (JMU-RTK-2) transfected with or without siRNAs for KREMEN2 and/or KREMEN1, for 96 h. The experiments were repeated twice independently with similar results. m Schematic models of the proposed molecular mechanism explaining synthetic lethality upon simultaneous inhibition of CBP and p300 in SMARCB1-deficient cancers. In SMARCB1-proficient cells, the SMARCB1-containing SWI/SNF complex suppresses transcription of KREMEN2 ; this suggests that SMARCB1-proficient cells are not dependent on CBP/p300 and KREMEN2. n In SMARCB1-deficient cancer cells, SMARCB1 deficiency increases expression of KREMEN2 mediated by both CBP and p300 in collaboration with the SMARCA1 chromatin remodeling complex and transcription factors, resulting in suppression of KREMEN1 due to homodimerization and culminating in activation of anti-apoptotic signaling pathways. o In SMARCB1-deficient cancer cells treated with a CBP/p300 dual inhibitor, downregulation of KREMEN2 via inhibition of CBP/p300 leads to monomerization of KREMEN1, followed by induction of apoptotic cell death via suppression of anti-apoptotic signaling pathways.

Article Snippet: The ability of CP-C27 to inhibit p300 HAT activity was evaluated using a SensoLyte HAT (p300) Assay Kit (ANASPEC, AS-72172).

Techniques: Two Tailed Test, Western Blot, Derivative Assay, Expressing, Transfection, Incubation, Inhibition, Activation Assay, Protein-Protein interactions