Fig 1: Conformational model of AU-15330 target interaction and activity profile in diverse cell lines.(a) Docking model of AU-15330 (cyan sticks) with the SMARCA2 and VHL complex. AU-15330 is suggested to fit into the pocket of SMARCA2 and VHL and capture several key interactions. Key hydrogen bond interactions with protein residues (pink sticks in SMARCA2, white sticks in VHL) are shown by yellow dashes. (b) Effects of AU-15330 (1 μM, 4h) on the proteome of VCaP cells. Data plotted Log2 of the fold change (FC) versus DMSO (dimethyl sulfoxide) control against –Log10 of the p-value per protein (FDR, false discovery rate) from n = 3 independent experiments. All t-tests performed were two-tailed t-tests assuming equal variances. TMT, tandem mass tag. (c) Heatmap showing TMT-based MS abundance of detectable SWI/SNF components after 4h of treatment with AU-15330 at 1 μM. Data from three independent replicates are shown. (d) Heatmap of relative abundance of several bromodomain-containing proteins detected via Tandem Mass Tag (TMT)-based quantitative MS upon 4h AU-15330 treatment. DMSO, dimethyl sulfoxide (vehicle). (e) Heatmap of mammalian SWI/SNF (BAF) complex subunits split into three constituent modules detected in SMARCC1 (also known as BAF155) nuclear co-immunoprecipitation followed by MS. Direct AU-15330 targets are in bold. (f) Dose-response curves of cells treated with AU-15330 and AU-16235 (inactive epimer of AU-15330). Data are presented as mean +/− SD (n = 6) from one-of-three independent experiments. (g) Crystal violet staining showing the effect of AU-15330 on colony formation. This experiment was repeated independently twice. (h) Dose-response curves and IC50 of cells treated with AU-15330, ACBI1, and BRM014. Data are presented as mean +/− SD (n = 6) from one-of-three independent experiments. (i) Immunoblots of noted proteins in VCaP cells treated with AU-15330, ACBI1, or BRM014 at increasing concentrations for 24h. Vinculin is the loading control probed on all immunoblots. This experiment was repeated independently twice. (j) Representative immunohistochemistry images showing expression of indicated proteins in patient-derived breast cancer cell lines. (k) Immunoblots of noted proteins in WA-72-P or WA-72-As breast cancer cells treated with DMSO or AU-15330 at noted concentrations for 24h, Vinculin is the loading control probed on a representative immunoblot. This experiment was repeated independently twice. Source data
Fig 2: AU-15330 is well tolerated in mice and induces on-target degradation of SMARCA2, SMARCA4, and PBRM1.(a) Immunoblots of indicated proteins in B16F10 and MC38 cells treated with DMSO or AU-15330 (100 nM or 1 μM). Vinculin is the loading control probed on a representative immunoblot. This experiment was repeated independently twice. (b) Schematic outlining the AU-15330 in vivo study in non-tumor bearing CD-1 mice. Male mice were treated with vehicle (control) or AU-15330 at the indicated concentration throughout the experiment. (c) Pharmacokinetics profile of AU-15330 following intravenous (IV) injection in CD-1 mice. Mice received a single injection at indicated concentration of AU-15330, and plasma levels were determined by HPLC. Data are presented as mean +/− SD (n = 6, biological replicates). (d) Immunohistochemistry staining of SMARCA4/BRG1 was carried out using lung, small intestine, and prostate sections after necropsy to show on-target efficacy of AU-15330 in vivo (n = 2, biological replicates). (e) Body weight measurements showing AU-15330 does not affect weight of non-tumor bearing CD-1 mice. Data are presented as mean +/− SD (n = 6, biological replicates). (f) Major organ weight measurements (taken after necropsy) showing AU-15330 does not affect their weight in non-tumor bearing CD-1 mice. Data are presented as mean +/− SD (n = 6, biological replicates). (g) Complete blood count showing AU-15330 does not affect the hematologic system. Non-tumor bearing CD-1 mice were treated with vehicle or AU-15330 at the indicated concentration throughout the treatment period, and whole blood was then collected and processed. WBC, white blood cells; RBC, red blood cells; PLT, platelets. Data are presented as mean +/− SD (n = 6, biological replicates). Source data
Fig 3: A947-mediated inhibition of cell proliferation in SMARCA4-mutant NSCLC cell lines.a Viability of NCI-H1944 following 7 days of treatment with a dose response of A947 or binding-defective epimers. Error bars represent mean ± SD from n = 3 biologic replicates. b Immunoblots of SMARCA2, SMARCA4 and PBRM1 levels across a panel of NSCLC cell lines defined by SMARCA4 gene mutation status. ß-tubulin serves as a loading control. Data are representative of 2 independent experiments. c Effect of A947 treatment on the growth of 30 lung cancer cell lines defined by SMARCA4 or SMARCA2 status, represented as the concentration of A947 required to inhibit growth by 50% (IC50) following 7 days of treatment. Individual cell line IC50’s were determined from n = 3 biologic replicates. Median IC50’s across models defined by mutational status are indicated by the black line. Significance was assessed by a two-tailed, Mann Whitney test. Asterick indicates p = 0.0003 d Cell cycle distribution following 48 h treatment of a dose response of A947 across 4 SMARCA4-mutant, 2 SMARCA4-wt and one SMARCA2-deficient NSCLC cell lines. Aphidicolin (Aph, 1 µM) treatment served as a control to block entry into S phase for the SMARCA4-wt and SMARCA2-deficient models. Error bars represent mean ± SD from n = 3 biologic replicates. e Log2-transformed fold change in mRNA expression values, as measured by RNA-seq, in HCC2302 cells treated for 96 h with A947 compared to control DMSO treated cultures (x-axis), as well as HCC2302-shSMARCA2 cells treated with doxycycline for 168 h compared to HCC2302 cells expressing a non-targeted control shRNA (shNTC). RNAseq data is representative of triplicated cultures. The correlation was calculated by the Pearson coefficient. p = 2.2e-16. Source data are provided as a Source Data file.
Fig 4: AU-15330, a specific degrader of SWI/SNF ATPases, exhibits preferential cytotoxicity in enhancer-binding transcription factor-driven cancers.a, Structure of AU-15330 and schematic of SMARCA2, SMARCA4 and PBRM1 domains. AU-15330-targeted bromodomains (BD) are shown. QLQ, conserved Gln, Leu, Gln motif containing domain; HSA, helicase/SANT-associated domain; BRK, Brahma and Kismet domain; SnAC, Snf2 ATP coupling domain; BAH1, bromo-adjacent homology domain 1; BAH2, bromo-adjacent homology domain 2. b, Immunoblots of SMARCA2, SMARCA4 and PBRM1 on treatment of HEK 293 and HeLa cells with AU-15330 at increasing concentrations or time durations. Vinculin is used as a loading control, and is probed on a representative immunoblot. This experiment was repeated independently twice. c, IC50 of AU-15330 in a panel of human-derived cancer or normal cell lines after 5 days of treatment. Known SMARCA4 loss-of-function (LOF) alterations and multiple myeloma (MM) cell lines with MYC rearrangements (MYC-R'ed) are identified below the graph. AR and FOXA1 scores quantify their transcriptional activities using cognate multi-gene signatures.Source data
Fig 5: SWI/SNF ATPases, SMARCA2 and SMARCA4, mediate chromatin accessibility at numerous sites across the genome in PCa cells.(a, b) ATAC-seq read-density heatmaps from VCaP cells treated with DMSO (solvent control), AU-15330, or ZBC-260 (a BRD4 degrader) for indicated durations at genomic sites that are compacted (a) or remain unaltered (b) upon AU-15330 treatment. Immunoblots show loss of target proteins upon treatment of cancer cells with AU-15330 (1 μM) for increasing durations or ZBC-260 (10 nM) for 4h. Vinculin is the loading control probed on all immunoblots. This experiment was repeated independently twice. Barplot shows the changes in mRNA expression (RNA-seq) of AU-15330 (1 μM) target genes in VCaP cells treated for noted durations. (c) Schematic outlining the CRISPR/Cas9 and shRNA-based generation of LNCaP cells with either independent or simultaneous inactivation of SWI/SNF ATPases, SMARCA2 and SMARCA4. Immunoblots showing the decrease in target expression in the genetic models shown above. Vinculin is the loading control probed on a representative immunoblot. This experiment was repeated independently twice. (d) ATAC-seq read-density heatmaps from genetically engineered LNCaP cells with SMARCA2 and/or SMARCA4 functional inactivation at AU-15330-compacted genomic sites. (e) Binding analysis for the regulation of transcription (BART) prediction of specific transcription factors mediating the observed transcriptional changes upon AU-15330 treatment in LNCaP or VCaP cells. The top 10 significant and strong (z-score) mediators of transcriptional responses are labeled (BART, Wilcoxon rank-sum test). (f) Top ten de novo motifs (ranked by p-value) enriched within AU-15330-compacted genomic sites (HOMER, hypergeometric test) in VCaP cells. (g) De novo motif analysis with top 10 motifs (ranked by p-value) enriched within genomic sites that retain chromatin accessibility upon AU-15330 treatment in VCaP cells (HOMER hypergeometric test). Source data
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