cb 6644  (MedChemExpress)

Journal: iScience

Article Title: RUVBL1 and RUVBL2 are druggable MYC effector regulators in neuroblastoma cells

doi: 10.1016/j.isci.2026.115236

Figure Lengend Snippet: Pharmacological inhibition of RUVBL1/2 ATPase activity in NB cell lines (A) Violin plots comparing DepMap RUVBL1 and RUVBL2 dependency scores from 34 NB cell lines to sets of essential and non-essential genes. ∗∗∗∗ p value <0.0001, two-sided Wilcoxon rank-sum test. (B) Western blot showing transient siRNA-mediated knockdown of RUVBL1 and RUVBL2 after 3 days of transfection. Scrambled siRNA was used as control (siCtrl). (C) Time-dependent effect of siRNA-mediated knockdown of RUVBL1 and/or RUVBL2 on NB cell line proliferation, as monitored by live scanning for cell confluency at regular time intervals with IncuCyte S3 system. Cell growth was normalized relative to the first scan at time zero. Results are mean ± SEM of 3 independent biological replicates. (D) Cell viability dose dependency curves after treatment with the RUVBL1/2 inhibitor CB-6644 in 7 different NB cell lines, as indicated. Mean IC50 values are indicated for each cell line (2–6 biological replicates). Cell viability was determined by resazurin assay. (E) Time-dependent effect of CB-6644 (250 nM or 500 nM) treatment on NB cell line proliferation, monitored as in C. (F) Long-term (14 days) effect of CB-6644 (250 nM) on NB cell growth. Cells were stained with crystal violet. See for western blot source data.

Article Snippet: CB-6644 , MedChemExpress , Cat# HY-114429.

Techniques: Inhibition, Activity Assay, Western Blot, Knockdown, Transfection, Control, Resazurin Assay, Staining

Journal: iScience

Article Title: RUVBL1 and RUVBL2 are druggable MYC effector regulators in neuroblastoma cells

doi: 10.1016/j.isci.2026.115236

Figure Lengend Snippet: Transcriptomic response to CB-6644 treatment of NB cells CLB-BAR and SK-N-AS NB cells were treated for 72 h with CB-6644 250 nM and differential gene expression (DGE) was determined. (A) Volcano plot for both cell lines as indicated. Differentially expressed genes (threshold log2FoldChange of ±1 at 1% FDR) indicated in blue with top up/downregulated and genes discussed in main text labeled. p values calculated using the Wald Statistic, as implemented in the DESeq2 package. (B) GSEA running score plots for 2 Hallmark gene sets in CLB-BAR cells as indicated. p values calculated using a permutation test as implemented in the fgsea package. (C) GSEA results with heatmap comparing normalized enrichment values (NES, color key) for both cell lines after treatment with CB-6644 (CB; 250 nM) and elimusertib (Eli; 50 nM) as indicated. Gene sets that were not significant (Padj <0.05) were left blank. Columns (gene sets) were hierarchically clustered as indicated in dendrogram. See for detailed DGE and GSEA results.

Article Snippet: CB-6644 , MedChemExpress , Cat# HY-114429.

Techniques: Gene Expression, Labeling

Journal: iScience

Article Title: RUVBL1 and RUVBL2 are druggable MYC effector regulators in neuroblastoma cells

doi: 10.1016/j.isci.2026.115236

Figure Lengend Snippet: Experimental validation of transcriptomic responses upon CB-6644 treatment of NB cells Western blots showing the effect of different CB-6644 (250 nM) treatment duration on (phospho-) protein expression in 4 NB cell lines as indicated. N/C-MYC denotes MYCN for CLB-BA, CLB-GA, and NB1, and MYC for SK-N-AS and were detected by two independent antibodies. Cl, cleaved. See for western blot source data.

Article Snippet: CB-6644 , MedChemExpress , Cat# HY-114429.

Techniques: Biomarker Discovery, Western Blot, Expressing

Journal: iScience

Article Title: RUVBL1 and RUVBL2 are druggable MYC effector regulators in neuroblastoma cells

doi: 10.1016/j.isci.2026.115236

Figure Lengend Snippet: RUVBL2 and MYCN CUT&RUN results in CLB-BAR NB cells (A) MYCN (top, blue) and RUVBL2 (bottom, gray) CUT&RUN peaks at the RUVBL2 , MYCN , and MYC gene location in CLB-BAR cells, as indicated. (B) Venn diagram indicating the number of significant MYCN and RUVBL2 CUT&RUN peaks (targets) for 3 replicates. The targets that were identified by minimally 2 out of 3 replicates were used for further analysis (reported in panels C, E, and F). (C) Venn diagram indicating overlap between protein-coding gene targets determined with MYCN and RUVBL2 CUT&RUN. (D) Heatmaps showing peaks scores at the transcription start (TSS) ±4 kb of RUVBL2 and MYCN targets for all RUVBL2 binding sites. (E) Venn diagram showing overlap between the protein-coding RUVBL2 CUT&RUN targets and up- or downregulated genes following 72 h of CB-6644 (250 nM) in CLB-BAR cells, as indicated. (F) Enrichment for RUVBL2 CUT&RUN targets of up- and downregulated genes following different CB-6644 (250 nM) treatment durations as indicated. Odds ratios and corresponding p values calculated using Fisher’s exact test. See for detailed MYCN and RUVBL2 CUT&RUN results. CUT&RUN performed in triplicate. Results in A and D are from replicate 1. See for the results from replicates 2–3.

Article Snippet: CB-6644 , MedChemExpress , Cat# HY-114429.

Techniques: Binding Assay

Journal: iScience

Article Title: RUVBL1 and RUVBL2 are druggable MYC effector regulators in neuroblastoma cells

doi: 10.1016/j.isci.2026.115236

Figure Lengend Snippet: Clinical validation of the prognostic and putative therapeutic relevance of the RUVBL genes (A) Immunohistochemical staining for RUVBL1 and RUVBL2 in human NB tumor tissue sections as indicated. Normal pancreatic tissue was used as negative control for antibody specificity. Images are representative of 3 independent NB tumors and 2 independent pancreatic stained tissues. (B–E) Analysis of publicly available primary neuroblastoma data (data from Cangelosi et al. ; n = 364). (B) Correlation plots between RUVBL1 (top), RUVBL2 (bottom), and MYCN gene expression (log2 normalized counts) for MYCN amplified and MYCN wild-type tumors. Linear regression line and Pearson’s correlation coefficient indicated. (C) Boxplots comparing RUVBL1 (top) and RUVBL2 (bottom) expression between MYCN amplified and MYCN wild-type tumors. p value calculated using two-sided Wilcoxon rank-sum test. (D) Kaplan-Meier survival plots comparing overall survival between patients with high and low RUVBL1 and RUVBL2 as indicated. High/low RUVBL1/2 expression defined based on median gene expression. p value calculated using log-rank test. (E) Forest plots comparing hazard ratios ±95% confidence intervals for 4 variables as indicated. Results were obtained using a Cox proportional hazards multivariate regression analysis. (F) Time-dependent effect of CB-6644 (250 nM or 500 nM as indicated) on human NB PDX organoid growth. Organoid growth was monitored by scanning confluency at regular intervals with IncuCyte live cell analysis system. Results are mean ± SD of three technical replicates. Graph is representative of 3 biological repeats with different organoid seeding densities.

Article Snippet: CB-6644 , MedChemExpress , Cat# HY-114429.

Techniques: Biomarker Discovery, Immunohistochemical staining, Staining, Negative Control, Gene Expression, Amplification, Expressing, Cell Analysis

Journal: Gut

Article Title: Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2

doi: 10.1136/gutjnl-2023-331519

Figure Lengend Snippet: Expression of MYC and RUVBL1 correlate in pancreatic ductal adenocarcinoma (PDAC) and high levels are associated with aggressive tumours. (A) Scatter plot comparing expression of RUVBL1 and MYC target gene expression (mean of all HALLMARK MYC TARGET V1 genes after scaling expression (FPKM) across all TCGA patients) in patients with human PDAC from the TCGA database (r, Pearson’s correlation coefficient; p value, unpaired t-test, n=159). (B) Exemplary immunohistochemistry of RUVBL1 from a tissue core from a tissue microarray containing 31 individual human PDAC specimens. A section with high RUVBL1 expression and a zoom-in with PDAC (P) and stromal tissue (S) are shown (scale: 200 µm). The panel is also shown as part of . (C) Quantification of RUVBL1 expression in a tissue microarray containing 31 sections of human PDAC specimens and 24 sections of benign acinar tissue as in panel B. RUVBL1 expression was scored as negative, low, medium and high in PDAC, adjacent stroma and non-malignant ductal and acinar tissue. The ratio of tissues with high RUVBL1 expression is shown. n, sample size. (D) Kaplan-Meier survival curves for patients with PDAC stratified into groups of low and high expression of RUVBL1 and MYC target genes (mean of all MYC TARGET V1 genes after scaling expression (FPKM) across all TCGA patients). P value, log-rank test. See also .

Article Snippet: The RUVBL1/2 inhibitor CB-6644 (MedChemExpress) was administered two times per day at 25 mg/kg in 10% DMSO, 90% PBS.

Techniques: Expressing, Targeted Gene Expression, Immunohistochemistry, Microarray

Journal: Gut

Article Title: Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2

doi: 10.1136/gutjnl-2023-331519

Figure Lengend Snippet: RUVBL1 is essential for DNA replication and growth of pancreatic cancer cells. (A) Ruvbl1 knock-in strategy for auxin-inducible degron (AID) tagging showing the elements of the knock-in cassette and the architecture of Ruvbl1 . Arrows indicate the position of primers used to identify recombined cell clones by PCR (see ). (B) Immunoblots of wild-type KPC and KPC AID-Ruvbl1 cell lysates probed with antibodies against RUVBL1 or the AID tag. (C) Immunoblot of KPC AID-Ruvbl1; TIR1 cells treated with various concentrations of auxin for 3 or 6 hours. Vinculin, loading control. (D) Immunoblot of KPC AID-Ruvbl1; TIR1 cells treated with 1 µM auxin over time. TIR1 F74G was detected with an antibody against the MYC tag. Vinculin, loading control. (E) Quantification of immunoblots of RUVBL1 after 6 hours of 1 µM auxin treatment (n=5, mean±SD, unpaired t-test). rU, relative units. (F) Logarithmic growth curve of KPC AID-Ruvbl1 cells expressing or not expressing TIR1 F74G . Cells were treated daily with 1 µM auxin or vehicle, and growth was followed for 9 days in biological triplicates (n=3, mean±SD). (G) Immunoblot of KPC AID-Ruvbl1; TIR1 cells treated with 1 µM auxin or 1 µM CB-6644 for indicated time points. Vinculin, loading control. (H) BrdU-PI flow cytometry scatter plots of KPC AID-Ruvbl1; TIR1 cells after treatment with 1 µM auxin or 1 µM CB-6644 for 24 or 48 hours. Cells were labelled with BrdU for 1 hour. (I) Quantification of S-phase cells with high or low BrdU incorporation as shown in panel H. The experiment was performed in biological duplicates (n=2, mean). See also .

Article Snippet: The RUVBL1/2 inhibitor CB-6644 (MedChemExpress) was administered two times per day at 25 mg/kg in 10% DMSO, 90% PBS.

Techniques: Knock-In, Clone Assay, Western Blot, Control, Expressing, Flow Cytometry, BrdU Incorporation Assay

Journal: Gut

Article Title: Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2

doi: 10.1136/gutjnl-2023-331519

Figure Lengend Snippet: RUVBL1 redirects transcription from immune genes to growth genes. (A) Gene set enrichment analysis (GSEA) plots of selected RUVBL1-activated and RUVBL1-repressed gene sets. GSEA was performed on SLAM-seq data of KPC AID-RUVBL1; TIR1 cells. Cells were treated with 1 µM auxin or DMSO (Vehicle) for 15 hours, followed by 800 µM 4sU for 2 hours. The normalised enrichment score (NES) is positive for gene sets activated by and negative for those repressed by RUVBL1 (FDR, false discovery rate). (B) GSEA of KPC AID-Ruvbl1; TIR1 cells treated with 1 µM auxin or DMSO for 15 hours and 800 µM 4sU. The NES and the q-value are shown for all enriched gene sets (positive NES: gene sets activated by RUVBL1, negative NES: gene sets repressed by RUVBL1). (C) NES values for different hallmark gene sets and a gene set containing primary MYC targets defined in Muhar et al , compared between the 3 and 15-hour durations of RUVBL1 depletion. (D) Scatter plot comparing gene regulation after auxin-induced degradation and CB-6644 inhibition of RUVBL1. KPC AID-Ruvbl1; TIR1 cells were treated with 1 µM auxin for 15 hours (n=3) or 1 µM CB-6644 for 24 hours (n=3). Gene expression was analysed by SLAM-seq. Changes (log2FC) in total RNA versus DMSO (vehicle)-treated cells are shown (r, Pearson’s correlation coefficient; p value, unpaired t-test). (E) Genome browser track of RUVBL1 chromatin immune precipitation (ChIP)-seq signal in KPC AID-Ruvbl1; TIR1 cells treated with vehicle or 1 µM auxin for 15 hours. Binding to the Rpl8 gene is shown as spike-normalised reads and compared with the input signal as control. (F) Density plots of RUVBL1 ChIP-seq signals around transcription start sites (TSS). Averaged binding (RPKM) of RUVBL1 in KPC AID-Ruvbl1; TIR1 cells treated with DMSO (vehicle) or 1 µM auxin for 15 hours compared with the input signal. (G) Bar graph of the genomic distribution of RUVBL1 and MYC peaks. Chromatin binding of RUVBL1 and MYC was analysed by ChIP-seq and was compared with a set of random genomic intervals in promoters (TSS− 3000 bp to TSS+ 3000 bp), gene bodies (TSS+ 3000 bp to transcription end site (TES)), regions downstream of genes (TES to TES+ 2000 bp) and intergenic regions. (H) Scatter plot comparing the promoter occupancy of MYC and RUVBL1 (spike-normalised reads) as measured by ChIP-seq. r, Pearson’s correlation coefficient; p value, unpaired t-test. (I) Bin plot comparing gene regulation due to acute RUVBL1 depletion with RUVBL1 and MYC binding (spike-normalised reads) at gene promoters. Genes were binned into eight equally distant bins of gene regulation on 15 hours of RUVBL1 depletion (‘Regulation’). Mean regulation per bin was plotted against mean promoter occupancy by MYC and RUVBL1 in unperturbed KPC AID-Ruvbl1; TIR1 cells (mean±SEM). r, Pearson’s correlation coefficient; p value, unpaired t-test. NFkB, nuclear factor kappa B; TGFβ, transforming growth factor-beta; TNFα, tumour necrosis factor alpha. See also .

Article Snippet: The RUVBL1/2 inhibitor CB-6644 (MedChemExpress) was administered two times per day at 25 mg/kg in 10% DMSO, 90% PBS.

Techniques: Inhibition, Expressing, ChIP-sequencing, Binding Assay, Control

Journal: Gut

Article Title: Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2

doi: 10.1136/gutjnl-2023-331519

Figure Lengend Snippet: RUVBL1 is an essential cofactor of MYC. (A) Genome browser tracks of RUVBL1 and MYC chromatin immune precipitation (ChIP)-seq signal in A375 MYC-AID cells treated with 1 µM auxin for 3 hours or vehicle control. RNA polymerase II (RNAPII) tracks of U2OS cells (GSE162264) are shown. Binding to the EIF4A1 and TBRG4 genes is shown as spike-normalised reads and compared with the input signal as control. In the TBRG4 promoter, one of two RUVBL1 peaks located at an MYC-negative region does not decrease on auxin-mediated depletion of MYC. (B) Rank plot of RUVBL1 and MYC ChIP-seq signal in A375 MYC-AID cells treated with 1 µM auxin for 3 hours or vehicle control. All MYC and RUVBL1-bound promoters are sorted for decrease in RUVBL1 chromatin binding on acute MYC depletion and plotted as log2FC (y-axis, black). Mean MYC binding is plotted for 15 equally sized bins containing the same genes (y-axis, blue). (C) Scatter plot of SLAM-seq data comparing MYC-induced gene expression changes in the absence or presence of CB-6644. KPC MYC-ER cells were treated with DMSO or 1 µM CB-6644 for 20 hours followed by ethanol or 200 nM 4-hydroxytamoxifen (OHT) for 4 hours. Changes in total RNA (log2FC) are shown (n=3). The slope (m) and p value (p) of the linear regression (blue) are indicated as is the Pearson’s correlation coefficient (r). A line with slope m=1 is shown in black. (D) Heatmap of SLAM-seq data from cells treated as in panel C. Biological replicates are labelled 1, 2 and 3. Changes in total RNA (log2FC) are shown. (E) Sucrose gradient ultracentrifugation demonstrating a physical interaction between MYC and RUVBL1/2. Purified recombinant His6-MBP-MYC 1-163 (MYC 1-163 , top panel), RUVBL1/2 (middle panel) and all three proteins together (bottom panel) were subjected to sucrose gradient ultracentrifugation. Fractions were collected and analysed by SDS-PAGE, followed by Coomassie blue staining. AID, auxin-inducible degron. See also .

Article Snippet: The RUVBL1/2 inhibitor CB-6644 (MedChemExpress) was administered two times per day at 25 mg/kg in 10% DMSO, 90% PBS.

Techniques: ChIP-sequencing, Control, Binding Assay, Expressing, Purification, Recombinant, SDS Page, Staining

Journal: Gut

Article Title: Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2

doi: 10.1136/gutjnl-2023-331519

Figure Lengend Snippet: RUVBL1 is required for the maintenance and progression of pancreatic cancer. (A) Immunoblot of pancreatic tumour lysates. Native KPC cells and KPC AID-Ruvbl1; TIR1 cells were transplanted into one and seven mice, respectively. After 18 days, mice with KPC AID-Ruvbl1; TIR1 cell allografts were treated with 20 mg/kg auxin for up to 72 hours or with vehicle for 72 hours. Lysates of tumours were analysed using an anti-RUVBL1 antibody. Lysates of untreated and auxin-treated cultured KPC AID-Ruvbl1; TIR1 cells were loaded for comparison. The band between WT and AID-tagged RUVBL1 can be attributed to murine immunoglobulin G (IgG) in the tissue. Vinculin, loading control. (B) Schematic of the in vivo RUVBL1 depletion experiment. (C) Bioluminescence images of mice with pancreatic tumours. Luciferase-expressing KPC AID-Ruvbl1; TIR1 cells were transplanted into mice, and tumour size on day 7 was used to form pairs of animals with similar size tumours. Then, mice were treated daily with 20 mg/kg auxin (n=7) or vehicle (n=7). Tumour growth was assessed by bioluminescence imaging at the indicated time points. (D) Line plot of the relative volume of murine pancreatic tumours, estimated from bioluminescence as in panel C. Vehicle, n=7. Auxin, n=7. Values are mean±SEM. *n=6. (E) Kaplan-Meier survival curves for mice with pancreatic tumours treated with auxin (n=7) or vehicle (n=7). P value, Log-rank test. (F) Immunoblot of TIR1 F74G in lysates of tumours excised from mice that reached the humane endpoint. After KPC AID-Ruvbl1; TIR1 cell engraftment, mice were treated daily with 20 mg/kg auxin or vehicle for up to 28 days. TIR1 F74G was detected with an antibody against the MYC tag. GAPDH, loading control. See also .

Article Snippet: The RUVBL1/2 inhibitor CB-6644 (MedChemExpress) was administered two times per day at 25 mg/kg in 10% DMSO, 90% PBS.

Techniques: Western Blot, Cell Culture, Comparison, Control, In Vivo, Luciferase, Expressing, Imaging

Journal: Gut

Article Title: Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2

doi: 10.1136/gutjnl-2023-331519

Figure Lengend Snippet: RUVBL1 promotes immune evasion in pancreatic ductal adenocarcinoma (PDAC). (A) Immunohistochemical staining of RUVBL1, KI67, BrdU and CD3 in sections of KPC AID-Ruvbl1; TIR1 tumours from mice treated with vehicle (n=4) or 20 mg/kg auxin (n=4) for 5 days. Mice received an injection of BrdU and 2.5 hours later were killed to harvest the tumours. Quantification of positively stained cells is depicted below (n=4, unpaired t-test). (B) Kaplan-Meier survival curves of mice bearing KPC AID-Ruvbl1; TIR1 tumours treated with 10 mg/kg anti-PD-1 antibody (αPD-1) two times per week, alone (n=10) or with 20 mg/kg auxin daily (n=11). Vehicle-treated group (n=7) from . (C) Dose–response curves of CB-6644 on cell viability (resazurin assay) in native KPC cells and KPC cells overexpressing RUVBL1 WT or RUVBL1 A62T . Cells were treated with CB-6644 for 72 hours (n=3, mean±SD). (D) Immunoblot of MYC and RUVBL1 in a panel of murine PDAC cell lines with the indicated genetic mutations representing different PDAC subtypes. (E) Scatter plot of MYC and RUVBL1 protein levels as in panel D (n=2; r, Pearson’s correlation coefficient; p value, unpaired t-test). (F) Scatter plot of MYC/RUVBL1 protein levels as in panel D and sensitivity to treatment with CB-6644 for 72 hours (n=2; r, Pearson’s correlation coefficient; p value, unpaired t-test). (G) Bioluminescence images of mice with pancreatic tumours. Luciferase-expressing KPC AID-Ruvbl1; TIR1 cells were transplanted into mice, and tumour size on day 7 was used to form pairs of animals with similar size tumours. Then, mice were treated two times per day with 25 mg/kg CB-6644 (n=5) or vehicle (n=5). Tumour growth was assessed by bioluminescence imaging at the indicated times. (H) Line plot of the relative volume of pancreatic tumours in mice treated with CB-6644 (n=5) or vehicle (n=5), as in panel G. Values are mean±SEM. (I) Kaplan-Meier survival curves for mice with pancreatic tumours treated with CB-6644 (n=5) or vehicle (n=5), as in panel G. Log-rank test. (J) Line plot of the relative volume (mean±SEM) of pancreatic tumours in mice treated with CB-6644 (n=8), αPD-1 (n=8), a combination of αPD-1 and CB-6644 (n=8) or vehicle (n=8). KPC cells were transplanted into mice, and tumour size on day 7 was used to form pairs of animals with similar size tumours. Mice were treated two times per day with 25 mg/kg CB-6644, two times per week with 10 mg/kg αPD-1, a combination of both or vehicle for up to 28 days. (K) Kaplan-Meier survival curves for mice with pancreatic tumours treated with CB-6644 (n=8), αPD-1 (n=8) or a combination of both (n=8), as in panel J. Log-rank test. See also .

Article Snippet: The RUVBL1/2 inhibitor CB-6644 (MedChemExpress) was administered two times per day at 25 mg/kg in 10% DMSO, 90% PBS.

Techniques: Immunohistochemical staining, Staining, Injection, Resazurin Assay, Western Blot, Luciferase, Expressing, Imaging