rg 7388  (MedChemExpress)

Journal: bioRxiv

Article Title: Enhancement of colorectal cancer therapy through interruption of the HSF1-HSP90 axis by p53 activation or cell cycle inhibition

doi: 10.1101/2024.02.22.581507

Figure Lengend Snippet: (A) Cell viability matrices of HCT116 (left) and RKO (right) cells treated with Ganetespib – RG-7388 combination for 72 hrs at indicated concentrations. Color scheme represents changes in cell viability. Numbers within the matrix indicate the HSA synergy score. Synergy scores: < −10 antagonistic; −10 to 10 additive; > 10 synergistic. ≥ 3 biological replicates. (B) Relative confluence after 72 hrs treatment of HCT116 (top) and RKO (bottom) cells. Cell confluence was analyzed by Celigo imaging cytometer. Confluence relative to DMSO control, set at value 1. (C) Induction of cell death. PI/Hoechst/Annexin V staining of HCT116 (left) and RKO (right) cells treated for 72 hrs with Ganetespib and RG-7388 at the indicated concentrations. Percent dead cells include PI+ only, annexin V+ only and PI+Annexin V+ cells and were analyzed by Celigo imaging cytometer. (D) PARP-1 immunoblots of HCT116 (left) and RKO (right) cells treated for 48 hrs. HCT116 were treated with 50nM Ganet and 1μM RG. RKO were treated with 25nM Ganet and 1.5μM RG. Representative immunoblots shown from 3 replicates with 2 independent experiments each. (B-C) Mean ± SEM from ≥ 3 biological replicates, Student’s t-test, p*≤ 0.05, p**≤ 0.01, p***≤ 0.001; ns, not significant. Ganet: Ganetespib, RG: RG-7388.

Article Snippet: RG-7388 (Merck), Palbociclib (Sigma) and Ganetespib (Syntha Pharmaceuticals) were dissolved according to manufacturer’s guidelines and used as indicated.

Techniques: Imaging, Cytometry, Staining, Western Blot

Journal: bioRxiv

Article Title: Enhancement of colorectal cancer therapy through interruption of the HSF1-HSP90 axis by p53 activation or cell cycle inhibition

doi: 10.1101/2024.02.22.581507

Figure Lengend Snippet: (A, B) Cell viability matrices of (A) HCT116 and RKO cells and (B) isogenic HCT116 p53-/- and HCT116 p53+/+ cells treated with Ganetespib – RG-7388 combinations for 48 hrs at the indicated concentrations. ≥ 3 biological replicates. (C) Relative confluence of HCT116 (top) and RKO (bottom) cells treated for 48 hrs. Cell confluence was analyzed by Celigo imaging cytometer. Confluence relative to DMSO control, set at value 1. (D) Induction of cell death. PI/Hoechst/Annexin V staining of HCT116 (top) and RKO (bottom) cells treated for 48 hrs with Ganet plus increasing concentrations of RG-7388. Percent dead cells include PI+ only, annexin V+ only and PI+ Annexin V+ cells and were analyzed using a Celigo imaging cytometer. (E) Cell viability matrices of HCT116 and RKO cells treated with Onalespib – RG-7388 combinations for 48 hrs at the indicated concentrations. ≥ 2 biological replicates. (C-D) Mean ± SEM from ≥ 3 biological replicates, Student’s t-test, p*≤ 0.05, p**≤ 0.01, p***≤ 0.001; ns, not significant. Ganet: Ganetespib, RG: RG-7388. (A, B, E) Color scheme represents changes in cell viability. Numbers within the matrix indicate the HSA synergy score. Synergy scores: < −10 antagonistic; −10 to 10 additive; > 10 synergistic.

Article Snippet: RG-7388 (Merck), Palbociclib (Sigma) and Ganetespib (Syntha Pharmaceuticals) were dissolved according to manufacturer’s guidelines and used as indicated.

Techniques: Imaging, Cytometry, Staining

Journal: bioRxiv

Article Title: Enhancement of colorectal cancer therapy through interruption of the HSF1-HSP90 axis by p53 activation or cell cycle inhibition

doi: 10.1101/2024.02.22.581507

Figure Lengend Snippet: (A) mRNA expression levels of HSF1 target genes in HCT116 (top) and RKO (bottom) cells treated for 24 hrs. qRT-PCRs for the indicated mRNAs normalized to RPLP0 mRNA. Mean ± SEM from ≥ 3 biological replicates. Student’s t-test, p*≤ 0.05, p**≤ 0.01, p***≤ 0.001; ns, not significant. (B) GSEA enrichment plot for HSF1 target genes in HCT116 cells treated with drug combination (1µM RG-7388 and 50nM Ganetespib) versus Ganetespib-only (50 nM). HSF1 target gene list from Mendillo et al. . (C, D) Immunoblots for HCT116 and RKO cells treated with 1µM RG-7388, 50nM Ganetespib for HCT116, or 1.5µM RG-7388, 15nM Ganetespib for RKO cells, treated for 24 hrs (C) or for 48 hrs (D). (C) Blots were probed for phospho-Ser326 HSF1 (pHSF1) and total HSF1 (tHSF1). (D) Blots were probed for total AKT (tAKT) and cRAF. Densitometric expression levels normalized for actin are indicated. (E, F) GSEA pathway analysis for hallmark gene sets on RNAseq data from HCT116 cells treated for 24 hrs as in (B), comparing drug combination versus RG-7388 alone (E) or Ganetespib alone (F). Grey bars, NES (Normalized Enrichment Score) of RG-7388 (E) or Ganetespib (F) treatment relative to DMSO. Red bars, NES of combination treatment relative to either RG-7388 or Ganetespib treatment alone, respectively.

Article Snippet: RG-7388 (Merck), Palbociclib (Sigma) and Ganetespib (Syntha Pharmaceuticals) were dissolved according to manufacturer’s guidelines and used as indicated.

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Enhancement of colorectal cancer therapy through interruption of the HSF1-HSP90 axis by p53 activation or cell cycle inhibition

doi: 10.1101/2024.02.22.581507

Figure Lengend Snippet: (A) Cell viability matrix of murine CRC tumor organoids treated with Ganetespib – RG-7388 combination treatment for 48 hrs. Organoids generated from AOM/DSS treated C57BL6/J mice. Four independent biological replicates with 3 in-plate technical replicates each. (B) mRNA expression levels of HSF1 target genes in murine CRC tumor organoids generated from AOM/DSS mice treated with 10nM (left) and 15nM (right) Ganetespib in combination with 500 nM RG-7388 for 24 hrs. qRT-PCRs for the indicated mRNAs normalized to RPLP0 mRNA. Mean ± SEM from min. 2 independent biological replicates. Student’s t-test, p*≤ 0.05, p**≤ 0.01, p***≤ 0.001; ns, not significant. (C) Quantification of normal small intestinal mucosa-derived organoids treated with the indicated combinations for 48 hrs. Mean ± SEM from 3 independent biological replicates. Student’s t-test, p*≤ 0.05, p**≤ 0.01, p***≤ 0.001; when ns, p-value indicated. (D) left , Quantification of dead CRC tumor-derived organoids treated with drug combination for 48 hrs as in (C). Organoids were generated from AOM/DSS treated C57BL6/J mice. Mean ± SEM from 2 independent biological replicates. right , Representative brightfield images of CRC tumor organoids. Scale bars, 100 μm. Ganet: Ganetespib, RG: RG-7388. (E) Cell viability matrices of p53-proficient (wildtype p53, wtp53) patient-derived organoids (PDOs) treated with Ganetespib, RG-7388 or in combination for 48 or 72 hrs. A PDO case represents one PDO culture (PDO #). For each PDO culture, four replicates (different passages) with 3 in-plate technical replicates each were measured. Organoids were cultivated and treated as large organoids. Bottom , Representative brightfield images of large CRC tumor organoids. Scale bars, 200 μm. (F) Cell viability matrix of PDO #2 culture, cultivated and treated as small organoids with Ganetespib, RG-7388 or in combination for 3 or 5 days. Three replicates (different passages) with 3 in-plate technical replicates each were measured. Bottom , Representative brightfield images of small PDOs #2 culture. Scale bars, 200 μm. (A, E, F) Color scheme represents changes in cell viability. Numbers in the matrix are HSA synergy scores. Synergy scores: < −10 antagonistic; −10 to 10 additive; > 10 synergistic. Ganet: Ganetespib, RG: RG-7388.

Article Snippet: RG-7388 (Merck), Palbociclib (Sigma) and Ganetespib (Syntha Pharmaceuticals) were dissolved according to manufacturer’s guidelines and used as indicated.

Techniques: Generated, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Enhancement of colorectal cancer therapy through interruption of the HSF1-HSP90 axis by p53 activation or cell cycle inhibition

doi: 10.1101/2024.02.22.581507

Figure Lengend Snippet: (A) Treatment scheme for the p53-proficient AOM/DSS mouse model. After tumor visualization by repeat colonoscopy and tumor scoring for at least one S2-sized tumor and three S1-sized tumors per mouse, mice were treated with single drugs or combination treatment for 19 days. 4 hrs after the final dose, mice were dissected and analyzed. (B, C) Representative images of entire resected colons (B) and H&E-stained colon sections (C) from single or combination treated C57BL6/J mice at endpoint as described in (A). Mice received 50 mg/kg RG-7388 orally 5x per week, or 50 mg/kg Ganetespib intravenously 2x per week, or both. (B) scale bar 5 mm. (C) x20 magnification, scale bars, 100 µm. (D) Tumor surface area from AOM/DSS mice that had received single or combination treatment for 19 days as in (A). Cross-sections of swiss roles were H&E stained (Supp ). Tumor areas of all tumors per H&E-stained swiss role were measured using ImageJ and calculated as ellipsoid in mm 2 . Bar, mean. vehicle n = 35 tumors from 5 mice; RG-7388 and Ganetespib n = 33 tumors from 6 mice each; combination n = 45 tumors from 9 mice. (E, F) GSEA hallmark analysis of RNAseq data from HCT116 cells treated with drug combination or single drugs for 24 hrs. Enriched pathways are plotted according to NES. Grey bars: Enrichment in (F) RG-7388-only or (G) Ganetespib-only versus DMSO controls. Red bars: further enrichment with combination treatment relative to single treatment. NES: normalized enrichment score. (G-I) Multiplex immunohistochemistry of p53-proficient tumors from AOM/DSS mice receiving drug treatments for 19 days as in (A). (G) Representative images of indicated groups. Ly6G staining represents neutrophils and pan-Cytokeratin represents tumor epithelial cells. DAPI counter staining. Scale bars, 50 µm. (H) Quantification of Ly6G+CD11b+F4/80-neutrophils using the “Vectra Polaris” platform and the inForm Advanced Image Analysis software. n = 5 mice per group. All tumors from stained swiss roles were analyzed for indicated groups. (I) Plot of the neutrophil-lymphocyte-ratio (NLR) as marker of an inflammatory response. A low NLR indicates an inflammatory response. (J, K) Multiplex immunohistochemistry of tumor-bearing AOM/DSS mice who received a short drug treatment of 6 days. Quantification of Ly6G+CD11b+F4/80-neutrophils of the indicated groups as in (H). DMSO, Ganetespib and RG-7388 mice n = 3 each, combination treated mice n = 4. (C, H-K) Mean ± SEM. Student’s t-test, p*≤ 0.05, p**≤ 0.01, p***≤ 0.001; ns, not significant. Ganet: Ganetespib, RG: RG-7388

Article Snippet: RG-7388 (Merck), Palbociclib (Sigma) and Ganetespib (Syntha Pharmaceuticals) were dissolved according to manufacturer’s guidelines and used as indicated.

Techniques: Staining, Multiplex Assay, Immunohistochemistry, Software, Marker

Journal: bioRxiv

Article Title: Enhancement of colorectal cancer therapy through interruption of the HSF1-HSP90 axis by p53 activation or cell cycle inhibition

doi: 10.1101/2024.02.22.581507

Figure Lengend Snippet: (A) Representative images of H&E-stained cross-sections of rolled-up resected full-length colons (called swiss role) from single or combination treated C57BL6/J mice at endpoint. Mice received 50 mg/kg RG-7388 orally 5x per week, or 50 mg/kg Ganetespib intravenously 2x per week, or both. x2.5 magnification, scale bar 2 mm. (B) Lack of weight change in single or combination treated mice over the duration of the drug treatment for 19 days. Mean ± SEM from ≥ 5 mice per group. The weight of each mouse at the start of treatment was set to 100%. Y-axis starts at 90%. Ganet: Ganetespib, RG: RG-7388.

Article Snippet: RG-7388 (Merck), Palbociclib (Sigma) and Ganetespib (Syntha Pharmaceuticals) were dissolved according to manufacturer’s guidelines and used as indicated.

Techniques: Staining