Article Figures & Data
Figures
- Figure 1.
MLN8237 and chaetocin (CH) demonstrate a dose-dependent inhibition of PDAC cell growth. A, PDAC cell lines (5 × 104 per well of 96 well) were plated and treated with MLN8237 for 72 hours at a dose of 200 nmol/L. Treatment of BxPC-3 (B), Capan-2 (C), L3.6 (D), MIA PaCa-2 (E), PANC-1 (F), and Pan02 (G) PDAC cell lines with escalating doses of chaetocin (25, 50, 75, and 100 nmol/L) for 48 hours, demonstrated a dose-dependent inhibition of cell growth. Cell viability was measured by MTS, normalized to a vehicle control, and data (technical triplicate with biological n = 3) are represented as mean ± SEM. For MLN8237, treated cells were compared with control by Student t test. For chaetocin-treated cell lines, statistical significance was calculated by one-way ANOVA with multiple comparisons. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005.
- Figure 2.
Dual targeting of pan-H3K9 HMTs and AURKA synergizes to inhibit PDAC cell growth. The CI of chaetocin and MLN8237 was calculated for 1:2, 1:3, and 1:5 ratios as an average (A) and in the individual human BxPC-3 (B), Capan-2 (C), L3.6 (D), MIA PaCa-2 (E), PANC-1 (F), and mouse Pan02 PDAC cell lines (G). The CIs from at least three independent experiments were averaged to determine CI values at Fa 0.50, 0.75, 0.90, and 0.95 for each cell line. H, L3.6 cells were plated for a clonogenic cell survival assay and stained for cell density after 7 days of treatment with chaetocin or MLN8237 alone or in combination (dose 1: 7.5 nmol/L chaetocin, 22.5 nmol/L MLN8237; dose 2: 15 nmol/L chaetocin, 45 nmol/L MLN8237; dose 3: 22.5 nmol/L chaetocin, 67.5 nmol/L MLN8237; dose 4: 30 nmol/L chaetocin, 90 nmol/L MLN8237). Representative images of clonogenic staining for the vehicle (DMSO)-treated cells and dose escalation (left to right) are presented. I, Average density of the clonogenic assay (mean ± SEM, n = 3), normalized to vehicle for each treatment, and statistical significance calculated by Student t tests for each dose compared with vehicle (*, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005). J, The CI for the clonogenic assay indicated that the drugs act synergistically at high Fa values.
- Figure 3.
Combination treatment of 3D spheroids, organoids, and orthotopic xenografts increases efficacy of PDAC growth reduction. A, PANC-1 cells were grown in methylcellulose as spheroids to mimic a 3D tumor model and treated with various concentrations of vehicle, chaetocin or MLN8237 alone and in combination (dose 1: 50 nmol/L chaetocin, 150 nmol/L MLN8237; dose 2: 150 nmol/L chaetocin, 450 nmol/L MLN8237; dose 3: 300 nmol/L chaetocin, 900 nmol/L MLN8237; dose 4: 450 nmol/L chaetocin, 1,350 nmol/L MLN8237; dose 5: 600 nmol/L chaetocin, 1,800 nmol/L MLN8237). APH activity was measured by colorimetric readings and mean cell viability (mean ± SEM, n = 3) calculated as a percentage of vehicle-treated control. Each experiment was performed in triplicate and statistical significance compared with control was calculated by one-way ANOVA with multiple comparisons; ***, indicates P ≤ 0.0005. B, The CI for the spheroid model indicated that the drugs act synergistically. C, Representative images after 72 hours of treatment are presented. Yellow circle delineates edge of relatively dense core of the spheroid. Scale bar, 25 μm. D, Pancreatic duct cells were isolated from the pancreas of Ela-Kras mice and grown in Matrigel domes to form 3D organoids. Representative images after 72 hours of treatment (300 nmol/L chaetocin, 900 nmol/L MLN8237, and 300 nmol/L chaetocin + 900 nmol/L MLN8237) are presented. Scale bar, 25 μm. E, After 72 hours of treatment with vehicle, chaetocin (150 nmol/L, 300 nmol/L), MLN8237 (450 nmol/L, 900 nmol/L) and combination (150 nmol/L + 450 nmol/L or 300 nmol/L + 900 nmol/L, chaetocin + MLN8237 respectively), Ela-Kras organoid viability was assessed by CellTiter-Glo 3D luminescent assay readings normalized to the vehicle-treated organoids. Each experiment was performed in triplicate, results expressed as mean ± SEM, and statistical analyses were performed using one-way ANOVA and multiple comparisons; **, P ≤ 0.005; ***, P ≤ 0.0005. F, C57Bl/6 mice were orthotopically injected with syngeneic Pan02 cells constitutively expressing luciferase and allowed to recover and grow tumors for 1 week before initiating vehicle, chaetocin, or MLN9237 alone and combination treatments. To monitor in vivo tumor growth, mice were injected with d-luciferin at 0, 3, and 10 days of treatment and total flux (photons/second, p/s) measured for each mouse in the Xenogen IVIS-200 imaging system. Total flux was normalized to day 0 and averaged (mean ± SEM) across mice per condition and statistical significance was calculated by multiple t tests at each day using the Holm–Sidak correction for multiple comparisons. *, P ≤ 0.05 for comparison of combination treatment to vehicle control. Treatments with individual drugs did not produce statistically significant effects. G, Representative images of mice injected with d-luciferin at day 10.
- Figure 4.
Chaetocin–MLN8237 combination synergizes to trigger a G2–M shift and significant mitotic catastrophe. PANC-1 cells were plated and treated for 0, 24, 48, and 72 hours with vehicle, chaetocin (CH; 30 nmol/L), MLN8237 (90 nmol/L), or combination (30 + 90 nmol/L, chaetocin + MLN8237 respectively). Levels of cell viability (A) and apoptosis (B) were observed by protease and caspase-3/7 cleavage of fluorescent and luminescent substrates, respectively. Values were normalized to 0 hours vehicle for each treatment and statistical significance calculated by two-way ANOVA with Tukey multiple comparisons test. *, P ≤ 0.05; ***, P ≤ 0.0005. C, FACS analysis of PANC-1 cells treated for 48 hours with chaetocin, MLN8237, and combination treatments resulted in slightly increased G2–M arrest for single treatments and nearly complete G2–M arrest and cell death (sub-G1 events shown in light green) for the combination treatment. Representative FACS cell-cycle graphs illustrate the significant shift and average percentage (n = 2) of events in each phase of cell cycle, G1, S, or G2–M are graphed with relative percentages indicated in each segment (D). E, L3.6 cells were treated with vehicle, chaetocin, MLN8237, or combination for 48 hours, fixed, and stained antibodies to γ-tubulin (green), α-tubulin (red), and DNA stained with DAPI (blue) to consider mitotic progression. Over 150 mitotic cells were observed and quantified as normal, aberration, or catastrophe and resultant graph indicates the percentage of the total number of cells counted. F, Representative images of cells counted as normal mitosis (vehicle), multiple spindle poles (chaetocin and MLN8237 alone), and total disruption of the spindle apparatus in mitotic catastrophe during combination treatment. Scale bar, 2,000 nm. G, Electron microscopy images of PANC-1 cells after 72 hours of treatment demonstrated multinucleated cells (black arrows). Normal mitoses are indicated by (*). Evidence of mitotic cell death/mitotic catastrophe is shown in the combination treatment (red arrow). Scale bar, 10 μm. H, PANC-1 lysates after 48 hours of treatment indicate that the relative levels of P-S10 H3 and P-S139 H2A.X (indicators of mitotic catastrophe) are increased in the combination treatment by Western blot analysis, with H2B as loading control. Pancreatic tissue from orthotopic xenografts was stained by IHC for P-S139 H2A.X as a measure of mitotic catastrophe in vivo. I, Quantification of average positive staining cells per field are graphed ± SEM with 5 field views per sample. Statistical significance was performed using a Student t test to compare combination treatment to all other conditions, ***, P ≤ 0.0005. J, Representative fields illustrate the increased number of positively stained cells (black arrows) in the combination-treated animals as compared with vehicle, chaetocin, or MLN8237 alone. Scale bar, 0.1 mm.
- Figure 5.
RNA-seq defines molecular markers of the MLN8237–chaetocin response. RNA-seq was performed on PANC-1 cells that were treated for 24 hours with vehicle, chaetocin (CH; 30 nmol/L), MLN8237 (90 nmol/L) or combination (30 + 90 nmol/L, chaetocin + MLN8237, respectively). A, Hierarchical clustering of all genes identified across the four conditions indicates large clusters of genes increased after cells were treated with the combination of drugs. B, A Venn diagram of the two conditions that induced differential gene expression, chaetocin alone and combination, illustrates 548 unique genes that changed expression only in the combination therapy. C, Separation of the 548 DEG by ontological function demonstrates that genes include a large number of noncoding RNAs and transcriptional, chromatin, epigenetic elements. Red and green bars depict upregulated and downregulated genes in each category, respectively.
- Figure 6.
Combined AURKA and pan-H3K9-HMT inhibition triggers uncoordinated checkpoint responses and reduces the centromeric H3K9me mark. A, Combination of chaetocin and MLN8237 treatments triggered dysregulation of a variety of proteins involved in cellular checkpoint response. Protein levels were observed by Western blot analysis with α-tubulin illustrating equal loading of samples across all treatments. B, Pathway diagram illustrates the ATM/ATR–Chk1–p53 pathway for G2–M cell arrest. Increases and decreases in nodes affected by the combination treatment are marked with red and green stars, respectively. C, Treatments synergize to reduce the H3K9me mark in cells. Total levels of H3K9me2 and H3K9me3 are indicated by Western blots of the vehicle, chaetocin, MLN8237, and combination treatments of PDAC cells with total H3 levels shown as loading control. D, Representative images of H3K9me3 immunofluorescence staining in L3.6 cells demonstrated a global decrease of H3K9me3 upon treatment with the combination of drugs. Scale bar, 2,000 nm.
Additional Files
Supplementary Data
- Supplementary Figure 1 - Additional data on clonogenic cell survival assay, cell viability, apoptosis, quantification of mitotic catastrophe and global decrease of H3K9me3 upon combination treatment.
- Supplementary Methods - Detailed extended description of key methods.
- Supplementary Table 1 - List of Antibodies.
- Supplementary Table 2 - IC50 values for PDAC cell lines.
- Supplementary Table 3 - Fold change values for expression of individual DEGs found in response to combination treatment.
- Supplementary Table 4 - qPCR and RNA-seq expression values for cell cycle checkpoint gene.
Volume 15, Issue 8
