M344 decreases orthotopic pancreatic tumor growth when used as a treatment alone or in combination with gemcitabine.

(A) S2-013 cells were orthotopically implanted into the pancreas of female NU/J mice. After 8 days, the tumor volume for each mouse was monitored twice weekly with the VisualSonic Vevo 3100 Imaging System. At 15 days post-implantation of tumor cells, the mice were randomized into control or treatment groups with matched average tumor volumes. M344 was administered intraperitoneally at 10 mg/kg for 5 days per week (5 days on, 2 days off). Gemcitabine was given every 3 days intraperitoneally at 50 mg/kg. On Day 25 post-tumor implantation, the mice were euthanized and the tumors were resected and weighed. The changes in tumor volume over time are shown in (B) and representative images of tumors at 25 days post implantation are shown in (C). For statistical analysis, ordinary One-way ANOVA with Dunnett’s Multiple Comparisons test in GraphPad Prism Version 8.4.2 was used. The asterisks indicate the following p values: * p<0.05, ** p< 0.01, *** p<0.001.</p

Immunoblotting of pancreatic cancer cell line HLA class I heavy chains following treatment of the cells with M344.

The immunoblot data displayed here correspond to Fig 9. The proteins were transferred after electrophoresis to a membrane that was then divided. The top portion was probed with anti-HSC 70 (loading control) antibody and the bottom portion was probed with the HC10 antibody (for HLA-B and–C heavy chains). The blots were imaged, and a long exposure and a short exposure are shown (on the left and right, respectively). (TIF)</p

Example of flow cytometry gating for the caspase 3/7 activation assay.

Details of the experimental process can be found in the legend for Fig 4. (TIF)</p

M344 impairs viability in combination with gemcitabine.

S2-013 pancreatic cancer cells were treated with 0.1% DMSO, 10 μM M344, 100 nM gemcitabine, or 10 μM M344 + 100 nM gemcitabine. Viability was assessed by the trypan blue exclusion assay at 24, 48, and 72 hours and graphed as the number of live cells/total cells x 100. Each error bar represents the standard error of the mean. The results from 0.1% DMSO control treatment versus treatment with each M344 concentration were compared using Ordinary One-way ANOVA with Dunnett’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the p values: * p<0.05, ** p< 0.01, **** p<0.0001.</p

In pancreatic cancer cells, M344 causes cell cycle arrest in G₁.

Treatment of S2-013 cells with 1 or 10 μM M344 resulted in large increases in the populations accumulated in G1 at 24 hours (A), 48 hours (B), and 72 hours (C), as shown by propidium iodide staining and flow cytometry. Statistical comparisons were made using a Two-way ANOVA with Tukey’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the following p values: * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001.</p

M344-induced apoptosis is apparent by 48 hours and necrosis peaks at 72 hours.

S2-013 cells were treated with 0.1% DMSO control or M344 (1 μM or 10 μM) for 24, 48, or 72 hours. Caspase-3 and caspase-7 cleavage was simultaneously analyzed by using the CellEventTM Caspase-3/7 Green Flow Cytometry Assay Kit. The SYTOXTM AADvancedTM Dead Cell Stain included in the kit identified necrotic cells. Each error bar represents the standard error of the mean. Statistical comparisons of the results were done using a Two-way ANOVA with Tukey’s multiple comparisons test. The asterisks indicate the following p values: * <0.05, ** <0.01, *** <0.001, **** <0.0001.</p

Compared to vorinostat, M344 decreases pancreatic cancer cell proliferation more effectively for a longer duration.

The proliferation of S2-013 cells was assessed by the MTT assay following treatment with 0.1% DMSO control or with 1 μM, 5 μM, 10 μM, or 25 μM M344 or vorinostat for 48 hours or 72 hours. The effects of M344 versus vorinostat at 48 hours (A) and at 72 hours (B) are shown. The same data are displayed for M344 (C) and vorinostat (D) at 48 hours versus 72 hours. The error bars represent the standard error of the mean. The results were compared using a Two-way ANOVA with Tukey’s multiple comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the following p values: ** p< 0.01, *** p<0.001, **** p<0.0001.</p

Pancreatic cancer cell proliferation is decreased upon M344 treatment.

After treatment with M344 at various concentrations, the proliferation of (A) S2-013, (B) BxPC-3, (C) MIA PaCa-2, (D) T3M-4, and (E) CFPAC-1 cells was assessed by the MTT assay at 24, 48, and 72 hours. The graphs display the optical density (OD) at 570 nm relative to the OD for the 0.1% DMSO control. Each error bar represents the standard error of the mean. These results are representative of findings in 2–3 separate experiments for each cell line. The results from 0.1% DMSO control treatment versus treatment with each M344 concentration were compared using Ordinary One-way ANOVA with Dunnett’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the p values: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.</p

M344 treatment increases the expression of the human MHC class I heavy chains HLA-B and -C.

For immunoblot analysis, lysates of S2-013 pancreatic cancer cells treated for 24 or 48 hours with 0.1% DMSO (vehicle control) or with 1, 5, 10, or 25 μM M344 were subjected to electrophoresis, and the proteins were transferred to a membrane that was cut and one section was probed with an antibody for HSC70 (as a loading control) and another section was probed with an antibody to detect the MHC class I heavy chain (specifically, with the HC10 antibody that binds to HLA-B and HLA-C heavy chains). The exposure for the HLA-B/C heavy chains that is shown in the figure is longer than the exposure for the HSC70 control. Similar results were also obtained using the same M344 concentrations, time points, and antibodies in a separate experiment using different S2-013 lysates.</p

MHC class I expression on S2-013 pancreatic cancer cells is increased following M344 treatment.

After 24- or 48-hour treatments with 0.1% DMSO (vehicle control) or with M344 at 5 or 10 μM concentrations, cell surface expression was monitored using flow cytometry with the BB7.2 antibody for peptide-occupied HLA-A2 and the B1.23.2 antibody that detects HLA-B/C. The bar graphs depict the median fluorescence intensity (MFI) fold change relative to the 0.1% DMSO control treatment for (A) 24-hour M344 treatment and (B) 48-hour M344 treatment. Triplicate wells for each concentration and time point were analyzed. Each error bar represents the standard error of the mean. Statistical comparison of the results from the 0.1% DMSO control treatment versus treatment with each M344 concentration was performed using Ordinary One-way ANOVA with Dunnett’s Multiple Comparisons Test in GraphPad Prism Version 8.4.2. The asterisks indicate the p values: * pC) HLA-A2 expression at 24 hours post-treatment with M344, (D) HLA-A2 at 48 hours post-treatment with M344, (E) HLA-B/C at 24 hours post-treatment with M344, and (F) HLA-B/C at 48 hours post-treatment with M344. In the histograms, solid lines represent 0.1% DMSO treatment, dashed lines represent 5 μM M344 treatment, and solid gray areas represent 10 μM M344 treatment.</p