Marburgvirus Hijacks Nrf2-Dependent Pathway by Targeting Nrf2-Negative Regulator Keap1

Marburg virus (MARV) has a high fatality rate in humans, causing hemorrhagic fever characterized by massive viral replication and dysregulated inflammation. Here, we demonstrate that VP24 of MARV binds Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of nuclear transcription factor erythroid-derived 2 (Nrf2). Binding of VP24 to Keap1 Kelch domain releases Nrf2 from Keap1-mediated inhibition promoting persistent activation of a panoply of cytoprotective genes implicated in cellular responses to oxidative stress and regulation of inflammatory responses. Increased expression of Nrf2-dependent genes was demonstrated both during MARV infection and upon ectopic expression of MARV VP24. We also show that Nrf2-deficient mice can control MARV infection when compared to lethal infection in wild-type animals, indicating that Nrf2 is critical for MARV infection. We conclude that VP24-driven activation of the Nrf2-dependent pathway is likely to contribute to dysregulation of host antiviral inflammatory responses and that it ensures survival of MARV-infected cells despite these responses

The histone deacetylase inhibitor M344 as a multifaceted therapy for pancreatic cancer.

The histone deacetylase (HDAC) inhibitor vorinostat, used with gemcitabine and other therapies, has been effective in treatment of experimental models of pancreatic cancer. In this study, we demonstrated that M344, an HDAC inhibitor, is efficacious against pancreatic cancer in vitro and in vivo, alone or with gemcitabine. By 24 hours post-treatment, M344 augments the population of pancreatic cancer cells in G1, and at a later time point (48 hours) it increases apoptosis. M344 inhibits histone H3 deacetylation and slows pancreatic cancer cell proliferation better than vorinostat, and it does not decrease the viability of a non-malignant cell line more than vorinostat. M344 also elevates pancreatic cancer cell major histocompatibility complex (MHC) class I molecule expression, potentially increasing the susceptibility of pancreatic cancer cells to T cell lysis. Taken together, our findings support further investigation of M344 as a pancreatic cancer treatment

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

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

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

M344 treatment of pancreatic cancer cells increases global histone H3 acetylation more than vorinostat treatment.

The inhibition of histone H3 deacetylation in S2-013 cells by M344 versus vorinostat was compared by monitoring the percentage of acetylated histone H3 following 48-hour treatment with M344 or vorinostat (at 1 μM or 10 μM) or with the 0.1% DMSO vehicle alone. Global H3 acetylation was assessed using the EpiQuikTM Global Histone H3 Acetylation Assay Kit. The data are displayed in (A) for 1 μM M344 and vorinostat (and 0.1% DMSO control), and in (B) for 10 μM M344 and vorinostat (and 0.1% DMSO control). These data were compiled from two biological replicates with triplicate samples in the first assay and quintuplet samples in the second assay. The graphs display the percentage global histone H3 acetylation, calculated by this formula: OD (treated sample–blank) / OD (untreated control–blank) X 100%. Error bars represent the standard error of the mean. Statistical comparisons were made with One-Way ANOVA with Tukey’s multiple comparisons tests. The asterisks indicate the p values: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.</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