Fig 2: Increase in pERK by recombinant SPIKE protein is suppressed by MEK inhibition.(A) Calu-3 human NSCLC type II alveolar cells were pretreated with 10 µM MEK inhibitor (VS-6766, trametinib or selumetinib) for 1 hr before incubation with 4.7 µM recombinant SARS-CoV-2 spike protein subunit 1 (S1, 0.118 µg/mL) or subunit 2 (S2, 0.275 µg/mL) for 2 hr. ACE2 protein detected with sc-390851 was increased following cell incubation with S1 and S2. All three MEK inhibitors suppressed increased ACE2. (B) HT-29 human colorectal cancer cells and BEAS-2B normal human bronchial epithelial cells were pretreated with 10 µM RAF/MEK inhibitor VS-6766 for 1 hr before incubation with 4.7 µM recombinant SARS-CoV-2 spike protein subunit 1 (S1, 0.118 µg/mL) and subunit 2 (S2, 0.275 µg/mL) for 2 hr. Recombinant SARS-CoV-2 spike protein subunits increased pERK expression which was completely abrogated by VS-6766 treatment.

Fig 3: Reduced phospho-ERK and ACE2 expression by the combination of MEK inhibitor and remdesivir.(A) Effects of 5 µM remdesivir (RDV), RAF/MEK inhibitor VS-6766, or the combination on ACE2, ERK1/2, and pERK protein expression. HCT116 human colorectal cancer cells, H1975 human NSCLC cells and BEAS-2B normal human bronchial epithelial cells were treated with 5 µM VS-6766 or/and 5 µM Remdesivir for 48 hr. Remdesivir alone increased pERK protein expression in all three cell lines, which was completely depleted by VS-6766 treatment. Effects of 5 µM RDV, VS-6766, or the combination on ACE2, ERK1/2, and pERK protein expression in (B) normal human lung cells and (C-E) human lung cancer cells are shown (B-E left 2 panels). Serum-deprived cells were plated and cultured in medium containing 1% FBS for the indicated amount of time. For serum-stimulated cells, FBS was added to a final concentration of 10% for 24 hours (E, right panel). Cells were plated in 10% serum for 16 hours, then media was removed and replaced with 1% FBS for serum-deprived cells. For serum-stimulated cells, FBS was added to a final concentration of 10% for 4 hours. ACE2 (PAB), ACE2 (CS), ERK1/2, and pERK were probed with Abnova PAB13444, Cell Signaling 4355, Cell Signaling 9102, and Cell Signaling 4370 antibodies. Ran was probed with BD Biosciences 610341 antibody as a loading control. (F) Modulation of pRb and ACE2 expression in H1299 cells with serum starvation, stimulation, and MEKi treatment. Control cells were grown with the normal 10% serum throughout the treatment. Serum starved cells were plated in 10% serum and incubated for 16 hours, then grown in media containing 0% serum for 72 hours. Serum stimulated cells were similarly starved for 72 hours, then stimulated with media containing 20% serum for 48 hours. All drug treated cells received 5 mM RDV, VS-6766, or the combination 48 hours before harvesting. (G) pRb as a fraction of total Rb was quantified in ImageJ. (H) Modulation of ACE2 expression with serum starvation and stimulation. Four different cell lines (2 lung, 1 colorectal, and 1 breast cancer) were grown in 10% serum for 16 hours then were starved in 0% FBS for 0 (no starvation), 24, 48, or 72 hours. Cells were stimulated with 20% FBS and harvested either 0 (no stimulation), 12, or 24 hours later. An increase in pRb relative to total Rb was seen upon stimulation and this correlated with ACE2 levels. pERK correlation with ACE2 was heterogeneous, with a correlation seen in MCF7 cells but not H1299 or HCT-116 cells (three left-most panels). pRb relative to total Rb decreased upon starvation and increased upon stimulation, which correlated with ACE2 expression (H460 and HCT-116 in right-most panels).

Fig 4: Increase in pERK by recombinant SPIKE protein is suppressed by MEK inhibition.(A) Calu-3 human NSCLC type II alveolar cells were pretreated with 10 μM MEK inhibitor (VS-6766, trametinib or selumetinib) for 1 hr before incubation with 4.7 μM recombinant SARS-CoV-2 spike protein subunit 1 (S1, 0.118 μg/mL) or subunit 2 (S2, 0.275 μg/mL) for 2 hr. ACE2 protein detected with sc-390851 was increased following cell incubation with S1 and S2. All three MEK inhibitors suppressed increased ACE2. (B) HT-29 human colorectal cancer cells and BEAS-2B normal human bronchial epithelial cells were pretreated with 10 μM RAF/MEK inhibitor VS-6766 for 1 hr before incubation with 4.7 μM recombinant SARS-CoV-2 spike protein subunit 1 (S1, 0.118 μg/mL) and subunit 2 (S2, 0.275 μg/mL) for 2 hr. Recombinant SARS-CoV-2 spike protein subunits increased pERK expression which was completely abrogated by VS-6766 treatment.

Fig 5: MEK inhibitors alone or in combination with remdesivir suppress ACE2 expression.The effects of chloroquine, hydroxychloroquine, remdesivir and MEK inhibitors (VS-6766, selumetinib and trametinib) on ACE2, TMPRSS2 and IL-6 in human lung and colon cells are shown. (A) H1975 human NSCLC cells were treated with the indicated drugs and doses for 48 hours. Glycosylated ACE2, ACE2, TMPRSS2 (full-length and Serine Protease-domain), IL-6 were probed with the cell signaling 4355, Abnova PAB13444, Sigma MABF2158, and Sigma SAB1408591 antibodies. β-Actin was probed with Sigma A5441 as a loading control. (B) Effect of remdesivir and VS-6766 on ACE2(−1119)-Luc reporter and cell viability. HCT116 human colorectal cancer cells were transiently transfected with ACE2(−1119)-Luc reporter for 24 hours followed by remdesivir and VS-6766 treatment for 24 hours at the indicated doses. First D-Luciferin was added to acquire ACE2-Luc reporter bioluminescence images (left panel) with the Xenogen IVIS system. After the bioluminescence signal decayed, CellTiter-Glo was added to acquire cell viability images (right panel; “CTG”) with the IVIS system. (C) Remdesivir alone increased ACE2 protein expression. H1299 human NSCLC cells, MSTO-211H human mesothelioma cells, and Calu-3 human NSCLC type II alveolar cells were treated with remdesivir and VS-6766 for 48 hours. ACE2 was probed with Abnova PAB13444 antibody, and β-Actin was probed with Sigma A5441 antibody as a loading control.