Mitochondrial complex I inhibitor deguelin induces metabolic reprogramming and sensitizes vemurafenib-resistant BRAFV600E mutation bearing metastatic melanoma cells

Abstract

Treatment with vemurafenib, a potent and selective inhibitor of mitogen-activated protein kinase signaling downstream of the BRAF^V600E oncogene, elicits dramatic clinical responses in patients with metastatic melanoma. Unfortunately, the clinical utility of this drug is limited by a high incidence of drug resistance. Thus, there is an unmet need for alternative therapeutic strategies to treat vemurafenib-resistant metastatic melanomas. We have conducted high-throughput screening of two bioactive compound libraries (Siga and Spectrum libraries) against a metastatic melanoma cell line (A2058) and identified two structurally analogous compounds, deguelin and rotenone, from a cell viability assay. Vemurafenib-resistant melanoma cell lines, A2058R and A375R (containing the BRAF^V600E mutation), also showed reduced proliferation when treated with these two compounds. Deguelin, a mitochondrial complex I inhibitor, was noted to significantly inhibit oxygen consumption in cellular metabolism assays. Mechanistically, deguelin treatment rapidly activates AMPK signaling, which results in inhibition of mTORC1 signaling and differential phosphorylation of mTORC1's downstream effectors, 4E-BP1 and p70S6 kinase. Deguelin also significantly inhibited ERK activation and Ki67 expression without altering Akt activation in the same timeframe in the vemurafenib-resistant melanoma cells. These data posit that treatment with metabolic regulators, such as deguelin, can lead to energy starvation, thereby modulating the intracellular metabolic environment and reducing survival of drug-resistant melanomas harboring BRAF ^V600E mutations.

Keywords: BRAFV600E; metabolic reprogramming; metastatic melanoma; vemurafenib resistance.

Figure 1.. High-throughput screening identifies Deguelin and Rotenone as effective against metastatic melanoma.

(A) Scatterplot depicting hits in green from the primary screen of A2058 cells using the Spectrum (circles) and Siga (squares) compound libraries. Hits were considered compounds that resulted in viabilities 3.5 standard deviations (red dotted lines) below the average of the screen (black dotted line) following 48 hours treatment. Circled are the lead compounds selected from the secondary screen in colors corresponding to panel B. 48-hour dose-response curves for the secondary screen (B) and validation in vemurafenib-resistant cell lines A375R (C) and A2058R (D). Data represent mean±SD from triplicate values (B) or from three independent experiments performed in triplicate (C,D).

Figure 2.. Deguelin and Rotenone significantly inhibit oxygen consumption in vemurafenib-resistant A375R and A2058R metastatic melanoma cell lines.

Seahorse XF24 rate graphs depicting OCR of A375 and A375R (A) or A2058 and A2058R (B) cell lines. Dotted line indicates the timepoint at which Deguelin was injected into the well. (C) Bar graph depicting the area under the curve of the rate graphs after compound injection. Baselined rate graphs depicting percentage change in ECAR of A375 and A375R (D) or A2058 and A2058R (E) cell lines. The baseline value for each treatment was considered the ECAR value immediately preceding compound injection into the well. Solid line indicates baseline value. (F) Bar graph depicting the average percentage change in ECAR from baseline for all the timepoints following compound injection. OCR and ECAR were normalized to cell counts. Data represent mean±SEM from three independent experiments (A,B,D,E) or mean±SD from three independent experiments for Deguelin and two independent experiments for Rotenone (C,F). All assays were performed in triplicates. Statistical significance was determined by two-tailed Student’s t-test (C) or by 2-way ANOVA (F). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3.. Deguelin inhibits AMPK-mediated MTORC1 activity as exemplified in the differential regulation of downstream effectors p70S6K and 4E-BP1.

(A) Representative western blotting to probe the phosphorylation status of AMPK and p70S6K as well as overall expression of 4E-BP1. Bar graphs depicting densiometric quantification of the preceding immunoblots relative to β-actin loading control for P-AMPK (B), P-p70S6K (C), and α/β isoforms of 4E-BP1 (D). Data represent the mean±SD from two independent experiments. Statistical significance was determined by one-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4.. Deguelin inhibits MAP kinase signaling and proliferation.

(A) Representative western blotting to probe for phosphorylation status of ERK. (B) Bar graph depicting densiometric quantification of the bands obtained from immunoblotting for relative intensity of phospho-protein over total protein for ERK. (C) Immunofluorescence to detect expression of the proliferation marker Ki67 following compound or DMSO treatment. (D) Counts of percentage Ki67+ cells obtained from immunofluorescence staining. Scale bar represents 50 µm. Data represent mean±SD from two independent experiments. Statistical significance was determined by one-tailed Student’s t-test. * p < 0.05.

Figure 5.. Mechanistic scheme portraying the partial activity of Deguelin and Rotenone in BRAF inhibitor-resistant metastatic melanoma.

Deguelin and Rotenone inhibit oxidative phosphorylation in the mitochondria which results in an elevation of the AMP to ATP ratio in the cell. This increase of AMP results in activation of AMPK signaling due to phosphorylation of the AMPKα2 subunit at T172 by LKB1. AMPK signaling activates the TSC complex which in turn inhibits MTORC1. Inhibition of MTORC1 results in a reduction in phosphorylation of p70S6 kinase, inhibiting its downstream activity. Additionally, the hyperphosphorylation of 4E-BP1 is inhibited resulting in its increased activity. The consequence of this is reduced cell survival and proliferation. Inhibition of MTORC1 can be reversed by addition of compound C (Dorsomorphin) which blocks AMPK signaling.