Pyruvate dehydrogenase inactivation causes glycolytic phenotype in BAP1 mutant uveal melanoma

Abstract

Effective therapeutic options are still lacking for uveal melanoma (UM) patients who develop metastasis. Metastatic traits of UM are linked to BRCA1-associated protein 1 (BAP1) mutations. Cell metabolism is re-programmed in UM with BAP1 mutant UM, but the underlying mechanisms and opportunities for therapeutic intervention remain unclear. BAP1 mutant UM tumors have an elevated glycolytic gene expression signature, with increased expression of pyruvate dehydrogenase (PDH) complex and PDH kinase (PDHK1). Furthermore, BAP1 mutant UM cells showed higher levels of phosphorylated PDHK1 and PDH that was associated with an upregulated glycolytic profile compared to BAP1 wild-type UM cells. Suppressing PDHK1-PDH phosphorylation decreased glycolytic capacity and cell growth, and induced cell cycle arrest of BAP1 mutant UM cells. Our results suggest that PDHK1-PDH phosphorylation is a causative factor of glycolytic phenotypes found in BAP1 mutant UM and propose a therapeutic opportunity for BAP1 mutant UM patients.

Figure 1.. BAP1 mutant UM tumors have elevated mRNA levels related to PDC and PDHK1.

UVM RNA-seq V2 gene expression data from the TCGA were retrieved from the latest Broad GDAC Firehose data run (stddata_2016_01_28). Based on BAP1 mutation and copy loss, samples were stratified into BAP1 mutant and wild type groups. Differential expression analysis was performed between BAP1 mutant (n=40) and wild type (n=40) and used for performing GSEA. a. GSEA enrichment plots of the glycolysis hallmark gene set for comparison in BAP1 mutant vs wild type group. b. PDH complex is composed of PDH (E₁), DLAT (E₂) and DLD (E₃). Phosphorylation of PDH inactivates its activity, elevating aerobic glycolysis. c. UVM RNA sequencing (RNA-seq) V2 gene expression of PDH (E₁), DLAT(E₂), DLD (E₃) and PDHK1 d. A heatmap showing z-scores for the average expression of genes in malignant cells from single cell RNA-seq of UM tumors.

Figure 2.. BAP1 mutant UM cells have elevated PDHK1-PDH phosphorylation.

a. Average expression of PDH (E₁), DLAT (E₂) and PDHK1 between BAP1 wild type (92.1, OMM1.3, UM001, UM004, UM002B and MM66) and mutant (MP46, MP65, MP38, MM28, PDX4 and WM3618F) UM cells were analyzed by RPPA analysis. RPPA data were used to determine antibodies that are significantly different between mutant and wild-type cell lines. Comparison of average normalized log₂ values were performed by the two-sample t-test method and assumed unequal variance. Data points are shown as individual lysate collections **p<0.01. b. Validation of protein expression by Western blot.

Figure 3.. BAP1 mutant UM cells have increased glycolytic profiles

a. Glycolytic capacity of MM66, MP46 and MP65 cells was compared by ECAR using the Seahorse analyzer. Data were normalized to protein level and analyzed via Agilent Seahorse XF report generators. Data are shown as mean ± SEM (n=12). b. Levels of metabolite of MM66, MP46 and MP65 cells in glycolysis and TCA cycle are shown. Data from whole cell extractions (n=6) are displayed as mean ± SEM (n=6). *p<0.05, **<0.01, ***<0.001, ****<0.0001 and not significant (ns).

Figure 4.. Suppression of PDH phosphorylation decreased the glycolytic capacity of BAP1 mutant UM cells

a. MP46 and MP65 cells were pre-treated with DCA (5mM) for 24 hours and glycolytic capacity was measured using the Seahorse instrument. Efficiency of DCA was determined by detecting downregulation of p-PDH in western blot analysis. b. Effect of siPDHK1 on MP65 cells glycolytic ability was measured by the Seahorse instrument. Knockdown of PDHK1 was confirmed by western blot. Data were normalized to protein level and analyzed via Agilent Seahorse XF report generators. Data are shown as mean ± SEM (n=12). *p<0.05, **<0.01, ***<0.001, ****<0.0001 and not significant (ns).

Figure 5.. Inhibition of PDH phosphorylation reduces cell survival of BAP1 mutant UM cells.

a. Efficiency of DCA in UM cells was determined by detecting downregulation of p-PDH after 5mM DCA treatment for 48 hours. b. BAP1 wild type (92.1 and MM66) and mutant (MP46, MP65, MP38 and MM28) cells were treated with DCA (5, 7.5 or 10 mM) for 3 days. Changes of cell viability were measured by crystal violet staining. Quantification bar graph represents relative fold change of cell viability with DCA treatments in cells. Representative images of crystal violet are shown. Scale bar: 100 μm. Data are shown as mean ± SEM from biological replicate experiments (n=4). c. Effect of siPDHK1 on MP65 cell growth was analyzed through Incucyte Live Cell Analysis Imaging System. Data are shown as mean ± SEM from biological replicate experiments. *p<0.05, **<0.01, ***<0.001, ****<0.0001 and not significant (ns).

Figure 6.. Suppression of PDH phosphorylation leads to the cell cycle arrest in BAP1 mutant UM cells.

Knockdown of PDHK1 was confirmed by western blot. MP38 and MM28 cells were treated with DCA (10mM) for 24, 48, and 72 hours. Then, cells were collected for a. a nalysis of protein expressions related to cell cycle progression, and b. EdU incorporation assay (72hr, n=3). c. MP38 cells were treated with DCA (10mM) for 72 hours. Cell viability assays of 3D tumor spheroids were performed. The representative figures of MP38 cells 3D spheroids are shown. For the quantification, 3D tumor spheroids were stained with calcein-AM (7μM) and propidium iodide (PI, 10μg/mL) for live cells and necrotic/dead cells, respectively (n=3). Magnification: 200X, Scale bar: 100μm. Data are shown as mean ± SEM from biological replicate experiments. *p<0.05, **<0.01, ***<0.001, ****<0.0001 and not significant (ns).

Figure 7.. UM patient survival curve based on PDC and PDHK1 expression.

a. Analysis of TCGA data retrieved from the TCGA Pan-Cancer Clinical Data Resource (TCGA-CDR) for UM patient disease specific survival based on PDH (E₁), DLAT (E₂), DLD (E₃) and PDHK1 expression are shown. Expression is stratified into high (n=40) or low (n=40) by median expression of each gene. Logrank test was used to determine significance in overall survival (https://wiki.nci.nih.gov/plugins/servlet/mobile#content/view/24279961). b. Cox proportional hazards (PH) model analysis for the association between disease-specific survival, PDHK-PDC score and BAP1 alteration status are shown. Patients were stratified into BAP1 altered groups based on BAP1 mutation and copy loss data. PDHK-PDC scores were calculated by averaging the z-score value for PDH, DLAT, DLD and PDHK1. c. A summary mechanism of BAP1 mutant-driven glycolysis in UM. BAP1 mutations induces phosphorylation of PDHK1-PDH axis, elevating aerobic glucose utilization of BAP1 mutant UM cells. Inhibition of PDH phosphorylation reduces BAP1 mutant UM cell growth via cell cycle arrest.