Volume 69, Issue 4, Pages 734-744 (April 2016) Metabolic Modulation of Clear-cell Renal Cell Carcinoma with Dichloroacetate, an Inhibitor of Pyruvate Dehydrogenase Kinase  Adam Kinnaird, Peter Dromparis, Bruno Saleme, Vikram Gurtu, Kristalee Watson, Roxane Paulin, Sotirios Zervopoulos, Trevor Stenson, Gopinath Sutendra, Desmond B. Pink, Katia Carmine-Simmen, Ronald Moore, John D Lewis, Evangelos D. Michelakis  European Urology  Volume 69, Issue 4, Pages 734-744 (April 2016) DOI: 10.1016/j.eururo.2015.09.014 Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 1 Pyruvate dehydrogenase kinase 1 (PDK1) is upregulated in clear-cell renal cell carcinoma (ccRCC) cells and tumors from patients compared to healthy kidney tissues removed from the same patient. (A) Real-time reverse-transcription polymerase chain reaction and confocal immunofluorescence imaging show that PDK1 is upregulated in ccRCC tumor samples compared to normal kidney tissue removed from the same patient during radical nephrectomy (PDK1, red; nuclear stain, blue; n=6 patients per group; * p<0.01 vs normal kidney). Patient information is shown in the inset. (B) The human ccRCC cell line 786-O expresses significantly more PDK1 compared to healthy proximal tubules, the cell type from which ccRCC is derived (n=3 per group; * p<0.01 vs proximal tubules). European Urology 2016 69, 734-744DOI: (10.1016/j.eururo.2015.09.014) Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 2 Dichloroacetate (DCA) increases pyruvate dehydrogenase (PDH) activity and reactivates mitochondrial function in clear-cell renal cell carcinoma (ccRCC) cells. (A) Schematic showing the mechanism of action for DCA. (B) DCA reduces pyruvate dehydrogenase kinase (PDK)-dependent phosphorylation of serine 293 on PDH (n=4 per group; * p<0.05 vs ccRCC). (C) DCA increases PDH activity measured by a dipstick immunocapture assay (n=3 per group; * p<0.05 vs ccRCC). (D) DCA increases the production of 13C-labeled acetyl-CoA when the medium is supplemented with 13C-labeled pyruvate (n=4 per group; * p<0.01 vs ccRCC). (E) DCA reduces both lactate and pyruvate levels measured by mass spectroscopy (n=5 per group; * p<0.05 vs ccRCC). (F) ccRCC cells consume more oxygen after treatment with DCA (n=4 per group; p<0.05 vs ccRCC). (G) ccRCC cells have a more hyperpolarized mitochondrial membrane potential (left; tetramethylrhodamine methyl ester [TMRM], red; nuclear stain, blue) and lower production of mitochondrial reactive oxygen species (mROS) (right; Mitosox stain, red; nuclear stain, blue) compared to normal proximal tubule (PT) cells at baseline. DCA treatment repolarizes the mitochondrial membrane potential and increases mROS in ccRCC but not normal PT cells (n=100 cells per group; * p<0.01 vs PT, # p<0.01 vs ccRCC). European Urology 2016 69, 734-744DOI: (10.1016/j.eururo.2015.09.014) Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 3 Dichloroacetate (DCA) inhibits hypoxia-inducible factor (HIF) transcriptional activity and increases p53 activity in clear-cell renal cell carcinoma (ccRCC). (A) DCA does not alter HIF2α protein levels measured by immunoblotting (n=4 per group). (B) DCA reduces HIF activity measured using a dual-reporter HIF luciferase assay (n=3 per group; * p<0.01 vs ccRCC) and decreases expression of HIF target genes VEGF and PDGFβ (n=4 per group; * p<0.05 vs ccRCC). (C) Schematic showing our hypothetical model of two mechanisms for inhibition of HIF activity without altering protein levels: (1) DCA increases p53 activation via mROS and thus reduces the availability of p300 for binding to HIF; and (2) DCA increases the activity of the α-ketoglutarate (αKG)-dependent asparagine hydroxalase factor inhibiting HIF (FIH), which directly inhibits HIF activity. (D) DCA increases nuclear localization of p53 (p53, green; DAPI nuclear stain, blue; n=150 cells per group; * p<0.01 vs ccRCC) and increases p53 activity as measured using a dual-reporter p53 luciferase assay (n=4 per group; * p<0.01 vs. ccRCC). (E) DCA treatment increases overall p53 protein levels and expression of the p53 target gene p21 (n=3 per group; * p<0.05 vs. ccRCC). (F) Mass spectroscopy data show that DCA treatment reduces multiple intermediary metabolites in the pentose phosphate pathway (n=6 per group; * p<0.01 vs. ccRCC). DAPI = 4’,6-diamidino-2-phenylindole. European Urology 2016 69, 734-744DOI: (10.1016/j.eururo.2015.09.014) Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 4 Dichloroacetate (DCA) inhibits hypoxia-inducible factor (HIF) in a factor inhibiting HIF (FIH)-dependent manner and reduces angiogenic signaling in vitro. (A) siRNA treatment reduces FIH mRNA by >90% (n=3 per group; * p<0.01 vs scrambled [Scr]). Knockdown of FIH increases HIF activity by more than 2.5-fold, but DCA treatment no longer reduces HIF activity (n=4 per group; * p<0.01 vs Scr). (B) DCA treatment increases intracellular α-ketoglutarate (αKG) levels (n=6; * p<0.05). (C) A Matrigel assay shows that clear-cell renal cell carcinoma (ccRCC) supernatant induces microvasculature endothelial-cell tubule formation and increases the number of complete structures, while supernatant from DCA-treated ccRCC cells does not (n=3; # p<0.01 vs medium only, * p<0.01 vs ccRCC supernatant). European Urology 2016 69, 734-744DOI: (10.1016/j.eururo.2015.09.014) Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 5 Dichloroacetate (DCA) reduces tumor formation, tumor size, and angiogenesis in vivo in fertilized egg xenograft models. (A) Schematic showing in ovo xenograft prevention and regression protocols. The red arrow shows a day-18 tumor; multiple blood vessels perfuse the tumor. (B) DCA reduced tumor uptake (left) and tumor size in both the prevention (middle) and regression (right) protocols (n≥8 eggs per group; * p<0.05 vs ccRCC). (C) An ex ovo onplant angiogenesis assay shows that both DCA treatment and reintroduction of functional von Hippel-Lindau (VHL) factor in ccRCC cells (via stable transfection) reduced the angiogenic index (n>75 onplants per group; * p<0.01 vs ccRCC, # p<0.05 vs DCA). The microphotographs show the grid with the ccRCC cells in situ (top) and after lectin injection. ccRCC = clear-cell renal cell carcinoma. European Urology 2016 69, 734-744DOI: (10.1016/j.eururo.2015.09.014) Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 6 Dichloroacetate (DCA) reduces tumor size, proliferation, and angiogenesis in a nude mouse xenograft model. (A) Representative photographs of flank xenograft tumors. (B) DCA reduced tumor volume (n≥10 mice per group; * p<0.01 and ** p<0.001 vs clear-cell renal cell carcinoma [ccRCC]) and weight after 1 mo of DCA treatment in drinking water (n≥10 mice per group; * p<0.001 vs ccRCC). (C) Immunofluorescent staining shows a reduction in the proliferative marker ki67 (n=4 mice per group; * p<0.01 vs ccRCC), an increase in the amount of apoptosis (TUNEL; n=3 mice per group; * p<0.01 vs ccRCC), and a reduction in the amount of VEGF (n=3 mice per group; * p<0.01 vs ccRCC) in tumors from DCA-treated animals. (D) Three-dimensional stereotactic analysis of stacked confocal imaging of lectin-injected tumors shows reduced vascular density in tumors from DCA-treated animals (n=3 mice per group; * p<0.01 vs ccRCC). (E) Proposed mechanism for the effect of DCA in ccRCC. TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling. European Urology 2016 69, 734-744DOI: (10.1016/j.eururo.2015.09.014) Copyright © 2015 European Association of Urology Terms and Conditions