Supplementary Figures from Targeting the Mevalonate Pathway Suppresses VHL-Deficient CC-RCC through an HIF-Dependent Mechanism
Authors: Thompson Jordan M. , Alvarez Alejandro , Singha Monika K. , Pavesic Matthew W. , Nguyen Quy H. , Nelson Luke J. , Fruman David A. , Razorenova Olga V. 
Published: April 2023

<p>Supplementary Figure 1. VHL re-expression blocks HIF expression. Western blot showing the effect of VHL re-expression in CC-RCC cell lines on HIF1α, HIF2α, and LDHA (HIF target gene) expression. α- tubulin serves as a loading control. Supplementary Figure 2. Simvastatin and Fluvastatin treatment causes synthetic lethality with VHL loss in multiple CC-RCC cell lines. Treatment with Simvastatin (a-c) and Fluvastatin (d-f) reduces the colony forming ability of VHL-deficient CC-RCC. Representative plates from the clonogenic assays quantified in Figure 1 are shown for the indicated compound concentrations for RCC4{plus minus}VHL (a, d), RCC10{plus minus}VHL (b, e), 786-O{plus minus}VHL (c, f) isogenic cell line pairs. (g-i) Treatment with Fluvastatin reduces VHL-deficient CC-RCC''s proliferation as shown by the decrease in cell number making up each colony. Representative colonies from the clonogenic assays quantified in Figure 1d-f are shown for the indicated Fluvastatin concentrations for RCC4{plus minus}VHL (g), RCC10{plus minus}VHL (h), and 786-O{plus minus}VHL (i) matched cell lines. Supplementary Figure 3. Lovastatin treatment causes synthetic lethality with VHL loss in the RCC4 matched cell line, while Pravastatin does not. Treatment with Lovastatin (a) reduces the colony forming ability of VHL-deficient RCC4. (b) Treatment with Pravastatin does not reduce the colony forming ability of either RCC4 or RCC4VHL. Statistical analysis was performed using a paired t-test between the matched cell lines at each dose (* p < 0.05, ** p < 0.01), SEMs are shown. (c) Western blot showing that Fluvastatin, Simvastatin, and Lovastatin treatments cause the appearance of unprenylated Rap1a, while Pravastatin treatment does not. α-tubulin serves as a loading control. Supplementary Figure 4. Statin treatment is cytostatic and cytotoxic in VHL-deficient CC-RCC. LIVE/DEAD assay measuring live cell numbers via Calcein staining (a) and dead cell numbers via PI staining (b) reveals that Simvastatin treatment inhibits 786-O cell proliferation and triggers cell death at low micromolar doses. 786-O{plus minus}VHL cells were treated with Simvastatin or vehicle control (80%DMSO/20%Ethanol) for 6 days. Calcein-positive cells in (a) were normalized to the vehicle control. Normalized % viability in (b) was calculated as described in Figure 2b. Statistical analysis in (a-b) was performed using a paired t-test between the matched cell lines at each dose (* p < 0.05, ** p < 0.01), SEMs are shown. (c) Representative images of LIVE/DEAD assay at three Simvastatin doses. Supplementary Figure 5. The mevalonate synthesis pathway. Statins target HMG-CoA reductase, the rate-limiting enzyme in the mevalonate synthesis pathway. Inhibition of mevalonate synthesis decreases the concentration of the isoprenoid geranylgeranyl pyrophosphate, which is required for isoprenylation and intracellular trafficking of GTPases (shown in red). Disruption of GTPase isoprenylation prevents their ability to activate their downstream targets like ROCK. Inhibition of mevalonate synthesis also decreases cholesterol synthesis. Supplementary Figure 6. The cytostatic and cytotoxic effects of Simvastatin can be rescued with Mevalonate. LIVE/DEAD assay measuring live cell numbers via Calcein staining (a) and viability via PI staining (b) reveals that the coadministration of mevalonate with Simvastatin results in a partial rescue of proliferation in RCC4 cells and a complete rescue in RCC4VHL cells even at high 10 and 20µM doses. LIVE/DEAD assay measuring viability via PI staining (b) reveals that the addition of mevalonate completely rescues both RCC4 and RCC4VHL cells from the cytotoxic effects of Simvastatin. (c) Representative images of LIVE/DEAD assay with the indicated treatments. RCC4 and RCC4VHL cells were treated with Simvastatin, Simvastatin and mevalonate, or vehicle control (80%DMSO/20%Ethanol) for 6 days. Calcein-positive cells in (a) were normalized to the vehicle control. Normalized % viability in (b) was calculated as described in Figure 2b. Statistical analysis in (a-b) was performed using a paired t-test between the matched cell lines at each dose (* p < 0.05, ** p < 0.01, *** p < 0.001), SEMs are shown. Supplementary Figure 7. Statin treatment in VHL-deficient CC-RCC cells disrupts cell cycle progression and increases apoptotic/debris cell population. (a) BrdU assay reveals that statin treatment is both cytotoxic and cytostatic in RCC4. RCC4 cells increase G0 cells, increase apoptotic/debris population, and reduce S phase population upon treatment with Fluvastatin for 6 days as opposed to RCC4VHL. The graph shows the representative experiment of two experiments performed. Treatment with 2µM Staurosporine for 4 hours serves as a positive control. (b) Representative flow cytometry plots quantitated in a. Supplementary Figure 8. The effect of statins on cholesterol synthesis is not involved in synthetic lethality with VHL loss. (a) Addition of up to 1000μM Squalene does not rescue the effect of Fluvastatin on colony forming ability of RCC4 cells. Each dose of Fluvastatin or vehicle control (DMSO) within each experiment was tested in duplicate, and the experiment was repeated two times. Statistical analysis was performed using a paired t-test between the matched cell lines at each dose (* p < 0.05), SEMs are shown. (b) Squalene is incorporated and converted to cholesterol in RCC4 cells as shown by increased Filipin III stain intensity in comparison to the DMSO control. Propidium Iodide (PI) was used to label cell nuclei. U-18666A is a cholesterol trafficking inhibitor (positive control) with the chemical structure shown in (c). Supplementary Figure 9. Treatment with statins inhibits ROCK substrate phosphorylation. Western blot showing that 24h treatment with Fluvastatin is sufficient to inhibit phosphorylation of LIMK1/2 in the RCC10{plus minus}VHL (a) and 786-O{plus minus}VHL (b). Unprenylated RAP1a is used as a readout for the disruption of GTPase isoprenylation by treatment with Fluvastatin. α-tubulin serves as a loading control. Supplementary Figure 10. Fluvastatin causes synthetic lethality with VHL loss in 786-OT1 cells similar to 786-O. In a clonogenic assay Fluvastatin shows selective toxicity towards VHL-deficient 786-OT1 and 786-O while sparing 786-OVHL cells expressing VHL. Each dose within each experiment was tested in duplicate, and each experiment was repeated three times. IC50s are indicated. Statistical analysis was performed using a General Linear Model comparing cell line and treatment followed by Tukey''s Post Hoc analysis. A statistically significant difference was observed between 786-OVHL and the two VHL-deficient 786-O and 786-OT1 cell lines (*** p < 0.001), SEMs are shown. Supplementary Figure 11. 15mpk Fluvastatin inhibits tumor cell proliferation and increases apoptotic cell death in vivo. Representative images of (a) Ki67 staining and (b) TUNEL staining. 786-O cells treated with 2µM Staurosporine for 4 hours were used as a positive control in b. Images were obtained at 20x.</p>