Supplementary Figures S1-S23 from Convergent Genetic Adaptation in Human Tumors Developed Under Systemic Hypoxia and in Populations Living at High Altitudes

Supplementary Figure S1. Description of the CCHD-PPGL cohort of 27 individuals with CCHD who developed 38 PPGL tumors. Supplementary Figure S2. Summary of the clinical features of patients with CCHD-PPGL. Supplementary Figure S3. EPAS1 mutations are enriched in the hypoxic cohort of patients with CCHD-PPGL (88.8%) compared to the normoxic cohort of patients without CCHD-PPGL (4.5%). Supplementary Figure S4. EPAS1 clonality in PPGL tumor samples from TCGA(1–4) (patients without CCHD with PPGL in normoxic conditions). Supplementary Figure S5. No EPAS1 mutations were detected in carotid body PGL tumors. Supplementary Figure S6. Hierarchical cluster analysis of RNA sequencing data from EPAS1-mutated CCHD-PPGL tumors. Supplementary Figure S7. Convergent Evolution in Natural Populations and Human Tumors. Supplementary Figure S8. Evolutionary speed of EPAS1 selection in different cohorts. Supplementary Figure S9. Hematoxylin and eosin staining and immunohistochemistry for Chromogranin A (neuroendocrine tumors maker) and for mismatch repair (MMR) proteins in PPGL tumors. Supplementary Figure S10. Acquisition of EPAS1 mutation and subsequent selection in sympathetic PPGL tumors developed under systemic hypoxia. Supplementary Figure S11. Assessment of apoptosis marker in pheochromocytoma-derived cells upon expression of mutant EPAS1 genes or exposure to hypoxic conditions. Supplementary Figure S12. Increase levels of fumarate metabolite in EPAS1MUT PPGL tumors and increase levels of succinate metabolite in SDHxMUT PPGL tumors. Supplementary Figure S13. Decrease of fumarate hydratase/fumarase (FH) mRNA expression and increment of SDHB mRNA expression in EPAS1MUT/VHLMUT PPGL tumors when compared to EPAS1WT/VHLWT PPGL tumors, as well as to SDHxMUT PPGL tumors. Supplementary Figure S14. Decrease of FH mRNA expression in missense EPAS1-mutated tumors (n = 8) when compared to EPAS1-wild-type tumors (n = 170). Supplementary Figure S15. Elevated fumarate and succinate levels in EPAS1-Overexpressing PPGL cells. Supplementary Figure S16. Oxygen consumption and supply balance in the mitochondrial electron transport chain as an advantageous survival and proliferative mechanism. Supplementary Figure S17. Gene-level extremely positive selection on EPAS1 gene in systemic hypoxia. Supplementary Figure S18. Time-dependent effects of systemic hypoxia on oxygen-sensing tissues. Supplementary Figure S19. Specialization process of the HIF2α gene for adaptation to systemic hypoxia. Supplementary Figure S20. Proliferative advantage and increased sensitivity to HIF2α inhibition in HEK293T cells expressing the EPAS1 P405A/P531A double mutant. Supplementary Figure S21. Authentication of PPGL-derived PC12 cell line. Supplementary Figure S22. Full western blots shown in Fig. 3B. Supplementary Figure S23. Full western blots shown in Supplementary Fig S11.