1. Neurodegeneration or Hereditary Tumor Syndrome? The Butterfly Effect of Mutated Genes Encoding Mitochondrial Enzymes * Prasanna G Vibhute MD, * Vivek Gupta MD, ** Girish M Fatterpekar MD, *** James Henry MD Departments of * Radiology, Mayo Clinic, Jacksonville, FL * * Radiology, NYU, New York, NY * * * Pathology, Univ. Texas HSC, San Antonio, TX

2. Poster # eEdE-03 Control # 2060 Disclosures: –Prasanna Vibhute, MDnothing to disclose –Vivek Gupta, MDnothing to disclose –Girish Fatterpekar, MDnothing to disclose –James Henry, MDnothing to disclose Neurodegeneration or Hereditary Tumor Syndrome? The Butterfly Effect of Mutated Genes Encoding Mitochondrial Enzymes

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4. Learning Objectives To understand how mitochondrial enzymes defects can lead to hereditary tumor syndromes and neurodegenerative disorders. To describe the cytogenetics, molecular signaling and the molecular biology related to these mitochondrial disorders To correlate the imaging findings to histopathology and phenotype characteristics

5. Butterfly Effect A relatively small change in the initial conditions of a deterministic nonlinear system can evolve into large differences in the final states: the possibility that a large storm in New England may be caused by fluttering of a butterfly in China, a slight global warming can cause widespread climate fluctuation, and slight variation in biomolecule concentration can change the outcome from cell death to uncontrolled cell proliferation.

6. Introduction: Salient Krebs Cycle Enzymes Succinate dehydrogenase (SDH) and Fumarate Hydratase (FH) catalyse two sequential steps of the mitochondrial Krebs or Tricarboxylic Acid (TCA) cycle. Succinate Fumarate Malate TCA cycle SDH FH

7. Introduction: SDH and FH Germline Mutations HPGL: Hereditary paraganglioma syndrome HLRCC: Hereditary cutaneous & uterine leiomyoma, & renal cell carcinoma syndrome Germ line mutations Heterozygous Homozygous Neurodegenerative diseases: Leigh Syndrome FHSDH HPGL syndrome HLRCC syndrome SDH/FH

8. Cell Biology of Mitochondria Outer membrane: Relatively permeable and composed predominantly of phospholipids Inner membrane: –Highly impermeable with high protein content –Has protein complexes of electron transport chain (ETC) Central matrix: –Contains components of Krebs i.e.Tricarboxylic acid (TCA) cycle, mitochondrial DNA and ribosomes. View ETC Animation

9. Span the mitochondrial inner membrane Active component of TCA cycle Projects into mitochondrial matrix 4 subunits Each encoded by a different gene AC and D Anchors A to C and D B Succinate Dehydrogenase (SDH) = ETC Complex II transmembrane subunits matrix subunits

10. Succinate Dehydrogenase (SDH) = ETC Complex II Only ETC complex encoded by nuclear DNA Mutations that disrupt the complex will compromise its function These mutations include not only genes encoding four subunits (A, B, C, and D) of the SDH complex, but several others involved in assembly and function of the complex.

11. Proposed Models of Tumorigenesis in HPGL Model 1: ETC dysfunction - Increased reactive oxygen species (ROS) Model 2: TCA dysfunction -Accumulation of succinate/fumarate with competitive inhibition of -KG-dependent dioxygenases.

12. Model 1: Increase Reactive Oxygen Species View ROS Animation Damages macromolecules – Lipids, Proteins & DNA Increased ROS ETC II dysfunction HIFs PI3K Metabolic adaptations Tumorigenesis Increased Proliferation, Survival, Mobility Oxidative Stress HIFs (Hypoxia inducible factors): allow tumor to adapt to diminished oxygen environment. PI3K (phosphoinositide 3-kinase): an important growth factor response pathway hyper- activated in many cancers

13. Model 2: Inhibition of -KG-dependent dioxygenases by Succinate & Fumarate HO-HIFs Histones DNA Mature collagen L-Carnitine DNA RNA HIFs Methylated histones Methylated DNA Immature collagen N-Trimethyllysine Damaged DNA m6A RNA O 2 + α-KG CO 2 + Succ Dioxygenases Examples of Dioxygenases and targets Prolyl hydroxlase: Hypoxia inducible factor (HIF) proteins Histone demethylases: Methylated histones DNA demethylases: Methylated DNA Normal cytosolic conditions

14. Model 2: Inhibition of -KG-dependent dioxygenases by Succinate & Fumarate HO-HIFs Histones DNA Mature collagen L-Carnitine DNA RNA Increased cytosolic Succinate/Fumarate O 2 + α-KG CO 2 + Succ Dioxygenases Tumorigenesis HIFs Methylated histones Methylated DNA Immature collagen N-Trimethyllysine Damaged DNA m6A RNA

15. SuccinateFumarate Malate -SDH- FH HPGL Syndrome HLRCC Syndrome Increase in HIF transcriptional activity. Inhibition of -KG-dependent dioxygenases Increase in ROS Hypoxia Inducible Transcription Factor (HIF) Induction

16. HIF Pathway & Hydroxylases ? Final mediator in neo- angiogenesis/tumorigenesis Active Hydroxylases O 2 High O 2 Low Inactivation of HIF-α through the pVHL mediated proteolysis InactiveHydroxylases Stabilization of HIF-α and increase in HIF transcriptional activity NeoangiogenesisTumorigenesis gradientgradient

17. Succinate Fumarate HPGL syndrome Loss of Succinate dehydrogenase Inhibition of prolyl hydroxylases Increase in HIF ROS ETC Dysfunction Hypervascular paraganglioma Ex: Carotid body tumor DSA Succinate, ROS and HIF

18. Homozygous SDHA & FH mutations Neurodegeneration Ex. Leigh syndrome Severe energy deficits Loss of mitochondrial synthetic capacity and ↑↑ ROS Death of cells with high energy requirements & O2 consumption under minor hypoxic-ischemic stresses (e.g. neurons) before activation of metabolic adaptations Total loss of enzymatic function Genophenotype Characteristics

19. Heterozygous SDH A, B, C, D mutations Partial loss of enzymatic function, ↑ ROS Metabolic adaptive mechanism activated (↑ glycolysis etc.) Mitochondrial synthetic activity intact, normal development Inherited HPGL syndrome Loss or mutation of the wild allele in cells of neural crest origin Complete loss of SDH function Active adaptive mechanisms prevent cell death ↑↑ ROS ↑ Expression of antioxidant proteins ↑HIF stabilization 2nd Hit Genophenotype Characteristics

20. Cytostatic ROS Generation ROS Scavenger (Cellular adaptive mechanisms) (mutations, hypoxia) Neoplasia (HPGL, HLRCC) Cytotoxic Neurodegeneration (Leigh syndrome) ROS Generation: Scavenger Equilibrium

21. Imaging Spectrum & Rad-Path Correlation Hereditary neurodegenerative disorder - usually autosomal recessive Usually affects young children and inevitably leads to death. Neuropathologic endpoint of disordered cerebral mitochondrial energy production Caused by gene mutations affecting ETC proteins and pyruvate dehydrogenase. Leigh Syndrome

22. Pathology Leigh Syndrome: Histology Leigh Syndrome : Early necrosis with focal sparing of neurons (arrows), attenuation of the neuropil and vascular proliferation (arrowheads). (H &E) Low powerHigh power

23. Pathology Leigh Syndrome: Gross A: Bilateral necrosis of lentiform nuclei (arrow heads) B: Bilateral perithird ventricular (black arrows) and putaminal necrosis (arrow heads); note sparing of the mamillary bodies (white arrows) C: Symmetric involvement of the midbrain (arrows) AB Putaminal involvement and sparing of the mamillary bodies are consistent but not constant features. C

24. Symmetrical lesions mainly in the basal ganglia, brain stem, white matter and posterior columns of the spinal cord. Putaminal involvement and sparing of the mamillary bodies are consistent but not constant features. MRI: Leigh Syndrome (Subacute necrotizing encephalomyelopathy) Axial T2-WIDiffusion-WI Case courtesy of Dr. Hemant Telkar, Mumbai, India

25. Hereditary Paraganglioma (HPGL) Syndrome WHO Definition: Pheochromocytoma: (Intra-adrenal paraganglioma): A tumor arising from chromaffin cells in adrenal medulla. Extra-adrenal paraganglioma: A tumor in extra-adrenal sympathetic & parasympathetic paraganglia. Sporadic Vs familial(30%) Familial: MEN 2, NF 1, VHL, HPGL syndrome

26. Pathology PGL: Microscopy Paraganglioma: Vascular tumor with nestling and clustering of chief cells – characteristic “ Zellballen ” (cell ball) growth pattern. The delicate fibrovascular network (arrows) surrounds each "ball" of cells.

27. Three siblings, two brothers and one sister (not shown) all with head and neck paragangliomas. As no sibling had stigmata of Neurofibromatosis type 1, VHL disease or MEN2, these were probably related to SDHD mutations Familial PGL Example # 1 Axial T2-WI demonstrates bilateral carotid body tumors (A) and left vagal paraganglioma (B) Axial post contrast T1-WI demonstrates a large right carotid body tumor in second sibling A B C Sibling # 1Sibling # 2

28. Familial PGL Example # 2 DSA in another patient: Multiple head and neck hypervascular paragangliomas (arrows). SDHD mutations and partial inactivation lead to head and neck paragangliomas. SDHB mutations and complete disassembly of complex II predisposes to pheochromocytoma. RTLT

29. True or false: Any mutation that abolishes SDH function should have the same outcome in susceptible tissues? SDH Genotype-Phenotype Mystery FALSE!

30. SDH Genotype-Phenotype Mystery Different kinds of SDH loss-of-function molecular pathologies lead to different tumor phenotypes! Probably due to mutation that reduces but doesn't eliminate a gene’s functionality (hypomorphic alleles)

31. SDH Genotype-Phenotype SDHASDHBSDHCSDHD GENE 5p15.331p36.1-p351q2111q23 PENETRANCE Very low77%less penetrant Highly penetrant PHENOTYPE5 PGL & 1 PheoPheoH/N PGLMultiple H/N PGL Very rareRareMost common Often aggressive Often benign Leigh syndrome (recessive) RCC Pheo: Pheochromocytoma; PGL: Paraganglioma; RCC: Renal cell carcinoma

32. Other Tumors Associated with SDH Gene Mutations 3 autosomal dominant hereditary syndromes: 1.~ 7.5% Gastrointestinal stromal tumor (GIST) SDHA (most common), B, C and D germline mutations and epigenetic silencing 2.GIST with PGL (Carney-Stratakis dyad) SDHB, SDHC or SDHD germline mutations 3.GIST with PGL and pulmonary chondroma (Carney triad) SDHC gene locus epigenetic silencing

33. Carney Triad: GIST, PGL and pulmonary chondroma As in most cases this 26 year old female had 2 out of 3 tumors at presentation. Pulmonary chondromaGastric GIST with hepatic metastasis

34. HLRCC Solitary papillary RCC type 2, Collecting duct RCC Multiple uterine benign & malignant smooth muscle neoplasms Poor prognosis Courtesy Dr. Peter Choyke, NIH

35. Not due to ROS or Succinate production Mutation or deletion of RET (10%), VHL genes (4%); Allelic losses at 1p, 3p (45%), 17p, & 22q chromosomes Similar hypervascular imaging appearance Retain full SDH activity Utilize HIF Pathway CT and DSA: Left Carotid Body Tumor A B Sporadic Paragangliomas

36. Paradox Neurodegeneration Vs Neoplasia Early childhood death before development of neoplasia HIF pathway in severe hypoxia induces pro-apoptotic genes: cell deat ? No neoplasia Neurodegeneration

37. Paradox Neurodegeneration Vs Neoplasia Neoplasia Altered mitochondrial membrane composition by mutant SDH prevents release of mediators of energy independent apoptosis HIF induced transcription of oncogenic genes Mutant SDH & FH ? Loss of tumor suppression function Insufficient energy dependent apoptosis ROS induced Oncogenic mitogen activated protein kinase pathway (MAPK)

38. Summary A fascinating group of mitochondrial disorders show remarkable biological diversity despite sharing a common gene defect. Homozygous gene mutations (that encode SDH/FH enzymes) result in neurodegenerative disorders, heterozygous mutations of the same genes cause inherited neoplastic syndromes. Improved understanding of these disorders may help understand the imaging manifestations, develop new imaging survellence protocols and treatment options.

39. References: Eng C, et al; A role for mitochondrial enzymes in inherited neoplasia and beyond. Nature reviews/Cancer (2003), 3: 193-202 Gottlieb E and Tomlinson Ian; Mitochondrial tumour suppressors: A genetic and biochemical update. Nature reviews/Cancer (2005), 5: 857- 859 Selak MA, et al; Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-α prolyl hydroxylase. Cancer Cell (2005), 7: 77-85 Pollard PJ, et al; The TCA cycle and tumorigenesis: the examples of fumarate hydratase and succinate dehydrogenase. Ann Med (2003), 35: 632-639 Plouin P, Gimenez-Roqueplo A; The genetic basis of pheochromocytoma: who to screen and how? Nature Clinical Practice (2006), 2(2): 60-61 Sena and Chandel; Physiological roles of mitochondrial reactive oxygen species. Mol Cell. 2012 October 26; 48(2): 158–167 Sullivan and Chandel; Mitochondrial reactive oxygen species and cancer. Cancer & Metabolism 2014, 2:17 Yeng F. Her and L. James Maher III; Succinate Dehydrogenase Loss in Familial Paraganglioma: Biochemistry, Genetics, and Epigenetics. International Journal of Endocrinology, Volume 2015, Article ID 296167, 14 pages

40. II I IIIIV Electron Tranfer Chain (ETC) Contains series of four protein complexes embedded in the mitochondrial inner membrane. Electrons captured from the donor molecules of the TCA cycle are transferred through these complexes and finally accepted by the molecular oxygen. KREBS/TCA Cycle e-e- e-e- O2O2 H 2 O + CO 2 Inter-Membrane Space Outer Membrane Matrix Inner Membrane Cytosol Small energy packets released during the electron transfer between neighbouring complexes are utilized to pump protons (hydrogen ions) from the matrix into the inter- membrane space, creating a gradient across the inner membrane. H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ The protons can only flow back into the matrix through the ATP synthetase. ATP Synthetase Potential energy released by these protons is harvested by the ATP synthetase to form the high energy phosphate bonds of the ATP ADP ATP

41. FAD: flavin adenine dinucleotide; Q: ubiquinone; ROS: reactive oxygen species A B CD FAD FADH 2 Heme b Q QH 2 3 x [Fe-S] Succinate Fumarate Malate SDH FH TCA cycle Complex II ETC Complex III Cytochrome c Q Cycle Q O2O2 O 2. (ROS) O2O2 Q ROS Production O 2. (ROS) QH 2 Q INNER MEMBRANE MATRIX