1 Karel Pacak [email protected]Basic and clinical research in pheochromocytoma (PHEO): a winning combination National Institutes of Health Disclosure Nothing to disclose 1. Summarize current basic-clinical discoveries that represent a winning combination in how we diagnose PHEO patients today: a/ genetics b/ biochemistry c/ metabolomics d/ functional imaging Lecture outline The lecture will focus on the main discoveries over the course of the past 4 years. 2. Promising future therapeutic approaches derived from basic research discoveries 3. Conclusions/perspectives Biochemical Diagnosis 2000 2011 1990 1950 Improved understanding of catecholamine metabolism Colorimetric Assays HPLC Assays LC-MS/MS (routine) Shift from catecholamines to metanephrines Methoxytyramine 2000 2007 1990 1950 New PET Ligands: 18 F-FDA, 18 F-FDOPA MRI & CT MIBG scintigraphy PET DOTA analogs ( 68 Ga-DOTA-TATE/TOC/NOC) Imaging Genetics 2000 2013 1990 1886 1950 NF1 SDHD SDHC VHL RET SDHB SDHAF2 TMEM127 PGL syndromes Fränkel NF1 MEN 2 VHL 2012 2014 PHD2 IDH1 SDHA MAX HIF2A H-RAS FH PHD1 MDH2 rule-of-ten defunct Adapted from G. Eisenhofer PGL: paraganglioma PHD1: prolyl hydroxylase 1 MDH2: malate dehydrogenase 2 PHEO/PGL: Continuing progress
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Basic and clinical research in pheochromocytoma(PHEO): a winning combination
National Institutes of Health
Disclosure
Nothing to disclose
1. Summarize current basic-clinical discoveries thatrepresent a winning combination in how we diagnosePHEO patients today:a/ geneticsb/ biochemistryc/ metabolomicsd/ functional imaging
Lecture outlineThe lecture will focus on the main discoveries over the course of the past 4 years.
2. Promising future therapeutic approaches derived frombasic research discoveries
• In over 50% of PHEOs/PGLs, a genetic defect is known(35-40% have germline and 15-25% harbor somaticmutations); 19 PHEO/PGL susceptibility genes arecurrently known
Current important facts:
• Biochemical dx. and localization are highly successful
• There is no satisfactory cure for metastatic PHEO/PGL
• International studies and consortia are evolving
SDHC SDHD
SDHB
SDHA
Mitochondrial innermembrane Complex III
3x[Fe‐S]
Fumarate
FADFADH2
Cytochrome C
QH2
Q
QH2
Ubiquinol
Ubiquinone
Qcycle
O‐2O2
O‐2
Mitochondrial matrix
O2
Succinate
e‐
Q
QQQQ
Q
ROSROS
ROS
HIF‐αstabilization
Genomic instability, oncogene activation
Lipid, protein, nucleotide synthesis
Krebs cycle
Cell proliferation, cell migration, invasiveness and survival
Apoptosis AngiogenesisGlycolysis
SDHxmutations
Adapted from Vicha, Taieb, Pacak, Endocrine Relat. Cancer 2014; 21:R261Opocher & Schiavi, Endocr. Pract. 2011; Suppl. 3:64Baysal et al. Science 2000; 287:848
Hereditary SDHx PHEO/PGL
nucleus
Glucose
GLUTs
Glucose 6‐phosphate
Hexokinase I‐II
Phosphoenolpyruvate
Pyruvate
PKM2
LDHA
Lactate
Glucose
Acetyl‐CoA
Oxalacetate
Malate
Fumarate
SuccinateSuccinyl‐CoA
Citrate
Isocitrate
α‐Ketoglutarate
ROS
Succinate
Succinate
H+
H+
H+
H+
H+
Glutamine
GutamateGlutamate
Glutamate
Glutaminolysis
HIF‐1/2αProteasomedegradationHIF‐1/2α
OH OH
VHL
Succinate
PHDs
HIF‐1/2α
HIF‐2α
HIF‐1β
HIF‐1α
HIF‐1β
OXPHOS dysfunction
DNA instability=
HRE HRE
Target mRNAs
HIF‐1α
HIF‐2α
ABL2EPOPOU5F1OCT4CCND1DLL4PDGFVEGF
Upregulation ofHIF‐related genes
pH
Adapted from Vicha, Taieb, Pacak, Endocrine Relat. Cancer 2014; 21:R26Castro-Vega et al. Hum. Mol. Genet. 2014; 23: 2440Ladrue et al. NEJM 2008; 359:2685Robledo: personal communication; Yang et al. J. Mol. Med.; in press
Toledo et al., 201310 C1591A P531T Y47 C1591T P531S Y72 C1592T P531L Y? C212A S71Y Y
Buffet et al., 201416 T1586C L529P Y2 C1625T L542P Y/N
Toyoda et al., 201415 C1589A A530E Y
HIF2A mutations: more patients are being discovered
Favier et al. NEJM 2012; 367:2161 Taieb et al. JCEM 2013; 98:E908Comino-Mendez et al. 2013; 22:2169Toledo et al. 2013; 20:349Buffet et al. JCEM 2014; 99:E369Toyoda et al. Pediatrics 2014; 133:e1787
New germline PHD1 (EGLN2) mutation
Young et al. J. Mol. Med. in press
• Some patients with polycythemia and PHEO do not have the HIF2A mutationand they present with normal or slightly elevated EPO levels
• Germline PHD2 mutations described previously in patients withpolycythemia and in one patient also with PGL
SDHx: A hypermethylator phenotype145 PHEOs/PGLs were included (17 SDHx, 21 VHL, 30 NF1, 13 RET and 64 others).
Immortalized mouse chromaffin cellsharboring a complete defect in SDH
• Increased migration, well inhibited by decitabine(inhibits DNA methyltransferase activity)
• Histone methylation was increased (also inhuman samples)
Letouze et al. Cancer Cell 2013; 23: 739
8
PHEO and somatostatin receptors: imaging
• SSTR imaging can be performedwith PET/CT, improving spatialresolution; also provides morerapid and whole-body tomographicimaging for precise anatomiclocalization
• Available 3 DOTA-coupled peptides include: 68Ga-DOTATATE, 68Ga-DOTATOC, 68Ga-DOTANOC
• PHEOs express 5 somatostatin receptors (SSTRs), allowing for the use of Octreoscan scintigraphy (relatively poor spatial resolution)
[68Ga]-DOTATATE [18F]-FDG [18F]-DOPA [18F]-FDA
[68Ga]-DOTATATE [111In]-Pentetreotide [123I]-MIBG NIH group & Taieb et al.; ongoing studies
68Ga-DOTA analogs: A future imaging approach?
Future treatment options: HIF- inhibitors
HIF-αtranslation
mTOR &topoisomerase
inhibitorsCardiac glycosides
PX-478
HIF-αdimerizationstabilization
HSP90 & HDACinhibitors
AcriflavineAntioxidantsAmphotericin
HIF-αDNA bindingdegradation
AnthracyclinesHSP90 inhibitorsPHD activation
Semenza et al. Trends Pharmacol. Sci. 2012; 18:534Powers et al. PLoS One 2014; 9:e87807
SDHB missense mutations: it is about protein degradation, not production
mRNA
protein
Protein+HDACi
Yang et al. FASEB J. 2012; 26:4506
9
Other future promising targets for the treatment of metastatic PHEO/PGL
Shlomi et al. Plos Comp. Biol. 2011; 7: e1002018Lopez-Lazaro Mol. Med. 2010; 16:144Chae et al. Nar. Commun. 2013; 4:2139Letouze et al. Cancer Cell 2013; 23:739Nolting et al. Endocrinology 2014; 155:2377Fliedner et al. PLoS One 2014; 9:e97712Baudin et al. Eur. J. Endocrinol. 2014; 171:R111Powers et al. PLoS One 2014; 9:e87807
1. Mitochondrial proton pump modulators
2. SDHB stabilization (HDAC inhibitors)
Future therapeutic targets
4. Topoisomerases inhibitors
3. Inhibition of cholesterol synthesis
5. Demethylating agents (5-Azacytidine)
CONCLUSIONS/PERSPECTIVES
Together we have a chance; separately we fail…
• Further promote tight collaboration, informationexchange, and unique teamwork between patients,clinicians, and scientists across various institutions.
• Bench-to-bedside and bedside-to-bench projects aremost valuable to our success in better treating patients inthe near future.
Acknowledgements
Many thanks to all the members of my laboratory, attendings,and endocrine, oncology, and surgery fellows for their longhours, dedication, and passion for helping those who suffer.
“Patients are our passion and we are their hope”
Many thanks to outside NIH co-investigators:G. Eisenhofer, J. Lenders, D. Taieb, S. Fliedner, A. Tischler,H. Timmers, M. Robledo, L. Mercado-Asis, A. Vicha, A. Grossman, H. Ghayee, R. Lechan, Z. Frysak, H. Lehnert, J. Breza,TCGA members, PheoPara Alliance, VHL Alliance and many others
AASNT
10
HIF-2 signaling in cluster 1 hereditary PHEO/PGL: Turning the rudder in the right direction?
HSP90
p300 CBP
HIF‐β HRE
HIF‐αtranscriptional
activity
HIF‐α
NUCLEUS
HIF‐αPHD HSP90
HIF‐α
α‐ketoglutarate + O2
Ascorbate + Fe2+
SSAT2
E3 ubiquitinligase complex
UQ
OH OH
E2
HIF‐α
ElonginB
pVHL
Cul2Rbx1Elongin C
Proteasomaldegradation
CYTOSOL
Cluster 1
ΔHIF‐α
Oxaloacetate
L‐Malate Fumarate
Succinate
Succinyl CoA
α‐ketoglutarate Isocitrate
Citrate
SDHxFHMDH2
CS
ACOIDH OGDH
SUCLG
Mitochondrial complex II
Krebs cycle
MITOCHONDRIA
HIF-1/2 signaling in cluster 2 hereditary PHEO/PGL: Turning the rudder in the right direction?
HSP90
p300 CPB
HIF‐β HRE
HIF‐α transcriptionalactivity
HIF‐α
HIF promoterNF‐κB
Induction of HIFαexpression
NUCLEUS
Growth factors(EGF, TGFα, IGF, NGF,…)
Receptor tyrosine kinase( ET , IGF1‐R, FGF‐R,
VEGF‐R, EGF‐R)RET
cMyc
PI3K
Ras
Akt
TSC1/2
mTORC1
Rheb
eIF‐4E S6K
mTORC2
NF1
MAX
TMEM127
Raptor
HIFα translation (protein synthesis)
HIF‐α
Cluster 2
CYTOSOLEXTRACELLULAR
HIFαmRNA
The Cancer Genome Atlas
2. Announced in 2009 to map the genomes of at least 20 cancers by 2014
3. Nine rare cancers included – PHEO/PGL is one of them (2012)
TCGA facts:
5. Researchers across the US and other nations to collaborate
1. A comprehensive, collaborative effort led by the NIH
4. Data types: whole exome, mRNA, and miRNA sequencing, DNAmethylation, and DNA copy number
6. The PHEO/PGL project is expected to be completed by 2015
Understanding genomicsto improve cancer care
The glory of medicine is that it is constantly moving forward, that there is always more to learn.