1
Karel [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 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
3. Conclusions/perspectives
Biochemical Diagnosis
2000 201119901950
Improved understanding ofcatecholamine metabolism
Colorimetric Assays
HPLC Assays LC-MS/MS (routine)
Shift from catecholamines to metanephrines
Methoxytyramine
2000 200719901950
New PET Ligands: 18F-FDA, 18F-FDOPA
MRI & CTMIBG scintigraphy PET
DOTA analogs (68Ga-DOTA-TATE/TOC/NOC)
Imaging
Genetics
2000 201319901886 1950
NF1SDHDSDHC
VHLRET SDHB SDHAF2
TMEM127PGL syndromes
Fränkel
NF1
MEN 2VHL
2012 2014
PHD2IDH1SDHA
MAXHIF2A
H-RASFH
PHD1MDH2
rule-of-ten defunct
Adapted from G. Eisenhofer
PGL: paragangliomaPHD1: prolyl hydroxylase 1MDH2: malate dehydrogenase 2
PHEO/PGL: Continuing progress
2
PHEO/PGL: Facts
• 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
FH
Increasedglycolysis
Tumorigenesis
FH: fumarate hydrataseMDH2: malate dehydrogenaseHIF2A: HIF‐2PHD1: prolyl hydroxylase
New genes: A story of metabolic/oxidative stress and hypoxia continues
MDH2
HIF2APHD1
OH
HIF‐1/2α
Goodacre et al. Metabolomics, 2005; 1:1
Proteomics & metabolomics: The way to new discoveries
Improve understanding of PHEO/PGL cell biology
Improve clinical care for afflicted patients
3
Methoxytyramine (MTY) as a new biomarker in the diagnosis of PHEO/PGL
We have found that about 70% of SDHB/DPHEOs and 20-30% of head and neck PGLs secrete MTY.
Eisenhofer et al. Clin. Chem. 2011; 57:411
VHL SDHB SDHD SDHA/C/AF2
NE NE/DA NE/DA NE/DA
17%17% 72%72% LowLow
Adapted from G. Eisenhofer & Fauci et al. Harrison’s Principles of Internal Medicine
Cluster 1
RET NF1 TMEM127 MAX
EPI/NE EPI/NE EPI/NE NE>EPI
<5%<5% 8%8% <5%<5% 10%10%
Cluster 2
Methoxytyramine in cluster 1 & 2 PHEOs/PGLs
67%67%
Methoxytyramine (MTY), tumor size, extra-adrenal location, and SDHB as independent predictors of metastatic PHEO/PGL
365 patients with PHEO, including 105 with metastases, 846 subjects without tumors
SIZE & LOCATIONMTY & METASTASIS4.7-fold higherin patients with thanwithout metastases;independent of tumorburden and NMNlevels
Zelinka et al. Eur.J. Clin. Invest.; 2011; 41:1121 Eisenhofer et al. Eur. J. Cancer; 2012; 48:1739
Eisenhofer et al. Endocr. Relat. Cancer 2011; 18:97
Epinephrine Norepinephrine Dopamine
156 tumors included for genotype-specific catecholamine profiles
Strategy for new genetic screening
• There is a need for a more cost-effective approach • Immunostaining approaches are less expensive than
genetic screening
Immunohistochemistry can be used to triage genetic testing;also to detect recently described a SDHA gene mutation in PHEO/PGL
van Nederveen et al. Lancet Oncol. 2009; 10:764
SDHB SDHC SDHD
VHL MEN NF-1
SDHB immunohistochemistry detects patients with SDHx-related PHEO/PGL
4
Lendvai et al. Endocrinology, 2014; 155:27
PHEO/PGL & metabolomics (1) PHEO/PGL & metabolomics (2)• 234 PHEOs/PGLs from 233 pts (45 had SDHx gene mutations). • Training set: 50 tumors; validation set: 184 tumors. • Metabolite extraction from fresh-frozen tumors, HPLC-tandem MS used.
1 2
Suc
cina
te:fu
mar
ate
ratio
100
101
102
103
104
105
SDHB/D(n=11)
non-SDH(n=39)
p<0.001
1 2
Suc
cina
te:fu
mar
ate
ratio
10-1
100
101
102
103
104
105
SDHB/C/D(n=32)
non-SDH(n=150)
p<0.001
Succinate to fumarate ratio as a predictor for SDHx mutations
97.797.7
Training set Validation set
Table 1. Diagnostic sensitivity and specificity for succinate:fumarate ratios to identify SDHx mutations
All PPGLs PPGLs excl. HNP
HNP
Validation set
Sensitivity [%] 90.9 (30/33) 100 (15/15) 83.3 (15/18)
Specificity [%] 97.3 (145/149)
97.9 (141/144)
80.0 (4/5)
Training and validation set
Sensitivity [%] 93.2 (41/44) 100 (25/25) 84.2 (16/19)
Specificity [%] 96.8 (181/187)
97.3 (177/182)
80.0 (4/5)
HNP- head and neck paraganglioma 1 - Specificity
0.0 0.2 0.4 0.6 0.8 1.0
Sen
sitiv
ity
0.0
0.2
0.4
0.6
0.8
1.0
succinate/fumarate, A = 0.94
High diagnostic performance Krebscycle
Oxaloacetate
Lactate
100
1000
10000
SDHx non-SDH
Pyruvate
0.1
1
10
100
SDHx non-SDH
Acetyl-CoA
Glycolysis
Malate
0.1
1
10
100
1000
SDHx non-SDH
Succinate
1
10
100
1000
10000
SDHx non-SDH
p<0.001
Fumarate
0.01
0.1
1
10
100
1000
10000
SDHx non-SDH
p<0.001
Ketoglutarate
0.001
0.01
0.1
1
10
100
SDHx non-SDH
Citrate
0.1
1
10
100
1000
10000
SDHx non-SDH
p<0.001
Cis-aconitate
0.001
0.01
0.1
1
10
100
SDHx non-SDH
p<0.001
Isocitrate
0.001
0.01
0.1
1
10
100
SDHx non-SDH
p<0.001
Krebs cycle changes beyond succinate and fumarate
Richter et al. JCEM, 2014; 99:3903
SDHx mutations of unknown significance
IHC: SDHB
Modelling
HRMAS
18FDOPA
Metabolomics to predict the pathogenicity of unknown SDHx mutations
MRI
Taieb et al; personal communication
• MYC-associated factor X regulates cell proliferation, apoptosis, differentiation; dimerization of MYC-MAX
• Malignancy in about 10-15% of patients
• PHEO, bilateral; rarely PGL
• Produce NE + EPI
MAX: Tumor suppressor gene
• Paternal transmission (similar to SDHD, SDHAF2)
Camino-Menendez et al. Nat. Genet. 2011; 43:663Burnichon et al. Clin. Cancer Res. 2012; 15:2828
5
Opposing effects of HIF-1 and HIF-2 on chromaffin cell phenotypic features: Insights from MYC-associated factor X (MAX)
Qin et al. Int. J. Cancer 2014; 135:2054Comino-Mendez et al. Nat. Genet. 2011; 43:663
• MAX plays an important role in the control of the MYC/MAX pathway and contributes to numerous neoplastic conditions, including neuroblastoma
• HIF-2 enhances MYC interaction with MAX to stimulate cancer cell progression; HIF-1 has the opposite effect
• Recently, MAX mutations have been found in some PHEOs/PGLs
Opposing effects of HIF-1 and HIF-2 on chromaffin cell phenotypic features: Insights from MYC-associated factor X (MAX)
Two PHEO cell lines were used: PC12 (- MAX) and MPC (-HIF2A)
e
FIT
C-A
FIT
C-A
Brd
U
DNA
FIT
C-A
24±0.55% PC12wt
FIT
C-A
16±0.73% PC12+MAX
*
23±0.39% MPC+Hif2α
29±0.36% MPCwt
PE-A PE-A
*
PC12
PC12wt
Silence Hif1α 0.0
0.4
0.8
1.2
Pnmt Relative mRNA expression
*
a
PC12wt
Silence H
if2α 0
1
2
3
Pnmt Relative mRNA expression
*
HIF2α Plasmid
β-Actin HIF2α
- + e
0.0
1.0
2.0
Pnmt Relative mRNA expression
*
0.0
0.4
0.8
1.2
Pnmt Relative mRNA expression
*
0.0
2.0
4.0
PNMT Activity
*
MPCwt MPC Silence Hif1α
0.0
2.0
4.0
PNMT Activity
*
f g
d
MPCwt MPC + Hif2α
MPC
b
c
Fig. 4
Fig. 6
mTOR
Ras
HIF2α
VHL Succinate
SDHx Fumarate
PHD
TMEM127
NF1
Akt
RET
MYC MAX
Differentiation blocked
Immature phenotype
Differentiation permitted
Mature phenotype
Cluster 1 tumors Cluster 2 tumors
HIF2α
HIF2α HIF1α
HIF1α
HIF1α
HIF2α
HIF1α
HIF2α HIF1α
Chromaffin progenitors cells
Cluster 1 Cluster 2
HIF1α
Qin et al. Int. J. Cancer 2014; 135:2054
The new syndrome of PGL, somatostatinoma and polycythemia
Patient 1 Patient 2 URL
PolycythemiaRed cheeks/lipsBlue feet
At birth8 yrNo
At birthAt birth< 1 yr
NA
ErythrocytesHematocrit
Erythropoietin
7,780.00050.5150
7,850.00059.3180
5,220.00044.936.7
Multiple PGLsNormetanephrine
14 yr4,834
18 yr858
NA112
Multiple SOMs 29 yr No (?) -
Zhuang et al. NEJM 2012; 367:922-930URL: upper reference limit
Patient 1 Patient 2 URL
PolycythemiaRed cheeks/lipsBlue feet
At birth8 yrNo
At birthAt birth< 1 yr
NA
ErythrocytesHematocrit
Erythropoietin
7,780.00050.5150
7,850.00059.3180
5,220.00044.936.7
Multiple PGLsNormetanephrine
14 yr4,834
18 yr858
NA112
Multiple SOMs 29 yr No (?) -
Zhuang et al. NEJM 2012; 367:922-930
The new syndrome of PGL, somatostatinoma and polycythemia
6
Patient 1 Patient 2 URL
PolycythemiaRed cheeks/lipsBlue feet
At birth8 yrNo
At birthAt birth< 1 yr
NA
ErythrocytesHematocrit
Erythropoietin
7,780.00050.5150
7,850.00059.3180
5,220.00044.936.7
Multiple PGLsNormetanephrine
14 yr4,834
18 yr858
NA112
Multiple SOMs 29 yr No (?) -
Zhuang et al. NEJM 2012; 367:922-930
The new syndrome of PGL, somatostatinoma and polycythemia
Patient 1 Patient 2 URL
PolycythemiaRed cheeks/lipsBlue feet
At birth8 yrNo
At birthAt birth< 1 yr
NA
ErythrocytesHematocrit
Erythropoietin
7,780.00050.5150
7,850.00059.3180
5,220.00044.936.7
Multiple PGLsNormetanephrine
14 yr4,834
18 yr858
NA112
Multiple SOMs 29 yr No (?) -
Zhuang et al. NEJM 2012; 367:922-930
The new syndrome of PGL, somatostatinoma and polycythemia
PHEO/PGL & polycythemia
PHD: prolyl hydroxylaseVHL: von Hippel-Lindau proteinHIF: hypoxia-inducible factorSDH: succinate dehydrogenase
Previous studies related to PHEO/PGL and polycythemia served as a clue to discovering a new syndrome
Kaelin Cold Spring Harb. Symp. Quant Biol. 2011; 76:335Kaelin Nat. Rev. Cancer 2008; 8:865Kelin et al. Mol. Cell 2008; 30:393Ladroue et al. NEJM 2008; 359:2685
Somatic gain-of-function HIF-2 (HIF2A) mutation
HIF-2IHC
N: normal adrenal medulla
Somatic mutation in HIF2Aexon 12
Pt. 1. A530T
Pt. 2. A530V
(PGL) (SOM)
7
Other genetic abnormalities in patients with HIF2Amutations
A gain of 2p chromosome
Comino-Mendez et al. Hum. Mol. Genet. 2013; 22:2169
Some patients without polycythemia: related to the time at which the mutation occurs?
Hematopoietic celldevelopment
Tumor development& progression
Oncogenic effects of HIF2A mutations in mice
Toledo et al. Endocrine Relat. Cancer 2013; 20:349
Age of onset Mutation Amino AcidSomatic mutation
Favier et al., 201224 C1591T P531S Y
Taieb et al. 201325 C1589T A530V Y
Comino-Mendez et al., 201318 G1588A A530T Y40 C1589T A530V Y13 C1591T P531S Y18 C1592T P531L Y46 C1599_1604del I533_P534del Y43 C1600_1608del P534_D536del Y78 G1615T D539Y Y
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.
Dr. William J. Mayo, 1928