1

Karel Pacakkarel@mail.nih.gov

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