24/10/2015 Pituitary signalling : from Zebrafish to Clinical - - PowerPoint PPT Presentation

SLIDE 1

24/10/2015 1

S.Melmed October 2015 UCSF CME

Pituitary signalling : from Zebrafish to Clinical Therapy

Li-Ng JCEM 2008 Hypothalamic releasing and inhibiting hormones Superior hypophysial artery Portal vein Inferior hypophysial artery Hypophysial vein Melmed NEJM 2006

Replace deficient hormone Shrink

• r

ablate mass Surgery Medical Rx Radiation

Pituitary compression Parasellar compression Hormone hypersecretion

Hypogonadism Thyroid failure Adrenal failure Visual field disturbance Headache Cranial nerve palsy Parasellar invasion GH Acromegaly ACTH Cushing PRL Prolactinoma FSH/LH Nonsecreting TSH TSHoma Mixed Co-morbidities Mortality

Melmed JCI 2003

Pituitary Tumorigenesis

Melmed Nat Rev Endocrinol 2011

Oncogene activation Tumor suppressor inactivation Cell cycle dysregulation Mutations Stromal and epigenetic events Mutations Unknown changes Normal pituitary Microadenoma Macroadenoma Aggressive Proliferative restraint Senescence Humoral factors Hyperplasia

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Pituitary Tumor Signalling Pathways

CDK1 Cyclin B G0 Mitogenic hormones and growth factors CDK2 Cyclin A E2Fs S M G1 G2 CDK2 CDK4 CDK6 Cyclin D Cyclin E Rb P P P P P P P P P P P P Proliferative constraints Senescence Chromosomal instability DNA damage p15, p16 p18, p19 p21, p27 p57 Cell cycle disruptors CDK inhibitors p53 GHRHR, CHRHR, GNRHR SSTR

Hormone

Cell proliferation Melmed Nat Rev Endocrin 2011 Melmed, JCI, 2009

Receptor subtype expression (%) Number of tumors SSTR1 SSTR2 SSTR3 SSTR4 SSTR5 GH 61 88 44 4 78 111

Ben-Shlomo, Trends Endocr Metab, 2010 Lamberts, JCEM, 1985 GH(µg/L) Control day Octreotide M M M 50 µg Octrotide or placebo 80 70 60 50 40 30 20 10 8am 10 12 2pm 4 6 8 10 12pm 2 4 6am

Before After

Acidophil Stem cell

Lactotroph Somatotroph Mammosomatotroph

PRL GH GH PRL

GH: Baseline 64 2-h OGTT 71 ng/mL IGF-1: 845 (<660) PRL: 8700 (<23) α subunit: 3.7 (<1.2)

Cell Origin and Receptor Profile

Gigantism

• 15-year-old female, height 179 cm
• Headaches, diplopia, sleepy
• Shoe size 11, tight rings
• Primary amenorrhea, no

galactorrhea

• Jaw prognathism, incisor gap

Acidophil Stem Cell Adenoma

GH PRL α subunit Acidophil Stem cell

Clonal expansion

Maheshwari JCEM 2000

4 M 1 2 3 5 6 Θ D2 Θ sstr2

Tumor receptor expression

Cabergoline SRL

Surgery 0.5 0.75 1.0 mg 30 mg

17.5 17 16.5 15.5 16 100 50 10 1200 400 8000 4000 1000 500

mg/L

Age (years) 18 23

GH IGF-1 PRL

SLIDE 3

24/10/2015 3

STAT3 Activates GH Promoter

Primer 1 Primer 2 Primer 3 ChIP Input 3 2 1 IgG STAT3 luc activity

S31-201 * *

• 4192/+167
• 1752/+167

50 100 150 µM luc activity

1 2 3 4

* *

pGL4.10

• 4192/+167
• 1752/+167

ZsGreen STAT3

40 30 20 10 Zhou JCI 2015

Rat Gh promoter

Potential STAT binding site

• 2500
• 2000
• 1500
• 1000
• 500

+1

• 119/+165

Primer 1 Primer 2 Primer 3

• 1118/-663
• 2040/-1806
• fold

control

*

ng/ml/103 GH3 cells

*

0.2 0.4 0.6 0.8

50 75 100 125 (µM) STAT3 inhibitor

* PRL GH

control

*

ng/ml/103 GH3 cells 0.5 1.5 2.5 3.5

50 75 100

*

STAT3 inhibitor 125 (µM)

STAT3 Inhibitor Suppresses GH

Zhou JCI 2015 STAT3 inhibitor (µM) 50 75 100 125

STAT3 GH PRL β-actin P-STAT3

STAT3 Inhibitor Suppresses hGH Secretion in vitro

RIA

P<0.0001

N=13

2.0 1.0 0.8 0.6 0.4 0.2 GH STAT3 inhibitor (mM) 50 100 150 0.5 1.0 1.5 GH STAT3 inhibitor (mM) 50 100 150

N=8

2.0 1.5 1.0 0.5 GH 50 100 150 50 100 150 P<0.0001

N=15 Western

0.5 1.0 1.5 2.0 50 100 150

N=6

GH 1.5 1.0 0.5 GH mRNA 50 100 150

P<0.0001

N=17 RT-PCR

0.5 1.0 1.5 2.0 50 100 150

N=4

GH mRNA

GH mRNA

1.2 1.0 0.8 0.6 0.4 0.2

* ** ** GH

1.2 1.0 0.8 0.6 0.4 0.2 50 100 150

S3I-201 (mM) ** *** ***

*p<0.05; **p<0.01; ***p<0.001

Western

P-STAT3 STAT3 GH 50 100 150

S3I-201 (mM)

Zhou JCI 2015

STAT3 Inhibitor Suppresses GH-tumor Proliferation and GH in vivo

6.0 4.0 2.0 3.0 2.0 1.0 Control Rx Control Rx

Serum GH Serum IGF-1

• fold
• fold

** Zhou JCI 2015 mg 900 700 500 300 100 Days

2 4 6 8 10 12 14

* * * * * * mm3

treated control

Volume

20 140 100 60

Weight

** S3I-201 (µM) 1.2 1.0 0.6 0.2

• fold

Stat3

Control Rx * 120 80 40 Staining (%) BrdU 50 100 * * Control

S3I-201

1.2 0.8 0.4 WST-1 S3I-201 (µM) * *

• fold

Tumors

Control Rx

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JM

EGF

JM Y992 Y845 Y920 Y891 Y1045 Y1068 Y1086 Y1148 Y1173 Y992 Y845 Y920 Y891 Y1045 Y1068 Y1086 Y1148 Y1173 Y701 Y701

TK

JM

HRG

JM Y992 Y845 Y920 Y891 Y1045 Y1068 Y1086 Y1148 Y1173 Y992 Y845 Y920 Y891 Y1045 Y1068 Y1086 Y1148 Y1173 Y701 Y701

TK TK

HER2 HER3 EGFR

Targeted Prolactinoma Therapy

>1500 1000 500 100 50

Lactation Impotence Normal potency

–8 –4 4 8 12 14 Time (months) Bromocriptine PRL (ng/mL)

Vlotides Cancer Res 2009

HER2-GH3

• Lapatinib Decreases HER2CA Tumor Size

and Prolactin Levels

*P<0.05, **P<0.01 vs vehicle

20 40 60 80 100 120 140

vehicle gefitinib lapatinib

*

PRL mRNA

PRL mRNA (%) Vehicle Gefitinib Lapatinib

Tumor volume

1000 Tumor volume (mm3) 800 600 400 200 1 4 10 Time (days) Vehicle Gefitinib Lapatinib 6

* ** * PRL

350 PRL (ng/mL) 300 250 200 150 100 50 –5 2 7 Vehicle Gefitinib Lapatinib

* **

Time (days)

Fukuoka Mol Endocrinol 2011

* Fischer 344 rat

17β-estradiol Vehicle

Vehicle

Pituitary weight

Lapatinib Decreases Tumor Size and Prolactin Levels in Estrogen-induced Prolactinoma

1000 2000 3000 4000 5000 6000 Vehicle Lapatinib

* Serum PRL

PRL (ng/mL) Lapatinib

Lapatinib

20 40 60 80 100 Vehicle Lapatinib

Serum GH

GH (ng/mL) Weight (mg) 250 200 150 100 50

Fukuoka Mol Endocrinol 2011 *P<0.05

H&E PRL EGFR HER2

Lapatinib Suppresses Both Prolactin mRNA and Secretion in Human Prolactinomas

*P<0.05, **P<0.01 vs vehicle H&E, hematoxylin and eosin

Lapatinib Gefitinib

PRL mRNA

120 PRL mRNA (%) 80 60 40 20 0.1 10 (µM) 1

* **

100 10 Lapatinib Gefitinib 120 PRL secretion (%) 80 60 40 20 0.1 10 1 100 10

PRL secretion ** ** **

(µM)

Fukuoka Mol Endocrinol 2011

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24/10/2015 5

50 100 150 200 250 300 350 1 2 3 4 5 6 7 PRL (ng/mL) Time (months) 11988 mm3 998 mm3 937 mm3

Pre-Rx Rx 6 months

Aggressive Prolactinoma: Lapatanib 1250 mg/day

Cooper Endocr 2013

Minnie G.– 1910 Harvey Cushing, MD

Cushing Disease

Cushing Bull Johns Hopkins Hosp. 1932 Stewart Best Pract Res Clin Endocrinol Metab. 2009

Cushing Disease

Central mass effects Hormone hypersecretion

Pituitary hormone deficiencies Visual field disturbance Headache Parasellar invasion ACTH → cortisol

• Obesity
• Hypertension
• Diabetes
• Osteoporosis
• Hirsutism
• Muscle weakness
• Moon face
• Acne

Surgery Medical Rx Radiation

Melmed JCI 2003

Therapy

• Surgery: 60–70% initial remission

20–60% recurrence

• Pituitary radiation: Hypopituitarism
• Adrenalectomy or cortisol synthesis

inhibitor: Side effects and morbidity

Physical and biochemical diagnosis

• Signs , symptoms and

hypercortisolemia overlap with other illnesses

• Cyclic or intermittent hypercortisolemia

Imaging

• Average tumor size ~6 mm
• ~40% tumors not visible
• 10% of normal population micro-

adenoma

Median survival 4.6 years if inadequately controlled

Life expectancy no different than in 1930

Challenges in Diagnosis and Treatment

• f Cushing Disease

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24/10/2015 6

Medical Therapies for Cushing Disease

Adrenal glands Adrenal glands Hypothalamus

Cortisol

Pituitary tumor

ACTH

G2 receptors

(peripheral tissues)

Ketoconazole Cabergoline Mifepristone Pasireotide

Glucocorticoid receptor antagonist

Increased ACTH and UFC

levels

AEs -- hypokalemia and

endometrial thickening

Does not target underlying

corticotroph tumor Steroidogenesis inhibitors

Reduce cortisol levels Do not target underlying

corticotroph tumor

Safety concerns

Dopamine receptor agonists

Small studies reported

efficacy

Long-term data conflicting Therapeutic escape

Pivonello Endo Rev, 2015 Stroke Depression Impairment of cognitive function Systemic arterial hypertension Left ventricular hypertrophy Infectious disease Sepsis Thrombosis diathesis

METABOLIC SYNDROME

Visceral obesity Diabetes mellitus Dyslipidemia Myocardial infarction

Targeted Therapy in Cushing Disease

JM

EGF

JM Y992 Y845 Y920 Y891 Y1045 Y1068 Y1086 Y1148 Y1173 Y992 Y845 Y920 Y891 Y1045 Y1068 Y1086 Y1148 Y1173 Y701 Y701

TK TK

HER2 EGFR Lapatinib (Tykerb) Gefitinib (Iressa)

JM

EGF

JM Y992 Y845 Y920 Y891 Y1045 Y1068 Y1086 Y1148 Y1173 Y992 Y845 Y920 Y891 Y1045 Y1068 Y1086 Y1148 Y1173 Y701 Y701

TK

JM

HRG

JM Y992 Y845 Y920 Y891 Y1045 Y1068 Y1086 Y1148 Y1173 Y992 Y845 Y920 Y891 Y1045 Y1068 Y1086 Y1148 Y1173 Y701 Y701

TK TK

HER2 HER3 EGFR

Tyrosine kinase inhibitors targeting EGFR and/or HER2

Fukuoka H et al. Mol Endocrinol 2011;25:92–103

• Omental fat

Omental fat

EGFR-AtT20

• Vehicle

Gefitinib

Gefitinib Action on ACTH Tumors in vivo

Fukuoka JCI 2011 *P<0.05, **P<0.01 700 ACTH (ng/mL) 500 400 300 200 Control EGFRWT 600

ACTH

100 600 500 400 300 200 Control EGFRWT

Corticosterone

100 Corticosterone (ng/mL) 23 Body weight (g) 21 20 19 18 15 Control EGFRWT 22

Body weight

17 450 350 300 250 200 Control EGFRWT 400

Glucose

Glucose (mg/dL) 16 Body weight (g) Control EGFRWT

Tumor weight

23 21 20 19 18 15 22 17 16

** * * * * * ** **

SLIDE 7

24/10/2015 7

Dog 4

** ** ** ** ** *

120 100 80 60 40 20 ACTH (%) 0.1 1 10 µM

* **

120 100 80 60 40 20 POMC (%)

* **

0.1 1 10 µM

Dog 1 Dog 2 Dog 3 Dog 5

POMC

Gefitinib Suppresses ACTH in Canine Cushing’s Tumors

*P<0.05, **P<0.01 POMC, proopiomelanocortin

ACTH

Gefitinib Gefitinib

POMC

Fukuoka JCI 2011

Pituitary Tumor Signalling Pathways

CDK1 Cyclin B G0 Mitogenic hormones and growth factors CDK2 Cyclin A E2Fs S M G1 G2 CDK2 CDK4 CDK6 Cyclin D Cyclin E Rb P P P P P P P P P P P P Proliferative constraints Senescence Chromosomal instability DNA damage p15, p16 p18, p19 p21, p27 p57 Cell cycle disruptors CDK inhibitors p53 GHRHR, CHRHR, GNRHR SSTR

Hormone

Cell proliferation Melmed Nat Rev Endocrin 2011

72 hpf

QuickTime™ and a decompressor are needed to see this picture.

POMC-GFP/PRL-RFP

32 hpf

Transgenic Zebrafish Pituitary Lineage Tracing

Liu Mol Endocrinol 2006

αGSU.PTTG: Pituitary Hyperplasia and Adenoma

4X

Abbud, Mol Endocrinol 2005

αGSU promoter IRES EGFP Poly A hPTTG 1 αGSU. PTTG

αGSU.PTTG

10 20 (relative to WT)

WT

Serum LH

SLIDE 8

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Hypercortisolism in Tg:POMC-PTTG Zebrafish

Liu PNAS 2011

Tg WT Adrenal steroidogenic cells Posterior cardinal vein

0.4 0.8 1.2 1.6

Tg Wt

Cortisol

* P<0.05 n=24

Cortisol (µg/L)

WT

Cardiac Hypertrophy in Tg:Pomc-Pttg Fish

WT Sibling Tg

Liu PNAS 2011

0.1 0.2 0.3 0.4 0.5 Tg WT

Weight (mg)

n=12, P<0.01

*

100 200 300 400 500

• 1

2 4 6 30 40 WT Tg:pomc-pttg

Ad lib low-fat, high-carbohydrate diet

N=72 AUC, P<0.0001 Blood glucose (mg/dL) Time (hours)

WT Tg

Hepatic steatosis

Metabolic Phenotype in Tg:POMC-PTTG Zebrafish

Liu PNAS 2011

Tg:POMC-PTTG Weight (mg)

Phenotype

Tg:pomc-pttg zebrafish Cushing’s disease

High cortisol High cortisol Neoplastic pituitary ACTH cells Corticotroph adenoma Intra-renal steroidogenic cell hyperplasia Adrenal hyperplasia Hepatic steatosis Central obesity Glucose intolerance Glucose intolerance Cardiac hypertrophy Concentric cardiomyopathy

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PTTG Upregulates Corticotroph Cyclin E

Liu PNAS 2011

AtT20 cells Zebrafish pituitary

mRNA (fold) 2.5 2.0 1.5 1.0 WT Tg:POMC-PTTG POMC Cyclin DCyclin E p21 p27 Rb 0.5

Cdk1/cyclin A Cdk1/cyclin B Cdk2/cyclin A Cdk4/cyclin D Cdk6/cyclin D Cdk2/cyclin E S M G1 G2 Cyclin E Cyclin D Proliferation Transcription S pRb

E2F CDK4 CDK2 p19, p15, p16, p18 p21, p27, p57 p p p p p p

G1

3.0

PTTG Cyclin E p27 p21 β-actin siRNA C PTTG

0.2 *

CDK Inhibitor Screening in Tg:POMC-PTTG/POMC-GFP Embryos

Liu PNAS 2011

1.2 Fluorescence (fold) 0.8 0.6 0.4 1.0

R-roscovitine (CYC202): Transcriptional CDK Inhibitor

2-(R)-(1-ethyl-2-hydroxyethylamino)- 6-benzylamino-9-isopropylpurine As a CDK inhibitor Primary CDK targets: CDK2, CDK7, CDK9

Validation of R-roscovitine Action in Mouse Corticotroph Tumors

Liu PNAS 2011 **P<0.01

Plasma ACTH (pg/mL) 1600 800 400 1200 ** Vehicle Roscovitine 1600 800 400 1200 Serum corticosterone (ng/mL) **

Vehicle Roscovitine

Tumor ACTH PCNA β-actin Vehicle Roscovitine V3 V1 V2 V6 R6 R4 R2 R3

SLIDE 10

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Cyclin E Tpit 0 10 20 Roscovitine (µM) ACTH Ponceau

R-roscovitine Suppresses Human Corticotroph Tumor Signaling

ACTH (pg/mL) 1 2 3 4 5 6

0.4 0.8 1.2

Roscovitne 0 uM Roscovitine 20 uM

*P<0.05

* * * * * Roscovitine 0 20 µM *

0.6 1.2 1.8

POMC alpha-GSU PRL GH

1.2 0.8 0.4 1.2 0.8 0.4 1 2 3 4 5 6

Tumor 6

Liu JCEM 2015

R-roscovitine Inhibits Corticotroph Tumor Gene Expression

Roscovitine (µM) 0 10 20

PitX NeuroD1 Tpit LAMIN ACTH Ponceau

Roscovitine

Tpit ACTH α-MSH NeuroD1 LH FSH TSH PRP-1 SF1 GATA-2 GATA-2 Pit1 TSH GH PRL Pituitary progenitor

Araki and Cuevas-Ramos

PTTG Cyclin E CDK2 G1 – S

Proliferation Hyperplasia/tumor

Rb-E2F p27

Tpit

ACTH overexpression

Cushing disease Roscovitine p21 p27 P57 Roscovitine

Senescence Cell cycle exit

E2F1

Corticotroph-specific R-roscovitine Targets

hPOMC gene

Acknowledgements

Cedars-Sinai

Ning-Ai Liu Vera Chesnokova Svetlana Zonis Cuiqi Zhou Kolja Wawrowsky Anat Ben-Shlomo Takako Araki Song-Guang Ren Hide Fukuoka Daniel Cuevas-Ramos Odelia Cooper Hide Fukuoka Serguei Bannykh Adam Mamelak

• St. Michael’s Hospital, Toronto

Kalman Kovacs Fabio Rotondo

SLIDE 11

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Lucas Cranach 1546

…… “Fons Juventutis” ….Elixir of Youth

43

Sustained GH and IGF-1 Suppression

Median GH control markedly superior to injections, median IGF-1 control comparable to injections

Response <2.5 ng/mL

3.0 2.5 2.0 1.5 1.0 0.5 Baseline

+

First RX

+ + +

13 mo

GH (ng/mL)

Growth Hormone

+

End Dose Esc 7 mo

Response <1.3 ×ULN

2.5 2.0 1.5 1.0 0.5 7 mo

+

End Dose Esc.

+

First Rx

+

Baseline

+

IGF-1 × × × × ULN

IGF-1

+

13 mo