general session: the art and science of endometriosis research (didactic… · 2020-01-30 ·...

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AAGLAdvancing Minimally Invasive Gynecology Worldwide

General Session:The Art and Science of Endometriosis Research

(Didactic)

Asgi T. Fazleabas, PhD Robert N. Taylor, MD Linda G. Griffi th, PhD

CHAIRVictor Gomel, MD

Professional Education Information Target Audience This educational activity is developed to meet the needs of residents, fellows and new minimally invasive specialists in the field of gynecology. Accreditation AAGL is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AAGL designates this live activity for a maximum of 1.50 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity. DISCLOSURE OF RELEVANT FINANCIAL RELATIONSHIPS As a provider accredited by the Accreditation Council for Continuing Medical Education, AAGL must ensure balance, independence, and objectivity in all CME activities to promote improvements in health care and not proprietary interests of a commercial interest. The provider controls all decisions related to identification of CME needs, determination of educational objectives, selection and presentation of content, selection of all persons and organizations that will be in a position to control the content, selection of educational methods, and evaluation of the activity. Course chairs, planning committee members, presenters, authors, moderators, panel members, and others in a position to control the content of this activity are required to disclose relevant financial relationships with commercial interests related to the subject matter of this educational activity. Learners are able to assess the potential for commercial bias in information when complete disclosure, resolution of conflicts of interest, and acknowledgment of commercial support are provided prior to the activity. Informed learners are the final safeguards in assuring that a CME activity is independent from commercial support. We believe this mechanism contributes to the transparency and accountability of CME.

Table of Contents

Course Description ........................................................................................................................................ 1 Disclosure ...................................................................................................................................................... 3 Endometriosis in the Baboon – A Model to Understand This Enigmatic Disease A.T. Fazleabas ................................................................................................................................................ 4 Endometriosis Lesions Are Reported to Be “Estrogen Sensitive and Progesterone Resistant” What Does This Mean for Our Clinical Management? R.N. Taylor ..................................................................................................................................................... 9 Systems Biology Approaches to Endometriosis L.G. Griffith .................................................................................................................................................. 14 Cultural and Linguistics Competency ......................................................................................................... 21

General Session: The Art and Science of Endometriosis Research

Endometriosis in the Baboon – A Model to Understand This Enigmatic Disease

Asgi T. Fazleabas

Endometriosis results in chronic pelvic pain and infertility and affects 10% of women of reproductive age. The initiation of endometriosis is difficult to evaluate because at the time of clinical diagnosis the disease has been prevalent for 8‐11 years in women. Therefore, identification of molecules involved with the early pathogenesis of endometriosis, non‐invasive diagnosis and strategic therapies for treatment is critical. Using a baboon model endometriosis is induced via laparoscopic inoculation of menstrual tissue into the peritoneal cavity. Peritoneal endometriosis develops within one month and in turn markedly alters the gene signature of the eutopic endometrium during the window of uterine receptivity. During disease progression there is a transitory dominance of an estrogenic phenotype which eventually results in a more permanent progesterone resistant phenotype. Accompanying changes in miRNA’s also regulate target genes that contribute to increased proliferation, decreased apoptosis and inhibition of progesterone signaling. Learning Objectives: At the conclusion of this course, the participant will be able to: 1) Recognize the importance of developing appropriate animal models to study human disease and evaluate the potential consequences of endometriosis on fertility.

Endometriosis Lesions Are Reported to Be “Estrogen Sensitive and Progesterone Resistant” What Does This Mean for Our Clinical Management?

Robert N. Taylor

This talk will provide the practicing physician and educator with a comprehensive review of evolving concepts about the derivation, pathogenesis and hormone responsiveness of endometriosis lesions. Abnormalities within the eutopic endometrium of women with endometriosis also will be defined, particularly emphasizing their impact on infertility and pelvic pain. Surgery continues to be the mainstay of endometriosis therapy, but ongoing research promises to offer new adjuvant approaches to enhance efficacy, improve compliance, or reduce side‐effects of medical treatments. The rationale and potential advantages of these emerging strategies will be illustrated. Learning Objectives: At the conclusion of this course, the participant will be able to: 1) Recognize the endocrine responsiveness of endometriosis lesions; and 2) interpret the rationale of emerging adjuvant medical therapies for symptomatic endometriosis.

Systems Biology Approaches to Endometriosis

Linda G. Griffith

This talk provides a basic introduction to how approaches from systems biology and tissue engineering are being applied to understand the etiology and pathophysiology of endometriosis, with the aim of aiding development new ways to diagnose and treat the disease. Computational systems biology

1

approaches integrate multiple kinds of data from various –omics measurements made on samples relatively easily obtained in surgical practice (e.g., peritoneal fluid) to provide insight on cellular crosstalk and possible points of intervention in inflammatory networks. Tissue engineering approaches allow cells from patients to be cultured in a physiological environment for analysis of disease processes and responses to potential therapeutics. Learning Objectives: At the conclusion of this course, the participant will be able to: 1) Explain how computational systems biology approaches are being used to classify disease processes in endometriosis patients; and 2) identify opportunities to improve communication between surgeons and scientists about how to describe endometriosis disease processes.

2

PLANNER DISCLOSURE The following members of AAGL have been involved in the educational planning of this workshop and have no conflict of interest to disclose (in alphabetical order by last name). Art Arellano, Professional Education Manager, AAGL* Viviane F. Connor Consultant: Conceptus Incorporated Kimberly A. Kho* Frank D. Loffer, Executive Vice President/Medical Director, AAGL* Linda Michels, Executive Director, AAGL* M. Jonathan Solnik* Johnny Yi*

SCIENTIFIC PROGRAM COMMITTEE Ceana H. Nezhat Consultant: Ethicon Endo-Surgery, Lumenis, Karl Storz Other: Medical Advisor: Plasma Surgical Other: Scientific Advisory Board: SurgiQuest Arnold P. Advincula Consultant: Blue Endo, CooperSurgical, Covidien, Intuitive Surgical, SurgiQuest Other: Royalties: CooperSurgical Linda D. Bradley* Victor Gomel* Keith B. Isaacson* Grace M. Janik Grants/Research Support: Hologic Consultant: Karl Storz C.Y. Liu* Javier F. Magrina* Andrew I. Sokol* FACULTY DISCLOSURE The following have agreed to provide verbal disclosure of their relationships prior to their presentations. They have also agreed to support their presentations and clinical recommendations with the “best available evidence” from medical literature (in alphabetical order by last name). Asgi Fazleabas* Victor Gomel* Linda G. Griffith* Robert N. Taylor Consultant: AbbVie, Allere Asterisk (*) denotes no financial relationships to disclose.

ENDOMETRIOSIS IN THE BABOON: A MODEL TO UNDERSTAND THIS ENIGMATIC DISEASE

Asgi T. FazleabasProfessor and Associate Chair

Department of Ob, Gyn & Reprod BiolDirector, Center for Women’s Health

ResearchCo-Director, Reproductive &

Developmental Sciences ProgramMichigan State University

[email protected]

Mechanisms of Hormone Resistance 42nd Global Congress on Minimally

Invasive Gynecology November 13th, 2013

DISCLOSURE

I have no financial relationships to disclose.

OBJECTIVES

Learning Objectives:

1) Recognize the importance of developing appropriate animal models to study human disease

1) Evaluate the potential consequences of endometriosis on fertility.

LIMITATIONS IN OUR UNDERSTANDING OF ENDOMETRIOSIS

Does the presence of endometriotic lesions affect the uterine endometrium?

The spontaneous evolution of endometriosis ANDwhether an inherently defective endometrium contributes to

the growth of endometriotic lesions?

What is the pathophysiology of endometriosis-associated infertility?

Endometriosis was first described in 1690 by Daniel Shroen

In 1927, Sampson first proposed his theory of retrograde menstruation

However, we still do not understand the underlying mechanisms associated with:

PROGESTERONE RESISTANCE

WOI

Normal

Endometriosis

Uterus

Serum

ESR Regulated

GenesPGR

Regulated Genes

Fazleabas AT, Progesterone Resistance in a Baboon Model of Endometriosis Semin Reprod Med. 2010; 28:75-80

ADVANTAGES OF THE USE OF PRIMATES AS A MODEL FOR ENDOMETRIOSIS

Reproductive anatomy and physiology are similar to humans

Non-human primates develop endometriosis spontaneously and the disease can also be induced which is histologically similar to the human disease – absent in non primate models

Ability to induce the disease permits the study of its etiology and progression from the time of initiation of the disease

Long term and invasive studies and multiple surgical procedures are possible under experimentally controlled conditions

Close phylogenetic relationship permits the use of humanmolecular probes and antibodies

Evaluation of treatment modalities for the human disease can be tested for treatment and long term safety

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EXPERIMENTAL DESIGN INDUCTION OF ENDOMETRIOSIS

Menses Menses

Laparoscopy & Innoculationwith Menstrual Tissue

Control

1 3-4 6-7 9-10 12

Laparotomies

Months

Innoculated Baboons

Harvest Tissues15

Pelvic cavity during to inoculation

Induced disease in the baboon

Spontaneous diseasein women

TIME COURSE OF GENE DYSREGULATION

EUTOPIC

38-40% of the genes thatare altered at 1 month in the baboon correspond to those reported in women

Afshar et al., BOR 2103; 88: 44

Inoculation

1 3 6 9 12 15

Early Responses

Late Responses

EMMPRINPgr

Calcitonin

Progression of Disease (months)

FKBP52

HOXA10

CYR61FOS

CHANGES IN ENDOMETRIAL GENE EXPRESSION DURINGTHE WINDOW OF UTERINE RECEPTIVITY

7026 3 month 7026 15 month

ESTROGEN DOMINANCE

PROGESTERONERESISTANCE

Hastings & FazleabasReprod Biol & Endocrin4 (Supp1) S7 (10-9-06)

Jones et al., Hum Repro21: 3098, 2006

miRNA’s play a significant role in normal physiological development and pathologic conditions. Biologically plausible miRNA targets have the clinical utility of being selected

for the development of agonists an antagonists.

MICRO RNA ‘s – A SIGNIFICANT ROLE IN ENDOMETRIOSIS?

<1/2 >21Treatment / Control Ratio

miRNAs differentially expressedAgilent - 8X15K miR microarray

miRNADisease vs.

Control Fold Change

P-Value

hsa-miR-451 -14.94 0.000036

hsa-miR-181a -1.46 0.068

hsa-miR-200a 1.88 0.037

hsa-miR-19b 2.19 0.073

hsa-miR-424 2.43 0.027

hsa-miR-21 2.49 0.014

hsa-miR-29c 4.46 0.009

hsa-miR-141 5.19 0.032

MIR-29c EXPRESSION PATTERN Targetscan analysis predicted 1078 conserved targets of miR-29c

3’ UTR of FKBP4(Position 426-432)…UGUGGCCUCCAUGUGGGUGCUAG...Seed sequence of miR-29c ………...… AUUGGCUAAAGUUUACCACGAU…...

• Predicted interaction between of miR-29c and FKBP4 mRNA

WHAT ARE THE TARGETS OF MIR-29c ?

5

Studies in the rodents have established thatprogesterone receptor mediated uterine receptivity iscontrolled by FKBP4.FKBP4 knockout mice have an implantation failurephenotype.Absence of FKBP4 may be associated withprogesterone resistance observed in endometriosis.

WHY FKBP4 ?FKBP4 gene codes for the FKBP52 protein FKBP4 is a co-chaperone associated with the steroid hormone receptor complex

Tranguch et al., PNAS 102: 14326, 2005 & JCI 117:1824, 2007: Jackson et al., Reprod Sci 14:137, 2007

FKBP4

β-Actin

Control 6 Months 15 Months

LaparoscopyConfirmation

HES cellsNo Yes No Yes Yes

Endometriosis Stage -- IV -- III mild

Baboon

Human

+ve Control Endometriosis

FKBP4

WESTERN BLOT SHOWING FKBP52 PROTEIN EXPRESSION IN MID-SECRETORY PHASE IN EUTOPIC ENDOMETRIUM OF BABOON AND HUMAN

Control Endometriosis

FKBP4 IMMUNOLOCALIZATION AND QUANTIFICATION IN WOMEN WITH ENDOMETRIOSIS

DECIDUALIZATION AND OPTIMAL PREGNANCY OUTCOME

• Decidualization is compromised as a consequence of endometriosis.

- Impaired decidualization is associated with implantation failure- Defective decidualization leads to recurrent pregnancy loss- Absent or deficient decidualization is associated with

potentially catastrophic hemorrhage seen in patients with ectopic pregnancies or placenta accreta (abnormal deep placental invasion)

- Decidual senescence contributes to pre-term labor and premature fetal delivery

CORRELATION OF FKBP4 EXPRESSION AND IMPAIRED DECIDUALIZATION IN ENDOMETRIOSIS

HOXA 10 Expression in decidualized stromal cells Control

↑ miR-451 ↓ YWHAZNo Proliferation

and Apoptosis

Endometriosis↓ miR-451 ↑ YWHAZ

Altered ProliferationDecreased Apoptosis

↓ miR-29c ↑ FKBP52 Progesterone Response

↑ miR-29c ↓ FKBP52 Progesterone Resistance

Pregnancy

Infertility

↑miR‐29c

miR‐29c

POSTULATED MECHANISM OF miR 29c AND FKBP52INTERACTION IN THE PATHOGENESIS OF ENDOMETRIOSIS

x

6

CYTOSKELETAL REORGANIZATION AND NOTCH 1EXPRESSION ARE COUPLED WITH DECIDUALIZATION

a-SMA

Notch 1

• Notch1 transcriptionally regulates the expression of SMA, which is essential for decidualizationNoseda, M, et al. Circ Res, 2006; Kim et al., 1999

• Notch 1 expression in stromal cells is critical for the initiation of decidualizationAfshar et al. Endo 2012

• SMA expression is blunted in the endometrium of baboons with endometriosis Sherwin et al., Endo, 2010

• Inhibition of Notch1 in stromal cells prevents cells from undergoing decidualizationAfshar et al. Endo 2012 &; FASEB J 2012

CG

Notch 1

aSMA, FOXO1 & FOXO3a IL11 COX-2

Inhibit Apoptosis & Initiate Differentiation

Stromal Fibroblast

DecidualCell

Progesterone

WORKING HYPOTHESIS

• Notch is an evolutionary conserved arbiter of cell fate and regulates diverse cellular functions, including survival, proliferation, and differentiation.

ER

Inhibition ofApoptosis

Data summarized from Afshar et al., 2012

ALTERED EXPRESSION OF THE NOTCH SIGNALTRANSDUCTION CASCADE IN ENDOMETRIOSIS

Human EndometriumBaboon Spontaneous Disease

ALTERED EXPRESSION OF THE NOTCH SIGNALTRANSDUCTION CASCADE DURING IN VITRO DECIDUALIZATION

INHIBITION OF NOTCH SIGNALING AT THE INITIATION OF DECIDUALIZATION INHIBITS STROMAL CELL DIFFERENTIATION

WORKING MODEL: NOTCH 1 AS AN INITIATOR OF DECIDUALIZATION

eEeE

ProgesteroneCG

NOTCH1

Survival Proliferation ERα signaling

EndometriosismiR 200a (?)

X

7

Stromal fibroblast

CG& Progesterone

CONCEPTUS MEDIATED TRANSFORMATION OF THE STROMAL FIBROBLASTS

ConceptusIL-1

SMA & Notch-1

IGFBP-1 & Prl

Decidual cell

cAMP

ENDOMETRIOSIS

Progesterone Resistance

Inhibition of Decidualization Cascade

ACKNOWLEDGEMENTSCollaborators

Supported by the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement U54 HD 40093 as part of the Specialized Cooperative Centers Program in Reproduction

and Infertility Research and RO1 HD 42280

Fazleabas Laboratory Zuzana Strakova Paula CameoJulie Hastings Kevin JacksonAndrea BraundmeierPatty Mavrogianis Yalda AfsharPrajna BanerjeeAnna JasinskaKavita Sapru

Sue FergusonMark Olson Niraj JoshiKirsten Parkin Ren-Wei SuSharra PoncillAngela MengRachael Radnor Emma Giuliani Danielle PeterseGeorgina Cerchi

Linda Giudice – UCSFHugh Taylor – Yale Bruce Lessey – Greenville HospitalSteve Young – UNC Lucio Miele – Univ of MississippiShuk-Mei Ho – Univ Cincinnati Rabindranath De La Fuentes – U GeorgiaAndrew Sharkey – Cambridge Rob Sherwin – Cambridge/WhittenhamDharani Hapangama – Univ LiverpoolIan Fraser – Univ SydneyFranco Demayo – Baylor Jae Wook Jeong - MSUSerdar Bulun – NorthwesternJulie Kim – Northwestern Carolyn Jones – Univ ManchesterElke Winterhager – Univ Essen Jan Brosens – Imperial College Idhaliz Flores – Ponce School of Med

REFERENCES1. Kim, J.J., R.C. Jaffe and A.T. Fazleabas (1999). Insulin-like Growth Binding Protein 1 in Baboon Endometrial Stromal Cells: Regulation by Filamentous Actin and Requirement for De Novo Protein Synthesis. Endocrinology 140:997-1004. PMID:9927334

2. Kim J.J., H.S. Taylor, G.E. Akbas, I. Foucher, A. Trembleau, R.C. Jaffe, A.T. Fazleabas and T.G. Unterman (2003). Regulation of Insulin-like Growth Factor Binding Protein-1 (IGFBP-1) Promoter Activity by FKHR and HOXA-10 in Primate Endometrial Cells. BiolReprod 68: 24-30. PMID:12493691

3. Jones C.J.P., J. Denton and A.T. Fazleabas (2006). Morphological and Glycosylation Changes Associated with the Endometrium in a Baboon Model of Endometriosis. Hum Reprod 21: 3068-3080. PMID:170185334. Hastings J.M. and A.T. Fazleabas (2006). A Baboon Mo del for Endometriosis: Implications for Fertility. Reprod Biol & Endocrin 4 (Supp1) S7

5. Jackson K..S., A. Brudney, Hastings J.M., Kim J.J. and A.T. Fazleabas (2007). The altered distribution of the steroid hormone receptors and the chaperone immunophilin FKBP52 in a baboon model of endometriosis during the window or uterine receptivity.

Reprod Sciences 14: 137-150. PMID:176362256. Kim J.J, H. S. Taylor, Z. Lu, O. Ladhan, Y. Wu, S-W. Guo and A. T. Fazleabas (2007). Alterations in HOXA10 expression in endometriosis: potential role in decidualization. Mol Hum Reprod 13: 323-332. PMID:173509637. Afshar Y., A. Stanculescu, L. Miele and A.T. Fazleabas (2007). The Role of Chorionic Gonadotrophin and Notch 1 in Implantation. J Assisted Reprod & Genetics 24: 296-302. PMID:17616802

8. Sherwin J.R.A., J.M. Hastings, P.A Mavrogianis, A.M. Sharkey and A.T. Fazleabas (2010). The Endometrial Response to Embryo Derived Signals is Blunted in a Baboon Model of Endometriosis. Endocrinology 151: 4982-4993. PMID:206680309. Fazleabas A.T. (2010). Progesterone Resistance in a Baboon Model of Endometriosis. In: Seminars in Reproductive Medicine (B.Carr Ed., Serdar Bulun, Guest Editor). 28:75-80, 2010.

10. Afshar Y., J-W Jeong, D. Roqueiro, F. DeMayo, J. Lydon, F. Radtke, L. Miele and A.T. Fazleabas (2012). Notch 1 Mediates Cell Fate Decisions in the Mouse Uterus and is Critical for Complete Decidualization. FASEB J. 26: 282-294. PMID:2199037211. Afshar Y., Miele L and A.T. Fazleabas (2012). Notch 1 is Regulated by Chorionic Gonadotropin and Progesterone in Endometrial Stromal Cells and Modulates Decidualization in Primates. Endocrinology 153 2884-2896. PMID:22535768

12, Tranguch S, H. Wang, T.Daikoku, H. Xie, D.F.Smith and S.K. Dey (2007) FKBP52 deficiency-conferred uterine progesterone resistanceis genetic background and pregnancy stage specific. J Clin Invest. 117:1824-1834. PMID: 17571166 .

13. Tranguch S, J. Cheung-Flynn, T. Daikoku, V. Prapapanich, M.B. Cox, H. Xie H. Wang, S.K.Das D.F. Smith and S.K. Dey (2005) Cochaperone immunophilin FKBP52 is critical to uterine receptivity for embryo implantation. Proc Natl Acad Sci U S A. 102:14326-14331.

14. Noseda M, Y. Fu, K. Niessen, F. Wong, L. Chang, G.McLean and A. Karsan (2006). Smooth Muscle alpha-actin is a direct target of Notch/CSL. Circ Res.98:1468-1470. PMID: 16741155

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Robert N. Taylor, MD PhDDepartment of Obstetrics and Gynecology

Wake Forest School of Medicine

Endometriosis Lesions are Reported to be ‘Estrogen Sensitive and Progesterone

Resistant’: What Does this Mean for Our Clinical Management?

Disclosures

Consultant: AbbVie, Allere

Learning Objectives

At the conclusion of this course, the participant will be able to:

1) recognize the endocrine responsiveness of endometriosis lesions; and

2) interpret the rationale of emerging adjuvant medical therapies for symptomatic endometriosis.

Classical Hormone Induced Changes inEndometrial Morphology

Noyes et al, 1950

Markee, 1978

E2 P4

How is Endometrial and Endometriosis Growth Regulated?

Effects of Steroid Hormones on Endometrial and Endometriosis Cells

Estrogen: activation of the cell cycleepithelial & stromal mitogenesis angiogenesis

Progesterone: epithelial secretion receptivity biomarker inductionstromal edema & decidualizationpreparation for apoptosis

9

Estrogen Receptor Action, 2013

ER ER

GPERMAPKSTAT

RNA pol IItranscription

complex

geneactivation

-- 10 kb

-- 1.2 kb

-- 8 kb

Estrogen Receptor β isOver-expressed in Endometriosis Cells

ER

GAPDH

ER/ER increased 3x in endometriosis vs. normal endometrial stromal cells (Brandenberger et al, 1999)

ER/ER increased >30x in endometiosis vs. normal endometrial tissues (Xueet al, 2007)

-- 4 kb

Cell Cycle Regulation

G1 / S checkpoint

Estradiol Stimulates PCNA in Endometriosis Stromal Cells

Lebovic et al., 2002

β-actin (42 kDa)

PCNA (36 kDa)

0h 8h 24h

Proliferating Cell Nuclear Antigen: an 36 kDa homotrimeric DNA binding protein increases the processivity of DNA polymerase δ and leading strand synthesis during DNA replication

10 nM E2

Medical Treatments Reduce PCNAAnd Cell Proliferation in Lesions

Gomes et al, 2009

ERα

PCNA

PCNA Expression in Murine Endometriosis is ER and PR Dependent

Fang et al, 2004

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Return of Postoperative Pain in Endometriosis

Stratton et al, 2008

Progesterone Receptor Mechanism of Action

PR-A PR-B

RNA pol IItranscription

complex

Progesterone Receptor, ergo Response, is Reduced in Endometriosis Cells

Attia et al, 2000

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.

Yu et al, under review

E

- hormones + hormones

Stromal Cells from Eutopic Endometriumof Endometriosis Patients show Impaired

Decidualization

fertile control

endometriosis

Apoptosis: “The ‘Dropping Off’ of Leaves from Trees”

caspase

“programmed cell death”

Dmowski et al, 2001

endometriosis normal

proliferative

secretory

Endometrial Apoptosis:Reduced TUNEL in Eutopic Endometrium

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“Reflux Redux”: Disposition of Shed Menstrual Endometrium in Women

antegrade

menstruation

retrograde

menstruationBax, Bcl-xS, Fas

90%

10%

apoptosisattachment,invasion,

vascularization,inflammation

Vercellini et al, 2012

Norethindrone Acetate (2.5 mg/d)is Partly Effective in Relief of Pelvic Pain

Natural Herbal Chemotherapeutics? Curcumin has Multiple Relevant Cellular Targets in Endometriosis

Curcuma longa (haldi)

Curcumin

O

MeO

HO

OMe

OH

O

Oxidation

Cell cycle

Inflammation

Apoptosis

NF-B

Novel Curcumin Analogs have Improved Potency and Bioavailabilty

Curcuma longa (haldi) Turmeric

Curcumin

O

MeO

HO

OMe

OH

O

Curcumin and EF-24 Induce ApoptosisIn Endometriosis Cells

Bcl-2 (28kDa)

Cleaved Caspase-3 (17/19kDa)

Bcl-2 (28 kD)

cleavedCaspase-3(17/19 kD)

Anti- and pro-apoptosismarkers

con

Curcum

EF-24MTS proliferation assays:Curcumin (IC50 = 32 µM)EF-24 (IC50 = 5 µM)

12

Summary•Endometriosis cells are estrogen sensitive and show time- and

dose-dependent PCNA induction; P4 resistance may render these cells less prone to cell cycle blockade

•Selective estrogen receptor modulators, to date, have not demonstrated much clinical efficacy for pain relief

•Programmed cell death in endometrial stromal cells is progesteronedependent and dysregulated in endometriosis

•While moderately effective, P4 resistance due to low PR expressionmay limit the clinical utility of progestins for endometriosis pain

•Natural herbal compounds, including curcumin, can induce stromalcell apoptosis via NF-κB, relatively independent of PR

•Synthetic curcuminoids (eg, EF-24) have good bioavailability, high potency and are promising new agents for non-hormonal therapies of endometriosis symptoms

Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol. 1975;122:262-3Markee JE. Menstruation in intraocular endometrial transplants in the Rhesus monkey. Am J Obstet Gynecol. 1978;131:558-9.Brandenberger AW, Lebovic DI, Tee MK, Ryan IP, Tseng JF, Jaffe RB, Taylor RN. Oestrogen receptor (ER)-alpha and ER-beta isoforms in normal endometrial and endometriosis-derived stromal cells. Mol Hum Reprod. 1999;5:651-5.Xue Q, Lin Z, Cheng YH, Huang CC, Marsh E, Yin P, Milad MP, Confino E, Reierstad S, Innes J, Bulun SE. Promoter methylation regulates estrogen receptor 2 in human endometrium and endometriosis. Biol Reprod. 2007;77:681-7.Gomes MK, Rosa-e-Silva JC, Garcia SB, de Sá Rosa-e-Silva AC, Turatti A, Vieira CS, Ferriani RA. Effects of the levonorgestrel-releasing intrauterine system on cell proliferation, Fas expression and steroid receptors in endometriosis lesions and normal endometrium. Hum Reprod. 2009;24:2736-45.Fang Z, Yang S, Lydon JP, DeMayo F, Tamura M, Gurates B, Bulun SE. Intact progesterone receptors are essential to counteract the proliferative effect of estradiol in a genetically engineered mouse model of endometriosis. Fertil Steril. 2004;82:673-8.Stratton P, Sinaii N, Segars J, Koziol D, Wesley R, Zimmer C, Winkel C, Nieman LK. Return of chronic pelvic pain from endometriosis after raloxifene treatment: a randomized controlled trial. Obstet Gynecol. 2008;111:88-96.Attia GR, Zeitoun K, Edwards D, Johns A, Carr BR, Bulun SE. Progesterone receptor isoform A but not B is expressed in endometriosis. J Clin Endocrinol Metab. 2000;85:2897-902.Dmowski WP, Ding J, Shen J, Rana N, Fernandez BB, Braun DP. Apoptosis in endometrial glandular and stromal cells in women with and without endometriosis. Hum Reprod. 2001;16:1802-8.Vercellini P, Somigliana E, Consonni D, Frattaruolo MP, De Giorgi O, Fedele L. Surgical versus medical treatment for endometriosis-associated severe deep dyspareunia: I. Effect on pain during intercourse and patient satisfaction. Hum Reprod. 2012;27:3450-9.

References

Endometriosis Study CollaboratorsIndrani Bagchi, PhDMilan Bagchi, PhDSarah Berga, MDAimee Browne, MD MSCRAntônio Francisco, MD PhDChristopher James, BSErika Johnston-MacAnanny, MDBenita Katzenellenbogen, PhDDennis Liotta, PhDCeana Nezhat, MDStacey Schutte, PhDNeil Sidell, PhDAnne Wofford, BSFritz Wieser, MD MSCRTamer Yalcinkaya, MDJie Yu, MD MS

Venha Brasil!

São Paulo

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Systems Biology of Endometriosis

Linda Griffith, MIT

Disclosure

I have no financial relationships to disclose.

Why are endometriosis patients different from other women -and different from each other in how they respond to therapies?

Genes?

Nutrition (gut microbiota)?

Exposure to environmental chemicals?

Parental/Grandparental Exposure to (environmental) chemicals/radiation?

Infections?

benign

malignant (cancer)

EndometriosisIs not a benign disease

incidental finding during surgery for something else

symptomatic (minimum/mild)

symptomatic (moderate / severe / deep infiltrating)

1. How to stratify patients for treatment?

‐pre‐surgery – imaging, response to OCP (biomarkers)

‐post‐surgery – geographic & morphological appearance of lesions (molecular biomarkers?)

2. Molecular mechanisms of disease progression – new drugs

3. Disease Etiology and prevention

Sauter et al, J Clin Oncol, 2009

Her2+ (IHC)

Consensus Molecular Markers for Stratification of Breast Cancer Patients

ERPRHER2

Her2+ (FISH) Her2‐ (FISH)

surgery

surgery + radiation

surgery + standard chemotherapy (eg AC/T) + radiation

surgery + dose‐dense chemotherapy + radiation

surgery + standard chemotherapy + herceptin + radiation

Similar Markers for endometriosis?

Risk Classification

Most breast cancers are spontaneous – genetic markers like BRCA1 may guide facets of treatment, but for only a small fraction of patients

Human Genome

Over 3 billion base pairs

Only 2% of total codes for proteins (~25,000 genes)

Cost to sequence a complete human genome$10M (2007) $3K (2013)

>30,000 individuals completely sequenced to date

Maps of regions that vary most enable clinical researchers to use lower-cost (~$300) partial sequencing methods to compare patients

Chromosome 1 Locus Map

4

14

4 Linda Griffith, 10/13/2013

15

Chromosome 1

‐ 249 million base pairs (8.5 cm long), encodes ~2000 proteins & 134 micro‐RNAs

‐ 4.4 million variations among sequenced genomes

Variant SequenceAt least 1% of the population

Common SequenceMost of the population

T

A

G

C

Single Nucleotide Polymorphism (“SNPs”)The most common kind of variation

(other variants: deletions, insertions, transpositions)

SNP SNP Array (~$300/analysis)~500,000 SNPs

3

Are genetic tests on the horizon?…..for “endometriosis” susceptibility?**

……..for “progesterone-resistant (or any other subtype) of endometriosis?

(Unlikely)

• E.g. BRCA‐1 gene mutation is associated with increased risk of breast cancer – but for any individual risk is a spectrum modulated by many unknown factors.

• Most breast cancer is spontaneous, arising from no known genetic risk factors!

**

6

Genome-Wide Association Studies (“GWAS”)

Identify candidate genetic loci associated with disease risk

Complex traits require very large populations with careful phenotyping

Patient DNA Control DNA

Statistical Comparisons

Replication

Are genetic tests on the horizon?

Rahmioglu, Missmer, Montgomery, and Zondervan Curr Obstet Gynecol Rep “Insights into the Genetics of Endometriosis,” 1: 124‐137 (2012)

Endometriosis GWAS results have identified 3 possible loci but explain very little of the heritability

• Separate studies in Japanese and European populations yielded different top candidates

• Relatively small population sizes and crude phenotyping (i.e., endometriosis vs not) limits analytical power

• need much better symptomatic/clinical stratification of patients to improve resolution

5000+ individuals in population

5

Epigenetic Modification in EndometriosisDNA & histone modification alter gene transcription

histone

DNAHistone modification

T

AG

C

DNA methylation (often in promoter regions)

Active (transcribed) genes

InflammationDevelopment/differentiationdiet

Silenced genes

Munro, Farquhar, Mitchell, Ponnampalam, “Epigenetic Regulation of Endometrium During the Menstrual Cycle,” Mol Hum Repro, 16, 297‐310 (2010)

Drugs modifying the enzymes in these processes are being studied for cancer & several inflammatory diseases, including endometriosis – but they are relatively non‐specific

Histone de‐acetylase inhibitors “HDACS”Valproic acidRomidepsinsuberoylanilide hydroxamic acid DNA methylation reversal

decitabine and azacitidine

7

Epigenetic Modification in EndometriosisDNA & histone modification alter gene transcription

histone

DNAHistone modification

TAGC

DNA methylation (often in promoter regions)

Active (transcribed) genes

InflammationDevelopment/differentiationdiet

Silenced genes

Munro, Farquhar, Mitchell, Ponnampalam, “Epigenetic Regulation of Endometrium During the Menstrual Cycle,” Mol Hum Repro, 16, 297‐310 (2010)

Need epigenomics to understand the whole picture – inherently an inflammatory process involving multiple processes

Igarashi, Bruner‐Tran, Yeaman, Lessey, Edwards, Esienberg, Osteen, Fert Stert 84, 67 (2005)

Example – treatment of endometrial cells with environmental chemical reducedrelative PR‐B:PR‐A expression

8

Genomic Insights are only the tip of the iceberg

Caution: targeted drug discovery should not be disconnected from mechanistic understanding of dynamic signaling networks

16


Slide 8


Slide 9


Slide 10


Slide 11


17

10/22/201313

DNA RNA Protein Metabolites

Genomics- GWAS/SNP- ChIP-Seq- Deep sequencing- Methylation

Transcriptomics- mRNA expression- miRNAexpression- RNA-Seq

Proteomics & Multiplex Immunoassays

- PPI- 2DGE - LC/MS- PTMs

Metabolomics- Lipids- Glycoproteins

Biological InsightClinical Management

Integrative Analysis

Wish list(quantitative annotated data base of surgical findings, symptoms)

Protein Activity States

Phopho-proteomics

Protease-omics

Kinase-omics

“Systems Biology” integrates information flow over multiple hierarchies

Signaling Networks as Cell “Information Processing Circuits”,in regulation and dysregulation of cell behavior by microenvironment

extracellular‘cues’

network of‘signals’

Phenotypic ‘effectors’ and‘responses’‐‐ e.g., geneexpression,metabolicactivities,cytoskeletalprocesses,

…

MIT BE

Can we learn how to intervene, in predictive manner, in complexmulti-cellular, multi-cytokine/chemokine, multi-pathway system?

e.g., for therapeutic targets (likely combinatorial)-- small molecule pathway inhibitors

-- cytokines/chemokines or blocking antibodies-- cell additions or depletions

[Tariverdian, Sem Immunopathol (2007)]

Can we learn how to intervene, in predictive manner, in complexmulti-cellular, multi-cytokine/chemokine, multi-pathway system?

Notion: a multitude of molecular and cellular constituents are all important; the problem is not presuming to ascertain which is “singularly important” (i.e., the ‘magic bullet’ target), but rather what happens when any/some of these in particular are perturbed

[Tariverdian, Sem Immunopathol (2007)]

Multiplex Molecular Profiling of Inflammatory Cytokines

Peritoneal Fluid

Beste et al, in revision

Consensus SignaturePatients and Controls

Mathematical analysis

Unsupervised Molecular Classification of Endometriosis Patients using Highly Multiplexed Molecular Profiling of Peritoneal Cytokines

S

Cluster 1 Cluster 2

IL-8IL-6

RANTES MIF

MCP-1IL-16

PDGF-bbG-CSFEotaxin

HGF FGFbVEGF

MIG GROaIL-1raIL-10

IL-12(p70)ICAM-1

IL-1bIL-3

Signature Cytokines

PainSymptoms

NoneMild

Severe

Beste et al, in revision

18

Hematopoietic Expression Profile Databases

HSC

CMP CLP

GMPMEP pre-B

Baso NK

TNaïve

TCM TEM TH1 TH2 TγδBmDC(-) lDCEos(-) M(-)

mDC(+)Eos(+) M(+)

Mast(-)

Mast(+)

N(-)

N(+)

MegaEry

Lymphocytes

Dendritic CellsGranulocytesMacrophagesMast Cells

Megakaryocytes

Erythrocytes

Consortium Species NCBI GEO Accession

# Profiles # Populations

Garvan Institute Human GSE3982 32 17

Genentech Human GSE22886 110 22

Broad Institute Human GSE24759 221 38

Immunological Genome Project Mouse GSE15907 559 221

Predicted Immunologic Network

In vitro Evidence for Peritoneal Macrophage Activation

MΦ

Peritoneal Mononuclear Cells

Granulocyte Depletion

24 hr Macrophage

culture

Peritoneal Leukocytes

LymphocyteDepletion

Controls

IL-15VEGFIL-12IL-16IL-2TNFaG-CSFIL-10FGFbEotaxinIL-4IL-9IL-5RANTESMIP1aMIP1bIL-1bIL-6

Endometriosis

Insights into new therapies targeting activation pathways

In vitro Evidence for Peritoneal Macrophage Activation

Controls

IL-15VEGFIL-12IL-16IL-2TNFaG-CSFIL-10FGFbEotaxinIL-4IL-9IL-5RANTESMIP1aMIP1bIL-1bIL-6

Endometriosis

*

**

**

*

*

*

•facilitate large-scale, cross-centre, longitudinal, epidemiologically robust, biomarker and treatment target discovery research in endometriosis

• Consensus format for detailed clinical phenotyping (phenome) data to be collected from women with endometriosis and controls to allow collaborative sub-phenotype discovery and validation analyses;

• Consensus Standard Operating Procedures (SOPs) for banking of biological samples from women with endometriosis and controls

EPHect Project :Endometriosis Phenome and BiobankingHarmonisation Project

Essential connection between the clinical and basic research communities

Prof. Stacey Missmer Prof. Krina Zondervan

PrincipalInvestigators

Prof. Frans Kaashoek

Prof. John Guttag

MIT Computer Science and Artificial Intelligence Lab

Dr Mauricio Abrão – AAGL Surgery APP

Building an Endometriosis “APP” for patient management and research data collection(beyond RedCap)

Dr Keith IsaacsonLinda Griffith

• Open source• Data encryption via Mylar• Web-based platform for mobile or other formats• Future Blue-tooth device integration

19

http://endogentest.meteor.com

• (will log on to APP web site here to demo)

Quiz

• Genome‐wide association studies of endometriosis patients have resulted in a genetic test for endometriosis susceptibility in European women (yes/no)

Answer = No

Acknowledgements

@MIT

Douglas Lauffenburger, PhD

John Guttag, PhD

Frans Kaashoek, PhD

@NWH/Harvard Medical School

Keith Isaacson, MD

Stephanie Morris, MD

Griffith/Lauffenburger/Isaacson LabMichael BesteNicole Pfäffle-Doyle, MD, PhDEmily PrenticeAmelia BaileyChristi CookNathan TedfordGrinia NogueiraKristen NaegleDave ClarkeShannon AlfordJustin PritchardHsinhwa LeeMary Rhoads

Collaborating Investigators

Paula Hammond, MIT Chemical Engineering

Barbara Imperiali, MIT Chemistry

Stacey Missmer, BWH and Harvard Medical

Asgerally Fazleabas, Michigan State

Kevin Osteen, Vanderbilt Univ.

Frank Gertler, MIT

Mauricio Abrão, São Paolo

Hilde Jørgenson, Oslo

Financial Support

M.I.T. Center for Gynepathology Research

M.I.T. Center for Cell Decision Processes

Newton-Wellesley Hospital

NIH Transformative R01 – NBIIB

Anonymous Foundation

Mauricio Abrãoin Granja JulietaSee you at WCE 2014!

Three Facets of System Integration Needed

“Horizontal” – greater number of molecular components considered together“Vertical” – greater number of space and time scales considered together

“Operational” – greater number of properties considered together

“horizontal”

“operational”

“vertical”

“horizontal”

“vertical”

“operational”

[D Lauffenburger, Integrative Biology (2012)]

MIT BE

20

CULTURAL AND LINGUISTIC COMPETENCY Governor Arnold Schwarzenegger signed into law AB 1195 (eff. 7/1/06) requiring local CME providers, such as

the AAGL, to assist in enhancing the cultural and linguistic competency of California’s physicians

(researchers and doctors without patient contact are exempt). This mandate follows the federal Civil Rights Act of 1964, Executive Order 13166 (2000) and the Dymally-Alatorre Bilingual Services Act (1973), all of which

recognize, as confirmed by the US Census Bureau, that substantial numbers of patients possess limited English proficiency (LEP).

California Business & Professions Code §2190.1(c)(3) requires a review and explanation of the laws

identified above so as to fulfill AAGL’s obligations pursuant to California law. Additional guidance is provided by the Institute for Medical Quality at http://www.imq.org

Title VI of the Civil Rights Act of 1964 prohibits recipients of federal financial assistance from

discriminating against or otherwise excluding individuals on the basis of race, color, or national origin in any of their activities. In 1974, the US Supreme Court recognized LEP individuals as potential victims of national

origin discrimination. In all situations, federal agencies are required to assess the number or proportion of LEP individuals in the eligible service population, the frequency with which they come into contact with the

program, the importance of the services, and the resources available to the recipient, including the mix of oral

and written language services. Additional details may be found in the Department of Justice Policy Guidance Document: Enforcement of Title VI of the Civil Rights Act of 1964 http://www.usdoj.gov/crt/cor/pubs.htm.

Executive Order 13166,”Improving Access to Services for Persons with Limited English

Proficiency”, signed by the President on August 11, 2000 http://www.usdoj.gov/crt/cor/13166.htm was the genesis of the Guidance Document mentioned above. The Executive Order requires all federal agencies,

including those which provide federal financial assistance, to examine the services they provide, identify any

need for services to LEP individuals, and develop and implement a system to provide those services so LEP persons can have meaningful access.

Dymally-Alatorre Bilingual Services Act (California Government Code §7290 et seq.) requires every

California state agency which either provides information to, or has contact with, the public to provide bilingual

interpreters as well as translated materials explaining those services whenever the local agency serves LEP members of a group whose numbers exceed 5% of the general population.

~

If you add staff to assist with LEP patients, confirm their translation skills, not just their language skills.

A 2007 Northern California study from Sutter Health confirmed that being bilingual does not guarantee competence as a medical interpreter. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2078538.

US Population

Language Spoken at Home

English

Spanish

AsianOther

Indo-Euro

California

Language Spoken at Home

Spanish

English

OtherAsian

Indo-Euro

19.7% of the US Population speaks a language other than English at home In California, this number is 42.5%

21

http://www.imq.org/

http://www.usdoj.gov/crt/cor/pubs.htm

http://www.usdoj.gov/crt/cor/13166.htm

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2078538

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