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Animal Models Ad Advances in Endometrial Cancer Epidemiology and Biology i E d i l C E id i l d Bi l Harvard School of Public Health 2014 Diego H. Castrilln, M.D., Ph.D. Department of Pathology and Department of Pathology and Simmons

SLIDE 1

Animal Models

Ad i E d i l C E id i l d Bi l Advances in Endometrial Cancer Epidemiology and Biology Harvard School of Public Health 2014 Diego H. Castrillón, M.D., Ph.D.

Department of Pathology and Department of Pathology and Simmons Comprehensive Cancer Center University of Texas Southwestern Medical Center D ll T Dallas, Texas

SLIDE 2

DISCLOSURES

dasdf

Consultant / Scientific Advisor / Inventor on IP licensed to

dasdf

/ / Molecular MD (LKB1 and oncology biomarker diagnostics)

SLIDE 3

Genetically-engineered d l f h mouse models of human cancer

Cancer is defined by complex tumor/host cell interactions

y p /

Cancer cannot be fully modelled in vitro
Diversity of genetic tools and alleles is needed
Useful for hypothesis testing and hypothesis generation
Preclinical models / therapy individualization
TCGA  many new endometrial cancer genes

SLIDE 4

The uterus: mouse vs. human

Mouse = “duplex type” uterus Human = “simplex type” uterus p yp

SLIDE 5

4000 5000 1000 2000 3000

s c l e

Neonatal Ovary Adult Ovary Eggs Cumulus Cells ES Cells Testis E11 Embryo Adrenal Gland Placenta Uterus

B. Marrow

Spleen Thymus Brain Eye

Sk. Muscle

Heart Intestine Kidney Liver Lung

Discovery of Sprr2f as endometrial specific gene

S k i n T

g u e L u n g H e a r t S k e l e t a l M u s c S p l e e n T h y m u s I n t e s t i n e L i v e r S t

a c h K i d n e y B r a i n T e s t i s U t e r u s 0.8 -

endometrial-specific gene

Day 4 (Estrus) O v a r y MG Uterus Vagina E2 3 Wk Uterus Cycle Day: 0.8 -

1 2 3 4 5

( s us)

+ -

SLIDE 6

Sprr2f RNA ISH Sprr2f RNA ISH

In situ Endometrinin

Contreras et al Dis Model Mech 2010 3:181

SLIDE 7

Generation and validation f S 2f C T ll l

f Sprr2f-Cre Tg allele

S 2f C x R26R Sprr2f-Cre x R26R

Contreras et al Dis Model Mech 2010 3:181

SLIDE 8

Stereotypical tumor progression in Sprr2f-Cre; Lkb1L/L ♀ Sprr2f-Cre; Lkb1 / ♀

WT Cre + Lkb1 L/L Cre + Lkb1 L/L Cre + Lkb1 L/L Cre + Lkb1 L/L 16 weeks 6 weeks 12 weeks 16 weeks 20 weeks

0.46g 0.19g 0.14g 1.17g

* * * *

0.11g bladder

cervix extra uterine fat

* * *

peritoneum bladder

vary

cervix extra-uterine fat peritoneum

*

SLIDE 9

60 80 100 Cre; Lkb1L/L (n=25) +/+ (n=10) Cre; Lkb1L/+ (n=12)

vival

S 2f C Lkb1L/L

20 40

p < 0.0001 % surv

Sprr2f-Cre; Lkb1L/L tumors are highly lethal

2.0 2.5 200 400 600 800 1000

days

highly lethal

1.0 1.5

wt Cre; Lkb1L/L weight (g)

3 5 6 8 4 1 1 2 1 4 1 6 8 1 9 6 0.0 0.5

Cre; Lkb1 days days

SLIDE 10

mTOR inhibitors for cancer therapy py

Mixed results

Everolimus (RAD001) Temsirolimus (CCI-779) Rapamycin

Mixed results
No benefit: melanoma, GBM, etc.
T

i li FDA d f RCC

Temsirolimus: FDA approved for RCC
Responses in endometrial ca

N di i

No predictive tests

SLIDE 11

Rapamycin sensitivity in endometrial cancer cell lines

A B

Cell line Rank Sensitivity (10 µM) AN3CA 4 0 086

endometrial cancer cell lines

AN3CA 4 0.086 MFE-319 11 0.157 MFE-296 16 0.195 HEC-1 24 0.228 Ishikawa 86 0.395 Heraklio 02 ER- 103 0.419 SNG-M 113 0.428 EN 149 0.474

p=3x10-5

28 e

EN 149 0.474 EFE-184 155 0.48 MFE-280 374 0.635

16 20 24 f a particular tumor type 4 8 12 f responding cell lines of endometrial cancer 10 20 30 40 50 60 70 80 90 100 % of responding cell lines of a particular tumor type # of

Jeff Settleman

SLIDE 12

mTOR inhibitor trial: early disease y Sprr2f-Cre; Lkb1L/L ♀ Sprr2f Cre; Lkb1 ♀ age: 12 weeks rapamycin vehicle rapamycin vehicle

(2mg/kg qD)

4 weeks

autopsy

SLIDE 13

1.0 p<0.0001 0.4 0.6 0.8 erine weight (g) Lkb1 pa wt wt + Rapa Lkb1 Lkb1 + Rapa 0.0 0.2 ute Lkb1+Rap

150 200 250 positivity 100 150 200 positivity

p=0.013

200 300

sitivity

p=0.004 p<0.0001

Rapamycin halts tumor progression

wt Lkb1 Lkb1+Rapa 50 100 p-S6K p wt Lkb1 Lkb1+Rapa 50 TUNEL p wt Lkb1 Lkb1+Rapa 100 Ki67 po

p-S6K p wt Lkb1 Lkb1 + Rapa

SLIDE 14

mTOR inhibitor trial: advanced disease Sprr2f-Cre; Lkb1L/L ♀ p ; ♀ age >20 wks (advanced disease) rapamycin

(2mg/kg qD)

serial MRI (q 2 wks) se a M (q w s)

Contreras et al Dis Model Mech 2010 3:181

SLIDE 15

Rapamycin results in tumor regression

1800 1200 1500 1800

† ↓

mm 3) 2.0 2.5 ge

600 900

† ↓

volume (m 0.5 1.0 1.5

↓ ↓

fold chang

2 4 6 8 1 300

weeks

Pre-tx (0 wks) 2 wks Rapa 4 wks Rapa 6 wks Rapa 8 wks (2 wks off)

2 4 6 8 10 0.0 weeks

Pre tx (0 wks) 2 wks Rapa 4 wks Rapa 6 wks Rapa 8 wks (2 wks off)

Contreras et al Dis Model Mech 2010 3:181

SLIDE 16

Oophorectomy prolongs survival in Lkb1-driven endometrial cancer

100

Lkb1 (n=11) Lkb1 + oophorectomy (n=13)

ival (%)

surv

50 100 150 200 250 300 350 400 450

p = 0.0004 Age (days)

Dennis Ruder

SLIDE 17

Validation of Lkb1 mAB

(Cell Signalling clone #D60C5)

in mosaic mice (6 weeks)

40 60 80 100 20 3wks 6wks 12wks 20wks

Lkb1L/L 6 weeks Sprr2f-Cre; Lkb1L/L 6 weeks 6 weeks 6 weeks

Nakada et al PLOS ONE 2013 8:e73449

SLIDE 18

Telomere shortening in Type II cancers?

TELO-CISH Type II << Type I

Akbay et al Am J Pathol 2008 173:536

Type II << Type I

SLIDE 19

Endometrial cancer in Pot1a/p53 mice

Akbay et al Oncogene 2013 32:2219

SLIDE 20

Type II features in Pot1a/p53 tumors /

Akbay et al Oncogene 2013 32:2219

SLIDE 21

Tetraploidy in Pot1a/p53 Pot1a/p53 tumors

Akbay et al Oncogene 2013 32:2219

SLIDE 22

Summary/outlook Summary/outlook

Mouse models rich potential resource for

understanding biology of endometrial carcinogenesis understanding biology of endometrial carcinogenesis (Type I vs. Type II)

Untapped potential in the ultimate goal of validation

and individualization of cancer therapy and individualization of cancer therapy

SLIDE 23

Acknowledgments

Castrillon Lab
MGH Cancer Center Harvard

– Cristina Contreras – Shana Wingo – Esra Akbay*** Teresa Gallardo – Nabeel Bardeesy – Jeff Settleman – John Schorge – Teresa Gallardo – Marshall Haynie – George John – Meredith Shidler

DFCI

– Kwok Kin-Wong – David Kwiatkowski

Y l S h l f M di i

– Lane Shirley – Jose Michel – Yuji Nakada – Chris Peña

Yale School of Medicine

– Sandy Chang

UNC Lineberger

Norman Sharpless Chris Peña – Dennis Ruder – Ileana Cuevas – Norman Sharpless – Neil Hayes

MD Anderson

– Russel Broaddus

Support

– NCI MMHCC UO1 – NCI RO1 – CPRIT Individual Investigator Award Russel Broaddus

UTSW Advanced Imaging Center

Animal Models

Ad i E d i l C E id i l d Bi l Advances in Endometrial Cancer Epidemiology and Biology Harvard School of Public Health 2014 Diego H. Castrillón, M.D., Ph.D.

Department of Pathology and Department of Pathology and Simmons Comprehensive Cancer Center University of Texas Southwestern Medical Center D ll T Dallas, Texas

DISCLOSURES

dasdf

dasdf

/ / Molecular MD (LKB1 and oncology biomarker diagnostics)

Genetically-engineered d l f h mouse models of human cancer

y p /

The uterus: mouse vs. human

Mouse = “duplex type” uterus Human = “simplex type” uterus p yp

Discovery of Sprr2f as endometrial specific gene

endometrial-specific gene

Sprr2f RNA ISH Sprr2f RNA ISH

In situ Endometrinin

Contreras et al Dis Model Mech 2010 3:181

Generation and validation f S 2f C T ll l

S 2f C x R26R Sprr2f-Cre x R26R

Contreras et al Dis Model Mech 2010 3:181

Stereotypical tumor progression in Sprr2f-Cre; Lkb1L/L ♀ Sprr2f-Cre; Lkb1 / ♀

* * * *

* * *

*

S 2f C Lkb1L/L

Sprr2f-Cre; Lkb1L/L tumors are highly lethal

highly lethal

mTOR inhibitors for cancer therapy py

i li FDA d f RCC

N di i

Rapamycin sensitivity in endometrial cancer cell lines

endometrial cancer cell lines

Jeff Settleman

mTOR inhibitor trial: early disease y Sprr2f-Cre; Lkb1L/L ♀ Sprr2f Cre; Lkb1 ♀ age: 12 weeks rapamycin vehicle rapamycin vehicle

(2mg/kg qD)

4 weeks

autopsy

Rapamycin halts tumor progression

mTOR inhibitor trial: advanced disease Sprr2f-Cre; Lkb1L/L ♀ p ; ♀ age >20 wks (advanced disease) rapamycin

(2mg/kg qD)

serial MRI (q 2 wks) se a M (q w s)

Contreras et al Dis Model Mech 2010 3:181

Rapamycin results in tumor regression

Contreras et al Dis Model Mech 2010 3:181

Oophorectomy prolongs survival in Lkb1-driven endometrial cancer

Dennis Ruder

Validation of Lkb1 mAB

(Cell Signalling clone #D60C5)

in mosaic mice (6 weeks)

Lkb1L/L 6 weeks Sprr2f-Cre; Lkb1L/L 6 weeks 6 weeks 6 weeks

Nakada et al PLOS ONE 2013 8:e73449

Telomere shortening in Type II cancers?

TELO-CISH Type II << Type I

Akbay et al Am J Pathol 2008 173:536

Type II << Type I

Endometrial cancer in Pot1a/p53 mice

Akbay et al Oncogene 2013 32:2219

Type II features in Pot1a/p53 tumors /

Akbay et al Oncogene 2013 32:2219

Tetraploidy in Pot1a/p53 Pot1a/p53 tumors

Akbay et al Oncogene 2013 32:2219

Summary/outlook Summary/outlook

understanding biology of endometrial carcinogenesis understanding biology of endometrial carcinogenesis (Type I vs. Type II)

and individualization of cancer therapy and individualization of cancer therapy

Acknowledgments

– Cristina Contreras – Shana Wingo – Esra Akbay*** Teresa Gallardo – Nabeel Bardeesy – Jeff Settleman – John Schorge – Teresa Gallardo – Marshall Haynie – George John – Meredith Shidler

– Kwok Kin-Wong – David Kwiatkowski

Y l S h l f M di i

– Lane Shirley – Jose Michel – Yuji Nakada – Chris Peña

– Sandy Chang

Norman Sharpless Chris Peña – Dennis Ruder – Ileana Cuevas – Norman Sharpless – Neil Hayes

– Russel Broaddus

– NCI MMHCC UO1 – NCI RO1 – CPRIT Individual Investigator Award Russel Broaddus

– Masaya Takahashi g – NIH Mouse Pathobiologist Award (K26) – Sidney Kimmel Foundation – ACS