Application of murine models of osteosarcoma to preclinical target discovery and

validation

Carl Walkley

St.Vincent’s Institute, Australia

Singapore Sarcoma Symposium, 12-13 Sept 2015

Osteosarcoma of the thigh bone – Egyptian mummy – 5th century

Osteosarcoma – Then

And now

Improving outcomes in OS

Osteosarcoma

• most common cancer of bone

• peak incidence at age 15 years

• affects lower long bones

• current treatment - surgical resection and chemotherapy

• standard of care has not significantly improved in 30 years

• survival - primary only 70% at 5 yrs, metastatic or recurrent disease 20% at 5 yrs

• 5th leading cause of childhood cancer and a high morbidity and mortality rate

Osteosarcoma

Challenges in the Clinical Study of OS

Novel approaches to drug and drug target discovery, with particular attention to drug development for

metastatic osteosarcoma, are needed

Translation of basic research in OS is hampered by:

• relatively rare tumour

• multi-institutional national clinical trials require years of enrolment

• standard of care includes amputation, complicating Phase II/III trials

• in last 20 years only one significant advance in treatment to standard upfront

chemotherapy resulting in a modest improvement in overall survival

Genetics of Osteosarcoma

Chen et al. Cell Reports 7, 104–112, April 10, 2014

Perry et al. PNAS E5564–E5573, doi: 10.1073/pnas.1419260111

Mouse Models of Human Cancer

A valid and biologically meaningful mouse model of human cancer should recapitulate

• genetic basis of disease

• appropriate age of disease development

• tissue restricted nature of disease

• metastasis to same sites

• gene expression signatures

• histology

Provide “robust pre-clinical infrastructure through molecularly defined model systems that are

directly reflective of their human counterpart and sufficient models numbers for one to

approach the disease diversity found in humans” (W Sellers, A blueprint for advancing genetics-based cancer therapy.

Cell 2011,147;26-31)

Bedside to Bench: Osteosarcoma Modelling

Known genetic lesions in familial OS predisposition and sporadic OS: • Li-Fraumeni syndrome (p53+/-)

• Hereditary retinoblastoma (RB+/-)

• Rothmund-Thomson Syndrome (Recql4 mutated; Ng et al PLoS Genetics 2015 Apr 10;11(4):e1005160)

Osx-Cre

Models of two histological subtypes of human OS

Conventional human OS:

60% osteoblastic OS

15% fibroblastic OS

15% chondroblastic OS

10% various rare forms (upto 8 types)

p53 shRNA OS Normal bone p53fl/fl OS

Mutsaers et al., Bone. 2013 Jul;55(1):166-78

Walkley et al., Genes & Dev. 2008 22: 1662-1676

Using models of human OS

Paired primary and metastatic cell cultures at early passage – not available from human biopsy material Use these for:

• define new therapeutic strategies – chemical/drug screen

• identify interactions with chemotherapy that could be used therapeutically – siRNA screen

• test new therapies based on hypothesis driven understanding of OS

Identifying OS susceptibility pathways

Integrated, parallel phenotypic and drug screening approach

Ankita Gupte, Kate Gould, Dr Kaylene Simpson

Whole Genome Screening

siRNA +Doxorubicin

siRNA alone

Gupte et al., Clin Can Res. 2015 Jul 15;21(14):3216-29

Primary Screen Data from mouse OS cells

Ankita Gupte, Kate Gould, Alistair Chalk Gupte et al., Clin Can Res. 2015 Jul 15;21(14):3216-29

The unknown knowns – new roles for old genes?

Pathways disproportionately enriched as increasing OS cell death

Ankita Gupte, Alistair Chalk Gupte et al., Clin Can Res. 2015 Jul 15;21(14):3216-29

siRNA screen – chemical validation of results

Ankita Gupte, Dr Emma Baker, Scott Taylor

Secondary and tertiary screens completed - validated candidates identified

Established if any chemicals/drugs were available that targeted these pathways

Could identify inhibitors for 3 of the 4 pathways of highest interest (1 a drug, 2 chemical/preclinical)

Integrated, parallel phenotypic and drug screening approach

Drug/Chemical Screening - HTS

Dr Emma Baker, Dr Kurt Lackovic, Prof David Huang, Dr Chris Burns, Soo San Wan

Chemical/Drug screen – with Prof David Huang, WEHI

3 primary and metastatic paired cell lines from mouse fibroblastic OS model

Screened against 131 kinase inhibitors in an 11 point dose response in duplicate

Library composed of clinically approved, preclinical and agents with known activity that didn’t progress

Can use to identify pathways that may be tractable or for rapid translation of the research with agents already in clinical use.

Drug/Chemical Screening - HTS

Dr Emma Baker, Dr Kurt Lackovic, Prof David Huang, Dr Chris Burns, Soo San Wan Gupte et al., Clin Can Res. 2015 Jul 15;21(14):3216-29

Dual targeting of the PI3K and mTOR axis is a species conserved OS vulnerability

Dr Emma Baker, Scott Taylor, Ankita Gupte, Prof Mike Dyer & Drs E Stewart/A Loh/A Shelat/A Karlstrom (St Jude’s)

Gupte et al., Clin Can Res. 2015 Jul 15;21(14):3216-29

Dual PI3K/mTOR inhibitors cause on target inhibition and cell cycle modulation

Dr Emma Baker, Scott Taylor, Ankita Gupte Gupte et al., Clin Can Res. 2015 Jul 15;21(14):3216-29

Dual PI3K/mTOR inhibitors cause apoptosis of primary OS derived cells

Dr Emma Baker, Scott Taylor, Ankita Gupte Gupte et al., Clin Can Res. 2015 Jul 15;21(14):3216-29

Dual targeting of the PI3K and mTOR axis – PI3Ka dependent effects

Dr Emma Baker, Scott Taylor, Ankita Gupte Gupte et al., Clin Can Res. 2015 Jul 15;21(14):3216-29

Combining clinically relevant PI3Ka and mTOR inhibitors

Dr Emma Baker, Scott Taylor, Ankita Gupte, Dr Jayesh Desai Gupte et al., Clin Can Res. 2015 Jul 15;21(14):3216-29

The intersection of chemical and genetic biology – PI3Kca/mTOR inhibitors in OS

Independent screening approaches have converged on the PI3K/mTOR pathway as a tractable candidate in OS

PI3K specific or mTOR specific agents have limited activity in OS

Dual pathway inhibition appears to be required for maximal activity

Activity requires inhibition of PIK3CA specifically

Pathway is conserved across murine and human OS and is subtype independent

The intersection of chemical and genetic biology – PI3Kca/mTOR inhibitors in OS

Proc Natl Acad Sci U S A. 2014 Dec 23;111(51):E5564-73. doi: 10.1073/pnas.1419260111. Epub 2014 Dec 15.

The intersection of chemical and genetic biology – PI3Kca/mTOR inhibitors in OS

Independent screening approaches have converged on the PI3K/mTOR pathway as a tractable candidate in OS

PI3K specific or mTOR specific agents have limited activity in OS in clinical trials to date

Dual pathway inhibition appears to be required for maximal activity

Activity requires inhibition of PIK3CA specifically – BYL719 now in clinical trial in BrCa

Pathway is conserved across murine and human OS and is subtype independent

Demonstrates efficacy of applying GEMM to preclinical target identification and validation

Collectively the data warrant further testing of PI3Kca / mTOR inhibitors in OS

Acknowledgements

St. Vincent’s Institute

Ankita Gupte

Scott Taylor

Dr Alvin Ng

Dr Alistair Chalk

Dr Mannu Walia

Dr Emma Baker

Dr Anthony Mutsaers (U Guelph)

St. Vincent’s Hospital

Dr Meaghan Wall

A/Prof John Slavin

Funding

AACR-Aflac Inc

Colin North

Australian Sarcoma Study Group

Zig Inge Foundation

Walter and Eliza Hall Institute

Prof David Huang

Dr Chris Burns

Dr Kurt Lackovic

Soo San Wan

Victorian Centre for Functional Genomics

A/Prof Kaylene Simpson

Kate Gould

Dr Piyush Madhamshettiwar

St Jude’s Children’s Hospital

Prof Mike Dyer

Dr Elizabeth Stewart

Dr Amos Loh

Dr Anang Shelat

Dr Asa Karlstrom

Peter MacCallum Cancer Centre

Dr Jayesh Desai

SAHMRI, University of Adelaide

Prof Andrew Zannettino

Dana-Farber Cancer Institute, Boston

Prof Stuart Orkin

Dr Katherine Janeway

Dr Jen Perry