INTEGRATED ANALYSIS OF THE MOLECULAR DEREGULATIONS OF THE MDM2:P53 AXIS IN DDLPSKATE LYNN BILL, JEANNINE GARNETT, CHAD CREIGHTON, XIAOYAN MA, ISABELLE MEAUX, LAURENT DEBUSSCHE, THERESA NGUYEN, DAVIS INGRAM, SVETLANA BOLSHAKOV, ROMAN BELOUSOV, DINA LEV, ALEXANDER J. LAZAR, RAPHAEL POLLOCK

MD ANDERSON CANCER CENTER, Houston Tx

Anaya et al. Ann Surg 2009; 16(3)667-75Lahat et al. Ann Surg Oncol 2008; 15(6):1585-1593

WDLPS

DDLPSWDLPS

DDLPS

Local recurrence Disease specific survival

DDLPS: marked propensity for local recurrence and dismal outcome

Disclosures

• Provided drug (SAR405838) for studies under MTA

• Sanofi reviewed this presentation – no significant changes

• Referred to as “inhibitor” in this talk

MDM2/p53 axis

Wang, S. et al., Pharm Taxicol, 2009.

MDM2 amplifications in Cancer

Tumor type MDM2 amplification (na) (%)Brain tumors 6.7 (239)

Astrocytomas 8.1 (37)Glioblastomas 6.8 (191)Medulloblastomas 0 (8)Other 0 (3)

Breast carcinomas 5.9 (1774)Cervical carcinomas 1.1 (88)Esophageal carcinomas 13 (96)Leukemias/lymphomas 0 (304)Hepatoblastomas 0 (38)Lung 5.7 (88)

Lung cancers (NSCLC) 6.0 (83)Lung (not specified) 0 (5)

Nasopharyngeal carcinomas 2.1 (46)Neuroblastoma 2.0 (51)Osteosarcomas 16 (207)Ovarian carcinomas 3.1 (190)Pancreatic carcinomas 0 (25)Soft tissue tumors 20 (479)

Ewing’s sarcomas 10 (30)Leiomyosarcomas 0 (46)Lipomas (benign) 30 (43)Liposarcomas 29 (87)Undifferentiated pleomorphic sarcoma

21 (163)

Malignant Schwannomas 19 (16)

Sarcomas (non-specific)b 13 (85)

Variousc 33 (9)Testicular tumors 4.6 (64)Thyroid carcinomas 0 (22)Urothelial carcinomas 2.2 (137)Wilms’ tumors 0 (40)

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MDM2: potential target for cancer therapy

Small molecule inhibitors:• Nutlin• RG7112• SAR405838

Wang, S. et al., Pharm Taxicol, 2009.

MDM2 Inhibitor: SAR405838 Orally bioavailable small

molecule Single-digit nM binding to

hMDM2, high selectivity and specificity

Blocks interaction of MDM2 & P53

Inactive in p53 mut cell lines

Phase I initiated mid-2012 Safety and efficacy Biomarker development

NHCl

NH

F

ClO

NH

OH

O

SAR299155

MW: 562.51

(Formerly: SAR299155 or MI-77301)

MDM2 Amplification and Overexpression in DDLPS

PA A 224

246

863

141

815

SW87

2

PLS-

1DDLPSS

MDM2

β -actin

gDNA RNA

**

*

*

Systematic characterization of DDLPS cell lines 4 DDLPS cell lines were treated with

SAR405838

ProteomicsmRNAmiRNADNA copy number

variation*DNA

Sequencing*

*Previously reported data

Experimental Outlinetwo independent biological

replicates

224 815 246 863

cell lines were either treated with 0.003% DMSO,

1 µM or 3 µM SAR405838 (24hrs)

Analysis for differential gene

expression/miRNA array

Differential Gene Expression with SAR405838 Treatment

Pathway/processenrichment

p53 regulated targets

12q13~15 amplified targets

>2 fold upregulated or downregulated genes in at least 3 cell lines

Top Deregulated Pathways: Ingenuity Pathway Analysis

P53 signaling / Cell cycle / DNA damage

Upregulated mRNA in DDLPS Downregulated by the MDM2:p53 Axis

UPREGULATED TRANSCRIPTS IN

DEDIFFERENTIATED LIPOSARCOMA CELLS

523

DOWNREGULATED TRANSCRIPTS WITH

MDM2i

238183

~35% of transcripts that are upregulated in DDLPS when compared with normal controls are downregulated in response to MDM2 inhibition

These data suggest that a 1/3 of all upregulated transcripts in DDLPS are regulated by the MDM2:p53 axis

~35%

Downregulated mRNA in DDLPS Upregulated by the MDM2:p53 Axis

UPREGULATED TRANSCRIPTS WITH

MDM2i

234

DOWNREGULATED TRANSCRIPTS IN

DEDIFFERENTIATED LIPOSARCOMA

CELLS

41119

~5% of transcripts that are downregulated in DDLPS when compared with normal controls are upregulated in response to MDM2 inhibition

These data suggest that a small number of transcripts that are downregulated in DDLPS are regulated in a MDM2/p53 axis-dependent manner

~5%

p53 and Mitotic Pathway Connections

SAR405838SAR405838 uM 0 0.1 0.3 1 3 10

Aurora A

PTTG

Actin

Proposed signaling pathway for PTTG1 action at the G2 DNA damage checkpoint

DNA Damage

PTTG1

PTTG1

p53

Aurora AG2 M

Cytoplasm

Nucleus

Top Canonical Pathways Potentially Affected by miRNAs in DDLPS

Potential miRNA Regulation of mRNA with SAR405838: ABI SOliD platform

Top 18 miRNAs differentially

expressed in 3 cell lines vs controls

MaxA > 50P < 0.05FC > 2

Top 472 genes differentially

expressed genes in 4 DDLPS cell lines

treated with SAR405838 vs DMSO controls

FC > 2P < 0.05

14 miRNAs potentially regulating 93 differentially

expressed mRNAs with SAR405838 treatment

~20%

Top Molecular Pathways Affected by miRNAs in DDLPS

1. Cell Cycle: G2/M DNA Damage Checkpoint

2. p53 Signaling

3. Mitotic Roles of Polo-Like Kinase 1

Summary

Gene expression arrays identified multiple targets / pathways of potential interest

Identify on and off effects, regulatory loops

Studies focus on validating specific pathway/targets

Acknowledgements Dina Lev Raphael E.

Pollock Jeannine Garnett Chad Creighton David Pollock Svetlana Bolshakov XiaoYan Ma Davis Ingram Roman Belousov Theresa Nguyen Gonzalo Lopez Danielle Braggio

Sanofi-Aventis Isabelle Meaux Laurent Debussche Cedric Barriere

Amschwand Sarcoma Cancer Foundation