Dual PTEN/P53 suppression promotes high grade sarcomas by activating Notch Eva Hernando, Ph.D.

Challenges in the study of high grade sarcoma and leiomyosarcoma Genomic complexity Low number of known recurrent genetic mutations Limited enrolment of patients in clinical trials better understanding of the molecular basis mouse models that recapitulate human disease Preclinical testing (immunotherapy)

Targeted sequencing revealed low incidence of mutations in classical ‘cancer genes’ N=40 specimens (NYU School of Medicine and Northwestern University) Platform: OncoMap3 core and extended panels (1047 mutations in 116 genes) 73 candidate mutations on 44 genes 6 confirmed mutations on 2 genes: TP53 (one V157F and two R273C amino acid substitutions). a low-frequency germ-line single-nucleotide polymorphism in EGFR (S703F) Guijarro et al., AJP 2013

Partial PTEN and TP53 deletions are common in HGUPS and LMS Barretina et al., Nat Gen 2010 Taking into account those molecular alterations associated with LMS, we tried to mimic the constitutive activation of the AKT mtor pathways, inactivating some components of the PI3K and p53 pathways Guijarro et al., AJP 2013

PTEN and TP53 expression is commonly downregulated in HGUPS and LMS Guijarro et al., AJP 2013

Smooth-muscle specific conditional inactivation of Pten and p53 Tagln-cre+/+ PtenL/L p53L/L F1 Tagln-cre+/- /PtenL/+p53L/+ Tagln-cre+/- /PtenL/+ p53L/+ Tagln-cre/ Ptenwtp53wt Tagln-cre/ PtenL/+p53L/+ Tagln-cre/ PtenL/+ p53wt Tagln-cre/ Ptenwtp53L/+ Tagln-cre/ PtenL/Lp53wt Tagln-cre/ Ptenwtp53L/L F2

PtenD/+p53D/+ mice have shorter overall survival than Pten+/+p53D/+ Guijarro et al., AJP 2013 Hernando et al., Nat Med 2007

PtenD/+p53D/+ mice have increased sarcoma incidence compared to Pten+/+p53D/+ Guijarro et al., AJP 2013

PtenD/+p53D/+ HGS histologically resemble the equivalent human lesions Guijarro et al., AJP 2013

Tagln-cre marks smooth muscle cells Tagln-cre+/+ x ROSA26-loxP-STOP-loxP-LacZ Guijarro et al., 2013

Tagln-cre is also expressed in mesenchymal stem cells

Murine HGUPS have complex karyotypes

PtenD/+p53D/+ HGUPS are highly metastatic Liver Pancreas Lung

Pten is haploinsufficient for sarcoma tumor progression PtenD/+p53D/+ Pten+/+p53D/+ No mutation

Which advantage does Pten downregulation confer to p53 deficient sarcomas?

PtenD/+/p53D/+ bone-marrow mesenchymal stem cells display higher clonability and proliferation PtenL/+/p53L/+ and Pten+/+/p53L/+ bone marrow-derived mouse mesenchymal stem cells + Adeno-cre infection Colony formation assay Proliferation assay Both, acute and genetically induced concomitant inactivation of Pten and P53 leads to increased clonogenic and proliferative capacity of mesenchymal stem cells

Pten suppression in Pten+/+/p53D/+ tumor cells increases clonogenic capacity and invasion

The Notch signaling pathway is up-regulated in PtenΔ/+Tp53Δ/+ murine HGUPS

The Notch signaling pathway is up-regulated in PtenΔ/+Tp53Δ/+ murine HGUPS

Gamma-secretase inhibition suppresses the clonogenic and invasive potential of tumor cells GSI Colony formation assay

shPten pro-oncogenic effects are counteracted by Notch inhibition Colony formation assay invasion assay

Model of molecular classification of high grade sarcoma patients for targeted therapies

Conclusions PTEN/TP53 deficient HGUPS may be susceptible Conditional inactivation of Pten and p53 in mouse mesenchymal progenitors recapitulates the histological and cytogenetic features of human HGUPS and LMS Pten deficiency confers increased clonogenic and invasive potential to p53 heterozygous sarcoma cells Pten suppression in p53 deficient MSCs and tumor cells triggers Notch signaling Gamma-secretase inhibitors negate the pro-oncogenic effects of Pten suppression in p53 deficient sarcoma cells PTEN/TP53 deficient HGUPS may be susceptible to Notch inhibition

Hernando Lab Maria V. Guijarro Laura Danielson Miguel Segura Martha Vega Avital Gaziel Silvia Menendez Luca Paoluzzi Doug Hanniford Raffaella Di Micco Lisa Koetz Barbara Fontanals Elena Sokolova Vivien Low Olivia Blackburn Rana Moubarak Veronica Davalos Praveen Agrawal Collaborators Sonika Dahiya Jian Jun Wei Carlos Cordon-Cardo Pier Paolo Pandolfi Funding American Cancer Society Edna´s Foundation of Hope Liddy Shriver Sarcoma Intiative Sarcoma Foundation of America Melanoma Research Alliance DOD NIH/NCI NIH/NIAMS