dual pten/p53 suppression promotes high grade sarcomas by activating notch

Dual PTEN/P53 suppression promotes high

grade sarcomas by activating Notch

Eva Hernando, Ph.D.

• Genomic complexity • Low number of known recurrent genetic

mutations• Limited enrolment of patients in clinical

trials

Challenges in the study of high grade sarcoma and

leiomyosarcoma

better understanding of the molecular basismouse models that recapitulate human disease

Preclinical testing (immunotherapy)

• 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)

Targeted sequencing revealed low incidence of mutations in classical

‘cancer genes’

Guijarro et al., AJP 2013

Partial PTEN and TP53 deletions are common in HGUPS and LMS

Barretina et al., Nat Gen 2010

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/ Ptenwtp53wt

Tagln-cre/ PtenL/+p53L/+

Tagln-cre/ PtenL/+

p53wt

Tagln-cre/ Ptenwtp53L/+

Tagln-cre/ PtenL/Lp53wt

Tagln-cre/ Ptenwtp53L/L

Tagln-cre+/+ PtenL/L p53L/L

Tagln-cre+/- /PtenL/+p53L/+F1 Tagln-cre+/- /PtenL/+ p53L/+

F2

F0

PtenD/+p53D/+ mice have shorter overall survival than Pten+/+p53D/+

Guijarro et al., AJP 2013Hernando 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

Guijarro et al., 2013

Tagln-cre+/+ x ROSA26-loxP-STOP-loxP-LacZ

Tagln-cre is also expressed in mesenchymal stem cells

Murine HGUPS have complex karyotypes

Liver

Pancreas Lung

PtenD/+p53D/+ HGUPS are highly metastatic

Pten is haploinsufficient for sarcoma tumor progression

No mutation

PtenD/+p53D/+ Pten+/+p53D/+

Which advantage does Pten downregulation

confer to p53 deficient sarcomas?

PtenD/+/p53D/+ bone-marrow mesenchymal stem cells display higher clonability and

proliferationPtenL/+/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 toincreased 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• 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 cellsPTEN/TP53 deficient HGUPS may be susceptible

to Notch inhibition

Hernando LabMaria V. GuijarroLaura DanielsonMiguel SeguraMartha VegaAvital GazielSilvia Menendez

Luca PaoluzziDoug HannifordRaffaella Di MiccoLisa KoetzBarbara FontanalsElena SokolovaVivien LowOlivia BlackburnRana MoubarakVeronica DavalosPraveen Agrawal

CollaboratorsSonika DahiyaJian Jun WeiCarlos Cordon-CardoPier Paolo Pandolfi

Funding

American Cancer SocietyEdna´s Foundation of HopeLiddy Shriver Sarcoma IntiativeSarcoma Foundation of AmericaMelanoma Research AllianceDODNIH/NCINIH/NIAMS