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