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

2. 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)

3. 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

4. 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

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

6. 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

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

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

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

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

11. Tagln-cre is also expressed in mesenchymal stem cells

12. Murine HGUPS have complex karyotypes

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

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

15. Which advantage does Pten downregulation
confer to p53 deficient sarcomas?

16. 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

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

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

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

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

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

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

23. 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

24. 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