radiation-associated and sporadic ups/mfh : searching for novel therapeutic strategies

Sarcoma Research LaboratoryUniversity of Texas, MD Anderson Cancer Center

Radiation-associated and sporadic UPS/MFH: Searching

for novel therapeutic strategies

Keila Enitt Torres

UPS/MFH Pleomorphic malignant fibrous

histiocytoma (MFH) unclassified pleomorphic sarcoma

Current technology ? definable line of differentiation

UPS Peak incidence in the 6th and 7th decades of

life Typically large, deep, aggressive tumors 5% - metastases at presentation (lung) <3% radiation-associated

UPS Treatment

Radical excision with negative margins Neo-Adjuvant RT is offered widely negative

margins is not possible. Chemotherapy Unresectable

disease/metastatic disease

5-year disease-specific survival 44- 66%*

Approach Low throughput: PI3K/AKT signaling pathway

Determine if AKT/mTOR axis is a relevant target for UPS therapy

High throughput: genomic/proteomic analysis Understand genetic and molecular de-regulations UPS progression

PI3K/AKT pathway

Nature Medicine 13, 748 - 753 (2007)

HGF p-c-Met p-MEK p-AKTc-Met

Goal #1 Confirm whether the AKT/mTOR axis is a potential

target for UPS/MFH therapy Determine whether this pathway is differentially

activated in radiation-associated versus sporadic UPS

Radiation-associated sarcoma Diagnosis of RAS based on the criteria

published by Cahan, et al. in 1948.* Previous radiation tx for different tumor Development of the sarcoma - radiation field Histologically different from the primary cancer Minimum latent period of 5 years between the

radiation and development of the sarcoma Arlen et al. 1971- modified definition

Latency period of 3 years

*Cahan et al. Cancer 1948;1:3-29.

Bioresources in the laboratory Cell lines Animal models Human Tissue

Frozen FFPE

Cell strain/cell line bank

> 60 human tumors

13 Sporadic UPS cell strains

6 Radiation-associated

UPS cell strains

RIS-819.1 RIS-023

UPS-060UPS-485 UPS-186

UPS-060

AKT/mTOR pathway is activated in UPS

UPS-186

RIS-D

L620

RIS-D

L618

hMSC

p4EBP1

pAKT

4EBP1

AKT

S6K

b-actin

UPS-060

UPS-356

UPS-DL5

11

RIS-02

3

RIS-81

9

Radiation-associated Sporadic

pS6K

Rapamycin

0 10 100

0 10 100

0 10 100

RIS -819RIS -023 UPS-485UPS-060

Radiation-associated Sporadic

0 10 100

p4EBP1

pAKT

4EBP1

AKT

S6K

b-actin

pS6K

Rapamycin

Control 0.05uM 0.1 uM 0.5 uM 1 uM 5 uM0.0

20.040.060.080.0

100.0120.0140.0

RIS-819

% C

ell P

rolif

erati

on

Con 0. 0. 0.1 u

M5 u

M0.0

20.040.060.080.0

100.0120.0140.0

RIS-023

%Ce

ll Pr

olife

ratio

n

Concentration (mM)

0 0.05 0.1 0.5 1 5

0 0.05 0.1 0.5 1 5

Radi

ation

-ass

ocia

ted

Concentration (mM)

0 0.05 0.1 0.5 1 50.0

50.0

100.0

150.0UPS-060

% C

ell P

rolif

erati

on

0 0.05 0.1 0.5 1 50.0

50.0

100.0

150.0UPS-485

% C

ell P

rolif

erati

on

Spor

adic

Concentration (mM)Concentration (mM)

pAKT

AKT

pS6KS6Kb-actin

p4EBP14EBP1

mM/4hr 0 0.05 0.1

0 0.05 0.1

UPS-060 RIS-819

PI103

PI103

0 0.05 0.1 0.5 10

20

40

60

80

100

120

RIS-819

% C

ell p

rolif

erati

on

0 0.05 0.1 0.5 10

20

40

60

80

100

120

UPS-485

% C

ell p

rolif

erati

on

0 0.05 0.1 0.5 10

20

40

60

80

100

120

UPS-060

% C

ell p

rolif

erati

on

Concentration (mM)

Radi

ation

-ass

ocia

ted

Concentration (mM)

Spor

adic

Concentration (mM)Concentration (mM)

0 0.05 0.1 0.5 1 50

20

40

60

80

100

120

RIS-023%

Cel

l pro

lifer

ation

Control PI1030

1020304050607080

G1SG2/M

Control PI1030

102030405060708090

G1SG2/M

RIS-819

Control PI103

UPS-060

Control PI103

Cell Cycle

Migration

Control PI103

RIS-

819

Control PI1030

20

40

60

80

100

120

% M

igra

tion

UPS

-060

Control PI1030

20

40

60

80

100

120

% M

igra

tion

Invasion

Control PI103

UPS-

060

Control PI1030

20

40

60

80

100

120

% In

vasio

n

RIS-

819

Control PI1030

20

40

60

80

100

120

% In

vasio

n

In vivo studies Orthotopic Xenograft Model

1-2mm pieces of tumor

0 4 8 12 16 200

100200300400500600700800900

1000ControlPI 50 mg/mL

Days

Tum

or V

olum

e (m

m3)

*

Control PI1030

0.20.40.60.8

11.21.41.61.8

2

Tum

or w

eigh

t (g)

*

RIS-819 tumor growth

pAKT p4EBP1 pS6RP Ki67H&E

PI10

3Co

ntro

lRIS-819 Immunohistochemistry

1 4 8 12 16 24 32 40 480

50

100

150

200

250

300

350

Control

PI103 (50 mg/Kg)

Days

Tum

or V

olum

e (m

m3)

UPS-060.1 tumor growth

PI103Control

*

pAKT p4EBP1 pS6RP Ki67H&E

PI10

3Co

ntro

lUPS-060.1 Immunohistochemistry

Summary PI3K/AKT pathway is activated in radiation-

associated and sporadic UPS RA-UPS and sporadic UPS are sensitive to

PI103 Effects in vivo model => Cell death

Current studies

ProteomicsmiRNA/

non-coding RNAs

mRNAMethylation

DNA copy number variation

DNA Sequencing

Sequenome

Exon Sequencing

Array-comparative

genomic hybridization

miRNA analysis

RPPA

TMA

Reverse-phase protein lysate array for cell signaling analysis

EIF4E

29 molecules differentially

expressed

HER-2 SignalingERK/MAPK

PI3K/AKT/mTORErbB AMPK

HGF signalingIL-15 Signaling -3.5058 5.1031

(Log 2)

Mesenchymal cells

UPS

>50%P value <0.01

Unsupervised hierarchical clustering

Unsupervised hierarchical clusteringRA-UPS versus sporadic UPS

-3.5058 5.1031

(Log 2)

RA-UPS

Sporadic-UPS

Sarcoma Research LaboratoryUniversity of Texas, MD Anderson Cancer Center

Dina Lev Raphael E. Pollock Billy Wang Alexander J. Lazar Vinod Ravi

SARC (Sarcoma Alliance for Research through Collaboration)

Acknowledgments