SIMULTANEOUS AUTOPHAGY INDUCTION AND INHIBITION INDUCES CELL DEATH THROUGH

NECROPTOSIS IN SARCOMAS THAT LACK ARGININOSUCCINATE SYNTHETASE 1 EXPRESSION

Philip A. Boone1, Dean Weich1, Shunqiang Li1, Munir R. Tanas3, Robert Kitchens1, David Y. Chen1, Douglas R. Adkins1, John Bomalaski2, Brian P. Rubin3, Brian A. Van Tine1

1. Medical Oncology, Washington University in St. Louis, St. Louis, MO, United States. 2. Polaris Group, San Diego, CA, United States. 3. Anatomic Pathology, Cleveland Clinic, Cleveland, OH, United States.

Disclosures

• Caris• Bayer• DFINE

Original Observation in Osteosarcoma

Kobayashi et. al. Mol Cancer Ther 2010;9:535-544

Argininosuccinate Synthetase 1

• The protein encoded by this gene catalyzes the penultimate step of the arginine biosynthetic pathway.

• There are approximately 10 to 14 copies of this gene, the only functional copy is on chromosome 9.

• Mutations in ASS1 cause citrullinemia.

CitrullineCarbamylPhosphate

N-acetylglutamateCPSI

HCO3 + NH4 + 2ATP

Argininosuccinate

ASS1

Aspartate

ArginineFumarate

ASL

Ornithine

Urea

ARG

OTC

Soft tissue tumors Malignant Peripheral Nerve Sheath Tumor (NF1 Related) 3/44 Leiomyoma 0/20Malignant Peripheral Nerve Sheath Tumor (Non-NF1) 3/31 Myxofibrosarcoma (Myxoid MFH) 0/7Neurofibroma 0/19 Well-Differentiated Liposarcoma 0/6Plexiform Neurofibroma 0/24 Dedifferentiated Liposarcoma 1/9Diffuse-Type Neurofibroma 0/11 Myxoid Liposarcoma 10/12Cellular Schwannoma 0/7 Pleomorphic Liposarcoma 1/3 Clear Cell Sarcoma 0/7 Desmoid Fibromatosis 0/23Desmoplastic Melanoma 1/10 Dermatofibrosarcoma Protuberans 1/6 Fibrosarcomatous Dermatofibrosarcoma Protuberans 0/9 Desmoplastic Small Round Cell Tumor 1/6 Perineurioma 2/4 Endometrial Stromal Sarcoma 0/7Schwannoma 0/36 Epithelioid Sarcoma 2/3 Synovial Sarcoma 14/36 Low-grade Fibromyxoid Sarcoma 1/3Gastrointestinal Stromal Tumor 3/95 Epithelioid Hemangioendothelioma 0/2Sarcoma, NOS/Malignant Fibrous Histiocytoma 7/60 Angiosarcoma 0/5Embryonal Rhabomdyosarcoma 1/3 Extraskeletal Myxoid Chondrosarcoma 5/7 Alveolar Rhabomdyosarcoma 0/2 Nodular Fasciitis 0/6Pleomorphic Rhabomdyosarcoma 2/8Solitary Fibrous Tumor/Hemangiopericytoma 16/19Tenosynovial Giant Cell Tumor 2/34 Bone TumorsAngiomyolipoma 3/8 Giant Cell Tumor of Bone 0/7Glomus Tumor 1/5 Fibrous Dysplasia 0/9Granular Cell Tumor 0/4 Non-Ossifying Fibroma 1/6Myxoma 2/5 Osteosarcoma 3/10Leiomyosarcoma 8/56 Ewing Sarcoma/PNET 1/7

H&E ASS1 3+ H&E ASS1 0

ASS1 Immunohistochemistry on 701 Soft Tissue Tumors

MPNST ASS+ MPNST ASS -

619/701

88.3%-

ASS1 Expression in Cell Lines

Expression of ASS1 in sarcoma cell lines. 3/5 osteosarcoma cell lines lack strong expression of ASS1, with the MG63 cell line expressing the highest amount. All 3 LMS cell lines lack high ASS1 expression, as do Ewing’s,Chondrosarcoma, Sunovial and Avelolar Soft Parts Sarcoma cell lines. All expression is normalized to MG63 and then to Actin.

13/15

86.7%-

0 1 2 30

5

10

15

20

25

30

35

40

45

50

Days

SKLMS1 ASS1 -

ADI-PEG20 Treatment

0 1 2 30

5

10

15

20

25

30

35

400

0.01

0.05

0.1

0.5

1

Days

# o

f C

ell

(10,

000)

MNNG/HOSASS Low

0 1 2 30

5

10

15

20

25

Days

MG63ASS1 High

IC50 ug/ulCell LineMNNG 0.047MG63 N/ASKLMS1 0.046U2OS 0.019SKUT1 0.064SKUT1B 0.102E2 0.062E11 0.057SKES 0.056NOS N/AHuO9N2 0.056ASPS1 0.041SY0-1 0.259FUTJI 0.123HCH-1 0.042

• High ASS1 expression renders sarcoma cells resistant arginine deprivation caused by ADI-PEG20.

• Sarcoma cell lines are arginine auxotrophs

shG

FP

shLU

C

shASS1

#1

shASS1

#40

10

20

30

40

50

MG63 ASS1 high-shRNA ADI Treatment

un-treated

x1

0^

4 C

ell

s

puro wt ASS10

5

10

15

20

25

30

35

40

SKLMS1 ASS1(-) ASS1 ADI Treatment

x1

0^

4 C

ells

Knockdown and Re-expression Experiments Demonstrate thatASS1 Is Necessary and Sufficient for Arginine Auxotrophy in

Sarcoma

ASS1

ACTIN

Puro-MSCV MCSV-ASS1

ASS1

ACTIN

shGFP shLuC shASS1-1 shASS1-2

Cell Cycle Inhibition is caused by treatment of ASS1 low cells with ADI-PEG20

MG63 MNNG SKLMS1 SKES0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

NT ADI

%B

RD

U p

ositi

ve

Autophagy• Autophagy is a catabolic process involving the

degradation of a cell's own components through the lysosomal machinery.

• It is a tightly regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products.

• It is a major mechanism by which a starving cell reallocates nutrients from unnecessary processes to more-essential processes.

Arginine Deprivation Induces Autophagy

Autophagy. The arginine depletion using ADI-PEG20 induces autophagy by day 2 as seen by in increased LC3 cleavage and p62 alterations in ASS1 low cell lines.

Duel Induction and Inhibition of Autophagy leads to cell Death.

A B

Induction of autophagy with ADI-PEG20. A. Cell counts over three days in cells treated with control, ADI-PEG20, chloroquine of the combination. B. Annexin V FACS on day 3 of cells treated with control, ADI-PEG20, chloroquine of the combination.

Colony Formation Assaysp=0.003 p=0.001

Colony formation: Colony formation assays for the ASS1 Low SKLMS1 cell line (Left) and the ASS1 High (Right) cell lines. Cells are treated with drug for 7 days and then released so that viable cells can grow out. The combination of ADI-PEG20 and Chloroquine is superior in ASS1 low cells.

MNNG/HOS ASS1 Low Xenografts

The osteosarcoma cell line MNNG/HOS was xenografted into the back fat pad of nude mice. Mice we treated daily with chloroquine and biweekly with ADI-PEG20. Tumors were measured starting on day 6. Mice were treated with PBS (Green) ADI-PEG20 (red), Chloroquine (Blue) or the combination of ADI-PEG20 and Chloroquine (Purple). The combination demonstrated statistical significance.

ASS1 - SKLMS1 Xenografts

21 25 29 33 37 41 45 49 53 57 61 650.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0

1800.0

PBS

Chloroquine

ADI-PEG20

ADI-PEG20/Chloroquine

Days

Tum

or V

olum

e m

m3

PBS Chloroquine

ADI-PEG20 ADI + Chloro

Cell Death Pathways NT ADI CQ ADI+CQ

FL RIP1

Cleaved RIP1

CASP 8

BCL2

cIAP

Cleaved CASP3

CASP3

Cleaved PARP

Actin

Combination Treatment and Necroptosis

The cell death induced by the combination of ADI-PEG20 and Chloroquine is best inhibited by necrostatin implicating necroptosis as the mechanism of cell death.

FADD

Casp-8

cIAP

Ub-RIP1

26S

Pro

teas

ome

Casp-3

Apoptosis

RIP1RIP1

Cleaved Casp-3

Necroptosis is activated by the combination of ADI-PEG20 and Chloroquine

Pre-IP RIP1-IP

Con ADI Chloro ADI/Chloro Con ADI Chloro ADI/Chloro

RIP1

Caspase 8

RIP3

Actin

RIP3 immunoprecipitates with RIP1 after treatment with chloroquine and ADI-PEG20 as part of necroptosis

FADD

Casp-8

cIAP

Ub-RIP1

26S

Pro

teas

ome

Casp-3

Apoptosis

ROS

Necroptosis

RIP1

FADD RIP3

RIP1

Cleaved Casp-3

Casp-8

NEC-1 Z-VAD

Acknowledgements

• Van Tine Laboratory– Greg Bean, Ph.D.– Dean Weich– Philip Boone– David Chen, M.D., Ph.D.

• Cleveland Clinic– Brian P. Rubin, M.D., Ph.D.– Munir R. Tanas, Ph.D.

• Polaris– John Bomalaski

• Shunqiang Li• Tom Kitchens

• Loren Michel• Dwight Towler • Douglas Adkins• Matthew Ellis

Postdoctoral position available !!!!