1. 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.

2. Disclosures
Caris
Bayer
DFINE

3. Original Observation in Osteosarcoma
Kobayashi et. al. Mol Cancer Ther 2010;9:

4. 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.

5. HCO3 + NH4 + 2ATP
Carbamyl
Phosphate
N-acetylglutamate
CPSI
Argininosuccinate
ASS1
Aspartate
Citrulline
OTC
Ornithine
Urea
ARG
Arginine
Fumarate
ASL

6. 619/701 88.3%- ASS1 Immunohistochemistry on 701 Soft Tissue Tumors
Malignant Peripheral Nerve Sheath Tumor (NF1 Related) 3/ Leiomyoma /20
Malignant Peripheral Nerve Sheath Tumor (Non-NF1) / Myxofibrosarcoma (Myxoid MFH) /7
Neurofibroma / Well-Differentiated Liposarcoma 0/6
Plexiform Neurofibroma / Dedifferentiated Liposarcoma 1/9
Diffuse-Type Neurofibroma / Myxoid Liposarcoma /12
Cellular Schwannoma / Pleomorphic Liposarcoma 1/3
Clear Cell Sarcoma / Desmoid Fibromatosis 0/23
Desmoplastic Melanoma / Dermatofibrosarcoma Protuberans 1/6
Fibrosarcomatous Dermatofibrosarcoma Protuberans / Desmoplastic Small Round Cell Tumor 1/6
Perineurioma / Endometrial Stromal Sarcoma 0/7
Schwannoma / Epithelioid Sarcoma 2/3
Synovial Sarcoma / Low-grade Fibromyxoid Sarcoma 1/3
Gastrointestinal Stromal Tumor / Epithelioid Hemangioendothelioma 0/2
Sarcoma, NOS/Malignant Fibrous Histiocytoma / Angiosarcoma 0/5
Embryonal Rhabomdyosarcoma / Extraskeletal Myxoid Chondrosarcoma 5/7
Alveolar Rhabomdyosarcoma / Nodular Fasciitis 0/6
Pleomorphic Rhabomdyosarcoma /8
Solitary Fibrous Tumor/Hemangiopericytoma /19
Tenosynovial Giant Cell Tumor / Bone Tumors
Angiomyolipoma / Giant Cell Tumor of Bone 0/7
Glomus Tumor / Fibrous Dysplasia /9
Granular Cell Tumor / Non-Ossifying Fibroma 1/6
Myxoma / Osteosarcoma 3/10
Leiomyosarcoma / Ewing Sarcoma/PNET 1/7
619/701
88.3%-
MPNST ASS MPNST ASS -
H&E ASS H&E ASS1 0

7. ASS1 Expression in Cell Lines
13/15
86.7%-
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.

8. ADI-PEG20 Treatment
IC50
ug/ul
Cell Line
MNNG
0.047
MG63
N/A
SKLMS1
0.046
U2OS
0.019
SKUT1
0.064
SKUT1B
0.102 E2
0.062
E11
0.057
SKES
0.056
NOS
HuO9N2
ASPS1
0.041
SY0-1
0.259
FUTJI
0.123
HCH-1
0.042
High ASS1 expression renders sarcoma cells resistant arginine deprivation caused by ADI-PEG20.
Sarcoma cell lines are arginine auxotrophs

9. Knockdown and Re-expression Experiments Demonstrate that
ASS1 Is Necessary and Sufficient for Arginine Auxotrophy in Sarcoma
shGFP shLuC shASS1-1 shASS1-2
Puro-MSCV MCSV-ASS1
ASS1
ACTIN
ASS1
ACTIN

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

11. 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.

12. 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.

13. 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.

14. Colony Formation Assays
p= 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.

15. 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.

16. ASS1 - SKLMS1 Xenografts
PBS Chloroquine
ADI-PEG20 ADI + Chloro

17. Cell Death Pathways FL RIP1 Cleaved RIP1 CASP 8 BCL2 cIAP Cleaved PARP
NT ADI CQ ADI+CQ
FL RIP1
Cleaved RIP1
CASP 8
BCL2
cIAP
Cleaved PARP
CASP3
Cleaved CASP3
Actin

18. 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.

19. FADD
RIP1
RIP1
Casp-8
cIAP
Ub-RIP1
Proteasome
26S
Casp-3
Cleaved
Casp-3
Apoptosis

20. 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

21. FADD
FADD
RIP3
RIP1
RIP1
Casp-8
Casp-8
cIAP
Ub-RIP1
Proteasome
26S
NEC-1
Z-VAD
Casp-3
ROS
Cleaved
Casp-3
Apoptosis
Necroptosis

22. Acknowledgements Shunqiang Li Tom Kitchens Loren Michel Dwight Towler
Douglas Adkins
Matthew Ellis
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
Postdoctoral position available !!!!