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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 !!!!