pubmed.ncbi.nlm.nih.gov

A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas - PubMed

  • ️Fri Jan 01 2016

A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas

Gregory R Bean et al. Cell Death Dis. 2016.

Abstract

Sarcomas comprise a large heterogeneous group of mesenchymal cancers with limited therapeutic options. When treated with standard cytotoxic chemotherapies, many sarcomas fail to respond completely and rapidly become treatment resistant. A major problem in the investigation and treatment of sarcomas is the fact that no single gene mutation or alteration has been identified among the diverse histologic subtypes. We searched for therapeutically druggable targets that are common to a wide range of histologies and hence could provide alternatives to the conventional chemotherapy. Seven hundred samples comprising 45 separate histologies were examined. We found that almost 90% were arginine auxotrophs, as the expression of argininosuccinate synthetase 1 was lost or significantly reduced. Arginine auxotrophy confers sensitivity to arginine deprivation, leading temporarily to starvation and ultimately to cell survival or death under different circumstances. We showed that, in sarcoma, arginine deprivation therapy with pegylated arginine deiminase (ADI-PEG20) maintains a prolonged state of arginine starvation without causing cell death. However, when starvation was simultaneously prolonged by ADI-PEG20 while inhibited by the clinically available drug chloroquine, sarcoma cells died via necroptosis and apoptosis. These results have revealed a novel metabolic vulnerability in sarcomas and provided the basis for a well-tolerated alternative treatment strategy, potentially applicable to up to 90% of the tumors, regardless of histology.

PubMed Disclaimer

Conflict of interest statement

Dr. Brian Van Tine has a basic research grant from Polaris Pharmaceuticals. Dr. John Bomalaski is an employee of Polaris Pharmaceuticals, Inc. and holds stock options in Polaris Group. The remaining authors declare no conflict of interest.

Figures

Figure 1
Figure 1

ASS1 expression level across sarcoma subtypes. (a) A summary of immunohistochemical detection of ASS1 in 701 primary sarcoma tumors. The tumors comprising 45 soft tissue and 5 bone sarcoma histological subtypes were examined. In all, 572/662 (86.4%) primary soft tissue and 34/39 (87.2%) bone sarcoma tumors did not exhibit strong ASS1 signal. (b) Typical examples of IHC. MPNST ASS1+ and ASS1- tumor samples were stained with hematoxylin and eosin (left panels) or with anti-ASS1 and counter stained with hematoxylin (right panel). Magnification x40. Scale bar=50 μm. (c) Immunoblotting for ASS1 in a representative panel of sarcoma cells lines. In total, 13/15 (86.7%) lacked strong ASS1 expression. The osteosarcoma cell line MG-63 had the highest ASS1 expression. All ASS1 expression was normalized relative to the ASS1 high MG-63 cell line and ASS1-negative cell line SK-LMS-1. Actin serves as a loading control to ensure accurate relative ASS1 expression across cell lines

Figure 2
Figure 2

Arginine depletion causes growth arrest in ASS1low cell lines. (a) Effects of ADI-PEG20 treatment on the growth of MNNG/HOS, SK-LMS-1 and MG-63 cell at a range of concentrations from 0 to 1 μg/ml. ADI-PEG20 induced cytostasis in a dose-dependent manner in ASS1low MNNG/HOS and SK-LMS-1 cells, but not in the ASS1high MG-63 cells. (N=3). Data represented as mean±S.D. (b) IC50s of ADI-PEG20 in a panel of sarcoma cell lines. ND, not determined, as ADI-PEG20 had no effect on proliferation. (c) Correlation between ASS1 expression levels and ADI-PEG20 IC50s illustrate a higher expression level of ASS1 correlates with decreased susceptibility to growth inhibition by ADI-PEG20 treatment. (d) Indirect fluorescence detection of BrdU incorporation into cellular DNA. Growth inhibition as revealed by a reduction in BrdU-positive nuclei in ADI-PEG20 (at 1 μg/ml) treated ASS1low MNNG/HOS, SK-LMS-1 and U-2 OS cells. ASS1high MG-63 cells were not affected by ADI-PEG20. These data indicate ADI-PEG20 treatment of ASS1low cell lines causes cell cycle arrest. Magnification x40. Scale bar=20 μm. (e) Quantification of BrdU-positive cells before and after treatment with ADI-PEG20 in MNNG/HOS, SK-LMS-1, U-2 OS and MG-63. (N=3). Data represented as mean±S.D.

Figure 3
Figure 3

ADI-PEG20 treatment inhibits tumor growth until ASS1 re-expression confers resistance. (a) Flow cytometry analysis of cell death induction, as measured by percentage of cells staining positive for PI, upon treatment with ADI-PEG20. As a single agent, ADI-PEG20 does not induce cell death in these sarcoma cell lines. (N=3). Data represented as mean±S.D. (b) Tumor growth of ASS1low SK-LMS-1 cells xenografted into nude mice with or without ADI-PEG20 treatment. Significant tumor growth inhibition was observed when mice were treated with ADI-PEG20 as compared with tumor growth in PBS-treated mice. Shown below is a western blot of five tumor lysates from five mice after ADI-PEG20 treatment showing re-expression of ASS1 in tumors, which had gained resistance to ADI-PEG20 mediated growth inhibition. (N=5 mice per arm). Data represented as mean tumor volume±S.E.M. (c) Re-expression of ASS1 in SK-LMS-1 cells after treatment with 1 μg/ml ADI-PEG20, 5 μM 5-aza-dC or both, as compared with untreated cells. After 48 h of combination treatment, ASS1 expression levels have significantly increased above wild-type conditions, as well as either drug individually. (N=3)

Figure 4
Figure 4

Arginine deprivation leads to a dependence on autophagy for continued cellular survival. (a) Cells were treated with ADI-PEG (3 days) or rapamycin (500 nM for 5 h) with or without bafilomycin A1 (100 nM) for an additional 4 h. Cell lysates were analyzed by immunoblot analysis. A representative western and bar graph, presented as means±S.D., are shown; n=3 (***P<0.0001). (b) Autophagosome formation as revealed by GFP-LC3 puncta. Lentiviral expression of GFP-LC3 was transduced into SK-LMS-1 cells that were then treated with 1 μg/ml ADI-PEG20 for 3 days. A significant increase in LC3-GFP puncta demonstrates the induction of autophagy by 1 μg/ml ADI-PEG20 mediated arginine deprivation in ASS1 low cells. Magnification x60. Scale bar=10 μm. (c) SK-LMS-1 cell counts over a course of 3 days when left untreated, as well as treatment with 1 μg/ml ADI-PEG20, 20 μM chloroquine and both. Either agent retarded cell growth; the combination treatment significantly reduced the cell number by day 3 (P=0.001). (N=3). Data represented as mean±S.D. (d) Measurements of cell death upon treatment of SK-LMS-1 cells with the agents individually and together. Cell death was measured on day 3 by propidium iodide staining followed by flow cytometric analyses of treated or untreated cells. Only treatment with both agents induced significantly cell death (P<0.001). (N=3). Data represented as mean±S.D. (e) Measurements of cell death upon treatment of SK-LMS-1 cells with 1 μg/ml ADI-PEG20, 50 μM pepstatin A and 25 μM E64D, individually and together. Cell death was measured on day 3 by propidium iodide staining followed by flow cytometric analyses of treated or untreated cells. Only treatment with both agents induced significantly cell death. (N=3). Measurements of cell death of SK-LMS-1 cells with shRNA knockdowns upon treatment with 1 μg/ml ADI-PEG20 for 3 days. (N=3). Data represented as mean±S.D. (f) Measurements of cell death of SK-LMS-1 cells transduced with Luc, ATG5 or ATG7 stable knockdowns. Cell death was measured by propridium iodide staining followed by flow cytometric analyses on day 3 of ADI-PEG20 treatment

Figure 5
Figure 5

Induction of necroptosis upon simultaneous arginine deprivation and chloroquine treatment. (a) Cell death as measured by FACS analysis after propidium iodide uptake. MNNG/HOS (left) and SK-LMS-1 (right) cells treated with 1 μg/ml ADI-PEG20, 20 μM chloroquine, both, or in combination with 100 μM ZVAD (an apoptosis inhibitor) or 10 μM necrostatin (a necroptosis inhibitor). Protection of cell death was more effective with necrostatin, indicating cell death is occurring primarily via necroptosis. (N=3). Data represented as mean±S.D. (b) Cell death as measured by FACS analysis after propidium iodide uptake in wild type, shRIP1 or shRIP3 SK-LMS-1 cells after treatment with 1 μg/ml ADI-PEG20 with or without 20 μM chloroquine. RIP kinase knockdown protected from induction of cell death, indicating necroptosis induction upon dual agent treatment. Data represented as mean±S.D. (N=2). (c) Western blots of SK-LMS-1 and MNNG/HOS cells untreated, or treated with ADI-PEG20 and chloroquine for 24, 48 or 72 h. In the presence of chloroquine and ADI-PEG20, the loss of the proapoptotic cleaved caspase 3 and the anti-necroptotic cIAP1 increases the threshold for apoptosis signaling while priming cells for death by necroptosis. Decrease in levels of cleaved RIP1 further suggest necroptosis induction (d). RIP1 co-IP. SK-LMS-1 ASS1low cells were treated with ADI-PEG20, chloroquine or both for 3 days. A significant RIP3 co-precipitation was observed upon exposure to 1 μg/ml ADI-PEG20 and 20 μM chloroquine, whereas caspase 8, which is negative regulator of ripoptosome formation, was reduced. Collectively, these observations are indicative of active ripoptosome formation and subsequent cell death executed preferentially by necroptosis

Figure 6
Figure 6

In vivo efficacy of synthetic lethal therapeutic targeting strategy. (a and b) Colony formation upon treatment with 1 μg/ml ADI-PEG20, 10 μM or 20 μM chloroquine or both. (a) Combination treatment of SK-LMS-1 ASS1low cells significantly inhibited long-term colony formation and colony size, especially when the chloroquine was used at 20 μM (P=0.001). (N=3). Data represented as mean±S.D. (b) The effects on MG-63 ASS1high cells were moderate and were attributable to chloroquine alone, whereas ADI-PEG20 had little effect. (N=3). Data represented as mean±S.D. (c) Tumor volumes of ASS1low MNNG/HOS osteosarcoma cells xenografted into nude mice. Combination ADI-PEG20 and chloroquine treatment significantly inhibited tumor growth versus PBS, ADI-PEG20, or chloroquine treatment alone. Data represented as mean tumor volume±S.E.M. (N=5 mice per arm)

Similar articles

Cited by

References

    1. Taylor BS, Barretina J, Maki RG, Antonescu CR, Singer S, Ladanyi M. Advances in sarcoma genomics and new therapeutic targets. Nat Rev Cancer 2011; 11: 541–557. - PMC - PubMed
    1. Latres E, Drobnjak M, Pollack D, Oliva MR, Ramos M, Karpeh M et al. Chromosome 17 abnormalities and TP53 mutations in adult soft tissue sarcomas. Am J Pathol 1994; 145: 345–355. - PMC - PubMed
    1. Das P, Kotilingam D, Korchin B, Liu J, Yu D, Lazar AJ et al. High prevalence of p53 exon 4 mutations in soft tissue sarcoma. Cancer 2007; 109: 2323–2333. - PubMed
    1. Hoe KK, Verma CS, Lane DP. Drugging the p53 pathway: understanding the route to clinical efficacy. Nat Rev Drug Discov 2014; 13: 217–236. - PubMed
    1. Frith AE, Hirbe AC, Van Tine BA. Novel pathways and molecular targets for the treatment of sarcoma. Curr Oncol Rep 2013; 15: 378–385. - PubMed

MeSH terms

Substances