Author response: Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma (2015)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Current therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets by selective targeting of the molecular networks that support sarcoma cell proliferation. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and disruption of Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Asparagine reliance of sarcoma cells may represent a metabolic vulnerability with potential anti-sarcoma therapeutic value. https://doi.org/10.7554/eLife.09436.001 eLife digest Sarcoma is a type of cancer that forms in the connective tissues of the body, such as bone, cartilage, muscle and fat. Usually, treatment involves surgical removal of the tumor and/or radiation to kill the tumor cells. However, if sarcomas spread to other parts of the body, the treatment options are limited. Genetic studies have revealed several genetic changes that contribute to the formation of sarcomas. Many sarcomas have a mutation in a gene that encodes a protein called Ras. In 2011, researchers found that injecting Ras mutant muscle cells into the muscles of mice could lead to the formation of sarcomas. Next, the researchers compared gene expression in the mouse sarcoma cells with gene expression in normal mouse muscle cells and found that certain genes appeared to be more highly expressed in the sarcoma cells. These genes were also hyperactive in human sarcoma cells and may promote the growth of sarcomas carrying mutant forms of Ras. Now, Hettmer et al. – including some of the same researchers involved in the earlier work – show that targeting one of these hyperactive genes can slow sarcoma growth. The experiments made use of a technique called ribonucleic acid interference (or RNAi for short) to specifically switch off the expression of the hyperactive genes and then observed how this affected sarcoma growth. Hettmer et al. found that blocking the expression of one particular gene, which encodes an enzyme called asparagine synthetase, slowed down the growth of the sarcoma the most. Asparagine synthetase makes the amino acid asparagine, which is needed to make proteins in cells. Further experiments showed that reducing the amount of asparagine in human and mouse sarcoma cells slowed down the growth of these cells. A drug that lowers the amount of asparagine in cells is already used to treat some blood cancers. Hettmer et al.’s findings suggest that drugs that alter the availability of asparagine in the body might also be useful to treat sarcomas with mutant forms of Ras. https://doi.org/10.7554/eLife.09436.002 Introduction Soft-tissue sarcomas (STS) are a heterogeneous group of non-hematopoietic, mesodermal cancers. Certain STS types present with tissue-specific features, such as skeletal muscle differentiation in rhabdomyosarcoma (RMS) (Parham and Barr, 2013). For most STS tumors, cure depends on radical resection and/or radiation of the tumor, and therapeutic options for tumors that have spread regionally and/or systemically are limited (Linch et al., 2014). The genetic spectrum of STS is heterogeneous. Many tumors carry complex karyotypes with variable genetic changes; others express specific oncogenic mutations or exclusive chromosomal translocations within a relatively simple karyotype (Bovee and Hogendoorn, 2010). For RMS, two main genotypes have been described: those characterized by expression of the fusion oncogenes PAX3:FOXO1 or PAX7:FOXO1 and those that lack these fusions. The most common oncogenic mutations in the latter group of fusion-negative RMS tumors are in the Ras pathway (Shern et al., 2014; Chen et al., 2013). We previously reported rapid sarcoma induction by intramuscular implantation of Kras (G12V)-expressing, Cdkn2a (p16p19) deficient mouse myofiber-associated (MFA) cells into the extremity muscles of NOD. SCID mice (Hettmer et al., 2011). Transcriptional profiling of Kras; p16p19null sarcomas identified a cluster of sarcoma-relevant candidate genes. These genes are enriched in mouse sarcomas and in human RMS as compared to normal mouse or human skeletal muscle (Hettmer et al., 2011), and may include transcripts of fundamental importance for sarcoma growth. To examine the contributions of each of these candidate genes to sarcoma growth, we performed a customized shRNA-based proliferation screen. The strongest inhibitory effect on sarcoma cell proliferation was observed after silencing of asparagine synthetase (ASNS), the enzyme that catalyzes cellular synthesis of the non-essential amino acid asparagine. We found that adequate availability of asparagine is required in rapidly proliferating sarcomas cells, likely to support nascent polypeptide synthesis, and that asparagine starvation impedes sarcoma growth. Thus, small molecules targeting asparagine availability (Richards and Kilberg, 2006) could be useful as anti-sarcoma therapeutics. Results Kras;p16p19null mouse sarcomas identify a cluster of sarcoma-relevant genes In prior work, we showed that ex-vivo lentiviral transduction with oncogenic Kras (G12v) of Cdkn2a (p16p19)-deficient myofiber-associated (MFA) cells, isolated by fluorescence activated cell sorting (FACS) from muscle tissue of Cdkn2a-/- mice, drives rapid sarcoma formation upon transplantation of these cells into the muscle of immunocompromised mice (Hettmer et al., 2011) (Figure 1—figure supplement 1). The myogenic differentiation status of the Ras-driven sarcomas generated in this system depends largely on the cell type transduced, also known as the “cell-of-origin”: Kras; p16p19null satellite cells typically gave rise to RMS, whereas the identical oncogenetic lesions introduced into fibroadipogenic precursors within the MFA cell pool almost always produced sarcomas lacking myogenic differentiation features (non-myogenic sarcomas, NMS) (Hettmer et al., 2011)(Figure 1—figure supplement 1). We previously showed that Kras;p16p19null mouse sarcomas from each of these cellular origins recapitulate transcriptional profiles across the entire spectrum of human RMS and used this information to identify 141 genes of potential significance for sarcoma growth (Hettmer et al., 2011). To evaluate the functional contributions of each of the previously identified sarcoma-relevant genes, we designed a customized shRNA proliferation screen, using 5 distinct shRNAs per candidate gene (Supplementary files 1–4). The screen was carried out in one Kras;p16p19null RMS and one Kras;p16p19null NMS cell line, and shRNAs were delivered in puromycin-selectable pLKO lentiviral vectors. Correlation coefficients of 0.8348 and 0.9501 between puromycin-treated and untreated cells confirmed adequate transduction efficiencies (Figure 1A,F). As shRNA mediated silencing of Kras (G12v)-IRES-GFP, the driver oncogene used to initiate the mouse sarcomas, markedly inhibits the growth of Kras;p16p19null sarcoma cells (Figure 2A–B, Figure 2—figure supplement 1A–B), shRNAs directed against either GFP or KRAS served as positive controls in this screen and showed clear growth-inhibitory effects (Figure 1C–D, 1H-I). Control shRNAs (cntrl-shRNA) directed against LACZ, red fluorescent protein (RFP) and luciferase (LUC) served as negative controls, and showed no growth-inhibitory effects (Figure 1C–D, 1H-I). Receiver operator curve (ROC) analysis, using an external control set of shGFP-infected and cntrl-shRNA-infected RMS and NMS cells (Supplementary files 3–4), validated the ability of this system to distinguish between shRNAs with and without growth-inhibitory effects on sarcoma cell proliferation (Figure 1B,G). ROC analysis also determined a false discovery rate of <30% for shRNAs associated with a reduction in proliferation to <52% of the average of cntrl-shRNA-infected RMS cells (Figure 1B–C) and <40% of cntrl-shRNA-infected NMS cells (Figure 1G–H). In both RMS and NMS cells, silencing of ASNS (Asparagine Synthetase) produced by far the strongest anti-proliferative effect (p<0.0001, q<0.01, 4–5 of 5 shRNAs with FDR<30%; Figure 1D,I and Supplementary files 5–6). ASNS silencing reduced the growth of Kras;p16p19null RMS and NMS cells to 30.16% and 6.69% of the average of control RMS and NMS cells, respectively. Effective depletion of ASNS protein by the target-specific shRNAs employed in our screen was confirmed by Western Blot (Figure 1E,J). Figure 1 with 1 supplement see all Download asset Open asset Functional genomic screening identified asparagine synthetase (ASNS) as a high-priority sarcoma target. 141 sarcoma-relevant genes were identified by prior transcriptional profiling of genetically engineered Kras;p16p19null mouse rhabdomyosarcomas (RMS) and non-myogenic sarcomas (NMS) (Hettmer et al., 2011). (A–D, F–I) The contributions of each of the 141 sarcoma-relevant genes to sarcoma cell proliferation were determined by customized shRNA screening. (B–D, G–I). The screen contained a control set, including cells exposed to shLUC, shRFP, shLACZ (cntrl; predicted to have no effect on cell proliferation) and cells exposed to shGFP (GFP; predicted to silence Kras (G12V)-IRES-GFP and reduce cell proliferation). (B,G) Receiver operator curve analysis using cntrl-shRNA-infected cells as negative and shGFP-infected cells as positive controls determined a false discovery rate of <30% for shRNAs associated with a reduction in proliferation to <52% of the average of cntrl-shRNA-infected RMS cells (grey line in panel C) and to <40% of cntrl-shRNA-infected NMS cells (grey line in panel H). (D, I) The shRNA screen included cells exposed to shLUC, shRFP, shLACZ (cntrl), shKRAS and shRNAs directed against each of the 141 candidate genes (5 shRNAs per gene). ShRNAs directed against the gene encoding Asparagine Synthetase (Asns) showed the strongest effect on NMS and RMS proliferation (p<0.0001, q<0.01, 4–5 of 5 shRNAs with FDR<30%). (E–J) Effective ASNS knockdown by the shRNAs used in the screen was confirmed by Western blot. See Supplementary files 1–4 for raw data from the shRNA screen, and Supplementary files 5–6 for scores for each of the 141 candidate genes. Significance levels were defined as follows: 1, 5 shRNAs with FDR<30%; 2, 4 shRNAs with FDR<30%; 3, 3 shRNAs with FDR<30%. https://doi.org/10.7554/eLife.09436.003 Figure 2 with 3 supplements see all Download asset Open asset Reduced growth of mouse Kras;p16p19null RMS cells after Asns silencing is associated with inhibition of polypeptide synthesis. (A–B) ShRNA-mediated silencing of Asns and Kras in a mouse Kras;p16p19null RMS cell line reduced proliferation activity compared to shLUC-infected control cells as measured by MTT uptake. Asparagine supplementation (100 mg/L) in the tissue culture medium reversed the anti-proliferative effects of shASNS but not shKRAS. (C–F) Asns silencing increased the (C–D) percentage of apoptotoc (PI-/Annexin5+) cells and reduced the (E–F) percentage of S phase cells as determined by BrdU staining, compared to shLUC-infected control cells. Both effects were reversed by exogenous Asparagine supplementation (100 mg/L). (G) Polypeptide synthetic activity was determined by OP-puromycin staining. Absent OP-puromycin staining in cells treated with cycloheximide (right panels), an inhibitor of protein translation, validated the experimental approach. Asns silencing reduced polypeptide synthesis in Kras;p16p19null RMS cells (top left panel), and polypeptide synthesis was restored in shASNS RMS cells by Asparagine supplementation (bottom left panel). (A–F) Data were evaluated for statistical significance by T-tests (ns p≥0.05, *p<0.05, **p<0.01, ***p <0.001). See Figure 2—figure supplement 1 for similar effects of Asns silencing in mouse Kras;p16p19null NMS cells. https://doi.org/10.7554/eLife.09436.005 ASNS silencing inhibits growth of mouse Kras;p16p19null sarcoma cells by asparagine starvation ASNS encodes the enzyme asparagine synthetase, which converts aspartate into asparagine using glutamine as a nitrogen donor. Therefore, we next tested if the growth-inhibitory effects of genetic ASNS inhibition (Figure 1D,I) on mouse sarcoma growth were reversed by exogenous supplementation with the ASNS product asparagine. Supplementation of the culture media with 100 mg/L asparagine reversed the growth inhibition observed in shASNS-infected Kras;p16p19null RMS (Figure 2A-2B) and NMS (Figure 2—figure supplement 1A-1B) cells. Dose response analysis revealed that reversal of growth inhibiton of shASNS-transduced Kras;p16p19null RMS and NMS cells was also observed at 10 mg/L asparagine, whereas 0.01, 0.1 or 1 mg/L asparagine were insufficient (Figure 2—figure supplement 2). Asparagine supplementation did not reverse the growth inhibitory effect of shRNA-mediated silencing of Kras in Kras;p16p19null RMS cells (Figure 2A-2B). Taken together, we conclude that the growth-inhibitory effects of ASNS silencing on mouse sarcoma cells result from cellular asparagine starvation. Asparagine starvation reduces cell proliferation, increases cell death and impedes nascent polypeptide synthesis in mouse Kras;p16p19null sarcoma cells Asns silencing in mouse Kras;p16p19null RMS cells caused an increase in the fraction of sarcoma cells undergoing apoptosis, as determined by staining with propidium iodide (PI) and Annexin V (p<0.001, Figure 2C-2D). Moreover, Asns silencing reduced the percentage of BrdU + cells in S phase (p<0.001, Figure 2E-2F). Both effects were reversed by exogenous asparagine supplementation (Figure 2D,F). To evaluate whether cellular asparagine starvation due to Asns silencing impedes sarcoma cell proliferation by interfering with the cells’ ability to generate nascent polypeptide chains, Kras;p16p19null RMS cells were exposed to O-propargyl-puromycin (OP-puromycin). OP-puromycin forms covalent conjugates with newly synthesized polypeptides, which can be visualized by azide-alkyne cycloaddition. Rapidly proliferating shLUC-infected Kras;p16p19null RMS cells exhibited strong OP-puromycin staining, indicating brisk polypeptide synthesis (Figure 2G, middle panels). However, blockade of protein translation by exposure to cycloheximide abrogated OP-puromycin staining (Figure 2G, far right panels). Similarly, shASNS-infected cells exhibited only minimal OP-puromycin staining (Figure 2G, upper left panel), while synthesis of new polypeptides was restored in shASNS-infected sarcoma cells grown in medium supplemented with asparagine (Figure 2G, lower left panel). Similar effects of ASNS silencing on apoptosis, cell cycle and synthesis of nascent peptide chains were observed in Kras;p16p19null NMS cells (Figure 2—figure supplement 1). Asparagine starvation impedes human RMS growth and polypeptide synthesis ASNS expression was evaluated in primary human sarcoma tissue by immunohistochemistry (IHC) using a commercially available tissue array (US Biomax SO2081). ASNS was detected in 16 of 22 (73%) human RMS cores (Figure 4—figure supplement 1A) and in 12 of 27 (44%) human leiomyosarcoma cores (Figure 4—figure supplement 1B). Also, increased expression of ASNS compared to normal human muscle was detected in 9 of 9 human sarcoma cell lines analyzed by PCR (Figure 4—figure supplement 1C), including the PAX3:FOXO1-positive human RMS cell line Rh30. To evaluate the impact of ASNS silencing on human RMS cells, we transduced Rh30 cells with lentiviruses encoding shASNS or control (shLACZ) shRNAs (Figure 3). ShRNA-mediated knockdown of ASNS in Rh30 cells (Figure 3A) reduced proliferation (p<0.001; Figure 3B–C), increased the percentage of apoptotic cells (p<0.01; Figure 3D–E), reduced the percentage of cells in S phase (p<0.001; Figure 3F–G) and impeded nascent polypeptide synthesis (Figure 3H). The effects of ASNS silencing on Rh30 growth and peptide synthesis were reversed by asparagine supplementation (Figure 3B–H). Thus, ASNS silencing in human Rh30 cells recapitulated the inhibitory effects on cell growth and polypeptide synthesis observed in mouse Kras;p16p19null sarcoma cells. Figure 3 Download asset Open asset Reduced growth of human Rh30 RMS cells after ASNS silencing is associated with reduced polypeptide synthesis. (A) ShRNA-mediated silencing of ASNS in Rh30 cells was validated by Western Blot. (B–C) ASNS silencing reduced proliferation compared to shLACZ-infected control cells as measured by MTT uptake. Asparagine supplementation (100 mg/L) in the tissue culture medium reversed the anti-proliferative effects of shASNS. (D–G) ASNS silencing increased the (D–E) percentage of apoptotic (PI-/Annexin5+ ) cells and reduced the (F–G) percentage of S-phase cells , as compared to shLACZ-infected control cells. (F–G) Exogenous Asparagine supplementation reversed shASNS effects on cell cycle progression. (H) Polypeptide synthetic activity was determined by OP-puromycin staining. Absent OP-puromycin staining in Rh30 cells treated with cycloheximide (right panels) validated the experimental approach. ASNS silencing reduced polypeptide synthesis (top left panel), and polypeptide synthesis was restored in shASNS RMS cells by Asparagine supplementation (bottom left panel). (B–G) Data were evaluated for statistical significance by T-tests (ns p≥0.05, * p<0.05, ** p<0.01, *** p <0.001). https://doi.org/10.7554/eLife.09436.009 Chemical targeting of Asparagine availability reduces sarcoma growth Asparagine homeostasis represents an actionable cellular process. Amino sulfoximines directly inhibit ASNS activity (Ikeuchi et al., 2012; Richards and Kilberg, 2006), whereas asparaginase, an FDA-approved drug widely used in the treatment of leukemia, hydrolyzes asparagine to aspartate and ammonia. Both amino sulfoximine 5 (AS5) and asparaginase reduced the proliferation of mouse and human sarcoma cell lines in vitro (Figure 4A–C). For asparaginase, EC50 concentrations were estimated at 0.2–0.5 IU/ml in mouse Kras;p16p19null sarcoma cells, 0.8–0.9 IU/ml in human HT1080, Rh30 and Rh41 cells and 6 IU/ml in human RD cells (Figure 4A–B). For the ASNS inhibitor AS5, EC50 concentrations were estimated at 80–150 μM in mouse Kras;p16p19null sarcoma cells and 200–300 μM in the human sarcoma cell lines tested (Figure 4A,C). Similar to previous observations in cells that underwent genetic inhibition of ASNS, the growth-inhibitory effects of chemical ASNS inhibition by AS5 were reversed by exogenous asparagine supplementation (Figure 4D). These findings strongly suggest that the growth inhibitory effects of AS5 result from asparagine deprivation of sarcoma cells. Figure 4 with 1 supplement see all Download asset Open asset Inhibition of mouse and human sarcoma cell growth in vitro by chemical compounds interfering with Asparagine homeostasis. (A–C) Proliferation assays of mouse (Ms RMS, Ms NMS) and human (HT1080, RD, Rh41, Rh30) sarcoma cell lines exposed to the indicated doses of chemical modulators of Asparagine homeostasis: (A–B) Asparaginase or (A,C) AS5. Both chemicals were diluted in 0.9% NaCl (Normal Saline (NS)) as vehicle. (D) Chemical and genetic ASNS inhibition was reversed by exogenous supplementation with 100 mg/L asparagine in the tissue culture medium (100 mg/L, which corresponds to 757 μM; compared to normal asparagine concentrations in mouse and human plasma of 29 μM and 55 μM, respectively (Cooney et al., 1970)). Data were evaluated for statistical significance by T-tests (ns p≥0.05, * p<0.05, ** p<0.01, *** p <0.001). See Figure 4—figure supplement 1 for ASNS expression in human sarcoma cells. https://doi.org/10.7554/eLife.09436.010 Asparagine depletion impedes mouse sarcoma growth in vivo Due to the poor cell permeability of AS5, its growth-inhibitory effects on sarcoma cells required EC50 concentrations greater than 80 μM, making in vivo testing infeasible. Thus, to determine the effects of reduced ASNS activity on sarcoma growth in vivo, 100 shASNS-infected and 100 shLUC-infected Kras; p16p19null RMS cells were implanted into the cardiotoxin-preinjured gastrocnemius muscles of 1- to 3-months old NOD. SCID mice (Figure 5). IHC showed that tumors arising from shASNS cells expressed less ASNS than tumors arising from shLUC cells (Figure 5A). However, there was no difference in latency of shASNS- and shLUC-tumors (p = 0.3; Figure 5B). Figure 5 with 1 supplement see all Download asset Open asset Asns silencing delayed Kras;p16p19null RMS growth in Asparagine-depleted mice. (A) Tumors arising from shASNS RMS cells expressed less ASNS than tumors arising from shLUC cells as shown by IHC staining. Of 6 tumors arising from shASNS cells, cytoplasmic ASNS positivity was observed in 50–75% of cells in one tumor, in 25–50% of cells in 3 tumors and in <25% of cells in 2 tumors. Of 5 tumors arising from control shLUC-cells, cytoplasmic ASNS staining was detected in >75% of cells in 3 tumors and in 25–50% of cells in 2 tumors. Representative images are shown. (B) Effects of Asns silencing on tumor growth in vivo were evaluated by transplantation. One subgroup of recipient mice was treated with Asparaginase (Elspar) by daily intraperitoneal (IP) injections at a dose of 1500 IU/kg. ShASNS silencing delayed tumor onset in recipient mice treated with Asparaginase compared to shLUC-infected RMS cells (p<0.0001). (C) Asparaginase-treated mice maintained their weight over the course of a 19-day exposure. Each experimental group included 5 mice, and findings were replicated in 2 independent transplantation experiments. (D) Daily IP injection of Asparaginase results in a 13-fold reduction in serum Asparagine levels from 52 ± 7 to 4 ± 1 μmol/L. Differences in tumor latency were evaluated for statistical significance by logrank (Mantel-Cox) test. Differences in serum amino acid concentrations were determined by T-test (ns p≥0.05, *p<0.05, **p<0.01, ***p <0.001). See Figure 5—figure supplement 1 for similar effects of Asns silencing in Kras;p16p19null NMS cells on secondary tumor induction and Supplementary file 7 for changes in serum amino acid levels in Asparaginase-treated versus control mice. https://doi.org/10.7554/eLife.09436.012 Our in vitro data suggested that growth inhibition induced by ASNS silencing can be rescued by provision of exogenous aparagine at concentrations between 1 and 10 mg/L (Figure 2—figure supplement 2). As normal asparagine concentrations in mouse and human plasma were previously reported to be between 3.8 mg/L and 7.3 mg/L (Cooney et al., 1970), these data suggest that freely available asparagine in mouse serum and tissue might counteract the effects of tumor-specific ASNS silencing. To examine this possibility, we treated subgroups of animals transplanted with shASNS- or shLUC-tumor cells with asparaginase (1500 IU/kg; (Szymanska et al., 2012)) by daily intraperitoneal (IP) injection. Asparaginase treatment was initiated on the day of tumor cell injection and continued for 35–41 days. This dosage was well tolerated by the animals without significant weight loss (Figure 5C). Serum asparagine levels were reduced 13-fold in asparaginase-treated mice (0.53 mg/L (4 μM) versus 6.87 mg/L (52 μM) in untreated mice, p<0.001; Figure 5D and Supplementary file 7). Daily exposure to asparaginase did not change the latency of shLUC-tumors (p = 0.5; Figure 5B); however, asparaginase treatment significantly tumor latency in mice implanted with cells (p<0.001; Figure 5B). Moreover, 2 out of mice in this experimental group did not tumors 4 of after tumor cell injection. Similar effects were observed NOD. SCID mice were transplanted with shASNS-infected and shLUC-infected Kras; p16p19null NMS cells (Figure 5—figure supplement 1). Thus, ASNS inhibition combined with depletion of plasma asparagine reduces sarcoma growth in vivo. Discussion cells on that adequate provision of and precursors to support cell growth. Functional genomic screening of genetically engineered mouse sarcomas revealed that asparagine synthetase (ASNS) the strongest observed effect on sarcoma cell proliferation within a small group of genes in both mouse and human sarcomas. ASNS is the amidotransferase that converts acid into in an and a nitrogen that is in cells (Richards and Kilberg, of functional ASNS in both mouse and human RMS cells reduced the of cells in S phase and impeded synthesis of nascent Similarly, ASNS silencing in cells was reported to result in cell cycle et al., The effects of ASNS reduction in sarcoma cells were reversed by exogenous supplementation with asparagine, the that rapidly sarcoma cells on adequate availability of or asparagine to support tumor growth. Moreover, ASNS inhibition significantly slowed mouse sarcoma growth in vivo only combined with depletion of plasma asparagine, likely the ability of asparagine in the tumor to asparagine availability after ASNS We that asparagine reliance of sarcoma cells represents a metabolic vulnerability that could be to inhibit rapid tumor growth. metabolic profiling studies have identified a of that are by cancer cell including and asparagine et al., starvation the phase of the cell cycle and reduced proliferation, in of this amino acid are required to support in rapidly cells. synthesis, protein synthesis in cells relatively et al., In we found that asparagine starvation of mouse and human sarcoma cells impedes synthesis of nascent polypeptide chains cell proliferation. These results are with observations in and asparagine levels in with protein levels at et al., Moreover, the or blocking the synthesis of amino is known to translation via of and of the translation et al., Thus, asparagine in the tumor cell may have in the synthesis and of protein in sarcoma cells. peptide may not be the only to the asparagine of sarcoma cells in this For our data not the that deprivation and/or aspartate from changes in asparagine impact cell and proliferation. However, aspartate reducing in the et al., and increased aspartate levels after ASNS knockdown might be predicted to the of sarcoma cells. In been suggested that increased expression in cancer cells can in a et al., 2014). with this we found that expression was increased by to in mouse sarcoma cells compared to normal mouse muscle (Figure 2—figure supplement that this may be used by sarcoma cells to counteract that may result from ASNS silencing. a that asparagine supplementation can cell death in cells et al., asparagine in cellular to metabolic such as glutamine This study also that asparagine is the amino acid synthesized in the cycle and that its depends on glutamine et al., 2014). Amino acid availability is known to of complex 1, which and to cell growth et al., Taken together, these observations suggest that asparagine may a as a cellular of cycle reduced nitrogen availability as a metabolic of cell our studies a clear of sarcoma cell growth and on cellular asparagine not the that adequate availability of