Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth

Abstract

Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. Under conditions of elevated ROS, endogenous L-cysteine (L-Cys) production is insufficient for GSH synthesis. This necessitates uptake of L-Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(-) transporter. We show that administration of an engineered and pharmacologically optimized human cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet this treatment results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in both prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53^-/- mice, which develop disease resembling human chronic lymphocytic leukemia. It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies.

Figure 1. Engineered human Cyst(e)inase for cancer treatment

(a) Schematic of the therapeutic effect of Cyst(e)inase treatment on cancer cells. Mutations in the transsulfuration pathway and/or elevated ROS production in cancer outstrips the normal l-Cys production capacity needed for GSH and protein synthesis, causing tumors to rely instead upon l-Cys import predominantly via the xCT(−) antiporter mediated exchange of l-Glu for extracellular CSSC. Therapeutic depletion of extracellular l-Cys and CSSC selectively targets cancers cells causing potent growth arrest and killing, increased levels of ROS, decreased levels of GSH, and induction of autophagy. (Abbr. l-Met = l-methionine, l-Hcy = l-homocysteine, MAT = methionine adenosyl transferase, SAHH = S-adenosylhomocysteine hydrolase, GCL = glutamate-cysteine ligase, GS = glutathione synthase). (b) Michaelis-Menten parameters for Cyst(e)inase and CGL. Results are from experiments performed in triplicate, data is expressed as mean ± s.d. (c) Structural analysis of Cyst(e)inase reveals the formation of a geminal diamine reaction intermediate with substrate l-Cys. (d) Time course of total serum Cysteine (free l-Cys + reduced CSSC + liberated protein-bound l-Cys) following single dose administration of Cyst(e)inase at 8 mg/kg in two cynomolgus monkeys.

Figure 2. Effect of Cyst(e)inase in PCa cells

(a–c) HMVP2 cells were treated with indicated concentrations of Cyst(e)inase. (a) Cell viability as assessed by alamar blue assay at indicated time points. (b) Relative cellular ROS levels 4 h post-treatment as assessed by 2′,7′-Dichlorofluorescin diacetate (DCFDA) fluorescence; (c) Relative GSH levels 24 h post-treatment as assessed by spectrophotometric analysis. (d–i) HMVP2 cells were treated with indicated concentrations of Cyst(e)inase for 24 hours. (d,e,f,h,i) Metabolic stress markers and regulatory cell cycle proteins were measured by immunoblot. Immunoblots were performed at least three times with β-actin controls for each experiment. Numbers above blots indicate band intensities relative to untreated conditions. Images have been cropped for presentation, uncropped images are shown in Supplementary Fig. 14. (g) Cell-cycle phase distribution was analyzed by Guava-based flow cytometry 24 h post treatment. (j) DU145 PCa cells were treated with indicated concentrations of Cyst(e)inase and/or N-Acetyl-L-Cysteine (NAC) for 48 h and cell viability was assessed by crystal violet assay. For (a–c, g, j): data are expressed as mean ± s.e.m; *P<0.05; ***P<0.001; ****P<0.0001; one-way ANOVA followed by Tukey’s multiple comparison test (a–c, g) or Bonferroni’s multiple comparison test (j).

Figure 3. Pharmacokinetics, pharmacodynamics and efficacy of Cyst(e)inase administration in mice

(a,b) Following single dose Cyst(e)inase administration at 50 mg/kg in FVB mice, (a) apparent Cyst(e)inase concentration in serum as measured by dot blot assay; and (b) relative concentrations of l-Cys and CSSC as assessed by MS as a function of time (n = 5 per group). * P<0.05; ** P<0.01; *** P<0.001; two-sided Student’s t-test. (c–d) Following treatment with active Cyst(e)inase or controls in male FVB/N mice bearing allograft tumors of HMVP2 PCa spheroids; (c) quantification of tumor volume; and (d) average body weight and food consumption per mouse per day for each treatment group. (PBS, n = 5; heat inactivated Cyst(e)inase, n = 6; 50 mg/kg Cyst(e)inase, n = 7; 100 mg/kg Cyst(e)inase, n = 7). (e–f) Following treatment with active Cyst(e)inase or controls, quantification of tumor volume in male nude mice bearing xenograft tumors of (e) DU145 PCa cells (n = 8 per group); and (f) PC3 PCa cells (n = 7 per group). For all studies dosing was terminated when control tumors reached an endpoint. For c, e, f *P<0.05; **P<0.001; *** P<0.0001; repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test; all data are expressed as mean ± s.e.m.

Figure 4. In vitro and in vivo efficacy of Cyst(e)inase in the TCL1-Tg:p53^−/− mouse model and primary CLL cells

(a) Cell viability 48 h after treatment with fludarabine, Cyst(e)inase, or their combination in splenocytes isolated from TCL1-Tg:p53^−/− mice co-cultured with murine stromal Kusa-H cells (n = 6 per group). (b) Survival curve (Kaplan-Meier) of TCL1-Tg:p53−/− mice following either no treatment (n = 47); or treatment with fludarabine (n = 10); Cyst(e)inase (n=10); or their combination (n = 10). (c–d) Cell viability 48 h after treatment with fludarabine, Cyst(e)inase, or their combination in (c) primary 17p wt CLL cells; and (d) primary 17p- CLL cells either cultured alone or co-cultured with stromal NKTert cells (n = 6 different CLL patient samples per phenotype). (e) Relative GSH levels as assessed by spectrophotometric analysis 24 h after treatment with Cyst(e)inase in primary 17p wt, 17pCLL cells, and mouse splenocytes isolated from TCL1-Tg:p53^−/− mice (n = 6 per group). (f) Cell viability 48 h after treatment with Cyst(e)inase in normal lymphocytes isolated from healthy donors co-cultured with NKTert cells (n = 4). For (a, c, d, f) cell viability was assessed by flow cytometry following double staining of cells with Annexin-V and PI. Data are expressed as mean ± s.d. (a, c–f). *P<0.05; **P<0.01; ***P<0.001; two-sided Student’s t-test (a, c–f) or log-rank (Mantel-Cox) test (b).