Arginine Depletion Therapy with ADI-PEG20 Limits Tumor Growth in Argininosuccinate Synthase-Deficient Ovarian Cancer, Including Small-Cell Carcinoma of the Ovary, Hypercalcemic Type

Abstract

Purpose: Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes.

Experimental design: We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT.

Results: Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo.

Conclusions: Preclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.

Figure 1.. Global proteomics identifies decreased ASS1 expression in rare ovarian cancer subtypes.

Proteomics data for six cases each of CCOC, ENOC and HGSC were obtained from a previous publication (25). An additional six cases each of SCCOHT and HGSC were analyzed using SP3-CTP global proteome profiling. Volcano plot showing differentially expressed proteins comparing A, ENOC to HGSC; B, CCOC to HGSC; and C, SCCOHT to HGSC. Significantly differentially expressed proteins are highlighted in red, and were defined as log2 fold change larger than 1 or smaller than −1, and a FDR-adjusted p < 0.05. Boxplot showing ASS1 protein expression z-score in each case of D, CCOC, ENOC and HGSC; and E, SCCOHT compared to HGSC. The statistical significance in multiple group comparisons is calculated with a Kruskal-Wallis test with a post-hoc dunn’s test with Benjamini-Hochberg correction. Pairwise comparison in figure 1E was calculated using a Mann-Whitney U test.

Figure 2.. ASS1 immunohistochemistry demonstrates differential expression in ovarian cancer subtypes.

Representative immunohistochemical stains in TMA cores of A, epithelial ovarian cancer subtypes including differential expression seen in CCOC, and C, non-epithelial subtypes. Corresponding boxplots depicting ASS1 histoscore distribution in B, epithelial subtypes and D, non-epithelial subtypes. Histoscore was calculated by multiplying the average staining intensity by the percentage of tumor cells staining positive. The statistical significance in multiple group comparisons is calculated with a Kruskal-Wallis test with a post-hoc dunn’s test with Benjamini-Hochberg correction.

Figure 3.. ASS1 deficient ovarian cancer are sensitive to arginine deprivation through ADI-PEG20.

A, western blot showing differential ASS1 expression in representative ovarian cancer cell lines. B, cell proliferation of ASS1 negative SCCOHT cell line (COV434) and CCOC cell line (JHOC 5) compared to ASS1 expressing CCOC cell line (JHOC 7), when treated with 0.63μg/mL of ADI-PEG20; and C, bar graph of IC50 of ASS1 deficient cell lines representing various ovarian cancer subtypes, IC50 (95% CI) are labeled for each cell line. ASS1 expressing cell lines (JHOC 7, JHOC9, OVCAR3, OVISE) all had IC50s above 2ug/mL. D, Treatment with 0.63μg/mL of ADI-PEG20 induced apoptosis as shown by a caspase 3/7 cleavage assay in ASS1 deficient, but not in ASS1 expressing cell lines. E, ADI-PEG20 treatment abolished the clonogenic potential of ASS1 deficient cell lines, while decreased the clonogenic potential in ASS1-expressing cell line.

Figure 4.. ASS1 is required for survival in arginine deplete conditions and its deficiency disrupts urea cycle function.

A, Argininosuccinate and arginine intracellular measurements in ASS1-deficient cells (JHOC 5 and COV434), compared to ASS1- proficient cells (JHOC 7 and JHOC 9). B, western blot showing the knockout efficiency of two CRISPR ASS1 knockout clones in a JHOC 7 background (A8: clone 8 using guide A, and B6: clone 6 using guide B, NTC30: empty vector negative control). and the specificity of ADI-PEG20 was confirmed in the ASS1 knockout clones A8 and B6. C, Western blot showing the stable over-expression of ASS1 in JHOC5, rescuing its growth when treated with 0.63μg/mL of ADI-PEG20. All p-values were generated from student t-test. Error bars represent standard error of mean.

Figure 5.. ADI-PEG20 is effective in inhibiting SCCOHT tumor growth in cell line and patient derived xenograft based in-vivo models.

A, Average tumor volume of the COV434 cell line model and PDX465 patient derived xenograft model. Mice were treated once a week (indicated by arrows). B, average weight of tumors at study termination. C, average tumor volume of an additional PDX-465 experiment where subjects received three weeks of 30mg/kg (black arrows), followed by 3 weeks of 15mg/kg (magenta arrows). D, histology and Ki67 immunohistochemistry of representative tumors from the control and treatment groups from the COV434 model. Ki67 score were determined based on the area with the most intense staining. Arrows represent mitotic figures; E, Average mitotic count on whole sections indicating significantly dampened mitotic activity in both treatment groups in the COV434 model. F, average Ki67 score between groups showing significantly decreased proliferation in the 30mg/kg group in the COV434 model. For multiple group comparisons, significance was calculated using ANOVA, followed by a post-hoc Tukey’s test. Error bars represent standard error of mean.