Search

IC50 curves for ADI-PEG20 in ovarian cancer cell lines. A, ASS1-deficient ovarian cancer cell lines and B, ASS1-proficient ovarian cancer cell lines.

ASS1 in ovarian cancer cell lines. A, Re-expression of SMARCA4 does not affect the expression of ASS1. SCCOHT cells were infected with Lenti-GFP or Lenti-SMARCA4 for 24 hours followed by puromycin selection for 72 hours. Cells were then harvested for protein quantification using mass spectrometry. B, ASS1 immunohistochemistry on FFPE cell line pellets showing positivity in JHOC 7 and JHOC 9, and lack of expression in ES2, JHOC 5, and TOV112D. C, ASS1 immunohistochemistry on FFPE cell line pellets for CRISPR knock out (JHOC 7 ASS1 CRISPR A8) and over-expression (JHOC 5 ASS1 CMV) cell lines compared to their corresponding vector controls (JHOC 7 CRISPR NTC30 and JHOC5 vector control, respectively). D, qPCR showing differential ASS1 mRNA expression levels between ovarian cancer cell lines. Difference of relative mRNA expression between cell lines is determined by one-way ANOVA with post-hoc Tukey test. * p

ASS1 expression does not correlate with survival in CCOC and ENOC. Kaplan-Meier plots with risk table comparing intense ASS1 expression (histoscore = 300, red line) to low/moderate ASS1 expression (histoscore

ASS1 immunohistochemistry in 15 cases of SCCOHT. Different areas (small cell, large cell, rhabdoid) are represented for some cases. Representative cores for each area for each case are shown. Final histoscore for each case is the highest score among all areas.

Table containing proteins differentially expressed in at least two rarer ovarian cancer subtypes when compared to HGSC.

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.

Decreased ASS1 expression correlates with CTNNB1 mutation in ENOC and EEC. A, ASS1 mRNA Z-score in 26 cases of ENOC cases described in publication by Wang et al (30). B, ASS1 histoscore distribution in CTNNB1 mutated and wildtype endometroid endometrial and endometrioid ovarian cancers. C, ASS1 mRNA raw counts in the TCGA endometrial cohort as related to CTNNB1 mutation status. P values were calculated using a Wilcoxin rank sum test.

Supplemental material and methods

Mouse models of SCCOHT cell lines. A, Representative histology of kidney and liver from COV434-implanted mouse in the control group, and in the 30mg/kg ADI-PEG20 group which exhibited abnormal organs upon macroscopic examination at necropsy. B, BRG1 IHC for the masses above the heart found in 2 mice in the treatment groups. Top image: mass only (mouse DGH12-2-14); bottom image, mass adjacent to normal heart (mouse DGH12-2-15). C, tumor volume and weight of SCCOHT1 mouse model. Significance was calculated using ANOVA, followed by a post-hoc Tukey's test. Error bars represent standard error of mean. D, ASS1 re-expression in SCCOHT1 cells in both saline control group and 30mg/kg treated groups. E, Representative tunnel assay images from each treatment group indicating no significant increase in apoptosis in tumors from the ADI-PEG20 treated compared to saline-treated groups, and F, COV434 subcutaneous xenograft showing foci of ASS1 re-expression in treated groups. G, average mouse weight of each mouse model, and H, plots reflecting the tumour weight adjusted by mouse weight (tumour weight/mouse weight).

ADI-PEG20 efficacy in an extended panel of ovarian cancer cell lines. Cells were treated with 0.63 µg/mL ADI-PEG20. First row represents ASS1 expressing cell lines, whereas the rest of the cells are ASS1 deficient, therefore sensitive to ADI-PEG20 treatment. OVCAR3: HGSC cell line; JHOC 9, OVTOKO: CCOC cell lines. BIN67, SCCOHT1: SCCOHT cell lines; OVISE and ES2 represent atypical CCOC/ENOC based on molecular characterizations; TOV112D: de-differentiated ovarian cancer cell line. Error bars represent standard error of mean.

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.