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Purpose: We recently reported that the transcription factor NFATC4, in response to chemotherapy, drives cellular quiescence to increase ovarian cancer chemoresistance. The goal of this work was to better understand the mechanisms of NFATC4-driven ovarian cancer chemoresistance. Experimental Design: We used RNA sequencing to identify NFATC4-mediated differential gene expression. CRISPR-Cas9 and FST (follistatin)-neutralizing antibodies were used to assess impact of loss of FST function on cell proliferation and chemoresistance. ELISA was used to quantify FST induction in patient samples and in vitro in response to chemotherapy. Results: We found that NFATC4 upregulates FST mRNA and protein expression predominantly in quiescent cells and FST is further upregulated following chemotherapy treatment. FST acts in at least a paracrine manner to induce a p-ATF2–dependent quiescent phenotype and chemoresistance in non-quiescent cells. Consistent with this, CRISPR knockout (KO) of FST in ovarian cancer cells or antibody-mediated neutralization of FST sensitizes ovarian cancer cells to chemotherapy treatment. Similarly, CRISPR KO of FST in tumors increased chemotherapy-mediated tumor eradication in an otherwise chemotherapy-resistant tumor model. Suggesting a role for FST in chemoresistance in patients, FST protein in the abdominal fluid of patients with ovarian cancer significantly increases within 24 hours of chemotherapy exposure. FST levels decline to baseline levels in patients no longer receiving chemotherapy with no evidence of disease. Furthermore, elevated FST expression in patient tumors is correlated with poor progression-free, post–progression-free, and overall survival. Conclusions: FST is a novel therapeutic target to improve ovarian cancer response to chemotherapy and potentially reduce recurrence rates.

Supplementary Figure 2. RNA-seq analysis. A. Schematic of the RNA-seq experimental design. B. Waterfall plot of RNA-seq data showing the 141 genes which were DE at all time points compared to luciferase control in HEY1 and SKOV3 cell lines. C. Unsupervised clustering analysis of the 141 DE genes. D. Box plots demonstrating MYC expression following NFATC4 activation. E. STRING analysis of the 141 DE genes.

Supplementary Figure 1. Histological analysis of PT340 cell line. PT340 cells were injected into NSG mice, and the resulting tumors were harvested for histological analysis, H&E staining showed features of Clear Cell Ovarian Carcinoma.

Purpose:We recently reported that the transcription factor NFATC4, in response to chemotherapy, drives cellular quiescence to increase ovarian cancer chemoresistance. The goal of this work was to better understand the mechanisms of NFATC4-driven ovarian cancer chemoresistance.Experimental Design:We used RNA sequencing to identify NFATC4-mediated differential gene expression. CRISPR-Cas9 and FST (follistatin)-neutralizing antibodies were used to assess impact of loss of FST function on cell proliferation and chemoresistance. ELISA was used to quantify FST induction in patient samples and in vitro in response to chemotherapy.Results:We found that NFATC4 upregulates FST mRNA and protein expression predominantly in quiescent cells and FST is further upregulated following chemotherapy treatment. FST acts in at least a paracrine manner to induce a p-ATF2–dependent quiescent phenotype and chemoresistance in non-quiescent cells. Consistent with this, CRISPR knockout (KO) of FST in ovarian cancer cells or antibody-mediated neutralization of FST sensitizes ovarian cancer cells to chemotherapy treatment. Similarly, CRISPR KO of FST in tumors increased chemotherapy-mediated tumor eradication in an otherwise chemotherapy-resistant tumor model. Suggesting a role for FST in chemoresistance in patients, FST protein in the abdominal fluid of patients with ovarian cancer significantly increases within 24 hours of chemotherapy exposure. FST levels decline to baseline levels in patients no longer receiving chemotherapy with no evidence of disease. Furthermore, elevated FST expression in patient tumors is correlated with poor progression-free, post–progression-free, and overall survival.Conclusions:FST is a novel therapeutic target to improve ovarian cancer response to chemotherapy and potentially reduce recurrence rates.

Supplementary Figure 4. The effect of ATF2 siRNA on gene expression and PT340 FST induced chemoresistance. A. Schematic detailing the transwell experiments. CellTrace Violet cells were sorted for rapidly dividing (Dim) or quiescent cells (Bright) which were then plated in transwell chambers either as Dim:Dim or Bright:Dim. Cells were untreated or treated with taxol alone or in combination with IgG or anti-FST antibody. Cell counts were performed on the dim cells and data was expressed as fold chnage. B. Relative ATF2 gene expression of PT412 and PT340 treated with scrambled siRNA or two independent ATF2 siRNAs (ATF2 siRNA #1 and #2). C. Viable cell number of PT340 cells treated with control scrambled siRNA or knocked down with ATF2 siRNA and treated with cisplatin +/- FST. *P

Supplementary Figure 3. The effect of FST or taxol treatment ovarian cancer proliferation and FST secretion, and the ability of a FST neutralization antibody to inhibit FST's effect on proliferation. A. PT340 and PT412 cells were treated with 200 ng/mL FST for 72h before cell counts were performed. B (i) IHC images and (ii) FST+ cell bodies in OVSAHO tumor xenografts treated with or without taxol and stained with both Ki67 (Forangi Blue) and FST (Fast Red). C. Titer of the ability of the anti-FST neutralizing antibody to inhibit the effect of FST on cell growth.

Supplementary Table 1. Primers used in this study.