Table S1 - Antibody Details Table S2 - Primer Sequences Table S3 - AP1 shRNA Screen Details Table S4 - Patient Characteristics from Tissue Microarray Table S5 - Transcription factor overlap Figure S1 - Dose response, AP1 activity and AP1 subunit expression. Figure S2 - ATF6 Immunohistochemistry, KM Curve, and AP1 transcriptional targets Figure S3 - P38 Inhibitor Dose Response Figure S4 - DNA Damage in OVCA420 cell line and qPCR expression of BRCA1 and PARP1 Figure S5 - Controls for animal study

Abstract High-grade serous ovarian cancer (HGSOC) is the deadliest ovarian cancer histotype due in-part to the lack of therapeutic options for chemotherapy-resistant disease. PARP inhibitors (PARPi) represent a targeted treatment. However, PARPi resistance is becoming a significant clinical challenge. There is an urgent need to overcome resistance mechanisms to extend disease-free intervals. We established isogeneic PARPi-sensitive and -resistant HGSOC cell lines. In three PARPi-resistant models, there is a significant increase in AP-1 transcriptional activity and DNA repair capacity. Using RNA-sequencing and an shRNA screen, we identified activating transcription factor 6 (ATF6) as a mediator of AP-1 activity, DNA damage response, and PARPi resistance. In publicly available datasets, ATF6 expression is elevated in HGSOC and portends a poorer recurrence-free survival. In a cohort of primary HGSOC tumors, higher ATF6 expression significantly correlated to PARPi resistance. In PARPi-resistant cell lines and a PDX model, inhibition of a known ATF6 regulator, p38, attenuated AP-1 activity and RAD51 foci formation, enhanced DNA damage, significantly inhibited tumor burden, and reduced accumulation of nuclear ATF6. Implications: This study highlights that a novel p38-ATF6-mediated AP-1 signaling axis contributes to PARPi resistance and provides a clinical rationale for combining PARPi and AP-1 signaling inhibitors.

Supplementary Data from Targeting DUSP Activity as a Treatment for High-Grade Serous Ovarian Carcinoma

Ovarian germ cell tumors (GCT) account for 2% to 3% of malignant ovarian neoplasms in Western countries and typically occur within the first 2 decades. When presenting later in life, GCTs may be associated with epithelial malignancies. In these circumstances, it has been theorized that these tumors may originate from a somatic, rather than germ cell origin, especially in the postmenopausal setting; however, the true derivation is not fully understood. Our database was searched for primary ovarian GCTs associated with a malignant epithelial component in patients above 35 yr of age, from 2006 to 2021. Two cases were identified and in each case, slides were reviewed and targeted next-generation sequencing was utilized to identify and compare gene mutation variants in morphologically distinct components. Patient A is a 58-yr-old, with choriocarcinoma and minor component of mucinous adenocarcinoma, and patient B is a 43-yr-old, with yolk sac tumor and minor component of endometrioid adenocarcinoma. The morphologically distinct areas in each case showed disparate staining patterns; however, next-generation sequencing demonstrated identical mutation variants within both the germ cell and epithelial components. Variants in CDKN2A , PIK3CA , PIK3R1 , and TP53 were present in patient A's tumor, while patient B's tumor showed CTNNB1 , PIK3R1 , and 2 PTEN variants. These mutational patterns are similar to those seen in pure epithelial counterparts, suggesting somatic derivation of the germ cell component. These rare tumors portend a poor prognosis and understanding their origin has clinical and therapeutic implications.

Abstract Identifying novel, durable treatments for high-grade serous ovarian cancer (HGSOC) is paramount to extend both progression-free survival (PFS) and overall survival (OS) in patients afflicted with this disease. Dual-specificity phosphatase 1 (DUSP1) was identified as one of seven genes that may significantly affect prognosis in patients with HGSOC; however, the role of DUSP inhibition (DUSPi) in the treatment of HGSOC remains largely unknown. In this study, we show that DUSP1 is highly expressed in HGSOC and confers worse PFS and OS. Further, we corroborate data that show DUSP1 expression is directly associated with therapy resistance. Using a tissue microarray of 137 different serous ovarian carcinomas, we demonstrate the high expression of DUSP1 in primary and recurrent serous ovarian cancer. In both acquired and de novo therapy HGSOC-resistant models, DUSPi both inhibited cellular proliferation and promoted cell death. RPPA analysis of HGSOC cells revealed DUSPi led to the differential regulation of several pathways, including AMPK and mTORC. Further, in a patient-derived xenograft HGSOC model, DUSPi significantly inhibited tumor progression.

Objectives: Dual Specificity Phosphatase (DUSP) proteins regulate signaling cascades involved in tumor progression, but there are limited studies of DUSPs in epithelial ovarian carcinoma, especially in high-grade serous ovarian carcinoma (HGSOC). The study objective was to elucidate DUSP1’s contribution to HGSOC progression and examine a DUSP1/6 inhibitor (DUSPi) in therapy-resistant HGSOC. Methods: Bioinformatic analyses were conducted using The Cancer Genome Atlas (TCGA) Ovarian Serous Cystadenocarcinoma Firehose Database and PanCancer Atlas. qPCR was completed to determine the mRNA expression of DUSP1. Fifty percent inhibitory concentration of DUSPi was determined using colony formation assays. Reverse-phase protein array (RPPA) examined the effect of DUSPi on cellular signaling. The antitumoral effect of DUSPi was examined using a patient-derived xenograft (PDX) model. Quantitative data were expressed as mean +/- standard error of the mean. RPPA relative protein levels were determined for each and designated as log2 intensities and subsequently median-centered. Analysis of variance with Tukey correction was used for multiple comparisons. The level of significance was set at 0.05. Results: Of 24 DUSP proteins examined in HGSOC, DUSP1 mRNA was significantly upregulated (p<0.001, one-way ANOVA). Progression-free survival (PFS) and overall survival (OS) were significantly higher in patients with low DUSP1 expression (PFS: 15.41 vs 22.22 months, Log-rank test, p=0.0010, HR: 1.65, 95% CI: 1.213-2.259; OS: 41.52 vs 52.40 months, Log-rank test, p=0.0183, HR: 1.462, 95% CI: 1.064-2.00). Elevated DUSP1 protein expression was noted in our tissue microarray of 137 serous ovarian carcinoma primary and recurrent tumors. Olaparib-resistant HGSOC cells had significantly increased DUSP1 expression compared to olaparib-sensitive HGSOC cells (unpaired t-test, p<0.0001). The RPPA analysis revealed 34 proteins with significantly (p<0.05) altered expression after treatment with DUSPi. DUSPi led to the significant downregulation of WEE1 and ATR. Phosphor-AMPK (AMPK-a2_pS345), part of the mTORC pathway that can serve both tumor-suppressive and oncogenic roles, was elevated and dual phosphorylated ERK (MAPK_pT202_Y204). RPPA findings were confirmed in TCGA data and by immunoblotting. A therapy-resistant PDX model was used to examine DUSPi in vivo activity. DUSPi led to a decrease in total flux (photons/sec), indicating tumor regression, in the mice treated with DUSPi compared to control. At the end of the study, there was no evidence of ascites or tumor burden in mice treated with DUSPi. Conclusions: DUSP1 expression is associated with poor progression-free and overall survival. After treatment with a novel DUSP1/6 inhibitor (DUSPi), cell viability was significantly reduced. We defined the differential activation of signaling following DUSPi via RPPA. DUSP inhibition significantly resulted in altering the expression of multiple pathway proteins, including increasing AMPK phosphorylation. Finally, we identified a robust in vivo antitumor response with DUSPi in PDX models. Objectives: Dual Specificity Phosphatase (DUSP) proteins regulate signaling cascades involved in tumor progression, but there are limited studies of DUSPs in epithelial ovarian carcinoma, especially in high-grade serous ovarian carcinoma (HGSOC). The study objective was to elucidate DUSP1’s contribution to HGSOC progression and examine a DUSP1/6 inhibitor (DUSPi) in therapy-resistant HGSOC. Methods: Bioinformatic analyses were conducted using The Cancer Genome Atlas (TCGA) Ovarian Serous Cystadenocarcinoma Firehose Database and PanCancer Atlas. qPCR was completed to determine the mRNA expression of DUSP1. Fifty percent inhibitory concentration of DUSPi was determined using colony formation assays. Reverse-phase protein array (RPPA) examined the effect of DUSPi on cellular signaling. The antitumoral effect of DUSPi was examined using a patient-derived xenograft (PDX) model. Quantitative data were expressed as mean +/- standard error of the mean. RPPA relative protein levels were determined for each and designated as log2 intensities and subsequently median-centered. Analysis of variance with Tukey correction was used for multiple comparisons. The level of significance was set at 0.05. Results: Of 24 DUSP proteins examined in HGSOC, DUSP1 mRNA was significantly upregulated (p<0.001, one-way ANOVA). Progression-free survival (PFS) and overall survival (OS) were significantly higher in patients with low DUSP1 expression (PFS: 15.41 vs 22.22 months, Log-rank test, p=0.0010, HR: 1.65, 95% CI: 1.213-2.259; OS: 41.52 vs 52.40 months, Log-rank test, p=0.0183, HR: 1.462, 95% CI: 1.064-2.00). Elevated DUSP1 protein expression was noted in our tissue microarray of 137 serous ovarian carcinoma primary and recurrent tumors. Olaparib-resistant HGSOC cells had significantly increased DUSP1 expression compared to olaparib-sensitive HGSOC cells (unpaired t-test, p<0.0001). The RPPA analysis revealed 34 proteins with significantly (p<0.05) altered expression after treatment with DUSPi. DUSPi led to the significant downregulation of WEE1 and ATR. Phosphor-AMPK (AMPK-a2_pS345), part of the mTORC pathway that can serve both tumor-suppressive and oncogenic roles, was elevated and dual phosphorylated ERK (MAPK_pT202_Y204). RPPA findings were confirmed in TCGA data and by immunoblotting. A therapy-resistant PDX model was used to examine DUSPi in vivo activity. DUSPi led to a decrease in total flux (photons/sec), indicating tumor regression, in the mice treated with DUSPi compared to control. At the end of the study, there was no evidence of ascites or tumor burden in mice treated with DUSPi. Conclusions: DUSP1 expression is associated with poor progression-free and overall survival. After treatment with a novel DUSP1/6 inhibitor (DUSPi), cell viability was significantly reduced. We defined the differential activation of signaling following DUSPi via RPPA. DUSP inhibition significantly resulted in altering the expression of multiple pathway proteins, including increasing AMPK phosphorylation. Finally, we identified a robust in vivo antitumor response with DUSPi in PDX models.

Cryptococcus species are ubiquitous in the environment with a global distribution. While causing disease predominantly in immunocompromised hosts such as those with advanced HIV, HIV-uninfected patients are increasingly recognized as being affected. The most common forms of infection are cryptococcal pneumonia and meningitis. HIV-uninfected patients and extrapulmonary infections have worse outcomes, likely due to delayed diagnosis and treatment. Cryptococcus infections involving chylothorax or chyloabdomen have rarely been reported in humans. We describe a case of fulminant disseminated cryptococcosis with fungemia, peritonitis, and empyema in a patient with chronic chylothorax treated with an indwelling pleurovenous shunt. Key autopsy findings included cryptococcal organisms identified on calcified lymphadenopathy, pleural adhesions, and pericardium. We discuss the importance of identifying patients with nontraditional risks factors for cryptococcal disease, such as lymphopenia and hypogammaglobulinemia, and the potential implications of pleurovenous catheters in Cryptococcus dissemination.

Paragangliomas are rare neuroendocrine neoplasms derived from sympathetic or parasympathetic paraganglia and have the ability to secrete catecholamines. We present the case of a 37-year-old asymptomatic female who underwent right ovarian cystectomy for a mature cystic teratoma and was found to have an intra-tumoral paraganglioma. More research is needed to determine metastatic potential as well as the likelihood of recurrence of these unique neoplasms.