Figure S5 shows that NMN supplementation during cisplatin-induced senescence of ovarian cancer cells promotes cancer stem-like cells.

Figure S8 shows that a cisplatin and NAMPT inhibitor FK866 combination reduces ALDH activity in vivo.

Figure S2 shows that NAMPT inhibition suppresses the SASP induced by carboplatin.

Legends for supplementary figures 1-8

Figure S1 shows that NAMPT inhibition suppresses the SASP in OVCAR5 cell line.

Figure S4 shows that genetically knocking down NAMPT suppresses ovarian cancer stem-like cells.

Figure S7 shows that high NAMPT expression correlates with a poorer survival.

Figure S3 shows that small molecule NAMPT inhibitors suppress ovarian cancer stem-like cells and promote apoptosis.

Figure S6 shows that conditioned media is not sufficient to induce senescence.

In response to DNA double-strand breaks, MAD2L2-containing shieldin complex plays a critical role in the choice between homologous recombination (HR) and non-homologous end-joining (NHEJ)-mediated repair. Here we show that EZH2 inhibition upregulates MAD2L2 and sensitizes HR-proficient epithelial ovarian cancer (EOC) to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor in a CARM1-dependent manner. CARM1 promotes MAD2L2 silencing by driving the switch from the SWI/SNF complex to EZH2 through methylating the BAF155 subunit of the SWI/SNF complex on the MAD2L2 promoter. EZH2 inhibition upregulates MAD2L2 to decrease DNA end resection, which increases NHEJ and chromosomal abnormalities, ultimately causing mitotic catastrophe in PARP inhibitor treated HR-proficient cells. Significantly, EZH2 inhibitor sensitizes CARM1-high, but not CARM-low, EOCs to PARP inhibitors in both orthotopic and patient-derived xenografts.

Abstract Epithelial ovarian cancer (EOC) is the most lethal of gynecologic malignancies. The standard-of-care treatment for EOC is platinum-based chemotherapy such as cisplatin. Platinum-based chemotherapy induces cellular senescence. Notably, therapy-induced senescence contributes to chemoresistance by inducing cancer stem-like cells (CSC). However, therapeutic approaches targeting senescence-associated CSCs remain to be explored. Here, we show that nicotinamide phosphoribosyltransferase (NAMPT) inhibition suppresses senescence-associated CSCs induced by platinum-based chemotherapy in EOC. Clinically applicable NAMPT inhibitors suppressed the outgrowth of cisplatin-treated EOC cells both in vitro and in vivo. Moreover, a combination of the NAMPT inhibitor FK866 and cisplatin improved the survival of EOC-bearing mice. These phenotypes correlated with inhibition of the CSCs signature, which consists of elevated expression of ALDH1A1 and stem-related genes, high aldehyde dehydrogenase activity, and CD133 positivity. Mechanistically, NAMPT regulates EOC CSCs in a paracrine manner through the senescence-associated secretory phenotype. Our results suggest that targeting NAMPT using clinically applicable NAMPT inhibitors, such as FK866, in conjunction with platinum-based chemotherapy represents a promising therapeutic strategy by suppressing therapy-induced senescence-associated CSCs. Significance: This study highlights the importance of NAMPT-mediated NAD+ biosynthesis in the production of cisplatin-induced senescence-associated cancer stem cells, as well as tumor relapse after cisplatin treatment.

ARID1A inactivation causes mitotic defects. Paradoxically, cancers with high ARID1A mutation rates typically lack copy number alterations (CNAs). Here, we show that ARID1A inactivation causes defects in telomere cohesion, which selectively eliminates gross chromosome aberrations during mitosis. ARID1A promotes the expression of cohesin subunit STAG1 that is specifically required for telomere cohesion. ARID1A inactivation causes telomere damage that can be rescued by STAG1 expression. Colony formation capability of single cells in G2/M, but not G1 phase, is significantly reduced by ARID1A inactivation. This correlates with an increase in apoptosis and a reduction in tumor growth. Compared with ARID1A wild-type tumors, ARID1A-mutated tumors display significantly less CNAs across multiple cancer types. Together, these results show that ARID1A inactivation is selective against gross chromosome aberrations through causing defects in telomere cohesion, which reconciles the long-standing paradox between the role of ARID1A in maintaining mitotic integrity and the lack of genomic instability in ARID1A-mutated cancers.

New plasma and tissue biomarkers of epithelial ovarian cancer (EOC) could improve early diagnosis and post-diagnosis clinical management. Here we investigated tissue staining and tissue secretion of CLIC1 and CLIC4 across EOC subtypes. CLIC1 and CLIC4 are two promising biomarkers we previously showed were elevated in EOC patient sera. Individually, CLIC1 or CLIC4 stained larger percentages of malignant tumors across all EOC subtypes compared with CA125, particularly early stage and mucinous tumors. CLIC4 also stained benign tumors but staining was limited to nuclei; whereas malignant tumors showed diffuse cellular staining of stromal and tumor cells. Both proteins were shed by all EOC subtypes tumors in short term organ culture at more consistent levels than CA125, supporting their potential as pan-subtype serum and tissue biomarkers. Elevated CLIC4 expression, but not CLIC1 expression, was a negative indicator of patient survival, and CLIC4 knockdown in cultured cells decreased cell proliferation and migration indicating a potential role in tumor progression. These results suggest CLIC1 and CLIC4 are promising serum and tissue biomarkers as well as potential therapeutic targets for all EOC subtypes. This justifies development of high throughput serum/plasma biomarker assays to evaluate utility of a biomarker panel consisting of CLIC1, CLIC4 and CA125.

Despite the importance of programmed cell death-1 (PD-1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT-rich sequence-binding protein-1 (Satb1) restrains PD-1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40-fold increase in PD-1 expression. Tumor-derived transforming growth factor β (Tgf-β) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1-NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1-mediated repression, Satb1-deficient tumor-reactive T cells lost effector activity more rapidly than wild-type lymphocytes at tumor beds expressing PD-1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor-infiltrating T cells results in diminished anti-tumor immunity.

Despite the importance of Programmed Cell Death-1 (PDCD1/PD-1) in inhibiting T-cell effector activity, the mechanisms regulating its expression in anti-tumor lymphocytes remain poorly defined. Here we show that the chromatin organizer Special AT-rich Sequence-Binding Protein-1 (Satb1) is required to restrain activation-induced PD-1 expression. We demonstrate that, mechanistically, Satb1 physically interacts with a nucleosome remodeling deacetylase (NuRD) complex, which it recruits to Pdcd1 regulatory regions. This molecular complex thus drives histone de-acetylation and results in PD-1 repression in T cells. Accordingly, T-cell-specific Satb1 deficiency results in a 40-fold increase in PD-1 expression. Intriguingly, tumor-derived Transforming growth factor (Tgf)-β decreases Satb1 expression in T cells through binding of Smad Family Member (Smad) proteins to the Satb1 promoter, while Smad also competes with Satb1/NuRD for binding to Pdcd1 enhancers, cooperatively unleashing PD-1 expression in a Satb1-dependent manner. Consequently, Satb1-deficient tumor-reactive T cells lose their effector activity more rapidly than wild-type T cells at PD-L1+ tumor beds, but these differences are abrogated by sustained PD-L1 blockade. Therefore, we demonstrate that Satb1 is an epigenetic controller of PD-1 expression, and that Tgf-β signaling contributes to T cell dysfunction within the tumor microenvironment by inhibiting Satb1-mediated repression of PD-1.

Special AT-rich sequence-binding protein 1 (Satb1) governs genome-wide transcriptional programs. Using a conditional knockout mouse, we find that Satb1 is required for normal differentiation of conventional dendritic cells (DCs). Furthermore, Satb1 governs the differentiation of inflammatory DCs by regulating major histocompatibility complex class II (MHC II) expression through Notch1 signaling. Mechanistically, Satb1 binds to the Notch1 promoter, activating Notch expression and driving RBPJ occupancy of the H2-Ab1 promoter, which activates MHC II transcription. However, tumor-driven, unremitting expression of Satb1 in activated Zbtb46(+) inflammatory DCs that infiltrate ovarian tumors results in an immunosuppressive phenotype characterized by increased secretion of tumor-promoting Galectin-1 and IL-6. In vivo silencing of Satb1 in tumor-associated DCs reverses their tumorigenic activity and boosts protective immunity. Therefore, dynamic fluctuations in Satb1 expression govern the generation and immunostimulatory activity of steady-state and inflammatory DCs, but continuous Satb1 overexpression in differentiated DCs converts them into tolerogenic/pro-inflammatory cells that contribute to malignant progression.

Mortality from ovarian cancer may be dramatically reduced with the implementation of attainable prevention strategies. The new understanding of the cells of origin and the molecular etiology of ovarian cancer warrants a strong recommendation to the public and health care providers. This document discusses potential prevention strategies, which include 1) oral contraceptive use, 2) tubal sterilization, 3) risk‐reducing salpingo‐oophorectomy in women at high hereditary risk of breast and ovarian cancer, 4) genetic counseling and testing for women with ovarian cancer and other high‐risk families, and 5) salpingectomy after childbearing is complete (at the time of elective pelvic surgeries, at the time of hysterectomy, and as an alternative to tubal ligation). The Society of Gynecologic Oncology has determined that recent scientific breakthroughs warrant a new summary of the progress toward the prevention of ovarian cancer. This review is intended to emphasize the importance of the fallopian tubes as a potential source of high‐grade serous cancer in women with and without known genetic mutations in addition to the use of oral contraceptive pills to reduce the risk of ovarian cancer. Cancer 2015;121:2108–2120. © 2015 American Cancer Society .

ObjectiveThe objective of this video is to present a case of a uterine didelphys and demonstrate the use of robotic-assisted laparoscopy to perform a hysterectomy safely and efficiently.DescriptionUterine didelphys is a mullerian anomaly which is caused by a failure of the mullerian ducts to fuse in embryogenesis. As a result, two separate uteri and cervices develop. A vertical vaginal septum can be present as well. This video describes a 42 year old G2P1102 who presented with dysfunctional uterine bleeding. She had completed child-bearing and requested definitive surgical management. She had a known uterine didelphys with a vertical vaginal septum diagnosed on prior hysterosalpingogram. She had two successful pregnancies in the right uterus. A RUMI uterine manipulator was placed in the dominant right endometrial cavity with a Koh ring placed around the right cervix. Two working robotic ports, a 12mm camera port and a right upper quadrant assistant port were placed. In addition to the Koh ring around the right cervix, a Briesky vaginal retractor was placed in the left fornix to aid in making a colpotomy around the left cervix. The specimen (both uteri and cervices) were removed intact through the right vagina. The colpotomy was closed using 0-Vicryl suture with interrupted figure of eight stiches.ConclusionMinimally invasive techniques such as robotic-assisted laparoscopy are safe and efficient methods of performing a hysterectomy in patients with mullerian anomalies including a uterine didelphys.DISCLOSURE OF RELEVANT FINANCIAL RELATIONSHIPS:Tanya P. Hoke: Nothing to discloseMark E. Borowsky: Nothing to discloseNima Patel: Nothing to disclose ObjectiveThe objective of this video is to present a case of a uterine didelphys and demonstrate the use of robotic-assisted laparoscopy to perform a hysterectomy safely and efficiently. The objective of this video is to present a case of a uterine didelphys and demonstrate the use of robotic-assisted laparoscopy to perform a hysterectomy safely and efficiently. DescriptionUterine didelphys is a mullerian anomaly which is caused by a failure of the mullerian ducts to fuse in embryogenesis. As a result, two separate uteri and cervices develop. A vertical vaginal septum can be present as well. This video describes a 42 year old G2P1102 who presented with dysfunctional uterine bleeding. She had completed child-bearing and requested definitive surgical management. She had a known uterine didelphys with a vertical vaginal septum diagnosed on prior hysterosalpingogram. She had two successful pregnancies in the right uterus. A RUMI uterine manipulator was placed in the dominant right endometrial cavity with a Koh ring placed around the right cervix. Two working robotic ports, a 12mm camera port and a right upper quadrant assistant port were placed. In addition to the Koh ring around the right cervix, a Briesky vaginal retractor was placed in the left fornix to aid in making a colpotomy around the left cervix. The specimen (both uteri and cervices) were removed intact through the right vagina. The colpotomy was closed using 0-Vicryl suture with interrupted figure of eight stiches. Uterine didelphys is a mullerian anomaly which is caused by a failure of the mullerian ducts to fuse in embryogenesis. As a result, two separate uteri and cervices develop. A vertical vaginal septum can be present as well. This video describes a 42 year old G2P1102 who presented with dysfunctional uterine bleeding. She had completed child-bearing and requested definitive surgical management. She had a known uterine didelphys with a vertical vaginal septum diagnosed on prior hysterosalpingogram. She had two successful pregnancies in the right uterus. A RUMI uterine manipulator was placed in the dominant right endometrial cavity with a Koh ring placed around the right cervix. Two working robotic ports, a 12mm camera port and a right upper quadrant assistant port were placed. In addition to the Koh ring around the right cervix, a Briesky vaginal retractor was placed in the left fornix to aid in making a colpotomy around the left cervix. The specimen (both uteri and cervices) were removed intact through the right vagina. The colpotomy was closed using 0-Vicryl suture with interrupted figure of eight stiches. ConclusionMinimally invasive techniques such as robotic-assisted laparoscopy are safe and efficient methods of performing a hysterectomy in patients with mullerian anomalies including a uterine didelphys.DISCLOSURE OF RELEVANT FINANCIAL RELATIONSHIPS:Tanya P. Hoke: Nothing to discloseMark E. Borowsky: Nothing to discloseNima Patel: Nothing to disclose Minimally invasive techniques such as robotic-assisted laparoscopy are safe and efficient methods of performing a hysterectomy in patients with mullerian anomalies including a uterine didelphys.