Targeting LxCxE cleft pocket of retinoblastoma protein in M2 macrophages inhibits ovarian cancer progression.

Ovarian cancer remains a major health threat with limited treatment options available. It is characterized by immunosuppressive tumor microenvironment (TME) maintained by tumor- associated macrophages (TAMs) hindering anti-tumor responses and immunotherapy efficacy. Here we show that targeting retinoblastoma protein (Rb) by disruption of its LxCxE cleft pocket, causes cell death in TAMs by induction of ER stress, p53 and mitochondria-related cell death pathways. A reduction of pro-tumor Rb high M2-type macrophages from TME in vivo enhanced T cell infiltration and inhibited cancer progression. We demonstrate an increased Rb expression in TAMs in women with ovarian cancer is associated with poorer prognosis. Ex vivo, we show analogous cell death induction by therapeutic Rb targeting in TAMs in post-surgery ascites from ovarian cancer patients. Overall, our data elucidates therapeutic targeting of the Rb LxCxE cleft pocket as a novel promising approach for ovarian cancer treatment through depletion of TAMs and re-shaping TME immune landscape. Statement of significance: Currently, targeting immunosuppressive myeloid cells in ovarian cancer microenvironment is the first priority need to enable successful immunotherapy, but no effective solutions are clinically available. We show that targeting LxCxE cleft pocket of Retinoblastoma protein unexpectedly induces preferential cell death in M2 tumor-associated macrophages. Depletion of immunosuppressive M2 tumor-associated macrophages reshapes tumor microenvironment, enhances anti-tumor T cell responses, and inhibits ovarian cancer. Thus, we identify a novel paradoxical function of Retinoblastoma protein in regulating macrophage viability as well as a promising target to enhance immunotherapy efficacy in ovarian cancer.

EZH2 Inhibition Sensitizes CARM1-High, Homologous Recombination Proficient Ovarian Cancers to PARP Inhibition.

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.

NAMPT Inhibition Suppresses Cancer Stem-like Cells Associated with Therapy-Induced Senescence in Ovarian Cancer.

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 promotes genomic stability through protecting telomere cohesion.

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.

CLIC1 and CLIC4 complement CA125 as a diagnostic biomarker panel for all subtypes of epithelial ovarian cancer.

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.

Malignant Transformation of Endometriosis in the Ischioanal Fossa.

We present the case of a 28-year-old nulliparous female with malignant transformation of an ectopic focus of endometriosis in the right ischioanal fossa. A 28-year-old nulliparous patient with a past medical history of polycystic ovarian syndrome (PCOS) was diagnosed with endometrioid adenocarcinoma in her right ischioanal fossa. Initially, patient presented to an emergency department and underwent a CT scan of the appendix to rule out appendicitis. A multiloculated cystic lesion adjacent to the right obturator internus muscle was found. She underwent surgical resection of the mass, which confirmed FIGO grade 2 endometrioid adenocarcinoma, followed by localized radiation therapy. Malignancy arising in endometriosis is rare, and the influence of PCOS on the rate of malignant transformation is not well established.

Follicle-Stimulating Hormone Receptor Is Expressed by Most Ovarian Cancer Subtypes and Is a Safe and Effective Immunotherapeutic Target.

PURPOSE: To define the safety and effectiveness of T cells redirected against follicle-stimulating hormone receptor (FSHR)-expressing ovarian cancer cells. EXPERIMENTAL DESIGN: FSHR expression was determined by Western blotting, immunohistochemistry, and qPCR in 77 human ovarian cancer specimens from 6 different histologic subtypes and 20 human healthy tissues. The effectiveness of human T cells targeted with full-length FSH in vivo was determined against a panel of patient-derived xenografts. Safety and effectiveness were confirmed in immunocompetent tumor-bearing mice, using constructs targeting murine FSHR and syngeneic T cells. RESULTS: FSHR is expressed in gynecologic malignancies of different histologic types but not in nonovarian healthy tissues. Accordingly, T cells expressing full-length FSHR-redirected chimeric receptors mediate significant therapeutic effects (including tumor rejection) against a panel of patient-derived tumors in vivo In immunocompetent mice growing syngeneic, orthotopic, and aggressive ovarian tumors, fully murine FSHR-targeted T cells also increased survival without any measurable toxicity. Notably, chimeric receptors enhanced the ability of endogenous tumor-reactive T cells to abrogate malignant progression upon adoptive transfer into naïve recipients subsequently challenged with the same tumor. Interestingly, FSHR-targeted T cells persisted as memory lymphocytes without noticeable PD-1-dependent exhaustion during end-stage disease, in the absence of tumor cell immunoediting. However, exosomes in advanced tumor ascites diverted the effector activity of this and other chimeric receptor-transduced T cells away from targeted tumor cells. CONCLUSIONS: T cells redirected against FSHR+ tumor cells with full-length FSH represent a promising therapeutic alternative against a broad range of ovarian malignancies, with negligible toxicity even in the presence of cognate targets in tumor-free ovaries. Clin Cancer Res; 23(2); 441-53. ©2016 AACR.

Tumor Cell-Independent Estrogen Signaling Drives Disease Progression through Mobilization of Myeloid-Derived Suppressor Cells.

The role of estrogens in antitumor immunity remains poorly understood. Here, we show that estrogen signaling accelerates the progression of different estrogen-insensitive tumor models by contributing to deregulated myelopoiesis by both driving the mobilization of myeloid-derived suppressor cells (MDSC) and enhancing their intrinsic immunosuppressive activity in vivo Differences in tumor growth are dependent on blunted antitumor immunity and, correspondingly, disappear in immunodeficient hosts and upon MDSC depletion. Mechanistically, estrogen receptor alpha activates the STAT3 pathway in human and mouse bone marrow myeloid precursors by enhancing JAK2 and SRC activity. Therefore, estrogen signaling is a crucial mechanism underlying pathologic myelopoiesis in cancer. Our work suggests that new antiestrogen drugs that have no agonistic effects may have benefits in a wide range of cancers, independently of the expression of estrogen receptors in tumor cells, and may synergize with immunotherapies to significantly extend survival. SIGNIFICANCE: Ablating estrogenic activity delays malignant progression independently of the tumor cell responsiveness, owing to a decrease in the mobilization and immunosuppressive activity of MDSCs, which boosts T-cell-dependent antitumor immunity. Our results provide a mechanistic rationale to block estrogen signaling with newer antagonists to boost the effectiveness of anticancer immunotherapies. Cancer Discov; 7(1); 72-85. ©2016 AACR.See related commentary by Welte et al., p. 17This article is highlighted in the In This Issue feature, p. 1.

Postmenopausal Bleeding Resulting from Acute Myeloid Leukemia Infiltration of the Endometrium.

Postmenopausal bleeding can be the result of numerous etiologies including endometrial carcinoma, vaginal atrophy, and endometrial polyps. Manifestation of a hematologic disease, such as acute myeloid leukemia (AML), is a rare occurrence. A 65-year-old woman with pancytopenia and postmenopausal bleeding was diagnosed with AML. Endometrial biopsy following dilation and curettage of the uterus revealed extensive mononuclear cell infiltrate consistent with AML. The patient was a poor surgical candidate and subsequently underwent treatment with chemotherapy, hormonal therapy, pelvic radiation, and uterine artery embolization to control her vaginal bleeding. A multi-disciplinary approach is necessary for treatment of post-menopausal bleeding resulting from AML infiltration of the endometrium.

Microbially driven TLR5-dependent signaling governs distal malignant progression through tumor-promoting inflammation.

The dominant TLR5(R392X) polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.

Tissue-based immune monitoring I: tumor core needle biopsies allow in-depth interrogation of the tumor microenvironment.

We sought to assess the feasibility and reproducibility of performing tissue-based immune characterization of the tumor microenvironment using CT-compatible needle biopsy material. Three independent biopsies were obtained intraoperatively from one metastatic epithelial ovarian cancer lesion of 7 consecutive patients undergoing surgical cytoreduction using a 16-gauge core biopsy needle. Core specimens were snap-frozen and subjected to immunohistochemistry (IHC) against human CD3, CD4, CD8, and FoxP3. A portion of the cores was used to isolate RNA for 1) real-time quantitative (q)PCR for CD3, CD4, CD8, FoxP3, IL-10 and TGF-beta, 2) multiplexed PCR-based T cell receptor (TCR) CDR3 Vß region spectratyping, and 3) gene expression profiling. Pearson's correlations were examined for immunohistochemistry and PCR gene expression, as well as for gene expression array data obtained from different tumor biopsies. Needle biopsy yielded sufficient tissue for all assays in all patients. IHC was highly reproducible and informative. Significant correlations were seen between the frequency of CD3+, CD8+ and FoxP3+ T cells by IHC with CD3ε, CD8A, and FoxP3 gene expression, respectively, by qPCR (r=0.61, 0.86, and 0.89; all p< 0.05). CDR3 spectratyping was feasible and highly reproducible in each tumor, and indicated a restricted repertoire for specific TCR Vß chains in tumor-infiltrating T cells. Microarray gene expression revealed strong correlation between different biopsies collected from the same tumor. Our results demonstrate a feasible and reproducible method of immune monitoring using CT-compatible needle biopsies from tumor tissue, thereby paving the way for sophisticated translational studies during tumor biological therapy.

Tissue-based immune monitoring II: multiple tumor sites reveal immunologic homogeneity in serous ovarian carcinoma.

The presence of tumor-infiltrating lymphocytes (TILs) in epithelial ovarian cancer indicates a host antitumor response and is associated with improved survival. We wished to determine the extent to which TIL density differs from site to site within a given patient. We initially studied multiple paired metastases from serous ovarian carcinoma obtained at the time of primary debulking. The expression of genes in specific immune-related pathways was profiled on a pilot set of five patients. We then used immunohistochemistry and quantitative PCR to estimate the density of CD3+, CD8+, and FoxP3+ TILs in these same tumors. To extend the findings to a larger cohort, we semiquantitatively measured intraepithelial and stromal TILs in a tissue microarray (TMA) containing both primary tumors and metastases from 50 patients. In the pilot group, genes related to antimicrobial signaling and TGF-beta signaling showed between-site heterogeneity, whereas cytokines and antigen presentation transcripts were more homogeneous in any given patient. IHC and qPCR for T cell markers were concordant. In the TMA cohort, 2-way ANOVA showed that TIL heterogeneity between sites was present in some but not all patients. The stroma of extra-ovarian metastases showed significantly greater TIL infiltration than ovarian sites. A simulation showed that at clinically meaningful levels of precision, up to 3% of patients will be misclassified for intraepithelial TILs by a single biopsy. In conclusion, between-site heterogeneity exists in some patients with metastatic serous ovarian cancer. The predictive value of biopsies should be considered in clinical trial design.

Intraepithelial T cells and tumor proliferation: impact on the benefit from surgical cytoreduction in advanced serous ovarian cancer.

BACKGROUND: The aim of the study was to determine whether tumor-infiltrating lymphocytes and/or tumor mitotic activity could identify subgroups of patients with advanced serous epithelial ovarian cancer who would maximally benefit from aggressive surgical cytoreduction. METHODS: Snap-frozen specimens from 134 consecutive patients with stage III or IV serous or poorly differentiated ovarian adenocarcinoma undergoing primary debulking surgery from a single US institution were characterized based on CD3(+), CD8(+), FoxP3(+) tumor-infiltrating lymphocytes, and Ki67 expression. Kaplan-Meier survival curves were estimated and compared using a log-rank statistic. A multivariate Cox model was used to estimate adjusted hazard ratios. Interactions were modeled using recursive partitioning based on maximal prognostic differentiation. RESULTS: Brisk intraepithelial CD8(+) cells (P = .035) and low Ki67 expression (P = .042) portended prolonged survival. The T-cell infiltration was more likely to occur in tumors with high proliferation index. Patients whose tumors exhibited low Ki67 expression and high intraepithelial CD8(+) frequency had a 5-year survival rate of 73.3%. Patients with aggressive tumor behavior, that is, whose tumors exhibited low frequency of intraepithelial CD8(+) T cells or high Ki67 expression were more likely to draw benefit from aggressive surgical cytoreduction. Survival was similar for patients with brisk CD8(+) T cells who had optimal or suboptimal debulking. Likewise, survival was similar for patients with low Ki67 expression who had optimal or suboptimal debulking. CONCLUSIONS: For the first time, these novel interactions of T cells, tumor proliferation index, and surgical treatment reveal that biological prognosticators may be useful for surgical decision making in ovarian cancer.

Intraepithelial T cells and prognosis in ovarian carcinoma: novel associations with stage, tumor type, and BRCA1 loss.

Intraepithelial tumor-infiltrating T cells have been correlated with improved outcomes in ovarian carcinoma, however, it is not known whether there is an association with disease stage, histological subtype, or BRCA mutation/expression. Two case series of ovarian carcinomas were included in the study; a retrospective series of 500 patients, and 40 prospectively collected cases fully characterized for BRCA1 mutation status and expression. Intraepithelial immune cells were assessed as present or absent by immunohistochemical staining of tissue microarrays. In the retrospective case series, the presence of intraepithelial CD8(+) T-cells correlated with improved disease-specific survival (P=0.027), whereas intraepithelial CD3(+) T cells did not (P=0.49). For serous ovarian carcinomas, the presence of intraepithelial CD3(+) and CD8(+) T-cells correlated with improved disease-specific survival (P=0.0016 and P

Integrative genomic analysis of protein kinase C (PKC) family identifies PKCiota as a biomarker and potential oncogene in ovarian carcinoma.

The protein kinase C (PKC) family plays a key regulatory role in a wide range of cellular functions as well as in various cancer-associated signal transduction pathways. Here, we investigated the genomic alteration and gene expression of most known PKC family members in human ovarian cancer. The DNA copy number of PKC family genes was screened by a high-resolution array-based comparative genomic hybridization in 89 human ovarian cancer specimens. Five PKC genes exhibited significant DNA copy number gains, including PKCiota (43.8%), PKCbeta1 (37.1%), PKCgamma (27.6%), PKCzeta (22.5%), and PKCtheta (21.3%). None of the PKC genes exhibited copy number loss. The mRNA expression level of PKC genes was analyzed by microarray retrieval approach. Two of the amplified PKC genes, PKCiota and PKCtheta, were significantly up-regulated in ovarian cancer compared with normal ovary. Increased PKCiota expression correlated with tumor stage or grade, and PKCiota overexpression was seen mostly in ovarian carcinoma but not in other solid tumors. The above results were further validated by real-time reverse transcription-PCR with 54 ovarian cancer specimens and 24 cell lines; overexpression of PKCiota protein was also confirmed by tissue array and Western blot. Interestingly, overexpressed PKCiota did not affect ovarian cancer cell proliferation or apoptosis in vitro. However, decreased PKCiota expression significantly reduced anchorage-independent growth of ovarian cancer cells, whereas overexpression of PKCiota contributed to murine ovarian surface epithelium transformation in cooperation with mutant Ras. We propose that PKCiota may serve as an oncogene and a biomarker of aggressive disease in human ovarian cancer.

Surgical resection of recurrent endometrial carcinoma.

OBJECTIVE: Chances of survival after the diagnosis of recurrent endometrial cancer are poor. Although total pelvic exenteration has been described as a treatment for a select subset of patients with recurrent endometrial cancer, the use of other surgical procedures in this setting has not been well described. The objective of this study was to review our experience with non-exenterative surgery for recurrent endometrial cancer. METHODS: We reviewed the medical records of all patients who underwent non-exenterative surgery for recurrent endometrial cancer between 1/91 and 1/03. Survival was determined from the time of surgery for recurrence to last follow-up. Survival was estimated using Kaplan-Meier methods. Differences in survival were analyzed using the log-rank test. The Fisher's exact test was used to compare optimal versus suboptimal cytoreduction against possible predictive factors. RESULTS: Twenty-seven patients were identified. Fifteen patients (56%) had disease limited to the retroperitoneum, 10 patients (37%) had intraperitoneal disease, and 2 patients (7%) had both intra- and retroperitoneal disease. Cytoreduction to 2 cm. There were no major perioperative complications or mortalities. The median hospital stay was 7 days (range, 1-18 days). Additional therapies included intraoperative radiation therapy in 9 patients (33%), radiation therapy in 12 patients (44%), and chemotherapy in 10 patients (37%). The median follow-up for the entire cohort was 24 months (range, 5-84 months). The median progression-free survival was 14 months (95% CI, 6-23), and the median disease-specific survival was 35 months (95% CI, 24-not reached). Size of residual disease was the only significant predictor for both progression-free and disease-specific survival. Patients with residual disease 2 cm residual (P = 0.01). CONCLUSIONS: Surgical resection for recurrent endometrial cancer may provide an opportunity for long-term survival in a select patient population. The only factor associated with improved long-term outcome was the size of residual disease remaining at the end of surgical resection.