Joan Brozick

Immunobiology of human mucin 1 in a preclinical ovarian tumor model

Epithelial ovarian cancer is an aggressive malignancy, with a low 5-year median survival. Continued improvement on the development of more effective therapies depends in part on the availability of adequate preclinical models for in vivo testing of treatment efficacy. Mucin 1 (MUC1) glycoprotein is a tumor-associated antigen overexpressed in ovarian cancer cells, making it a potential target for immune therapy. To create a preclinical mouse model for MUC1-positive ovarian tumors, we generated triple transgenic (Tg) mice that heterozygously express human MUC1+/− as a transgene, and carry the conditional K-rasG12D oncoallele (loxP-Stop-loxP-K-rasG12D/+) and the floxed Pten gene (Pten/loxP/loxP). Injection of Cre recombinase-encoding adenovirus (AdCre) in the ovarian bursa of triple (MUC1KrasPten) Tg mice triggers ovarian tumors that, in analogy to human ovarian cancer, express strongly elevated MUC1 levels. The tumors metastasize loco-regionally and are accompanied by high serum MUC1, closely mimicking the human disease. Compared with the KrasPten mice with tumors, the MUC1KrasPten mice show increased loco-regional metastasis and augmented accumulation of CD4+Foxp3+ immune-suppressive regulatory T cells. Vaccination of MUC1KrasPten mice with type 1 polarized dendritic cells (DC1) loaded with a MUC1 peptide (DC1–MUC1) can circumvent tumor-mediated immune suppression in the host, activate multiple immune effector genes and effectively prolong survival. Our studies report the first human MUC1-expressing, orthotopic ovarian tumor model, reveal novel MUC1 functions in ovarian cancer biology and demonstrate its suitability as a target for immune-based therapies.

PD-L1 biology in response to chemotherapy in vitro and in vivo in ovarian cancer

Meeting abstracts PD-L1 is an immune checkpoint molecule expressed by a variety of tumors, including ovarian, which binds to circulating PD-1 expressing effector T cells allowing for tumor escape from the immune system. PD-L1 blockade prevents PD-L1/PD-1 interaction and is currently explored as

MUC1 Positive, Kras and Pten Driven Mouse Gynecologic Tumors Replicate Human Tumors and Vary in Survival and Nuclear Grade Based on Anatomical Location

Activating mutations of Kras oncogene and deletions of Pten tumor suppressor gene play important roles in cancers of the female genital tract. We developed here new preclinical models for gynecologic cancers, using conditional (Cre-loxP) mice with floxed genetic alterations in Kras and Pten. The triple transgenic mice, briefly called MUC1KrasPten, express human MUC1 antigen as self and carry a silent oncogenic KrasG12D and Pten deletion mutation. Injection of Cre-encoding adenovirus (AdCre) in the ovarian bursa, oviduct or uterus activates the floxed mutations and initiates ovarian, oviductal, and endometrial cancer, respectively. Anatomical site-specific Cre-loxP recombination throughout the genital tract of MUC1KrasPten mice leads to MUC1 positive genital tract tumors, and the development of these tumors is influenced by the anatomical environment. Endometrioid histology was consistently displayed in all tumors of the murine genital tract (ovaries, oviducts, and uterus). Tumors showed increased expression of MUC1 glycoprotein and triggered de novo antibodies in tumor bearing hosts, mimicking the immunobiology seen in patients. In contrast to the ovarian and endometrial tumors, oviductal tumors showed higher nuclear grade. Survival for oviduct tumors was significantly lower than for endometrial tumors (p = 0.0015), yet similar to survival for ovarian cancer. Oviducts seem to favor the development of high grade tumors, providing preclinical evidence in support of the postulated role of fallopian tubes as the originating site for high grade human ovarian tumors.

Effects of Kras activation and Pten deletion alone or in combination on MUC1 biology and epithelial-to-mesenchymal transition in ovarian cancer.

Mucin1 (MUC1) is an epithelial glycoprotein overexpressed in ovarian cancer and actively involved in tumor cell migration and metastasis. Using novel in vitro and in vivo MUC1-expressing conditional (Cre-loxP) ovarian tumor models, we focus here on MUC1 biology and the roles of Kras activation and Pten deletion during cell transformation and epithelial-to-mesenchymal transition (EMT). We generated several novel murine ovarian cancer cell lines derived from the ovarian surface epithelia (OSE) of mice with conditional mutations in Kras, Pten or both. In addition, we also generated several tumor-derived new cell lines that reproduce the original tumor phenotype in vivo and mirror late stage metastatic disease. Our results demonstrate that de novo activation of oncogenic Kras does not trigger increased proliferation, cellular transformation or EMT, and prevents MUC1 upregulation. In contrast, Pten deletion accelerates cell proliferation, triggers cellular transformation in vitro and in vivo, and stimulates MUC1 expression. Ovarian tumor-derived cell lines MKP-Liver and MKP-Lung cells reproduce in vivo EMT and represent the first immune competent mouse model for distant hematogenous spread. Whole genome microarray expression analysis using tumor and OSE-derived cell lines reveal a 121 gene signature associated with EMT and metastasis. When applied to n=542 cases from The Cancer Genome Atlas (TCGA) ovarian cancer dataset, the gene signature identifies a patient subset with decreased survival (P=0.04). Using an extensive collection of novel murine cell lines we have identified distinct roles for Kras and Pten on MUC1 and EMT in vivo and in vitro. The data has implications for future design of combination therapies targeting Kras mutations, Pten deletions and MUC1 vaccines.

Abstract 4793: MUC1 immunogenicity in a triple transgenic mouse model for epithelial ovarian cancer

Background: Ovarian cancer continues to be associated with a very high mortality rate. Improvements on the development of new therapeutic approaches, including antigen-specific immune therapies, depend in part on the availability of adequate preclinical models for in vivo testing of treatment efficacy. MUC1 mucin is a tumor associated antigen and a potential target for cancer immunotherapy. Human and murine mucin 1 share little homology in the extracellular region and transgenic mouse models with de novo lesions expressing the human antigen are needed. Objective: To identify MUC1 antigen-specific immune regulation in mice with de novo malignant ovarian lesions. Methods: In order to generate a human MUC1 antigen expressing ovarian cancer mouse model, we crossed the MUC1 transgenic mice which express human MUC1 under the endogenous promoter with the previously described KrasG12D/+PtenloxP/loxP mice that develop orthotopic ovarian tumors. Results: The newly generated MUC1+/−KrasG12D/+ PtenloxP/loxP triple transgenic mice constitute a valuable Cre-loxP conditional in vivo model for MUC1 expressing ovarian tumors. Upon injection of Cre-encoding adenovirus under the ovarian bursa, the triple transgenic mice develop aggressive ovarian tumors with clear cell histology. The lesions express high levels of MUC1 and are estrogen receptor and cytokeratin 7-positive, markers classically used to diagnose malignant epithelial proliferation. The tumors are accompanied by ascites and numerous peritoneal implants, mimicking late stage disease clinically seen in women. We identified increased accumulation of Treg cells in the para-aortic (draining) lymph nodes and presence of Th17 cells in ascites, mimicking closely the immune phenotype of the human disease. In addition, using quantitative PCR T cell gene profiler arrays, we identified 5 significantly up- and 16 down-regulated genes in the spleen T cells of tumor-bearing mice. One of the upregulated genes was osteopontin (Spp1), recently validated as a marker in detecting human recurrent ovarian cancer. Among the down-regulated genes we identified, Cd4, Cd40ligand (Cd40lg), Interferon-g (Ifnγ), IL-23 receptor (IL-23ra) all suggestive of a shift in immune effectors, some of which may promote the chronic inflammatory background. Conclusions: Our results reveal important immune regulatory mechanisms in mice with de novo MUC1-expressing ovarian tumors and provide unique opportunities for future in vivo testing of MUC1 vaccines as ovarian cancer therapy and potentially immune prevention. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4793.

Multiplex profiling identifies distinct local and systemic alterations during intraperitoneal chemotherapy for ovarian cancer: An NRG Oncology/Gynecologic Oncology Group Study

OBJECTIVES Ovarian cancer leads to abdominal carcinomatosis and late stage (III/IV) diagnosis in 75% of patients. Three randomized phase III trials have demonstrated that intraperitoneal (IP) chemotherapy improves outcomes in epithelial ovarian cancer. While IP treatment is validated by clinical trials, there is a poor understanding of the mechanism(s) leading to the survival advantage other than the increased concentration of cytotoxic drugs within the tumor microenvironment. A better understanding of this process through analysis of dynamic biomarkers should promote novel approaches that may enhance tumor clearance. We propose this pilot study to confirm the feasibility of collecting serial peritoneal samples from implanted catheters in women receiving IP chemotherapy. We believe these specimens may be used for multiplex analysis to reveal unique biomarker fluctuations when compared to peripheral blood. METHODS From 13 women participating on GOG 252, 30 whole blood, 12 peritoneal fluid (PF), and 20 peritoneal wash (PW) with 30mL saline were obtained. Samples were requested prior to the first three chemotherapy cycles. Samples were assessed for volume, cell populations, protein, RNA, and miRNA content changes. RESULTS Median volume for PF was 1.6mL and 3.1mL for PW. PW is a dilution of PF capable of capturing measurable biomarkers. Peritoneal aspirates contain a unique profile of biomarkers distinct from blood. miRNA undergo earlier alteration with chemotherapy than genes. Flow cytometry does not adequately capture biomarker fluctuations. CONCLUSIONS As a proof of principle study, this trial provides evidence that sampling the peritoneal cavity can be adapted for biomarker analysis.

Abstract A59: PD-L1 biology in response to chemotherapy in vitro and in vivo in ovarian cancer.

Objective: PD-L1 is an immune checkpoint molecule expressed by a variety of tumors, including ovarian, which binds to circulating PD-1 expressing effector T cells allowing for tumor escape from the immune system. PD-L1 blockade prevents PD-L1/PD-1 interaction and is currently explored as therapy of solid tumors. Ovarian cancer patients receive combination cisplatin/taxane chemotherapy as standard of care. Chemo-induced effects on tumor PD-L1 expression have been only partially addressed. We studied here the effect of platinum/taxane exposure on PD-L1 expression in vitro and in vivo. Methods: Human (OVCA 420 and OVCA432) and mouse (2F8) ovarian cancer cell lines were exposed to increasing doses of cisplatin and paclitaxel for different time periods. PD-L1 expression was analyzed with flow cytometry and western blot. Through continuous exposure in vitro of mouse 2F8 ovarian cancer cells to increasing doses of cisplatin we have derived a new cisplatin-resistant line (2F8-Cis). In vivo, we have challenged n=37 mice IP with 0.8 million 2F8 cells . Tumor-bearing mice were treated with cisplatin, anti-PD-L1 antibody, both drugs, or isotype control every two weeks for three doses starting at day 14 post-inoculation. Tumor- and ascites-derived cancer cells were analyzed with flow cytometry. Result: Exposure of OVCA420 and OVCA432 to cytotoxic doses of cisplatin or paclitaxel trigger PD-L1 up-regulation. Similarly, 2F8-Cis cells show increased cell surface PD-L1 compared to parental 2F8 cells, providing the rationale for combination therapy with PD-L1 blockade. In vivo treatment of mice with aggressive 2F8 tumors respond well to cisplatin and anti-PD-L1 individually with increased survival (median 45 days versus 24 days for isotype control, p=0011). At necropsy, anti-PD-L1 therapy significantly reduced tumor burden (1.48 g versus 0.25 g, p=0.0294). Tumor cells cultured from cisplatin-only treated mice expressed higher levels of PD-L1, in line with our in vitro results. A higher percentage of PD-1 expressing cells were found amongst the tumor cells in these cultures versus cisplatin/anti-PD-L1 treated mice. Although high dose anti-PD-L1 immediately following cisplatin administration can control tumor burden (0.48 g), it does not significantly prolong survival (median 29 days). We are currently testing an alternative therapeutic schema exploring a lower anti-PD-L1 dose and a different timing post-chemo. Conclusion: Tumor cells upregulate PD-L1 in response to chemotherapy exposure and combination PD-L1 blockade in conjunction with chemotherapy effectively controls tumor burden. Optimization of timing and dosage for this combination therapy will likely increase its therapeutic benefit. Citation Format: Shannon Grabosch, Feitianzhi Zeng, Lixin Zhang, Mary Strange, Joan Brozick, Robert P. Edwards, Anda Vlad. PD-L1 biology in response to chemotherapy in vitro and in vivo in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A59.

Abstract POSTER-THER-1434: Intraperitoneal anti-PD-L1 increases survival in a novel ovarian cancer model and its in vivo efficacy is influenced by baseline anti-tumor immunity of the host

Purpose: Immunotherapy with monoclonal antibodies that block inhibitory immune checkpoint molecules and enhance antitumor responses has recently showed clinical promise in advanced solid tumors. However, most of the preliminary evidence on therapeutic efficacy comes from melanoma, prostate and lung cancer. Further studies on other tumor types (including ovarian cancer), and on additional, potentially more effective regimens that take into consideration the administration route and baseline immune status of the host, are currently needed. Approach: We recently generated a triple transgenic MUC1+/-KrasG12D/+PtenloxP/loxP (MKP) mouse model that carries a conditional, oncogenic Kras mutation, Pten deletion and express human MUC1 as self. Upon AdCre injection under the ovarian bursa, the MKP mice progress to endometrioid ovarian tumors that overexpress MUC1 tumor-associated antigen. Using an orthotopic MKP ovarian tumor, we generated a new ovarian cancer cell line (2F8). MUC1.Tg mice injected IP with 8x10E5 of completely syngeneic 2F8 cells develop IP tumors. Mice wild-type for MUC1 were similarly challenged with 2F8 cells, thus creating an isogenic system, in which the hosts are syngeneic to all other tumor antigens, except for human MUC1. Results: At baseline, the cells express low PD-L1 but expression increases after in vivo growth. Anti-PDL1 was administered IP every 2 weeks (200 mg/dose) for a total of 3 doses. Treatment was started 21 days post-tumor challenge, a time point which corresponds to a late tumor stage. The anti-PD-L1 treated MUC1.Tg mice showed substantial T cell infiltration within the tumor and significantly increased survival (p= 0.001) compared to isotype control- treated mice. In contrast, wild type mice with isogenic 2F8 tumors developed spontaneous, high titer anti-MUC1 antibodies and did not respond to anti-PD-L1 therapy. However, earlier treatment (at day 11, instead of day 21) post tumor challenge, higher frequency of IP injections (weekly) and higher overall dose (seven doses, 200 μg/dose) increased treatment efficacy in these mice and led to increased survival. Anti-PDL1 therapy did not change the amplitude of MUC1 specific antibodies in responding mice. In addition to the above quantitative and qualitative changes in systemic and intratumoral T cells, broad range profiling using microarrays are ongoing and will reveal the hallmarks of response in anti-PD-L1 treated mice. Conclusions: These results demonstrate that targeting PD-L1 is a viable therapeutic strategy in ovarian cancer and can increases survival despite (1) treatment initiation at late stage, (2) low dose/low frequency of administration and (3) low PD-L1 expression on tumors. By employing an isogenic animal model that favors anti-tumor humoral immunity, we show here that tumor-bearing hosts with antibody-driven immune imbalance at time of treatment initiation are non-responsive and may require a more dose intensive regimen. This is in line with our previous findings showing inverse correlation between survival and pre-existing MUC-1 specific antibody levels in ovarian cancer patients receiving IP Interleukin -2 (IL-2). Our studies have high translational potential and support IP administration of PD-L1 blockers in ovarian cancer patients, while taking into consideration pre-existent, anti-tumor antibodies. Citation Format: Jyothi Mony, Lixin Zhang, Tejas Tirodkar, Esther Elishaev, Joan Brozick, Robert P. Edwards, Anda M. Vlad. Intraperitoneal anti-PD-L1 increases survival in a novel ovarian cancer model and its in vivo efficacy is influenced by baseline anti-tumor immunity of the host [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1434.

Abstract 260: In vivo efficacy of intraperitoneal anti-PD-L1 therapy in ovarian cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Objectives: Programmed cell death ligand 1 (PD-L1) is an immune checkpoint cell surface molecule expressed on many types of cancers including ovarian cancer. Its interaction with the programmed death (PD-1) receptor on T cells essentially halts the immune response and allows tumor escape. Monoclonal antibodies to either PD-L1 or PD-1 block this interaction and show efficacy in a variety of cancers, though to a lesser degree in ovarian cancer. We have evaluated the efficacy of intraperitoneal anti-PD-L1 therapy in two ovarian cancer mouse models. Methods: Murine 2F8 tumor cells derived from an orthotopic tumor isolated from a triple transgenic MUC1+/−KrasG12D/+PtenloxP/loxP (MKP) mouse as we recently described. MUC1 transgenic (MUC1.Tg) mice received an intraperitoneal inoculation of 8×105 syngeneic 2F8 cells. Untreated mice develop extensive tumor burden and expire around 29 days. Mice were treated with 200 μg anti-PD-L1 antibody (or rat IgG as control) every two weeks for three doses starting at 21 days post-inoculation, thus corresponding with late tumor stage. C57BL/6 mice were inoculated via intraperitoneal (IP) injection with 1×106 IG10 (spontaneously transformed mouse ovarian surface epithelial) tumor cells. Treatment with anti-PD-L1 (or rat IgG as control) started 22 days following inoculation when the mice began showing ascites. Immunohistochemistry for perforin was performed on tumor tissue sections. Immune gene profiling of spleen cells collected at necropsy was performed via Nanostring, using 561 mouse immunology-related genes. Results: The 2F8 tumors are aggressive with little T cell infiltration. The anti-PD-L1 treated MUC1.Tg mice showed substantial infiltration of perforin positive cells within the tumor and significantly increased survival (p = 0.001) compared to isotype control- treated mice. Splenocyte profiling of 561 immune genes via Nanostring revealed a treatment-induced immune gene signature that points to T cell functions and cytotoxic anti-tumor immune responses. A similar gene signature favoring cytotoxic anti-tumor activity was also revealed in IG10 tumor bearing C57Bl/6 mice treated with anti-PD-L1. Conclusion: These preclinical results from two different ovarian cancer tumor models demonstrate that targeting PD-L1 is a viable therapeutic strategy in ovarian cancer and increases survival despite (1) non-immunogenic tumors, (2) late treatment initiation, (3) low dose and administration frequency. The tumors respond well to anti-PD-L1 blockade, due to increased systemic T cell responses and intratumoral T cell accumulation, without changing tumor-specific antibody levels. Our studies have high translational potential and support IP administration of PD-L1 blockers in ovarian cancer patients. Citation Format: Shannon Grabosch, Jyothi T. Mony, Lixin Zhang, Tianzhou Ma, Tejas Tirodkar, Joan Brozick, George Tseng, Esther Elishaev, Robert P. Edwards, Xin Huang, Anda M. Vlad. In vivo efficacy of intraperitoneal anti-PD-L1 therapy in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 260. doi:10.1158/1538-7445.AM2015-260

Abstract 4081: Lung metastasis of ovarian cancer in a transplantable MUC1.Tg mouse model is accompanied by upregulation of MUC1 expression and epithelial to mesenchymal transition

Introduction: Patients with ovarian cancer are usually diagnosed late, when tumors have already spread to the surrounding tissues. Hematogenous dissemination of ovarian cancer cells can also occur and lung metastasis can be found in 12% of ovarian cancer patients. Human mucin1 (MUC1) is a large transmembrane glycoprotein with roles in cell adhesion and migration. More than 80% of ovarian cancer overexpress MUC1, regardless of histology. MUC1 is a widely used vaccine target for numerous adenocarcinomas, including ovarian tumors. Approach and results: We have recently described the triple transgenic (Tg) mice with conditional (cre-loxP) mutations in Kras and Pten pathways that progress to human MUC1-expressing endometrioid ovarian tumors. Using orthotopic tumors from these mice we generated here several new murine cell lines. The MKP-L cell line was derived from an ovarian tumor implant attached to the liver surface. Intraperitoneal (IP) injection of MKP-L cells into syngeneic mice triggers IP tumors and multiple lung metastatic masses. Cells isolated from lung metastases (MKP-Lung cell line) triggered peritoneal tumors as well as lung metastases, suggesting that they retain the original homing capacity. Compared to the parental, endometrioid-looking and low-MUC1 expressing MKP-L cells, the metastatic MKP-Lung cells showed morphological changes consistent to epithelial-to-mesenchymal transition (EMT) and are homogeneously high in MUC1. Mouse WG6 microarray analysis of MKP-L and MKP-Lung cells revealed expression changes of several EMT-associated transcription factors as well as their downstream proteins. Western blot confirmed upregulation of ZEB1, vimentin and downregulation of cytokeratins. To dissect the mechanistic roles of Kras and Pten pathways in EMT and MUC1 regulation, we also generated a series of ovarian surface epithelial (OSE)-derived cell lines with conditional (Cre-loxP) oncogenic mutations in Kras (MKOSE), deletion in Pten (MPOSE) or both (MKPOSE). Using these cell lines we recapitulated in vitro the EMT phenotype and MUC1 upregulation acquired by MKP-Lung cells in vivo. Furthermore, small molecule inhibitors of oncogenic Kras (AZD6244) and Pi3K/Pten pathway (BEZ235) demonstrated that MUC1 expression is significantly upregulated in response to Pten deletion and that Pi3K inhibitor (BEZ235) downregulated MUC1. In contrast, Kras activation slightly lowered MUC1 expression while cell exposure to AZD6244 upregulated MUC1. Conclusions: We report here the first transgenic mouse metastatic ovarian cancer model that expresses human MUC1.Usig this model we identified a gene signature consistent with lung metastasis and EMT and identified mechanisms of MUC1 upregulation during these processes. These findings have important consequences for MUC1 immunobiology in metastatic ovarian tumors with mutations in Kras, Pten/Pi3K or both. Citation Format: Lixin Zhang, Tianzhou Ma, Joan Brozick, Kathlene Babalola, Raluca Budiu, George Tseng, Anda Vlad. Lung metastasis of ovarian cancer in a transplantable MUC1.Tg mouse model is accompanied by upregulation of MUC1 expression and epithelial to mesenchymal transition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4081. doi:10.1158/1538-7445.AM2015-4081