Dual Fluorescence Isogenic Synthetic Lethal Kinase Screen and High-Content Secondary Screening for MUC16/CA125-Selective Agents.

Significant strides have been made in the development of precision therapeutics for cancer. Aberrantly expressed glycoproteins represent a potential avenue for therapeutic development. The MUC16/CA125 glycoprotein serves as a biomarker of disease and a driver of malignant transformation in epithelial ovarian cancer. Previously, we demonstrated a proof-of-principle approach to selectively targeting MUC16+ cells. In this report, we performed a synthetic lethal kinase screen using a human kinome RNAi library and identified key pathways preferentially targetable in MUC16+ cells using isogenic dual-fluorescence ovarian cancer cell lines. Using a separate approach, we performed high-content small-molecule screening of six different libraries of 356,982 compounds for MUC16/CA125-selective agents and identified lead candidates that showed preferential cytotoxicity in MUC16+ cells. Compounds with differential activity were selected and tested in various other ovarian cell lines or isogenic pairs to identify lead compounds for structure-activity relationship (SAR) selection. Lead siRNA and small-molecule inhibitor candidates preferentially inhibited invasion of MUC16+ cells in vitro and in vivo, and we show that this is due to decreased activation of MAPK, and non-receptor tyrosine kinases. Taken together, we present a comprehensive screening approach to the development of a novel class of MUC16-selective targeted therapeutics and identify candidates suitable for further clinical development.

Expression of the Carboxy-Terminal Portion of MUC16/CA125 Induces Transformation and Tumor Invasion.

The CA125 antigen is found in the serum of many patients with serous ovarian cancer and has been widely used as a disease marker. CA125 has been shown to be an independent factor for clinical outcome in this disease. In The Cancer Genome Atlas ovarian cancer project, MUC16 expression levels are frequently increased, and the highest levels of MUC16 expression are linked to a significantly worse survival. To examine the biologic effect of the proximal portion of MUC16/CA125, NIH/3T3 (3T3) fibroblast cell lines were stably transfected with the carboxy elements of MUC16. As few as 114 amino acids from the carboxy-terminal portion of MUC16 were sufficient to increase soft agar growth, promote matrigel invasion, and increase the rate of tumor growth in athymic nude mice. Transformation with carboxy elements of MUC16 was associated with activation of the AKT and ERK pathways. MUC16 transformation was associated with up-regulation of a number of metastases and invasion gene transcripts, including IL-1ß, MMP2, and MMP9. All observed oncogenic changes were exclusively dependent on the extracellular "ectodomain" of MUC16. The biologic impact of MUC16 was also explored through the creation of a transgenic mouse model expressing 354 amino acids of the carboxy-terminal portion of MUC16 (MUC16c354). Under a CMV, early enhancer plus chicken ß actin promoter (CAG) MUC16c354 was well expressed in many organs, including the brain, colon, heart, kidney, liver, lung, ovary, and spleen. MUC16c354 transgenic animals appear to be viable, fertile, and have a normal lifespan. However, when crossed with p53-deficient mice, the MUC16c354:p53+/- progeny displayed a higher frequency of spontaneous tumor development compared to p53+/- mice alone. We conclude that the carboxy-terminal portion of the MUC16/CA125 protein is oncogenic in NIH/3T3 cells, increases invasive tumor properties, activates the AKT and ERK pathways, and contributes to the biologic properties of ovarian cancer.

Dual-fluorescence isogenic high-content screening for MUC16/CA125 selective agents.

Most of the currently used cancer chemotherapies are based on compounds that inhibit general cellular mechanisms, such as DNA replication or tubulin function, and lack specificity in relation to features of the cancer cell. Recent advances in genomic studies have increased our knowledge of tumor cell biology, and a panoply of new targets have been postulated. This has provided an opportunity to develop and validate drugs that specifically target cancer cells through their unique genetic characteristics. Identification of MUC16/CA125 both as a marker and a driver of transformation led us to design a target-based high-content screen to identify and classify compounds that exhibit differential effect on MUC16-expressing cells. We developed a coculture assay in 384-well plate containing isogenic ovarian cancer cells that are positive or negative for the MUC16 protein. High-throughput screening of our small molecule pilot library led to the identification of compounds preferentially cytotoxic to MUC16(+) or MUC16(-) cells, using a Preferential Score analysis. We compared screening results in both A2780 and SK-OV-3 ovarian cancer cells in single and coculture settings. We also identified compounds that were cytotoxic for both types of ovarian cancer cells regardless of the MUC16 status. Compounds that were preferentially targeting MUC16 cells were subsequently confirmed by caspase-induction assays. The isogenic, dual-color fluorescence strategy is an innovative approach that can effectively identify novel drug candidates, selectively targeting cancer cells that have unique molecular properties.

Successful eradication of established peritoneal ovarian tumors in SCID-Beige mice following adoptive transfer of T cells genetically targeted to the MUC16 antigen.

PURPOSE: Most patients diagnosed with ovarian cancer will ultimately die from their disease. For this reason, novel approaches to the treatment of this malignancy are needed. Adoptive transfer of a patient's own T cells, genetically modified ex vivo through the introduction of a gene encoding a chimeric antigen receptor (CAR) targeted to a tumor-associated antigen, is a novel approach to the treatment of ovarian cancer. EXPERIMENTAL DESIGN: We have generated several CARs targeted to the retained extracellular domain of MUC16, termed MUC-CD, an antigen expressed on most ovarian carcinomas. We investigate the in vitro biology of human T cells retrovirally transduced to express these CARs by coculture assays on artificial antigen-presenting cells as well as by cytotoxicity and cytokine release assays using the human MUC-CD(+) ovarian tumor cell lines and primary patient tumor cells. Further, we assess the in vivo antitumor efficacy of MUC-CD-targeted T cells in SCID-Beige mice bearing peritoneal human MUC-CD(+) tumor cell lines. RESULTS: CAR-modified, MUC-CD-targeted T cells exhibited efficient MUC-CD-specific cytolytic activity against both human ovarian cell and primary ovarian carcinoma cells in vitro. Furthermore, expanded MUC-CD-targeted T cells infused through either i.p. injection or i.v. infusion into SCID-Beige mice bearing orthotopic human MUC-CD(+) ovarian carcinoma tumors either delayed progression or fully eradicated disease. CONCLUSION: These promising preclinical studies justify further investigation of MUC-CD-targeted T cells as a potential therapeutic approach for patients with high-risk MUC16(+) ovarian carcinomas.