Ritonavir Repositioning from HIV-AIDS to Treatment of Ovarian Cancer
HIV-AIDS patients have very high prevalence of cancers. Protease Inhibitors (PI) are a class of drugs widely used to treat HIV-AIDS. In last two decades, numerous small and large observational studies have shown that patients using PIs have a drastic reduction in HIV-AIDS related cancers. These observations raised a question-do PIs have anticancer properties?
We took a widely used and well tolerated PI, Ritonavir (already FDA approved for HIV-AIDS and used worldwide) and tested it against several ovarian cancer cell lines and published that ritonavir exhibits potent anticancer activity against ovarian cancer in vitro. These anticancer effects are brought about by cell cycle arrest, activating apoptosis and inhibiting invasion and migration of ovarian cancer cells. Meanwhile other groups of investigators have reported similar data in other cancers (breast, lung, prostate and sarcoma) and have started clinical trials utilizing PIs (references included in grant application text).
Based on our robust published in-vitro data, we propose to conduct in-vivo studies to treat ovarian cancer in mice with ritonavir in the present MOCA grant application. If funded, completion of the in-vivo mice studies will pave the way to start human trials of ritonavir in ovarian cancer.
Lymphodepleting Chemotherapy and T-Cell Suppression Followed by Allogeneic Natural Killer Cells and Interleukin-2 in Patients with Recurrent Ovarian Cancer
Our preliminary clinical Phase II study at the University of Minnesota using allogeneic (derived from healthy donors) NK cell infusions to treat ovarian cancer has shown exciting potential clinical efficacy with the ability to detect donor-derived NK cells up to 35 days following infusion. It is from this observation that stems our current clinical trial and ongoing basic science research which addresses how to optimize NK cell expansion, which we hypothesize, is required for therapeutic efficacy. To understand the mechanism of NK mediated tumor cell killing and NK cell expansion we will create Green Fluorescent Protein (GFP) expressing MA148 and OVCAR3 mouse models to test clinically relevant endpoints.
Two aims were chosen, each of which will provide better insight into how to proceed with our clinical trial
Aim 1.To determine whether intravenous (IV) or intraperitoneal (IP) delivery of activated NK cells is the optimal route of delivery to promote ovarian cancer cell kill.
Aim 2. To determine if extended dosing of bortezomib in combination with IL-2 activated allogeneic NK cells can potentiate cell kill in a mouse model of ovarian cancer by:
a.) sensitizing ovarian cancer to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and Fas mediated NK cell killing
b.) decreasing tumor MHC-1 expression
c.) decreasing tumor volume
Sub Aim 2a. To determine the above in a mouse model of platinum sensitive ovarian cancer
Sub Aim 2b. To determine the above in a mouse model of platinum resistant ovarian cancer
Specifically, in both aims we will be testing tumor cell kill and tumor infiltration by NK cells with and without bortezomib treatment. The outcomes of this research will have significant implication because we expect the knowledge will suggest development of novel immunotherapy treatment strategies and therapeutic interventions needed for women with ovarian cancer. The research will be of additional significance, because what is learned will contribute to the broader understanding of how immune based therapies can be utilized as an approach to treatment in women with this devastating disease. My preliminary clinical data and in vitro and mouse model findings demonstrate the importance of integrating these entities to understand novel clinical strategies and to improve efficacy.
Targeting the Tumor Microenvironment in Ovarian Cancer
Following optimal cytoreductive surgery, ovarian cancer patients are commonly treated with carboplatin/cisplatin and paclitaxel , with only ~10 15% of >stage 3 patients remaining disease-free at five years. The rest ultimately develop chemotherapy-resistant disease, relapse, and eventually succumb – often within 1-2 years of relapse. Hence, there is considerable need to develop improved up-front and salvage treatment strategies for ovarian cancer. Although various targeted therapeutics have been explored that include monoclonal antibodies directed towards vascular endothelial growth factor , VEGF (bevacizumab) and folate receptor, small molecule tyrosine kinase inhibitors, PARP and mTOR inhibitors, none of them specifically target the tumor microenvironment and inhibit several pathways mediated by various growth factors simultaneously. Extracellular matrix (ECM) heparan sulfate (HS) glycosaminoglycans are complex polysaccharides that are ubiquitous in nature and play important roles in the regulation of several aspects of cancer biology, including angiogenesis, tumor progression and metastasis. We are proposing to test the efficacy of a novel compound, a HS mimetic, PG545 that has never been tested in ovarian cancer that targets heparanase and heparin binding growth factors overexpressed in the tumor microenvironment. PG545 has been shown to inhibit solid tumor progression of breast, prostate and liver cancer xenografts. This selective inhibition of the heparanase enzyme influences cellular extravasation through the basement membranes and remodels tissues during tumor growth. More importantly, heparan sulfate proteoglycans, the substrate for heparanase act as coreceptors for pro-angiogenic and pro-metastatic growth factors such as FGF2 and VEGF.
Therefore targeting HSPG- mediated signaling using heparan sulfate (HS) mimetic is a very innovative approach.
Hypothesis and Objectives: We hypothesize that HS mimetics that target heparanase and multiple growth factors in the tumor microenvironment will function as an inhibitor of multiple signaling pathways simultaneously resulting in inhibition of tumor growth, angiogenesis and metastatic spread. Based on our in vitro preliminary data presented in figure 2 that indicate increased cisplatin -induced cytotoxicity when combined with PG545, while PG545 alone had minimal anti-proliferative effects indicating synergy, we further hypothesize thatPG545 might represent a candidate ovarian cancer therapeutic either as a single agent or in combination with cytotoxic chemotherapy. The objective of the proposed study is to test the efficacy of the HS mimetic PG545 in inhibiting angiogenesis and peritoneal metastasis in vivo both alone and in combination with cisplatin and/or paclitaxel using two different ovarian cancer models.
Specific Aims and Study Design: In order to test our hypotheses, we propose three specific aims.
In Aim 1, we will assess the in vitro effects of PG545 in potentiating cisplatin and or paclitaxel induced cytotoxicity in several ovarian cell lines using colony formation assays (CFA), and cell cycle analysis by FACS.
In Aim 2, we will examine of the in vivo efficacy of PG545 in potentiating cisplatin and or paclitaxel induced cytotoxicity of ovarian xenografts in athymic immunodeficient mice and examine the effect of PG545 on angiogenesis by Matrigel Plug Assay. While experiments in immune-compromised mice bearing human cancers have merit, such models ignore effects of PG545 that may require either i) an intact immune systems or ii) syngeneic tumor-stroma interactions.
Therefore in Aim 3, we will assess the therapeutic efficacy of PG545 in a syngeneic ID8 murine ovarian cancer model.
Innovation: Due to the highly refractory nature of recurrent ovarian tumors, standard treatment options lead to short term survival rates. Thus, the pursuit of novel therapeutics is of utmost importance if treatment advancements are to be made to improve overall survival and disease free survival of patients with ovarian cancer. With this award, we will establish whether one of the novel agents, HS mimetics, that is known to inhibit heparanase and growth factor mediated signaling are also involved in modulating chemoresponse and or metastatic spread of cancer. Despite advances of testing multiple targeting agents in ovarian cancer, there are limitations with each one of the targeting agent due to the lack of understanding of redundant pathways that exist in cell signaling and the mechanism of resistance. There are no preclinical studies of PG545 in ovarian cancer. Testing the effect of PG545 in cell lines both in vitro and in vivo is innovative and will provide important preclinical data on the potential use of PG545 in future clinical trials.
Impact: Our preliminary data suggests that PG545 targets multiple pathways simultaneously and thus may have a better advantage over other targeting agents since the target also includes heparanase, an extracellular enzyme that is over expressed in ovarian cancer and is associated with poor prognosis. Based on the report that HS mimetics sensitize cells to chemotherapy induced cytotoxicity in patients with prostate cancer lends additional support to testing this agent in preclinical models of ovarian cancer. If HS mimetics are successful in inhibiting carcinomatosis and sensitize cells to drug induced cytotoxicity, the impact of this research to patients with ovarian cancer will be substantial.
Novel Bispecific Ligand-Directed Toxins for the Treatment of Ovarian Cancer
Ovarian cancer is the most lethal malignancy of the female reproductive tract. While most women will achieve complete remission after treatment (surgery and chemotherapy), the majority will relapse within two years, highlighting the need for novel therapies. Cancer stem cells (CSC) have been identified in a number of different types of solid tumors (including ovarian cancer) as a small population of cells that are relatively quiescent, can self-renew, form “spheroids” or multicellular aggregates when grown on a nonadhesive surface, and maintain the tumor by differentiating into cells that make up the tumor bulk. Since conventional chemotherapies target only the rapidly dividing “bulk tumor” cells, CSC may be the primary source of tumor recurrence.
The hypothesis driving this study is that ovarian cancer tumors can be eradicated by use of liganddirected toxins that specifically target each of the two populations of cancer cells within the tumor simultaneously; in effect eliminating both the CSC and the bulk tumor cells and thereby preventing recurrence of the disease. To this end, several ovarian cancer cell lines will be evaluated for the presence of CD133, a CSC marker, on their cell surface. A novel specific CD133-directed toxin will be tested for its efficacy in killing ovarian cancer cells in vitro and in vivo, alone and in combination with other monospecific and bispecific ligand-directed toxins that target the more differentiated tumor cells.
Aim #1 is to characterize the expression levels of CD133 on the surface of several ovarian cancer cell lines and normal surface epithelial cell lines by flow cytometry. CD133+ and CD133- cells will be segregated and collected by fluorescent activated cell sorting.
Aim #2 is to correlate the expression of CD133 with the CSC phenotype by perform in vitro functional assays on the CD133+ and CD133- cell populations. In addition, CD133+ and CD133- cell populations will be tested for their tumorigenicity in nude mice.
Aim #3 is to test the sensitivity of CD133+ and CD133- cells to our novel CD133-directed toxin (dCD133KDEL), as well as other monospecific and bispecific ligand-directed toxins vs. EGFR, IL-4R, IL-13R, and uPAR that we developed and found to be successful at eradicating other solid tumors.
Aim #4 is to test the CD133+ and CD133- cells for their sensitivity to the toxins from Aim #3 in an in vivo mouse model.
The results of this study will shift the paradigm for ovarian cancer treatment from one that treats patients solely with chemotherapy that targets the rapidly proliferating cells to one that targets both the rapidly proliferating “bulk tumor” cells and the quiescent CSC. This will have a significant impact on disease recurrence and thereby increase the rate of survival for ovarian cancer patients.