Current treatments for cancer are manifestly unsatisfactory in the extreme. Development of improved therapies will be accelerated by the identification of compounds that specifically target novel cellular proteins that contribute to tumr cell survival. The endoplasmic reticulum (ER) resident prolyl isomerase Cyclophilin B (CypB) is highly up-regulated in multiple types of cancers. We recently discovered that it plays an essential role in supporting the proliferation and survival of glioblastoma multiforme (GBM), an important type of brain tumor, as well as several other types of malignancies. Other groups have independently confirmed our observations on the importance of CypB for cancer cell survival. Biochemical analysis revealed that knockdown of CypB enhanced ER stress, and led to elevated reactive oxygen species (ROS) and decreased levels of Chk1 and mutant p53, as well as decreased activation of Stat3, all of which have been implicated in regulation of cancer cell proliferation, suggesting a mechanism underlying the requirement for CypB in these tumor cells. Inhibiting CypB to kill cancer cells would not be useful if normal tissues were equally dependent upon it for viability. Importantly, while suppression of CypB is deleterious for tumor cell surviva, we found that mice completely lacking CypB have normal development and fertility with a minor phenotype involving collagen processing, demonstrating that loss of CypB is not required for viability of the organism. We propose, therefore, that CypB is a novel target in cancer that can be inhibited therapeutically. There exist some small molecule inhibitors of cyclophilins, and several have been tested in clinical trials (thus far for the treatment of hepatitis). Using these inhibitors in vitro, we find that several of these compounds efficiently killed GBM cell lines and tumor cells freshly isolated from mice. However, there are a multitude of highly related cyclophilins in cells, and the known inhibitors typically bind and block the action of most of them Global inhibition of cyclophilins likely engenders multiple off-target and potentially conflicting effects on tumor cells, particularly in the case of the D isoform (CypD), which is known to participate in necrotic cell death. In addition, the abundance of cellular cyclophilins requires tht very high amounts of inhibitor be used in order to interfere with the activity of the single target CypB. These finding lead to our central hypothesis that potent compounds with the ability to selectively inhibit CypB will have improved efficacy and reduced toxicity in the clinical treatment of GBMs and other cancers. We propose here to leverage our novel findings and expertise in the cyclophilin system, in synergy with the high-throughput screening and medicinal chemistry expertise at the Conrad Prebys Center for Chemical Genomics (CPCCG) at the Sanford-Burnham Medical Research Institute (SBMRI), to identify and refine compounds that specifically target CypB and that do not bind to CypD. The proposed critical path testing funnel is already in place, so we anticipate that we can rapidly obtain and evaluate selective in vitro hits for their activity against GBM cell lines and freshly isolated tumor cells, and ultimately their suitability s starting points for hit-to-lead studies and for future in vivo evaluation in animal models and eventually patients. The goals of this proposal are to perform a high throughput primary screen of a ~370,000-compound library based on binding to recombinant CypB, through the use of a protein thermal shift assay. Secondary screening will use GBM cell cytotoxicity and CypD binding assays. Hits will be validated and then tested for effects on intracellular signaling pathways. We anticipate that these novel chemical probes that selectively suppress CypB intracellular functions will lead to proof-of-concept anti-cancer compounds. These compounds will ultimately lead to establishing a novel approach for therapeutic treatment of GBM. In addition, they will have great utility as molecular probes to enhance studies defining the critical functions of CypB.
Current treatments for cancer are inadequate, as many tumors recur after some time period, or do not respond in the first place. Even when a cure can be effected, there are severe toxicities from exposure to radiation or chemotherapy drugs that cause long term disability or death. Preliminary studies from our group, and verified by others, indicate that the Cyclophilin B protein (CypB) would be an effective therapeutic target for many tumor types, particularly including Glioblastoma multiforme. Cyclosporine is a drug that can target CypB, but it also binds to most of the other forms of cyclophilin, including Cyclophilin D, which tends to oppose the action of CypB. These conflicting effects of cyclosporine and other non-specific cyclophilin inhibitors are thought to increase their toxicity and to blunt their anti-cancer effects. The proposed work will use high throughput screening to discover new compounds that target CypB specifically. These new compounds will be tested for efficacy in killing GBM cells in culture. Successful accomplishment of this work should lead rapidly to the development of new drugs for treatment of human malignancies.
| Choi, Jae Won; Schroeder, Mark A; Sarkaria, Jann N et al. (2014) Cyclophilin B supports Myc and mutant p53-dependent survival of glioblastoma multiforme cells. Cancer Res 74:484-96 |
