Author : Adrianne Cecelia Wallace-Povirk
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (137 download)
Book Synopsis Targeting Metabolic Vulnerabilities in Epithelial Ovarian Cancer Via Folate Transporters by : Adrianne Cecelia Wallace-Povirk
Download or read book Targeting Metabolic Vulnerabilities in Epithelial Ovarian Cancer Via Folate Transporters written by Adrianne Cecelia Wallace-Povirk and published by . This book was released on 2021 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Novel therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. In addition, therapies which target a unique vulnerability in the tumor microenvironment (TME) of EOC have largely been unrealized. It is known the major infiltrating immune population in EOC represents tumor-activated macrophages, yet it remains an enigma how the tumor has 0́−educated0́+ the niche to allow for tumor tolerance. To elucidate mechanisms for targeting EOC, we must discover metabolic vulnerabilities and establish mechanisms of selective delivery of therapeutic agents to tumors as well as immune cells, and investigate how the tumor has altered the metabolism of infiltrating macrophages, thus shaping the permissive environment for EOC. One strategy to achieve tumor cell and microenvironment selective drug delivery to EOC is by uptake via folate receptor (FR) and PCFT proteins, with internal targeting at mitochondrial and cytosolic one-carbon (C1) metabolism, pathways elevated in EOC patient tumors. FRÎł is highly expressed on up to 90% of EOC tumor cells, whereas FRÎø is expressed on activated 0́−M20́+ macrophages. PCFT is constitutively expressed in EOC. Thus, great potential exists to target both tumor and the TME with agents delivered via selective transport by FRs and/or PCFT. In this dissertation work, we discovered 6-substituted thieno[2,3-d]pyrimidine compounds, typified by AGF102, AGF103 and AGF131 with 3-4 bridge carbons and side-chain thiophene or furan rings for dual targeting C1 metabolism in FR expressing cancers. From patterns of growth inhibition toward Chinese hamster ovary cells expressing FRÎł or FRÎø, the proton-coupled folate transporter or reduced folate carrier, specificity for uptake by FRs was confirmed. Anti-proliferative activities were demonstrated toward FRÎł-expressing KB tumor cells and NCI-IGROV1 ovarian cancer cells. Inhibition of de novo purine biosynthesis at both 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and glycinamide ribonucleotide formyltransferase (GARFTase) was confirmed by metabolite rescue, metabolomics and enzyme assays. X-ray crystallographic structures were obtained with compounds AGF102, AGF103 and AGF131 and human GARFTase. Our studies identify first-in-class C1 inhibitors with selective uptake by FRs and dual inhibition of enzyme targets in de novo purine biosynthesis, resulting in anti-tumor activity. This series affords an exciting new platform for selective multi-targeted anti-tumor agents. In Aim 2, we discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF347, AGF359, and AGF362) as targeted agents for EOC. In vitro inhibition in the nanomolar range was seen with the FRÎł- and PCFT-expressing cisplatin-sensitive EOC cell line IGROV1 and cisplatin-resistant SKOV3 cells. Uptake by PCFT was demonstrated in IGROV1 and SKOV3 cells. shRNA knockdown of FRÎł (~95%) in IGROV1 cells resulted in a significantly decreased intracellular accumulation of [3H]AGF347, highlighting an important role for FRÎł in drug uptake. [3H]AGF347 treatment of IGROV1 EOC resulted in substantial drug accumulation in cytosol and mitochondria. Inhibition of C1 metabolism in mitochondria at serine hydroxymethyltransferase 2 (SHMT2), and de novo purine biosynthesis in the cytosol were implicated by metabolite 0́−rescue0́+ studies. Targeted metabolomics, using L-[2,3,3-2H]serine as a tracer in wild-type or SHMT2 knockdown SKOV3 cells, verified inhibition of mitochondrial C1-metabolism (at SHMT2) and de novo purine biosynthesis (at glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase). In vitro assays with purified recombinant proteins confirmed these intracellular enzyme targets. Secondary effects of SHMT2 inhibition were observed with all agents including depletion of reduced glutathione and mitochondrial NADH. Depletion of glutathione was accompanied by synergistic in vitro efficacy with AGF347 and cisplatin in SKOV3 cells. In vivo results against SKOV3 EOC xenografts with AGF347 and cisplatin in SCID mice confirmed cisplatin resistance, while AGF347 displayed potent anti-tumor efficacy. We extended our studies to investigate the impact of a cytosolic C1 inhibitor has upon the microenvironment in EOC, using a high-grade serous (HGS) syngeneic tumor model (BR-Luc). Previous studies revealed an important role for our previously characterized GARFTase inhibitor and FR and PCFT transport substrate, AGF94. Potent in vitro cytotoxicity was observed against the BR-Luc syngeneic model of HGS ovarian cancer. In vitro activity was extended to in vivo activity in both early and late stage trials of BR-Luc with AGF94. In late stage disease, our data revealed a direct impact upon M2-like FRÎø-expressing macrophages in syngeneic mice bearing HGS ovarian tumors and an increase in CD+ T cells, whereas CD4+ and CD8+ T cells were unaffected by AGF94 treatment. Collectively, our studies describe the development of novel mitochondria and cytosolic C1-metabolic inhibitors that are selectively delivered via FRs and PCFT, resulting in in vitro and in vivo activity against EOC. Our studies additionally lay an important framework to elucidate the complex TME of EOC, highlighting unique drug delivery mechanisms and metabolic vulnerabilities that EOC tumors employ, thus contributing to an immunosuppressive TME.
