Abstract 3809: Modulation of MRP1 activity reverses chemotherapy resistance in adult cancer cells
Authors: Hanssen Kimberley M. , Gana Christine C. , Wheatley Madeleine S. , Yu Denise M. , Flemming Claudia L. , Young Richard , Wright Gavin M. , Conseil Gwenaelle , Kennedy Catherine J. , deFazio Anna , Solomon Ben , Cole Susan P. , Norris Murray D. , Haber Michelle , Fletcher Jamie I. 
Published: August 2019

Abstract
Multidrug resistance protein 1 (MRP1) is an ATP-dependent efflux pump that extrudes chemotherapeutic drugs from cancer cells, preventing their intracellular accumulation and thus their efficacy. MRP1 also regulates intracellular levels of glutathione (GSH), an antioxidant that when elevated in cancer provides resistance against chemotherapy- and radiotherapy-induced oxidative damage. The different binding sites for drugs and GSH on MRP1 has allowed the development of small molecule MRP1 modulators that block chemotherapy efflux whilst simultaneously enhancing GSH efflux to deplete GSH. As no therapeutic options are available to combat MRP1-mediated multidrug resistance, we have been investigating MRP1 modulators for their efficacy in sensitizing MRP1-expressing cancers to treatment. MRP1 immunohistochemistry staining in ovarian and non-small cell lung cancer (NSCLC) patient samples was assessed to determine the frequency of high MRP1 expression. High MRP1 staining was frequently observed in the subcellular compartment (37%, 47%) and, to a lesser extent, plasma membrane (7%, 16%) in ovarian and NSCLC patient tumour samples respectively. In NSCLC, subcellular MRP1 was associated with poorer overall survival in late stage disease (p=0.0095) and membrane MRP1 with poorer disease-free survival in Stage 4 cancer (p=0.01449). Analysis for ovarian cancer is ongoing. As MRP1 is therefore an attractive target for inhibition, we used membrane vesicle uptake assays to compare the ability of modulators to inhibit [3H]-labelled MRP1 substrate transport. 7-(difluoromethyl)-N-4-morpholinyl-5-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7914321) was identified as an analogue of the MRP1 inhibitor Reversan with improved potency. Interestingly, this inhibition was GSH dependent. Exploring further this relationship with GSH, the high MRP1-expressing A549 lung and SKOV3 ovarian cancer cell lines were treated with 7914321 alone or in combination with the GSH synthesis inhibitor buthionine sulfoximine (BSO), then GSH levels were determined by glutathione recycling assay. 7914321 stimulated GSH efflux in both cell lines in an MRP1-dependent manner and strongly synergised with BSO to cause near complete GSH depletion. To determine the potential of this synergistic GSH depletion to chemosensitize cells, the viability of A549 and SKOV3 cells treated with chemotherapy, 7914321, and BSO was assessed by clonogenic and resazurin-based cytotoxicity assays. 7914321/BSO significantly diminished clonogenicity in high but not low MRP1-expressing cells, and further sensitized cancer cell lines to MRP1-substrate chemotherapeutics compared to the single agents. Together, these findings provide preliminary evidence that 7914321 is a potent MRP1 modulator whose dual effects on MRP1 place it in a unique position to potentially enhance the therapeutic window to treat chemoresistant MRP1-overexpressing cancers.
Citation Format: Kimberley M. Hanssen, Christine C. Gana, Madeleine S. Wheatley, Denise M. Yu, Claudia L. Flemming, Richard Young, Gavin M. Wright, Gwenaelle Conseil, Catherine J. Kennedy, Anna deFazio, Ben Solomon, Susan P. Cole, Murray D. Norris, Michelle Haber, Jamie I. Fletcher. Modulation of MRP1 activity reverses chemotherapy resistance in adult cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3809.