Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

Division Of Basic Sciences - Nci

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Abstract

Resistance to chemotherapy occurs in cancer cells because of intrinsic or acquired changes in expression of specific proteins due to expression of the multidrug efflux pumps ABCB1 (P-glycoprotein) and ABCG2. We have studied resistance to natural product chemotherapeutic agents such as doxorubicin, Vinca alkaloids, and taxol and more recently, histone deacetylase inhibitors, targeted kinase inhibitors, antibody-drug conjugates, and PROTACs. In most cases, cells become simultaneously resistant to multiple drugs because of reductions in intracellular drug concentrations. For the natural product drugs, this cross-resistance is frequently due to expression of an energy-dependent drug efflux system (ABC transporter) known as P-glycoprotein (P-gp), the product of the MDR1 or ABCB1 gene, or to other members of the ABC transporter family, including ABCG2 and ABCB5. In collaboration with Jean-Pierre Gillet (University of Nemours, Belgium), we have explored the role of ABCB5 in cancer drug resistance, in the malignant phenotype in melanoma (where it is frequently mutated), and as a partner as a heterodimer with other ABC B-type transporters. In pigmented cells that express ABCB5 there are both full-length and partial transcripts generated from different promoters. The partial transcripts encode a half-transporter which appears to be able to dimerize with other half transporters (notably ABCB6 and ABCB9). The localization and potential function of these heterodimeric transporters is under study. Mice in which ABCB5 is knocked out are viable and fertile, but have a complex metabolic phenotype suggesting a normal physiological role for ABCB5 in transporting as yet undetermined metabolites. Ferroptosis is a non-apoptotic form of cell death caused by lethal lipid peroxidation. Several small molecule ferroptosis inducers (FINs) have been reported, yet little information is available regarding their interaction with the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp, ABCB1) and ABCG2. We thus sought to characterize the interactions of FINS with P-gp and ABCG2 which may provide information regarding oral bioavailability and brain penetration and predict drug-drug interactions. P-gp overexpression conferred resistance to FIN56 and the erastin derivatives imidazole ketone erastin and piperazine erastin. P-gp-mediated resistance to imidazole ketone erastin and piperazine erastin was also reversed in UO-31 renal cancer cells by CRISPR-mediated knockout of ABCB1. The FINs ML-162, GPX inhibitor 26a, and PACMA31 at 10 micromolar were able to increase intracellular rhodamine 123 fluorescence over 10-fold in P-gp-expressing MDR-19 cells. GPX inhibitor 26a was able to increase intracellular purpurin-18 fluorescence over 4-fold in ABCG2-expressing R-5 cells. We conclude that expression of P-gp may reduce the efficacy of these FINs in cancers that express the transporter and may prevent access to sanctuary sites such as the brain. The ability of some FINs to inhibit P-gp and ABCG2 suggests potential drug-drug interactions. To characterize the role of ABC transporters at the blood-brain barrier, we have explored the ability of ABCB1 and ABCG2 to transport substrates for the nanoluciferase (NanoLuc) enzyme, which produces light in response to these compounds. We transfected HEK-293 cells with either ABCB1 and ABCG2 as well as NanoLuc. From a panel of ten NanoLuc substrates, we identified one ABCB1 substrate and six ABCG2 substrates. Antibody Antibody-drug conjugates (ADCs) feature an antibody recognizing a specific protein joined to a potent toxic payload. Numerous antibody-drug conjugates have received FDA approval; however, clinical resistance arises. P-gp is known to confer resistance to some ADC payloads, leading us to collaborate with Matt Hall at NCATS who performed a high-throughput screen with 27 common ADC payloads using cells lines expressing ABC transporters P-gp or ABCG2. Confirmatory assays were also performed using cells transfected to express P-gp, ABCG2, or MRP1 (encoded by ABCC1). Several commonly used ADC payloads were substrates of P-gp, including calicheamicin gamma1, monomethyl auristatin E, DM1, and DM4. All the pyrrolobenzodiazepines tested-SJG136, SGD-1882, SG2057, and SG3199-were substrates of P-gp, ABCG2, and MRP1. The modified anthracyclines nemorubicin and its metabolite PNU-159682 were poorly transported by both ABCB1 and ABCG2 and displayed nanomolar to picomolar toxicity. Further, we found that the efficacy of the FDA-approved ADC mirvetuximab soravtansine, with DM4 as the toxic payload, was decreased in cell lines expressing P-gp. Thus, ABC transporter expression may arise as a mechanism of resistance when using these treatments.PROTACs (proteolysis targeting chimeras) are another novel treatment modality which consist of two linked protein binding molecules; one that binds a target of interest and one that binds ubiquitin ligase. This hybrid molecule leads to degradation of the targeted protein via the proteasome.We have found that several PROTACs are substrates of P-gp and a few are substrates of both P-gp and ABCG2. This could mean decreased oral bioavailability, decreased brain penetration of these compounds and could be a mechanism of resistance of cancer cells to some PROTACs.