Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants

Abstract

Resistance to cancer therapy is a major obstacle in the long-term treatment of cancer. A greater understanding of drug resistance mechanisms will ultimately lead to the development of effective therapeutic strategies to prevent resistance from occurring. Here, we exploit the mutator phenotype of mismatch repair defective yeast cells combined with whole genome sequencing to identify drug resistance mutations in key pathways involved in the development of chemoresistance. The utility of this approach was demonstrated via the identification of the known CAN1 and TOP1 resistance targets for two compounds, canavanine and camptothecin, respectively. We have also experimentally validated the plasma membrane transporter HNM1 as the primary drug resistance target of mechlorethamine. Furthermore, the sequencing of mitoxantrone-resistant strains identified inactivating mutations within IPT1, a gene encoding inositolphosphotransferase, an enzyme involved in sphingolipid biosynthesis. In the case of bactobolin, a promising anticancer drug, the endocytosis pathway was identified as the drug resistance target responsible for conferring resistance. Finally, we show that that rapamycin, an mTOR inhibitor previously shown to alter the fitness of the ipt1 mutant, can effectively prevent the formation of mitoxantrone resistance. The rapid and robust nature of these techniques, using Saccharomyces cerevisiae as a model organism, should accelerate the identification of drug resistance targets and guide the development of novel therapeutic combination strategies to prevent the development of chemoresistance in various cancers.

miR-1266 Contributes to Pancreatic Cancer Progression and Chemoresistance by the STAT3 and NF-κB Signaling Pathways

“… 33 Other mechanisms of resistance, including insensitivity to drug-induced apoptosis, upregulation of DNA repair enzymes, induction of drug-detoxifying mechanisms, stromal proliferation, and reduced angiogenesis, play a crucial role in drug resistance. 34 , 35 , 36 , 37 , 38 , 39 …”

Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

“…However, its use has mostly relied on the availability of rapid methods for mapping the resistance locus to a given chromosomal position. The advent of whole-genome tiling arrays and whole-genome sequencing has made the method more practical in other single-celled organisms such as Mycobacterium tuberculosis bacilli 35 , malaria parasites 36 , trypanosomes 37 , and yeast 38 39 40 . Unlike other essential gene products that are often described as possible drug targets, drug targets discovered through directed evolution are known from the outset to be druggable and are considered “chemically validated.” The method has been successfully used to identify the targets of several antimalarial compounds initially found through phenotypic screens, as well as to identify genes contributing to the resistome 2 41 42 .…”

Identification and Mode of Action of a Plant Natural Product Targeting Human Fungal Pathogens

“…A forward genetic screen in S. cerevisiae was finally undertaken as an unbiased way to discover the target(s) of tomatidine. The strain chosen in this approach lacked PDR5 and MSH2 to avoid multidrug transporter-dependent resistance mechanisms and to increase the rates at which resistance mutations may occur, respectively ( 28 , 29 ). A pdr5 Δ msh2 Δ strain (strain P1) was plated on YEPD medium containing 10 μM tomatidine, and after a screening of more than 2 × 10 8 cells, one resistant mutant (mutant R1) was isolated.…”

“…A pdr5 Δ msh2 Δ strain (strain P1) was plated on YEPD medium containing 10 μM tomatidine, and after a screening of more than 2 × 10 8 cells, one resistant mutant (mutant R1) was isolated. To obtain additional resistant mutants, a second strategy inspired by Ojini and Gammie ( 29 ) was developed and consisted of submitting cells to two drug exposure periods in liquid medium (72 and 48 h) interspersed by a period of drug-free growth (48 h). Cultures with robust growth at the end of the experiment were further plated on solid YEPD medium containing 10 μM tomatidine, which resulted in the isolation of four resistant mutants (mutants R2, R3.1, R3.2, and R3.4) from two different cultures.…”

“…One pop-out mutant (mutant R1) was identified, and resistance to tomatidine was confirmed using the MIC broth dilution method. The other tomatidine-resistant strains were identified as described previously by Ojini and Gammie ( 29 ) with few modifications. The msh 2Δ prd5 Δ strain (P1) was grown to saturation in 5 ml of YEPD medium at 30°C.…”