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. 2015 Jul 21;5(9):1925-35.
doi: 10.1534/g3.115.020560.

Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants

Irene Ojini  1 , Alison Gammie  2
Affiliations

Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants

Irene Ojini et al. G3 (Bethesda). .

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.

Keywords: DNA mismatch repair; cancer; drug resistance; mutator; whole genome sequencing.

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Figures

Figure 1
Figure 1
Examples of the effects of compounds of interest from the small molecule screening. Representative growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence of a drug (DMSO control, No Drug), in the presence of a drug that inhibited the growth of both strains (sensitive), in the presence of 400 μM etoposide (resistant, short lag) or 100 μM camptothecin (resistant, long lag), or in the presence of 60 μM bactobolin (synthetic growth defect). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr using a Biotek Plate Reader. The number of compounds remaining in each category after the screening process is indicated in the upper left of each panel of the msh2Δ curves. The three rightmost panels are representative of molecules resulting in an msh2∆-specific growth difference.
Figure 2
Figure 2
Verification of the resistance discovery method using canavanine. (A) The chemical structure of canavanine. The structure was rendered using ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence (No Drug) and presence of 200 μM canavanine (Can). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. (C) Schematic representation of the frameshift positions within the CAN1 locus on chromosome V (chrV) conferring resistance to canavanine. The numbers indicate the chromosomal position. The mutations all resulted in frameshifts at homopolymers detailed in the bottom panel. (D) A table listing the mutations in CAN1 conferring resistance to canavanine. The nucleotide position for each mutation is shown along with the region mutated. Because CAN1 is in the opposite orientation (chrV:33466-31694) within the W303 reference genome, the sequence shown is the reverse complement of the reference genome for the given interval. The insertion or deletion at the homopolymeric run (highlighted in red) is indicated for each isolate, for example, a deletion at an A6 repeat followed by a C (AAAAAAC) would be designated A6C > A5C, whereas an insertion would be CA6 > CA7.
Figure 3
Figure 3
Verification of resistance discovery method using camptothecin. (A) Chemical structure of camptothecin. The structure was rendered using ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence (No Drug) and presence of 9 μM camptothecin (Camp). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. (C) Schematic representation of the frameshift positions within the TOP1 locus on chromosome XV (chrXV) conferring resistance to camptothecin. The numbers indicate the chromosomal position. The mutations all resulted in frameshifts at homopolymers detailed in the bottom panel. (D) A table listing the mutations in TOP1 conferring resistance to camptothecin. The nucleotide position for each mutation is shown along with the region mutated. The sequence given corresponds to the strand in the W303 reference genome. Because TOP1 is in the opposite orientation (chrXV:315387-313078) within the W303 reference genome, the sequence shown is the reverse complement of the reference genome for the given interval. The nucleotide numbers differ slightly from the S288C draft genome. The specific insertion or deletion at the homopolymeric run (indicated in red) is indicated in a format described in Figure 2.
Figure 4
Figure 4
Inactivation of the Hnm1 transporter is the major cause of resistance to mechlorethamine. (A) Chemical structure of mechlorethamine. The structure was rendered using ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence (No Drug) and presence of 70 μM mechlorethamine (Mech). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. (C) Schematic representation of the frameshift positions within the HNM1 locus on chromosome VII (chrVII) conferring resistance to mechlorethamine. The numbers indicate the chromosomal position. The mutations were frameshifts at homopolymers, a missense mutation (GGA > AGA), and a nonsense mutation (TAT > TAA), and are detailed in the bottom panel. (D) A table listing the mutations in HNM1 conferring resistance to mechlorethamine. The nucleotide position for each mutation is shown along with the region mutated. The sequence given corresponds to the strand in the W303 reference genome. Because HNM1 is in the opposite orientation (chrVII: 363916–362225) within the W303 reference genome, the sequence shown is the reverse complement of the reference genome for the given interval. The nucleotide numbers differ slightly from the S288C draft genome. The mutated nucleotides for the point mutations are underlined. The mutated codons and homopolymers are indicated in red. The specific insertion or deletion at the homopolymeric run is indicated in a format described in Figure 2.
Figure 5
Figure 5
Mutations in the endocytosis pathway confer resistance to bactobolin. (A) Chemical structure of bactobolin. The structure was rendered using ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence (No Drug) and presence of 100 μM bactobolin (Bact). Optical density readings at 600 nm (OD600) were taken every 15 min for 72 hr. (C) The table lists the genes and mutations conferring resistance to bactobolin. The coding strand nucleotide sequence and mutation for each isolate is shown. Five caused inactivating frameshifts and one (within YSC84) resulted in a mutation in the splice donor consensus sequence (SDC). (D) A schematic drawing of the clathrin-dependent endocytic pathway is adapted from a previously published model (Weinberg and Drubin 2012). The black line represents a membrane undergoing endocytosis. The endocytosis components that were identified are represented with colors: Ede1 (red); Ysc84 (green); Vrp1 (orange); Sla2 (purple); and Inp52 (blue). Other endocytosis components are in gray.
Figure 6
Figure 6
Inactivation of Ipt1 is the major cause of resistance to mitoxantrone. (A) Chemical structure of mitoxantrone. The structure was rendered with ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence and presence of mitoxantrone (50 μM). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. The OD600 offset observed in the presence of the drug is due to the colored nature of mitoxantrone. (C) Schematic representation of the frameshift positions within the IPT1 locus on chromosome IV (chrIV) conferring resistance to mitoxantrone. The numbers indicate the chromosomal position. The mutations all resulted in frameshifts at homopolymers detailed in the bottom panel. (D) A table listing the mutations in IPT1 conferring resistance to mitoxantrone. The nucleotide position for each mutation is shown along with the region mutated. The sequence given corresponds to the strand in the W303 reference genome. Because IPT1 is in the opposite orientation (chrIV: 591344–589761) within the W303 reference genome, the sequence shown is the reverse complement of the reference genome for the given interval. The nucleotide numbers differ slightly from the S288C draft genome. The specific insertion or deletion at the homopolymeric run is indicated in a format described in Figure 2.
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