Using transcriptome sequencing to identify mechanisms of drug action and resistance

Abstract

Determining mechanisms of drug action in human cells remains a major challenge. Here we describe an approach in which multiple-drug-resistant clones are isolated and transcriptome sequencing is used to find mutations in each clone. Further analysis of mutations common to more than one clone can identify a drug's physiological target and indirect resistance mechanisms, as indicated by our proof-of-concept studies of the cytotoxic anticancer drugs BI 2536 and bortezomib.

Figure 1. Characterization of BI 2536-resistant clones

(a) Structure of PLK1 inhibitor BI 2536. (b) Median lethal dose (LD₅₀s) measured for the parental cell line and two drug-resistant clones (LD₅₀s: 3.9 ± 2.8 nM (parental), 33.5 ± 2.6 nM (Clone B), 14.2 ± 7.8 nM (Clone D); n = 3, mean ± sem). (c) Graph-based analysis of similarities (0 = low similarity, 2 = high similarity) between BI 2536-resistant clones allows clones D, E, and F to be grouped together (group 4). (d) Proliferation assay showing the effects of BI 2536 exposure on hTERT-RPE1 cells, transfected with GFP-PLK1 wt, GFP-PLK1 G63S, or GFP-PLK1 R136G. Median lethal dose (LD₅₀s) measured for each transfected cell line (LD₅₀s: 44 ± 5 nM (GFP-PLK1 wt), 83 ± 9 nM (GFP-PLK1 G63S), 76 ± 8 nM (GFP-PLK1 R136G); n = 6, mean ± sem, p < 0.01 for both, two-tailed paired t-test). (e) Proliferation assay showing the effect of 20 nM taxol on HCT-116 parental cells and clones A, B, and C, normalized to untreated cells (n = 3, mean ± s.d.).

Figure 2. Characterization of bortezomib-resistant clones

(a) Structure of proteasome inhibitor bortezomib. (b) Median lethal dose (LD₅₀s) measured for the parental cell line and two drug-resistant clones (LD₅₀s: 6.3 ± 0.9 nM (parental), 19.4 ± 6.7 nM (Clone A), 41.5 ± 9.5 nM (Clone E); n = 3, mean ± sem). (c) Graph-based analysis of similarities (0 = low similarity, 2 = high similarity) between bortezomib-resistant clones groups clones A, B, C, and D (group 1). (d) Proliferation assay showing the response of hTERT-RPE1 cells, transfected with GFP-PSMB5 wt, GFP-PSMB5 M104V, or GFP-PSMB5 A108T, to treatment with bortezomib. Median lethal dose (LD₅₀s) measured for each transfected cell line (LD₅₀s: 5.55 ± 0.09 nM (GFP-PSMB5 wt), 28.7 ± 2.8 nM (GFP-PSMB5 M104V, p < 0.05, two-tailed paired t-test), 19.2 ± 2.8 nM (GFP-PSMB5 A108T, p < 0.05, two-tailed paired t-test); n = 3, mean ± sem). (e) Schematic highlights the key steps of the approach: Selecting and expanding drug-resistant clones from a heterogeneous parental population. Massively parallel sequencing of mRNA from multiple drug-resistant clones and parental (untreated) cells. Bioinformatics analyses to identify genes that are mutated with high frequency. A subset of sequencing reads for a BI 2536-resistant clone (clone A) and the HCT-116 parental cells are shown. The ~50% mutation frequency indicates heterozygosity.