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. 2016 May;151(1):10-22.
doi: 10.1093/toxsci/kfw032. Epub 2016 Mar 22.

Identification of Genes That Modulate Susceptibility to Formaldehyde and Imatinib by Functional Genomic Screening in Human Haploid KBM7 Cells

Hua Shen  1 , Cliona M McHale  1 , Syed I Haider  1 , Cham Jung  1 , Susie Zhang  1 , Martyn T Smith  1 , Luoping Zhang  2
Affiliations

Identification of Genes That Modulate Susceptibility to Formaldehyde and Imatinib by Functional Genomic Screening in Human Haploid KBM7 Cells

Hua Shen et al. Toxicol Sci. 2016 May.

Erratum in

Abstract

Though current functional genomic screening systems are useful for investigating human susceptibility to chemical toxicity, they have limitations. Well-established, high-throughput yeast mutant screens identify only evolutionarily conserved processes. RNA interference can be applied in human cells but is limited by incomplete gene knockout and off-target effects. Human haploid cell screening is advantageous as it requires knockdown of only a single copy of each gene. A human haploid cell mutant library (KBM7-Mu), derived from a chronic myeloid leukemia (CML) patient, was recently developed and has been used to identify genes that modulate sensitivity to infectious agents and pharmaceutical drugs. Here, we sought to improve the KBM7-Mu screening process to enable efficient screening of environmental chemicals. We developed a semi-solid medium based screening approach that cultures individual mutant colonies from chemically resistant cells, faster (by 2-3 weeks) and with less labor than the original liquid medium-based approach. As proof of principle, we identified genetic mutants that confer resistance to the carcinogen formaldehyde (FA, 12 genes, 18 hits) and the CML chemotherapeutic agent imatinib (6 genes, 13 hits). Validation experiments conducted on KBM7 mutants lacking each of the 18 genes confirmed resistance of 6 FA mutants (CTC1, FCRLA, GOT1, LPR5, M1AP, and MAP2K5) and 1 imatinib-resistant mutant (LYRM9). Despite the improvements to the method, it remains technically challenging to limit false positive findings. Nonetheless, our findings demonstrate the broad applicability of this optimized haploid approach to screen toxic chemicals to identify novel susceptibility genes and gain insight into potential mechanisms of toxicity.

Keywords: KBM7; formaldehyde; functional genomics; haploid; imatinib..

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Figures

FIG. 1.
FIG. 1.
Selection of critical screening concentrations of formaldehyde (FA) and imatinib. Viable cell density as a percentage of untreated controls 72 h after treatment in liquid culture medium are shown for (A) 20–120 μM FA and (B) 0.01–10 μM imanitib. Data and SD are shown from 2 FA experiments and 4 imatinib experiments, each with 2 replicates per chemical concentration.
FIG. 2.
FIG. 2.
Validation of resistance to FA of select mutant clones. A, Viable cell density of lipoprotein receptor-related protein 5 (LRP5), glutamic-oxaloacetic transaminase 1 (GOT1), and meiosis 1 associated protein (M1AP) mutants and wild-type KBM7 cells, 72 h after treatment with FA. Average (and SD) of 3 replicate experiments shown as a percentage of untreated controls cells; B, Viable cell density of mitogen-activated protein kinase kinase 5 (MAP2K5), CTC1, and FCRLA mutants and wild-type KBM7 cells 72 h after treatment with FA. Average and SD of 2 replicate experiments shown as a percentage of untreated controls cells; C, Representative data (from 1 of 2 experiments, 2 replicates per experiment) showing live/dead LRP5, GOT1, and M1AP mutants and wild-type KBM7 cells, after treatment with FA (90 µM) for 72 h, compared with untreated cells; D, Validation of knockdown of LRP5 and M1AP mRNA in the mutant cells by RT-PCR normalized to the housekeeping gene ribosomal protein L13a. Data (fold change and SD) are averaged from 4 experiments and 3 experiments, respectively, 2 replicates per experiment; E, Validation of LRP5 protein knockdown in the mutant cells by Western blot. Representative data from 1 of 2 experiments shown. *P < .05 by Student’s t test.
FIG. 3.
FIG. 3.
Validation of resistance to imatinib of select mutant clones. A, Viable cell density of Caspase 10 (CASP10), LYR motif containing 9 (LYRM9), CUX1, and neurofibromin 1 mutants and wild-type KBM7 cells 72 h after treatment with imatinib. Average (and SD) of 2 replicate experiments, 2 replicates per experiment, shown as a percentage of untreated controls cells. B, Viable cell density of LRP5 mutant cells and wild-type KBM7 cells 72 h after treatment with imatinib. Average (and SD) of 2 replicate experiments, 2 replicates per experiment, shown as a percentage of untreated controls cells. *P < .05 by Student’s t test.
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