title: Identification of Genes That Modulate Susceptibility to Formaldehyde and Imatinib by Functional Genomic Screening in Human Haploid KBM7 Cells.
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.
authors.affiliation: Superfund Research Program, School of Public Health, University of California, Berkeley, California 94720.
authors.forename: Hua
authors.initials: H
authors.lastname: Shen
authors.affiliation: Superfund Research Program, School of Public Health, University of California, Berkeley, California 94720.
authors.forename: Cliona M
authors.initials: CM
authors.lastname: McHale
authors.affiliation: Superfund Research Program, School of Public Health, University of California, Berkeley, California 94720.
authors.forename: Syed I
authors.initials: SI
authors.lastname: Haider
authors.affiliation: Superfund Research Program, School of Public Health, University of California, Berkeley, California 94720.
authors.forename: Cham
authors.initials: C
authors.lastname: Jung
authors.affiliation: Superfund Research Program, School of Public Health, University of California, Berkeley, California 94720.
authors.forename: Susie
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Superfund Research Program, School of Public Health, University of California, Berkeley, California 94720.
authors.forename: Martyn T
authors.initials: MT
authors.lastname: Smith
authors.affiliation: Superfund Research Program, School of Public Health, University of California, Berkeley, California 94720 luoping@berkeley.edu.
authors.forename: Luoping
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005557:Formaldehyde / Q000494:pharmacology*; D015973:Gene Expression Regulation, Leukemic; D055785:Gene Knockdown Techniques; D006238:Haploidy; D057166:High-Throughput Screening Assays; D006801:Humans; D000068877:Imatinib Mesylate / Q000494:pharmacology*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D009154:Mutation; D034622:RNA Interference; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D005557:Formaldehyde; D000068877:Imatinib Mesylate
keywords: KBM7; formaldehyde; functional genomics; haploid; imatinib.