title: Immunotoxin resistance via reversible methylation of the DPH4 promoter is a unique survival strategy.
abstract: HA22 is a recombinant immunotoxin composed of an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A. HA22 produced a high rate of complete remissions in drug-resistant hairy cell leukemia and has a lower response rate in pediatric acute lymphoblastic leukemia (ALL). To understand why patients with ALL have poorer responses, we isolated an ALL cell line that is resistant to killing by HA22. The resistance is unstable; without HA22 the cells revert to HA22 sensitivity in 4 mo. We showed that in the resistant cell line, HA22 is unable to ADP ribosylate and inactivate elongation factor-2 (EF2), owing to a low level of DPH4 mRNA and protein, which prevents diphthamide biosynthesis and renders EF2 refractory to HA22. Analysis of the promoter region of the DPH4 gene shows that the CpG island was hypomethylated in the HA22-sensitive cells, heavily methylated in the resistant cells, and reverted to low methylation in the revertant cells. Our data show that immunotoxin resistance is associated with reversible CpG island methylation and silencing of DPH4 gene transcription. Incubation of sensitive cells with the methylation inhibitor 5-azacytidine prevented the emergence of resistant cells, suggesting that this agent in combination with HA22 may be useful in the treatment of some cases of ALL.
authors.affiliation: Laboratory of Molecular Biology and Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Wei
authors.forename: Laiman
authors.initials: L
authors.lastname: Xiang
authors.forename: Alan S
authors.initials: AS
authors.lastname: Wayne
authors.forename: Oleg
authors.initials: O
authors.lastname: Chertov
authors.forename: David J
authors.initials: DJ
authors.lastname: FitzGerald
authors.forename: Tapan K
authors.initials: TK
authors.lastname: Bera
authors.forename: Ira
authors.initials: I
authors.lastname: Pastan
mesh_terms: D001374:Azacitidine / Q000494:pharmacology; D001427:Bacterial Toxins / Q000494:pharmacology*; D001483:Base Sequence; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation / Q000187:drug effects; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D005098:Exotoxins / Q000494:pharmacology*; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000276:immunology
chemical_list: D001427:Bacterial Toxins; C497867:CD22 protein, human; D004273:DNA, Neoplasm; C572774:DNAJC24 protein, human; D005098:Exotoxins; D050956:HSP40 Heat-Shock Proteins; D000922:Immunotoxins; D020652:Peptide Elongation Factor 2; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D051918:Sialic Acid Binding Ig-like Lectin 2; C541974:immunotoxin HA22; D001374:Azacitidine