title: A modified form of diphthamide causes immunotoxin resistance in a lymphoma cell line with a deletion of the WDR85 gene.
abstract: HA22 is a recombinant immunotoxin that kills CD22-expressing cells by ADP-ribosylating and inactivating elongation factor-2 (EF2). HA22 is composed of an Fv that binds to CD22 fused to a portion of Pseudomonas exotoxin A. HA22 is very active in drug-resistant hairy cell leukemia but is less active in children with acute lymphoblastic leukemia. To understand why some patients do not respond to HA22, we isolated an HA22-resistant lymphoma cell line and showed that resistance was due to the inability of HA22 to ADP-ribosylate and inactivate EF2. We analyzed the diphthamide synthesis genes and found that the WDR85 gene was deleted. We show that WDR85 knockdown conferred HA22 resistance to sensitive cells and that sensitivity was restored by introduction of a WDR85 cDNA into resistant cells. Analysis of EF2 in the mutant cells revealed a novel form of diphthamide with an additional methyl group that prevented ADP-ribosylation and inactivation of EF2. The abnormal methylation appeared to be catalyzed by DPH5. Inactivation of the WDR85 gene could be a mechanism of immunotoxin resistance in patients undergoing immunotoxin therapy.
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Wei
authors.forename: Tapan K
authors.initials: TK
authors.lastname: Bera
authors.forename: Alan S
authors.initials: AS
authors.lastname: Wayne
authors.forename: Laiman
authors.initials: L
authors.lastname: Xiang
authors.forename: Simona
authors.initials: S
authors.lastname: Colantonio
authors.forename: Oleg
authors.initials: O
authors.lastname: Chertov
authors.forename: Ira
authors.initials: I
authors.lastname: Pastan
mesh_terms: D002265:Carboxylic Ester Hydrolases; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D017353:Gene Deletion; D006639:Histidine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D008223:Lymphoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D008745:Methylation / Q000187:drug effects; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D020652:Peptide Elongation Factor 2 / Q000235:genetics / Q000378:metabolism*; D011506:Proteins
chemical_list: D000922:Immunotoxins; D009363:Neoplasm Proteins; D020652:Peptide Elongation Factor 2; D011506:Proteins; D006639:Histidine; C027527:diphthamide; C000706694:DPH7 protein, human; D008780:Methyltransferases; D002265:Carboxylic Ester Hydrolases