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.DNA methylation | drug resistance | ADP-ribosylation | diphthamide synthesis | epigenetic regulation

Phase 1 study of the anti-CD22 immunotoxin moxetumomab pasudotox for childhood acute lymphoblastic leukemia

“…10 Notably, intermittent exposure to sublethal doses of moxetumomab pasudotox in vitro can lead to reversible epigenetic changes that confer resistance in ALL cell lines. 24,25 This potential mechanism of resistance has not yet been explored in patients. Finally, we recently demonstrated wide interpatient variability in the exposure time required to induce cytotoxicity of ALL blasts by moxetumomab pasudotox.…”

A Modified Form of Diphthamide Causes Immunotoxin Resistance in a Lymphoma Cell Line with a Deletion of the WDR85 Gene

“…However, some patients do not respond, whereas others relapse after initially responding to the agent. We have undertaken studies to understand possible mechanisms that can lead to resistance and recently reported that silencing of the DPH4 gene in the HAL-01 ALL cell line results in immunotoxin resistance (13). Here, we have selected CA46 cells, which are immunotoxin-resistant, and observed that the resistant cells have a deletion of the WDR85 gene, which causes a novel modification of diphthamide in EF2 and immunotoxin resistance.…”

“…We have recently reported that immunotoxin resistance in the HAL-01 ALL cell line is mediated by reversible DNA methylation in the CpG island of the DPH4 promoter region (13). To study the WDR85 promoter in CA46-R cells, we attempted to amplify the promoter region (spanning Ϫ1588 to ϩ17 bp) by PCR using three primer pairs and were unable to do so, suggesting that the region was deleted in the resistant cell line.…”

Pseudomonas aeruginosa exotoxin T induces potent cytotoxicity against a variety of murine and human cancer cell lines

“…Although these new recombinant immunotoxins have shown high potency in killing tumour cells with high specificity, they too share the limitation of targeting a single cellular substrate, eEF-2 (Jørgensen et al, 2008). In fact, mutations in eEF-2 have been shown to emerge easily, conferring resistance to these toxins in cancer cells (Foley et al, 1995;Jørgensen et al, 2008;Wei et al, 2012). & Barbieri, 2003;Garrity-Ryan et al, 2004), which play important roles in survival, proliferation, metastasis and angiogenesis in cancer.…”