Decreased Expression of Diphthamide Genes Is Responsible for the Resistance to the Anti-CD22 Immunotoxin Moxetumomab Pasudotox (m. pasudotox) in Acute Lymphoblastic Leukemia (ALL)/Non-Hodgkin Lymphoma (NHL) Cell Lines.

Complete remissions have been observed in some but not all children in an ongoing Phase 1 trial of the anti-CD22 recombinant immunotoxin moxetumomab pasudotox (m. pasudotox, HA22, CAT-8015) for relapsed and refractory ALL and NHL.

To develop ALL/NHL cell lines resistant to m. pasudotox in attempt to uncover possible mechanisms of immunotoxin resistance and to use this information to try to improve clinical outcomes.

Using the ALL cell line HAL-01 and the Burkitt NHL cell line CA46, we generated two cell lines (HAL-01-R and CA46-R, respectively) that were resistant to killing by m. pasudotox by repeated exposure to sub-lethal doses of m. pasudotox. We studied the basis of their resistance.

Cytotoxicity was markedly reduced in the resistant cell lines (Table) and m. pasudotox was unable to ADP-ribosylate and inactivate elongation factor-2 (EF2). In HAL-01-R this was due to a low level of DPH4 mRNA and protein, which prevented diphthamide biosynthesis and rendered EF2 refractory to m. pasudotox. Analysis of the promoter region of the DPH4 gene showed that the CpG island was heavily methylated. Incubation of sensitive cells with the methylation inhibitor 5-azacytidine prevented the emergence of resistant cells. A second resistance mechanism in the diphthamide pathway was identified in the CA46-R line. There was a small decrease in CD22 site density and internalization rate in CA46-R cells, which were insufficient to account for m. pasudotox resistance. In contrast, protein synthesis measured by ³H-leucine incorporation was not reduced in CA46-R cells suggesting a defect in diphthamide modification of EF2. Consistent with this hypothesis was the finding that m. pasudotox catalyzed the incorporation of ADP-ribose into EF2 in extracts of sensitive CA46 cells, but not in CA46-R cells, although EF2 protein levels were similar in the two cell lines. We examined the expression of the six genes required for diphthamide synthesis (DPH1–5 and WDR85). Only the levels of WDR85 mRNA and protein were substantially decreased in CA46-R cells. We used shRNA to knock down WDR85 mRNA and protein levels and tested the cells for sensitivity to m. pasudotox. The IC50 increased 10-fold from 0.6 ng/ml to 6.0 ng/ml. Conversely, we restored WDR85 protein expression into CA46-R cells by transduction of resistant cells with a WDR85 expressing vector, which partially restored sensitivity to m. pasudotox. Altogether, these findings show that a reduction in WDR85 protein is sufficient to cause m. pasudotox resistance. To study why WDR85 expression is reduced in CA46-R cells, a high resolution SNP array analysis of the WDR85 gene was performed. A homozygous deletion at Chr9:140,468,104–140,610,581 (Build: GRCh37/19) was detected, which accounts for the inability of the cells to make WDR85 protein.

We isolated two immunotoxin-resistant ALL/NHL cell lines and found that resistance was associated with reduced expression of diphthamide synthesis genes required to correctly post-translationally modify EF2. As a consequence, m. pasudotox was unable to ADP-ribosylate and inactivate EF2. Reduced expression of DPH4 in HAL-01-R was related to hypermethylation and could be prevented by 5-azacytidine. Reduced expression of WDR85 in CA46-R was due to a deletion of the gene. These represent novel mechanisms of immunotoxin resistance that will be of interest to explore in future studies.

This study was supported by the Intramural Research Program of the NIH, National Cancer Institute (NCI), Center for Cancer Research and by a Cooperative Research and Development Agreement between MedImmune, LLC and the NCI.

No relevant conflicts of interest to declare.