Pretreatment of Low-Dose Decitabine to Leukemia Cells Markedly Enhances the Cytotoxicity of Gemtuzumab Ozogamicin

Abstract 5000

Prognosis of relapsed/refractory acute myeloid leukemia (AML) patients has been poor due to its drug resistance. Although gemtuzumab ozogamicin (GO) targets CD33 antigen, which is overexpressed on most AML cells, the efficacy of GO alone for relapsed/refractory patients remains to be unsatisfied. Therefore we aimed to improve GO effects by combination with the other drugs.

GO needs target cells intact to complete several steps for its cytotoxicity: GO binding to CD33 antigen, internalization of GO with CD33 antigen, fusion of the endosome to lysosome, digestion of the linker by lysozyme and release of calicheamicin, and intercalation of activated calicheamicin to DNA. GO could exert its cytotoxicity only when these cellular steps of target cells are intact. This paradoxical mechanism prompted us to study decitabine (DAC), azacitidine (AZA) and, valproic acid (VPA), which modify epigenetic alterations in leukemia cells with modest cytotoxicity. Low-dose DAC, AZA, or VPA have been reported to differentiate AML cells, with the increased expression of myeloperoxidase and lysozyme. Differentiation of AML cells by some agents, for example G-CSF, has been reported to reduce the expression of P-glycoprotein. Thus, we hypothesized that DAC, AZA or VPA could activate the cellular functions of target cells and enhance the cytotoxicity of GO.

As refractory leukemic cells, we used SKK-1 and SKM-1 cells derived from patients with leukemia progressed from myelodysplastic syndrome, and SKNO-1, KO52, and Kasumi3 cells with complex karyotypes. We also used an acute promyelocytic leukemia cell line (NB4) and an acute myelomonocytic leukemia cell line (U937).

Low-dose of DAC (100nM), AZA (2μM), or VPA (1mM) showed negligible cytotoxic effects. Simultaneous administration of GO (2.5 μg/ml) and DAC did not enhance cytotoxicity of GO. In contrast, GO cytotoxicity was enhanced only when cells were pretreated with DAC prior to GO. Pretreatment with DAC for 48 hours showed maximum enhancing effects. AZA also required pretreatment for enhancing effect. It was suggested that DAC and AZA induced some cellular response which enhance the cytotoxic steps of GO during pretreatment. VPA enhanced the cytotoxicity added either simultaneously or antecedently. The enhancing effect varied in each cell line. Pretreatment with DAC showed the most prominent enhancing effects among the three agents.

DAC treatment changed neither the expression of CD33 antigens nor internalization of GO. DAC enhanced GO cytotoxicity partly by both promoting intercalation of calicheamicin to DNA and suppressing the activity of a drug-efflux pump, MRP-1.

DAC-enhanced GO cytotoxicity in vitro was also observed in primary leukemic blasts of four (29%) among 14 relapsed/refractory AML patients.

A large phase III study about GO, S0106 trial conducted by SWOG, revealed no significant improvement either in CR rate or in LFS for de novo AML patients, whereas significant increase of early complication death, in concurrent use of chemotherapy and GO. The result of SWOG trial and the mechanism of GO cytotoxicity might suggest that concurrent use of strongly-cytotoxic agents is not the best way of combination for GO. In contrast to SWOG trial, the present study targeted relapsed/refractory AML cells, employed pretreatment of low-dose DAC, and demonstrated preclinical possibility that the combination of DAC and GO would be useful for relapsed/refractory AML.