Overexpression of Carboxylesterase-2 Results in Enhanced Efficacy of Topoisomerase I Inhibitor, Irinotecan (CPT-11), for Refractory Multiple Myeloma.

Abstract

[Background] Multiple myeloma (MM) still remains an incurable disease even after the development of novel agents including immunomodulatory drugs and proteasome inhibitors, thus needs alternative agents especially for the refractory subtypes harboring t(4;14)(p16;q32). Since constitutive expression of topoisomerase I (TopoI) in MM cells and the efficacy of SN-38, an active metabolite of irinotecan (CPT-11), have been reported (Br J Haematol 83:68, 1993; Cancer Res 64: 8749, 2004), we investigated on the therapeutic potential of the CPT-11.[Method] Eight MM together with lymphoma cell lines were examined for cellular proliferation and viability in the presence of various concentrations of CPT-11 or SN-38 (gift from Yakult Inc., Tokyo) by means of 3H-thymidine uptake and MTS assays. Total RNA prepared from purified plasma cells derived from MM and reactive plasmacytosis patients was quantified for the mRNA levels of carboxylesterase-2(CE-2), CE-1, TopoI, UGT-1A and ABCG2, which had been reported to be associated with either sensitivity or toxicity to CPT-11. To ensure responsive roles of CE-2 in drug sensitivity, we established U266-derived stable transfectants overexpressing CE-2 (U266/CE-2) using hCE-2 cDNA kindly provided by Dr M.E. Dolan (Univ. of Chicago). To make sure in vivo effect, SCID mice were inoculated subcutaneously with 3×107 KMS-11 cells together with Matrigel. At the day of (prophylactic model) and at the 13 days after inoculation (therapeutic model), mice began to be treated by weekly intra-peritoneal injection with CPT-11 (25 mg/kg) or saline as control. Tumor size was validated until 28 days after treatment. Serum free-light-chain (FLC) levels were also measured on day 28.[Results and Discussion] CPT-11 and SN-38 inhibited DNA synthesis of most MM cells to <15% and to <10% with 2 μg/mL and 2 ng/mL, respectively. In 4/8 MM cell lines, IC50 was <2 μg/mL for CPT-11 and <2 ng/mL for SN-38. The efficacy of CPT-11 was observed in MM cells with any types of chromosomal translocations including t(4;14) or with resistance against any types of chemotherapeutic agents. Such efficacy was partly explained by expression level of CE-2, which plays a major role in catalyzing CPT-11 to its bioactive form, SN-38, being higher in MM than lymphoma cells. The highest CE-2 level was observed in reactive plasma cells, i.e., normal counterpart of the MM cells. IC50 for CPT-11 was 3.0 and 0.13 μg/mL for U266/mock and U266/CE-2 cells, respectively, indicating the importance of the CE-2 in terms of the sensitivity to CPT-11. On the other hand, expression level of CE-1, TopoI, UGT-1A and ABCG2 did not seem to be associated with its sensitivity in MM. In murine xenograft model, CPT-11 significantly reduced the tumor volume at day 26 when compared to the controls from 3,986±1,338 to 0±0 mm3 in prophylactic and from 10,397±2,881 to 5,375±3,594 mm3 in therapeutic models. Serum kappa FLC produced by KMS-11 cells was lower in the treatment group as well. Taken together, efficacy of the CPT-11 for MM can be validated in the clinical trial setting, as Cmax after 100 mg/m2 infusion reaches to 1.87 μg/mL, probably followed by efficient generation of intracellular SN-38 in MM cells by highly expressed CE-2.