Markedly additive antitumor activity with the combination of a selective survivin suppressant YM155 and alemtuzumab in adult T-cell leukemia

Abstract

Adult T-cell leukemia (ATL) is an aggressive malignancy of CD4(+)CD25(+) lymphocytes caused by human T-cell lymphotropic virus type 1. Currently, there is no accepted curative therapy for ATL. In gene expression profiling, the antiapoptotic protein survivin (BIRC5) demonstrated a striking increase in ATL, and its expression was increased in patient ATL cells resistant to the anti-CD52 monoclonal antibody alemtuzumab (Campath-1H). In this study, we investigated the antitumor activity of a small-molecule survivin suppressant YM155 alone and in combination with alemtuzumab in a murine model of human ATL (MET-1). Both YM155 alone and its combination with alemtuzumab demonstrated therapeutic efficacy by lowering serum soluble IL-2Rα (sIL-2Rα) levels (P < .001) and prolonged the survival of tumor-bearing mice (P < .0001). Moreover, the combination of YM155 with alemtuzumab demonstrated markedly additive antitumor activity by significantly lowering serum sIL-2Rα levels and improving the survival of leukemia-bearing mice compared with monotherapy with either YM155 (P < .001) or alemtuzumab (P < .05). More significantly, all mice that received the combination therapy survived and were tumor free >6 months after treatment. Our data support a clinical trial of the combination of YM155 with alemtuzumab in ATL. This trial was registered at www.clinicaltrials.gov as #NCT00061048.

Figures

Figure 1

Increased survivin expression in ATL patients’ PBMCs, MET-1 ATL tumor cells, and HTLV-1–infected cell lines. (A) Western blot analysis of survivin protein levels in PBMCs of ATL patients. (B) Western blot analysis of survivin protein levels in MET-1 ATL tumor cells and in HTLV-1–infected cell lines MT-2, Hut102, and CaGT1.

Figure 2

Increased survivin expression in the PBMCs of ATL patients after alemtuzumab treatment. (A) The expression of survivin mRNA levels was measured by real-time reverse transcription–polymerase chain reaction in the ATL PBMCs before and after alemtuzumab treatment (patients ATL8 and ATL11, 1 week after treatment; patients ATL9 and ATL10, 5-6 months after treatment). The fold change was calculated based on the survivin mRNA levels in the normal CD4 cells. (B) Western blot analysis of survivin protein expression levels in patients ATL9 and ATL10 before and after alemtuzumab treatment. (C) Flow cytometry analysis of leukemic cell population (CD3+CD25+) in ATL patients before and after alemtuzumab treatment. (D) Expression of transcription factor TCF4 mRNA in ATL10 and ATL11. Fold change was calculated based on TCF4 mRNA expression before treatment. (E) Expression of β-catenin mRNA in ATL10 and ATL11. Fold change was calculated based on β-catenin mRNA expression before treatment.

Figure 3

YM155 downregulated survivin expression, inhibited cell proliferation, and induced cell apoptosis in ATL leukemic cell lines ATL-43b and ATL-55(+). (A) ATL-43b and ATL-55(+) cells were cultured with medium alone or 1 nM, 10 nM, or 100 nM YM155 for 48 hours. Cells were harvested and analyzed for survivin expression by western blot analysis. (B) MTT cell proliferation assay of ATL-43b and ATL-55(+) cells treated with a serial dilution of concentrations of YM155 for 72 hours. (C) ATL-43b and ATL-55(+) cells were cultured with medium alone or 1 nM, 10 nM, 100 nM, or 1000 nM YM155 for 48 hours. The cells were then labeled with fluorochrome inhibitor of caspases reagent and analyzed by flow cytometry to detect cells with active caspase 3 and caspase 7. (D) ATL-43b and ATL-55(+) cells were cultured with medium alone or 1 nM, 10 nM, 100 nM, or 1000 nM YM155 for 48 hours. The cells were then fixed and labeled with antibody to cleaved PARP.

Figure 4

YM155, alemtuzumab, and the combination of YM155 with alemtuzumab treatment inhibited the growth of MET-1 ATL tumor cells in the MET-1 mouse model of human ATL. (A) The mean concentrations of tumor surrogate marker, human sIL-2Rα levels in MET-1–bearing mice before and after YM155, alemtuzumab, and the combination of YM155 with alemtuzumab treatment (2 and 4 weeks posttherapy). The animals treated with YM155 alone, alemtuzumab alone, and the combination of YM155 with alemtuzumab had significantly decreased values of sIL-2Rα when compared with those of the PBS control group 4 weeks posttherapy (YM155, P < .01; alemtuzumab, P < .01; the combination, P < .001). Furthermore, there were no detectable levels of sIL-2Rα in the mice that received the combination therapy 4 weeks posttherapy. (B) The mean concentration of tumor surrogate marker, human sIL-2Rα levels in MET-1–bearing mice 8 weeks after treatment with YM155, alemtuzumab, and the combination of YM155 with alemtuzumab. The animals receiving the combination of YM155 with alemtuzumab had no detectable levels of sIL-2Rα 8 weeks posttherapy. N.D., not detected.

Figure 5

YM155 and its combination with alemtuzumab significantly prolonged the survival of MET-1 leukemia-bearing mice. The animals treated with YM155 alone, alemtuzumab alone, and the combination of YM155 with alemtuzumab had significantly prolonged survivals when compared with the PBS control group (P < .0001). The combination of YM155 with alemtuzumab significantly prolonged the survival of leukemia-bearing mice when compared with YM155 alone (P < .001) or alemtuzumab alone (P < .05). *Two tumor-unrelated deaths excluded from the analysis are described in the “Materials and methods.”