YM155, a Small-Molecule Survivin Suppressant, Mainly Targets Primary CLL Cells Actively Proliferating and Overcomes Microenvironment-Mediated CLL Cell Protection

Abstract 3868

Chronic lymphocytic leukemia (CLL) has been historically considered an accumulative disease. However, recent studies demonstrate the relevance of proliferation centers found in lymphoid organs (lymph nodes [LN] and bone marrow [BM]),which provide survival and proliferation signals to CLL cells. In CLL, immunohistochemical analyses show that survivin is mainly expressed in CLL cells that are actively proliferating in proliferation centers. Interestingly, increased expression of survivin in peripheral blood CLL cells correlates with worse response to treatment. YM155 (Astellas®), a survivin suppressant, has demonstrated promising antitumoral activity in a wide variety of human cancer cell lines and xenograft models, and nowadays it is being evaluated in several phase II clinical trials. As survivin is usually expressed in proliferating cells, herein we tested the effects of YM155 on CLL cells co-cultured in a system mimicking the conditions that they are encountering in the proliferation centers. Primary CLL cells from 20 patients were co-cultured with BM stromal cells along with CD40 ligand and oligodeoxynucleotides-CpG. Optimal proliferative response was observed after 48 hours of co-culture (mean % Ki-67 expression, 1.52±0.06 in cells in suspension vs. 11.78±6.73 in co-culture, p<0.001). Therefore, CLL cells were subsequently treated with YM155 and/or fludarabine at this time point. By Western Blot, increasing survivin expression in co-culture, was completely abrogated by treatment with YM155 50nM for 24 hours. Induction of apoptosis was assessed by flow cytometry (FC) measurement of annexin V and propidium iodide (PI). Mean LD50 of YM155 was 430.4 nM (95%CI 227.3–815.3 nM) for CLL cells cultured in suspension, whereas it was 7.7 times lower (56.25nM, 95%CI 39.71–79.7 nM) in proliferative conditions, revealing that apoptotic cell death induced by inhibition of survivin is mainly affecting proliferating CLL cells. Unlike treatment with YM155, CLL cells treated with 5μg/ml fludarabine showed significantly higher viability in co-culture than in suspension (normalized mean of viability: 60.2%±16.9% vs. 23.4%±7.1%, p<0.001), thus confirming the microenvironment-induced chemoresistance. Of note, the addition of 50nM YM155 overcame this chemoresistance by significantly increasing fludarabine-induced cytotoxicity (normalized mean of viability: 42.1%±7.6% for fludarabine vs. 5.2%±1.9% for the combination; p<0.05). Prior to any treatment, 98.54%±0.12% of cells in suspension were arrested at G0/G1, whereas the co-culture increased the number of CLL cells in S/G2/M phase from 0.89%±0.31% on day 0 to 44.96%±23% at 72 hours (p=0.05). The addition of fludarabine, YM155, or the combination of both drugs after 48 hours of co-culture induced a significant G1 arrest at 72 hours (% cells in G1 phase: control, 55.04%±13.28%; fludarabine, 88.26%±7.94%; YM155, 81.61%±8.47%; combination, 90.75±4.68; p<0.01). Finally, YM155 treatment caused a drastic reduction of 23.31%±9.52% in Ki67 expression, whereas the combination of YM155 with fludarabine caused a significant decrease of 49.64%±4.12% (p<0.001), an effect that fludarabine alone could not achieve. Following this, we also analyzed the effect of YM155 on the proliferative compartment defined by Calissano, C et al (Mol Med, 2011; CD5high/CXCR4dim). Firstly, we observed that the co-culture induced an increase in this fraction (from 3.28%±1.12% to 9.66%±2.83%, p<0.05). Treatment with YM155 alone or combined with fludarabine reduced this proliferative fraction (control: 13.29%±4.21%; YM155: 5.82%±1.24%[p<0.05]; combination: 4.12%±1.14%[p<0.01]), whereas the proportion of the non-proliferative compartments where not affected by these treatments. Altogether, these data show that reproducing signals from the microenvironment in ex vivo co-cultures can promote proliferation and protect CLL cells from apoptosis induced by cytotoxic agents such as fludarabine. In this setting, YM155 treatment is able to efficiently overcome microenvironment-mediated cell protection and proliferation and has specific effect on actively proliferating CLL cells. Combination experiments revealed that YM155 strongly potentiates fludarabine cytotoxicity, especially in proliferating CLL cells chemoresistants to fludarabine alone. Finally, these results open the door to future clinical trials inhibiting survivin in CLL.