Rational for the Use of a Targeted-Therapy Using ABT-737 In Mantle-Cell Lymphoma

Abstract 770

Mantle-cell lymphoma (MCL) is still considered as incurable, thus new therapeutic approaches are needed. The anti-apoptotic protein Bcl-2 is known to be over-expressed in MCL and implicated in drug resistance. Therefore, there is a strong rational for the use of a Bcl-2-targeted therapy in MCL. The BH3-only mimetic ABT-737 (Abbott) is a potent specific inhibitor of Bcl-2, Bcl-x_L and Bcl-w but not of Mcl-1. ABT-737 have been investigated in several hematologic malignancies cell lines, such as chronic lymphoid leukemia (CLL) and diffuse large B cell lymphoma, and showed promising results. In this study, we investigated the anti-tumoral effect of ABT-737 in the setting of MCL using MCL cell lines and MCL patients' samples. Our aims were also to identify prognostic biomarkers that may predict MCL tumor cells sensitivity to ABT737 and to study the anti-MCL activity of ABT737 alone or in combination.

Five well characterized MCL cell lines were used: JEKO-1, MINO, REC-1, GRANTA-519 and UPN-1. Cytotoxicity of ABT-737 was assessed by flow cytometry using APO 2.7 staining. MINO and GRANTA 519 cell lines were highly sensitive (EC50= 20 nM after 24 hours of treatment) and apoptosis occurred rapidly within 2 hours following treatment as demonstrated by caspase 3 cleavage. In contrast, the three other cell lines (JEKO-1, REC-1 and UPN-1) were resistant (EC50> 4μM) to ABT-737. Western Blot analysis revealed that a major difference between sensitive cell lines (MINO and GRANTA-519) compared to other cell lines was their Bcl-2^high/Mcl-1^low profile. We also investigated ABT-737-induced apoptosis in primary MCL tumor cells (n=12). Seven patients' samples were sensitive to ABT-737 (median EC50 of 20 nM), whereas the 5 others samples were resistant (EC₅₀ not achieved). As observed in MCL cell lines, western blot analysis revealed that primary tumor cells showing a Bcl-2^high/Mcl-1^low profile were associated with sensitivity to ABT-737. In contrast a Mcl-1^high profile was associated with resistance to ABT-737. Taken together our investigation in both MCL cell lines and primary tumor cells from patients suggested that expression level Mcl-1 could influence ABT-737-induced apoptosis. Flavopiridol is a cyclin dependant kinase inhibitor and known to induce a transcriptional down-regulation of Mcl-1 in multiple myeloma and CLL. We therefore hypothesized that addition of Flavopiridol to ABT-737 could enhance induced-apoptosis. We first confirmed that two hours of treatment with flavopiridol induced a strong down-regulation of Mcl-1 at both mRNA and protein levels. Using suboptimal dose of ABT-737 (25 nM) and Flavopiridol (100 nM) for 24 hours, we observed a strong synergistic anti-MCL as evidence by a combination indice <1 according to the Chou-Talalay method.

In conclusion, ABT-737 at low nanomolar concentration induces a strong apoptosis in the subgroup of Bcl-2^high/Mcl-1^low MCL cells profile which represents around 50% of the MCL patients. Thus, this subgroup of patients appears to be good candidate for clinical trials evaluating ABT-737 alone. In the subgroup of Mcl-1^high patients, a specific down-regulation of Mcl-1 overcomes Mcl-1-induced resistance and synergizes with ABT-737. Indeed, our results strongly support the use of ABT-737 in MCL based on the concept of a targeted therapy according to the Bcl-2/Mcl-1 tumor cell profile. ABT-263, an orally bioavailable BH3 mimetic compound of the same class than ABT-737, is currently under investigation in various hematological malignancies, thus our investigation provides a biological rational for future clinical trials evaluating ABT-263 in combination or not with Flavopiridol in MCL patients.