Tel : (33) 2 40 08 32 63; Fax : (33) 2 40 08 32 50 and Dr Martine Amiot, INSERM UMR 892 équipe 10, Nantes, France. Email: Martine.Amiot@univ-nantes.fr. Tel. : (33) 2 40 08 47 68; Fax : (33) 2 40 08 47 78 Running title: ABT-737 in mantle cell lymphoma TRANSLATIONAL RELEVANCE: Prognosis of MCL is considered to be inferior to most of the other NHLs and new therapeutic approaches are warranted. The rational to target Bcl-2 proteins in MCL has been investigated using ABT-737, a BH3 mimetic that

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... ts Bcl-2, Bcl-x L but not Mcl-1. We show that ABT-737 alone induce apoptosis only in Bcl-2 high /Mcl-1 low MCL cells. By the use of siMcl-1 transfection, we demonstrate the key role of Mcl-1 in ABT-737 resistance. To overcome Mcl-1 resistance in clinical practice, we investigated two different strategies using three different drugs in combination with ABT-737: (i) to down-regulate Mcl-1 expression using flavopiridol or ara-C and (ii) to neutralize Mcl-1 anti-apoptotic function by increasing Noxa with Bortezomib. Our investigations confirm the synergistic effect of these combinations in ABT-737-resistant cell lines. In conclusion, the present work brings the rationale for the use of ABT-737 alone or in combination according to the apoptotic profile of MCL cells. Research. on July 15, ABSTRACT: Purpose Mantle cell lymphoma (MCL) is considered to be incurable. ABT-737 is a BH3 mimetic that targets Bcl-2, which is over-expressed in MCL and implicated in drug resistance. The present work investigated the anti-tumor effect of ABT-737. Experimental Design Six MCL cell lines and primary MCL cells (n=13) were used. Sensitivity to ABT-737 was assessed and expression levels of Bcl-2 and Mcl-1 were analyzed. Finally, ABT-737 was combined with other cytotoxic agents to promote tailored therapy. Results MINO and GRANTA-519 cell lines were highly sensitive to ABT-737 (LD 50 = 20 and 80 nM, respectively), whereas other cell lines were resistant. In primary MCL cells, 46% of patients' samples were sensitive to ABT-737. Analysis of protein expression levels revealed that both sensitive cell lines and primary MCL cells are characterized by a Bcl-2 high /Mcl-1 low profile, whereas resistant MCL cells contain high levels of Mcl-1. ABT-737 induced a rapid disruption of both Bcl-2/Bax and Bcl-2/Bik complexes. In addition, silencing of Mcl-1 by siRNA sensitized MCL cell lines to ABT-737. Similarly, flavopiridol which induces Mcl-1 down-regulation resulted in combination with ABT-737 in a synergistic anti-MCL effect in ABT-737 resistant cell lines. This synergy was also observed when ABT-737 was combined with either bortezomib or cytarabine. Conclusions The present work demonstrates that ABT-737 induces strong apoptosis in MCL cells expressing a Bcl-2 high /Mcl-1 low profile. In ABT-737-resistant MCL cells, down-regulation of Mcl-1 overcomes Mcl-1-induced resistance and synergizes ABT-737 effects. Our results strongly support the use of ABT-737 according to the Bcl-2/Mcl-1 tumor cell profiles in the treatment of MCL. Research. on July 15, studies have also shown that ABT-737 is effective as a single agent against some leukemia/lymphoma cell lines both in vitro and in vivo (11, (15) (16) (17) . This present study was focused on the anti-tumoral effect of ABT-737 in MCL. We also aimed to identify prognostic biomarkers that may predict MCL tumor cell responses to ABT-737. Using this approach, we have developed a rationale for ABT-737-based tailored therapeutic strategies in MCL. Research. on July 15, 18 24. de Guibert S, Jaccard A, Bernard M, Turlure P, Bordessoule D, Lamy T. Rituximab and DHAP followed by intensive therapy with autologous stem-cell transplantation as firstline therapy for mantle cell lymphoma. Haematologica. 2006;91:425-6. 25. Goy A, Bernstein SH, Kahl BS, Djulbegovic B, Robertson MJ, de Vos S, et al. Bortezomib in patients with relapsed or refractory mantle cell lymphoma: updated time-toevent analyses of the multicenter phase 2 PINNACLE study. Ann Oncol. 2009;20:520-5. 26. Gressin R, Caulet-Maugendre S, Deconinck E, Tournilhac O, Gyan E, Moles MP, et al. Evaluation of the (R)VAD+C regimen for the treatment of newly diagnosed mantle cell lymphoma. Combined results of two prospective phase II trials from the French GOELAMS group. Haematologica. 2010;95:1350-7. al. ABT-737, an inhibitor of Bcl-2 family proteins, is a potent inducer of apoptosis in multiple myeloma cells. Leukemia. 2007;21:1549-60. 30. Khoury JD, Medeiros LJ, Rassidakis GZ, McDonnell TJ, Abruzzo LV, Lai R. Expression of Mcl-1 in mantle cell lymphoma is associated with high-grade morphology, a high proliferative state, and p53 overexpression. et al. Flavopiridol, fludarabine, and rituximab in mantle cell lymphoma and indolent B-cell lymphoproliferative disorders. J Clin Oncol. 2010;28:418-23. 34. Lin TS, Ruppert AS, Johnson AJ, Fischer B, Heerema NA, Andritsos LA, et al. Phase II study of flavopiridol in relapsed chronic lymphocytic leukemia demonstrating high response rates in genetically high-risk disease.