Cotargeting of PIM, PI3K and Mtor in Mantle Cell Lymphoma (MCL)

Introduction: Mantle cell lymphoma (MCL) comprises about 6% of all non-Hodgkin's lymphoma with a median survival of 3-5 years. Constitutional activation of the mTOR/AKT pathway has been identified in the majority of cases (Rudelius, Blood 2006). The pro-viral insertion in murine (PIM) lymphoma proteins are serine/threonine kinases which play a critical role in cell survival as well as proliferation and identifies a high risk patient cohort with MCL (Hsi, Leuk Lymphoma 2008). In this study we evaluated the efficiency and mode of action of a dual PIM/PI3K (IBL-202) and a triple PIM/PI3K/mTOR inhibitor (IBL-301) in MCL cell lines and primary cells.

Methods: MCL cell lines (Granta 519, Jeko-1, Rec-1 and Mino), as well as primary MCL cells were exposed to the combined PIM-kinase/PI3K (IBL-202) and the PIM-kinase/PI3K/mTOR Inhibitor (IBL-301). Cell proliferation (trypan blue staining), cell apoptosis (Annexin V PE/7-AAD staining) and cell cycle (FACS) were investigated. Protein expression and phosphorylation status of different downstream proteins (Akt, GSK-3β, 4EBP1) as well as markers of apoptosis (PARP, Caspase 9) were analysed after 1h, 4h, 8h and 24h. Cell viability was assessed by CellTiter-Glo® assay after 48h.

Results: Both inhibitors led to G1 arrest. At 500 nM, the triple inhibitor IBL-301 (19,8%) is in average slightly more efficacious than the dual-inhibitor IBL-202 (13,5%). Accordingly, IBL-301 had a much higher impact on cell proliferation than IBL-202 in all tested MCL cell lines (reduction by 48 - 93% vs 22 - 87%), possibly due to its mTOR inhibitory potential, although it may be also a more potent inhibitor of PIM and PI3K kinases. In addition, treatment with IBL-202 and IBL-301 induced cell apoptosis in Jeko-1, Rec-1 and Mino. Again, rate of apoptosis by IBL-301 was much higher (e.g. JEKO: 56% vs 13%) and could be achieved at lower concentrations in comparison to IBL-202. The differential impact on apoptosis could be confirmed based on PARP and Caspase 9 cleavage, which was higher after treatment with IBL-301 after 24h. In Jeko-1, Granta-519 and Mino both agents led to de-phosphorylation of Akt. Interestingly, this effect was more prominent in IBL-301 treated cell lines, supporting the mode of action via the PI3K-AKT pathway of both inhibitors. De-phosphorylation of GSK-3β was observed in all tested MCL cell lines with both inhibitors already during the first hour of exposure and was reversible thereafter.

Primary MCL cells of 2 patients were treated with 62.5 nM IBL-202, 31.25 nM IBL-301 and single inhibitors of PIM (2.5 µM AZD1208), PI3K (1.25 µM idelalisib) and mTOR (5 nM temsirolimus). Viability after 48h was reduced by about 70% following IBL-301 exposure compared to 39% in IBL-202 treated samples. Both combined inhibitors were more potent than any of the single inhibitors. IBL-301 and IBL-202 decreased viability in a similar way as the combination of AZD1208, idelalisib +/- temsirolimus. Normal lymphocytes tolerated both inhibitors in various concentrations (62,5 - 500 nM).

Conclusions: Triple inhibition of PIM kinases, PI3K and mTOR is very efficient in MCL cell lines as well as in primary MCL cells, exceeding dual inhibition of PIM kinases and PI3K. Thus, cotargeting PIM kinases, PI3K and mTOR may be a promising novel approach for clinical development in MCL.