Primary Waldesntrom Macroglobulinemia Cells Harbor Constitutive Activation of Akt, mTOR, Rictor and Raptor: Rational for Testing a Dual Inhibitor of the PI3K/Akt and mTOR Pathways in This Disease.

The PI3K/Akt and mTOR pathways play a pivotal role in the initiation and progression of malignancies, enhancing cell survival by stimulating cell proliferation and inhibiting apoptosis. Therefore, it is critical to examine therapeutic agents that explicitly target this pathway, specifically in tumors that harbor activation of the PI3K/Akt pathway, such as Waldenstrom macroglobulinemia (WM).

Primary-CD19+ bone marrow-derived WM cells, -bone marrow stromal cells, WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL), and primary normal CD19+ peripheral blood-derived (CD19+ PB)cells were used. Gene-expression and microRNA profiling have been performed on primary WM cells, as compared to CD19+ PB cells. Cytotoxicity, DNA synthesis, cell cycle and apoptosis were measured by thymidine uptake, MTT, PI staining, and Apo2.7/flow cytometry analysis, respectively. Cell signaling and apoptotic pathways were delineated by Western Blot and immunofluorescence analysis. In vivo homing has been assessed by in vivo flow cytometry.

Primary bone-marrow derived WM cells are characterized by lower expression of PTEN gene and protein; higher expression of pospho(p)-Akt, p-mTOR, rictor and raptor, as compared to their normal cellular counterpart (CD19+ PB cells). We also observed that microRNA-542-3p and -494 are more highly expressed in primary WM cells as compared to normal CD19+ PB cells (P<.01); and they both target PTEN, as predicted using TargetScan, PicTar, and miRanda algorithms, suggesting their role in inhibiting PTEN expression. We next assessed the effect of the dual PI3K/Akt and mTOR inhibitor NVP-BEZ235 (Novartis, Basel, Switzerland). This agent induced cytotoxicity and inhibited DNA synthesis (IC50 20-25nM) in BCWM.1 at 48 hours. Similar effects were demonstrated in all IgM secreting cell lines and in primary CD19+ WM cells (IC50 20-50nM). No cytotoxicity was observed on CD19+ PB cells, indicating selective toxicity of the compound on the malignant lymphoplasmacytic clone. NVP-BEZ235 inhibited p-Akt and p-mTOR, as well as the downstream Akt- targeted proteins GSK3a/b, p-S6R and p-p70S6, in a dose-dependent manner. Akt and mTOR in vitro kinase activity was also inhibited by NVP-BEZ235 treatment. In addition, NVP-BEZ235 inhibited both rictor and raptor, thus abrogating the rictor-induced Akt phosphorylation in WM cells. NVP-BEZ235 also induced significant cytotoxicity in WM cells in a caspase-dependent and -independent manner, through targeting the forkhead box transcription factors. Finally, NVP-BEZ235 targeted WM cells in the context of bone marrow microenvironment evidenced by significant inhibition of migration, adhesion in vitro and homing in vivo.

These studies therefore show that dual targeting of the PI3K/mTOR pathway represents a promising therapy for tumors that harbor activation of the PI3K/mTOR signaling cascade such as WM.

Ghobrial:Millennium : Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.