RAD001 Exerts Anti-Tumor Activity in Waldenstrom Macroglobulinemia.

Waldenstrom Macroglobulinema (WM) is an incurable B-cell disorder characterized by the presence of IgM monoclonal gammopathy and bone marrow infiltration of lymphoplasmacytic cells. Apart from promising advances in therapeutic strategy, response rates and treatment-free survival remain inconsistent across prescribed regimens. As we have previously shown, the PI3K/Akt pathway actively mediates tumor growth, survival, migration, and cell cycle within primary WM cells. An active target of this pathway includes the downstream mammalian target of rapamycin (mTOR). By inhibiting the mTOR pathway with RAD001 (Afinitor™, Novartis Pharmaceuticals), we hoped to constructively regulate aforementioned cellular function, and also understand from a preclinical perspective its specificity of action in our microenvironmental models, both alone and the presence of monoclonal antibody against CD20 and proteasome inhibitor.

WM cell lines (BCWM1) and IgM secreting cell lines MEC1 and RL were used. Bone marrow stromal cells (BMSC) were obtained from patients with WM. Peripheral blood mononuclear cells were harvested from healthy donors. Agents tested included RAD001, Rituximab and Bortezomib. Cytotoxicity, DNA synthesis, and cell cycle were measured using MTT assay, [³H]-thymidine uptake, and flow cytometry/PI staining, respectively. Antibody-dependent cellular cytoxicity (ADCC), transwell, and Matrigel assays were preformed to measure cell lysis, SDF-1 chemotaxis-induced migration, and angiogenesis, respectively. Adhesion to fibronectin has been evaluated with WM cells and BMSC in the presence of RAD001, with and without Bortezomib.

RAD001 induced cytotoxicity and inhibition of DNA synthesis with an IC₅₀ of 1-10 nM in BCWM1 cells at 48 hours. Similar effects were seen in IgM cell lines with an effective dose of 0.1-1 nM. In contrast, at IC₅₀ treatment level donor cells displayed no significant cytotoxicity (>85% survival). Cell cycle analysis showed corresponding G1 arrest, and angiogenesis was also inhibited. RAD001, Bortezomib, and Rituxan combinations showed synergistic cytotoxicity, which was attenuated when WM cells were co-cultured with BMSC. Migration of BCWM1 and adhesion of BCWM1 to BMSC was reduced under presence of RAD001.

These functional assays therefore show that RAD001 has significant and synergistic antitumor activity in WM which will inform the design of future clinical trials.

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