Novel Agent Perifosine Enhances Antitumor Activity of Bortezomib, Rituximab and Other Conventional Therapies in Waldenstrom’s Macroglobulinemia.

Background: Waldenstrom’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with a median overall survival of 5 to 6 years, and most symptomatic patients succumb to disease progression. Current therapies used in upfront or relapsed settings include the monoclonal antibody rituximab, alkylator agents (e.g. chlorambucil), and nucleoside analogues (cladribine or fludarabine). With those agents, the overall response rates (ORR) ranges from 30 to 70%. The proteasome inhibitor bortezomib has recently demonstrated about 50% ORR in patients with relapsed WM. Therefore, new therapies are needed in WM to enhance the activity of current therapeutic regimens. Perifosine (NSC 639966; Keryx Biopharmaceuticals, NY) is a novel Akt inhibitor that demonstrated significant activity in vitro and in vivo in WM. Here, we examined the effect of combinations of perifosine with other conventional therapies for WM. Methods: chlorambucil (Sigma Aldricht), dexamethasone (Sigma Aldricht), doxorubicin (Sigma Aldricht), fludarabine (Berlex), rituximab (Genentech), bortezomib (Millenium), and melphalan (Sigma Aldricht) were tested alone or in combination with perifosine in WM cell lines (BCWM.1 and WM-WSU). Cytotoxicity was measured using the MTT growth inhibition assay. ADCC was measured by calcein-AM release on a Wallack Spectrafluor (485 nm excitation/535 nm emission) in the presence or absence of effector mononuclear cells for 4hrs (pretreated 24hrs with IL-2 100UI/mL, ratio effector:target (E:T) cells 40:1). Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method, with a combination index (CI)<1.0 indicating synergism. Results: Perifosine induced significant cytotoxicity, with an IC50 of 5–20uM. The following doses of single agent bortezomib and cytotoxic agents induced 50% growth inhibition (IC50): bortezomib 15–20nM, fludarabine 5ug/mL, doxorubicin 0.5–1nM, and melphalan 6–10uM. Dexamethasone 100nM and chlorambucil 100uM inhibited cell growth by 20%. Rituximab (10ug/mL, 1 hr) induced 121% specific lysis by ADCC in presence of effector cells. We then studied low doses of perifosine (5uM) in combination with these drugs. Perifosine 5uM induced 20% cytotoxicity at 48hrs, that was increased to 72% with fludarabine 5ug/mL (CI=0.521), to 60% with bortezomib 10nM (CI=0.49), to 67% with doxorubicin 0.1nM (CI=0.765), to 41% with dexamethasone 100nM (CI=0.335), to 50% with melphalan 5uM (CI=0.359), and to 42% with chlorambucil 50uM (CI=0.477). Finally, perifosine 10uM in combination with rituximab (10ug/mL, 1 hour) induces 193% specific lysis by ADCC mechanism in the presence of effector cells (p<0.001). Conclusion: The combination of perifosine with bortezomib, rituximab and other conventional agents used in the therapy of WM has synergistic activity. Those results provide the framework for clinical evaluation of combination of perifosine with other therapies in WM. XL and GO are co-first authors. Supported in part by an ASH Scholar Award.