Effect of bendamustine on apoptosis and colony-initiating precursors in Hodgkin lymphoma cells.

Abstract

e18559 Background: Hodgkin lymphoma (HL) recurring after stem cell transplantation is incurable and active agents are needed in this setting. Bendamustine is a bi-functional alkylator active in non-Hodgkin lymphoma. Activity of bendamustine towards tumor cells of HL has never been previously described. Methods: A panel of human cell lines (L1236, L428, KMH2, HDLM2, L540), validated models for HL, was studied. Cells were exposed to increasing concentrations of bendamustine (12.5 to 100 µmol/L) and analyzed for proliferation, apoptosis and cell cycle changes. Effect of bendamustine on HL clonogenic precursors was assessed by primary/secondary methylcellulose assays and colony cells characterization (CD30, aldehyde dehydrogenase [ALDH] or Hoechst [Hcst]-33342). Results: Bendamustine displayed a significant time- and dose-dependent antiproliferative effect on all HL cell lines, with an observed IC50 between 25-50 µmol/L at 48 hrs. In all lines, at least 50% of cells showed Annexin-V binding following 24 hrs of exposure to concentrations lower than 100 µmol/L, with 70% to 90% of apoptotic cells at longer times (48-72 hrs). Sensitivity to Bendamustine of HL cells was independent from the presence of p53 mutations. In all HL lines, exposure to a concentration as low as 25 µmol/L induced >50% of cells to accumulate in the G2/M phase of the cell cycle with presence of tetraploid nuclei witnessing mitotic defects. Finally, a 24 hr-exposure to very low concentrations (IC50: 17-38 µmol/L) of bendamustine was able to inhibit primary colony formation in all HL cell lines irrespective of p53 status. Intriguingly, a significantly lower concentration of the agent (IC50: 3.1-15.0 µmol/L) was able to suppress formation of secondary colonies, which were sustained, in L428 and KMH2 cells, by CD30+/ALDH)hi or Hcsthi precursor cells. Conclusions: Bendamustine displays marked antiproliferative and pro-apototic effects on cultured HL cells, irrespective of p53 status, by promoting G2/M arrest and changes compatible with mitotic catastrophe. HL colony-initiating precursors are highly sensitive to extremely low concentrations of the agent. Clinical testing of bendamustine in HL both as single agent and in combination is warranted.