Treosulfan is an effective inducer of cell death in myeloma cells

Meinhardt, G. H.; Eppinger, E.; Jahrsdoerfer, B.

Multiple myeloma (MM) is a malignant B-cell disorder that remains incurable despite recent advances utilizing high-dose chemotherapy followed by autologous stem cell transplantation. Median survival after relapse is usually short with myeloma cells being largely resistant to conventional therapeutic strategies. Thus, we attempted to identify cytotoxic compounds that could represent alternatives to current mephalan-based frontline or salvage treatment strategies in MM. The alkylating agent treosulfan, with has been used in the treatment of ovarian cancer for over twenty years, demonstrated strong cytotoxicity against myeloma cells in in vitro assays. NCI-H929 and U-266 myeloma cells were incubated with various concentrations of melphalan or treosulfan. Cell death was independently assayed by flow cytometry using unfixed cells (Annexin V/PI staining) or fixed cells (hypodiploid DNA content). Increasing concentrations and exposure time of treosulfan strongly altered the ratio of viable (Annexin-/PI-) vs. apoptotic/necrotic (Annexin+) cells. Whereas this ratio was approximately 72:25 in untreated NCI-H929 control cells, 30uM and 100uM treosulfan lead to a ratio of 41:50 and 1:83 after a two day exposure, respectively. Moreover, a four day incubation resulted in more than 61% cells being apoptotic or necrotic with treosulfan concentrations of 10uM and 89% with 30uM. Using subG1 phase analysis of fixed, propidium iodide stained cells, the amount of dead cells was 15% and 47% after 2 days and 40% and 72% after 4 days of treatment with 25uM or 100uM treosulfan, respectively. In comparison, a two day incubation with 10uM or 30uM melphalan resulted in 37% or 56% and a four day course in 47% and 67% cell death using subG1 phase analysis of fixed cells. Similar results were obtained with U-266 cells. Melphalan, used as reference compound, induced cell death with a ratio of 77:21 (viable vs. apoptotic/necrotic) and 56:41 after a 2 day incubation with 10 and 30uM melphalan, respectively. In comparison, analysis of subG1 phase cells demonstrated 8% and 41% cell death after 2 days and 15% and 42% cell death after 4 days of 10 and 30uM melphalan, respectively. Treosulfan treatment resulted in 6% and 18% subG1 phase cells after 2 days and 20% and 50% after 4 days of a dose level of 25uM and 100uM. Moreover, treosulfan induced the appearance of 19% and 21% Annexin V positive cells after a 2 day incubation and of 43% and 63% after a 4 day incubation with 10uM and 30uM treosulfan, respectively. In this context, it has to be noted, that very high plasma levels of treosulfan can be achieved at a conventional dose level of e.g. 8g/m2, although recently the mean tolerated i.v. dose was determined to be 47g/m2. Preliminary data were also obtained from bone marrow samples of patients with MM. A 48h incubation of mononuclear cells from bone marrow aspirates with 100uM treosulfan lead to a moderate increase in the number of propidium iodide (PI) positive CD138- cells, indicating cell death of non-myeloma cells. In contrast, the fraction of CD138+ cells displaying PI positivity was 2-3 fold higher reaching up to 90% in individual patients. Interestingly, treosulfan seems to be at least as potent as melphalan in these experiments. Mature data including statistical analysis and further studies with drug combinations will be presented at the meeting. Taken together, treosulfan has promising potential as a candidate drug for clinical studies in myeloma patients.