CDDO and CDDO-Im Reduce Tumor Burden in a Transgenic Mouse Model of CLL. (2004)

Abstract Transgenic mice over-expressing in B lymphocytes both Bcl-2 and a TRAF2 mutant lacking the N-terminal RING and zinc finger domains (TRAF2DN), which mimics TRAF1, develop small B cell lymphoma and leukemia that have remarkably similar characteristics to human chronic lymphocytic leukemia (CLL). TRAF2DN/Bcl-2 mice develop over time leukemia, severe splenomegaly, and lymphadenopathy, which are associated with monoclonal and oligoclonal B cell neoplasms. The lifespan of TRAF2DN/Bcl-2 mice is markedly reduced compared to Bcl-2 and TRAF2DN single transgenics or wild-type littermates. The expanded B cell population in the blood of leukemic TRAF2DN/Bcl-2 double transgenic mice is primarily comprised of small-medium size, non-cycling B220M/IgMH/IgDL/CD21L/CD23−/CD11b+/CD5+ cells that were Bcl-6 negative, consistent with a B-1 phenotype, closely resembling their human CLL counterparts. Indeed, these B cells showed comparable proliferation rates to normal B-cells, but exhibited markedly increased survival and were resistant to apoptosis induced by chemotherapeutic agents and glucocorticoids. We studied the effects of synthetic triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid (CDDO) and its imidazolide derivative (CDDO-Im) on cultured B-cells from the TRAF2DN/Bcl-2 transgenic mice. Both CDDO and CDDO-Im efficiently induced apoptosis of these cells in vitro, although CDDO-Im was approximately 10-times more potent than CDDO (LD50: 0.35μM CDDO-Im vs 3.8 μM CDDO). To study the effect of CDDO and CDDO-Im in vivo, groups of TRAF2DN/Bcl-2 mice that had developed leukemia were injected i.v. with liposomes alone or liposomes containing either CDDO or CDDO-Im, at a dose of 20 mg/kg/day. Each mouse received a total of nine injections administered over a period of 22 days. The concentration of B cells in the blood of these mice was monitored daily after each injection, using a mini-FACS (Guava Technologies, Inc.). CDDO-treated mice showed a steady reduction in the number of leukemic cells in blood during the treatment and this tendency was maintained 10 days after the last treatment. In contrast, CDDO-Im treated mice showed a striking increase in the concentration of B cells in blood (B220+ events) immediately after the first inoculation. One mouse of this group died after the first injection, and 2 more mice died after 5 injections. Only 2 mice treated with CDDO-Im survived the full treatment, showing a striking reduction of leukemic cells in blood by the end of the treatment. Administration of empty liposomes had no inhibitory effect on the leukemia, and mice in this control group had massive splenomegaly (1431±323 mg; n=3) and severe disseminated lymphadenopathy. In contrast, CDDO-treated mice had less severe splenomegaly (938±234; n=4) but still had severe lymphadenopathy. CDDO-Im treated mice showed a dramatic reduction in the spleen size that was evident also in those mice that died after 5 injections (474±185 mg; n=4) and had no signs of lymphadenopathy. Although preliminary, these results indicate that in vivo administration of CDDO and CDDO-Im reduced the tumor burden in a transgenic model of CLL, and illustrate the potential of triterpenoids as single agents for the treatment of CLL.