No abstract

Chronic lymphocytic leukemia (CLL) cells express high-levels of Bcl-2-family proteins that inhibit apoptosis, enhancing leukemia-cell survival and drug-resistance. AT-101 (Ascenta Therapeutics, Inc.) is an orally active BH3-mimetic that inhibits the anti-apoptotic activity of Bcl-2, Bcl-XL, and Mcl-1, and induces CLL cells to undergo apoptosis. (Prada et. al., ASH 2005). AT-101 also enhanced the cytotoxicity of rituximab for CLL cells in vitro (James et. al., ASH 2005). These in vitro effects of AT-101 were concentration and time-dependent. We are conducting a phase 2 trial to evaluate the safety and activity of AT-101 by two dosing schedules, used in combination with rituximab to treat patients with relapsed/refractory CLL. We previously reported (Castro et. al., ASH 2006) on 12 patients who received up to 12 weeks of AT-101, 30 mg daily for 3 out of every 4 weeks, with rituximab, 375 mg/m2 for 12 doses (total dose = 4,500 mg/m2) on days 1, 3, 5, 8, 15, 22, 29, 31, 33, 40, 57, 59, 61. Here we describe initial results from a second cohort (n=6) treated with intermittent, “pulse” AT-101, 80 mg/d on days 1–3 and 15–17 of each 28-day cycle, in combination with weekly rituximab, 375 mg/m2/week. To date, 6 patients have received “pulse” AT-101. Patient demographic characteristics and risk prognostic status (ZAP70, IgVH mutational status, and cytogenetics / FISH) are comparable between the two dose cohorts. Gastrointestinal (GI) toxicity, the most notable adverse effect of AT-101 with daily administration, appears reduced with intermittent AT-101; 2/6 patients have had NCI-CTCAE Grade 1–2 GI toxicity, and 0/6 have had Grade 3–4 ileus, compared with 11/12 and 2/12 patients, respectively, in the daily dose cohort. Apoptosis of CLL cells evaluated by Annexin V FACS at the time of maximum AT-101 concentration, was present in 18–45% of cells in 4 of the 6 patients after a single 80 mg dose of AT-101. By comparison, apoptosis after a 30 mg AT-101 dose appeared lower and was detected in approximately 1–15% of cells. After 80 mg of AT-101, plasma concentrations of up to 6.6 μM have been observed compared with concentrations of approximately 0.8–1.8 μM after a 30 mg dose in the daily dose cohort. In the “pulse” AT-101 cohort we have observed partial responses (PR) in 3 patients while the other 3 are still receiving treatment. Five (5) out of 12 patients had a PR in the previously reported AT-101 continuous administration group. Intermittent administration of AT-101 with a “pulse” dose regimen appears associated with an increased pro-apoptotic effect in vivo and higher plasma concentrations, as well as reduced toxicity, when compared with daily dosing. Enrollment continues to confirm these observations and assess whether clinical activity in combination with rituximab is increased with “pulse” dosing of AT-101.

No abstract

Chronic lymphocytic leukemia (CLL) is a disease characterized by the monoclonal accumulation of well-differentiated CD5 B cells. Previously, we reported that CLL cells that use unmutated immunoglobulin VH (IgVH) genes and/or express ZAP-70 are more responsive to signaling induced by ligation of surface IgM compared to cells that use mutated IgVH and lack expression of ZAP-70 (Chen et al. Blood 2005) Because signaling through the Ig receptor appears to play a role in the pathogenesis or progression of CLL, targeting the Ig signal transduction pathway in CLL might have therapeutic utility, particularly for those patients with high-risk disease. Dasatinib (Sprycel) is a tyrosine kinase inhibitor (TKI) with antiproliferative activity against hematological and solid tumor cell lines and it is FDA approved for the treatment of patients with CML and Ph+ ALL. Contrary to Imatinib (Gleevec), Dasatinib is a potent TKI not only of the Abl family of kinases but also of Src kinases, which regulate Ig-receptor signal transduction and govern the early events following Ig receptor ligation. Because Dasatinib TKI profile and its potential role inhibiting BCR signaling we studied its in vitro activity in CLL. Primary leukemia cells from 40 different CLL patients were evaluated. We found that Dasatinib, but not Imatinib, induced apoptosis in CLL cells at doses that were pharmacologically achievable. The IC50 for Dasatinib was in the 30–100 nM range. Interestingly, the pro-apoptotic activity of Dasatinib in CLL cells was not observed in normal B, T cells or blood mononuclear cells of healthy donors, suggesting that Dasatinib has a specific effect on CLL cells. Dasatinib induced apoptosis in CLL cells in a time and concentration dependent manner. Peak apoptosis occurred after 2 hours of in vitro exposure. Leukemia cells that expressed ZAP-70 were significantly more sensitive to Dasatinib-induced apoptosis than CLL cells lacking expression of ZAP-70. In addition, Dasatinib enhanced in vitro the pro-apoptotic activity of Rituximab and Fludarabine in CLL cells. Dasatinib treatment of CLL cells induced changes in the expression profile of apoptosis related genes as well as changes in apoptosis related proteins such as cleavage of PARP-1 and Caspases. Moreover, CLL cells treated in vitro with Dasatinib showed reduced tyrosine kinase activity measured by ELISA and also by immunoblots of CLL-cell lysates using specific phospho-TK antibodies. In addition, Ig receptor signaling following surface IgM ligation was decreased in CLL cells that were pre-treated with Dasatinib relative to that of untreated CLL cells. In conclusion, Dasatinib induces apoptosis of CLL cells at low nanomolar concentrations that do not appear to affect the viability of B cells, T cells, or blood mononuclear cells of healthy adults. CLL cells that expressed ZAP-70 were significantly more sensitive to Dasatinib than CLL cells that lacked expression of ZAP-70. This process was associated with impairment of BCR signaling, decrease TK activity and regulation of genes and proteins related to apoptosis. In addition, treatment of CLL cells with Dasatinib enhanced the in vitro activity of Rituximab and Fludarabine. These results reveal that Dasatinib is potentially active in CLL, providing a rationale for clinical trials evaluating its clinical activity in the treatment of patients with this disease.