Preclinical Studies of Cytotoxicity, Drug Synergy and Biological Correlates of Clofarabine Against Infant Leukemia Cells

Primary leukemic cells, cell lines derived from iALL patients and cell lines carrying the molecular abnormalities commonly found in iALL, were used in this study (n=10). Karyotypic abnormalities of these cells include: t(11;19) (q23;p13), t(9;11)(p21;q23) and t(4;11)(q21;q23). With respect to Flt-3 expression, cell lines demonstrating wild-type, internal tandem duplication (ITD) and over-expression phenotypes were also included in this panel. Primary infant AML samples (n=2) and the infant AML cell line TIB-202 (THP-1) containing the t(9;11)(p21;q23) rearrangement and MLL-AF9 fusion gene were also included. ALL cell lines derived from pediatric patients (n= 5) were evaluated in parallel. Stromal cells established from normal bone marrow specimens and peripheral blood mononuclear cells were evaluated under identical conditions for assessment of non-specific toxicity. An increasing concentration of clofarabine was added to leukemic and control cells (10⁴ cells per well, in 96 well plates) cell lines. Over the following four days, cell growth inhibition was measured by the Alamar blue assay. For drug combination studies, leukemia cells were incubated with a panel of conventional and targeted therapeutic agents (n=12) alone or in combination with clofarabine (IC₁₀ or IC₂₅ concentrations). Growth inhibition under each condition was measured and combination indices were calculated according to established methods. Induction of apoptosis and the release of mitochondrial mediators were measured by Western blot analysis. Alteration in mitochondrial integrity was evaluated by immunocytochemistry for fluorescent labeled anti-mitochondrial Hsp70 and real-time imaging.

Clofarabine inhibited growth of all iALL cells tested with IC₅₀ values ranging from 0.1 μ M to 0.01 μ M. Primary iAML cells were found to be most sensitive to clofarabine. For iALL cell lines the highest IC₅₀ value was found in Bel-1 cells, expressing a t(4;11)(q21;q23) karyotype. Drug combination studies showed significant synergy with 17-AAG (Hsp90 inhibitor, CI 0.7), sorafenib (CI 0.12), bortezomib (CI 0.3) and rapamycin (CI 0.2). No drug combinability was noted, with conventional alkylating agents and antimetabolites. Interestingly, the therapeutic opioid methadone (D,L-methadone hydrochloride), used extensively in the treatment of cancer pain and opioid addiction, showed significant synergy with clofarabine at low concentrations (CI 0.74, range 0.66 – 0.79 μ M). Incubation of cells with clofarabine (IC₂₅) for 48 hours resulted in detectable activation of caspase 9 and cleavage of PARP.

We demonstrate the ability of clofarabine to induce cytotoxicity against a panel of leukemia cells that carry the molecular aberrations and growth properties seen in iALL. We also present data on the biological correlates and synergistic effects of clofarabine with other anti-leukemic agents. Of particular interest is the synergy with methadone, which has been shown previously to affect mitochondrial activity in leukemia cells. Data presented in study provide key initial data to construct effective xenograft studies and to formulate a clofarabine based treatment protocol for iALL in the near future.

Narendran:Genzyme Canada: Research Funding.