Cellular Aminopeptidase Inhibition as a Target for the Therapy of AML by the Novel Agent CHR 2797.

CHR 2797 is one of a new class of enzyme inhibitors with pleiotropic effects against cancer cells which is currently in Phase II clinical trials. It inhibits a number of the M1 family of metalloenzymes that include the Zn⁺⁺-dependent aminopeptidases. Aminopeptidases catalyze the hydrolysis of the terminal amino acids from short chain polypeptides, and are involved in the continuous cycle of protein formation and degradation in cells. As malignant cells may be more dependent on protein cycling than normal cells, interrupting this pathway is, therefore, a potential therapeutic target for novel agents. The effect of CHR 2797 on AML cells in-vitro was investigated to determine its effectiveness and mode of action. Treatment of leukemic cells with CHR 2797 was performed under standard culture conditions at a concentration range of 0.0002 - 20μM. The WST-1 reagent was used to determine cell viability at the end of the culture and the IC₅₀ values were calculated. Primary diagnostic AML samples (n=52) were analyzed and an IC₅₀ range of between 0.01 and >40μM was established, with a median of 1.2μM. There were no significant differences between the different classes of AML, although there was a trend towards a better response in the monocytic leukemias and the good prognostic cytogenetic groups. The comparative effects of CHR 2797 on normal bone marrow samples (n=10) were also analyzed. Under identical culture conditions, an IC₅₀ range between 6.2 and >40μM was found, with a median of 15μM. This 13-fold higher result demonstrates a significant potential therapeutic window for leukemic versus normal cells. The level of synergy or antagonism with conventional therapeutic agents was calculated using a combination index. Synergy was demonstrated in 69% of cell samples in combination with cytarabine, and 78% with bortezomib. Synergy was also shown in 62% of cells samples with ATRA, even in non-promyelocytic leukemia types. Annexin V staining and cell cycle analysis demonstrated apoptosis after treatment with CHR 2797 in many cases. A degree of differentiation of acute promyelocytic cells to mature myeloid cells was also stimulated with the treatment. To determine the mode of drug action, the effect of CHR 2797 on intact cell aminopeptidase activity was also measured. The activity of these enzymes can be measured by their conversion of the membrane-permeant substrate ala-MCA to MCA, which can be fluorimetrically detected. CHR 2797 was demonstrated to reduce intact cell aminopeptidase activity in a time- and dose-dependent manner. A reduction in aminopeptidase activity was shown with a concentration of CHR 2797 of less than 0.5μM; with 10μM drug the activity in many AML samples was reduced by >90%. CD13 is a cell surface protein which is expressed selectively on myeloid cells and is also classified as an aminopeptidase N. By comparison with the results obtained with the CD13 blocking antibody WM15, CHR 2797 was demonstrated to fully inhibit CD13 activity. Further investigations proved that other extracellular, cytoplasmic and nuclear aminopeptidase enzymes were also inhibited. In conclusion new treatments are needed for acute myeloid leukemia to improve survival and circumvent the toxicity of conventional therapy. This study demonstrates that CHR 2797 might be an effective molecular therapy for AML, either alone or in combination with other chemotherapeutic agents.