Aldehyde dehydrogenases inhibition eradicates leukemia stem cells while sparing normal progenitors

Abstract

The vast majority of patients with acute myeloid leukemia (AML) achieve complete remission (CR) after standard induction chemotherapy. However, the majority subsequently relapse and die of the disease. A leukemia stem cell (LSC) paradigm has been invoked to explain this failure of CR to reliably translate into cure. Indeed, LSCs are highly enriched in CD34+CD38- leukemic cells that exhibit positive aldehyde dehydrogenase activity (ALDH+) on flow cytometry, these LSCs are resistant to currently existing treatments in AML such as cytarabine and anthracycline that, at the cost of great toxicity on normal cells, are highly active against the leukemic bulk, but spare the LSCs responsible for relapse. To try to combat the LSC population selectively, a well-characterized ALDH inhibitor by the trivial name of dimethyl ampal thiolester (DIMATE) was assessed on sorted CD34+CD38- subpopulations from AML patients and healthy patients. ALDH activity and cell viability were monitored by flow cytometry. From enzyme kinetic studies DIMATE is an active enzyme-dependent, competitive, irreversible inhibitor of ALDH1. On cells in culture, DIMATE is a powerful inhibitor of ALDHs 1 and 3, has a major cytotoxic activity on human AML cell lines. Moreover, DIMATE is highly active against leukemic populations enriched in LSCs, but, unlike conventional chemotherapy, DIMATE is not toxic for healthy hematopoietic stem cells which retained, after treatment, their self-renewing and multi-lineage differentiation capacity in immunodeficient mice, xenografted with human leukemic cells. DIMATE eradicates specifically human AML cells and spares healthy mouse hematologic cells.

Figure 1

Inhibition of ALDH by DIMATE is cytotoxic on human AML cell lines by promoting apoptogenic aldehyde accumulation inducing apoptosis. (a) Viability assay of AML cells to increasing concentration of DIMATE. All experiments were performed in triplicates (n=3). Values for all cell lines are showed in Supplementary Table 2. (b) Monitoring of aldehyde dehydrogenase activity in flow cytometry in HL-60. Graphic representations of average percentages of ALDH+ cells at H0, and after 24 h of cell culture, without and with DIMATE 5 μmol/l. (n=3). (c) Quantification of MDA and HNE adduct in HL-60 cells treated with DIMATE 5 μmol/l during 24 h. Adduct formation is higher in cells treated than with the vehicle. (d) Monitoring of caspase 3/7 activity of HL-60 cells treated with DIMATE 5 μmol/l. After 6 h of treatment, activation of caspase activity is observed.

Figure 2

Cytotoxicity profile of DIMATE, daunorubicine, cytarabine and azacytidine on CD34+CD38−ALDH+ leukemic cells and CD34+CD38− healthy hematopoietic stem cell. (a) Cytotoxicity profiles of DIMATE, daunorubicine, cytarabine and azacytidine after 48 h of treatment for the CD34+CD38−ALDH+ leukemic cells population enriched in LSCs (n=10). (b) Healthy HSCs proliferation, in percentage, after 48 h of treatment with DIMATE, daunorubicine, cytarabine and azacytidine. Drugs were used at a concentration equal to the IC50 values determined for the different drugs in LSCs (n=51). (c) LSCs and healthy HSCs survival according to the different concentration of DIMATE (n=10). Doted lines (5–9 μmol/l) determine a therapeutic window within DIMATE eradicated all LSCs (100% of lethality) and showed low toxicity (under 3% of lethality) on normal HSCs.

Figure 3

DIMATE shows potent activity against primary AML cells transplanted in NOG mice. (a) Experimental scheme and gaiting strategy in flow cytometry for hCD45+ and mCD45+ cells monitoring in blood, spleen and bone marrow for study of the antileukemic activity of DIMATE in immunodeficient mice engrafted with primary human AML cells. Mice were randomized and treatment with DIMATE (14, 28 mg/kg) and vehicle started for 4 weeks. Weekly monitoring of hCD45+ and mCD45+ circulating cells were performed during the treatment. After treatment, mice were killed and bone marrow and spleen were harvested. CD45+ have been sorted and monitored in spleen and bone marrow. Spleen weighing was performed. (b) Human CD45+ cells (hCD45+) count in peripheral blood during the treatment with vehicle or DIMATE. (c) Mouse CD45+ cells (mCD45+) count in peripheral blood during the treatment with vehicle or DIMATE. (d) Absolute human AML cells in the peripheral blood of NOG mice engrafted with AML cells before and after treatment with vehicle and different concentrations of DIMATE after mice euthanasia. (e) Counts of human CD45 cells in spleen in mice engrafted with AML cells after treatment with vehicle and different concentrations of DIMATE. (f) Counts of human CD45 cells in bone marrow in mice engrafted with AML cells after treatment with vehicle and different concentrations of DIMATE. (g) Spleen weight of mice transplanted with human AML cells treated with vehicle or DIMATE. In panels (d–f) each symbol denotes a single animal (n=3–6 per group). ** Mean P<0.001 and * mean P<0.05.

Figure 3

DIMATE shows potent activity against primary AML cells transplanted in NOG mice. (a) Experimental scheme and gaiting strategy in flow cytometry for hCD45+ and mCD45+ cells monitoring in blood, spleen and bone marrow for study of the antileukemic activity of DIMATE in immunodeficient mice engrafted with primary human AML cells. Mice were randomized and treatment with DIMATE (14, 28 mg/kg) and vehicle started for 4 weeks. Weekly monitoring of hCD45+ and mCD45+ circulating cells were performed during the treatment. After treatment, mice were killed and bone marrow and spleen were harvested. CD45+ have been sorted and monitored in spleen and bone marrow. Spleen weighing was performed. (b) Human CD45+ cells (hCD45+) count in peripheral blood during the treatment with vehicle or DIMATE. (c) Mouse CD45+ cells (mCD45+) count in peripheral blood during the treatment with vehicle or DIMATE. (d) Absolute human AML cells in the peripheral blood of NOG mice engrafted with AML cells before and after treatment with vehicle and different concentrations of DIMATE after mice euthanasia. (e) Counts of human CD45 cells in spleen in mice engrafted with AML cells after treatment with vehicle and different concentrations of DIMATE. (f) Counts of human CD45 cells in bone marrow in mice engrafted with AML cells after treatment with vehicle and different concentrations of DIMATE. (g) Spleen weight of mice transplanted with human AML cells treated with vehicle or DIMATE. In panels (d–f) each symbol denotes a single animal (n=3–6 per group). ** Mean P<0.001 and * mean P<0.05.