XIAP inhibitors induce differentiation and impair clonogenic capacity of acute myeloid leukemia stem cells

Abstract

Acute myeloid leukemia (AML) is a neoplasia characterized by the rapid expansion of immature myeloid blasts in the bone marrow, and marked by poor prognosis and frequent relapse. As such, new therapeutic approaches are required for remission induction and prevention of relapse. Due to the higher chemotherapy sensitivity and limited life span of more differentiated AML blasts, differentiation-based therapies are a promising therapeutic approach. Based on public available gene expression profiles, a myeloid-specific differentiation-associated gene expression pattern was defined as the therapeutic target. A XIAP inhibitor (Dequalinium chloride, DQA) was identified in an in silico screening searching for small molecules that induce similar gene expression regulation. Treatment with DQA, similarly to Embelin (another XIAP inhibitor), induced cytotoxicity and differentiation in AML. XIAP inhibition differentially impaired cell viability of the most primitive AML blasts and reduced clonogenic capacity of AML cells, sparing healthy mature blood and hematopoietic stem cells. Taken together, these results suggest that XIAP constitutes a potential target for AML treatment and support the evaluation of XIAP inhibitors in clinical trials.

Figure 1. XIAP inhibitor treatment induces cytotoxicity and differentiation on AML cell lines

A. Cytotoxicity in HL-60, MonoMac-1 (MM) and Kasumi-1 (K-1) AML cell lines resulting from treatment with 5 μM DQA for 48 h in the absence (upper panel) or presence of HS-5 stroma cells (lower panel). Y-axis: relative number of live cells as assessed by flow cytometry (7-AAD⁻). B. Up-regulation of CD15 surface expression, measured by flow cytometry in AML cell lines (HL-60, KG-1, MonoMac-1 and Kasumi-1) treated with 5 μM DQA. Data from all AML cell lines are presented combined. Frequency of CD15-positive population normalized against control-treated samples is represented. CD15 surface expression representative plot of HL-60 untreated (left) or treated with 5 μM DQA (right). C. HL-60, Kasumi-1, MonoMac-1 and KG-1 AML cells were treated with different concentrations of Embelin for 48 h. Cell viability (upper left panel) and CD15 surface expression (upper right panel) were measured by flow cytometry. Representative flow cytometry plot of HL-60 untreated (left) or treated with 10 μM Embelin (right). D. XIAP protein was detected by Western blot upon DQA (D) and Emb (E) treatment of HL-60 cells. GAPDH was used as loading control. MFI refer to GAPDH and vehicle-treated control is represented. E. HL-60 cells were treated for 18 h with 5 μM DQA (left) and 10 μM embelin (right). Colonies were counted at day 7. * p<0.05; ** p<0.005; *** p<0.0005. Error bars correspond to SEM.

Figure 2. DQA treatment induces cell cycle arrest and downregulation of P-Akt, P-Erk and P-Stat3

HL-60, KG-1, MonoMac-1 and Kasumi-1 were treated with 5 μM DQA and cell cycle was analyzed by flow cytometry 48 h after treatment. A. Relative frequency of G0/G1, S and G2/M phases in control- vs. DQA-treated AML cells. Bars represent the mean value of all AML cell lines and error bars represent SEM. B. Representative DNA content flow profile of control- (left) and DQA-treated (right) HL-60 (green represents G0/G1 phase; yellow, S phase; blue, G2/M phase). P-Akt and P-Erk expression levels by flow cytometry in AML cell lines after treatment with 5 μM DQA for 24 h. C. Mean fluorescence intensity of each staining was normalized against vehicle-control treated sample and data from all AML cell lines tested is represented. D. Representative flow histograms of P-Akt (left), P-Erk (centre) and P-Stat3 (right) intracellular staining of DQA-treated HL-60 AML cells. Shadow, negative control; solid line, control treated sample; dashed line, DQA-treated sample. * p<0.05; ** p<0.005.

Figure 3. DQA and embelin treatment induces cell death in AML primary blasts by preferentially affecting LSC population and reduces clonogenic capacity

AML primary blasts A. or healthy myeloid blood cells B. were treated with different concentration of DQA (0.05, 0.5, 5 μM) and embelin (0.1, 1, 10 μM). Cell viability was analysed at day 1 (upper panels) and 3 (lower panels) after treatment. Each symbol corresponds to a single AML patient sample, specified in the graph legend. Bulk population corresponds to AML blast cells and the primitive fraction corresponds to a CD34+CD38- blast population. C. Primary AML patient samples were treated for 24 h with 5 mM DQA. Cell viability was measured by flow cytometry (volumetric counts on live 7-AAD⁻ cells). Each symbol corresponds to an AML patient sample. Green, favourable risk group; blue, intermediate risk group; red, unfavourable risk group. * p<0.05; ** p<0.005; *** p<0.0005.

Figure 4. XIAP inhibitor treatment reduced the clonogenic capacity of AML cells with little effect on primitive healthy blood cells

D. AML primary cells or E. lineage-depleted umbilical cord blood cells were treated with 5 μM DQA or 10 μM embelin for 18 h. Colonies were screened at day 14 based on morphological criteria. Each symbol represents primary sample. * p<0.05; ** p<0.005; *** p<0.0005.