Concurrent Targeting Akt and Sphingosine Kinase 1 by A-674563 in Acute Myeloid Leukemia Cells
Keywords: Acute myeloid leukemia, A-674563, Akt, Sphingosine kinase 1
Abstract
Akt signaling plays a pivotal role in acute myeloid leukemia (AML) development and progression. In this study, we evaluated the potential anti-AML activity of the novel Akt kinase inhibitor A-674563. Our results showed that A-674563 dose-dependently inhibited survival and proliferation of U937 AML cells and six lines of human AML progenitor cells, while sparing human peripheral blood mononuclear leukocytes (PBMCs). A-674563 activated caspase-3/9 and induced apoptosis in AML cells. Conversely, the pancaspase inhibitor z-VAD-CHO dramatically alleviated A-674563-induced AML cell apoptosis and cytotoxicity. Molecular studies demonstrated that A-674563 blocked Akt activation in U937 cells and human AML progenitor cells. Furthermore, A-674563 decreased sphingosine kinase 1 (SphK1) activity in AML cells, depleting pro-survival sphingosine-1-phosphate (S1P) and boosting pro-apoptotic ceramide production. This effect on SphK1 signaling appeared independent of Akt inhibition. Notably, K6PC-5, a novel SphK1 activator, or supplementation with S1P, attenuated A-674563-induced ceramide production and subsequent U937 cell death and apoptosis. Importantly, intraperitoneal injection of A-674563 at well-tolerated doses suppressed U937 leukemic xenograft tumor growth in nude mice and significantly improved animal survival. Our results demonstrate that A-674563 exerts potent anti-leukemic activity in vitro and in vivo, possibly via concurrent targeting of Akt and SphK1 signaling.
1. Introduction
Acute myeloid leukemia (AML) is one of the most common and lethal malignancies among children and young adults. Epidemiologic studies show that AML accounts for at least 3% of all human cancers, causing over 250,000 cancer-related deaths annually. The incidence of AML has been increasing, especially in China and other Eastern countries. Despite recent advances in research and clinical treatment, prognosis remains poor, with a five-year overall survival rate of less than 30%. Therefore, developing novel anti-AML agents is a major focus.
Numerous AML cell growth and survival signaling molecules have been identified. Among them, Akt signaling plays vital roles in cancer development and progression. This pathway is often constitutively activated in AML cells, promoting proliferation, survival, apoptosis resistance, transformation, and metastasis. Thus, Akt represents an important molecular target for AML therapy. Several classes of Akt inhibitors have been investigated, with some showing promising results.
In this study, we analyzed the anti-AML activity of the newly developed Akt kinase inhibitor A-674563, focusing on associated signaling mechanisms. Our preclinical results demonstrate that A-674563 efficiently inhibits human AML cells in vitro and in vivo. Intriguingly, both Akt-dependent and Akt-independent (SphK1) mechanisms participate in A-674563-induced anti-leukemic activity.
2. Materials and Methods
2.1. Chemicals and Reagents
A-674563 was provided by AdooQ Bioscience (Beijing, China). K6PC-5 was a gift from Dr. Cao’s Lab at Nanjing Medical University. The PI3K-Akt inhibitor Wortmannin, pancaspase inhibitor z-VAD-CHO, and sphingosine-1-phosphate (S1P) were purchased from Sigma. All antibodies were from Cell Signaling Technology.
2.2. Cell Lines
Human AML cell line U937 was obtained from the Cell Bank of Fudan University (Shanghai, China) and cultured in RPMI 1640 with 10% FBS and 1% L-glutamine at 37°C in a humidified 5% CO₂ atmosphere.
2.3. Primary Culture of Human AML Progenitor Cells and PBMCs
Leukemic blasts were collected from six AML patients at Xiangya Hospital, Central South University (see Table 1 for characteristics). Blasts were separated by centrifugation and cultured in DMEM with rhGM-CSF, rhIL-3, and rhG-CSF. PBMCs from healthy donors were isolated by density gradient centrifugation and cultured as described. All protocols were IRB-approved, and informed consent was obtained.
2.4. Cell Viability (MTT) Assay
Cells (5 × 10³/well) were plated in 96-well plates. After treatment, 20 μL of MTT solution (5 mg/mL) was added and incubated for 3 h. Formazan was dissolved in 150 μL DMSO, and absorbance was measured at 570 nm.
2.5. Cell Death Assay
Cell death was determined by trypan blue dye exclusion. Cells were stained and dead cells counted using a hemocytometer.
2.6. Clonogenicity Assay
U937 cells (1000/plate) were plated in 60 mm dishes, treated with A-674563 for 48 h, washed, and incubated for 8 days. Colonies were fixed, stained with Coomassie Blue, and counted.
2.7. BrdU Proliferation Assay
Cells (1 × 10⁴/well) were exposed to A-674563, then incubated with BrdU (5 μM) for 12 h. BrdU incorporation was measured using an ELISA kit.
2.8. Caspase-3/-9 Activity Assay
Cytosolic extracts (20 μg/sample) were incubated with caspase assay buffer and substrates for caspase-3 or -9. After 2 h at 37°C, AFC release was quantified by fluorescence.
2.9. Annexin V Apoptosis Assay
Apoptosis was measured using Annexin V/PI FACS analysis. The percentage of Annexin V⁺/PI⁻ cells was calculated.
2.10. SphK1 Activity and S1P Content
After A-674563 treatment, SphK1 activity and S1P content were measured using radiolabeled ATP and TLC separation, followed by autoradiography and scintillation counting.
2.11. Ceramide Content Measurement
Ceramide content was analyzed using the DAG kinase method and expressed as fold change compared to control.
2.12. Mice Xenograft Assay
U937 cells (5 × 10⁶) were injected into the flanks of 4–5-week-old male nude mice. After tumors reached 100 mm³, mice were randomized to receive A-674563 (15 or 40 mg/kg) or saline by daily intraperitoneal injection for 28 days. Tumor volumes and body weights were recorded weekly.
2.13. Statistical Analyses
Data are mean ± SD. Statistical significance (p < 0.05) was determined by two-way ANOVA with Bonferroni post hoc test using GraphPad Prism 5.0.
3. Results
3.1. Anti-AML Activity of A-674563 In Vitro
A-674563 dose-dependently inhibited U937 cell viability (MTT assay), but had no significant effect on PBMC viability, indicating selectivity for AML cells. Trypan blue and clonogenicity assays confirmed increased cell death and decreased colony formation. BrdU incorporation showed dose-dependent inhibition of U937 cell proliferation.
A-674563 also decreased viability in six lines of human AML progenitor cells (L1–L6). L1–L3 (PTEN mutant, high basal Akt activation) were more susceptible to A-674563 than L4–L6 (PTEN wild-type, low basal Akt activation). PBMCs had very low Akt phosphorylation and were not affected.
3.2. A-674563 Activates Caspase-3/-9 and Induces Apoptosis
A-674563 increased caspase-3 and -9 activity and Annexin V⁺ apoptosis in U937 cells. The pancaspase inhibitor z-VAD-CHO blocked A-674563-induced apoptosis, viability reduction, and cell death, indicating caspase-dependent apoptosis. Caspase-3 activation and apoptosis were also observed in all six AML progenitor cell lines, with higher activation in PTEN mutant lines.
3.3. A-674563 Inhibits SphK1 Activity Independently of Akt
A-674563 was more potent than the PI3K-Akt inhibitor Wortmannin in inhibiting U937 cells. While both blocked Akt activation, A-674563 retained anti-survival and pro-death effects even in the presence of Wortmannin. Wortmannin had little effect on PTEN wild-type AML progenitor cells, suggesting Akt-independent mechanisms for A-674563.
A-674563 dose-dependently decreased SphK1 activity in U937 cells, leading to increased ceramide and decreased S1P. The SphK1 activator K6PC-5 or exogenous S1P blocked A-674563-induced ceramide production, apoptosis, and viability reduction. In all AML progenitor cell lines, SphK1 inhibition and ceramide accumulation were observed after A-674563 treatment, regardless of PTEN/Akt status. Wortmannin did not affect SphK1 activity, indicating SphK1 inhibition by A-674563 is independent of Akt.
3.4. Anti-AML Activity of A-674563 In Vivo
In a U937 xenograft mouse model, daily intraperitoneal injection of A-674563 (15 or 40 mg/kg) significantly inhibited tumor growth and improved survival compared to saline controls. The effect was dose-dependent. Mice tolerated the treatment well, with no significant difference in body weight between groups.
4. Discussion
This study demonstrates that A-674563, a novel Akt kinase inhibitor, potently inhibits survival and proliferation of U937 cells and human AML progenitor cells by activating caspase-dependent apoptosis. Mechanistically, A-674563 blocks Akt activation and simultaneously inhibits SphK1 activity, leading to increased pro-apoptotic ceramide and decreased pro-survival S1P in AML cells. In vivo, A-674563 suppressed leukemic tumor growth and improved survival in mice.
Sphingolipid metabolites, including ceramide, sphingosine, and S1P, play vital roles in cancer cell development. SphK1 regulates the balance between pro-apoptotic ceramide and pro-survival S1P. SphK1 is dysregulated in AML and is a promising therapeutic target. A-674563’s ability to inhibit SphK1 and increase ceramide appears to be independent of Akt inhibition and may explain its superior activity against AML cells. SphK1 inhibition and ceramide accumulation were most significant in certain AML progenitor cells, regardless of PTEN or Akt status. In non-cancerous PBMCs, both basal Akt and SphK1 levels were low, which may explain their resistance to A-674563.
Overall, A-674563 exerts potent anti-leukemic activity in vitro and in vivo, possibly via concurrent targeting of Akt and SphK1 signaling.