Primary Human AML Cells Research Articles

Histone Deacetylase Inhibitors LBH589 and LAQ824 Deplete Ezh2 and Associated Polycomb Repressive Complex 2/3 Proteins Resulting in Downregulation of HOXA9 and MEIS1 Expression in Human Acute Leukemia Cells.

Human enhancer of Zeste homolog (Ezh2) protein belongs to Polycomb Repressive Complex (PRC) 2, which also includes Eed and Suz12. Ezh2 has been shown to promote cellular transformation, and increased Ezh2 expression has been strongly correlated with the invasiveness of prostate and breast cancers. The enzymatically-active Ezh2-containing, PRC2 complex possesses histone methyl transferase (HMTase) activity mediated by the SET domain of Ezh2, which is involved in the methylation of histone (H) 3, lysine (K)-27 and −9. Through this, the PRC2 complex regulates the expression of homeobox domain containing HOX family of transcription factors including HOXA9 and MEIS1, which have been shown to be involved in human leukemogenesis. Co-expression of HOXA9 and MEIS1 is common in acute myeloid leukemia and collaborates in the leukemogenesis. In the present studies, we determined the effect of hydroxamate histone deacetylase inhibitors LBH589 and LAQ824 on Ezh2 and PRC2 complex proteins and their activity in the cultured (K562, LAMA-84, U937 and HL-60) and primary human AML cells. Exposure to 10 to 100 nM of LBH589 or LAQ824 for 24 hours, in a dose dependent manner depleted the protein levels of Ezh2, as well as reduced Suz12 and Eed levels in the cultured and primary leukemia cells. This was associated with decreased levels of the tri- and dimethylated K27 and increased acetylation of K27, both on H3. Correspondingly, these H3 modifications were associated with a significant decline in the levels of HOXA9 and MEIS1. As has been previously reported, treatment with LBH589 and LAQ824 induced p21, as well as caused cell cycle growth arrest in the G1 phase and apoptosis of the cultured and primary AML cells. We next determined whether knockdown of Ezh2 by siRNA to Ezh2 also induces growth inhibitory and cytotoxic effects against the leukemia cells. In the cultured and primary acute leukemia cells, knockdown of Ezh2 expression (by ~80%) by Ezh2 siRNA, but not by the control siRNA, was associated with depletion of Suz12 levels and increased H3K27 acetylation. This was associated with increase in the % of cells in the G1 phase of the cell cycle and significant inhibition of their clonogenic survival in colony growth assays. Co-treatment with LBH589 and siRNA to Ezh2 caused further decline in the Ezh2 levels and increased loss of clonogenic survival of the leukemia cells. These findings suggest that down modulation of Ezh2 and PRC2 complex and its HMTase activity may inhibit clonogenic survival of human acute myeloid leukemia cells. Additionally, combined effect of the knockdown of Ezh2 and its activity along with treatment with LBH589 or LAQ824 may have an improved anti-leukemia efficacy, especially against human AML where HOXA9 and MEIS1 genes are deregulated.

Motility, Proliferation and Egress of Human AML Cells in Transplanted NOD/SCID Mice Are Regulated by Membrane Bound and Secreted Elastase.

Neutrophil -or leukocyte- elastase is a serine protease stored in azurophilic granules of myeloid cells and is released upon activation and degranulation. Elastase degrades a number of extracellular matrix proteins as well as various cytokines and their receptors and may facilitate neutrophil motility. Lately, others and we have shown that elastase is also involved in G-CSF induced mobilization of stem cells. In the present study we examined the role of elastase in motility and proliferation of leukemic AML cells. We demonstrate a correlation between abnormal high levels of secreted elastase in the peripheral blood plasma of AML patients and the number of leukemic blast cells in the circulation. Interestingly, we found that AML cells not only secrete elastase, but also express constitutively membrane-bound elastase on their cell surface. Confocal microscopy revealed uniform localization of elastase on the cell surface. Of note, incubation of some AML cells with the chemokine SDF-1 increased elastase cell surface expression whereas treatment with neutralizing anti CXCR4 Abs decreased it. Pretreatment of primary human AML cells and cell lines with various elastase inhibitors and neutralizing anti elastase Abs led to reduced spontaneous and SDF-1-induced transwell migration of the leukemic cells. In vivo homing of AML mononuclear cells and CD34-enriched progenitors cells to the BM and spleen of transplanted NOD/SCID/B2mnull mice was significantly impaired by elastase inhibition. Moreover, when leukemic AML cells were pretreated with elastase inhibitors, the formation of spontaneous and SDF-1 induced protrusions was prevented suggesting that elastase participates in leukemic cell motility through direct regulation of cytoskeletal rearrangements and cell polarization. In addition, we found that the proliferation rate of AML cells was likewise elastase-dependent. In contrast, treatment of enriched human cord blood CD34+ progeniotrs with elastase inhibitors enhanced their proliferation rate, the percentage of undifferentiated CD34+/38− cells and their in vivo homing to the BM and spleen of immune deficient mice. Finally, we examined whether elastase may play a role in the abnormal AML cell egress from the BM to the circulation. Primary AML cells from different patients with different FAB subtypes were injected into sublethally irradiated NOD/SCID mice to establish human AML chimeras and 3–4 weeks later elastase inhibitor (1mg) was administrated daily for 4 consecutive days. Egress of AML cells to the blood circulation including blast cells was significantly decreased as compared to untreated chimeric mice. Taken together, these results demonstrate that elastase participates in the regulation of human AML progenitor cell proliferation and cell emigration from the BM to the circulation and that elastase inhibition can efficiently prevent AML cell egress in NOD/SCID chimeric mice.