Patient-derived Acute Myeloid Leukemia Blasts Research Articles

Upregulation of SOCS-1 by Nutlin-3 in acute myeloid leukemia cells but not in primary normal cells

OBJECTIVE:It has been shown that SOCS-1 plays an important role in the proper control of cytokine/growth factor responses and acts as a tumor suppressor in acute myeloid leukemias. Therefore, the objective of the present study was to evaluate the in vitro effect of treatment with Nutlin-3, a small molecule inhibitor of the MDM2/p53 interaction, on the expression of the suppressor of cytokine signaling 1 in primary acute myeloid leukemia cells and in myeloid cell lines with differential p53 status.METHOD:The expression of the suppressor of cytokine signaling 1 was quantitatively analyzed by real-time PCR in myeloid p53wild-type (OCI and MOLM) and p53null HL-60, leukemic cell lines, in patient-derived acute myeloid leukemia blasts, and in primary normal cell types, such as macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependence of the suppressor of cytokine signaling 1 upregulation that is induced by Nutlin-3 was analyzed in experiments performed using siRNA for p53, while the functional upregulation of the suppressor of cytokine signaling 1 was analyzed by assessing the levels of phosphorylated STAT-3.RESULTS:Nutlin-3 significantly upregulated the transcription of the suppressor of cytokine signaling 1 in p53wild-type OCI and MOLM but not in p53deleted p53null HL60, myeloid leukemic cell lines, as well as in primary acute myeloid leukemia blasts. Conversely, and somewhat unexpectedly, Nutlin-3 did not modulate the suppressor of cytokine signaling 1 expression in primary normal macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependent upregulation of the suppressor of cytokine signaling 1 by Nutlin-3 was associated with the downregulation of phosphorylated STAT-3, a major molecular target of the suppressor of cytokine signaling 1.CONCLUSION:Overall, our data suggest a potential role for the suppressor of cytokine signaling 1 as a therapeutic target of Nutlin-3 in p53 wild-type acute myeloid leukemias.

The delta Assay - A Suitable Tool To Detect the Cellular Immune Response Against Acute Myeloid Leukemia Blasts.

Adoptive immunotherapy with donor T-cells has been used successfully for the treatment of both relapsed chronic and acute myeloid leukemia after hematopoietic stem cell transplantation. Here we describe a method for the detection of T cell immunity by the inhibition of cytokine driven growth of blasts. According to the heterogeneous growth pattern of various cases of acute myeloid leukemia (AML) the culture requirements had to be determined individually. The basic cytokine cocktail consisted of SCF (50 ng/mL), GM-CSF (100 ng/mL), IL3 (50 ng/mL), G-CSF (100 ng/mL) and EPO (2 U/mL); this cocktail succeeded to induce blast-proliferation in 15 out of 16 AML samples tested. In a first step we determined optimal growth conditions regarding cell density and the day of linear growth. The peak proliferation was measured by the incorporation of tritium labeled thymidine at various time points. It ranged between days 2 and 5. In a second step, donor cells were stimulated for 10 days with irradiated AML-cell lines (MonoMac6, THP1) or patient derived AML blasts. After harvesting, the donor cells were irradiated (15 Gy) and co-cultured with the AML-cell lines, or the patients cytokine stimulated blast suspensions. Inhibition of growth was measured by the incorporation of tritium labeled thymidine at the previously determined day of linear proliferation of the AML-blasts. A significant inhibition of blast proliferation was observed in the co-culture of normal T cells with both AML-cell lines. In HLA-identical combinations T cells were primed by dendritic cells derived from AML blasts and re-stimulated on days 6 and 10. T cells primed by this method inhibited the growth of AML blasts in higher effector target ratios, in lower ratios no inhibition or even stimulation was observed. In summary, the delta-assay is a suitable tool to monitor specific anti-leukemic immune responses of donor lymphocytes even against very heterogeneous blast populations, if individualized culture conditions are considered.

Gefitinib induces myeloid differentiation of acute myeloid leukemia

AbstractCure rates for patients with acute myeloid leukemia (AML) remain low despite ever-increasing dose intensity of cytotoxic therapy. In an effort to identify novel approaches to AML therapy, we recently reported a new method of chemical screening based on the modulation of a gene expression signature of interest. We applied this approach to the discovery of AML-differentiation-promoting compounds. Among the compounds inducing neutrophilic differentiation was DAPH1 (4,5-dianilinophthalimide), previously reported to inhibit epidermal growth factor receptor (EGFR) kinase activity. Here we report that the Food and Drug Administration (FDA)-approved EGFR inhibitor gefitinib similarly promotes the differentiation of AML cell lines and primary patient-derived AML blasts in vitro. Gefitinib induced differentiation based on morphologic assessment, nitro-blue tetrazolium reduction, cell-surface markers, genome-wide patterns of gene expression, and inhibition of proliferation at clinically achievable doses. Importantly, EGFR expression was not detected in AML cells, indicating that gefitinib functions through a previously unrecognized EGFR-independent mechanism. These studies indicate that clinical trials testing the efficacy of gefitinib in patients with AML are warranted. (Blood. 2005;106: 2841-2848)