pep005 and Leukemia--Myeloid--Acute

The novel small molecule ingenol 3-angelate (PEP005) has been shown previously to induce apoptosis in leukaemic cell lines and primary AML cells, an effect that requires the expression of protein kinase C-delta (PKCdelta). Here we have investigated signalling events downstream of PKCdelta that determine sensitivity of AML cells to PEP005. We show that activation of ERK1/2 MAP kinase occurred in both sensitive and resistant cells and that induction of apoptosis required sustained signalling through the ERK1/2 pathway. Inhibition of ERK1/2 signalling using the MEK inhibitor PD98059 inhibited PEP005-induced apoptosis and activation of ERK1/2 was shown to occur downstream of PKC activation. The data show that PEP005-induced apoptosis is both PKC and ERK1/2 dependent and indicate that chronic activation of ERK1/2 in leukaemic cells delivers a pro-apoptotic rather than a proliferative or survival signal.

Topics: Animals; Antineoplastic Agents; Apoptosis; Diterpenes; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Humans; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Protein Kinase C-delta; Signal Transduction; Tumor Cells, Cultured

Acute myeloid leukaemia (AML) cells show constitutive release of several chemokines that occurs in three major clusters: (I) chemokine (C-C motif) ligand (CCL)2-4/chemokine (C-X-C motif) ligand (CXCL)1/8, (II) CCL5/CXCL9-11 and (III) CCL13/17/22/24/CXCL5. Ingenol-3-angelate (PEP005) is an activator of protein kinase C and has antileukaemic and immunostimulatory effects in AML. We investigated primary AML cells derived from 35 unselected patients and determined that PEP005 caused a dose-dependent increase in the release of chemokines from clusters I and II, including several T cell chemotactic chemokines. The release of granulocyte-macrophage colony-stimulating factor and hepatocyte growth factor was also increased. CCL2-4/CXCL1/8 release correlated with nuclear factor (NF)-kappaB expression in untreated AML cells, and PEP005-induced chemokine production was associated with further increases in the expression of the NF-kappaB subunits p50, p52 and p65. Increased DNA binding of NF-kappaB was observed during exposure to PEP005, and the specific NF-kappaB inhibitor BMS-345541 reduced constitutive chemokine release even in the presence of PEP005. Finally, PEP005 decreased expression of stem cell markers (CD117, CXCR4) and increased lineage-associated CD11b and CD14 expression. To conclude, PEP005 has a unique functional pharmacological profile in human AML. Previous studies have described proapoptotic and T cell stimulatory effects and the present study describes additional T cell chemotactic and differentiation-inducing effects.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Cell Differentiation; Chemokines; Diterpenes; Female; Flow Cytometry; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; NF-kappa B; Protein Kinase C; RNA Interference; RNA, Small Interfering; Statistics, Nonparametric; Tumor Cells, Cultured

Cytokines are released during T cell activation, including the potentially anti-leukemic interferon-gamma (IFNgamma), but also the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) that enhance proliferation and inhibit apoptosis of acute myelogenous leukemia (AML) cells. In the present study we investigated the release of IFNgamma and GM-CSF by circulating T cells in AML patients with chemotherapy-induced cytopenia. T cells were activated with anti-CD3 plus anti-CD28 in a whole-blood assay in the presence of their natural cytokine network. We examined 63 samples derived from 16 AML patients during 28 chemotherapy cycles. Activated T cells showed a broad cytokine release profile, but IFNgamma and GM-CSF levels showed a significant correlation and were generally higher than the other cytokine levels. Higher IFNgamma and GM-CSF responses were associated with a low CD4:CD8 ratio, older patient age and no ongoing chemotherapy indicating potential utility of T cell activation regimes for the older AML patient. The cytokine levels could be further increased by the novel protein kinase C agonist PEP005, which also induced significant production of IL2 and TNFalpha which could contribute to anti-tumor effects in AML patients. We conclude that remaining T cells after intensive AML therapy show a broad cytokine release profile including high and significantly correlated levels of potentially anti-leukemic IFNgamma and the AML growth factor GM-CSF. The final outcome of an AML-initiated T cell cytokine response will thus depend on the functional characteristics of the AML cells, in particular the relative expression of IFNgamma and GM-CSF receptors which differs between AML patients.

Topics: Adult; Aged; Antineoplastic Agents; Cells, Cultured; Cytokines; Diterpenes; Esters; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon-gamma; Leukemia, Myeloid, Acute; Lymphocyte Activation; Lymphopenia; Male; Middle Aged; Protein Kinase C; T-Lymphocyte Subsets; T-Lymphocytes