Celia Sigua

Cotreatment with Histone Deacetylase Inhibitor LAQ824 Enhances Apo-2L/Tumor Necrosis Factor-Related Apoptosis Inducing Ligand-Induced Death Inducing Signaling Complex Activity and Apoptosis of Human Acute Leukemia Cells

Present studies demonstrate that treatment with the histone deacetylases inhibitor LAQ824, a cinnamic acid hydroxamate, increased the acetylation of histones H3 and H4, as well as induced p21WAF1 in the human T-cell acute leukemia Jurkat, B lymphoblast SKW 6.4, and acute myelogenous leukemia HL-60 cells. This was associated with increased accumulation of the cells in the G1 phase of the cell cycle, as well as accompanied by the processing and activity of caspase-9 and -3, and apoptosis. Exposure to LAQ824 increased the mRNA and protein expressions of the death receptors DR5 and/or DR4, but reduced the mRNA and protein levels of cellular FLICE-inhibitory protein (c-FLIP). As compared with treatment with Apo-2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or LAQ824 alone, pretreatment with LAQ824 increased the assembly of Fas-associated death domain and caspase-8, but not of c-FLIP, into the Apo-2L/TRAIL-induced death-inducing signaling complex. This increased the processing of caspase-8 and Bcl-2 interacting domain (BID), augmented cytosolic accumulation of the prodeath molecules cytochrome-c, Smac and Omi, as well as led to increased activity of caspase-3 and apoptosis. Treatment with LAQ824 also down-regulated the levels of Bcl-2, Bcl-xL, XIAP, and survivin. Partial inhibition of apoptosis due to LAQ824 or Apo-2L/TRAIL exerted by Bcl-2 overexpression was reversed by cotreatment with LAQ824 and Apo-2L/TRAIL. Significantly, cotreatment with LAQ824 increased Apo-2L/TRAIL-induced apoptosis of primary acute myelogenous leukemia blast samples isolated from 10 patients with acute myelogenous leukemia. Taken together, these findings indicate that LAQ824 may have promising activity in augmenting Apo-2L/TRAIL-induced death-inducing signaling complex and apoptosis of human acute leukemia cells.

Superior activity of the combination of histone deacetylase inhibitor LAQ824 and the FLT-3 kinase inhibitor PKC412 against human acute myelogenous leukemia cells with mutant FLT-3.

Purpose: Mutant FLT-3 receptor tyrosine kinase is a client protein of the molecular chaperone heat shock protein 90 and is commonly present and contributes to the leukemia phenotype in acute myelogenous leukemia (AML). LAQ824, a cinnamyl hydroxamate histone deacetylase inhibitor, is known to induce acetylation and inhibition of heat shock protein 90. Here, we determined the effects of LAQ824 and/or PKC412 (a FLT-3 kinase inhibitor) on the levels of mutant FLT-3 and its downstream signaling, as well as growth arrest and cell-death of cultured and primary human AML cells. Experimental Design: The effect of LAQ824 and/or PKC412 treatment was determined on the levels of FLT-3 and phosphorylated (p)-FLT-3, on downstream pro-growth and pro-survival effectors, e.g., p-STAT5, p-AKT, and p-extracellular signal-regulated kinase (ERK) 1/2, and on the cell cycle status and apoptosis in the cultured MV4–11 and primary AML cells with mutant FLT-3. Results: Treatment with LAQ824 promoted proteasomal degradation and attenuation of the levels of FLT-3 and p-FLT-3, associated with cell cycle G1-phase accumulation and apoptosis of MV4–11 cells. This was accompanied by attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels. STAT-5 DNA-binding activity and the levels of c-Myc and oncostatin M were also down-regulated. Cotreatment with LAQ824 and PKC412 synergistically induced apoptosis of MV4–11 cells and induced more apoptosis of the primary AML cells expressing mutant FLT-3. This was also associated with more attenuation of p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5. Conclusions: The combination of LAQ824 and PKC412 is highly active against human AML cells with mutant FLT-3, which merits in vivo studies of the combination against human AML.

Cotreatment with 17-allylamino-demethoxygeldanamycin and FLT-3 kinase inhibitor PKC412 is highly effective against human acute myelogenous leukemia cells with mutant FLT-3

Presence of the activating length mutation (LM) in the juxtamembrane domain or point mutation in the kinase domain of FMS-like tyrosine kinase-3 (FLT-3) mediates ligand-independent progrowth and prosurvival signaling in approximately one-third of acute myelogenous leukemia (AML). PKC412, an inhibitor of FLT-3 kinase activity, is being clinically evaluated in AML. Present studies demonstrate that treatment of human acute leukemia MV4-11 cells (containing a FLT-3 LM) with the heat shock protein 90 inhibitor 17-allylamino-demethoxy geldanamycin (17-AAG) attenuated the levels of FLT-3 by inhibiting its chaperone association with heat shock protein 90, which induced the poly-ubiquitylation and proteasomal degradation of FLT-3. Treatment with 17-AAG induced cell cycle G1 phase accumulation and apoptosis of MV4-11 cells. 17-AAG-mediated attenuation of FLT-3 and p-FLT-3 in MV4-11 cells was associated with decrease in the levels of p-AKT, p-ERK1/2, and p-STAT5, as well as attenuation of the DNA binding activity of STAT-5. Treatment with 17-AAG, downstream of STAT5, reduced the levels of c-Myc and oncostatin M, which are transactivated by STAT5. Cotreatment with 17-AAG and PKC412 markedly down-regulated the levels of FLT-3, p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5, as well as induced more apoptosis of MV4-11 cells than either agent alone. Furthermore, the combination of 17-AAG and PKC412 exerted synergistic cytotoxic effects against MV4-11 cells. Importantly, 17-AAG and PKC412 induced more loss of cell viability of primary AML blasts containing FLT-3 LM, as compared with those that contained wild-type FLT-3. Collectively, these in vitro findings indicate that the combination of 17-AAG and PKC412 has high level of activity against AML cells with FLT-3 mutations.

Identification of a risk dependent microRNA expression signature in myelodysplastic syndromes

The myelodysplastic syndromes (MDS) display both haematological and biological heterogeneity with variable leukaemia potential. MicroRNAs play an important role in tumour suppression and the regulation of self‐renewal and differentiation of haematopoietic progenitors. Using a microarray platform, we evaluated microRNA expression from 44 patients with MDS and 17 normal controls. We identified a thirteen microRNA signature with statistically significant differential expression between normal and MDS specimens (P < 0·01), including down‐regulation of members of the leukaemia‐associated MIRLET7 family. A unique signature consisting of 10 microRNAs was closely associated with International Prognostic Scoring System (IPSS) risk category permitting discrimination between lower (Low/Intermediate‐1) and higher risk (Intermediate‐2/High) disease (P < 0·01). Selective overexpression of MIR181 family members was detected in higher risk MDS, indicating pathogenetic overlap with acute myeloid leukaemia. Survival analysis of an independent cohort of 22 IPSS lower risk MDS patients revealed a median survival of 3·5 years in patients with high expression of MIR181 family compared to 9·3 years in patients with low MIR181 expression (P = 0·002). Our pilot study suggested that analysis of microRNA expression profile offers diagnostic utility, and provide pathogenetic and prognostic discrimination in MDS.

Deletion of toll-like receptor-4 downregulates protein kinase C-ζ and attenuates liver injury in experimental pancreatitis

BACKGROUND Toll-like receptor-4 (TLR4) and protein kinase C-zeta (PKC-zeta) play a role in macrophage activation. We hypothesized that deletion of TLR4 downregulates PKC-zeta and attenuates liver cell apoptosis in experimental pancreatitis. METHODS Acute pancreatitis was induced by choline-deficient ethionine diet in C57/BL6 (TLR4+/+ and TLR4-/-) mice. RESULTS During pancreatitis, staining for TLR4 and PKC-zeta, which colocalized in Kupffer cells but not in hepatocytes, increased in TLR4+/+ mice and decreased in TLR4-/- mice. In TLR4+/+ mice, pancreatitis increased TLR4 protein and mRNA and PKC-zeta protein and activity, nuclear factor (NF)-kappaB, ERK1/2, caspase-3 cleavage, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining; all P < .01 versus controls. In TLR4-/- mice with pancreatitis, PKC-zeta mRNA and activity were reduced, ERK1/2 and caspase-3 did not increase, and NF-kappaB and TUNEL (mostly in hepatocytes) increased mildly (all P < .01 vs control). PKC-zeta did not interact directly with NF-kappaB; however, during pancreatitis, coimmunoprecipitation of PKC-zeta with ERK1/2 was increased in TLR4+/+ mice and was attenuated in TLR4-/- mice (all P < .01 vs control), indicating that PKC-zeta interacts with ERK1/2. CONCLUSION Acute pancreatitis upregulates TLR4, PKC-zeta, NF-kappaB, and ERK1/2, and increases apoptosis in mice livers. PKC-zeta induces nuclear translocation of NF-kappaB via ERK1/2-dependent mechanisms. Deletion of TLR4 downregulates PKC-zeta, NF-kappaB, and ERK1/2, and attenuates pancreatitis-induced liver cell apoptosis.