MEK and PI3K-AKT inhibitors synergistically block activated IL7 receptor signaling in T-cell acute lymphoblastic leukemia.

We identified mutations in the IL7Ra gene or in genes encoding the downstream signaling molecules JAK1, JAK3, STAT5B, N-RAS, K-RAS, NF1, AKT and PTEN in 49% of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). Strikingly, these mutations (except RAS/NF1) were mutually exclusive, suggesting that they each cause the aberrant activation of a common downstream target. Expressing these mutant signaling molecules-but not their wild-type counterparts-rendered Ba/F3 cells independent of IL3 by activating the RAS-MEK-ERK and PI3K-AKT pathways. Interestingly, cells expressing either IL7Ra or JAK mutants are sensitive to JAK inhibitors, but respond less robustly to inhibitors of the downstream RAS-MEK-ERK and PI3K-AKT-mTOR pathways, indicating that inhibiting only one downstream pathway is not sufficient. Here, we show that inhibiting both the MEK and PI3K-AKT pathways synergistically prevents the proliferation of BaF3 cells expressing mutant IL7Ra, JAK and RAS. Furthermore, combined inhibition of MEK and PI3K/AKT was cytotoxic to samples obtained from 6 out of 11 primary T-ALL patients, including 1 patient who had no mutations in the IL7R signaling pathway. Taken together, these results suggest that the potent cytotoxic effects of inhibiting both MEK and PI3K/AKT should be investigated further as a therapeutic option using leukemia xenograft models.

Chemical genomic screening identifies LY294002 as a modulator of glucocorticoid resistance in MLL-rearranged infant ALL.

Successful treatment results for MLL-rearranged Acute Lymphoblastic Leukemia (ALL) in infants remain difficult to achieve. Significantly contributing to therapy failure is poor response to glucocorticoids (GCs), like prednisone. Thus, overcoming resistance to these drugs may be a crucial step towards improving prognosis. We defined a gene signature that accurately discriminates between prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patient samples. In the current study, we applied Connectivity Map analysis to perform an in silico screening for agents capable of reversing the prednisolone-resistance profile and induce sensitivity. These analyses revealed that LY294002, a PI3K inhibitor, would potentially fulfill this task. Subsequent validation experiments demonstrated that indeed LY294002, and other known PI3K inhibitors, markedly sensitized otherwise resistant MLL-rearranged ALL cells to prednisolone in vitro. Using quantitative RT-PCR analyses, we validated the modulating effects of the PI3K inhibitors on the expression of the genes present in our prednisolone-resistance profile. Interestingly, prednisolone-sensitizing actions may be mediated by inhibition of FCGR1B. Moreover, only high-level expression of FCGR1B showed to be predictive for a poor prognosis and shRNA-mediated knock-down of FCGR1B led to in vitro prednisolone sensitization. Thus, implementing FDA-approved PI3K inhibitors in current treatments may potentially improve the GC response and prognosis in patients with MLL-rearranged ALL.

Src kinase-induced phosphorylation of annexin A2 mediates glucocorticoid resistance in MLL-rearranged infant acute lymphoblastic leukemia.

MLL-rearranged infant acute lymphoblastic leukemia (ALL) (<1 year of age) are frequently resistant to glucocorticoids, like prednisone and dexamethasone. As poor glucocorticoid responses are strongly associated with therapy failure, overcoming glucocorticoid resistance may be a crucial step towards improving prognosis. Unfortunately, the mechanisms underlying glucocorticoid resistance in MLL-rearranged ALL largely remain obscure. We here defined a gene signature that accurately discriminates between prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patient samples, demonstrating that, among other genes, high-level ANXA2 is associated with prednisolone resistance in this type of leukemia. Further investigation demonstrated that the underlying factor of this association was the presence of Src kinase-induced phosphorylation (activation) of annexin A2, a process requiring the adapter protein p11 (encoded by human S100A10). shRNA-mediated knockdown of either ANXA2, FYN, LCK or S100A10, all led to inhibition of annexin A2 phosphorylation and resulted in marked sensitization to prednisolone. Likewise, exposure of prednisolone-resistant MLL-rearranged ALL cells to different Src kinase inhibitors exerting high specificity towards FYN and/or LCK had similar effects. In conclusion, we here present a novel mechanism of prednisolone resistance in MLL-rearranged leukemias, and propose that inhibition of annexin A2 phosphorylation embodies a therapeutic strategy for overcoming resistance to glucocorticoids in this highly aggressive type of leukemia.

Elevated S100A8/S100A9 expression causes glucocorticoid resistance in MLL-rearranged infant acute lymphoblastic leukemia.

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants is characterized by a poor clinical outcome and resistance to glucocorticoids (for example, prednisone and dexamethasone). As both the response to prednisolone in vitro and prednisone in vivo are predictive for clinical outcome, understanding and overcoming glucocorticoid resistance remains an essential step towards improving prognosis. Prednisolone-induced apoptosis depends on glucocorticoid-evoked Ca(2+) fluxes from the endoplasmic reticulum towards the mitochondria. Here, we demonstrate that in MLL-rearranged infant ALL, over-expression of S100A8 and S100A9 is associated with failure to induce free-cytosolic Ca(2+) and prednisolone resistance. Furthermore, we demonstrate that enforced expression of S100A8/S100A9 in prednisolone-sensitive MLL-rearranged ALL cells, rapidly leads to prednisolone resistance as a result of S100A8/S100A9 mediated suppression of prednisolone-induced free-cytosolic Ca(2+) levels. In addition, the Src kinase inhibitor PP2 markedly sensitized MLL-rearranged ALL cells otherwise resistant to prednisolone, via downregulation of S100A8 and S100A9, which allowed prednisolone-induced Ca(2+) fluxes to reach the mitochondria and trigger apoptosis. On the basis of this novel mechanism of prednisolone resistance, we propose that developing more specific S100A8/S100A9 inhibitors may well be beneficial for prednisolone-resistant MLL-rearranged infant ALL patients.

Hypoxia contributes to development of recurrent endometrial carcinoma.

Tumor hypoxia can trigger the induction of angiogenesis. High microvessel density (MVD) as well as hypoxia-inducible factor-1alpha (HIF-1alpha) have been related to recurrent disease and tumor aggressiveness, respectively. In this study, MVD and hypoxic status were investigated in primary and recurrent endometrial carcinomas. A total of 65 primary tumors of patients with recurrent endometrial carcinoma (n = 40), and without recurrent endometrial carcinoma (n = 25) were studied. Immunohistochemical analysis was performed on paraffin-embedded tumor tissue. MVD was determined by quantitative analysis of CD31/FVIII positive vessels. Tumor hypoxia was estimated by evaluating the expression of the hypoxia-regulated gene HIF-1alphaand its target gene carbonic anhydrase IX (CA-IX). An additional 23 recurrent tumors were available for determination of MVD and HIF-1alpha expression. Effects of hypoxia on tumor protein p53 (TP53) expression were evaluated in the endometrial cancer cell lines (ECC-1), Ishikawa (derived from adenocarcinomas), and AN3CA (derived from a lymph node metastasis). MVD, CA-IX, and HIF-1alpha expression were not significantly different in primary tumors of patients with recurrence compared to the control tumors. The MVD was significantly lower, and HIF-1alpha expression was significantly higher in recurrent tumors when compared with their primary tumors (paired t test, P < 0.05). HIF-1alpha expression correlated well with TP53 expression levels in primary tumors, but not in recurrences. TP53 protein levels were highest in AN3CA cells. Hypoxic conditions induced TP53 protein in ECC-1 and Ishikawa, but not AN3CA cells. We conclude that MVD, CA-IX, and HIF-1alpha expression are not independent prognostic markers for recurrent endometrial carcinoma. The low MVD, increased HIF-1alpha protein levels, dissociation of hypoxia, and TP53 protein induction in the metastatic tumor cells (AN3CA) support a role for hypoxia in the development of recurrent endometrial carcinoma.

Antiviral potential of interferon-omega on hepatitis B virus replication in human hepatoma cells.

Interferon-alpha (IFN-alpha) and other cytokines are able to interfere with hepatitis B virus (HBV) replication. However, a sustained antiviral effect is achieved only in 25% to 40% of the patients with chronic HBV infection and clearance of the virus rarely occurs, stressing the need for developing therapeutic alternatives. In this study the antiviral potential of a new recombinant interferon, IFN-omega was investigated. IFN-omega was assessed in comparison with IFN-alpha 2c, IFN-gamma, and TNF-alpha with respect to production of HBV proteins and DNA in HepG2.2.15 cells, a HBV-DNA transfected hepatoma cell line which produces infectious viral particles. Cells were seeded at different states of confluence (20%-90%) and treated with increasing concentrations of interferons (5 to 5,000 U/ml), TNF-alpha (5 to 500 ng/ml), or combinations of both for one to three days. IFN-omega reduced the production of HBsAg down to 59% of the untreated controls, which was comparable to the reduction obtained by treatment with IFN-alpha (60%), the standard interferon used for the treatment of chronic HBV infections. The strongest inhibition, however, was achieved by treatment with 500 ng/ml TNF-alpha (42%). Likewise, production of HBeAg and synthesis of HBV DNA were inhibited to similar degrees by the different interferons. In non-replicating high-density cultures only TNF-alpha was effective. IFN-omega is of similar antiviral potential as IFN-alpha in this in vitro experimental system.

[Manual ultrasound of the knee joint. A general practice method for diagnosis of fresh rupture of the anterior cruciate ligament]. / Manuelle Sonometrie des Kniegelenks. Eine praxisnahe Methode zur Diagnostik der frischen Ruptur des vorderen Kreuzbandes.

A prospective, arthroscopic controlled study was performed to evaluate the usefulness of mancal sonometry of the knee in the diagnosis of acute rupture of the anterior cruciate ligament (ACL). In 58 patients with an acute knee injury, a maximum of 30 days elapsed between accident and clinical examination (Lachmantest, pivot-shift/ McIntosh, 90 degrees anterior translation), manual sonometry and functional X-ray examination. Afterwards all patients were examined by arthroscopy. Nine patients showed a partial, 38 patients a total rupture of the ACL. If the ACL was completely ruptured, the average difference in anterior translation between the contralateral and the injured knee was 3.3 mm (p < or = 0.001). Statistical analysis showed high sensitivity (85%), specificity (91%) and positive predictive value (98%) for manual sonometry. Clinical examination, apparative sonometry and functional X-ray were, overall, less effective than manual sonometry in detecting ligament rupture. Manual sonometry proved to be a good and practicable method for assessing acute rupture of the ACL.

pH-independent uptake of hepatitis B virus in primary human hepatocytes.

The replication cycle of the hepatitis B virus (HBV) is still incompletely understood. In particular, the early steps of the viral life cycle, such as absorption, penetration, uncoating, and nuclear translocation require further clarification. In this study we performed infection experiments with HBV in primary human hepatocyte cultures. To further elucidate the possible mechanism of virus uptake, infection experiments were performed at different pH levels, after pretreatment of viral particles with acidic buffers and in the presence of lysosomotropic agents (chloroquine and ammonium chloride, respectively). Using a selective PCR technique which discriminates between input virus DNA and the earliest replicative form, we could demonstrate viral replication 36 hr after inoculation. HBV was taken up most efficiently at a pH of 7.4. Infection was still successful after pretreatment of viral particles at low pH and was unaffected by the presence of lysosomotropic agents. In conclusion, this suggests HBV to be a pH-independent virus.

Myristylation of the large surface protein is required for hepatitis B virus in vitro infectivity.

The large surface protein (L) of the enveloped hepatitis B virus (HBV) is myristylated at glycine 2. To investigate whether this fatty acid moiety is required for HBV infectivity we made use of a point mutant in which the myristyl acceptor was mutated to a nonfunctional alanine. The mutant virus and a wild-type control were expressed in a human hepatoma cell line by transfection of genomic DNA constructs. Comparable amounts of virions were secreted by both strains as measured by the endogenous polymerase activity of immunoprecipitated virions. The presentation of an N-terminal epitope of L on the virion surface was not influenced by the mutation. To test the infectivity of this mutant virus primary human hepatocytes were incubated with the media of transfected cells. The covalently circular closed HBV DNA molecules generated after infection were discriminated from the open circular DNA genomes of inoculated virions by a sensitive PCR-based technique. The experiments demonstrated that the wild type was infectious but not the myristate negative mutant. This reflects the phenotype of an homologous duck hepatitis B virus mutant although the N-terminal L protein domains of this virus and of HBV show no primary sequence homology.

In vitro experimental infection of primary human hepatocytes with hepatitis B virus.

BACKGROUND/AIMS: Studies on the interaction of hepatitis B virus (HBV) with its host cell require a suitable tissue culture system. This study used primary adult hepatocytes from healthy human liver tissue to establish productive infection in vitro. METHODS: Hepatocytes were inoculated overnight with HBV. Production of viral proteins was assessed by radioimmunoassay and by [35S]methionine labeling, and production of viral DNA was assessed by Southern blotting and endogenous polymerase assay. RESULTS: Secretion of high levels of hepatitis B surface antigen (HBsAg) and low levels of hepatitis B virus e antigen (HBeAg) into the medium was detectable 6 days after infection and reached maximum values after 12 days. Metabolic labeling showed production of viral proteins to be a result of de novo synthesis. The appearance of single-stranded HBV DNA in the cytoplasm of infected cells, typically present in immature cores, indicated viral replication. HBV DNA containing particles possessing an active viral DNA polymerase could be immunoprecipitated from the medium 12 days after infection. An antiserum specific for the preS1 region of the viral envelope was capable to block infection. Presence of dimethyl sulfoxide in the medium greatly improved the yield of viral proteins. CONCLUSIONS: Primary adult human liver cells are competent for infection with HBV.

The humoral immune response to p53 in patients with hepatocellular carcinoma is specific for malignancy and independent of the alpha-fetoprotein status.

Recently, p53 gene aberrations have been recognized as a relevant factor in hepatocarcinogenesis, in tumors from both high-risk and low-risk areas. Because p53 gene mutations typically result in increased p53 levels in tumor cells, this cellular protein might become immunogenic during tumor development. To test this hypothesis, we have analyzed sera from 80 European patients with hepatocellular carcinoma for the presence of p53 antibodies. For this purpose we developed an immunoblot assay using recombinant p53 as antigen. Sixty-seven sera from patients with different acute and chronic liver diseases were used as controls. In addition, serum alpha-fetoprotein assays were performed. Circulating antibodies against p53 were found in 25% (20 of 80) of the sera from patients with hepatocellular carcinoma but not in various nonmalignant liver diseases. The association of p53 antibodies with malignancy was highly significant (p < 0.00003). In 73.8% (59 of 80) of the hepatocellular carcinoma sera the alpha-fetoprotein levels were elevated. Among the 21 alpha-fetoprotein-negative hepatocellular carcinoma sera, 5 were found to contain p53 antibodies (23.8%). In conclusion, an antibody response against p53 developed in a significant proportion of patients with hepatocellular carcinoma but not in those with nonmalignant liver diseases. Serological testing for p53 antibodies gives the opportunity to identify a subgroup of patients with hepatocellular carcinoma not detected by conventional tests for serum alpha-fetoprotein.