Susan Wittig

The expression of histone deacetylase 4 is associated with prednisone poor-response in childhood acute lymphoblastic leukemia

This study aimed at the identification of histone deacetylase (HDAC) isoforms relevant for childhood acute lymphoblastic leukemia (ALL). Expression of HDAC1-11 was determined in 93 primary ALL and eight healthy donor samples. HDAC1, HDAC2 and HDAC8 showed significantly higher expressions in ALL samples. Correlation analysis of HDAC expression with clinicopathological parameters revealed that high HDAC1, HDAC2, HDAC4 and HDAC11 levels were significantly associated with unfavorable prognostic factors. Particularly, high HDAC4 expression was associated with high initial leukocyte count, T cell ALL and prednisone poor-response. siRNA-mediated inhibition of HDAC4 sensitized a T-ALL cell line to etoposide-induced cell death. In conclusion, our data point to HDAC4 as drug target in childhood ALL, especially in prednisone poor-responders.

Anticancer effects of the p53 activator nutlin-3 in Ewing’s sarcoma cells

Mutation of p53 is rare in Ewings sarcoma (ES), suggesting that targeting and activation of wild-type p53 may be an effective therapeutic strategy for ES. The recently developed small-molecule MDM2 inhibitor nutlin-3 restores wild-type p53 function, resulting in the inhibition of cancer cell growth and the induction of apoptosis. In the present study, we explored the responsiveness of ES cell lines with wild-type or mutated p53 to nutlin-3. We found that treatment with nutlin-3 increased p53 level and induced p53 target gene expression (MDM2, p21, PUMA) in ES cells with wild-type p53, but not in ES cells with mutated p53. Consistently, nutlin-3 elicited apoptosis only in wild-type p53 cells, as assessed by caspase-3 activity assay and flow cytometric analyses of mitochondrial depolarisation and DNA fragmentation. In addition, we found nutlin-3 to evoke cellular senescence, indicating that nutlin-3 induces pleiotropic anticancer effects in ES. Furthermore, combined treatment with nutlin-3 and an inhibitor of NF-κB produced synergistic antineoplastic activity in ES cells. Our findings suggest that the direct activation of p53 by nutlin-3 treatment may be a useful new therapeutic approach for patients with ES.

Increased activity of histone deacetylases in childhood acute lymphoblastic leukaemia and acute myeloid leukaemia: support for histone deacetylase inhibitors as antileukaemic agents.

Butterfield, Y., Birol, I., Holt, R., Schein, J., Horsman, D.E., Moore, R., Jones, S.J., Connors, J.M., Hirst, M., Gascoyne, R.D. & Marra, M.A. (2011) Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature, 476, 298–303. Ngo, V.N., Young, R.M., Schmitz, R., Jhavar, S., Xiao, W., Lim, K.H., Kohlhammer, H., Xu, W., Yang, Y., Zhao, H., Shaffer, A.L., Romesser, P., Wright, G., Powell, J., Rosenwald, A., MullerHermelink, H.K., Ott, G., Gascoyne, R.D., Connors, J.M., Rimsza, L.M., Campo, E., Jaffe, E.S., Delabie, J., Smeland, E.B., Fisher, R.I., Braziel, R.M., Tubbs, R.R., Cook, J.R., Weisenburger, D. D., Chan, W.C. & Staudt, L.M. (2011) Oncogenically active MYD88 mutations in human lymphoma. Nature, 470, 115–119. Rinaldi, A., Mian, M., Chigrinova, E., Arcaini, L., Bhagat, G., Novak, U., Rancoita, P.M., De Campos, C.P., Forconi, F., Gascoyne, R.D., Facchetti, F., Ponzoni, M., Govi, S., Ferreri, A.J., Mollejo, M., Piris, M.A., Baldini, L., Soulier, J., Thieblemont, C., Canzonieri, V., Gattei, V., Marasca, R., Franceschetti, S., Gaidano, G., Tucci, A., Uccella, S., Tibiletti, M.G., Dirnhofer, S., Tripodo, C., Doglioni, C., Dalla Favera, R., Cavalli, F., Zucca, E., Kwee, I. & Bertoni, F. (2011) Genome-wide DNA profiling of marginal zone lymphomas identifies subtype-specific lesions with an impact on the clinical outcome. Blood, 117, 1595–1604. Wang, L., Lawrence, M.S., Wan, Y., Stojanov, P., Sougnez, C., Stevenson, K., Werner, L., Sivachenko, A., DeLuca, D.S., Zhang, L., Zhang, W., Vartanov, A.R., Fernandes, S.M., Goldstein, N.R., Folco, E.G., Cibulskis, K., Tesar, B., Sievers, Q.L., Shefler, E., Gabriel, S., Hacohen, N., Reed, R., Meyerson, M., Golub, T.R., Lander, E. S., Neuberg, D., Brown, J.R., Getz, G. & Wu, C. J. (2011) SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. New England Journal of Medicine, 365, 2497–2506. Xu, L., Sohani, A.R., Arcaini, L., Hunter, Z., Yang, G., Zhou, Y., Liu, X., Cao, Y., Manning, R., Patterson, C.J., Ioakimidis, L., Tripsas, C., Pinkus, G.S., Harris, N.L., Rodig, S.J. & Treon, S. (2011) A somatic variant in MYD88 (L265P) revealed by whole genome sequencing differentiates lymphoplasmacytic lymphoma from marginal zone lymphomas. Blood (ASH Annual Meeting Abstracts), 118, 261. Yan, Y., Huang, Y., Watkins, A.J., Kocialkowski, S., Zeng, N., Hamoudi, R.A., Isaacson, P.G., de Leval, L., Wotherspoon, A. & Du, M.Q. (2012) BCR and TLR signaling pathways are recurrently targeted by genetic changes in splenic marginal zone lymphomas. Haematologica, 97, 595–598.

The histone deacetylase inhibitor vorinostat induces calreticulin exposure in childhood brain tumour cells in vitro

PurposeIt has recently been recognised that anticancer chemotherapy can elicit an immunogenic form of apoptosis characterised by the exposure of calreticulin (CRT) on the surface of dying tumour cells, entailing an immune response that contributes to the therapeutic outcome. CRT exposure has been found to be induced by anthracyclins and oxaliplatin, but not by other proapoptotic antineoplastic agents including etoposide, camptothecin and cisplatin. In this study, we examined the histone deacetylase inhibitor vorinostat for its capability to stimulate CRT exposure in tumour cells.MethodsChildhood tumour cells, i.e. the brain tumour cell lines PFSK and DAOY and the Ewing’s sarcoma cell line CADO-ES-1, were treated with vorinostat, and CRT exposure was determined by flow cytometric analysis of CRT immunofluorescence. Combination effects of vorinostat/TRAIL and vorinostat/bortezomib were also assessed.ResultsVorinostat treatment induced CRT exposure in PFSK and DAOY cells, but not in caspase-8-deficient CADO-ES-1 cells. CRT exposure could be prevented by the pan-caspase inhibitor z-VAD-fmk and by brefeldin A, an inhibitor of Golgi-mediated transport.ConclusionVorinostat has the capacity to elicit CRT exposure, suggesting its usefulness as immunogenic antitumour agent.

Mesothelin, a possible target for immunotherapy, is expressed in primary AML cells

Background:  Mesothelin is a promising candidate for tumor‐specific therapy because of its limited expression in normal tissues and high expression in several cancers. The expression of the protein mesothelin in hematological malignancies has not yet been analyzed. SS1(dsFv)PE38 is a recombinant anti‐mesothelin immunotoxin which is undergoing clinical evaluation in patients with mesothelin‐expressing tumors.

Histone deacetylase inhibitor-mediated sensitization to TRAIL-induced apoptosis in childhood malignancies is not associated with upregulation of TRAIL receptor expression, but with potentiated caspase-8 activation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has great potential for the treatment of cancer because it targets tumor cells while sparing normal cells. Several cancers, however, fail to respond to TRAILs antineoplastic effects. These resistant tumors require cotreatment with sensitizing agents in order for TRAIL to exert anticancer activity. Histone deacetylase inhibitors (HDACi) have been recognized as potent TRAIL sensitizers. In searching for the determinants of TRAIL responsiveness, HDACi-mediated TRAIL sensitization has been predominantly attributed to TRAIL receptor upregulation. This explanation, however, has been challenged by a few studies. The aim of the present study was to explore the relevance of TRAIL receptor expression for HDACi-mediated TRAIL sensitization in childhood tumors, i.e., in medulloblastoma, Ewings sarcoma and osteosarcoma. In previous studies, we had shown that TRAIL and HDACi were synergistic in inducing apoptosis in medulloblastoma and Ewings sarcoma. In the present study, we demonstrate that HDACi cooperated with TRAIL in eliciting cell death in osteosarcoma. However, HDACi treatment did not alter or even reduced cell surface expression of TRAIL receptors in the three childhood tumors. In gaining insight into the apoptotic pathway involved in TRAIL sensitization, HDACi were found to potentiate TRAIL-induced caspase-8 activation. Taken together, our findings suggest that HDACi-mediated TRAIL sensitization is not the result of TRAIL receptor upregulation, but the result of a receptor-proximal event in childhood tumor cells.

Polymorphisms of Dectin-1 and TLR2 Predispose to Invasive Fungal Disease in Patients with Acute Myeloid Leukemia

Background Patients with acute myeloid leukemia (AML) who undergo induction chemotherapy are at high risk for invasive fungal disease (IFD). Dectin-1, a C-type lectin family member represents one of the most important pattern recognition receptors of the innate immune system and single nucleotide polymorphisms (SNPs) in the Dectin-1 gene have been associated with an increased risk of infectious complications. We sought to investigate the impact of three different Dectin-1 SNPs and one TLR2 SNP on developing IFD in 186 adult patients with newly diagnosed AML following anthracycline-based induction chemotherapy. Patients and methods Genotyping of Dectin-1 SNPs (rs16910526, rs3901533 and rs7309123) and TLR2 SNP (rs5743708) was performed by TaqMan method and pyrosequencing. IFD was defined according to the EORTC/MSG consensus guidelines. Multiple logistic regression analyses were applied to evaluate the association between the polymorphisms and the occurrence of pulmonary infections. Dectin-1 expression studies with SNP genotyped human monocytes were performed to elucidate susceptibility to IFD following chemotherapy. Results We could demonstrate that patients carrying the Dectin-1 SNP rs7309123 G/G (n = 47) or G/G and C/G (n = 133) genotype revealed a significant higher risk for developing both pneumonia in general (adjusted odds ratio (OR): 2.5; p = 0.014 and OR: 3.0, p = 0.004) and pulmonary IFD (OR: 2.6; p = 0.012 and OR: 2.4, p = 0.041, respectively). Patients carrying the TLR2 SNP rs5743708 (R753Q, GA/AA genotype, n = 12) also revealed a significantly higher susceptibility to pneumonia including IFD. Furthermore, Dectin-1 mRNA expression in human monocytes was lower following chemotherapy. Conclusion To our best knowledge, this study represents the first analysis demonstrating that harbouring polymorphisms of Dectin-1 (rs7309123) or TLR2 (rs5743708) represents an independent risk factor of developing IFD in patients with AML undergoing induction chemotherapy.

Prognostic impact of WT1 expression prior to hematopoietic stem cell transplantation in children with malignant hematological diseases

PurposeMalignant hematological diseases represent the most common pediatric cancer. As they cannot always be cured by chemotherapy alone, leukemia and myelodysplastic syndrome (MDS) are frequent medical indications for hematopoietic stem cell transplantation, yet even this treatment is not capable of preventing relapse for certain. Therefore, molecular markers are used to monitor minimal residual disease (MRD) to be enabled to react early to an impeding relapse. As specific markers are not always available, Wilms’ tumor gene 1 (WT1) has been suggested as a universal marker, but has not yet been established clinically.MethodsWe determined the level of WT1 gene expression in 130 children, adolescents and young adults with malignant hematological diseases prior to transplantation and evaluated its impact on patients’ outcome. A real-time quantitative RT-PCR was used for this purpose.ResultsThe relationship between a high level of WT1 and the cumulative incidence of relapse, event-free survival and overall survival proved to be highly significant in univariate and multivariate analyses. Forty-eight percent of all patients with high WT1 levels suffered from a relapse, whereas only eight percent showing normal WT1 levels before transplantation relapsed. The most convincing result was found for acute myeloid leukemia (AML) and MDS.ConclusionWe conclude that WT1 expression prior to transplantation qualifies as an independent prognostic factor and should be further evaluated for MRD monitoring. It might especially be useful for patients with AML or MDS missing specific markers.

Adiponectin serum concentrations in newborn at delivery appear to be of fetal origin

Abstract Objective: Adiponectin (APN) may play a role in adapting energy metabolism at the maternal-fetal unit. The aim of the study was to investigate the relationship between placental APN mRNA expression, maternal serum APN concentration and umbilical cord serum APN concentration in full-term healthy newborns. Methods: Serum APN levels were compared in 46 samples (23 from healthy newborns; gestational age 37.0 to 41.5 weeks) and their mothers (n=23). The APN concentration was measured using enzyme linked immunosorbent assay (ELISA). We analyzed the mRNA expression profile of APN in 22 placenta tissue samples using real time polymerase chain reaction (RT-PCR). Results: The highest APN serum concentrations were found in umbilical cord blood, these were significantly higher than maternal APN levels (mean concentration±SD; 38.48±12.8 vs. 6.6±2.3 μg/mL, p<0.001). Otherwise, there were no significant correlation between maternal APN and umbilical cord APN concentration. APN gene expression was very low and only found in 8 out of 22 placentas. There were no significant correlation between placental APN mRNA and umbilical cord serum APN or maternal serum APN concentration. Umbilical cord APN concentrations were positively associated with birth weight (r=0.535; p=0.012) and gestational age (r=0.559; p=0.013). Maternal APN concentration revealed a negative correlation between maternal body weight (r=–0.623; p=0.009) and body mass index (BMI) (r=–0.634; p=0.008) at delivery. Additionally, no significant correlation was found between newborn birth weight and maternal weight. Conclusions: This study suggests that high serum APN concentrations in umbilical cord blood are not regulated by placental APN mRNA gene expression. The high concentration of APN in cord blood is independent from maternal APN concentration, suggesting an important physiological role of APN and implicating that umbilical APN concentration reflects its exclusive production by fetal tissues.

RETRA exerts anticancer activity in Ewing’s sarcoma cells independent of their TP53 status

Mutant p53 can exert oncogenic activity by inhibitory interaction with p73. The small-molecule RETRA has been described to disrupt this interaction and to suppress carcinoma cells (Kravchenko et al., 2008). RETRAs anticancer activity was restricted to tumour cells bearing mutant p53; it was not active in p53 negative and in p53 wild-type cells. Here, we explored the responsiveness of Ewings sarcoma (ES) cells with mutant p53 to RETRA. For comparison, we also tested RETRA in p53 null and in p53 wild-type ES cells. We found RETRA to be effective in the three mutant p53 ES cell lines investigated. Strikingly, however, RETRA was similarly effective in the p53-deficient and in the two p53 wild-type ES cell lines examined. RETRA elicited apoptosis, as assessed by flow cytometric analyses of mitochondrial depolarisation and DNA fragmentation, caspase 3/7 activity assay and PARP-1 cleavage immunodetection, and G2/M cell cycle arrest completely independent of the cellular TP53 status. In contrast, various p53-deficient and -proficient carcinoma, osteosarcoma and leukaemia cells were unresponsive to RETRA. RETRA also induced gene expression of p53 target genes PUMA and p21 in ES cells irrespective of their TP53 status. These in vitro findings provide a rationale for an in vivo exploration of RETRAs potential as an effective therapeutic approach for patients with ES.