Alternative TEL-JAK2 fusions associated with T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia dissected in zebrafish

Background Chromosomal translocations resulting in alternative fusions of the human TEL (ETV6) and JAK2 genes have been observed in cases of acute lymphoblastic leukemia and chronic myelogenous leukemia, but a full understanding of their role in disease etiology has remained elusive. In this study potential differences between these alternative TEL-JAK2 fusions, including their lineage specificity, were investigated.

Design and Methods TEL-JAK2 fusion types derived from both T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia were generated using the corresponding zebrafish tel and jak2a genes and placed under the control of either the white blood cell-specific spi1 promoter or the ubiquitously-expressed cytomegalovirus promoter. These constructs were injected into zebrafish embryos and their effects on hematopoiesis examined using a range of molecular approaches. In addition, the functional properties of the alternative fusions were investigated in vitro.

Results Injection of the T-cell acute lymphoblastic leukemia-derived tel-jak2a significantly perturbed lymphopoiesis with a lesser effect on myelopoiesis in zebrafish embryos. In contrast, injection of the atypical chronic myelogenous leukemia-derived tel-jak2a resulted in significant perturbation of the myeloid compartment. These phenotypes were observed regardless of whether expressed in a white blood cell-specific or ubiquitous manner, with no overt cellular proliferation outside of the hematopoietic cells. Functional studies revealed subtle differences between the alternative forms, with the acute lymphoblastic leukemia variant showing higher activity, but reduced downstream signal transducer and activator of transcription activation and decreased sensitivity to JAK2 inhibition. JAK2 activity was required to mediate the effects of both variants on zebrafish hematopoiesis.

Conclusions This study indicates that the molecular structure of alternative TEL-JAK2 fusions likely contributes to the etiology of disease. The data further suggest that this class of oncogene exerts its effects in a cell lineage-specific manner, which may be due to differences in downstream signaling.

A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies.

Validation of DNA methylation biomarkers for diagnosis of acute lymphoblastic leukemia

DNA methylation biomarkers capable of diagnosis and subtyping have been found for many cancers. Fifteen such markers have previously been identified for pediatric acute lymphoblastic leukemia (ALL). Validation of these markers is necessary to assess their clinical utility for molecular diagnostics. Substantial efficiencies could be achieved with these DNA methylation markers for disease tracking with potential to replace patient-specific genetic testing.

Epigenetic deregulation in pediatric acute lymphoblastic leukemia

Similar to most cancers, genome-wide DNA methylation profiles are commonly altered in pediatric acute lymphoblastic leukemia (ALL); however, recent observations highlight that a large portion of malignancy-associated DNA methylation alterations are not accompanied by related gene expression changes. By analyzing and integrating the methylome and transcriptome profiles of pediatric B-cell ALL cases and primary tissue controls, we report 325 genes hypermethylated and downregulated and 45 genes hypomethylated and upregulated in pediatric B-cell ALL, irrespective of subtype. Repressed cation channel subunits and cAMP signaling activators and transducers are overrepresented, potentially indicating a reduced cellular potential to receive and propagate apoptotic signals. Furthermore, we report specific DNA methylation alterations with concurrent gene expression changes within individual ALL subtypes. The ETV6-RUNX1 translocation was associated with downregulation of ASNS and upregulation of the EPO-receptor, while Hyperdiploid patients (> 50 chr) displayed upregulation of B-cell lymphoma (BCL) members and repression of PTPRG and FHIT. In combination, these data indicate genetically distinct B-cell ALL subtypes contain cooperative epimutations and genome-wide epigenetic deregulation is common across all B-cell ALL subtypes.

Bardoxolone methyl induces apoptosis and autophagy and inhibits epithelial-to-mesenchymal transition and stemness in esophageal squamous cancer cells

Natural and synthetic triterpenoids have been shown to kill cancer cells via multiple mechanisms. The therapeutic effect and underlying mechanism of the synthetic triterpenoid bardoxolone methyl (C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; CDDO-Me) on esophageal cancer are unclear. Herein, we aimed to investigate the anticancer effects and underlying mechanisms of CDDO-Me in human esophageal squamous cell carcinoma (ESCC) cells. Our study showed that CDDO-Me suppressed the proliferation and arrested cells in G2/M phase, and induced apoptosis in human ESCC Ec109 and KYSE70 cells. The G2/M arrest was accompanied with upregulated p21Waf1/Cip1 and p53 expression. CDDO-Me significantly decreased B-cell lymphoma-extra large (Bcl-xl), B-cell lymphoma 2 (Bcl-2), cleaved caspase-9, and cleaved poly ADP ribose polymerase (PARP) levels but increased the expression level of Bcl-2-associated X (Bax). Furthermore, CDDO-Me induced autophagy in both Ec109 and KYSE70 cells via suppression of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. There were interactions between the autophagic and apoptotic pathways in Ec109 and KYSE70 cells subject to CDDO-Me treatment. CDDO-Me also scavenged reactive oxygen species through activation of the nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) pathway in Ec109 and KYSE70 cells. CDDO-Me inhibited cell invasion, epithelial-mesenchymal transition, and stemness in Ec109 and KYSE70 cells. CDDO-Me significantly downregulated E-cadherin but upregulated Snail, Slug, and zinc finger E-box-binding homeobox 1 (TCF-8/ZEB1) in Ec109 and KYSE70 cells. CDDO-Me significantly decreased the expression of octamer-4, sex determining region Y-box 2 (Sox-2), Nanog, and B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), all markers of cancer cell stemness, in Ec109 and KYSE70 cells. Taken together, these results indicate that CDDO-Me is a promising anticancer agent against ESCC. Further studies are warranted to explore the molecular targets, efficacy and safety of CDDO-Me in the treatment of ESCC.

Zebrafish as a model for leukemia and other hematopoietic disorders

Zebrafish is an established model for the study of vertebrate development, and is especially amenable for investigating hematopoiesis, where there is strong conservation of key lineages, genes, and developmental processes with humans. Over recent years, zebrafish has been increasingly utilized as a model for a range of human hematopoietic diseases, including malignancies. This review provides an overview of zebrafish hematopoiesis and describes its application as a model of leukemia and other hematopoietic disorders.

Leukemia risk associated with low-level benzene exposure

Background: Men who were part of an Australian petroleum industry cohort had previously been found to have an excess of lympho-hematopoietic cancer. Occupational benzene exposure is a possible cause of this excess.

Methods: We conducted a case-control study of lympho-hematopoietic cancer nested within the existing cohort study to examine the role of benzene exposure. Cases identified between 1981 and 1999 (N = 79) were age-matched to 5 control subjects from the cohort. We estimated each subject's benzene exposure using occupational histories, local site-specific information, and an algorithm using Australian petroleum industry monitoring data.

Results: Matched analyses showed that the risk of leukemia was increased at cumulative exposures above 2 ppm-years and with intensity of exposure of highest exposed job over 0.8 ppm. Risk increased with higher exposures; for the 13 case-sets with greater than 8 ppm-years cumulative exposure, the odds ratio was 11.3 (95% confidence interval = 2.85-45.1). The risk of leukemia was not associated with start date or duration of employment. The association with type of workplace was explained by cumulative exposure. There is limited evidence that short-term high exposures carry more risk than the same amount of exposure spread over a longer period. The risks for acute nonlymphocytic leukemia and chronic lymphocytic leukemia were raised for the highest exposed workers. No association was found between non-Hodgkin lymphoma or multiple myeloma and benzene exposure, nor between tobacco or alcohol consumption and any of the cancers.

Conclusions: We found an excess risk of leukemia associated with cumulative benzene exposures and benzene exposure intensities that were considerably lower than reported in previous studies. No evidence was found of a threshold cumulative exposure below which there was no risk.

Parenting behaviors and nutrition in children with leukemia

The aim of this study was to examine whether parenting behaviors are associated with child nutrition amongst pre-school children receiving treatment for acute lymphoblastic leukemia (ALL), and to determine whether this association differs from that observed amongst a healthy population. Participants were 73 parents of children aged 2–6 years. The children were either a) receiving treatment for ALL (n = 43), or b) had no major medical history (n = 30). Participants completed psychometrically validated questionnaires that assessed parenting behaviors and child diet. Increased parental overprotection was associated with higher fruit and vegetable consumption for the control group but lower fruit and vegetable consumption for the ALL group. Parental overprotection, inconsistent discipline and emotional feeding were positively associated with non-core (“junk”) food consumption for the ALL group, particularly those who had recently received steroid treatment. To the best of our knowledge, this is the first study to show that certain parenting behaviors may be associated with poor nutrition during treatment for ALL. In light of these results, parenting interventions, specifically targeting parenting behaviors such as assertive discipline, may be a mechanism for nutrition promotion amongst this vulnerable group.

Multiple pathways contribute to the hyperproliferative responses from truncated granulocyte colony-stimulating factor receptors

Mutations in the granulocyte colony-stimulating factor receptor (G-CSF-R) gene leading to a truncated protein have been identified in a cohort of neutropenia patients highly predisposed to acute myeloid leukemia. Such mutations act in a dominant manner resulting in hyperproliferation but impaired differentiation in response to G-CSF. This is due, at least in part, to defective internalization and loss of binding sites for several negative regulators, leading to sustained receptor activation. However, those signaling pathways responsible for mediating the hyperproliferative function have remained unclear. In this study, analysis of an additional G-CSF-R mutant confirmed the importance of residues downstream of Box 2 as important contributors to the sustained proliferation. However, maximal proliferation correlated with the ability to robustly activate signal transducer and activator of transcription (STAT) 5 in a sustained manner, whereas co-expression of dominant-negative STAT5, but not dominant-negative STAT3, was able to inhibit G-CSF-stimulated proliferation from a truncated receptor. Furthermore, a Janus kinase (JAK) inhibitor also strongly reduced the proliferative response, whereas inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) or phosphatidylinositol (PI) 3-kinase reduced proliferation to a lesser degree. These data suggest that sustained JAK2/STAT5 activation is a major contributor to the hyperproliferative function of truncated G-CSF receptors, with pathways involving MEK and PI 3-kinase playing a reduced role.

The role of the granulocyte colony-stimulating factor receptor (G-CSF-R) in disease

Granulocyte colony-stimulating factor (G-CSF) is a key regulator of granulopoiesis via stimulation of a specific cell-surface receptor, the G-CSF-R, found on hematopoietic progenitor cells as well as neutrophilic granulocytes. It is perhaps not surprising, therefore, that mutations of the G-CSF-R has been implicated in several clinical settings that affect granulocytic differentiation, particularly severe congenital neutropenia, myelodysplastic syndrome and acute myeloid leukemia. However, other studies suggest that signalling via the G-CSF-R is also involved in a range of other malignancies. This review focuses on the molecular mechanisms through which the G-CSF-R contributes to disease.

Somatostatin modulates G-CSF-induced but not interleukin-3-induced proliferative responses in myeloid 32D cells via activation of somatostatin receptor subtype 2

Somatostatin, originally identified as a peptide involved in neurotransmission, functions as an inhibitor of multiple cellular responses, including hormonal secretion and proliferation. Somatostatin acts through activation of G-protein-coupled receptors of which five subtypes have been identified. We have recently established that human CD34/c-kit expressing hematopoietic progenitors and acute myeloid leukemia (AML) cells exclusively express SSTR2. A major mechanism implicated in the antiproliferative action of somatostatin involves activation of the SH2 domain-containing protein tyrosine phosphatase SHP-1. While 0.1-1 x 10(-9) M of somatostatin, or its synthetic stable analog octreotide, can inhibit G-CSF-induced proliferation of AML cells, little or no effects are seen on GM-CSF- or IL-3-induced responses.
MATERIALS AND METHODS: To study the mechanisms underlying the antiproliferative responses of myeloblasts to somatostatin, clones of the IL-3-dependent murine cell line 32D that stably express SSTR2 and G-CSF receptors were generated. RESULTS: Similar to AML cells, octreotide inhibited G-CSF-induced but not IL-3-induced proliferative responses of 32D[G-CSF-R/SSTR2] cells. Somatostatin induced SHP-1 activity and inhibited G-CSF-induced, but not IL-3-induced, activation of the signal transducer and activator of transcription proteins STAT3 and STAT5.
CONCLUSION: Based on these data and previous results, we propose a model in which recruitment and activation of the tyrosine phosphatase SHP-1 by SSTR2 is involved in the selective negative action of somatostatin on G-CSF-R signaling.