Aberrant DNA hypermethylation of the ITIH5 tumor suppressor gene in acute myeloid leukemia

Epigenetic mechanisms such as DNA hypermethylation and modifications of histone amino acids are known to play an important role in the control of gene expression both in normal human development and tumorigenesis. Hypermethylation of CpG islands within promoter regions of tumor suppressor genes is associated with transcriptional inactivation and represents, in addition to genetic aberrations, an important mechanism of gene silencing in the pathogenesis of human cancer. Inter-α-trypsine inhibitors (ITIs) are a family of serine protease inhibitors consisting of one light chain (bikunin) and two heavy chains (ITI heavy chains, ITIHs). ITIHs stabilize the extracellular matrix (ECM) by interacting with hyaluronic acid, which is a major ECM component. Hypermethylation in the upstream region of the promoter-associated CpG island of ITIH5, the most recently described member of the ITIH family, has been previously detected in breast cancer and was associated with an adverse outcome. In this study, we determined the DNA methylation status of the promoter region near the transcription start site of the ITIH5 tumor suppressor gene in leukemia cell lines and primary samples from patients with acute myeloid leukemia (AML) as well as the potential use of demethylating agents to restore a demethylated state of the promoter. Aberrant ITIH5 promoter hypermethylation occurred in 15 of 104 (14.4%) diagnostic AML samples. There were no statistically significant correlations between the ITIH5 methylation status and clinical prognostic parameters. Our results indicate that aberrant ITIH5 promoter hypermethylation is a novel epigenetic event in AML

Germline mutations in mitochondrial complex I reveal genetic and targetable vulnerability in IDH1-mutant acute myeloid leukaemia

The interaction of germline variation and somatic cancer driver mutations is underinvestigated. Here we describe the genomic mitochondrial landscape in adult acute myeloid leukaemia (AML) and show that rare variants affecting the nuclear- and mitochondriallyencoded complex I genes show near-mutual exclusivity with somatic driver mutations affecting isocitrate dehydrogenase 1 (IDH1), but not IDH2 suggesting a unique epistatic relationship. Whereas AML cells with rare complex I variants or mutations in IDH1 or IDH2 all display attenuated mitochondrial respiration, heightened sensitivity to complex I inhibitors including the clinical-grade inhibitor, IACS-010759, is observed only for IDH1-mutant AML. Furthermore, IDH1 mutant blasts that are resistant to the IDH1-mutant inhibitor, ivosidenib, retain sensitivity to complex I inhibition. We propose that the IDH1 mutation limits the flexibility for citrate utilization in the presence of impaired complex I activity to a degree that is not apparent in IDH2 mutant cells, exposing a mutation-specific metabolic vulnerability. This reduced metabolic plasticity explains the epistatic relationship between the germline complex I variants and oncogenic IDH1 mutation underscoring the utility of genomic data in revealing metabolic vulnerabilities with implications for therapy.Mahmoud A. Bassal, Saumya E. Samaraweera, Kelly Lim, Brooks A. Bernard, Sheree Bailey, Satinder Kaur, Paul Leo, John Toubia, Chloe Thompson-Peach, Tran Nguyen, Kyaw Ze Ya Maung, Debora A. Casolari, Diana G. Iarossi, Ilaria S. Pagani, Jason Powell, Stuart Pitson, Siria Natera, Ute Roessner, Ian D. Lewis, Anna L. Brown, Daniel G. Tenen, Nirmal Robinson, David M. Ross, Ravindra Majeti, Thomas J. Gonda, Daniel Thomas, Richard J. D, Andre

High Erk-1 activation and Gadd45a expression as prognostic markers in high risk pediatric haemolymphoproliferative diseases

Studies on activated cell-signaling pathways responsible for neoplastic transformation are numerous in solid tumors and in adult leukemias. Despite of positive results in the evolution of pediatric hematopoietic neoplasias, there are some high-risk subtypes at worse prognosis. The aim of this study was to asses the expression and activation status of crucial proteins involved in cell-signaling pathways in order to identify molecular alterations responsible for the proliferation and/or escape from apoptosis of leukemic blasts. The quantitative and qualitative expression and activation of Erk-1, c-Jun, Caspase8, and Gadd45a was analyzed, by immunocytochemical (ICC) and western blotting methods, in bone marrow blasts of 72 patients affected by acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (ALL) and stage IV non-Hodgkin Lymphoma (NHL). We found an upregulation of Erk-1, Caspase8, c-Jun, and Gadd45a proteins with a constitutive activation in 95.8%, 91.7%, 86.2%, 83.4% of analyzed specimens, respectively. It is worth noting that all AML patients showed an upregulation of all proteins studied and the high expression of GADD45a was associated to the lowest DFS median (p = 0.04). On univariate analysis, only Erk-1 phosphorylation status was found to be correlated with a significantly shorter 5-years DFS in all disease subgroups (p = 0.033) and the lowest DFS median in ALL/NHL subgroup (p = 0.04). Moreover, the simultaneous activation of multiple kinases, as we found for c-Jun and Erk-1 (r = 0.26; p = 0.025), might synergistically enhance survival and proliferation potential of leukemic cells. These results demonstrate an involvement of these proteins in survival of blast cells and, consequently, on relapse percentages of the different subgroups of patients

Systemic mastocytosis associated with t(8;21)(q22;q22) acute myeloid leukemia

Although KIT mutations are present in 20–25% of cases of t(8;21)(q22;q22) acute myeloid leukemia (AML), concurrent development of systemic mastocytosis (SM) is exceedingly rare. We examined the clinicopathologic features of SM associated with t(8;21)(q22;q22) AML in ten patients (six from our institutions and four from published literature) with t(8;21) AML and SM. In the majority of these cases, a definitive diagnosis of SM was made after chemotherapy, when the mast cell infiltrates were prominent. Deletion 9q was an additional cytogenetic abnormality in four cases. Four of the ten patients failed to achieve remission after standard chemotherapy and seven of the ten patients have died of AML. In the two patients who achieved durable remission after allogeneic hematopoietic stem cell transplant, recipient-derived neoplastic bone marrow mast cells persisted despite leukemic remission. SM associated with t(8;21) AML carries a dismal prognosis; therefore, detection of concurrent SM at diagnosis of t(8;21) AML has important prognostic implications

Shared and Distinct Functions of the Transcription Factors IRF4 and IRF8 in Myeloid Cell Development

Interferon regulatory factor (IRF) 8 and IRF4 are structurally-related, hematopoietic cell-specific transcription factors that cooperatively regulate the differentiation of dendritic cells and B cells. Whilst in myeloid cells IRF8 is known to modulate growth and differentiation, the role of IRF4 is poorly understood. In this study, we show that IRF4 has activities similar to IRF8 in regulating myeloid cell development. The ectopic expression of IRF4 in myeloid progenitor cells in vitro inhibits cell growth, promotes macrophages, but hinders granulocytic cell differentiation. We also show that IRF4 binds to and activates transcription through the IRF-Ets composite sequence (IECS). Furthermore, we demonstrate that Irf8-/-Irf4-/- mice exhibit a more severe chronic myeloid leukemia (CML)-like disease than Irf8-/- mice, involving a disproportionate expansion of granulocytes at the expense of monocytes/macrophages. Irf4-/- mice, however, display no obvious abnormality in myeloid cell development, presumably because IRF4 is expressed at a much lower level than IRF8 in granulocyte-macrophage progenitors. Our results also suggest that IRF8 and IRF4 have not only common but also specific activities in myeloid cells. Since the expression of both the IRF8 and IRF4 genes is downregulated in CML patients, these results may add to our understanding of CML pathogenesis

A Differentiation-Based Phylogeny of Cancer Subtypes

Histopathological classification of human tumors relies in part on the degree of differentiation of the tumor sample. To date, there is no objective systematic method to categorize tumor subtypes by maturation. In this paper, we introduce a novel computational algorithm to rank tumor subtypes according to the dissimilarity of their gene expression from that of stem cells and fully differentiated tissue, and thereby construct a phylogenetic tree of cancer. We validate our methodology with expression data of leukemia, breast cancer and liposarcoma subtypes and then apply it to a broader group of sarcomas. This ranking of tumor subtypes resulting from the application of our methodology allows the identification of genes correlated with differentiation and may help to identify novel therapeutic targets. Our algorithm represents the first phylogeny-based tool to analyze the differentiation status of human tumors

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases

Regulation of the gene for CD34, a human hematopoietic stem cell antigen, in KG-1 cells

Abstract CD34 is one of the best characterized human hematopoietic stem cell antigens defined to date. It is expressed on 1% to 4% of normal bone marrow cells, including colony forming units of all lineages and their precursors. CD34 expression is lost during hematopoietic development and is not found on mature peripheral blood cells. The control of CD34 expression was studied in the myeloblast cell line KG-1 as a model for the regulation of stem cell genes. CD34 mRNA was expressed at high levels in uninduced KG-1 cells. Upon induction of the cells towards macrophages with tetradecanoylphorbol-13-acetate (TPA) and ionomycin, steady state levels of CD34 mRNA decreased rapidly. Nuclear run-on assays did not show a significant change in the rate of CD34 transcription upon induction. The half-life of CD34 mRNA was 4.5 hours in uninduced KG-1 cells and 2.25 hours in induced cells, demonstrating the involvement of post-transcriptional mechanisms in CD34 downregulation. Cycloheximide had no effect on the downregulation of CD34, suggesting that labile proteins are not required for this process. This model should allow the study of some of the regulatory mechanisms controlling early events in hematopoiesis.</jats:p

The human CD34 hematopoietic stem cell antigen promoter and a 3' enhancer direct hematopoietic expression in tissue culture

Abstract The human CD34 hematopoietic stem cell antigen is a highly glycosylated type 1 membrane protein of unknown function. CD34 is expressed on 1% to 4% of bone marrow cells, including pluripotent stem cells and committed progenitors of each hematopoietic lineage. CD34 has also been shown to be expressed on the small vessel endothelium of a variety of tissues and on a subset of bone marrow stromal cells. We have chosen to use the human CD34 gene as model to examine the transcription factors and cis-elements required for stem cell/progenitor cell-specific gene regulation. We show here that the CD34 gene is transcriptionally regulated in tissue culture cells. Using a luciferase reporter gene, we have isolated and characterized an active CD34 promoter. A CD34- luciferase construct, containing 4.5 kb of 5′ flanking DNA from a CD34 genomic clone, was 30-fold more active in CD34+ tissue culture cells than in HeLa cells. Sequences from the 3′ end of the CD34 gene were shown to have enhancing activity in CD34+ T-lymphoblastic RPMI-8402 cells and not in CD34- U937 cells or in nonhematopoietic HeLa cells. We also show that a cytidine-guanosine island in the 5′ end of the CD34 gene is heavily methylated in two CD34- hematopoietic cell lines and demethylated in two CD34+ cell lines. Analysis of the CD34 promoter should result in the identification of stem cell/progenitor cell- specific transcription factors and should provide a means to direct the expression of heterologous genes in hematopoietic stem cells and progenitors.</jats:p