Differential Hox expression in murine embryonic stem cell models of normal and malignant hematopoiesis

The Hox family are master transcriptional regulators of developmental processes, including hematopoiesis. The Hox regulators, caudal homeobox factors (Cdx1-4), and Meis1, along with several individual Hox proteins, are implicated in stem cell expansion during embryonic development, with gene dosage playing a significant role in the overall function of the integrated Hox network. To investigate the role of this network in normal and aberrant, early hematopoiesis, we employed an in vitro embryonic stem cell differentiation system, which recapitulates mouse developmental hematopoiesis. Expression profiles of Hox, Pbx1, and Meis1 genes were quantified at distinct stages during the hematopoietic differentiation process and compared with the effects of expressing the leukemic oncogene Tel/PDGFR;2. During normal differentiation the Hoxa cluster, Pbx1 and Meis1 predominated, with a marked reduction in the majority of Hox genes (27/39) and Meis1 occurring during hematopoietic commitment. Only the posterior Hoxa cluster genes (a9, a10, a11, and a13) maintained or increased expression at the hematopoietic colony stage. Cdx4, Meis1, and a subset of Hox genes, including a7 and a9, were differentially expressed after short-term oncogenic (Tel/PDGFR;2) induction. Whereas Hoxa4-10, b1, b2, b4, and b9 were upregulated during oncogenic driven myelomonocytic differentiation. Heterodimers between Hoxa7/Hoxa9, Meis1, and Pbx have previously been implicated in regulating target genes involved in hematopoietic stem cell (HSC) expansion and leukemic progression. These results provide direct evidence that transcriptional flux through the Hox network occurs at very early stages during hematopoietic differentiation and validates embryonic stem cell models for gaining insights into the genetic regulation of normal and malignant hematopoiesis

Oncogenic Properties of Apoptotic Tumor Cells in Aggressive B Cell Lymphoma

BACKGROUND: Cells undergoing apoptosis are known to modulate their tissue microenvironments. By acting on phagocytes, notably macrophages, apoptotic cells inhibit immunological and inflammatory responses and promote trophic signaling pathways. Paradoxically, because of their potential to cause death of tumor cells and thereby militate against malignant disease progression, both apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. We hypothesized that, in progression of malignant disease, constitutive loss of a fraction of the tumor cell population through apoptosis could yield tumor-promoting effects. RESULTS: Here, we demonstrate that apoptotic tumor cells promote coordinated tumor growth, angiogenesis, and accumulation of TAMs in aggressive B cell lymphomas. Through unbiased "in situ transcriptomics" analysis-gene expression profiling of laser-captured TAMs to establish their activation signature in situ-we show that these cells are activated to signal via multiple tumor-promoting reparatory, trophic, angiogenic, tissue remodeling, and anti-inflammatory pathways. Our results also suggest that apoptotic lymphoma cells help drive this signature. Furthermore, we demonstrate that, upon induction of apoptosis, lymphoma cells not only activate expression of the tumor-promoting matrix metalloproteinases MMP2 and MMP12 in macrophages but also express and process these MMPs directly. Finally, using a model of malignant melanoma, we show that the oncogenic potential of apoptotic tumor cells extends beyond lymphoma. CONCLUSIONS: In addition to its profound tumor-suppressive role, apoptosis can potentiate cancer progression. These results have important implications for understanding the fundamental biology of cell death, its roles in malignant disease, and the broader consequences of apoptosis-inducing anti-cancer therapy

Proteomic and transcriptional changes induced by Tel/PDGFbetaR

The TeIjPDGF~R {TP} chimeric protein is an activated tyrosine kinase, implicated in the pathogenesis of chronic myelomonocytic leukaemia {CMML}. In order to investigate in more detail the coupling of TP induced molecular signals to functional responses relating to haemopoiesis, we have used the tetracycline-regulated expression system in murine ES cells. The use of this system provides an ideal in vitro model to monitor the effects of expressing TP not only in primitive stem cells with pluripotent potential but also during the initiating and more definitive stages of haemopoiesis. Results indicate that expression of TP results in marked increase in differentiation of ES cells, decreasing the overall levels of transcription factors associated with pluripotency whilst increasing the expression of factors known to haemopoietic differentiation of ES cells TP promotes differentiation along the mesodermaljhaemangioblast/myelomonocytic route, resulting in a marked increase in myelopoiesis with on average 20% more myeloid colonies forming. TP also activates several major signalling pathways with tyrosines 579/581 playing a critical role in mediating signals via the RAS/ERK and STAT5 pathways, with dual targeting of the tyrosine kinase activity of TP and the MEK/ERK pathway completely preventing TP ind uced differentiation. Although activation of the Wnt/GSK3~/~- catenin pathway has been illustrated in other myeloid leukaemia's, in our study TP down regulated this pathway in our stem cell model. Activation of the pathway using three independent pharmacological compounds, supplementation with Wnt3a and expressing a dominant positive form of ~-catenin improved self-renewal v and reversed transcriptional changes induced following TP induction. Overall this study highlights an important role for the RAS/ERK, STATS and Wnt/GSK313/13- catenin pathways in leukaemic transformation by TP, and reinforces the principle that targeted disruption of key signalling pathways alongside tyrosine kinase oncogenes may result in more efficacious therapies for suppressing MPN.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Improved clonality assessment in germinal centre/post‐germinal centre non‐Hodgkin's lymphomas with high rates of somatic hypermutation

Background: PCR detects clonal rearrangements of the Ig gene in lymphoproliferative disorders. False negativity occurs in germinal centre/post-germinal centre lymphomas (GC/PGCLs) as they display a high rate of somatic hypermutation (SHM), which causes primer mismatching when detecting Ig rearrangements by PCR. Aims: To investigate the degree of SHM in a group of GC/PGCLs and assess the rate of false negativity when using BIOMED-2 PCR when compared with previously published strategies. Methods: DNA was isolated from snap-frozen tissue from 49 patients with GC/PGCL (23 diffuse large B cell lymphomas (DLBCLs), 26 follicular lymphomas (FLs)) and PCR-amplified for complete (VDJH), incomplete (DJH) and Ig kappa/lambda rearrangements using the BIOMED-2 protocols, and compared with previously published methods using consensus primers. Germinal centre phenotype was defined by immunohistochemistry based on CD10, Bcl-6 and MUM-1. Results: Clonality detection by amplifying Ig rearrangements using BIOMED-2 family-specific primers was considerably higher than that found using consensus primers (74% DLBCL and 96% FL vs 69% DLBCL and 73% FL). Addition of BIOMED-2 DJH rearrangements increased detection of clonality by 22% in DLBCL. SHM was present in VDJH rearrangements from all patients with DLBCL (median (range) 5.7% (2.5-13.5)) and FL (median (range) 5.3% ( 2.3-11.9)) with a clonal rearrangement. Conclusions: Use of BIOMED-2 primers has significantly reduced the false negative rate associated with GC/ PGCL when compared with consensus primers, and the inclusion of DJH rearrangements represents a potential complementary target for clonality assessment, as SHM is thought not to occur in these types of rearrangements

Canonical Wnt Signaling Promotes Early Hematopoietic Progenitor Formation and Erythroid Specification during Embryonic Stem Cell Differentiation

<div><p>The generation of hematopoietic stem cells (HSCs) during development is a complex process linked to morphogenic signals. Understanding this process is important for regenerative medicine applications that require <i>in vitro</i> production of HSC. In this study we investigated the effects of canonical Wnt/β-catenin signaling during early embryonic differentiation and hematopoietic specification using an embryonic stem cell system. Our data clearly demonstrates that following early differentiation induction, canonical Wnt signaling induces a strong mesodermal program whilst maintaining a degree of stemness potential. This involved a complex interplay between β-catenin/TCF/LEF/Brachyury/Nanog. β-catenin mediated up-regulation of TCF/LEF resulted in enhanced brachyury levels, which in-turn lead to Nanog up-regulation. During differentiation, active canonical Wnt signaling also up-regulated key transcription factors and cell specific markers essential for hematopoietic specification, in particular genes involved in establishing primitive erythropoiesis. This led to a significant increase in primitive erythroid colony formation. β-catenin signaling also augmented early hematopoietic and multipotent progenitor (MPP) formation. Following culture in a MPP specific cytokine cocktail, activation of β-catenin suppressed differentiation of the early hematopoietic progenitor population, with cells displaying a higher replating capacity and a propensity to form megakaryocytic erythroid progenitors. This bias towards erythroid lineage commitment was also observed when hematopoietic progenitors were directed to undergo myeloid colony formation. Overall this study underscores the importance of canonical Wnt/β-catenin signaling in mesodermal specification, primitive erythropoiesis and early hematopietic progenitor formation during hematopoietic induction.</p> </div

Activation of the canonical Wnt pathway maintains self-renewal in the absence of LIF.

<p>Parental E14 ES cells were cultured with or without BIO (5 µM) or XAV (5 or 10 µM) and DP-βC ES cells with or without Tet for 72 and 96 hours in the absence of LIF. (A) Protein extracts were immunoblotted for key signaling proteins involved in the Wnt pathway regulation; GSK-3, Active and total β-catenin with GAPDH used as a loading control, (Representative gel images shown, n=3). (B) Self-renewal potential was assessed by the percentage of colonies staining positive for alkaline phosphatase (AP) is given (Mean ⁺ SEMs, n=3 * p<0.05, ** p<0.005 by paired students t-test).</p

Active β-catenin induces an embryonic erythroid program during early differentiation.

<p>(A) Schematic diagram of the different differentiation stages examined. CONDITION 1 early differentiation, CONDITION 2 Hemangioblast/Early hematopoietic progenitors, CONDITION 3 MPP, MEP and GMP differentiation, CONDITION 4 Myeloid colony formation. (B) QRT-PCR demonstrating activation of the canonical Wnt signaling (DP-βC or E14 ES cells + BIO) up-regulates key genes important for establishing early hematopoietic commitment, primitive erythroid specification and embryonic/fetal globin genes during differentiation induction in the absence of LIF (CONDITION 1). Control cells (DP-βC + tet or E14 ES cells -BIO) were used as calibrators and the fold change was calculated using the 2 ^–ΔΔCT method (Mean of fold change +/- SEM, n=3). (C) Cells were directed to form EB’s and then cultured in M3434 to promote primitive erythroid colony formation and colonies scored. Active β-catenin signalling (DP-βC or BIO) resulted in a significant increase in primitive erythroid colonies (Mean %, +/- SEM, n=3).</p

Active β-catenin directs mesodermal differentiation.

<p>(A) Relative gene expression profile TaqMan® Mouse Stem Cell Pluripotency Array of mRNA harvested from DP-βC (+/- Tet) or E14 ES cells (+/- 5 µM BIO) following 72 and 96 hours without LIF. Relative expression of each gene calibrated to untreated controls (-DP-βC or -BIO) using the 2 ^–ΔΔCT method, plotted on a log scale with a relative expression of 1 representing no change to gene expression (Mean ⁺ SEMs, n=3). (B) Semi-quantitative RT-PCR for DP-βC (+/- Tet) and E14 ES cells (+/- 5 µM BIO, +/- 5 µM & 10 µM XAV) following 72 and 96 hours without LIF. (Representative gel images shown, n=3). (C) FACS plots confirming higher levels of Sox2, Oct3/4, Nanog or Brachyury over 72 and 96 hours following DP-βC or BIO treatment without LIF for 96 hours compared to control cells (Representative images shown, n=2).</p