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 in vitro 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.
Wnt signaling plays several roles in hematopoiesis, promoting hemopoietic stem cell (HSC) self-renewal, providing proliferative signals for immature progenitors and regulating lineage commitment. To ...ascertain which Wnt proteins and receptors are important during hematopoietic development, we used two systems; in vitro hematopoietic differentiation of embryonic stem (ES) cells and tissues isolated from sites specific for hematopoiesis during mouse embryogenesis. Initially genes involved in hematopoiesis were profiled and indicate differentiating ES cells undergo a wave of primitive hematopoiesis (Day 3.75) similar to the mouse yolk sac, followed by a wave of more definitive hematopoiesis (Day 7.75) comparable to the aorta-gonad-mesonephros (AGM) and E15.5 liver with lineage commitment by Day 15. A similar biphasic expression pattern occurred for Wnt/Fzd/LRP genes with Wnt 3, 5a, 8a, Fzd4, and LRP5 becoming upregulated during primitive hematopoiesis, followed by Wnt3a, 6, 7b, 10b, and 16 during more definitive hematopoiesis. High expression of Wnt5a, Fzd4, and LRP5 during the first phase of hematopoiesis suggests these genes are involved in early hematopoietic regulation. Wnt3a and 16 were also expressed at specific stages, with Wnt16 detected when the earliest lymphoid progenitors are formed (AGM and 2 degrees BC of ES differentiation). Wnt3a expression corresponded with the induction of definitive hematopoiesis a period, which involves rapid expansion of HSC (Day 7.75 of ES differentiation, AGM and E15.5 liver). Supplementation with Wnt3a during ES hematopoietic differentiation increased proliferation and appeared to promote stem cell expansion. Overall this study provides valuable information on the Wnt/Fzd/LRP involved in supporting embryonic hematopoiesis.
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.
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.
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.
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•Apoptotic lymphoma cells promote tumor growth, angiogenesis, and TAM accumulation•Unbiased “in situ transcriptomics” analysis shows TAMs promote pro-tumor pathways•Apoptotic tumor cells express and process matrix remodeling proteins•The oncogenic potential of apoptotic tumor cells extends beyond lymphoma
Apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. Ford et al. demonstrate apoptotic lymphoma cells can promote tumor growth, angiogenesis, TAM accumulation, and TAM activation to potentiate cancer progression. These results have important implications for apoptosis-inducing anti-cancer therapies.
Primary cilia regulate hedgehog (Hh) signal transduction; these sensory organelles being present on most mammalian cells. Aberrant Hh activity has been implicated in malignancy with defective primary ...cilia expression linked to disease. The role the Hh pathway plays in AML has not been fully elucidated and it is unclear whether AML cells express primary cilia. Our aims were (1) to determine the presence of primary cilia and (2) further evaluate the role of Hh signalling in AML.
Primary cilia were identified in all AML (n=23), and 20% of normal (n=10) bone marrow trephines (BMTs) by immunocytochemistry (ICC). Primary cilia were not identified in AML cell lines, primitive (n=4) or mature hematopoietic cells (n=6) isolated from peripheral blood, suggesting they are lost once cells migrate from the bone marrow (BM) microenvironment.
Despite the heterogeneity of AML, analysis of 76 primary AML samples demonstrated clear evidence of Hh pathway activity with up regulation of SMO (p<0.001), confirmed by ICC (n=8), and Gli-1 (p<0.05) in 55% of cases. Sanger sequencing (n=36) did not identify a mutation in SMO to account for the increased activity, although genomic instability was apparent with a high level of SNPs (mean 4; range 3-8), and silent and missense mutations (mean 1; range 1-3 per sample) identified. Samples with a >5-fold increase in SMO (n=28) showed a >5-fold up regulation of genes associated with chemoresistance and poor survival, including MECOM and FOXM1 (both p=0.0001), ABCC1, HOXA3, HOXA9, TWIST1 and SNAIL1 (all p<0.001), KIT (p<0.01), CD44, MMP2 and DNMT3b (all p<0.05); with a 4-fold reduction in pro-apoptotic genes BAK and BAX. Next Gen sequencing (n=9) confirmed an aggressive phenotype with 67% of patients having a c-Kit mutation and 46% (n=13), stratifying within the high risk category.
The BM microenvironment is important in AML. Immunohistochemistry (IHC) on human AML BMTs (n=37) enabled analysis within this unique environment. Proteins associated with poor prognosis and a more aggressive phenotype were up regulated 2-30 fold, including BCL-2 (p<0.0001), CD44 (p<0.001) and Vimentin (p<0.05). SHH was significantly down regulated (p<0.001) within the blast population. Secreted SHH, measured by ELISA, was up regulated suggesting paracrine activity. Impaired post translational modification of SHH was demonstrated with protein located within the nuclei by IHC and ICC. Nuclear expression of SHH was limited to primitive (CD34+) cells and absent from mature (CD14+, CD15+) cells. This correlated with a 20-fold reduction in HHAT the acetyltransferase involved in Hh processing in primitive cells compared to MNCs (p=<0.01).
In vitro, SMO inhibition with cyclopamine reduced cell proliferation in myelomonocytic cell lines (Kasumi-1 p<0.05, MOLM-13 p<0.01, MV4-11 p<0.05, OCI-AML3 p<0.001 and THP-1 p<0.01). No change in early or late apoptosis was seen. Cell cycle arrest was demonstrated with an increase in G0-G1 within OCI-AML3 (p<0.05) and a 3-fold reduction in cell division. Cells demonstrated a striking morphological change with increased cytoplasm, granules and loss of nucleoli. Reduction in cell proliferation and morphological changes were also observed when SMO was knocked down using a SMO siRNA. OCI-AML3 cells demonstrated increased expression of CD11b (p<0.001) and CD11c (p<0.0001) by FACS in keeping with a more mature phenotype. Gene expression correlated with a 2-fold reduction in early lineage markers, GATA1, SCL, NAB2 (p<0.05) and 3-4 fold up regulation of genes involved in differentiation and maturation, PU1, GMCSF and GCSF (all p<0.05), and IRF8 (p<0.01). Primary AML samples (n=6) were treated with the clinical grade SMO antagonist, PF-04449913, alone and in combination with cytarabine. Combination treatment caused a significant reduction in proliferation (p<0.05) with an increase in apoptosis. LTCIC showed a trend towards differentiation with a greater number of CFU-M and CFU-G than CFU-GM compared to no drug control (n=4).
This is the first report to demonstrate primary cilia on hematopoietic cells, with an increased frequency observed in AML. Their absence when cells migrate from the BM fits with their function and suggests a 'switching off' of the Hh pathway occurs on maturation. The ability of SMO inhibition to cause differentiation, in genetically diverse AML cell lines and primary AML is promising. SMO inhibition should continue to be explored as a potential therapy in AML.
Dobbin:Almac: Employment. Wheadon:GlaxoSmithKline: Research Funding. Copland:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Amgen: Honoraria; Shire: Honoraria.
Acute myeloid leukemia (AML) is a heterogeneous clonal disorder often associated with dismal overall survival. The clinical diversity of AML is reflected in the range of recurrent somatic mutations ...in several genes, many of which have a prognostic and therapeutic value. Targeted next-generation sequencing (NGS) of these genes has the potential for translation into clinical practice. In order to assess this potential, an inter-laboratory evaluation of a commercially available AML gene panel across three diagnostic centres in the UK and Ireland was performed.
DNA from six AML patient samples was distributed to each centre and processed using a standardised workflow, including a common sequencing platform, sequencing chips and bioinformatics pipeline. A duplicate sample in each centre was run to assess inter- and intra-laboratory performance.
An average sample read depth of 2725X (range 629-5600) was achieved using six samples per chip, with some variability observed in the depth of coverage generated for individual samples and between centres. A total of 16 somatic mutations were detected in the six AML samples, with a mean of 2.7 mutations per sample (range 1-4) representing nine genes on the panel. 15/16 mutations were identified by all three centres. Allelic frequencies of the mutations ranged from 5.6 to 53.3 % (median 44.4 %), with a high level of concordance of these frequencies between centres, for mutations detected.
In this inter-laboratory comparison, a high concordance, reproducibility and robustness was demonstrated using a commercially available NGS AML gene panel and platform.
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 Igκ/λ 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.
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 in vitro production of HSC. In this study we investigated the effects of canonical Wnt/ beta -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 beta -catenin/TCF/LEF/Brachyury/Nanog. beta -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. beta -catenin signaling also augmented early hematopoietic and multipotent progenitor (MPP) formation. Following culture in a MPP specific cytokine cocktail, activation of beta -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/ beta -catenin signaling in mesodermal specification, primitive erythropoiesis and early hematopietic progenitor formation during hematopoietic induction.
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/PDGFRβ. 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/PDGFRβ) 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.
Objective Fusion genes involving the platelet-derived growth factor receptor-β ( PDGFRβ ) are found in a subgroup of myeloproliferative neoplasms, with one such fusion, Tel/PDGFRβ found in a subset ...of chronic myelomonocytic leukemia patients. Tel/PDGFRβ results in constitutive activation of several signaling pathways and induces a myeloproliferative disease in mice, with signals via tyrosines 579/581 identified as being important for this phenotype. In this study, we have used a tetracycline-regulated system to express wild-type and the mutated F2 Tel/PDGFRβ to identify the key signaling pathways, which drive Tel/PDGFRβ-induced differentiation of embryonic stem (ES) cells. Materials and Methods The leukemic oncogene Tel/PDGFRβ and Tel/PDGFRβ-F2 were inducibly expressed in ES cells and their effects on self-renewal, signal transduction, and gene expression patterns analyzed. Results Tel/PDGFRβ activated several major signal transduction pathways (signal transducers and activators of transcription STAT 3, STAT5, mitogen-activated protein kinases, phosphatidylinositol-3 kinase) in ES cells, but only specific inhibition of the mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK/ERK) or STAT5 pathways was able to significantly prevent Tel/PDGFRβ-induced differentiation and restore ES-cell self-renewal. Inhibiting the tyrosine kinase activity of the oncogene using Gleevec or PDGFRβ inhibitor III also substantially prevented Tel/PDGFRβ-induced differentiation and its ability to upregulate key genes involved in myelopoiesis. Tyrosines 579/581 played a critical role in mediating signals via the Ras/ERK and STAT5 pathways, with dual targeting of the tyrosine kinase activity of Tel/PDGFRβ and the MEK/ERK pathway completely preventing Tel/PDGFRβ-induced differentiation. Conclusion These findings suggest that targeted disruption of key signaling pathways in combination with the tyrosine kinase activity of leukemic oncogenes, such as Tel/PDGFRβ, may result in more efficacious therapies for suppressing leukemic progression in the clinical setting.
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