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1
Kettyle, Laura M., Charles-Étienne Lebert-Ghali, Ivan V. Grishagin, Glenda J. Dickson, Paul G. O’Reilly, David A. Simpson, Janet J. Bijl, Ken I. Mills, Guy Sauvageau, and Alexander Thompson. "Pathways, Processes, and Candidate Drugs Associated with a Hoxa Cluster-Dependency Model of Leukemia." Cancers 11, no. 12 (December 17, 2019): 2036. http://dx.doi.org/10.3390/cancers11122036.
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High expression of the HOXA cluster correlates with poor clinical outcome in acute myeloid leukemias, particularly those harboring rearrangements of the mixed-lineage-leukemia gene (MLLr). Whilst decreased HOXA expression acts as a readout for candidate experimental therapies, the necessity of the HOXA cluster for leukemia maintenance has not been fully explored. Primary leukemias were generated in hematopoietic stem/progenitor cells from Cre responsive transgenic mice for conditional deletion of the Hoxa locus. Hoxa deletion resulted in reduced proliferation and colony formation in which surviving leukemic cells retained at least one copy of the Hoxa cluster, indicating dependency. Comparative transcriptome analysis of Hoxa wild type and deleted leukemic cells identified a unique gene signature associated with key pathways including transcriptional mis-regulation in cancer, the Fanconi anemia pathway and cell cycle progression. Further bioinformatics analysis of the gene signature identified a number of candidate FDA-approved drugs for potential repurposing in high HOXA expressing cancers including MLLr leukemias. Together these findings support dependency for an MLLr leukemia on Hoxa expression and identified candidate drugs for further therapeutic evaluation.
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Serio, Justin, Wei Chen, Maria Mysliwski, Lili Chen, James Ropa, Jingya Wang, and Andrew G. Muntean. "The PAF Complex Regulation of Prmt5 Facilitates Leukemic Progression." Blood 128, no. 22 (December 2, 2016): 3914. http://dx.doi.org/10.1182/blood.v128.22.3914.3914.
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Abstract Acute myeloid leukemias have been linked with dysregulated epigenetic landscapes sometimes attributed to altered functions of epigenetic regulators. The Polymerase-Associated Factor complex (PAFc) is an epigenetic regulator involved in transcriptional initiation, elongation and termination and directly interacts with the CTD of RNA Pol II. The complex is comprised of 6 subunits in human cells, Paf1, Cdc73, Ctr9, Leo1, Rtf1 and Ski8. Many of these subunits have key roles in a variety of cancers including acute myeloid leukemia (AML). We have previously shown the relevance of the PAFc in MLL-rearranged leukemias where its interaction with MLL fusion-proteins is required for leukemic progression in vitro and in vivo (Muntean et al. 2013 Blood, Muntean et al. 2010 Cancer Cell). However, little is known about the gene programs controlled by the PAFc and how these contribute to leukemogenesis. Here we identify Prmt5, an arginine methyltransferase, as a direct downstream target gene of the PAFc. Prmt5 is upregulated in variety of cancers and has been linked to cell cycle progression and activation of known oncoproteins. In addition, Prmt5 has been implicated in AML and is essential for normal hematopoiesis where loss of Prmt5 induces bone marrow aplasia due to impaired cytokine signaling (Tarighat et al. 2015 Leukemia, Liu et al. 2015 J Clin Invest). Our work establishes a major role for the PAFc in regulating Prmt5 expression in AML. We observe that excision of the Cdc73 subunit of the PAFc results in reduced proliferation, the induction of differentiation, cell cycle arrest, and a mild increase in apoptosis. Several key epigenetic marks are reduced globally upon loss of Cdc73 including H4R3me2s, a modification catalyzed by Prmt5. RNA sequencing and bioinformatics analysis using GSEA, revealed that loss of Cdc73 led to increased expression of a gene program associated with hematopoietic differentiation, in agreement with our cellular characterization. In addition, the downregulation of a methyltransferase gene program was detected upon Cdc73 excision. Included in this signature were several members of the Prmt family. Analysis of changes in expression following loss of Cdc73 and functional relevance in MLL-AF9 leukemic cells led us to Prmt5 as a gene critically important in AML cells and modulated by the PAFc. To interrogate the function of Prmt5 in AML cells, we performed shRNA knockdown experiments which resulted in reduced proliferation, reduced cell fitness, G1 cell cycle arrest and global reduction H4R3me2s. ChIP experiments revealed that the PAFc localizes to the Prmt5 locus in mouse and human derived leukemic cells. Further, preliminary data suggests the MLL-AF9 fusion protein also localizes to the Prmt5 locus and may enhance its transcriptional output. The enzymatic activity of Prmt5 is necessary for AML cell growth as wild type PRMT5 can rescue proliferation of Prmt5 knock-down cells while a catalytic dead mutant cannot. Furthermore, we have observed that knockdown of Prmt5 increases the disease latency of Hoxa9/Meis1 induced leukemia in vivo. Utilizing a commercially available inhibitor for Prmt5, EPZ015666 (Chan-Pembre et al. 2015 Nat Chem Bio), we show pharmacologic inhibition of PRMT5 reduces the growth of a spectrum of human leukemic cell lines, suggesting PRMT5 is important for multiple subtypes of AML. Overall, our findings elucidate the PAFc as a regulator of Prmt5 expression that is necessary for the maintenance of AML. Disclosures No relevant conflicts of interest to declare.
3
Gan, TE, PE Dadonna, and BS Mitchell. "Genetic expression of adenosine deaminase in human lymphoid malignancies." Blood 69, no. 5 (May 1, 1987): 1376–80. http://dx.doi.org/10.1182/blood.v69.5.1376.1376.
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Abstract Adenosine deaminase (ADA) is an enzyme in the purine catabolic pathway that has been used as an enzymatic marker of T cell lymphoblastic malignancies due to its high specific activity in thymocytes and immature T cells. We have investigated whether the level of ADA activity in lymphoid leukemic cells correlates with the amount of ADA- specific RNA and/or immunoreactive protein in these cells as an initial step toward characterizing the nature of the genetic regulation of ADA expression during differentiation. We have found a good correlation between the steady state levels of ADA-specific RNA and ADA- immunoreactive protein in T lymphoblastic leukemic cell lines, mature T cell lines, a B lymphoblast cell line, and leukemic cells directly isolated from four patients with acute lymphoblastic leukemia and three patients with chronic lymphocytic leukemia. Southern blot analysis of DNA from these cells shows no evidence for differences in ADA gene copy number or gene rearrangement to account for the variability in ADA expression. We conclude that levels of ADA in lymphoid leukemic cells are directly related to the amount of ADA-specific mRNA present. These findings imply that ADA expression in leukemic cells reflects either the transcriptional activity of the ADA gene or the stability of ADA mRNA in these cells.
4
Gan, TE, PE Dadonna, and BS Mitchell. "Genetic expression of adenosine deaminase in human lymphoid malignancies." Blood 69, no. 5 (May 1, 1987): 1376–80. http://dx.doi.org/10.1182/blood.v69.5.1376.bloodjournal6951376.
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Adenosine deaminase (ADA) is an enzyme in the purine catabolic pathway that has been used as an enzymatic marker of T cell lymphoblastic malignancies due to its high specific activity in thymocytes and immature T cells. We have investigated whether the level of ADA activity in lymphoid leukemic cells correlates with the amount of ADA- specific RNA and/or immunoreactive protein in these cells as an initial step toward characterizing the nature of the genetic regulation of ADA expression during differentiation. We have found a good correlation between the steady state levels of ADA-specific RNA and ADA- immunoreactive protein in T lymphoblastic leukemic cell lines, mature T cell lines, a B lymphoblast cell line, and leukemic cells directly isolated from four patients with acute lymphoblastic leukemia and three patients with chronic lymphocytic leukemia. Southern blot analysis of DNA from these cells shows no evidence for differences in ADA gene copy number or gene rearrangement to account for the variability in ADA expression. We conclude that levels of ADA in lymphoid leukemic cells are directly related to the amount of ADA-specific mRNA present. These findings imply that ADA expression in leukemic cells reflects either the transcriptional activity of the ADA gene or the stability of ADA mRNA in these cells.
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Rosenbauer, Frank, Steffen Koschmieder, Ulrich Steidl, and Daniel G. Tenen. "Effect of transcription-factor concentrations on leukemic stem cells." Blood 106, no. 5 (September 1, 2005): 1519–24. http://dx.doi.org/10.1182/blood-2005-02-0717.
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Abstract Increasing evidence suggests that leukemias are sustained by leukemic stem cells. However, the molecular pathways underlying the transformation of normal cells into leukemic stem cells are still poorly understood. The involvement of a small group of key transcription factors into this process was suggested by their frequent mutation or down-regulation in patients with acute myeloid leukemia (AML). Recent findings in mice with hypomorphic transcription-factor genes demonstrated that leukemic stem-cell formation in AML could directly be caused by reduced transcription-factor activity beyond a critical threshold. Most interestingly, those experimental models and the paucity of biallelic null mutations or deletions in transcription-factor genes in patients suggest that AML is generally associated with graded down-regulation rather than complete disruption of transcription factors. Here, we discuss the effects of transcription-factor concentrations on hematopoiesis and leukemia, with a focus on the regulation of transcription-factor gene expression as a major mechanism that alters critical threshold levels during blood development and cancer.
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Resar, Linda, Joelle Hillion, Surajit Dhara, Takita Felder Sumter, Mita Mukherjee, Francescopaolo Di Cello, Amy Belton, et al. "The HMGA1a-STAT3 axis: an “Achilles Heel” for Hematopoietic Malignancies Overexpressing HMGA1a?" Blood 112, no. 11 (November 16, 2008): 3810. http://dx.doi.org/10.1182/blood.v112.11.3810.3810.
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Abstract Although the high mobility group A1 (HMGA1) oncogene is widely overexpressed in high-risk hematopoietic malignancies and other aggressive cancers, the molecular mechanisms underlying transformation by HMGA1 are only beginning to emerge. The HMGA1 gene encodes the HMGA1a and HMGA1b protein isoforms, which function in regulating gene expression. We showed that HMGA1 induces leukemic transformation in cultured human lymphoid cells. Inhibiting HMGA1 expression blocks the transformed phenotype in cultured human leukemia and lymphoma cells. We also engineered HMGA1a transgenic mice and all mice develop aggressive lymphoid malignancy which closely models human T-cell acute lymphoblastic leukemia. Because HMGA1 participates in transcriptional regulation, we hypothesize that it drives leukemic transformation by dysregulating specific molecular pathways. To discover genes targeted by HMGA1 in leukemic transformation, we performed gene expression profile analysis. The signaltransducer andactivator oftranscription 3 (STAT3) gene was identified as a critical downstream target of HMGA1. STAT3 mRNA and protein are up-regulated in leukemic cells overexpressing HMGA1a and activated STAT3 recapitulates the transforming activity of HMGA1a. HMGA1a binds directly to a conserved region of the STAT3 promoter in vivo and activates transcription of the STAT3 promoter in human leukemia cells. Blocking STAT3 function with a small molecule, platinum compound inhibitor (CPA-7) induces apoptosis in leukemic cells from HMGA1 transgenic mice, but not in control cells. In primary, human leukemia samples, there is a positive correlation between HMGA1a and STAT3 mRNA. Moreover, blocking STAT3 function with a dominant-negative construct in human leukemia or lymphoma cells leads to decreased cellular motility and colony formation. We also showed that treatment with a small molecule, oligonucleotide inhibitor decreases the leukemic burden in the HMGA1a transgenic mice. Our results demonstrate that the HMGA1a-STAT3 axis is a potential “Achilles heel” that could be exploited therapeutically in selected hematopoietic malignancies.
7
Cheruku, Patali S., Marina Bousquet, Guoqing Zhang, Guangtao Ge, Wei Ying, Cong Meng, Andrew Shie, et al. "MiR-150 Inhibits MLL-AF9 Associated Leukemia By Suppressing Leukemic Stem Cells." Blood 122, no. 21 (November 15, 2013): 3764. http://dx.doi.org/10.1182/blood.v122.21.3764.3764.
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Abstract Leukemic stem cells (LSCs) are derived from hematopoietic stem or progenitor cells and often share gene expression patterns and specific pathways. Characterization and mechanistic studies of LSCs are critical as they are responsible for the initiation and potential relapse of leukemias, however the overall framework, including epigenetic regulation, is not yet clear. We previously identified microRNA-150 (miR-150) as a critical regulator of mixed lineage leukemia (MLL) -associated leukemias by targeting oncogenes. Our additional results suggest that miR-150 can inhibit LSC survival and disease initiating capacity by suppressing more than 30% of “stem cell signature genes,” hence altering multiple cancer pathways and/or stem cell identities. MLL-AF9 cells derived from miR-150 deficient hematopoietic stem/progenitor cells displayed significant proliferating advantage and enhanced leukemic colony formation. Whereas, with ectopic miR-150 expression, the MLL-AF9 associated LSC population (defined as Lin-ckit+sca1- cells) was significantly decreased in culture. This is further confirmed by decreased blast leukemic colony formation in vitro. Furthermore, restoration of miR-150 levels in transformed MLL-AF9 cells, which often display loss of miR-150 expression in AML patients with MLL-fusion protein expressing, completely blocked the myeloid leukemia development in a transplantation mouse model. Gene profiling analysis demonstrated that an increased level of miR-150 expression down regulates 30 of 114 stem cell signature genes by more than 1.5 fold, partially mediated by the suppressive effects of miR-150 on CBL, c-Myb and Egr2 oncogenes. In conclusion, our results suggest that miR-150 is a potent MLL-AF9 leukemic inhibitor that may act by suppressing the survival and leukemic initiating potency of MLL-AF9 LSCs. Disclosures: No relevant conflicts of interest to declare.
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Palmi, Chiara, Grazia Fazio, Ilaria Brunati, Valeria Cazzaniga, Valentina André, Silvano Sozzani, Antonello Villa, et al. "TEL-AML1 Affects the Regulation of Cytoskeleton and Causes Alteration In Cellular Adhesive and Migratory Properties In An In Vitro Model of Pre-Leukemia." Blood 116, no. 21 (November 19, 2010): 3624. http://dx.doi.org/10.1182/blood.v116.21.3624.3624.
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Abstract Abstract 3624 Introduction: The t(12;21) chromosome translocation generating TEL-AML1 chimeric fusion gene is a frequent initiating event in childhood leukaemia. Its impact is to generate a clone of covert, clinically silent pre-leukemic B cell progenitors. The leukemia arises only following second, post-natal hit/genetic events occurring years later. Moreover, relapse of leukemia is frequently arising from the pre-leukemic clone. Aim of our study is to investigate how TEL-AML1 expression can sustain this covert condition for many years. In a recent paper we described that the fusion gene rendered the B precursors resistant to the inhibitory activity of TGFbeta. Here we want to inquire into other factors that can explain the positive selection of the pre-leukemic clones over the normal counterpart. In particular, given the importance of the interaction with the microenvironment for survival signals for normal and leukemic stem cells, we question if the fusion gene causes changes in cellular adhesive and migratory properties. Methods: the study was performed by using two different models: i) a TEL-AML1 inducible expression system on the murine pro-B Ba/F3 cell line and ii) murine primary B lymphocytes (pre-BI cells) isolated from fetal liver, stably transduced with the pMIGR1-TEL-AML1-IRES-GFP construct. Gene expression assays were performed by using TaqMan (Applied Biosystems) and PCR Array technologies (SABioscences). Results: The expression of TEL-AML1 in Ba/F3 cell line causes over-expression of genes regulators of the cytoskeleton, specifically involved in cellular movement and in the regulation of actin dynamics. This gene expression alteration results in changes in the cellular morphology and phenotype: the cells acquire long extensions and several molecules involved in cell adhesion and migration are disregulated. Moreover, the TEL-AML1 positive cells present an increased ability to adhere to the ICAM1 substrate, but they also show a significant defect in the chemotactic response to CXCL12 in transwell migration assays in vitro, although the expression and the recycling of CXCR4 receptor are unaffected. This inability is not due to defects to migrate in general, as spontaneous motility is enhanced, but it is associated with a defect in CXCR4 signaling. In particular, CXCL12 calcium flux and ERK phosphorylation were inhibited. Those results have been confirmed in murine PreBI primary cells. Conclusions: in our murine models, TEL-AML1 affects the cytoscheleton regulation and causes alteration in cellular adhesive and migratory properties. We are now investigating how these alterations can give advantages to the pre-leukemic cells in the pathogenesis of TEL-AML1–expressing leukemia. Disclosures: No relevant conflicts of interest to declare.
9
Reikvam, Håkon. "Inhibition of NF-κB Signaling Alters Acute Myelogenous Leukemia Cell Transcriptomics." Cells 9, no. 7 (July 12, 2020): 1677. http://dx.doi.org/10.3390/cells9071677.
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Acute myelogenous leukemia (AML) is an aggressive hematological malignancy. The pathophysiology of the disease depends on cytogenetic abnormalities, gene mutations, aberrant gene expressions, and altered epigenetic regulation. Although new pharmacological agents have emerged during the last years, the prognosis is still dismal and new therapeutic strategies are needed. The transcription factor nuclear factor-κB (NF-κB) is regarded a possible therapeutic target. In this study, we investigated the alterations in the global gene expression profile (GEP) in primary AML cells derived from 16 consecutive patients after exposure to the NF-κB inhibitor BMS-345541. We identified a profound and highly discriminative transcriptomic profile associated with NF-κB inhibition. Bioinformatical analyses identified cytokine/interleukin signaling, metabolic regulation, and nucleic acid binding/transcription among the major biological functions influenced by NF-κB inhibition. Furthermore, several key genes involved in leukemogenesis, among them RUNX1 and CEBPA, in addition to NFKB1 itself, were influenced by NF-κB inhibition. Finally, we identified a significant impact of NF-κB inhibition on the expression of genes included in a leukemic stem cell (LSC) signature, indicating possible targeting of LSCs. We conclude that NF-κB inhibition significantly altered the expression of genes central to the leukemic process.
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Jiang, Xiaoyan, Yun Zhao, Wing-Yiu Chan, Suzanne Vercauteren, Emily Pang, Sean Kennedy, Frank Nicolini, Allen Eaves, and Connie Eaves. "Deregulated expression in Ph+ human leukemias of AHI-1, a gene activated by insertional mutagenesis in mouse models of leukemia." Blood 103, no. 10 (May 15, 2004): 3897–904. http://dx.doi.org/10.1182/blood-2003-11-4026.
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Abstract Ahi-1/AHI-1 (Abelson helper integration site-1) encodes a family of protein isoforms containing one Src homology 3 (SH3) domain and multiple tryptophan-aspartic acid 40 (WD40)–repeat domains. The function of these proteins is unknown, but involvement in leukemogenesis has been suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias. Here we show that in both mice and humans, Ahi-1/AHI-1 expression is highest in the most primitive hematopoietic cells with specific patterns of down-regulation in different lineages. Cells from patients with chronic myeloid leukemia (CML; n = 28) show elevated AHI-1 transcripts in all disease phases and, in chronic phase, in the leukemic cells at all stages of differentiation, including quiescent (G0) CD34+ cells as well as terminally differentiating cells. In the most primitive lin–CD34+CD38– CML cells, transcripts for the 2 shorter isoforms of AHI-1 are also increased. Although 15 of 16 human lymphoid and myeloid leukemic cell lines showed aberrant control of AHI-1 expression, this was not seen in blasts obtained directly from patients with acute Philadelphia chromosome–negative (Ph–) leukemia (n = 15). Taken together, our results suggest that down-regulation of AHI-1 expression is an important conserved step in primitive normal hematopoietic cell differentiation and that perturbations in AHI-1 expression may contribute to the development of specific types of human leukemia.
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Vitols, S., S. Norgren, G. Juliusson, L. Tatidis, and H. Luthman. "Multilevel regulation of low-density lipoprotein receptor and 3-hydroxy- 3-methylglutaryl coenzyme A reductase gene expression in normal and leukemic cells." Blood 84, no. 8 (October 15, 1994): 2689–98. http://dx.doi.org/10.1182/blood.v84.8.2689.2689.
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Abstract Altered cholesterol homeostasis has been noted in malignant cells, which led us to explore the regulation of cholesterol metabolism in normal and leukemic cells. The mean low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities were fivefold and threefold higher in mononuclear blood cells from 33 patients with leukemia, compared with cells from 23 healthy subjects, whereas elevations in RNA levels were twofold and 40% only. The activities of the two proteins correlated in normal cells (r = .46), whereas an inverse correlation was found in leukemic cells (r = -.40). Relatively weak correlations were found between LDL receptor RNA levels and receptor activity in normal (r = .48) and leukemic cells (r = .49), and HMG-CoA reductase RNA levels correlated (r = .53) with reductase activity in leukemic cells only. The ratios of protein activities to RNA levels in cells were constant during consecutive blood samplings and similar in leukemic blood and bone marrow cells from the same individual. During cholesterol deprivation, protein activities increased more than RNA levels, and leukemic cells with high LDL receptor activity showed a partial resistance to the suppressing effect of sterols on LDL receptor gene expression. The results demonstrate that LDL receptor RNA levels alone can not explain variation in receptor activity, suggesting post-RNA regulation of LDL receptor expression, similar to what has been described for HMG-CoA reductase. Taken together, the present results suggest multilevel regulation of both proteins and demonstrate that each cell clone, normal or malignant, has a unique ratio of protein activity to RNA level. Leukemic cells, in contrast to normal cells, can meet increased cholesterol requirements by either elevated LDL receptor activity or increased cholesterol synthesis, which is of potential interest for diagnosis and specific treatment of leukemia.
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Vitols, S., S. Norgren, G. Juliusson, L. Tatidis, and H. Luthman. "Multilevel regulation of low-density lipoprotein receptor and 3-hydroxy- 3-methylglutaryl coenzyme A reductase gene expression in normal and leukemic cells." Blood 84, no. 8 (October 15, 1994): 2689–98. http://dx.doi.org/10.1182/blood.v84.8.2689.bloodjournal8482689.
Full textAbstract:
Altered cholesterol homeostasis has been noted in malignant cells, which led us to explore the regulation of cholesterol metabolism in normal and leukemic cells. The mean low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities were fivefold and threefold higher in mononuclear blood cells from 33 patients with leukemia, compared with cells from 23 healthy subjects, whereas elevations in RNA levels were twofold and 40% only. The activities of the two proteins correlated in normal cells (r = .46), whereas an inverse correlation was found in leukemic cells (r = -.40). Relatively weak correlations were found between LDL receptor RNA levels and receptor activity in normal (r = .48) and leukemic cells (r = .49), and HMG-CoA reductase RNA levels correlated (r = .53) with reductase activity in leukemic cells only. The ratios of protein activities to RNA levels in cells were constant during consecutive blood samplings and similar in leukemic blood and bone marrow cells from the same individual. During cholesterol deprivation, protein activities increased more than RNA levels, and leukemic cells with high LDL receptor activity showed a partial resistance to the suppressing effect of sterols on LDL receptor gene expression. The results demonstrate that LDL receptor RNA levels alone can not explain variation in receptor activity, suggesting post-RNA regulation of LDL receptor expression, similar to what has been described for HMG-CoA reductase. Taken together, the present results suggest multilevel regulation of both proteins and demonstrate that each cell clone, normal or malignant, has a unique ratio of protein activity to RNA level. Leukemic cells, in contrast to normal cells, can meet increased cholesterol requirements by either elevated LDL receptor activity or increased cholesterol synthesis, which is of potential interest for diagnosis and specific treatment of leukemia.
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Hamid, Rizwan, Johnequia Patterson, Danko Martincic, and Stephen J. Brandt. "Complex Regulation of the Homeobox Transcription Factor TGIF: Differential Expression of Multiple 5′ Isoforms." Blood 106, no. 11 (November 16, 2005): 4254. http://dx.doi.org/10.1182/blood.v106.11.4254.4254.
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Abstract TG-interacting factor (TGIF) is a transcriptional repressor belonging to the TALE (three amino acid loop extension) class of homeobox proteins. In addition to its involvement by mutation or deletion in the inherited craniofacial disorder holoprosencephaly, we have shown that expression of TGIF is highly predictive of relapse and survival in acute myelogenous leukemia (AML). To better understand how TGIF expression is regulated, we characterized its genomic structure using expressed sequence tag analysis, reverse transcriptase-coupled PCR, and rapid amplification of cDNA ends. These studies revealed a complex pattern of gene expression, with 15 splice isoforms and 11 alternative 5′ exons spread over 40 kb of DNA, suggesting the presence of multiple promoters. Real-time and semi-quantitative PCR analysis showed these isoforms were differentially expressed in various adult tissues, leukemic cell lines, and AML blasts. Further, isoform C was found to be the major RNA product in hematopoietic cells, contributing significantly to total TGIF expression in leukemic cell lines TF1a, U937, AML-193, KG-1a, Kasumi-1, K562, GDM-1, HL-60 and AML blasts. This analysis suggests that altered splicing and/or expression of specific isoforms could be responsible for the reduced levels of TGIF message observed in AML blasts and cell lines. The unusually complex structure of the TGIF gene may enable its precise regulation during normal hematopoiesis and may be relevant to its reduced expression in myeloid leukemias.
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Starkova, Julia, Karolina Kramarzova, Karel Fiser, Ester Mejstrikova, Katerina Rejlova, Martina Slamova, Harry A. Drabkin, and Jan Trka. "Leukemic Pattern Of HOX Gene Expression Is Driven By Genetic Aberrations Through Epigenetic Modifiers." Blood 122, no. 21 (November 15, 2013): 2504. http://dx.doi.org/10.1182/blood.v122.21.2504.2504.
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Abstract Introduction Homeobox (HOX) genes encode transcription factors crucial in embryogenesis. They are often dysregulated in malignancies including leukemias. The aberrant HOX gene expression and its regulation in leukemic cells is neither completely described nor understood. Aims Our main aim was to determine whether the leukemic HOX gene expression pattern is driven by differentiation stage of hematopoietic cells or determined de novo during the process of malignant transformation. Consequentially, we aimed to study the role epigenetic modifiers in regulation of HOX gene expression in normal and malignant hematopoiesis. Methods The expression pattern of HOX genes (cluster of HOX A and B) and epigenetic modifiers (DNMT1, DNMT3a, DNMT3b, EZH2, BMI-1, MLL, JMJD3, UTX) was assessed by qPCR in 8 FACS-sorted subpopulations of healthy BM representing stages of myeloid differentiation (each sample representing a pool of cells sorted from five individuals). The leukemic expression pattern of these genes was analyzed in diagnostic BM samples of childhood AML patients with typical genotypic and morphological (FAB classification) characteristics (N=46). In vitro experiments were performed using NB4 cell line. Results As expected HOX genes were gradually downregulated during normal differentiation of granulocytic and monocytic lineages (assessed in four consecutive differentiation stages for each lineage). In AML samples, HOX gene expression patterns differed significantly among morphological subtypes. However, HOX gene expression did not correlate among subtypes of AML and their physiologically differentiated counterparts. Interestingly, unsupervised hierarchical clustering (HCA) divided AML patients into four main clusters characterized by the presence of prevalent gene rearrangement (PML-RARa, AML1-ETO, MLL rearrangements and NK-AML). The presence of PML/RARa rearrangement was strongly associated with the lowest expression of both HOXA and HOXB clusters, while the other groups had more variable expression of HOX genes. Moreover, the effect of genetic aberrations on HOX gene expression was clearly apparent within AML M2 and M4 subtypes, where AML1/ETO+ or CBFb/MYH11+ patients had significantly lower expression of HOX genes compared to patients with the same FAB classification but without the rearrangements. The expression pattern of epigenetic modifiers in sorted subpopulations of healthy BM followed their expected role in transcriptional regulation during differentiation. However, there was no relation of this pattern to HOX gene expression. On the contrary, in AML samples, the expression levels of epigenetic modifiers clearly correlated with expression profile of HOX genes. These results were supported by unsupervised HCA based on the expression of epigenetic modifiers that showed upregulation of histon demethylases JMJD3 and UTX together with downregulation of DNMT3b in concordance with high levels of HOX genes. Negative correlation between JMJD3 and DNMT3b expression was observed in all leukemic samples (p=0.03); most apparently in PML/RARa+ patients. Therefore we further studied the impact of genetic aberrations on the epigenetic regulation of HOX gene expression in vitrowith PML-RARa+ cell line. Treatment of NB4 cells with ATRA (8, 24hours, 1uM, 10uM) increased the levels of particular HOX genes (HOXA5, A7, B4, B7; FCA=2.8; 1.7; 4; 4 respectively) as well as JMJD3 (FCA=3) and UTX (FCA=1.6). Concordantly, the expression of DNMT3b (FCA=5) was downregulated. The hypothetical driving effect of PML-RARa on de novo determination of leukemic HOX gene expression is further supported by our Results. PML-RARa+ patients had the lowest HOX gene expression regardless of their FLT3/ITD status – previously shown to upregulate strongly HOX genes expression. Conclusion We conclude that the leukemic expression pattern of HOX genes does not reflect the differentiation stages of malignant cells. Our data also demonstrate different contribution of epigenetic modifiers to the HOX gene expression in healthy and malignant hematopoiesis. Moreover, HCA and expression data together with the results of in vitro experiments suggest that the specific molecular aberrations (as exemplified by PML-RARa) participate in regulation of leukemic HOX gene expression through epigenetic changes. Supported by GACR P304/12/2214, GAUK 568213, 00064203. Disclosures: No relevant conflicts of interest to declare.
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Abramowitz, Julia, Tzahi Neuman, Rania Samman, Riki Perlman, and Dina Ben-Yehuda. "Mdm2 and Mdmx Are Not the Major Negative Regulators of the p53 Pathway in Myeloid Leukemias." Blood 114, no. 22 (November 20, 2009): 5046. http://dx.doi.org/10.1182/blood.v114.22.5046.5046.
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Abstract Abstract 5046 Mutations in the p53 tumor suppressor gene are rare in human hematological malignancies, suggesting that aberrant p53 function may be due to alterations in its regulatory pathways. p53 is negatively regulated by MDM2 through ubiquitin-dependent degradation and by Mdmx through inhibition of transcriptional function. There is little information on the expression of Mdm2 and Mdmx in most myeloid leukemias. We determined the gene expression and protein levels of Mdm2 and Mdmx in bone marrow samples of leukemia patients. We first performed quantitative evaluation of Mdm2 and Mdmx gene expression in bone marrow samples of 144 leukemic patients at the time of diagnosis: 29 de novo AML patients (AML, M0-M7, M3 excluded), 30 de novo AML M3 patients (APL), 39 therapy-related AML patients (t-AML, M0-M7, M3 included) and 46 CML chronic phase (CML-CP) patients in comparison to 35 normal bone marrow samples from Hodgkin's disease patients. Quantitative Real-Time PCR analysis showed no global statistically significant over expression of Mdm2 or Mdmx in any of the tested leukemias. However, a number of patients in both de novo and therapy-related AML had elevated levels of Mdm2 or Mdmx. Significant down regulation of Mdm2, Mdmx and a splicing variant of Mdmx lacking exon 6 (Mdmx-S) was observed in CML-CP. We next performed IHC staining, evaluated by semi-quantitative score, to examine the levels of Mdm2 and Mdmx protein expression in 151 leukemic patients at the time of diagnosis: 40 AML patients, 23 APL, 47 t-AML and 41 CML-CP patients in comparison to 58 normal bone marrow samples from Hodgkin's disease patients. Protein expression analysis also showed no global statistically significant over expression of Mdm2 in any of the tested leukemias. Nonetheless, a number of patients in both de novo and therapy-related AML had elevated levels of Mdm2 protein. Specifically, APL patients segregated into 2 groups: while half of the patients did not express the Mdm2 protein, the other half over-expressed Mdm2. A significant down-regulation of Mdmx was observed in APL. In summary, while in some leukemic patients Mdm2 and Mdmx might play a role in the regulation of p53, our quantitative analysis indicates that these negative regulators do not seem to provoke the inactivation of the p53 pathway in most myeloid leukemias patients. These findings have implications on the possible use of Mdm2 antagonists like nutlin-3 in myeloid leukemias. To the best of our knowledge this is the largest group of myeloid leukemia patients studied for Mdm2 and Mdmx expression. Disclosures No relevant conflicts of interest to declare.
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Sorokina, Tamara, Irina Shipounova, Alexey Bigildeev, Nina I. Drize, Larisa A. Kuzmina, Elena N. Parovichnikova, and Valery G. Savchenko. "Modification of Gene Expression in Mesenchymal Stromal Cells of the Leukemia Patients during Chemotherapy." Blood 128, no. 22 (December 2, 2016): 5065. http://dx.doi.org/10.1182/blood.v128.22.5065.5065.
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Abstract Background In patients with acute leukemia the stromal microenvironment is deeply modified. Disturbances in signaling pathways, genetic abnormalities and functional changes in mesenchymal cells of these patients have been previously described. Chemotherapy also affect stromal progenitor cells. A damaged microenvironment might impair hematopoiesis in acute leukemia patients. Aims To investigate the relative expression level in MMSCs and CFU-Fs, derived from the bone marrow (BM) of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients before and over the course of chemotherapy. Methods 54 newly diagnosed cases (33 AML, 21 ALL) were involved in the study after informed consent. BM was aspirated prior to any treatment (time-point 0) and at days 37, 100 and 180 since the beginning of treatment of acute leukemia. MMSCs were cultured in aMEM with 10% fetal calf serum, CFU-Fs, in aMEM with 20% fetal calf serum. The relative expression level (REL) of different genes was measured by TaqMan RQ-PCR. As a control MMSCs and CFU-Fs from 88 healthy donors were used. Results At the time of the disease manifestation the analysis of gene expression in MMSCs from acute leukemia patients revealed a significant increase in the REL of genes which regulate immune system responses and thereby can influence on the leukemic cell proliferation and migration (IL-6, IL-8, IL-1b and IL-1R1) (Pic.1). Also at the time of the diagnosis an increase in REL of genes, that are responsible for hematopoiesis regulation, was observed. For example, the REL of CSF1 that can influence on leukemic cells proliferation was increased at the disease manifestation and became normal during the treatment. The same dynamics was observed in the REL of JAG1 that has an antiapoptotic effect on leukemic cells. The REL of LIF had been also significantly increased at the disease manifestation, reflecting the efforts of MMSCs to inhibit leukemic proliferation. Chemotherapy affected REL of the studied genes differently. The treatment lead to the downregulation of IGF, TGFB1 and TGFB2 (Pic.2). As far asTGFB1 and 2 inhibit the differentiation of mesenchymal stem cells, and IGF is associated with myelodysplastic changes in elderly bone marrow, so their downregulation may refer to the effectiveness of therapy. The REL of genes regulating MMSC proliferation (PDGFRa and PDGFRb, FGF2, FGFR1 and 2) increased during chemotherapy. Exploring cell adhesion molecules, the decrease in the REL of their encoding genes was observed. As far as VCAM facilitate the leukemic cell extravasation and ICAM was shown to depress the Th17 cell differentiation, the down-regulation of their genes may reflect the microenvironment restoration. The influence of chemotherapy lead to decrease in REL of genes, associated with MMSCs differentiation (BGLAP and SOX9 (Pic.3)), reflecting the mechanism of the blocking of MMSCs migration and differentiation under the stress conditions. The alterations of bone marrow stroma were more pronounced in patients who didn't achieve remission. The REL of 9 genes was studied in CFU-F colonies. There were no differences in gene expression in CFU-Fs before the treatment, except for an increase in the REL of PPARg in acute leukemia CFU-Fs. During the treatment, a decrease in the REL of SPP1 and an increase in the REL of FGFR1 and 2 were observed. Conclusion Therefore, chemotherapy used does not impair the functional ability of MMSCs and CFU-Fs, but influence on their gene expression profile. The two types of precursors are affected differently, indicating their different differentiation level and functions. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.
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Schulze, Isabell, Petra Tschanter, Christian Rohde, Annika Krause, Heinz Linhart, Katja Hebestreit, Martin Dugas, Frank Rosenbauer, Wolfgang E. Berdel, and Carsten Müller-Tidow. "Identification Of Leukemia Suppressive Genes By Inducible Overexpression Of The DNA Methyltransferase DNMT3B During Leukemogenesis In Mice." Blood 122, no. 21 (November 15, 2013): 2493. http://dx.doi.org/10.1182/blood.v122.21.2493.2493.
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Abstract DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. In previous studies using a tetracycline-inducible DNMT3B mouse model, we could show that overexpression of DNMT3B affected leukemia initiation and maintenance upon retroviral transduction and serial transplantation of hematopoietic stem and progenitor cells with MSCV-MLL-AF9-GFP and MSCV-cmyc-bcl2-mcherry oncogenic vectors, respectively. Sublethally irradiated recipient mice of DNMT3B overexpressing MLL-AF9 and cmyc/bcl2 leukemic cells developed leukemia with a prolonged latency when compared to recipients of wildtype cells. We performed serial transplantation assays of MLL-AF9 leukemic stem cells, which were sorted for high expression of ckit. The life-prolonging effect of DNMT3B expression was stem cell-specific, as the potential to initiate leukemia was maintained upon serial retransplantation and recipients of DNMT3B overexpressing leukemic stem cells also died significantly later in secondary (p<0.001) and tertiary transplantations (p<0.001). Analysis of global DNA methylation levels in MLL-AF9 ckit+ leukemic stem cells and cmyc/bcl2 leukemic cells via Reduced Representation Bisulfite Sequencing (RRBS) revealed a strong hypermethylation in DNMT3B overexpressing cells, independent of the oncogene used for leukemia induction. Differentially methylated CpG sites were defined as CpGs with at least 20% methylation difference between wildtype and DNMT3B overexpressing samples. Hypermethylation in MLL-AF9 leukemic cells directly correlated with observed hypermethylation in cmyc/bcl2 leukemic cells and inversely correlated with hypomethylation in cmyc/bcl2 cells, indicating that in both leukemias, the same sites are prone to DNMT3B induced DNA methylation. To investigate, if these changes in DNA methylation resulted in different gene expression patterns, we performed microarray analysis of the same MLL-AF9 leukemic wildtype and DNMT3B expressing samples which were also used for DNA methylation analysis. In microarray analyses, we could identify several genes differentially expressed in DNMT3B overexpressing cells when compared to wildtype samples. Interestingly, changes in expression levels could not be attributed to differential DNA methylation in promoter regions. Instead, hypermethylation in exons and gene bodies resulted in downregulation of the respective genes, whereas genes with hypomethylated exons and gene bodies showed higher expression levels. Genes downregulated in DNMT3B overexpressing cells, were mainly cancer-associated genes, which are known to have functions in cellular growth and proliferation, as well as in the hematopoietic system development and maintenance. Gene Set Enrichment Analysis (GSEA) of wildtype cells revealed a strong enrichment of genes upregulated in different stages of hematopoietic stem and progenitor cells as well as in leukemic stem cells, whereas DNMT3B overexpressing samples were enriched in genes which have been shown to be downregulated in hematopoietic and leukemic stem cells and upregulated in mature hematopoietic cells. This strengthens our hypothesis that DNMT3B induced DNA methylation mainly influences the phenotype and function of hematopoietic stem cells and thereby, exerts its inhibitory function on leukemia initiation and maintenance. Taken together, these findings demonstrate that DNMT3B exerts its anti-leukemic effect mainly via induction of aberrant DNA methylation in hematopoietic and leukemic stem cells, thereby changing expression patterns of genes known to be important for stem cell function. The identification of differentially expressed DNMT3B target genes could help to find promising targets for new therapeutic strategies in AML. Disclosures: No relevant conflicts of interest to declare.
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Godfrey, Laura, Nicholas T. Crump, Sorcha O’Byrne, I.-Jun Lau, Siobhan Rice, Joe R. Harman, Thomas Jackson, et al. "H3K79me2/3 controls enhancer–promoter interactions and activation of the pan-cancer stem cell marker PROM1/CD133 in MLL-AF4 leukemia cells." Leukemia 35, no. 1 (April 2, 2020): 90–106. http://dx.doi.org/10.1038/s41375-020-0808-y.
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AbstractMLL gene rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL-AF4 fusion protein. MLL-AF4 promotes leukemogenesis by activating key target genes, mainly through recruitment of DOT1L and increased histone H3 lysine-79 methylation (H3K79me2/3). One key MLL-AF4 target gene is PROM1, which encodes CD133 (Prominin-1). CD133 is a pentaspan transmembrane glycoprotein that represents a potential pan-cancer target as it is found on multiple cancer stem cells. Here we demonstrate that aberrant PROM1/CD133 expression is essential for leukemic cell growth, mediated by direct binding of MLL-AF4. Activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) leading to increased enhancer–promoter interactions between PROM1 and the nearby gene TAPT1. This dual locus regulation is reflected in a strong correlation of expression in leukemia. We find that in PROM1/CD133 non-expressing cells, the PROM1 locus is repressed by polycomb repressive complex 2 (PRC2) binding, associated with reduced expression of TAPT1, partially due to loss of interactions with the PROM1 locus. Together, these results provide the first detailed analysis of PROM1/CD133 regulation that explains CD133 expression in MLLr ALL.
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Yik, Mot Yee, Adam Azlan, Yaashini Rajasegaran, Aliaa Rosli, Narazah Mohd Yusoff, and Emmanuel Jairaj Moses. "Mechanism of Human Telomerase Reverse Transcriptase (hTERT) Regulation and Clinical Impacts in Leukemia." Genes 12, no. 8 (July 30, 2021): 1188. http://dx.doi.org/10.3390/genes12081188.
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The proliferative capacity and continuous survival of cells are highly dependent on telomerase expression and the maintenance of telomere length. For this reason, elevated expression of telomerase has been identified in virtually all cancers, including leukemias; however, it should be noted that expression of telomerase is sometimes observed later in malignant development. This time point of activation is highly dependent on the type of leukemia and its causative factors. Many recent studies in this field have contributed to the elucidation of the mechanisms by which the various forms of leukemias increase telomerase activity. These include the dysregulation of telomerase reverse transcriptase (TERT) at various levels which include transcriptional, post-transcriptional, and post-translational stages. The pathways and biological molecules involved in these processes are also being deciphered with the advent of enabling technologies such as next-generation sequencing (NGS), ribonucleic acid sequencing (RNA-Seq), liquid chromatography-mass spectrometry (LCMS/MS), and many others. It has also been established that TERT possess diagnostic value as most adult cells do not express high levels of telomerase. Indeed, studies have shown that prognosis is not favorable in patients who have leukemias expressing high levels of telomerase. Recent research has indicated that targeting of this gene is able to control the survival of malignant cells and therefore offers a potential treatment for TERT-dependent leukemias. Here we review the mechanisms of hTERT regulation and deliberate their association in malignant states of leukemic cells. Further, we also cover the clinical implications of this gene including its use in diagnostic, prognostic, and therapeutic discoveries.
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Tomasello, Luisa, Marzia Vezzalini, Christian Boni, Massimiliano Bonifacio, Luigi Scaffidi, Mohamed Yassin, Nader Al-Dewik, Paul Takam Kamga, Mauro Krampera, and Claudio Sorio. "Regulative Loop between β-catenin and Protein Tyrosine Receptor Type γ in Chronic Myeloid Leukemia." International Journal of Molecular Sciences 21, no. 7 (March 26, 2020): 2298. http://dx.doi.org/10.3390/ijms21072298.
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Protein tyrosine phosphatase receptor type γ (PTPRG) is a tumor suppressor gene, down-regulated in Chronic Myeloid Leukemia (CML) cells by the hypermethylation of its promoter region. β-catenin (CTNNB1) is a critical regulator of Leukemic Stem Cells (LSC) maintenance and CML proliferation. This study aims to demonstrate the antagonistic regulation between β-catenin and PTPRG in CML cells. The specific inhibition of PTPRG increases the activation state of BCR-ABL1 and modulates the expression of the BCR-ABL1- downstream gene β-Catenin. PTPRG was found to be capable of dephosphorylating β-catenin, eventually causing its cytosolic destabilization and degradation in cells expressing PTPRG. Furthermore, we demonstrated that the increased expression of β-catenin in PTPRG-negative CML cell lines correlates with DNA (cytosine-5)-methyl transferase 1 (DNMT1) over-expression, which is responsible for PTPRG promoter hypermethylation, while its inhibition or down-regulation correlates with PTPRG re-expression. We finally confirmed the role of PTPRG in regulating BCR-ABL1 and β-catenin phosphorylation in primary human CML samples. We describe here, for the first time, the existence of a regulative loop occurring between PTPRG and β-catenin, whose reciprocal imbalance affects the proliferation kinetics of CML cells.
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Yen, A., and S. Chandler. "Inducers of Leukemic Cell Differentiation Cause Down-Regulation of RB Gene Expression." Experimental Biology and Medicine 199, no. 3 (March 1, 1992): 291–97. http://dx.doi.org/10.3181/00379727-199-43359.
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Sassano, Antonella, Marco Lo Iacono, Giovanni Antico, Alison Jordan, Shahab Uddin, Raffaele A. Calogero, and Leonidas C. Platanias. "Regulation of leukemic cell differentiation and retinoid-induced gene expression by statins." Molecular Cancer Therapeutics 8, no. 3 (February 24, 2009): 615–25. http://dx.doi.org/10.1158/1535-7163.mct-08-1196.
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Yamochi, T., A. Kitabayashi, M. Hirokawa, AB Miura, T. Onizuka, S. Mori, and M. Moriyama. "Regulation of BCL-6 gene expression in human myeloid/monocytoid leukemic cells." Leukemia 11, no. 5 (May 1997): 694–700. http://dx.doi.org/10.1038/sj.leu.2400631.
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Rambaldi, A., M. Terao, S. Bettoni, ML Tini, R. Bassan, T. Barbui, and E. Garattini. "Expression of leukocyte alkaline phosphatase gene in normal and leukemic cells: regulation of the transcript by granulocyte colony- stimulating factor." Blood 76, no. 12 (December 15, 1990): 2565–71. http://dx.doi.org/10.1182/blood.v76.12.2565.2565.
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Abstract The levels of leukocyte alkaline phosphatase (LAP) messenger RNA (mRNA) are evaluated in B and T lymphocytes, monocytes, and polymorphonuclear cells (PMNs), and this transcript is found to be present only in PMNs. Precursors of the myelomonocytic pathway, represented by leukemic cells isolated from several cases of chronic myelogenous leukemia (CML) in its stable and blastic phase and acute myelogenous leukemia (AML), are devoid of LAP transcript. These data support the notion that LAP is a marker of the granulocyte terminal differentiation. Despite the absence of LAP mRNA in both the myeloid and the lymphoid precursors, nuclear run-on experiments show constitutive transcription of the LAP gene in leukemic cells obtained from AML, CML, as well as acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia (B-CLL). In CML and in chronic myelo-monocytic leukemia (CMML) PMNs, granulocyte colony- stimulating factor (G-CSF) specifically accumulates LAP mRNA without showing a substantial increase in the rate of transcription of the LAP gene. Once increased by G-CSF, LAP mRNA is very stable, showing a half- life of more than 4 hours in the presence of actinomycin-D. G-CSF is suggested to play a pivotal role in the modulation of LAP transcript in PMNs.
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Rambaldi, A., M. Terao, S. Bettoni, ML Tini, R. Bassan, T. Barbui, and E. Garattini. "Expression of leukocyte alkaline phosphatase gene in normal and leukemic cells: regulation of the transcript by granulocyte colony- stimulating factor." Blood 76, no. 12 (December 15, 1990): 2565–71. http://dx.doi.org/10.1182/blood.v76.12.2565.bloodjournal76122565.
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The levels of leukocyte alkaline phosphatase (LAP) messenger RNA (mRNA) are evaluated in B and T lymphocytes, monocytes, and polymorphonuclear cells (PMNs), and this transcript is found to be present only in PMNs. Precursors of the myelomonocytic pathway, represented by leukemic cells isolated from several cases of chronic myelogenous leukemia (CML) in its stable and blastic phase and acute myelogenous leukemia (AML), are devoid of LAP transcript. These data support the notion that LAP is a marker of the granulocyte terminal differentiation. Despite the absence of LAP mRNA in both the myeloid and the lymphoid precursors, nuclear run-on experiments show constitutive transcription of the LAP gene in leukemic cells obtained from AML, CML, as well as acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia (B-CLL). In CML and in chronic myelo-monocytic leukemia (CMML) PMNs, granulocyte colony- stimulating factor (G-CSF) specifically accumulates LAP mRNA without showing a substantial increase in the rate of transcription of the LAP gene. Once increased by G-CSF, LAP mRNA is very stable, showing a half- life of more than 4 hours in the presence of actinomycin-D. G-CSF is suggested to play a pivotal role in the modulation of LAP transcript in PMNs.
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Cantilena, Sandra, Nicholas Goulden, Owen Williams, and Jasper de Boer. "Drug Induced MLL Fusion Degradation Via Hsp90 Inhibition." Blood 126, no. 23 (December 3, 2015): 4850. http://dx.doi.org/10.1182/blood.v126.23.4850.4850.
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Abstract The survival rate for infants is less than 50%. The fast majority of infant acute leukemias are characterized cytogenetically by balanced chromosomal translocations involving the mixed lineage leukemia (MLL) gene. Leukemic therapies that degrade the driver oncogene are associated with loss of cancer cell self-renewal and excellent cure rates. Therefore, therapy that degrades the MLL fusion gene would offer new hope to these patients. Recently it was shown that the Drosophila Trithorax gene, an analogue of the human MLL, is degraded by Radicicol. Radicicol is a natural compound and a well-known Hsp90 inhibitor. Here, we show that Radicicol is able to induce a dose dependent degradation of the MLL-fusion protein in a panel of human MLL rearranged cell lines and in human cord blood-derived MLL-AF9 immortalised myeloid cells. This drug induced degradation of the MLL-fusion gene results in down-regulation of the expression of MLL target genes, including HOXA9, MEIS1 and c-MYB. Functionally, this results in a loss of self-renewal of the leukemic stem cells, as shown by methylcellulose colony forming assays. Radicicol proved ineffective and too toxic for in vivo use. One of the best tolerated Hsp90 inhibitors is Ganetespib. It is currently in phase II/III clinical trials. We extended our Radicicol data to Ganetespib. Like Radicicol, Ganetespib induces MLL-fusion protein degradation and downregulation of MLL target genes. Treatment of MLL rearranged leukemia with Ganetespib results in a loss of leukemic stem cell activity. In conclusion, we show how the inactivation of the MLL-fusion and down-regulation of MLL target genes results in a block of leukemic stem cell self- renewal. We will validate these findings in a pre-clinical in vivo model in the near future. Disclosures No relevant conflicts of interest to declare.
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Jiang, Yajian, Tianyuan Hu, Tao Wang, Xiangguo Shi, Ayumi Kitano, Kenneth Eagle, Kevin A. Hoegenauer, et al. "AMP-activated protein kinase links acetyl-CoA homeostasis to BRD4 recruitment in acute myeloid leukemia." Blood 134, no. 24 (December 12, 2019): 2183–94. http://dx.doi.org/10.1182/blood.2019001076.
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There is increasing evidence that the metabolic regulation of acute myeloid leukemia (AML) cell growth interacts with epigenetic pathways of gene expression and differentiation. Jiang et al link inhibition of glucose metabolism to epigenetic changes and altered transcriptional pathways in leukemic cells and demonstrate synergy between simultaneously targeting metabolism and chromatin modifiers in suppression of AML.
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Milani, L., A. Lundmark, J. Nordlund, A. Kiialainen, T. Flaegstad, G. Jonmundsson, J. Kanerva, et al. "Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation." Genome Research 19, no. 1 (October 29, 2008): 1–11. http://dx.doi.org/10.1101/gr.083931.108.
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Sun, Lei, Patricia A. Goodman, Carla M. Wood, Mya-Lisa Crotty, Martha Sensel, Harland Sather, Christopher Navara, et al. "Expression of Aberrantly Spliced Oncogenic Ikaros Isoforms in Childhood Acute Lymphoblastic Leukemia." Journal of Clinical Oncology 17, no. 12 (December 1999): 3753–66. http://dx.doi.org/10.1200/jco.1999.17.12.3753.
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PURPOSE: We sought to determine if molecular abnormalities involving the Ikaros gene could contribute to the development of acute lymphoblastic leukemia (ALL) in children. PATIENTS AND METHODS: We studied Ikaros gene expression in normal human bone marrow, normal thymocytes, normal fetal liver–derived immature lymphocyte precursor cell lines, eight different ALL cell lines, and leukemic cells from 69 children with ALL (T-lineage ALL, n = 18; B-lineage ALL, n = 51). Expression of Ikaros protein and its subcellular localization were examined by immunoblotting and confocal laser-scanning microscopy, respectively. Polymerase chain reaction (PCR) and nucleotide sequencing were used to identify the specific Ikaros isoforms expressed in these cells. Genomic sequencing of splice junction regions of the Ikaros gene was performed in search for mutations. RESULTS: In each of the ALL cases, we found high-level expression of a non–DNA-binding or aberrant DNA-binding isoform of Ikaros with abnormal subcellular compartmentalization patterns. In contrast, only wild-type Ik-1 and Ik-2 isoforms with normal subcellular localization were found in normal bone marrow cells and thymus-derived or fetal liver–derived normal lymphocyte precursors. In leukemic cells expressing the aberrant Ikaros coding sequences with the 30-base-pair deletion, genomic sequence analysis of the intron-exon junctions between exons 6 and 7 yielded the wild-type sequence. We identified a single nucleotide polymorphism (SNP) affecting the third base of the triplet codon for a proline (CCC or CCA) in the highly conserved bipartite activation region (viz, A or C at position 1002 numbering from the translation start site of Ik-1) within our Ikaros clones. Bi-allelic expression of truncated and/or non–DNA-binding isoforms along with wild-type isoforms was observed in leukemic cells, which implicates trans-acting factor(s) affecting splice site recognition. CONCLUSION: Our findings link specific molecular defects involving the Ikaros gene to childhood ALL. Posttranscriptional regulation of alternative splicing of Ikaros RNA seems to be defective in leukemic lymphocyte precursors from most children with ALL. Consequently, leukemic cells from ALL patients, in contrast to normal lymphocyte precursors, express high levels of non–DNA-binding Ikaros isoforms that are reminiscent of the non–DNA-binding Ikaros isoforms that lead to lymphoblastic leukemia in mice.
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Ashton, John M., Marlene Balys, Sarah Neering, Glenn Cowley, David E. Root, Peter G. Miller, Benjamin Ebert, et al. "Oncogene Cooperativity Analysis Reveals a Novel Set of Genes That Regulate the In Vivo Growth and Survival of Leukemia Stem Cells." Blood 118, no. 21 (November 18, 2011): 553. http://dx.doi.org/10.1182/blood.v118.21.553.553.
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Abstract Abstract 553 In order to increase our understanding of key biological properties governing the development of leukemia stem cells (LSCs), we employed a novel gene identification strategy based on cooperation between initiating oncogenes. Previous studies have demonstrated that genes whose expression is regulated in a synergistic manner as a consequence of two cooperating oncogenes (termed “cooperativity response genes”, or CRGs) are highly enriched for activity in tumor formation. Further, in contrast to the thousands of genes identified by differential expression analyses of normal vs. leukemic cell populations, CRGs represent a much smaller subset of targets; thereby, providing a defined set of genes to investigate. We adapted the CRG strategy to identify synergistically regulated genes in primitive leukemic cells. Using a mouse model of myeloid blast crisis leukemia induced through the cooperation of BCR-ABL and NUP98-HOXA9, we performed genome-wide transcriptional profiling comparing hematopoietic cells expressing each translocation alone or in combination. Using this system, we were able to model the genetic alterations induced as normal cells progressed towards LSC transformation, identifying 72 CRGs (50 aberrantly up-regulated and 22 down-regulated) with potential importance in leukemia development. To investigate the relevance of these CRGs in leukemia biology, an RNAi screen approach was employed. Primary leukemic progenitors were purified and transduced with a custom lentiviral RNAi library and subsequently transplanted into recipient animals to assess the engraftment potential upon perturbation of the individual CRGs. Our findings demonstrate that knock-down of expression in 35 of 50 (70%) leukemia CRGs reduced in vivo growth of primitive leukemia, a finding that was independently validated through single gene perturbation of several genes that scored in the RNAi screen (GJB3, EphA3, PMP22, Serinc2, SerpinB2, and CP). In particular, serpinB2, a gene that scored strongly in the RNAi analysis, was shown to directly effect the frequency of LSC in vivo. Given that the cooperative gene signature represented genes with many distinct cellular functions, we hypothesized that the CRG expression profile represents a key regulatory network in leukemia survival. To investigate our hypothesis we utilized the Broad Institute's Connectivity Map (CMAP) to identify pharmacological compounds with the ability to modulate multiple CRGs simultaneously. This analysis revealed that both Tyrophostin AG-825 (AG825) and 4-hydroxy-2-nonenol (4HNE) were predicted to reverse the gene expression induced as a consequence of leukemic transformation. To test the effect of these agents as selective toxicants to leukemia, we treated both normal and leukemia murine bone marrow cells with each compound. Both bulk and phenotypically primitive leukemic cells were eradicated in dose-responsive fashion upon treatment with either AG825 or 4HNE, while normal cells showed significantly reduced sensitivity. Progenitor function as measured by colony forming assays also showed a selective reduction in leukemia colony formation, suggesting that both these compounds are toxic to the majority of leukemic cell types. Interestingly, similar results were obtained when human normal and leukemic bone marrow specimens were treated with both drugs, suggesting the CRG signature represents an important class of genes with conserved function across species. To determine the level of conservation of the leukemia CRG signature between murine and human leukemia, we profiled eight normal and leukemic patient specimens for expression of the CRG signature. Of the 39 evaluable human CRG orthologs, 13 showed similar expression trends in human leukemia samples relative to normal controls. Intriguingly, both AG825 and 4HNE were predicted to inhibit this 13-gene signature by the CMAP database, suggesting that the compounds may act through these genes to influence leukemia cell death. Taken together, our findings demonstrate the importance of cooperative gene regulation in leukemogenesis and provide a novel platform for future research toward more effective therapeutic strategies to treat leukemia. Disclosures: No relevant conflicts of interest to declare.
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Pise-Masison, Cynthia A., Michael Radonovich, Kathleen Dohoney, John C. Morris, Deirdre O'Mahony, Min-Jung Lee, Jane Trepel, Thomas A. Waldmann, John E. Janik, and John N. Brady. "Gene expression profiling of ATL patients: compilation of disease-related genes and evidence for TCF4 involvement in BIRC5 gene expression and cell viability." Blood 113, no. 17 (April 23, 2009): 4016–26. http://dx.doi.org/10.1182/blood-2008-08-175901.
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Abstract Adult T-cell leukemia/lymphoma (ATL) is an aggressive and fatal disease. We have examined 32 patients with smoldering, chronic, lymphoma and acute leukemia using Affymetrix HG-U133A2.0 arrays. Using the BRB array program, we identified genes differentially expressed in leukemia cells compared with normal lymphocytes. Several unique genes were identified that were overexpressed in leukemic cells, including TNFSF11, RGS13, MAFb, CSPG2, C/EBP-α, and TCF4; 200 of the most highly overexpressed ATL genes were analyzed by the Pathway Studio, version 4.0 program. ATL leukemia cells were characterized by an increase in genes linked to “central” genes CDC2/cyclin B1, SYK/LYN, proliferating cell nuclear antigen, and BIRC5. Because of its potential therapeutic importance, we focused our studies on the regulation and function of BIRC5, whose expression was increased in 13 of 14 leukemia samples. TCF4 reporter assays and transfection of DN-TCF4 demonstrated that TCF4 regulates BIRC5 gene expression. Functionally, transfection of ATL cells with BIRC5 shRNA decreased BIRC5 expression and cell viability 80%. Clinical treatment of ATL patients with Zenapax or bortezomib decreased BIRC5 expression and cell viability. These experiments represent the first direct experimental evidence that BIRC5 plays an important role in ATL cell viability and provides important insight into ATL genesis and potential targeted therapies.
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Holyoake, Tessa L., Xiaoyan Jiang, Heather G. Jorgensen, Susan Graham, Michael J. Alcorn, Chris Laird, Allen C. Eaves, and Connie J. Eaves. "Primitive quiescent leukemic cells from patients with chronic myeloid leukemia spontaneously initiate factor-independent growth in vitro in association with up-regulation of expression of interleukin-3." Blood 97, no. 3 (February 1, 2001): 720–28. http://dx.doi.org/10.1182/blood.v97.3.720.
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Abstract It was previously shown that patients with chronic myeloid leukemia (CML) have a rare but consistently detectable population of quiescent (G0) leukemic (Philadelphia chromosome–positive and BCR-ABL–positive [BCR-ABL+]) CD34+ cells. In the study described here, most such cells expressed a primitive phenotype (CD38−, CD45RA−, CD71−, and HLA-DRlo) and cultures of these cells containing growth factors produced ultimately larger, but initially more slowly growing clones than do cultures of initially cycling CD34+ leukemic cells. Initially quiescent leukemic cells expressing BCR-ABLproliferated in single-cell cultures in the absence of added growth factors, thereby demonstrating their ability to spontaneously exit G0 and enter a continuously cycling state. Interestingly, on isolation, few of these quiescentBCR-ABL+ cells contained either interleukin-3 (IL-3) or granulocyte colony-stimulating factor (G-CSF) transcripts, whereas both were present in most cyclingBCR-ABL+ CD34+ cells. However, after 4 days of culture in the absence of added growth factors and in association with their entry into the cell cycle (as indicated by up-regulation of Ki-67 and cdc25 transcripts), IL-3 transcripts became detectable. These findings show that entry of leukemic (BCR-ABL–expressing) progenitors into a quiescent (G0) state in vivo is highest among the most primitive leukemic cell populations, associated with a down-regulation of IL-3 and G-CSF gene expression, and spontaneously reversible in association with up-regulation of IL-3 expression. These results highlight the potential physiologic relevance of quiescent CML progenitors, even in treated patients, in whom these cells would be predicted to have a proliferative advantage over their quiescent normal counterparts when cytokine concentrations are low.
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Sarmadi, Vahid Hosseinpour, Salma Ahmadloo, Mohadese Hashem Boroojerdi, Cini Mathew John, Satar Jabbar Rahi al-Graitte, Hamza Lawal, Maryam Maqbool, Ling King Hwa, and Rajesh Ramasamy. "Human Mesenchymal Stem Cells-mediated Transcriptomic Regulation of Leukemic Cells in Delivering Anti-tumorigenic Effects." Cell Transplantation 29 (January 1, 2020): 096368971988507. http://dx.doi.org/10.1177/0963689719885077.
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Treatment of leukemia has become much difficult because of resistance to the existing anticancer therapies. This has thus expedited the search for alternativ therapies, and one of these is the exploitation of mesenchymal stem cells (MSCs) towards control of tumor cells. The present study investigated the effect of human umbilical cord-derived MSCs (UC-MSCs) on the proliferation of leukemic cells and gauged the transcriptomic modulation and the signaling pathways potentially affected by UC-MSCs. The inhibition of growth of leukemic tumor cell lines was assessed by proliferation assays, apoptosis and cell cycle analysis. BV173 and HL-60 cells were further analyzed using microarray gene expression profiling. The microarray results were validated by RT-qPCR and western blot assay for the corresponding expression of genes and proteins. The UC-MSCs attenuated leukemic cell viability and proliferation in a dose-dependent manner without inducing apoptosis. Cell cycle analysis revealed that the growth of tumor cells was arrested at the G0/G1 phase. The microarray results identified that HL-60 and BV173 share 35 differentially expressed genes (DEGs) (same expression direction) in the presence of UC-MSCs. In silico analysis of these selected DEGs indicated a significant influence in the cell cycle and cell cycle-related biological processes and signaling pathways. Among these, the expression of DBF4, MDM2, CCNE2, CDK6, CDKN1A, and CDKN2A was implicated in six different signaling pathways that play a pivotal role in the anti-tumorigenic activity exerted by UC-MSCs. The UC-MSCs perturbate the cell cycle process of leukemic cells via dysregulation of tumor suppressor and oncogene expression.
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Chaturvedi, Nagendra K., Adam K. Ahrens, Ashima Shukla, Christine E. Gilling, Amit K. Mittal, Philip Bierman, Dennis D. Weisenburger, Runqing Lu, and Shantaram S. Joshi. "Stromal Tumor Microenvironment in CLL: Regulation of Leukemic Progression." Blood 120, no. 21 (November 16, 2012): 1781. http://dx.doi.org/10.1182/blood.v120.21.1781.1781.
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Abstract Abstract 1781 Chronic Lymphocytic Leukemia (CLL), the most prevalent adult B-cell malignancy in western countries, is a highly heterogeneous with a very variable clinical outcome. Emerging evidence indicates that the stromal tumor microenvironment (STME) play important roles in the pathogenesis of CLL. However, the precise mechanism and molecules of STME involved in this process remain unknown. In an attempt to explore the role of STME in this process, we examined the expression levels of stromal associated genes using gene expression profiling (GEP) of CLL cells from 53 patients’ lymph node (LN) (n=15), bone marrow (BM) (n =18), and peripheral blood (PB) (n=20). Using significant analyses of microarray (SAM), gene set enrichment analyses (GSEA), and ingenuity pathway analyses (IPA), among the major pathways associated with the differentially expressed genes, a cytoskeleton genes associated with stromal signatures are the focus of this report. Of these molecules, a significant number of molecules including: LUM, MMP9, MYLK, ITGA9, CAV1, CAV2, FBN1, PARVA, CALD1, ITGB5 and EHD2 were overexpressed and ITGB2, DLC1 and ITGA6 are under expressed in LN-CLL compared to BM-CLL and PB-CLL indicating a role of LN-mediated TME in CLL cell survival/progression. Among these genes, expression of myosin light chain kinase (MYLK), caveolin 1 (CAV1) and caveolin 2 (CAV2) correlated with clinical outcome (see adjacent Figure) as determined by time to treatment. We recently reported the role of a CAV1 in LN microenvironment-induced immune tolerance in CLL and possibility of their involvement in CLL cytoskeleton (Gilling et al, 2012). In the present study we report aberrant expression of other cytoskeleton genes such as MYLK and CAV2 are involved in the regulation of CLL cell survival in the stromal microenvironment affecting other members of the cytoskeletal signature via actin cytoskeleton signaling, integrin signaling and Pak signaling. In addition, MYLK and CAV2 are also involved in regulation of CLL proliferation. Together our studies show that members of the stromal signature particularly in the CLL cells from lymph nodes regulate the CLL cell survival and proliferation and thus leukemic progression. Figure: Association of MYLK expression with time to first treatment Figure:. Association of MYLK expression with time to first treatment Disclosures: No relevant conflicts of interest to declare.
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Quesnel, Bruno, Gaelle Guillerm, Rodolphe Vereecque, Eric Wattel, Claude Preudhomme, Francis Bauters, Michael Vanrumbeke, and Pierre Fenaux. "Methylation of the p15INK4b Gene in Myelodysplastic Syndromes Is Frequent and Acquired During Disease Progression." Blood 91, no. 8 (April 15, 1998): 2985–90. http://dx.doi.org/10.1182/blood.v91.8.2985.2985_2985_2990.
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p15INK4b gene is an inhibitor of cyclin-dependent kinase (CDK) 4 and CDK6 whose expression is induced by transforming growth factor (TGF)β. Recent reports suggest frequent methylation of the p15INK4b gene promoter in leukemias, and it has been proposed that this methylation could be necessary for leukemic cells to escape TGFβ regulation. We investigated the methylation status of p15INK4b gene in 53 myelodysplastic syndromes (MDS) cases, including nine that had progressed to acute myeloid leukemia (AML), using a recently described sensitive method where polymerase chain reaction (PCR) is preceded by bisulfite modification of DNA (methylation specific PCR). p15INK4b methylation was observed in 20 of 53 (38%) of the cases. Twenty of the 24 patients with greater than 10% bone marrow blasts had p15INK4bmethylation (including all nine patients who had progressed to AML) as compared with none of MDS patients with <10% bone marrow blasts. No correlation between karyotypic abnormalities and methylation status was found. Patients with p15INK4b methylation had a worse prognosis, but the prognostic significance of p15INK4bmethylation was no more found by multivariate analysis, due to its strong correlation to the percentage of marrow blasts. In 10 MDS cases, sequential DNA samples were available. In five of them, methylation of the p15INK4b gene was detected at leukemic transformation, but not at diagnosis. Our results showed that methylation of the p15INK4b gene in MDS is correlated with blastic bone marrow involvement and increases with disease evolution toward AML. It suggests that proliferation of leukemic cells might require an escape of regulation of the G1 phase of the cell cycle, and possibly of TGFβ inhibitory effect.
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Chen, Lili, Yuqing Sun, Jingya Wang, Hui Jiang, and Andrew G. Muntean. "Differential Regulation of c-Myc/Lin28 Discriminates Subclasses of Rearranged MLL Leukemia." Blood 126, no. 23 (December 3, 2015): 163. http://dx.doi.org/10.1182/blood.v126.23.163.163.
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Abstract Rearrangements of the 11q23 locus account for ~70% of infant ALL and ~50% of infant AML1 and about 10% of leukemia overall. The prognosis for 11q23 patients is generally poor, however, outcomes vary depending on the fusion partner2. Rearrangements fuse the N-terminus of MLL with one of >70 different partner genes that includes both nuclear and cytoplasmic proteins. Despite the different intracellular localization of these partner proteins, to date, all studied MLL fusion proteins (MLL-FPs) localize to chromatin in the nucleus and drive aberrant transcriptional activation. Recent seminal work by a number of groups has revealed that several of the most common nuclear translocation partner genes (including AF9, ENL, AFF1 (AF4), AFF4 (AF5q31), AF10, AF17 and ELL) assemble into a transcriptional activation complex that includes p-TEFb and/or the histone H3K79 methyltransferase DOT1l. Translocation of MLL with members of this complex results in deregulated transcriptional activation of target genes. Conversely, oligomerization motifs are necessary for transformation following 11q23 translocation with a cytoplasmic partner. However, these mechanisms fail to explain different survival outcomes observed in patients. Further, the transcriptional programs induced in these diverse 11q23 leukemias are currently not well understood. In this study, we examined the genome wide expression profiles in leukemic cells transformed by several MLL-FPs representative of nuclear translocations [t(9;11) (MLL-AF9), t(10;11) (MLL-AF10) and t(11;19) (MLL-ENL)] or cytoplasmic translocations [t(1;11) (MLL-AF1p), t(6;11) (MLL-AF6) and t(11;17) (MLL-Gas7)]. Leukemia cell lines established from mouse bone marrow cells expressing these MLL-FPs proliferated at different rates and mice transplanted with the transformed cells develop leukemia with different latencies remarkably consistent with differences observed in patients harboring different MLL translocations. To elucidate differences in the gene programs induced by different MLL-FPs we performed genome wide expression profiling by RNA-sequencing. These data demonstrated that while the MLL-AF9 and MLL-ENL fusion proteins induce very similar gene programs, the cytoplasmic fusion proteins (MLL-AF6, MLL-AF1p and MLL-GAS7) all possess unique gene signatures. We then performed a pathway analysis comparing nuclear fusion proteins and cytoplasmic fusion proteins and discovered the Myc transcription factor program as one of the top distinguishing features. Myc overexpression significantly increased the growth rate of slow-growing cells that also had low intrinsic Myc, while the growth rate change of more highly proliferative cells was minimal. While all leukemic cell lines were sensitive to the BET inhibitor JQ1 which regulates c-Myc expression, greater sensitivity was observed in those with low c-Myc expression demonstrating the universal importance of this gene program. The Myc target and micro RNA binding protein Lin28B is also differentially expressed between nuclear and cytoplasmic fusions. Negative regulation of miR-150 by Lin28B was observed in all MLL-FP cell lines, which is necessarily downregulated in 11q23 leukemias. We then investigated another Lin28B microRNA target let-7. Interestingly, let-7g expression was significantly increased in MLL-FP transformed cells associated with the longest disease latency. These data demonstrate that differential activation of the c-Myc/Lin28 program accounts for changes in let-7g expression and is associated with MLL-FP disease latency. These data also suggest that patients harboring different 11q23 rearrangements will respond differentially to therapeutic targeting of c-Myc expression dependent on fusion partner. References: 1. Krivtsov AV, Armstrong SA. MLL translocations, histone modifications and leukaemia stem-cell development. Nature reviews Cancer. 2007;7(11):823-833. 2. Balgobind BV, Raimondi SC, Harbott J, et al. Novel prognostic subgroups in childhood 11q23/MLL-rearranged acute myeloid leukemia: results of an international retrospective study. Blood. 2009;114(12):2489-2496. Disclosures No relevant conflicts of interest to declare.
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Buechele, Corina, Erin H. Breese, Dominik Schneidawind, Matthew H. Porteus, and Michael L. Cleary. "Genome Engineering to Prospectively Investigate the Pathogenesis of MLL-AF9 Acute Leukemia." Blood 124, no. 21 (December 6, 2014): 883. http://dx.doi.org/10.1182/blood.v124.21.883.883.
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Abstract Chromosomal rearrangements involving the MLL gene occur in both primary and treatment-related leukemias, and are associated with a poor prognosis. While animal models of MLL-AF9 translocations have improved our understanding of the role of MLL oncogenes in leukemia pathogenesis, in this study a more representative approach based on generation of endogenous activated oncogenes is used to more faithfully model the genomic events on human leukemia. This system allows for prospective investigation of the downstream effects of the initiating event on gene expression, epigenetic regulation, stem cell biology, and genome stability in order to understand the key steps critical for the pathogenesis of MLL-rearranged leukemias. We designed a set of TALENs that cut in intron 11 of the MLL gene (MLL-11 TALENs) to facilitate insertion of sequences encoding for AF9 and the fluorescent marker gene coding NeonGreen (knock-in approach). This resulted in MLL-AF9 expression controlled through the endogenous MLL promoter. Co-expression of MLL-11 TALENs with the AF9 knock-in template resulted in AF9 NeonGreen insertion both in K562 cells and in primary human hematopoietic stem cells isolated from umbilical cord blood. This approach showed a knock-in efficiency of ~20% (range 2-40%) in K562 and CD34+ cells based on NeonGreen expression detected by flow cytometry and confocal microscopy. Long-term culture of primary human hematopoietic cells showed increased frequency of knock-in cells suggesting a survival advantage. Remarkably, further enrichment of knock-in cells was promoted by colony-forming assays in semi-solid medium. Subsequent transplantation into NSG mice was performed to assess their leukemogenic potential. Strikingly, leukemia was induced within 8-14 weeks post transplantation. All mice presented with a similar disease profile that included peripheral blood blasts and splenomegaly. Histologic examination confirmed extensive replacement of bone marrow cells and splenic infiltration by leukemic blasts expressing MLL-AF9 detected by RT-PCR. The leukemic blasts showed increased levels of common MLL target genes (HoxA9, Meis1) compared to non-MLL leukemias and comparable levels to known MLL-AF9 cell lines (Mono Mac 6, THP-1). The blast cells displayed a pre-lymphoid phenotype with CD10+/CD19++/CD38++/CD34+ and were negative for mature B cell markers CD20 and IgM as measured by flow cytometry. Our studies establish a novel experimental model to generate MLL leukemia deriving from primary human hematopoietic stem cells expressing the fusion oncogene under control of the endogenous promoter. The model will allow for further prospective study of leukemia initiating and stem cell biology for a genetic subtype of poor prognosis leukemia. Disclosures No relevant conflicts of interest to declare.
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Pendino, Frédéric, Eric Nguyen, Inge Jonassen, Bjarte Dysvik, Abdulkader Azouz, Michel Lanotte, Evelyne Ségal-Bendirdjian, and Johan R. Lillehaug. "Functional involvement of RINF, retinoid-inducible nuclear factor (CXXC5), in normal and tumoral human myelopoiesis." Blood 113, no. 14 (April 2, 2009): 3172–81. http://dx.doi.org/10.1182/blood-2008-07-170035.
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Abstract Retinoids triggers differentiation of acute promyelocytic leukemia (APL) blasts by transcriptional regulation of myeloid regulatory genes. Using a microarray approach, we have identified a novel retinoid-responsive gene (CXXC5) encoding a nuclear factor, retinoid-inducible nuclear factor (RINF), that contains a CXXC-type zinc-finger motif. RINF expression correlates with retinoid-induced differentiation of leukemic cells and with cytokine-induced myelopoiesis of normal CD34+ progenitors. Furthermore, short hairpin RNA (shRNA) interference suggests for this gene a regulatory function in both normal and tumoral myelopoiesis. Interestingly, RINF localizes to 5q31.3, a small region often deleted in myeloid leukemia (acute myeloid leukemia [AML]/myelodysplasia [MDS]) and suspected to harbor one or several tumor suppressor gene.
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Frech, Miriam, Sabine Teichler, Christine Weber, Caroline Bouchard, Katharina Sorg, Lars Bullinger, Uta-Maria Bauer, and Andreas Neubauer. "Identification of the Oncogene SKI As a New Target Gene of the Myeloid Transcription Factor c-MYB in AML." Blood 128, no. 22 (December 2, 2016): 2719. http://dx.doi.org/10.1182/blood.v128.22.2719.2719.
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Abstract Background: Acute myeloid leukemia (AML) accounts for 80% of acute leukemias and arises through clonal expansion and arrest of differentiation of hematopoietic progenitor cells in the bone marrow. Depending on the AML subtype and age of the patient, AML patients have a 5-year survival rate of about 25%. AML is a genetically heterogenous disease, where chromosomal aberrations, point mutations in critical oncogenes as well as aberrant expression of key regulatory factors of hematopoiesis collude during transformation. c-MYB is an important hematopoietic transcription factor involved in proliferation and differentiation of progenitor cells of the myeloid and lymphoid lineages. It was first described as a viral oncogene of avian leukemia viruses and is upregulated or mutated in many leukemic subtypes as well as solid tumors. c-MYB interacts with other transcription factors or co-factors, which are essential for its transcriptional activity. In this regard, c-MYB transactivation ability is inhibited by a histone deacetylases recruiting corepressor complex containing SKI, TIF1BETA, NCOR and mSIN3A. c-SKI is a proto-oncogene and an inhibitor of TGFβ signalling. However, it acts not only as a transcriptional co-repressor but also as a transcriptional co-activator. Like c-MYB, c-SKI is upregulated in different solid tumors and leukemias. Though SKI activity is well described, transcriptional regulation of the SKI gene itself still remains unknown. Here, we deliver insight into the transcriptional regulation of the human SKI gene via the transcription factor c-MYB. Methods: In silico analyses were performed to identify potential MYB binding sites in the SKI regulatory region. Chromatin immunoprecipitation (ChIP) assays were used to validate the interaction between MYB and the potential SKI regulatory regions. Reporter gene assays were engaged to analyze MYB regulatory potential regarding SKI expression. RNAi experiments were performed to further examine transcriptional regulation of SKI by MYB. Since MYB is known to be downregulated by the histone deacetylase inhibitor (HDACi) valproic acid (VPA), MYB and SKI protein levels were analyzed in AML cell lines treated with VPA via Western Blot. Correlated protein levels of MYB and SKI were examined in the myeloid leukemia cell lines HL60, U937, THP1, NB4 and K562 as well as in primary cells of AML patients (n=27). Correlation of MYB and SKI transcript levels were performed in three different data sets of primary AML samples. The first cohort (n=7) was analyzed via RT-qPCR. Cohort 2 consists of cDNA microarray data of AML patients (n=17). Furthermore, principal component analysis (PCA) of the gene expression profile of MYB and SKI of AML patients (n=542) were performed with the leukemia gene atlas (LGA). Results: In silico analyses revealed four putative MYB binding sites (MBS1-4) in the SKI regulatory region. Direct binding of MYB to the regulatory sites MBS2-4 of the SKI gene could be confirmed via ChIP experiments. Dual luciferase reporter gene assays comprising c-MYB binding sites present in the SKI gene locus further show c-MYB-dependent transcriptional activation of the reporter. RNAi experiments depleting c-MYB in leukemic cell lines resulted in the decrease of SKI protein levels and thereby reveal that c-MYB is essential for the induction of SKI gene expression. Accordingly, treatment of the AML cell lines with the HDACi VPA led to a decrease of MYB and consequently SKI protein levels. Consistently, we observed a positive correlation of MYB and SKI protein expression in leukemic cell lines and in samples of AML patients. Moreover, a highly positive correlation of MYB and SKI transcript levels could be observed in three different cohorts of AML patients, further confirming regulation of SKI expression by the transcription factor MYB. Conclusion: Our findings provide new insights in the transcriptional regulation of the proto-oncogene c-SKI by the oncogenic transcription factor c-MYB during leukemogenesis. c-MYB and c-SKI expression and functions are highly positively correlated in human AML suggesting that c-SKI is a mediator of c-MYB oncogenic potential. So far, various therapeutic approaches targeting MYB failed to be transferred to patients. In this regard, c-MYB and c-SKI represent promising marker and target proteins for novel HDACi-based therapeutic approaches in AML. Disclosures No relevant conflicts of interest to declare.
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Ye, Haobin, Nabilah Khan, Marlene Balys, John M. Ashton, Biniam Adane, and Craig T. Jordan. "Leukemia Cells Resident in Adipose Tissue Display a Pro-Inflammatory Phenotype and Induce Lipolysis and Atrophy of Adipose Tissue." Blood 126, no. 23 (December 3, 2015): 2765. http://dx.doi.org/10.1182/blood.v126.23.2765.2765.
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Abstract Aberrant function of adipose tissue (AT) is seen in several diseases including cancer. Studies show that AT facilitates the progression of tumors through paracrine signaling of adipokines as well as regulation of cancer cell metabolism. However, the mechanism by which cancer cells corrupt the normal function of AT to gain proliferative and survival advantages is unknown. Using a murine model of blast crisis CML, we have shown enrichment of phenotypically primitive leukemia cells (Sca+/lin- leukemia cells, termed "PLCs") in the gonadal AT (GAT) as well as a fatty acid oxidation (FAO) regulatory role of AT. In this study, we evaluated the functional alteration of GAT in leukemic mice. We hypothesized that resident leukemia cells change the characteristics of GAT to obtain metabolic benefits. To test this hypothesis, we first examined whether PLCs in GAT differed from PLCs in other tissues including bone marrow, spleen and peripheral blood. To this end, we utilized RNA-seq to obtain a genome-wide transcriptional profile of PLCs in different tissues. We found PLCs in GAT had a distinct gene expression pattern with enrichment of inflammatory response genes. Specifically, pro-inflammatory cytokines and chemokines were highly expressed by PLCs in GAT (Figure 1). Furthermore, the expression of those genes was also increased in the stromal vascular fraction (SVF) of GAT, indicating resident leukemia cells induced inflammation in GAT. Collectively, these results suggest that leukemia cells found in GAT are distinct from leukemia cells in other tissues and may alter the function of GAT. Another characteristic observed in our model was atrophy of GAT (Figure 2) as well as loss of body weight during leukemia progression, indicating the presence of cancer cachexia. Loss of GAT was also found prior to loss of body weight, suggesting the presence of a pre-cachexia stage. We speculated that atrophy of GAT was due to lipolysis induced by inflammation. Indeed, leukemic GAT released more free fatty acid (FFA) and had a lipolytic pattern of adipokines compared to normal GAT. Elevated FFA and lipolytic adipokines were also detected in leukemic serum. Together, these observations demonstrate that GAT in leukemic mice is lipolytic. To gain insights into mechanisms involved in lipolysis of leukemic GAT, we examined expression of lipolysis-related genes (Figure 3). We found that leukemic GAT had increased expression of the adipose triglyceride lipase (Atgl), which is a rate-limiting lipase controlling lipolysis, and reduced expression of lipoprotein lipase (Lpl), whose expression correlates with the influx of fatty acids into adipocytes. Additionally, decreased expression of the cell death activator CIDE-A (Cidea), which is a lipid droplet (LD) associated protein that shields LDs from lipases and inhibits lipolysis, was found in leukemic GAT. Together, these findings suggest that regulation of lipid metabolism is disrupted in leukemic GAT, leading to lipolysis. To test whether resident leukemia cells contribute to the atrophy of GAT, we examined the lipolytic effect of the pro-inflammatory cytokines and chemokines that were highly expressed by PLCs in GAT. We found that IL-1β and CSF2 induced lipolysis and engendered similar gene expression patterns of lipolysis-related genes in 3T3-L1 adipocytes. Notably, palmitate induced the expression of IL-1β in leukemia cells while it had an opposite effect in naive hematopoietic cells, implying a positive feedback loop where inflammation induces lipolysis which induces IL-1β which in turn augments inflammation. Additionally, an increased amount of IL-1β was observed in leukemic serum. Taken together, these data suggest that resident leukemia cells contribute to the atrophy of GAT through paracrine signaling of pro-inflammatory agents. This phenomenon appears to benefit leukemia cells by fostering FAO and metabolic properties that enhance leukemia cell survival. Thus, targeting pathways that mediate inflammation and/or lipolysis may create a microenvironment that is less favorable to leukemia cells. Disclosures No relevant conflicts of interest to declare.
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Arai, Shunya, Akihide Yoshimi, Munetake Shimabe, Motoshi Ichikawa, Masahiro Nakagawa, Yoichi Imai, Susumu Goyama, and Mineo Kurokawa. "Evi-1 is a transcriptional target of mixed-lineage leukemia oncoproteins in hematopoietic stem cells." Blood 117, no. 23 (June 9, 2011): 6304–14. http://dx.doi.org/10.1182/blood-2009-07-234310.
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Abstract Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation, overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities, which indicates the presence of other mechanisms for Evi-1 activation. In this study, we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL–mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population, in which Evi-1 particularly contributes to the propagation of MLL-ENL–immortalized cells. Furthermore, gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell–like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell–derived MLL leukemic cells.
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Potluri, Sandeep, Salam A. Assi, Suyin Paulynn Chin, Anetta Ptasinska, Daniel Coleman, Peter Cockerill, and Constanze Bonifer. "Isoform Specific Regulation of Acute Myeloid Leukemia Maintenance By Wilms Tumor 1." Blood 134, Supplement_1 (November 13, 2019): 1232. http://dx.doi.org/10.1182/blood-2019-122533.
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The gene encoding Wilms Tumor 1 (WT1) is recurrently upregulated in Acute Myeloid Leukemia (AML). WT1 transcript burden is associated with primary refractory disease and with relapse when there is detectable WT1 minimal residual disease (Lambert et al., Oncotarget, 2014). We have recently described the gene regulatory networks in purified blasts from AML patients with various driver mutations (Assi et al., Nature Genetics, 2019) and found that WT1 was a key transcription factor node in all networks examined. Furthermore, in knockdown experiments as part of a shRNA depletion screen against transcription factors in vitro and in murine xenotransplantation experiments, we showed that WT1 was essential for leukemic maintenance (Martinez-Soria et al., Cancer Cell, 2018). WT1 produces 8 primary isoforms, all of which are expressed in hematopoietic cells by employing one of two transcription start sites and alternative splicing in exon 5 and exon 9. To further understand how WT1 maintains leukemia, we cloned each of the 8 isoforms into a doxycycline-inducible vector and transduced them into a t(8;21) AML cell line, Kasumi-1, which well recapitulates primary t(8;21) AML (Ptasinska et al., Leukemia, 2014). We carefully overexpressed each of the 8 WT1 isoforms to levels seen in AML patient samples and functionally and molecularly characterised them. Overexpression of isoforms lacking 3 amino acids in the zinc finger DNA binding domain (WT1 -KTS) significantly reduced cell growth and colony formation, increased apoptosis, caused a G1 cell cycle arrest and caused myeloid differentiation of leukemic blasts. However, overexpression of isoforms with the 3 amino acids (WT1 +KTS) in the DNA-binding domain had the opposite effects, thus having a major role in the maintenance of a leukemic phenotype. Also, we found that the expression of WT1 +KTS isoforms in purified blasts from patients was higher than WT1 -KTS isoforms and may therefore permit leukemic maintenance. We did not find any functional differences between isoforms employing alternate transcriptional start sites or between exon 5 splice variants. We then performed the first ever WT1 chromatin immunoprecipitation (ChIP-seq) in AML and show that WT1 -KTS shows increased binding as compared to WT1 +KTS with 9451 unique WT1 binding sites in WT1 -KTS expressing cells and only 1355 sites in WT1 +KTS expressing cells with 2391 sites overlapping between the isoforms. This finding suggests that WT1 isoforms compete for binding at multiple sites and may explain why the ratio of expression between WT1 isoforms is critical (Calabrese et al., Blood, 2012). We also correlated WT1 ChIP-seq peaks to transcription as assayed by RNA-seq. We found that differentially bound cis-regulatory elements are associated with altered expression of genes involved in proliferation, cell cycle, apoptosis and differentiation pathways, explaining many of the functional alterations in cellular behaviour that we see. Several vaccine studies and more recently a T cell receptor study targeted against WT1 have been undertaken but they have shown mixed efficacy in AML (Chapuis et al, Nature Medicine, 2019). Since we find that different isoforms of WT1 have antagonistic effects, we hypothesise that a more effective therapeutic strategy would be to selectively target only WT1 +KTS isoforms. Disclosures No relevant conflicts of interest to declare.
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Ropa, James, Nirmalya SAHA, and Andrew G. Muntean. "SETDB1 Represses Hox Gene Expression and Suppresses Acute Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 1320. http://dx.doi.org/10.1182/blood-2018-99-117690.
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Abstract Epigenetic regulators play an important role in normal and malignant hematopoiesis. Epigenetic deregulation of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia (AML), including those harboring MLL rearrangements and NPM mutations, as well as others. Expression of Hoxa9 and its co-factor Meis1 is sufficient to transform bone marrow into a lethal AML in mouse models. We previously demonstrated that the pro-leukemic genes Hoxa9 and Meis1 are critically regulated by the histone H3 Lysine 9 (H3K9) methyltransferase SETDB1. Recent studies show that SETDB1 is required for normal hematopoiesis and MLL-AF9 mediated leukemia (Koide, et al. Blood 2016). Our lab recently demonstrated that SETDB1 negatively regulates the expression of HoxA9 and Meis1 through deposition of promoter H3K9 methylation in MLL-AF9 AML cells (Ropa et al. Oncotarget 2018). Consistent with these data, HOXA9 and MEIS1 expression negatively correlates with SETDB1 expression in AML patient samples. Therefore, we investigated the biological impact of SETDB1 on AML. We first noted that expression of SETDB1 in AML patient samples is significantly lower compared to normal hematopoietic cells. Further, higher SETDB1 expression correlated with a significantly better overall survival (p=0.003) and lower expected hazard (HR=0.9/100RSEM; p=0.009) in AML patients compared with lower SETDB1 expression. These data are consistent with SETDB1 negatively regulating pro-leukemic genes and suggests that SETDB1 expression may be correlated with AML patient prognosis. We tested this directly by expressing high levels of SETDB1 in AML cells. Ex vivo assays show that retroviral overexpression of SETDB1 in MLL-AF9 AML cells leads to cell differentiation, decreased leukemia colony formation, and decreased cell proliferation. Consistent with the AML patient data, overexpression of SETDB1 significantly delays MLL-AF9 mediated leukemogenesis in vivo (p=0.01). Further, we observed a strong selective pressure against exogenous SETDB1 expression in moribund mice. Transcriptome analyses demonstrate that SETDB1 globally represses Hox and pluripotency gene programs. Strikingly, we found that SETDB1 represses many of the same genes that exhibit reduced promoter H3K9me3 in AML patient samples relative to CD34+ cells. These data point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing AML. We also explored how chemical and genetic inhibition of H3K9 methylation and Setdb1 affects AML initiation and maintenance. We first confirmed the previously reported requirement for Setdb1 in AML cell lines by genetically deleting both alleles of Setdb1 in MLL-AF9 cells, which resulted in a complete arrest of proliferation (Koide, et al. Blood 2016). Combined with our data presented above, these results suggest a narrow window of SETDB1 expression is maintained in AML cells. To achieve reduced (but not complete loss of) activity, we investigated how small molecule inhibition of H3K9 methylation (UNC0638) or shRNA mediated knock down of Setdb1 affects AML initiation. We observed increased ex vivo colony formation of normal ckit+ bone marrow cells upon shRNA mediated knockdown of Setdb1 or upon UNC0638 treatment. We hypothesized that this expansion of colony forming unit potential of hematopoietic cells may translate to increased transformation potential by leukemic oncogenes. Indeed, cells pretreated with UNC0638 followed by retroviral transduction with MLL-AF9 exhibit significantly higher capacity for leukemic colony formation than vehicle treated cells. These data are consistent with H3K9 methylation repressing genes required for AML transformation. Our data identified a narrow window of expression of SETDB1 in AML patient samples. SETDB1 expression is reduced in AML patients relative to normal cells and chemical inhibition of H3K9 methylation expands the pool of cells amenable to MLL-AF9 mediated transformation ex vivo. While inhibition of SETDB1 and other H3K9 methyltransferases has been suggested as a possible therapeutic strategy, our data suggests this may also prime bone marrow cells for transformation by inhibiting epigenetic processes that repress pro-leukemic target genes. Further investigation of the roles of SETDB1 and H3K9 methylation levels is necessary to determine the value of these epigenetic modifiers as therapeutic targets in AML and is currently ongoing. Disclosures No relevant conflicts of interest to declare.
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Gricks, Clair S., David Zahrieh, A. Jason Zauls, Gullu Gorgun, Daniela Drandi, Katja Mauerer, Donna Neuberg, and John G. Gribben. "Differential regulation of gene expression following CD40 activation of leukemic compared to healthy B cells." Blood 104, no. 13 (December 15, 2004): 4002–9. http://dx.doi.org/10.1182/blood-2004-02-0494.
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Abstract It is possible to differentiate malignant from healthy cells and to classify diseases based on identification of specific gene expression profiles. We hypothesized that gene expression profiling could also be used to identify differential activation of healthy and malignant cells, and as a model for this, we examined the molecular sequelae of CD40 activation of healthy and B-cell chronic lymphocytic leukemia (CLL) cells. Hierarchical clustering analysis of gene expression signatures grouped samples by CD40 activation status and further subclassified CD40-activated CLL cells from healthy B cells. Supervised analyses in healthy B cells compared to CLL cells identified differential regulation of genes governing cell cycle progression and apoptosis. CD40 signaling of CLL cells increases their susceptibility to immune recognition, but promotes survival and cell cycle arrest, making these cells potentially more resistant to chemotherapy. These results illustrate the utility of gene expression profiling to elucidate the molecular sequelae of signaling in healthy cells and altered signaling pathways in malignant cells. This type of approach should be useful to identify targets of therapy of malignant diseases. (Blood. 2004;104:4002-4009)
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Ge, Zheng, Jianyong Li, Baoan Chen, Sinisa Dovat, and Chunhua Song. "Targeting High Dynamin2 Expression By Restoring Ikaros Function in Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 2713. http://dx.doi.org/10.1182/blood.v128.22.2713.2713.
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Abstract Background: Dynamin-2 (DNM2) is a GTPase essential for intracellular vesicle formation and trafficking, cytokinesis and receptor endocytosis. Mutations in DNM2 are common in early T-cell precursor acute lymphoblastic leukemia (ALL). However, DNM2 expression in other types of ALL is not reported. Ikaros, encoded by IKZF1, is a transcriptor factor functioned as a tumor suppress gene, and its dysfunction is associated with poor survival and high relapse rate in ALL. Casein Kinase II (CK2) inhibition could restore Ikaros function in high-risk leukemia and CK2 inhibitor-CX4945 showed the therapeutic efficacy on high-risk leukemia with human-derived xenograft mouse model. It is still undetermined if Ikaros regulates DNM2 expression in the leukemic cells. Methods: The 151 patients' and 30 volunteers' BM samples were collected between June 2008 and June 2014 at the First Affiliated Hospital of Nanjing Medical University. The ALL diagnosis was made according to the morphologic, Immunophenotypic, cytogenetic, and molecular criteria of WHO Diagnosis and Classification of ALL (2008).Cytogenetic and molecular analyses as previously reported. The DNM2 expression was determined by qPCR in the patients. All the patients were divided into high or low DNM2 expression groups (Q4 vs Q1-3) and the cutoff was determined by SPSS 17.0. For quantitative parameters, overall differences between the cohorts were evaluated using a Mann - Whitney U -test. For qualitative parameters, overall group differences were analyzed using a χ2 test. All statistical analyses were performed using the SPSS 17.0 and P<0.05 was considered statistically significant. The effect of Ikaros on DNM2 gene expression was observed by qPCR in the leukemic cells expressed Ikaros or Ikaros ShRNA. Ikaros binding with promoter of DNM2 was evaluated by chromatin immunoprecipitation assay following quantitative real-time PCR in leukemic cells. The effect of DNM2 inhibitor on cell proliferation was performed by WST-1 cell proliferation assay, and the synergy of Casein Kinase inhibitor which restores Ikaros function with DNM2 inhibitor on cell proliferation of leukemic cells was analyzed by CalcuSyn. Results: We studied DNM2 mRNA level in adults with B- and T-cell ALL, and found DNM2 is more highly expressed compared with normals in both forms of ALL. High DNM2 expression is significantly associated with poor overall survival (OS), high relapse rate, and leukaemia cell proliferation markers particularly in B-ALL. DNM2 expression is significantly higher in the patients with IKZF1 deletion compared to that of without deletion. Ikaros directly binds the DNM2 promoter in Nalm6 (B-ALL) and CEM (T-ALL) leukemic cells. Ikaros suppresses the transcription of DNM2 with luciferase reporter assay. Retroviral transduction of Ikaros results in the down-regulation of DNM2 in the leukemic cells. CK2 inhibitor, TBB increases Ikaros binding to promoter of DNM2 and suppresses DNM2 expression in an Ikaros-dependent manner in both leukemic cell lines and primary cells. TBB induced-increase of H3K9me3 binding on the promoter of DNM2 was also observed in leukemic cell lines and primary cells. Finally, DNM2 inhibitor-MiTMAB significantly suppresses the cell proliferation of Nalm6 and CEM cells with the WST-1 cell proliferation assay and has significantly synergistic effect with Ck2 inhibitor, CX-4945 in the cells. Conclusion: High DNM2 expression is associated with Ikarosdys-regulation, revealing their potential roles on the development of ALL. DNM2 inhibitor MiTMAB inhibits cell proliferation and has synergistic effect with CK2 inhibitor CX4945 in leukemic cells. Disclosures No relevant conflicts of interest to declare.
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Kumar, Ashish R., and John H. Kersey. "The Expression of Genes 5′ in the Hox-A Cluster Is Co-Ordinated Both in Normal and Leukemic Hematopoiesis." Blood 104, no. 11 (November 16, 2004): 3560. http://dx.doi.org/10.1182/blood.v104.11.3560.3560.
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Hox genes are known to play critical roles in hematopoiesis and are probably important in the pathogenesis of leukemias. Disruption of the Hoxa9 gene in mice leads to defects in erythroid, lymphoid and myeloid hematopoiesis while over-expression of Hoxa9 leads to leukemia in mice. Transcription of Hoxa9 is partly regulated by the Mixed lineage leukemia gene (Mll) product. Bone marrow cells of mice carrying the leukemic Mll-AF9 fusion gene as a knock-in mutation (henceforth called Mll), display an increase in expression of several Hox genes - Hoxa5, Hoxa6, Hoxa7, Hoxa9 and Hoxa10, when compared to age matched wild type mice (Kumar et. al, 2004, Blood). Since the over-expressed Hox genes all belong to the same Hox-a cluster, we hypothesized that these genes might be co-regulated during normal hematopoiesis. Co-regulation of neighboring genes within the same cluster has been reported by others for the Hox-b and c clusters. To test this hypothesis for the 5′ Hox-a cluster genes, we compared expression levels of Hoxa5, Hox7 and Hoxa10 in Hoxa9 homozygous knockout (Hoxa9−/−) and wild type bone marrow by real-time quantitative RT-PCR using Taqman primers/probe sets (Applied Biosystems, CA). Expression levels of Hoxa5, Hoxa7, and Hoxa10 were all reduced by 65% ± 2% in Hoxa9−/− mice compared to wild type mice. In contrast, levels of Hoxb4 and Meis1 - homeobox genes that are not part of the Hox-a cluster - were identical in Hoxa9−/− and wild type mice. These results show no compensatory increases in expression of other 5′ Hox-a genes in the absence of Hoxa9, but instead demonstrate that disruption of Hoxa9 decreases the expression of neighboring genes in the Hox-a cluster. The hematopoietic defects seen in Hoxa9−/− mice (leucopenia, lymphopenia and blunted granulocytic response to G-CSF) might thus be attributable to the deficiency of multiple Hox-a gene products, rather than of Hoxa9 alone. To further evaluate the extent of this co-regulation, we studied the expression levels of these genes in Mll mice that lacked Hoxa9 (Hoxa9−/−/Mll-AF9+/−, henceforth called Mll/Hox). The Mll/Hox mice develop leukemia at the same rate and time course as the Mll mice, but with a phenotype that is relatively more immature. In Mll/Hox mice, the expression levels of Hoxa5 and Hoxa7 were increased 13 fold and 4 fold respectively, while those of Hoxa10 remained decreased at 35% of wild type. These results indicate that the decreased expression of neighboring Hox-a genes in Hoxa9−/− mice was reversed by Mll-AF9 for Hoxa5 and Hoxa7, but not for Hoxa10, suggesting a second level of co-regulation for Hoxa9 and Hoxa10. Overall, our findings demonstrate a co-regulated relationship between the 5′ Hox-a cluster genes during normal hematopoiesis, and provide evidence that deregulation of a single Hoxa9 gene significantly alters the expression of neighboring Hox-a cluster genes with implications for understanding the pathogenetic mechanisms of leukemia.
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Salvestrini, Valentina, Rossella Manfredini, Miriam Fogli, Elisa Bianchi, Simona Salati, Marilina Amabile, Francesco Bertolini, et al. "Molecular and Functional Analysis of Stem Cell Compartment of Chronic Myelogenous Leukemia Reveals the Presence of a CD34− cell Population with Intrinsic Resistance to IMATINIB Treatment." Blood 112, no. 11 (November 16, 2008): 4221. http://dx.doi.org/10.1182/blood.v112.11.4221.4221.
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Abstract We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin−CD34−) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular caryotyping and quantitative analysis of BCR-ABL transcript demonstrated that about one third of CD34− cells are leukemic. CML Lin−CD34− cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells, cell cycling, acquisition of clonogenic activity and increased expression of BCR-ABL transcript. Lin−CD34− cells showed hematopoietic cell engraftment rate in immunodeficient mice similar to Lin-CD34+ cells whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell cycle arrest genes, genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated when compared to normal counterparts. Flow cytometry analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin−CD34−cells. Imatinib mesilate did not reduce fusion transcript levels, BCR-ABL kinase activity and clonogenic efficiency of CML Lin− CD34− cells in vitro. Moreover, leukemic CD34− cells survived to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34− leukemic stem cell subset in CML with peculiar molecular and functional characteristics.
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Lemoli, Roberto M., Valentina Salvestrini, Elisa Bianchi, Francesco Bertolini, Miriam Fogli, Marilina Amabile, Agostino Tafuri, et al. "Molecular and functional analysis of the stem cell compartment of chronic myelogenous leukemia reveals the presence of a CD34− cell population with intrinsic resistance to imatinib." Blood 114, no. 25 (December 10, 2009): 5191–200. http://dx.doi.org/10.1182/blood-2008-08-176016.
Full textAbstract:
Abstract We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin−CD34−) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular karyotyping and quantitative analysis of BCR-ABL transcript demonstrated that approximately one-third of CD34− cells are leukemic. CML Lin−CD34− cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells and cell cycling, and increased clonogenic activity and expression of BCR-ABL transcript. Lin−CD34− cells showed hematopoietic cell engraftment rate in 2 immunodeficient mouse strains similar to Lin-CD34+ cells, whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell-cycle arrest genes and genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated compared with normal counterparts. Phenotypic analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin−CD34− cells. Imatinib mesylate did not reduce fusion transcript levels, BCR-ABL kinase activity, and clonogenic efficiency of CML Lin−CD34− cells in vitro. Moreover, leukemic CD34− cells survived exposure to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34− leukemic stem cell subset in CML with peculiar molecular and functional characteristics.
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Manfioletti, G., V. Gattei, E. Buratti, A. Rustighi, A. De Iuliis, D. Aldinucci, GH Goodwin, and A. Pinto. "Differential expression of a novel proline-rich homeobox gene (Prh) in human hematolymphopoietic cells." Blood 85, no. 5 (March 1, 1995): 1237–45. http://dx.doi.org/10.1182/blood.v85.5.1237.bloodjournal8551237.
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Proline-rich homeobox (Prh) is a novel human homeobox-containing gene recently isolated from the CD34+ cell line KG-1A, and whose expression appears mainly restricted to hematopoietic tissues. To define the pattern of Prh expression within the human hematopoietic system, we have analyzed its constitutive expression in purified cells obtained from normal hematopoietic tissues, its levels of transcription in a number of leukemia/lymphoma cell lines representing different lineages and stages of hematolymphopoietic differentiation, and its regulation during in vitro maturation of human leukemic cell lines. Prh transcripts were not detected in leukemic cells of T-lymphoid lineage, irrespective of their maturation stage, and in resting or activated normal T cells from peripheral blood and lymphoid tissues. In contrast, high levels of Prh expression were shown in cells representing early stages of B lymphoid maturation, being maintained up to the level of circulating and tissue mature B cells. Terminal B-cell differentiation appeared to be conversely associated with the deactivation of the gene, since preplasmacytic and plasmocytoma cell lines were found not to express Prh mRNA. Prh transcripts were also shown in human cell lines of early myelomonocytic, erythromegakaryocytic, and preosteoclast phenotypes. Prh expression was lost upon in vitro differentiation of leukemic cell lines into mature monocyte-macrophages and megakaryocytes, whereas it was maintained or upregulated after induction of maturation to granulocytes and osteoclasts. Accordingly, circulating normal monocytes did not display Prh mRNA, which was conversely detected at high levels in purified normal granulocytes. Our data, which show that the acquisition of the differentiated phenotype is associated to Prh downregulation in certain hematopoietic cells but not in others, also suggest that a dysregulated expression of this gene might contribute to the process of leukemogenesis within specific cell lineages.
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Hawley, Teresa S., Andrew Z. C. Fong, Henrik Griesser, Stewart D. Lyman, and Robert G. Hawley. "Leukemic Predisposition of Mice Transplanted With Gene-Modified Hematopoietic Precursors Expressing flt3 Ligand." Blood 92, no. 6 (September 15, 1998): 2003–11. http://dx.doi.org/10.1182/blood.v92.6.2003.
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Abstract flt3/flk-2 ligand (FL) is a cytokine that exhibits synergistic activities in combination with other early acting factors on subpopulations of hematopoietic stem/progenitor cells. In addition to normal hematopoietic precursors, expression of the FL receptor, flt3R, has been frequently demonstrated on the blast cells from patients with acute B-lineage lymphoblastic, myeloid, and biphenotypic (also known as hybrid or mixed) leukemias. Because many of these leukemic cell types express FL, the possibility has been raised that altered regulation of FL-mediated signaling might contribute to malignant transformation or expansion of the leukemic clone. In humans, FL is predominantly synthesized as a transmembrane protein that must undergo proteolytic cleavage to generate a soluble form. To investigate the consequences of constitutively expressing the analogous murine FL isoform in murine hematopoietic stem/progenitor cells, lethally irradiated syngeneic mice (18 total) were engrafted with post–5-fluorouracil–treated bone marrow cells transduced ex vivo with a recombinant retroviral vector (MSCV-FL) encoding murine transmembrane FL. Compared with control mice (8 total), MSCV-FL mice presented with a mild macrocytic anemia but were otherwise healthy for more than 5 months posttransplant (until 22 weeks). Subsequently, all primary MSCV-FL recipients observed for up to 1 year plus 83% (20 of 24) of secondary MSCV-FL animals that had received bone marrow from asymptomatic primary hosts reconstituted for 4 to 5 months developed transplantable hematologic malignancies (with mean latency periods of 30 and 23 weeks, respectively). Phenotypic and molecular analyses indicated that the tumor cells expressed flt3R and displayed B-cell and/or myeloid markers. These data, establishing that dysregulated expression of FL in primitive hematopoietic cells predisposes flt3R+ precursors to leukemic transformation, underscore a potential role of this cytokine/receptor combination in certain human leukemias. © 1998 by The American Society of Hematology.