The Polycomb Group Transcriptional Repressor Bmi-1 Is Constitutively Expressed in Multiple Myeloma (MM) Cells and Modulates Proliferation through a Mechanism Independent of the Ink4a/ARF Locus.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3346-3346
Author(s):  
Frank J. Desarnaud ◽  
Masood Shammas ◽  
Paola Neri ◽  
Hemanta Koley ◽  
Rao Prabhala ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Critical Role ◽  
Polycomb Group ◽  
Cytokine Signaling ◽  
Polycomb Group Protein ◽  
Hematopoietic Stem

Abstract The polycomb group protein Bmi-1 is required for self-renewal of adult hematopoietic stem cells by inhibiting cell senescence and apoptosis through repression of p16INK4a and p14/p19ARF expression. Based on gene expression profile studies showing absence of Bmi-1 expression in normal plasma cells versus upregulation in all MM cell lines and primary patient MM cells, we evaluated its role in MM pathobiology. Bmi-1 expression in MM cell lines is constitutive, and is not modulated by IL-6 and IGF-1. Immunoprecipitation and immunoblotting studies confirmed both Bmi-1 overexpression and nuclear localization. Bmi-1 was found to be tyrosine- and serine-phosphorylated. We next inhibited Bmi-1 expression in MM cell lines (ARP and MM.1S) by stably transfecting a PU6 vector expressing siRNA targeting Bmi-1. Following G418 selection, cell growth, as measured by H3 thymidine incorporation and MTT assay, was stimulated 1.4 fold in MM.1S and 2 fold in ARP cells compared to control cells transfected with a PU6 vector. Gene expression profiling showed: up-regulation of IL-6 and IGF-1 receptors, as well as Gab-1, which plays a critical role in MM cell proliferation and survival induced by IL-6; Phospholipase D1 and proteinase 3 which potentiate cell proliferation by repression and cleavage of p21cip1/waf1; stem cell growth factor, HOXA9, which modulate bone marrow stem cell survival and proliferation. In contrast, p21cip1/waf1 and GADD45a, GADD45b which moderate cell growth arrest were down regulated. However, no change in expression of either p16INK4a or p14/p19ARF was detected by microarray and immunoblotting. We therefore conclude that Bmi-1 is abnormally expressed in MM cells and contrary to its classic action, modulates MM cell growth and survival by modulating cytokine signaling rather than INK4a/ARF. Our studies further suggest that Bmi-1 represents a promising target for novel MM therapeutics.

scholarly journals Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1204-1204
Author(s):  
Xi Jin ◽  
Tingting Qin ◽  
Nathanael G Bailey ◽  
Meiling Zhao ◽  
Kevin B Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Double Mutant ◽  
Mutant Mice ◽  
Cytokine Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Single Mutant ◽  
Tet2 Mutation

Abstract Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown. In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/-(single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred. Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation. To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling. Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Latexin: A Potential Tumor Suppressor.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1805-1805
Author(s):  
Ying Liang ◽  
Gary Van Zant
Keyword(s):  
Cell Proliferation ◽  
Stem Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Negative Regulator ◽  
Stem Cell Population ◽  
Western Blots ◽  
Hematopoietic Stem ◽  
Cell Counts

Abstract We recently found that latexin is a negative regulator of the size of the hematopoietic stem cell population in mice. It acts by increasing apoptosis and decreasing cell proliferation. This 29 kD protein bears a strong structural resemblance to tazarotene-induced gene 1 (TIG1), a tumor suppressor down-regulated in a variety of cancers. The structural similarity and close genetic linkage led us to hypothesize that latexin also may have tumor suppressor properties. We found that latexin was down-regulated in a variety of human leukemias and lymphomas as determined by a survey of malignant cell lines and by analysis of CD34+ cells isolated from the blood and marrow of patients diagnosed with these malignancies and presenting with very high white cell counts. Bisulfite sequencing revealed that methylation of CpG dinucleotides in the latexin promoter at least partially accounted for latexin down-regulation. 5-aza-deoxycytidine treatment reinitiated or significantly increased latexin expression in K562, Molt4, CRF-CEM, Jurkat, U937, HL60, KG-1, and Sup B15 cell lines. To test the hypothesis that ectopic latexin expression in tumor cells would inhibit their growth, we developed a retrovirus-based expression vector with which we infected the murine lymphoma cell lines, WEHI231 and A20, neither of which contained significant latexin levels by Western blot. A vector containing GFP, but not latexin, was used to infect control cells. In triplicate experiments, the growth of both cell lines in vitro was inhibited an average 48% by infection with the latexin vector. Western blots revealed that latexin was durably expressed throughout the 2-week culture period at 2- to 4-fold the level expressed in normal T and B cells. As we found in our normal stem cell studies, latexin caused growth inhibition of lymphoma cells by significantly increasing apoptosis by 6-fold, and by suppressing cell proliferation by 2-fold. To test whether tumor inhibition extended to lymphomas in vivo, we injected either control or latexin vector-infected A20 cells subcutaneously in the flanks of BALB/c mice. Three weeks following adoptive transfer of identical numbers of cells, in duplicate experiments the latexin-expressing cells developed tumors only half the volume of those caused by the control cells. These results are consistent with a tumor suppressor role for latexin and suggest that latexin, or molecular mimics thereof, may have clinical efficacy in the treatment of malignancies.

CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4174-4174
Author(s):  
Jiaying Tan ◽  
Jay L. Hess
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Polycomb Group ◽  
Human Leukemia ◽  
Interaction Sites

Abstract Abstract 4174 Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

BMC Genomics ◽  
2006 ◽  
Vol 7 (1) ◽  
Author(s):  
Karin Richter ◽  
Valtteri Wirta ◽  
Lina Dahl ◽  
Sara Bruce ◽  
Joakim Lundeberg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Lines ◽  
Global Gene Expression ◽  
Hematopoietic Stem ◽  
Gene Expression Analyses

Inhibition of Human Plasmacytoma Cell Growth by a Novel JAK Kinase Inhibitor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 644-644
Author(s):  
Renate Burger ◽  
Steven Legouill ◽  
Yu-Tzu Tai ◽  
Reshma Shringarpure ◽  
Klaus Podar ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Cell Lines ◽  
Hormone Receptors ◽  
Critical Role ◽  
Myeloma Cell ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Jak Kinase ◽  
Dose Dependent

Abstract Novel strategies in cancer therapy aim at inhibiting distinct signal transduction pathways that are aberrantly activated in malignant cells. Protein tyrosine kinases of the JAK family are associated with a number of cytokine and cytokine-like hormone receptors and regulate important cellular functions such as proliferation, survival, and differentiation. Constitutive or enhanced JAK activation has been implicated in neoplastic transformation and abnormal cell proliferation in various hematological malignancies. In multiple myeloma (MM), JAK kinases play a critical role because of their association with cytokine receptors of the IL-6/gp130 family. A novel small-molecule inhibitor was developed that shows a 100 to 1,000-fold selectivity for JAK1, JAK2, JAK3, and TYK2 relative to other kinases including Abl, Aurora, c-Raf, FGFR3, GSK3b, IGF-1R, Lck, PDGFRa, PKBb, and Zap-70. Growth of MM cell lines and primary patient cells was inhibited by this compound in a dose-dependent manner. The IL-6 dependent cell line INA-6 and derived sublines were sensitive to the drug, with IC50’s of less than 1 mM, in [3H]-thymidine uptake and a colorimetric, tetrazolium compound (MTS) based assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI). Importantly, INA-6 and patient tumor cell growth was also inhibited in the presence of bone marrow stromal cells, which by themselves remained largely unaffected. Growth suppression of INA-6 correlated with a significant and dose-dependent increase in the percentage of apoptotic cells, as evaluated by Apo2.7 staining after 48 hours of drug treatment. In addition, the compound blocked IL-6 induced phosphorylation of STAT3, a direct downstream target of JAK kinases and important transcription factor triggering anti-apoptotic pathways. In other myeloma cell lines, the drug overcame the protective effect of gp130 cytokines on dexamethasone induced apoptosis. In MM1.S cells, it completely blocked IL-6 induced phosphorylation of SHP-2 and AKT, both known to mediate the protective effects of IL-6. In contrast, AKT phosphorylation induced by IGF-1 remained unchanged, demonstrating selectivity of the compound. These studies show that disruption of JAK kinase activity and downstream signaling pathways inhibits myeloma cell growth and survival as well as circumvents drug resistance, thereby providing the conceptual basis for the use of JAK kinase inhibitors as a novel therapeutic approach in MM.

A Subnuclear Targeting Defective Mutant of Runx1 Uncouples the Roles of Runx1 In Embryonic Development and Adult Definitive Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1387-1387
Author(s):  
Christopher R Dowdy ◽  
Dana Frederick ◽  
Sayyed K. Zaidi ◽  
Jane B. Lian ◽  
J. van Wijnen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Mouse Model ◽  
Hematopoietic Stem Cell ◽  
Globin Gene ◽  
Critical Role ◽  
Chromosomal Translocations ◽  
Common Mechanism ◽  
Human Leukemia ◽  
Hematopoietic Stem

Abstract Abstract 1387 As shown by gene ablation or replacement studies, Runx1, a master regulator of hematopoiesis, is critical for the emergence of the hematopoietic stem cell (HSC). Runx1 is also the most common target for point mutations and chromosomal translocations in human leukemia. Localization of Runx1 to distinct foci within the nucleus through a unique subnuclear targeting signal is linked to physiological roles of the protein. Many leukemia-related chromosomal translocations (e.g., 8;21 translocation that encodes for AML1-ETO protein) disrupt the Runx1 C-terminus and modify its biological activity as well as alter subnuclear localization. We hypothesize that altered subnuclear routing of leukemic proteins plays a key role and provides a common mechanism in human leukemias. We have previously developed a knock-in mouse model with a Runx1 C-terminal truncation (Runx1 Q307X), which similar to a complete ablation of Runx1 or a genetic knock-in of the leukemic AML1-ETO, causes mid gestation lethality. The Runx1 Q307X mutation also abolishes subnuclear targeting and alters Runx1-dependent myeloid gene expression. To further dissect the specific functional contributions of altered Runx1 subnuclear targeting, we developed a knock-in mouse model in which endogenous Runx1 is replaced with a triple point mutation (Runx1 HTY350-352AAA) that modifies the subnuclear targeting. In contrast to Runx1 Q307X or AML1-ETO knock-in mice, animals homozygous for Runx1 HTY350-352AAA bypass embryonic lethality. However, expression of key Runx1 target genes that regulate hematopoiesis is altered. In addition, bone marrow from Runx1 HTY350-352AAA animals shows increased proliferation ex vivo, resulting in an increase of myeloid and a decrease of B-lymphoid lineage cells. Importantly, red blood cell development, previously thought unrelated to Runx1 expression, is also modified in the Runx1 HTY350-352AAA homozygous animals, as shown by decreased mean corpuscle volume and selective effects on globin gene expression. Together, these findings show that altered subnuclear targeting uncouples the critical role of Runx1 during hematopoietic stem cell emergence and differentiation into various lineages in the adult. Disclosures: No relevant conflicts of interest to declare.

scholarly journals INPP4B is a Biomarker of Poor Prognosis in AML Which is Associated with EVI1 Overexpression and a LSC Signature

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3929-3929
Author(s):  
Irakli Dzneladze ◽  
John F Woolley ◽  
Youqi Han ◽  
Mark Sharobim ◽  
Ayesha Rashid ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Differential Expression ◽  
Cell Lines ◽  
Colony Formation ◽  
Promoter Region ◽  
Akt Signaling ◽  
Hematopoietic Stem ◽  
Hematopoietic Lineage

Abstract Acute myeloid leukemia (AML) is a highly heterogeneous cancer of the bone marrow. To better understand leukemogenesis and improve predictors of patient response to chemotherapy and overall survival (OS), we recently examined the role of inositol polyphosphate-4-phosphatase, type-II (INPP4B) in AML. Normally, INPP4B plays a role in PI3K/Akt signaling by regulating phosphorylation of phosphoinositides (PIs), critical membrane-bound second messenger molecules, by dephosphorylating the 4'-position of PI(3,4)P2 to generate PI(3)P. Because PI(3,4)P2, like PI(3,4,5)P3, is necessary for the activation Akt, INPP4B was first hypothesized and demonstrated to be a tumor suppressor protein, akin to PTEN, in several cancers including breast, prostate and ovarian. Recent work however, including our own study, has demonstrated a paradoxical tumor-promoting role of INPP4B in AML and estrogen receptor positive (ER+) breast cancer. Specifically, we demonstrated that INPP4Bhigh AML patients (25% of patients) had significantly shorter OS, lower response to induction therapy, and shorter event-free survival. Furthermore, INPP4B expression was found to be an independent prognostic marker for OS in AML outperforming FLT3-ITD and NPM1 mutation status. Overexpression of INPP4B in several AML cell lines results in enhanced colony formation potential, chemotherapy drug resistance, and increased proliferation. Though this previous work has shown that INPP4B plays a significant role in AML, it remains unclear why INPP4Bhigh AML differs from INPP4Blow AML, and what causes its upregulation. To address this question, we interrogated gene expression data from three independent datasets (n=942) to identify genes with differential expression between INPP4Bhigh and INPP4Blow AML. Gene expression analysis revealed that INPP4Bhigh AML was associated with differential expression of 233 genes. High INPP4B expression was associated with higher expression of genes related to the hematopoietic lineage, PI3K-Akt signaling, Jak-STAT signaling and ECM-receptor interaction pathways. Specifically, INPP4Bhigh AML has significantly higher expression of leukemic stem cell signature (LSC) genes CD34, RBPMS, GUCY1A3, KIAA0125, SOCS2, SPINK2, HTR1F, PPP1R16B, EVI1 (MECOM), DAPK1, BAALC, ABCB1, and PRKCH. Furthermore, INPP4B was found to be co-expressed with anti-apoptotic genes of the BCL2 family, namely BCL2 and BCL2L1. Further analysis revealed that INPP4B was co-expressed with the transcription factors EVI1, GATA2, NFATC2, ZEB1, GATA3 and ETS1, all of which having predicted binding sites within the INPP4B promoter region. Due to the observed enrichment of hematopoietic lineage/LSC genes in INPP4Bhigh AML, we wanted to validate the potential role of the EVI1 transcription factor in regulating INPP4B expression. Chromatin immunoprecipitation was used to demonstrate that EVI1 binding was enriched in the INPP4B promoter region of both EVI1high OCI/AML-4 and OCI/AML-6 cell lines. In addition, retroviral overexpression of EVI1 in EVI1low U937 cells resulted in subsequent upregulation of INPP4B transcript levels. Moreover, shRNA mediated knockdown of EVI1 in EVI1highOCI/AML-4 and UCSD-1 cells resulted in downregulation of INPP4B expression. Overall, our analysis reveals that INPP4Bhigh AML is characterized by upregulation of genes related to the hemotopoietic lineage, and LSC signature - consistent with the in vitro colony formation phenotype seen in INPP4B overexpressing AML cell lines. Furthermore, we demonstrate that one of the hematopoietic stem cell genes, EVI1 is a potentially key regulator of INPP4B expression in AML. Disclosures Jain: Roche Canada: Research Funding.

scholarly journals Polycomb Group Protein YY1 Is an Essential Regulator of Hematopoietic Stem Cell Quiescence

Cell Reports ◽  
2018 ◽  
Vol 22 (6) ◽  
pp. 1545-1559 ◽  
Author(s):  
Zhanping Lu ◽  
Courtney C. Hong ◽  
Guangyao Kong ◽  
Anna L.F.V. Assumpção ◽  
Irene M. Ong ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Polycomb Group ◽  
Polycomb Group Protein ◽  
Hematopoietic Stem ◽  
Stem Cell Quiescence ◽  
Cell Quiescence ◽  
Group Protein

scholarly journals The Polycomb group gene Ezh2 prevents hematopoietic stem cell exhaustion

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 2170-2179 ◽  
Author(s):  
Leonie M. Kamminga ◽  
Leonid V. Bystrykh ◽  
Aletta de Boer ◽  
Sita Houwer ◽  
José Douma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Chromatin Modification ◽  
Cellular Aging ◽  
Polycomb Group ◽  
Polycomb Group Protein ◽  
Hematopoietic Stem ◽  
Replicative Stress ◽  
Senescence Program

The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2), a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly, whereas normal HSCs were rapidly exhausted after serial transplantations, overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast, similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a “genetic genomics” screen, we identified novel putative Ezh2 target or partner stem cell genes that are associated with chromatin modification. Our data suggest that stabilization of the chromatin structure preserves HSC potential after replicative stress.

scholarly journals RSK Inhibition Suppresses AML Proliferation through Activation of DNA Damage Pathways and S Phase Arrest

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2894-2894 ◽  
Author(s):  
Ritika Dutta ◽  
Maria Castellanos ◽  
Bruce Tiu ◽  
Hee-Don Chae ◽  
Kara L. Davis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Cell Growth ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
S Phase ◽  
Hematopoietic Stem ◽  
Phase Arrest ◽  
S Phase Arrest

Abstract The 90 kDa Ribosomal S6 Kinase (RSK), downstream of the ERK signaling pathway, has recently been implicated in a wide variety of cancers, ranging from lung cancer to medulloblastoma, as a driver of cancer cell proliferation and survival. However, its role in Acute Myeloid Leukemia (AML) remains unknown. Thus, the goal of this study was to characterize RSK-dependent signaling pathways in AML, with the overall hypothesis that disruption of this pathway represents a potential strategy for the treatment of AML. The RSK family consists of four gene isoforms, RSK1-4 (RPS6KA1 (RSK1), RPS6KA2 (RSK3), RPS6KA3 (RSK2), RPS6KA4 (RSK4). Knockdown (KD) of RSK1 by shRNA in HL-60 and KG-1 cell lines resulted in reduced AML cell growth in vitro. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were injected with 2x106 HL-60 or KG-1 RSK1KD cells and vector control transduced cells in order to investigate the effects of RSK1 KD on AML cell growth and survival in vivo. Mice injected with RSK1 KD cells exhibited prolonged survival by 17 and 21 days respectively for HL-60 and KG-1 cell induced disease (p=0.0023 and 0.0018 respectively). These data indicate that RSK1 knockdown inhibits leukemia progression, and RSK1 is required for maximal proliferation of AML cells in vivo. Pharmacological inhibition of total RSK (RSK1-4) by the small molecule inhibitor BI-D1870 reduced AML cell growth and induced cell death in both AML cell lines and patient samples after treatment for 48 hours. The IC50 for growth inhibition was 1.8 uM for MOLM-13, 1.6 uM for MV-4-11, and 1.9 uM for HL-60 cells. In methylcellulose colony assays, normal hematopoietic stem and progenitor cell proliferation was not affected by RSK inhibition up to a concentration of 15 uM, establishing an approximately 10-fold therapeutic index. To elucidate the mechanism by which RSK inhibition suppresses AML proliferation, we performed cell cycle analysis with HL-60 cells. RSK inhibition by BI-D1870 resulted in delayed S-phase progression and accumulation of cells in late S-phase with increased pH2AX, cPARP, and CDK2/Cyclin A expression, as measured by flow cytometry. These data indicate that inhibition of RSK leads to activation of DNA damage pathways and arrest in S-phase, resulting in apoptosis. Inhibition of CDK activity rescued S-phase arrest, demonstrating that activation and dysregulation of CDK are crucial mediators of RSK inhibitor-induced S-phase arrest. In summary, this is the first study to demonstrate that RSK plays an important role in maintaining AML cell survival and proliferation and to position RSK as a promising target for treatment of AML. Disclosures No relevant conflicts of interest to declare.

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