Polycomb Group Ring Finger 5 (PCGF5) Is a Notch Transcriptional Target and Regulates Cell Size and Cell Cycle in Hematopoietic Progenitors.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1325-1325
Author(s):  
Chris L Cochrane ◽  
Hind Medyouf ◽  
Andrew P. Weng

Abstract The highly conserved Notch gene is activated by mutation in more than half of human T cell acute lymphoblastic leukemia (T-ALL) cases. The Notch protein is a transmembrane receptor which, upon binding of its ligand, is cleaved in a series of proteolytic steps releasing the intracellular portion (ICN) to translocate to the nucleus where it acts as a transcriptional activator for target genes such as HES1, Deltex, preTalpha, and c-Myc. To better understand the mechanism by which Notch causes leukemogenesis, microarray gene expression profiling experiments where conducted on five human Notch signaling-dependent T-ALL cell lines which where either mock-treated or treated with a gamma-secretase inhibitor (GSI) to prevent the release of Notch from the membrane. The Polycomb Group gene PCGF5 was identified as one of the genes most strongly downregulated upon Notch inhibition. This regulation was subsequently confirmed by quantitative RT-PCR in both human and mouse leukemia cell lines. Our interest in this gene was encouraged by its homology to the well-known oncogene Bmi-1, which acts to modify chromatin and silence expression of several genes including the cyclin-dependent kinase inhibitors p16 and p19ARF within the CDKN2a locus. Interestingly, we found that inhibition of Notch signaling by GSI treatment in both human and mouse leukemia cells resulted in an increase of both p16 and p19ARF at the mRNA and protein levels. This suggested that Notch may be responsible for maintaining expression of a transcriptional repressor that suppresses p16 and p19ARF. We hypothesize that PCGF5 may be acting in a manner analogous to Bmi-1 in this cellular context and thus mediating p16/p19ARF repression. Studies to test this hypothesis are currently in progress. To further investigate the role of PCGF5 in hematopoiesis, mouse bone marrow progenitors were transduced with retrovirus to express PCGF5 constitutively and transplanted into lethally irradiated recipients. Our results show long term reconstitution by PCGF5-expressing cells with as yet no evidence of PCGF5-induced hematopoeitic malignancy in a small cohort up to 6 months post-transplant. However, we did observe cells expressing high levels of PCGF5 to be skewed toward myeloid lineages, while mid-level expressing cells develop efficiently into lymphocytes. We detected no defects in B cell maturation; however, PCGF5-expressing T cell numbers were significantly lower than controls in the peripheral blood and spleen of recipient animals. Consistent with this observation, fetal thymic organ culture of PCGF5-transduced fetal liver hematopoietic progenitors showed accumulation in the early double negative thymocyte stages. Additionally, we found PCGF5-expressing B and T lymphocytes to be larger than control cells, and preliminary data suggests these cells may be arrested in G2/M phase of the cell cycle. Biochemical studies are also in progress to assess participation of PCGF5 in the Polycomb Repressive Complex PRC-1 and its effect on chromatin structure. In sum, these preliminary data suggest enforced PCGF5 expression, though not oncogenic, alters normal lymphoid/myeloid fate selection by hematopoietic progenitors and may affect lymphoid cell size by altering cell cycle progression.

Blood ◽  
2009 ◽  
Vol 113 (8) ◽  
pp. 1689-1698 ◽  
Author(s):  
Ila Joshi ◽  
Lisa M. Minter ◽  
Janice Telfer ◽  
Renée M. Demarest ◽  
Anthony J. Capobianco ◽  
...  

Abstract Notch signaling plays a role in normal lymphocyte development and function. Activating Notch1-mutations, leading to aberrant downstream signaling, have been identified in human T-cell acute lymphoblastic leukemia (T-ALL). While this highlights the contribution of Notch signaling to T-ALL pathogenesis, the mechanisms by which Notch regulates proliferation and survival in normal and leukemic T cells are not fully understood. Our findings identify a role for Notch signaling in G1-S progression of cell cycle in T cells. Here we show that expression of the G1 proteins, cyclin D3, CDK4, and CDK6, is Notch-dependent both in vitro and in vivo, and we outline a possible mechanism for the regulated expression of cyclin D3 in activated T cells via CSL (CBF-1, mammals; suppressor of hairless, Drosophila melanogaster; Lag-1, Caenorhabditis elegans), as well as a noncanonical Notch signaling pathway. While cyclin D3 expression contributes to cell-cycle progression in Notch-dependent human T-ALL cell lines, ectopic expression of CDK4 or CDK6 together with cyclin D3 shows partial rescue from γ-secretase inhibitor (GSI)-induced G1 arrest in these cell lines. Importantly, cyclin D3 and CDK4 are highly overexpressed in Notch-dependent T-cell lymphomas, justifying the combined use of cell-cycle inhibitors and GSI in treating human T-cell malignancies.


Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1419-1419 ◽  
Author(s):  
Robert M. Sutphin ◽  
Wendy Fang ◽  
Claudia Miller ◽  
Patrick A. Zweidler-McKay

Abstract Introduction: The Notch pathway regulates critical cell-fate decisions affecting the growth and development of human hematopoietic cells. Although Notch1 is a known T cell oncogene, we have discovered that Notch signaling behaves as a tumor suppressor in acute myeloblastic leukemia (AML) inducing growth arrest and apoptosis in both cell lines and patient samples. To characterize the mechanism of this effect we have evaluated the influence of the Notch pathway on key effectors of differentiation, cell cycle, and apoptosis in human AML. Results: Notch signaling induces rapid growth arrest and apoptosis in a panel of human AML cell lines representing a range of AML FAB subtypes (M2–M6). Specifically, activated Notch1 expression caused a 70–95% reduction in AML cells compared to controls (p<0.001) (Figure 1). Notch-mediated growth arrest occurred in 24–48 hours with cells accumulating in G0/G1. Apoptosis was demonstrated by a 3.8-fold increase in AnnexinV binding (p<0.004) and a 3-fold upregulation of caspase 3 activity (p=0.0002) within 24 hours. The caspase 3 activity was abolished by the caspase 8 inhibitor IETD (p<0.0001) suggesting a potential role for the extrinsic death pathway. We also found that all four Notch receptors (1–4) are capable of inducing this effect, as is the Notch target gene HES1, suggesting a generalized Notch tumor suppressor effect in AML. Furthermore, Notch signaling through HES1 modulates the expression of key regulators of myeloid differentiation and cell cycle progression including downregulation of CEBPα 2.5-fold (p<0.02) and upregulation of p21WAF1 6-fold (p<0.004) suggesting potential mechanisms. As a novel therapeutic approach, we synthesized Notch agonists which effectively induce Notch signaling with a >18-fold increase in HES1 expression (p<.0001). Exposure of human AML cell lines and primary patient AML samples to this Notch agonist for 24 hours led to a 3 to 9-fold increase in apoptosis (p<0.017) compared to controls (Figure 2). Conclusions: We report here that Notch signaling is a novel tumor suppressor pathway in human AML. We demonstrate how Notch agonists can be used to induce growth arrest and apoptosis in human AML cell lines and patient AML samples. As a regulator of cell fate, proliferation and differentiation, Notch effectively disrupts multiple pathways in AML. We propose that Notch agonists represent a novel and feasible therapeutic approach in AML. Pre-clinical evaluation is underway. Figure.1 Effect of Notch on growth of AML cells Figure.1. Effect of Notch on growth of AML cells Figure.2 Notch Agonist induces apoptosis in AML Figure.2. Notch Agonist induces apoptosis in AML


Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2525-2525
Author(s):  
Tetsuro Nakazato ◽  
Chie Ishikawa ◽  
Taeko Okudaira ◽  
Mariko Tomita ◽  
Naoki Mori

Abstract Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type I (HTLV-I) and remains incurable. Retinoid is a collective term for compounds, which bind to and activate retinoic acid receptors (RARα, β, γ and RXRα, β, γ), members of nuclear hormone receptor superfamily. It is involved in cell differentiation, morphogenesis, proliferation, and anti-neoplastic processes. The most important endogenous retinoid is all-trans-retinoic acid (ATRA), which is an RARα, β, and γ ligand. ATRA and its mimics have been in clinical use for treatment of acute promyelocytic leukemia (APL) and adult T-cell leukemia (ATL). Many synthetic retinoids have been developed and attempts to improve their medicinal properties have been made. Among them, a novel synthetic retinoid, Am80 (Tamibarotene) is an RARα- and RARβ-specific (but RARγ- and RXRs-nonbinding) synthetic retinoid that is expected to overcome ATRA resistance, because of several times more potent differentiation activity than ATRA and sustained plasma level during continuous administration due to a lower affinity for cellular retinoic acid binding protein. On this background, we examined the inhibitory effect of Am80 on HTLV-I-infected T-cell lines and primary ATL cells. Am80 showed little growth inhibition of peripheral blood mononuclear cells, but it markedly inhibited the growth of both HTLV-I-infected T-cell lines and primary ATL cells. Am 80 could arrest cells in the G1 phase of the cell cycle and induced apoptosis in HTLV-I-infected T-cell lines. The NF-κB pathway is critical for the immortalization and survival of HTLV-I-infected T cells. Therefore, NF-κB pathway was examined as potential targets of Am80 signaling. Am80 significantly inhibited phosphorylation of IκBα and NF-κB-DNA binding, in conjunction with the reduction of expression of proteins involved in the G1-S cell cycle transition and apoptosis. Furthermore, in animal studies, treatment with Am80 produced partial inhibition of growth of tumors of an HTLV-I-infected T-cell line transplanted subcutaneously in severe combined immunodeficient mice. These findings clearly demonstrate that Am80 is a potential inhibitor of NF-κB in ATL cells, and might be a useful therapeutic agent against ATL.


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2999-2999 ◽  
Author(s):  
Samantha Pozzi ◽  
Diana Cirstea ◽  
Loredana Santo ◽  
Doris M Nabikejje ◽  
Kishan Patel ◽  
...  

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4630-4630
Author(s):  
Samuel D Gusscott ◽  
Florian Kuchenbauer ◽  
Andrew P Weng

Abstract Abstract 4630 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of the Notch1 signaling pathway. Several studies have utilized mRNA expression profiling to identify downstream mediators of oncogenic Notch signaling in this context. Since microRNAs (miRNAs) have in recent years been shown to play important roles in hematological maliganancy, we performed a microarray-based screen for Notch-dependent miRNA expression in T-ALL. Jurkat and P12-Ichikawa cell lines were treated with gamma-secretase inhibitor to block Notch signaling vs. DMSO control for 4 days and profiled using Exigon miRCURY LNA miRNA microarrays. Surprisingly few miRNAs were found to be regulated by this approach; however, one of the hits, miR-223, showed consistent upregulation after gamma-secretase treatment in Jurkat cells and 5 additional human T-ALL cell lines assessed by miRNA qPCR. This observation was unique to human T-ALL as murine models of T-ALL showed no evidence for Notch-dependent miR-223 expression. Given that canonical Notch signaling results in transcriptional activation, our observation that Notch signaling is associated with reduced miR-223 expression suggests an intermediary repressor may be involved. miR-223 has been reported to play an important role in normal granulopoiesis, to be expressed relatively highly in T-ALL with myeloid-like gene features, and most recently to accelerate Notch-mediated T-cell leukemogenesis. To explore potential functional consequences for Notch-dependent miR-223 repression in T-ALL, candidate miR-223 targets identified by TargetScan software were analyzed with Ingenuity Pathway Analysis software, which indicated IGF-1, insulin receptor, PTEN, and ERK5 signaling pathways as the top hits. We recently reported IGF1R signaling to be important for growth and viability of bulk T-ALL cells as well as for leukemia-initiating cell activity. Additionally, we reported that Notch signaling directly upregulates IGF1R transcription by binding to an intronic enhancer which is present between exons 21/22 in the human, but not mouse IGF1R locus. As miR-223 has previously been reported to target IGF1R mRNA and reduce its translation, we hypothesized that Notch signaling may also upregulate net IGF1R protein expression by repressing miR-223. To test this hypothesis, we transduced several human T-ALL cell lines with miR-223 retrovirus and observed a modest decrease in total IGF1R protein levels by western blot; however, no significant change was observed in surface IGF1R levels as assessed by flow cytometry. Addtionally, knockdown of miR-223 by lentiviral expression miR-223 target sequences (miR-223 “sponge”) resulted in modestly increased total IGF1R protein levels, but again showed no demonstrable effect on surface IGF1R levels. Of note, we also observed no apparent effect of either overexpression or knockdown of miR-223 on bulk cell growth or viability. We interpret these findings to suggest that Notch signaling does not have major effects on the miR transcriptome, and that up- or down-modulation of miR-223 in established T-ALL cells does not have significant effects on overall cell growth/viability. Further studies will be required to determine if miR-223 may act in concert with other Notch target genes to modulate cell physiology. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4872-4872 ◽  
Author(s):  
Michela Boi ◽  
Paola Bonetti ◽  
Maurilio Ponzoni ◽  
Maria Grazia Tibiletti ◽  
Anastasios Stathis ◽  
...  

Abstract Abstract 4872 Background: ALCL, is clinically/biologically heterogeneous disease, including ALK+ and ALK- systemic forms. Despite the progresses in understanding the molecular pathogenesis of ALCL, the therapy is still based on chemotherapy, thus the identification of new treatment modalities is needed. Bromodomain-containing proteins are components of transcription factors complexes and determinants of epigenetic memory. Inhibitors of BRD2/3/4, members of the Bromodomain and Extraterminal (BET) family, have recently shown antitumor activity in different hematological malignancies models. Here, we report anti-proliferative activity of OTX015, a novel selective orally bioavailable BRD2/3/4 inhibitor, in a panel of ALCL cell lines. Material and Methods: Eight established human cell lines derived from ALK+ and ALK- anaplastic large cell lymphoma (ALCL) were treated with increasing doses of OTX015 (OncoEthix SA) and MTT assays were performed after 72h exposure. For cell cycle analysis, cells were treated and stained with Click-iT Edu Flow Cytometry Assay Kits (Invitrogen) and 7-AAD and analyzed for DNA content using a FACScan flow cytometer. Results were analyzed with FlowJo 7.6.3 software. RNA was extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit according to the manufacturer's instructions. RT-PCR was performed on using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For senescence detection, cells were stained using a b-Galactosidase Staining Kit (Calbiochem). Results: We assessed OTX-015 anti-proliferative activity in eight ALCL cell lines. The majority (5/8) of the cell lines were sensitive, with IC50 between 36 and 546 nM. There was no apparent difference between ALK+(6) and ALK- (2) cell lines. Cell cycle analyses revealed G1 arrest and a concomitant decrease of the S phase after 24h OTX015 exposure in 4/4 ALCL cell lines, without an increase in cell death, suggesting a cytostatic effect of OTX015. An increase in the percentage of senescent cells after treatment with the BRD-inhibitor was observed in the most sensitive ALK+ALCL cell line. To understand the mechanism of action of OTX015, we assessed MYC mRNA levels before and after treatment. We observed that OTX015 suppressed the transcription of MYCgene and some of its downstream target genes (such as NCL and CAD) in 4/4 ALCL cell lines, with less efficacy in the most resistant one. Conclusion: OTX015 is a new potent BRD-inhibitor with evident anti-proliferative activity in several ALCL cell lines. The down-regulation of MYC gene, followed by cell cycle G1 arrest and increase of cellular senescence, was observed after OTX015 treatment, appearing one of the possible mechanisms of action of the compound. The compound appears worth of further investigation as a new promising therapeutic agent in ALCL and in other mature T-cell tumors. Disclosures: Bonetti: OncoEthix SA: Research Funding. Cvitkovic:OncoEthix SA: Membership on an entity's Board of Directors or advisory committees. Inghirami:OncoEthix SA: Research Funding. Bertoni:OncoEthix SA: Research Funding.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 845-845
Author(s):  
Noriaki Yoshida ◽  
Kennosuke Karube ◽  
Atae Utsunomiya ◽  
Kunihiro Tsukasaki ◽  
Yoshitaka Imaizumi ◽  
...  

Abstract Introduction Adult T-cell leukemia/lymphoma (ATL) is a human T-cell leukemia virus type-1-induced neoplasm with four clinical subtypes; acute, lymphoma, chronic and smoldering. Although chronic and smoldering subtypes are regarded as indolent ATL, about half of these cases progress to acute type ATL and subsequent death. Therefore, cases of indolent ATL also have poor prognosis and acute transformation is a predictive indicator for patients with indolent ATL. However, the molecular pathogenesis of acute transformation remains unknown. In the present study, oligo-array comparative genomic hybridization (CGH) and comprehensive gene-expression profiling (GEP) were applied to 27 and 35 cases of chronic and acute type ATL, respectively, in an effort to delineate the molecular pathogeneses of ATL, and especially the molecular mechanism of acute transformation. Materials and Methods All DNA and RNA used in this study were extracted from purified CD4-positive cells. Oligo-array CGH analyses and comprehensive GEP analyses were performed on 27 and 35 cases of chronic and acute type ATL, respectively. Subsequently, we established Tet-OFF ATL cell lines for functional analyses. Results Oligo-array CGH revealed that genomic loss of 9p21.3 was significantly characteristic of acute type ATL, but not chronic type ATL (p-value= 0.039). Although the minimal common deleted region of 9p21.3 contained MTAP, CDKN2A and CDKN2B, the expression level of only CDKN2A was reduced with genomic loss of 9p21.3 (Figure 1). Moreover, analysis of serial samples of a chronic type ATL patient showing acute transformation also revealed that reduction of CDKN2A expression by 9p21.3 loss was associated with acute transformation in this case. CDKN2A contains two known variants, INK4a and ARF. Re-expression of INK4a and ARF suppressed proliferation of Tet-OFF ATL cell lines, while the suppression efficiency of INK4a was stronger than that of ARF (Figure 2). In cell-cycle assays, the induction of INK4a and ARF decreased the proportion of S-phase cells. Additionally, re-expression of INK4a also increased the amount of apoptotic cells in induced cell lines, while re-expression of ARF did not have this effect. Since CDKN2A is a well-known cell cycle regulator, deregulation of the cell-cycle might be involved in acute transformation of chronic type ATL. In fact, deregulation of the cell-cycle pathway has been reported as a predictive indicator for the outcome in diffuse large B-cell lymphoma patients (Cancer Cell, 22:359-372). Therefore, we examined whether chronic ATL patients had alterations in cell-cycle related genes and found that chronic ATL patients could be divided into two groups. The group possessing alterations in these genes (referred to as “Cell cycle Alteration”) showed poorer prognosis compared with the group lacking such alterations (referred to as “Clean”) (p-value= 0.037) (Figure 3). Additionally, patients with such alterations tended to have earlier progression to acute type ATL. Conclusion These findings indicated that cell cycle-related genes play an important role in acute transformation and should serve as good prognostic markers for chronic type ATL. Disclosures: No relevant conflicts of interest to declare.


Author(s):  
Andrea Ghelli Luserna Di Rorà ◽  
Martina Ghetti ◽  
Lorenzo Ledda ◽  
Anna Ferrari ◽  
Matteo Bocconcelli ◽  
...  

AbstractDoxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B−/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B−/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. Graphical abstract • Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. • ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. • The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. • The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.


Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-35
Author(s):  
Anna Luiza Facchetti Vinhaes Assumpcao ◽  
Guoping Fu ◽  
Zhanping Lu ◽  
Ashley Kuehnl ◽  
Renren Wen ◽  
...  

T cell development originates from hematopoietic stem and progenitor cells in the bone marrow, which migrate to the thymus and obtain T cell identification. Transcription factors play critical roles in regulating early T cell development. While Notch signals are critically required at the early stage of T cell development, the completion of T cell lineage commitment is far from the initial response to Notch signaling. Other transcription factors such as PU.1, Ikaros, and RUNX1 are required to enable progenitor cells to committee T cell lineage before Notch signaling. YY1 is a ubiquitous transcription factor and mammalian Polycomb Group Protein (PcG) with important functions to regulate lymphocytes development, stem cell self-renewal, cell proliferation, and survival. Previous study showed that YY1 can interact with the Notch1 receptor intracellular domain and regulate Notch1 transactivation activities in vitro. Thus, YY1 may also belong to the core T cell lineage regulatory factors and is required for progenitor cell commitment to T cell development. To test how loss-of-function of YY1 impacts early T cell development, we utilized a conditional Yy1 knockout allele Yy1f/f with loxP sites flanking the Yy1 promoter region and exon 1. Yy1f/fmice were crossed to the inducible Mx1-Cre. In Yy1f/fMx1-Cre mice, YY1 deletion was achieved after treatment with the pI-pC. Yy1-/- mice had significantly reduced numbers of lymphoid-primed multipotent progenitor, (LMPP), common lymphoid progenitor (CLP), and double-negative (DN) T cells compared to Yy1+/+ mice. YY1 deficiency resulted in an early T cell developmental blockage at the DN1 stage. In addition, Notch1 mRNA and protein expressions were significantly reduced in Yy1-/- thymocytes compared to Yy1+/+ thymocytes. In Yy1-/- thymocytes, Notch target gene Hes1 was also downregulated. Thus, YY1 is required for early T cell development and Notch1 signaling. YY1 mediates stable PcG-dependent transcriptional repression via recruitment of PcG proteins that catalyze histone modifications. Our previous results demonstrated that YY1 PcG function is required for Igκ chain rearrangement in early B cell development, however, it is not required for YY1 functions in promoting HSC self-renewal and maintaining HSC quiescence. Many questions remain unanswered regarding how cell- and tissue-specificity is achieved by PcG proteins. Herein, we utilized a YY1 REPO domain mutant (YY1ΔREPO). The small 25 amino acid REPO domain is necessary and sufficient for recruiting other PcG proteins to YY1-bound chromatin sites in Drosophila. While YY1ΔREPO is competent for DNA binding, transcriptional activation, transient transcriptional repression, and interaction with transcriptional coregulators such as HDACs, it is defective in all YY1 PcG functions and unable to recruit other PcG proteins to DNA. This mutant is therefore a powerful tool for dissecting mechanisms governing YY1 PcG-dependent versus -independent functions. Bone marrow cells from Yy1f/f Mx1-Cre mice were transduced retrovirally with MigR1-FlagYY1, MigR1-FlagYY1ΔREPO or MigR1 vector and transplanted into lethally irradiated CD45.1+ mice. In addition, Mx1-Cre bone marrow cells infected with MigR1 vector were used as the wild-type control and transplanted into CD45.1+ mice. While YY1 is required for DN1 to DN2 transition, YY1 PcG function/REPO domain is not required for DN1 transition. Instead, in mice lack of YY1 PcG function/REPO domain, early T cells had increased cell apoptosis and failed to survive. Interestingly, although YY1 PcG function/REPO domain is critical for early T cell survival, it is not required for YY1 regulation of Notch1 expression. We concluded that YY1 is a critical regulator for early T cell development and Notch signaling. There is a lineage-specific requirement for the YY1 PcG function/REPO domain for early T cell development. While YY1 PcG function is required for early T cell survival, it is not required for YY1 regulation of Notch1 expression. YY1 PcG and non-PcG functions promotes T cell development by unique mechanisms of promoting cell survival and Notch1 expression respectively. Disclosures No relevant conflicts of interest to declare.


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