Growth factor independence 1 (Gfi1) as a new target for leukemia therapy
ABSTRACT: Using (conditional) Gfi1 knock-out mice we show that ablation of the transcriptional repressor Gfi1 cures mice from lymphoid leukemia and reduces the expansion of primary human T-ALL xenografts in mice. We find that Gfi1 alters the p53 dependent transcriptional activation of a substantial subset of known p53 target genes and thus sets a threshold for cell death. We used Affymetrix mouse Gene-1.0-ST arrays to define the changes in the gene expression pattern of wt or Gfi1-KO thymocytes (Gfi1-fl/fl X MX-CRE, induced by pIpC) that were either untreated (WT, GfiKO thymocytes), irradiated (WT_irr, Gfi1KO_irr), ENU transformed (WT_Tum, Gfi1KO_Tum), or transformed by Notch1-CT and ENU-induced to enhance tumorigenesis (WT_Notch_Tum, Gfi1KO_Notch_Tum). Overall design: The study should determine how loss of Gfi1 alters the gene expression pattern in irradiated or tumor derived thymocytes
INSTRUMENT(S): [MoGene-1_0-st] Affymetrix Mouse Gene 1.0 ST Array [probe set (exon) version]
Project description:Using (conditional) Gfi1 knock-out mice we show that ablation of the transcriptional repressor Gfi1 cures mice from lymphoid leukemia and reduces the expansion of primary human T-ALL xenografts in mice. We find that Gfi1 alters the p53 dependent transcriptional activation of a substantial subset of known p53 target genes and thus sets a threshold for cell death. We used Affymetrix mouse Gene-1.0-ST arrays to define the changes in the gene expression pattern of wt or Gfi1-KO thymocytes (Gfi1-fl/fl X MX-CRE, induced by pIpC) that were either untreated (WT, GfiKO thymocytes), irradiated (WT_irr, Gfi1KO_irr), ENU transformed (WT_Tum, Gfi1KO_Tum), or transformed by Notch1-CT and ENU-induced to enhance tumorigenesis (WT_Notch_Tum, Gfi1KO_Notch_Tum). The study should determine how loss of Gfi1 alters the gene expression pattern in irradiated or tumor derived thymocytes
Project description:Most patients with acute lymphoblastic leukemia (ALL) fail current treatments highlighting the need for better therapies. Because oncogenic signaling activates a p53-dependent DNA damage response and apoptosis, leukemic cells must devise appropriate countermeasures. We show here that growth factor independence 1 (Gfi1) can serve such a function because Gfi1 ablation exacerbates p53 responses and lowers the threshold for p53-induced cell death. Specifically, Gfi1 restricts p53 activity and expression of proapoptotic p53 targets such as Bax, Noxa (Pmaip1), and Puma (Bbc3). Subsequently, Gfi1 ablation cures mice from leukemia and limits the expansion of primary human T-ALL xenografts in mice. This suggests that targeting Gfi1 could improve the prognosis of patients with T-ALL or other lymphoid leukemias.
Project description:Using Gfi1b conditional mice, deletion of gfi1b in the hematopietic system was induced by injecting MxCre tg Gfi1bfl/fl mice with pIpC. 30 days after injection, Cd150 pos, Cd 48 neg, Lin neg Sca and c-kit pos stem cells were sortrted from Gfi1bfl/fl and Mxcre tg Gfi1bfl/fl mice and analysed. We used the mouse Affymetrix Gene ST Array. The study should determine whether loss of Gfi1 alters the gene expression pattern in the hematopietic stem cells.
Project description:Using Gfi1b conditional mice, deletion of gfi1b in the hematopietic system was induced by injecting MxCre tg Gfi1bfl/fl mice with pIpC. 30 days after injection, Cd150 pos, Cd 48 neg, Lin neg Sca and c-kit pos stem cells were sortrted from Gfi1bfl/fl and Mxcre tg Gfi1bfl/fl mice and analysed. We used the mouse Affymetrix Gene ST Array. Overall design: The study should determine whether loss of Gfi1 alters the gene expression pattern in the hematopietic stem cells.
Project description:GFI1 is a transcriptional repressor that plays a critical role in hematopoiesis and has also been implicated in lymphomagenesis. It is still poorly understood how GFI1 expression is regulated in the hematopoietic system. We show here that GFI1 transcription was repressed by the tumor suppressor p53 in hematopoietic cells. Knockdown of p53 resulted in increased GFI1 expression and abolished DNA damage-induced GFI1 downregulation. In contrast, GFI1 expression was reduced and its downregulation in response to DNA damage was rescued upon restoration of p53 function in p53-deficient cells. In luciferase reporter assays, wild type p53, but not a DNA binding-defective p53 mutant, repressed the GFI1 promoter. Chromatin immunoprecipitation (ChIP) assays demonstrated that p53 bound to the proximal region of the GFI1 promoter. Detailed mapping of the GFI1 promoter indicated that GFI1 core promoter region spanning from -33 to +6 bp is sufficient for p53-mediated repression. This core promoter region contains a putative p53 repressive response element, mutation of which abolished p53 binding to and repression of GFI1 promoter. Significantly, apoptosis induced by DNA damage was inhibited upon Gfi1 overexpression, but augmented following GFI1 knockdown. Our data establish for the first time that GFI1 is repressed by p53 and add to our understanding of the roles of GFI1 in normal hematopoiesis and lymphomagenesis.
Project description:Here we demonstrate a mode of reciprocal regulation between GFI1 and p53 that controls the induction of apoptosis in T cells. We show that GFI1 prevents induction of p53 dependent apoptosis by recruiting LSD1 to p53, which leads to the demethylation of its C-terminal domain. This is accompanied by a decrease of the acetylation of lysine 117 within the core domain of the murine p53 protein, which is required for transcriptional induction of apoptosis. Our results support a model in which the effect of GFI1's regulation of methylation at the c-terminus of p53 is ultimately mediated through control of acetylation at lysine 117 of p53. We propose that GFI1 acts prior to the occurrence of DNA damage to affect the post-translational modification state and limit the subsequent activation of p53. Once activated, p53 then transcriptionally activates GFI1, presumably in order to re-establish the homeostatic balance of p53 activity. These findings have implications for the activity level of p53 in various disease contexts where levels of GFI1 are either increased or decreased.
Project description:BACKGROUND:In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable malignancy due to the emergence of drug-resistant clones. We previously showed that MM cells upregulate the transcriptional repressor, growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSCs) that induces prolonged inhibition of osteoblast differentiation. However, the role of Gfi1 in MM cells is unknown. METHODS:Human primary CD138+ and BMSC were purified from normal donors and MM patients' bone marrow aspirates. Gfi1 knockdown and overexpressing cells were generated by lentiviral-mediated shRNA. Proliferation/apoptosis studies were done by flow cytometry, and protein levels were determined by Western blot and/or immunohistochemistry. An experimental MM mouse model was generated to investigate the effects of MM cells overexpressing Gfi1 on tumor burden and osteolysis in vivo. RESULTS:We found that Gfi1 expression is increased in patient's MM cells and MM cell lines and was further increased by co-culture with BMSC, IL-6, and sphingosine-1-phosphate. Modulation of Gfi1 in MM cells had major effects on their survival and growth. Knockdown of Gfi1 induced apoptosis in p53-wt, p53-mutant, and p53-deficient MM cells, while Gfi1 overexpression enhanced MM cell growth and protected MM cells from bortezomib-induced cell death. Gfi1 enhanced cell survival of p53-wt MM cells by binding to p53, thereby blocking binding to the promoters of the pro-apoptotic BAX and NOXA genes. Further, Gfi1-p53 binding could be blocked by HDAC inhibitors. Importantly, inoculation of MM cells overexpressing Gfi1 in mice induced increased bone destruction, increased osteoclast number and size, and enhanced tumor growth. CONCLUSIONS:These results support that Gfi1 plays a key role in MM tumor growth, survival, and bone destruction and contributes to bortezomib resistance, suggesting that Gfi1 may be a novel therapeutic target for MM.
Project description:Double-positive (DP) thymocytes respond to intrathymic TCR signals by undergoing positive selection and lineage differentiation into single-positive (SP) mature cells. Concomitant with these wellcharacterized events is the acquisition of a mature T cell gene expression program characterized by the induction of effector molecules IL-7Ra, S1P1 and CCR7, but the underlying mechanism remains elusive. We report here that transcription repressor Gfi1 orchestrates the fidelity of DP gene expression program and developmental maturation into SP cells. Loss of Gfi1 resulted in premature induction of effector genes and transcription factors Foxo1 and Klf2 in DP thymocytes, and accumulation of post-selection intermediate populations and accelerated transition into SP cells. Strikingly, partial loss of Foxo1 function, but not restored survival fitness, rectified the dysregulated gene expression and thymocyte maturation in Gfi1-deficient mice. Our results establish Gfi1-Foxo1 axis and the transcriptional circuitry that actively maintain DP identity and shape the proper generation of mature T cells Overall design: We used microarrays to compare the global transcription profiles of CD4+CD8+ double-positive (DP) thymocytes isolated from wild-type mice, and thymocytes from hCD2-iCre x Gfi1fl/fl mice
Project description:Interleukin-7 receptor ? (IL-7R?) is essential for T cell survival and differentiation. Glucocorticoids are potent enhancers of IL-7R? expression with diverse roles in T cell biology. Here we identify the transcriptional repressor, growth factor independent-1 (Gfi1), as a novel intermediary in glucocorticoid-induced IL-7R? up-regulation. We found Gfi1 to be a major inhibitory target of dexamethasone by microarray expression profiling of 3B4.15 T-hybridoma cells. Concordantly, retroviral transduction of Gfi1 significantly blunted IL-7R? up-regulation by dexamethasone. To further assess the role of Gfi1 in vivo, we generated bacterial artificial chromosome (BAC) transgenic mice, in which a modified Il7r locus expresses GFP to report Il7r gene transcription. By introducing this BAC reporter transgene into either Gfi1-deficient or Gfi1-transgenic mice, we document in vivo that IL-7R? transcription is up-regulated in the absence of Gfi1 and down-regulated when Gfi1 is overexpressed. Strikingly, the in vivo regulatory role of Gfi1 was specific for CD8(+), and not CD4(+) T cells or immature thymocytes. These results identify Gfi1 as a specific transcriptional repressor of the Il7r gene in CD8 T lymphocytes in vivo.
Project description:To develop a mouse model of cardiac injury after partial heart irradiation (PHI) and to test whether dual energy (DE)-microCT and 4-dimensional (4D)-microCT can be used to assess cardiac injury after PHI to complement myocardial perfusion imaging using micro-single photon emission computed tomography (SPECT).To study cardiac injury from tangent field irradiation in mice, we used a small-field biological irradiator to deliver a single dose of 12 Gy x-rays to approximately one-third of the left ventricle (LV) of Tie2Cre; p53(FL/+) and Tie2Cre; p53(FL/-) mice, where 1 or both alleles of p53 are deleted in endothelial cells. Four and 8 weeks after irradiation, mice were injected with gold and iodinated nanoparticle-based contrast agents, and imaged with DE-microCT and 4D-microCT to evaluate myocardial vascular permeability and cardiac function, respectively. Additionally, the same mice were imaged with microSPECT to assess myocardial perfusion.After PHI with tangent fields, DE-microCT scans showed a time-dependent increase in accumulation of gold nanoparticles (AuNp) in the myocardium of Tie2Cre; p53(FL/-) mice. In Tie2Cre; p53(FL/-) mice, extravasation of AuNp was observed within the irradiated LV, whereas in the myocardium of Tie2Cre; p53(FL/+) mice, AuNp were restricted to blood vessels. In addition, data from DE-microCT and microSPECT showed a linear correlation (R(2) = 0.97) between the fraction of the LV that accumulated AuNp and the fraction of LV with a perfusion defect. Furthermore, 4D-microCT scans demonstrated that PHI caused a markedly decreased ejection fraction, and higher end-diastolic and end-systolic volumes, to develop in Tie2Cre; p53(FL/-) mice, which were associated with compensatory cardiac hypertrophy of the heart that was not irradiated.Our results show that DE-microCT and 4D-microCT with nanoparticle-based contrast agents are novel imaging approaches complementary to microSPECT for noninvasive assessment of the change in myocardial vascular permeability and cardiac function of mice in whom myocardial injury develops after PHI.