ALBERT

Description: Zinc finger (ZF) transcriptional repressor Gfi-1 plays an important role in hematopoiesis and inner ear development, and also functions as an oncoprotein that cooperates with c-Myc in lymphomagenesis. Gfi-1 represses transcription by directly binding to the consensus DNA sequence in the promoters of its target genes. We report here an alternative mechanism by which Gfi-1 represses CDKN2B encoding the cyclin-dependent kinase inhibitor p15INK4B. Gfi-1 did not directly bind to CDKN2B, but interacted with Miz-1 and, via Miz-1, was recruited to the core promoter of CDKN2B. The C-terminal zinc finger domains of Gfi-1 and Miz-1 are involved in the interaction. Miz-1 is a POZ-ZF transcription factor that has been shown to mediate transcriptional repression by c-Myc. Like c-Myc, upon recruitment to the CDKN2B promoter, Gfi-1 repressed transcriptional activation of CDKN2B by Miz-1 and in response to TGFb. Notably, Gfi-1 and c-Myc formed a ternary complex with Miz-1 and were both recruited to the CDKN2B core promoter via Miz-1, and acted in collaboration to repress CDKN2B. Consistent with its role in repressing CDKN2B transcription, knockdown of Gfi-1 in human leukemic cells resulted in augmented levels of p15INK4B, which was associated with attenuated cell proliferation. The expression of p15INK4B was also significantly higher in Gfi-1−/− mouse bone marrow (BM) cells than in Gfi-1+/+ BM cells. Our data reveal a novel mechanism of transcriptional repression by Gfi-1 and also identify CDKN2B as a new Gfi-1 target gene. The findings may have important implications for understanding the role of Gfi-1 in normal development and the cooperation between Gfi-1 and c-Myc in lymphomagenesis.

Description: Neutrophils and monocytes/macrophages are derived from hematopoietic stem cells that, through progressive commitment, give rise to granulocyte-monocyte progenitors that in turn develop into either neutrophils or monocytes/macrophages. Although it is well known that cell fate specification in the hematopoietic system depends on the expression of lineage specific transcription factors, the roles of cytokines in lineage commitment are less clear and two models have been proposed. According to the stochastic model, cell fate choice is stochastic and cytokines simply provide signals for the survival and proliferation of committed cells. The instructive model, on the other hand, proposes that cytokines stimulate intracellular signaling pathways that dictate cell fate decisions. G-CSF and M-CSF are two lineage-specific cytokines that play a dominant role in granulopoiesis and monopoiesis, respectively. Recent studies lend strong support to the roles of G-CSF and M-CSF in instructing lineage commitment. However, the signaling pathways that determine neutrophil versus monocyte cell fate following stimulation with G-CSF and M-CSF are unknown. Here we show that tyrosine (Y) 729 of the G-CSFR is involved in transducing signals that specify neutrophil cell fate. Substitution of Y729 with phenylalanine (F) results in monocytic differentiation in response to G-CSF in murine myeloid 32D and multipotent FDCP-mix A4 cells. G-CSF stimulated activation of Erk1/2 was prolonged in cells expressing G-CSFR Y729F mutant. Significantly, treatment of cells with Mek1/2 inhibitors U0126 or PD0325901 rescued neutrophilic differentiation. M-CSF has been shown to induce prolonged activation of Erk1/2, which is required for monocytic differentiation. Interestingly, the Mek1/2 inhibitors also promoted neutrophil cell fate at the expense of monocytic development in lineage marker negative (Lin-) primary bone marrow cells cultured in M-CSF. We further demonstrate that prolonged activation of Erk1/2 was associated with augmented activation of c-Fos and Egr1, both of which have previously been shown to promote monocytic development. Consistent with this, knockdown of c-Fos or Egr1 redirected 32D cells expressing G-CSFR Y729F mutant to develop into neutrophils in response to G-CSF. We propose that M-CSF stimulates more sustained activation of Erk1/2 than G-CSF does and that the duration of Erk1/2 signaling regulates neutrophil versus monocyte cell fate choices, likely through altering the activation statuses of c-Fos and Egr1. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1228 Gfi1 is a transcriptional repressor that plays a critical role in hematopoiesis. Gfi1 has also been implicated in lymphomagenesis when aberrantly activated as a result of deregulated expression. It is still poorly understood how Gfi1 expression is regulated and how it acts in the hematopoietic system. We show here that Gfi1 transcription was repressed by the tumor suppressor p53 in hematopoietic cells. Treatment of cells with doxorubicin (Doxo), which induces topoisomerase II-mediated DNA double strand breaks (DSB), led to a steady increase in p53 protein level, which was accompanied by a gradual decline in Gfi1 expression at both protein and mRNA levels. Knockdown of p53 resulted in increased Gfi1 expression and also abolished Doxo-induced Gfi1 downregulation. In contrast, Gfi1 expression was reduced and its downregulation in response to DNA damage was restored in p53-deficient cells transfected with the p53/estrogen receptor ligand binding domain fusion protein (p53ERTAM) in the presence of 4-hydroxytamoxifen (4-OHT). In luciferase reporter assays, Doxo treatment inhibited the activity of a ∼2.4-kb Gfi1 promoter fragment in p53+/+ cells, but not in p53−/− cells. Consistent with this, the wild type p53, but not a DNA binding-defective p53 mutant, repressed the Gfi1 promoter fragment. Chromatin immunoprecipitation (ChIP) assays demonstrated that p53 bound to the proximal region of the Gfi1 promoter. Detailed mapping of the Gfi1 promoter indicated that the core promoter region of Gfi1 spanning −33 to +6 bp is sufficient for p53-mediated repression. This core promoter region contains a putative p53 repressive response element (RRE) and, when the RRE was mutated, p53 failed to bind to and repress the Gfi1 promoter. Significantly, apoptosis induction by Doxo treatment was inhibited upon Gfi1 overexpression, but augmented following Gfi1 knockdown. Together, these data establish for the first time that Gfi1 is repressed by p53 and indicate that Gfi1 acts to protect hematopoietic cells from DNA damage-induced apoptosis. Our findings have important implications for understanding the roles of Gfi-1 in normal hematopoiesis and lymphomagenesis. Disclosures: No relevant conflicts of interest to declare.

Description: Mutations in ELANE encoding neutrophil elastase (NE) have been identified in the majority of patients with severe congenital neutropenia (SCN). SCN patients are predisposed to acute myeloid leukemia (AML), and progression from SCN to AML is accompanied by acquisition of mutations in CSF3R encoding the G-CSFR receptor (G-CSFR) in approximately 80% of the patients. The mutations, which cause receptor’s carboxy-terminal truncation, are highly specific for SCN/AML, but the reason for the prevalence of the CSF3R mutations in patients with SCN/AML is unknown. We show here that NE expression was induced by G-CSF in myeloid 32D cells expressing the wild type (WT) G-CSFR (32D/GR), but not in cells expressing an SCN-derived truncated G-CSFR, d715 (32D/d715). Unlike the WT G-CSFR, the d715 receptor was unable to activate a 1.8-kb promoter fragment of mouse Elane. The NE mutants associated with SCN have been shown to cause premature apoptosis of differentiating granulocytic precursors. To address whether the d715 receptor might abolish apoptosis induction by the NE mutants in part by suppressing their expression, we placed the cDNA encoding the SCN-derived NE G185R mutant under the control of the Elane promoter and the resultant expression construct was transfected into 32D/GR and 32D/d715 cells. G-CSF treatment resulted in increased expression of G185R mutant and premature apoptosis in 32D/GR cells, but had no effect on G185R expression and cell survival in 32D/d715 cells. We further addressed whether the d715 receptor was capable of directly suppressing apoptosis induced by the G185R mutant. As expected, forced expression of the G185R mutant in 32D/GR cells led to accelerated apoptosis in response to G-CSF, but did not negatively influence the viability of 32D/d715 cells cultured in G-CSF. To evaluate whether the d715 receptor inhibited apoptosis induction by the G185R mutant in other hematopoietic cells, we expressed the G185R mutant in murine multipotent FDCP-mix A4 cells transfected with WT G-CSFR (FDCP/GR) or d715 receptor (FDCP/d715). Although FDCP/GR cells expressing G185R mutant showed no significant increase in apoptotic cell death upon treatment with G-CSF, they exhibited increased sensitivity to tunicamycin-induced endoplasmic reticulum (ER) stress. In contrast, the G185R mutant failed to sensitize FDCP/d715 cells to apoptosis induction by tunicamycin. Thus, it appears that the d715 receptor may abolish G185R mutant-induced apoptosis by two distinct mechanisms, i.e., suppression of its expression and direct inhibition of its pro-apoptotic action. Our data may have implications for understanding the prevalence of CSF3R mutations in patients with SCN/AML. We propose that the CSF3R mutations might represent a mechanism by which myeloid cells carrying the ELANE mutations evade the pro-apoptotic effect of the NE mutants in SCN patients. Disclosures: No relevant conflicts of interest to declare.

Description: Granulocytic progenitor cells become progressively less capable of proliferation and survival with terminal differentiation. Granulocyte colony-stimulating factor (G-CSF) is the major regulator of granulopoiesis and stimulates the activation of multiple signaling pathways including the signal transducer and activator of transcription 5 (Stat5) pathway. Little is known about the activation status of G-CSF-stimulated signaling pathways at the distinct stages of granulocytic differentiation. When myeloid 32D cells transfected with the G-CSF receptor were induced to differentiate with G-CSF, Stat5 activation in response to G-CSF was gradually attenuated. Activation of other signaling molecules including Stat1, Stat3, Erk1/2, JNK and p38 was not altered significantly. Stat5 activation was also downregulated in multipotent FDCP-mix cells, which differentiated into mature granulocytes upon induction with G-CSF, but not in pro-B BaF/3 transfected with the G-CSF receptor, which showed no terminal granulocytic differentiation in response to G-CSF, suggesting that the effect of G-CSF is cell type specific. Attenuated activation of Stat5 correlated with reduced Stat5 protein levels, which was associated with expression of a protease activity capable of degrading Stat5 protein in vitro. The Stat5 protease activity was upregulated when myeloid cells were induced to differentiate with G-CSF, but its upregulation by G-CSF was blocked upon expression of leukemogenic proteins Bcr-Abl and Tel-Jak2. The activity of the Stat5 protease was inhibited partially by PMSF and completely by a1-antitrypsin, suggesting that it belongs to the serine family of protease. Our data provide the first evidence that a Stat5 protease activity is upregulated by G-CSF and may have important implications for understanding the molecular mechanism by which G-CSF orchestrates granulopoiesis.

Description: Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that plays a major role in granulopoiesis. G-CSF supports the proliferation, differentiation and survival of myeloid progenitor cells. Treatment of cells with G-CSF activates multiple intracellular signal transduction pathways leading to alterations in the activities of transcription factors involved in myeloid development. Gfi-1 is a zinc-finger transcriptional repressor that is required for granulopoiesis. Mutations in Gfi-1 have been associated with severe congenital neutropenia (SCN). How Gfi-1 acts in myeloid cells is still poorly understood. Expression of Gfi-1 was upregulated during granulocytic differentiation induced by G-CSF in myeloid 32D cells. Truncation of the carboxy terminus of the G-CSF receptor, as seen in patients with acute myeloid leukemia (AML) evolving from SCN, disrupted the ability of the G-CSF receptor to upregulate Gfi-1. Ectopic expression of a naturally occurring dominant negative Gfi-1 mutant, N382S, identified in patients with SCN, resulted in premature apoptosis and reduced proliferation of cells induced to differentiate with G-CSF. The expression of neutrophil elastase (NE) and C/EBPe was significantly increased in 32D cells expressing N382S. In contrast, overexpression of wild type Gfi-1 abolished G-CSF-induced upregulation of C/EBPe, but had no apparent effect on NE upregulation by G-CSF. Notably, G-CSF-dependent proliferation and survival were inhibited upon overexpression of C/EBPe, but not NE. These data suggest that a key role of Gfi-1 in granulopoiesis is to repress C/EBPe and that overt C/EBPe overexpression may be deleterious to the proliferation and survival of myeloid cells. Our data may explain why loss of Gfi-1 function, either as a result of gene knockout in mice or gene mutations in human, results in a block of granulocytic differentiation and severe neutropenia.

Description: Mutations in the G-CSF receptor gene causing carboxy terminal truncation of the G-CSF receptor have been associated with the development of acute myelogenous leukemia (AML) in patients with severe congenital neutropenia (SCN). The truncated G-CSF receptors mediate augmented cell proliferation and survival, but are defective in inducing granulocytic differentiation in myeloid cell lines and transgenic mice. Little is known about the mechanism by which the carboxy terminus of the G-CSF receptor regulates granulocytic differentiation. It has been shown that myeloid transcription factors including C/EBPα, C/EBPε and PU.1 are critically involved in granulopoiesis. Of the three transcription factors, expression of C/EBPε was dramatically upregulated upon G-CSF treatment of myeloid 32D cells expressing the wild type G-CSF receptor (32D/WT). C/EBPε upregulation by G-CSF was abolished in 32D cells expressing the G-CSF receptor mutant D715 (32D/D715), which lacked a region of 98 amino acids in the carboxy terminus. Forced expression of C/EBPε in 32D/D715 cells at levels comparable to those in G-CSF-treated 32D/WT cells resulted in markedly attenuated cell proliferation and survival, and rapid terminal granulocytic differentiation in response to G-CSF. These data indicated that the functional defect of the D715 mutant in mediating granulocytic differentiation may arise from its inability to upregulate C/EBPε expression. Notably, forced expression of C/EBPε failed to induce terminal granulocytic differentiation of 32D/D715 cells cultured in IL-3, suggesting that the D715 receptor acted in collaboration with C/EBPε to drive granulocytic differentiation. Of the four cytoplasmic tyrosine (Tyr) residues of the G-CSF receptor, the D715 mutant contains only Tyr 704 that is involved in Stat3 activation. Because Stat3 is an important regulator of granulopoiesis, experiments are under way to address whether Tyr 704 in the D715 mutant is required for granulocytic differentiation driven by C/EBPε.

Description: Severe congenital neutropenia (SCN) is characterized by early onset of bacterial infections and maturation arrest of myeloid cells at early stages of differentiation in the bone marrow. Point mutations in ELA2 encoding neutrophil elastase (NE) have been identified in 60% to 80% of patients with SCN. SCN patients are predisposed to acute myeloid leukemia (AML), which occurs in approximately 15 % of cases. With rare exceptions, leukemic cells from these patients carry mutations in CSF3R encoding the G-CSF receptor, leading to C-terminal truncation of the receptor. Notably, the nonsense mutations in CSF3R are present only in SCN/AML patients, particularly those with ELA2 mutations, but not in other types of neutropenias and de novo AML. The mechanism for the exclusive presence of the nonsense CSF3R mutations in SCN/AML is unknown. In myeloid 32D cells transfected with the wild type (WT) G-CSF receptor (32D/WT), G-CSF treatment induced the expression of NE. However, NE expression was not upregulated by G-CSF in 32D cells expressing the truncated G-CSF receptor d715, derived from an SCN patient. It has been shown that myeloid cells from patients with SCN/AML express both the wild type and the truncated G-CSF receptors. Indeed, the d715 mutant acted in a dominant negative manner to suppress NE upregulation by the WT G-CSF receptor. In luciferase reporter assays, the WT G-CSF receptor, but not the d715 mutant, activated a 1.8-kb fragment of the mouse Ela2 promoter. Significantly, forced expression of an SCN-associated NE mutant G185R caused premature apoptosis of differentiating 32D/WT cells in response to G-CSF with no significant effect on IL-3-stimulated survival. To address whether the d715 mutant may abolish the proapoptotic effect of the G185R mutant via suppressing its expression, we transfected 32D/WT and 32D/d715 cells with an expression construct in which the expression of the G185R mutant was driven by the 1.8-kb fragment of the Ela2 promoter. G-CSF treatment induced the expression of the G185R mutant and subsequent apoptosis in 32D/WT cells. In 32D/d715 cells, however, the expression of the G185R mutant was not induced by G-CSF and accordingly its proapoptotic activity was not evident. We propose that acquisition of the nonsense mutations in CSF3R may represent a mechanism utilized by the myeloid cells harboring the ELA2 mutations to evade the proapoptotic effect of the NE mutants. However, expression of the truncated G-CSF receptors has other biological consequences: they transduce strong proliferative signals but are defective in inducing granulocytic differentiation, which may initiate the leukemogenic process.

Description: Chronic myelogenous leukemia (CML) is a clonal hematopoietic disorder caused by the BCR/ABL fusion oncogene. CML typically evolves in three distinct clinical stages: chronic and accelerated stages and blast crisis. The progression of CML from chronic phase (CP) to blast crisis (BC) is characterized by the increasing failure of myeloid precursors to differentiate into mature granulocytes and aggressive proliferation of immature myeloid cells. It has been shown that CML progression from CP to BC is associated with downregulation of C/EBPs including C/EBPa and C/EBPe, critical regulators of myeloid development. Forced expression of C/EBPs suppresses transformation and restores granulocytic differentiation of BCR/ABL-expressing myeloid cells, suggesting that downregulation of C/EBPs may be involved in CML transition from CP to BC. The proto-oncogene Gfi-1 encodes a nuclear zinc-finger transcriptional repressor that is required for the survival and proliferation of myeloid cells. We recently showed that expression of a dominant negative Gfi-1 mutant, N382S, led to markedly increased expression of C/EBPe in myeloid cells, suggesting that Cebpe encoding C/EBPe is a Gfi-1 target. We investigated the possibility that inhibition of Gfi-1 function in BCR/ABL-expressing cells may increase C/EBPe expression, thereby exerting a negative effect on BCR/ABL-mediated transformation. Myeloid 32D cells transfected with BCR/ABL (32D/BCR/ABL) are independent of IL-3 for proliferation and survival. Expression of the N382S mutant in 32D/BCR/ABL cells (32D/N382S) had a significant inhibitory effect on proliferation and survival in the absence of IL-3, but only a weak effect in the presence of IL-3, suggesting that the N382S mutant specifically inhibited BCR/ABL-mediated proliferation and survival. The level of C/EBPe protein was markedly augmented in 32D/N382S cells. Interestingly, when cultured in medium containing no growth factors, 32D/N382S cells exhibited certain features of macrophage differentiation including increased cell size, adherence to the surface of culture flasks, spreading and increased surface expression of macrophage differentiation marker F4/80. 32D/N382S cells also became more sensitive to c-ABL kinase inhibitor imatinib. These data indicate that Gfi-1 may play an important role in BCR/ABL-mediated transformation and thus represent a potential therapeutic target in the treatment of CML.