Gurveen Saberwal

Evolving intricacies and implications of E2F1 regulation

E2F transcription factors may play a pivotal role in the transcriptional regulation of several cellular processes far beyond the originally described cell cycle and proliferation. Among the six E2F family members, only E2F1 is noted for its role in apoptosis. The pocket protein family members Rb, p107, and p130 act as the main regulators of E2F activity. Nonetheless, in recent years other protein‐protein interactions have been described for E2Fs. The post‐translational modifications resulting from such protein interactions may have significant implications in the stability, half‐life, and functional activity of E2Fs. In human diseases the significance of E2Fs is still under appreciated and is primarily recognized only as a consequence of the impairment in retinoblastoma gene product (Rb). However, with increasing knowledge of other protein interactions, the derailment of E2F activity could be anticipated to stem from an abnormality of any node in the complex network governing their availability and activity. The present review is intended to provide a perspective on the diversity of biochemical mechanisms underlying abnormal E2F expression and activity, understanding of which may have significant clinical implications.

Biological significance of proliferation, apoptosis, cytokines, and monocyte/macrophage cells in bone marrow biopsies of 145 patients with myelodysplastic syndrome.

Labeling index (LI), apoptosis, levels of 2 pro-apoptotic cytokines tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), and the number of monocyte/macrophage cells that are the likely source of the cytokines were simultaneously measured in plastic-embedded bone marrow (BM) biopsy sections of 145 patients with myelodysplastic syndromes (MDS). TNF-α was correlated with TGF-β (P = .001) and with monocyte/macrophage cells (P = .003). Patients with excess blasts in their marrows had a higher TGF-β level (P = .01) and monocyte/macrophage number (P = .05). In a linear regression model, TGF-β emerged as the most significant biological difference between patients who have excess of blasts and those who do not (P = .01). We conclude that in addition to TNF-α, TGF-β also plays a significant role in the initiation and pathogenesis of MDS, and that a more precise definition of its role will likely identify better preventive and therapeutic strategies.

Increased incidence of mitochondrial cytochrome c‐oxidase gene mutations in patients with myelodysplastic syndromes

Summary.  Mitochondria (mt) play an important role in both apoptosis and haem synthesis. The present study was conducted to determine DNA mutations in mitochondrial encoded cytochrome c‐oxidase I and II genes. Bone marrow (BM) biopsy and aspirate, peripheral blood (PB) and buccal smear samples were collected from 20 myelodysplastic syndrome (MDS) patients and 10 age‐matched controls. Cytochrome c‐oxidase I (CO I) and II (CO II) genes were amplified using polymerase chain reaction and sequenced. CO I mutations were found in 13/20 MDS patients and the CO II gene in 2/10 normal and 12/20 MDS samples, irrespective of MDS subtype. Mutations were substitutional, deletional and insertional. CO I mutations were most common at nucleotide positions 7264 (25%) and 7289 (15%), and CO II mutations were most common at nucleotide positions 7595 (40%) and 7594 (30%), suggesting the presence of potential ‘hot‐spots’. Mutations were not found in buccal smears of MDS patients and were significantly higher in MDS samples compared with age‐matched controls in all cell fractions (P < 0·05), with bone marrow high‐density fraction (BMHDF) showing a higher mutation rate than other fractions (P < 0·05). MDS marrows showed higher levels of apoptosis than normal controls (P < 0·05), and apoptosis in BMHDF was directly related to cytochrome c‐oxidase I gene mutations (P < 0·05). Electron microscopy revealed apoptosis affecting all haematopoietic lineages with highly abnormal, iron‐laden mitochondria. These results suggest a role for mt‐DNA mutations in the excessive apoptosis and resulting cytopenias of MDS patients.

Constitutive activation of SHP2 in mice cooperates with ICSBP deficiency to accelerate progression to acute myeloid leukemia

Myeloproliferative disorders (MPDs) are characterized by cytokine hypersensitivity and apoptosis resistance. Development of a block in myeloid differentiation is associated with progression of MPD to acute myeloid leukemia (AML) and portends poor prognosis. Identifying molecular markers of this transition may suggest targets for therapeutic intervention. Interferon consensus sequence binding protein (ICSBP, also known as IRF8) is an interferon-regulatory transcription factor that functions as a leukemia tumor suppressor. In mice, ICSBP deficiency induces an MPD that progresses to AML over time, suggesting that ICSBP deficiency is sufficient for myeloproliferation, but additional genetic lesions are necessary for AML. Since activity of ICSBP is influenced by tyrosine phosphorylation state, we hypothesized that mutations in molecular pathways that regulate this process might synergize with ICSBP deficiency for progression to AML. Consistent with this, we found that constitutive activation of SHP2 protein tyrosine phosphatase synergized with ICSBP haploinsufficiency to facilitate cytokine-induced myeloproliferation, apoptosis resistance, and rapid progression to AML in a murine bone marrow transplantation model. Constitutive SHP2 activation cooperated with ICSBP deficiency to increase the number of progenitors in the bone marrow and myeloid blasts in circulation, indicating a block in differentiation. Since SHP2 activation and ICSBP deficiency may coexist in human myeloid malignancies, our studies have identified a molecular mechanism potentially involved in disease progression in such diseases.

Leukemia-Associated, Constitutively Active Mutants of SHP2 Protein Tyrosine Phosphatase Inhibit NF1 Transcriptional Activation by the Interferon Consensus Sequence Binding Protein

ABSTRACT Deficiency in either the interferon consensus sequence binding protein (ICSBP) or neurofibromin 1 (Nf1) increases the proliferative response of myeloid progenitor cell to hematopoietic cytokines. Consistent with this, we previously demonstrated that ICSBP activates transcription of the gene encoding Nf1 (the NF1 gene). In the studies presented here, we determine that ICSBP tyrosine phosphorylation is necessary for the activation of NF1 transcription. Since ICSBP is tyrosine phosphorylated in response to hematopoietic cytokines, these studies identify a novel pathway by which cytokine-induced posttranslational modification of ICSBP results in NF1 transcription. Nf1 subsequently inactivates cytokine-activated Ras, thereby creating a negative feedback mechanism for cytokine-induced proliferation. In these studies, we also determine that ICSBP is a substrate for SHP2 protein tyrosine phosphatase (SHP2-PTP). We find that wild-type SHP2-PTP dephosphorylates ICSBP only in undifferentiated myeloid cells. In contrast, a leukemia-associated, constitutively activated mutant form of SHP2-PTP dephosphorylates ICSBP in both myeloid progenitors and differentiating myeloid cells. Activated SHP2-PTP mutants thereby inhibit ICSBP-dependent NF1 transcription, impairing this negative feedback mechanism on cytokine-activated Ras. Therefore, these studies suggest that leukemia-associated ICSBP deficiency cooperates with leukemia-associated activating mutants of SHP2-PTP to contribute to the proliferative phenotype in myeloid malignancies.

The Interferon Consensus Sequence-binding Protein Activates Transcription of the Gene Encoding Neurofibromin 1

Deficiency of the interferon consensus sequence-binding protein (ICSBP) is associated with increased myeloid cell proliferation in response to hematopoietic cytokines. However, previously identified ICSBP target genes do not indicate a mechanism for this “cytokine hypersen-sitivity.” In these studies, we identify the gene encoding neurofibromin 1 (Nf1) as an ICSBP target gene, by chromatin immunoprecipitation. Additionally, we find decreased Nf1 expression in bone marrow-derived myeloid cells from ICSBP–/– mice. Since Nf1 deficiency is also associated with cytokine hypersensitivity, our results suggested that NF1 is a functionally significant ICSBP target gene. Consistent with this, we find that the hyper-sensitivity of ICSBP–/– myeloid cells to granulocyte monocyte colony-stimulating factor (GM-CSF) is reversed by expression of the Nf1 GAP-related domain. We also find that treatment of ICSBP-deficient myeloid cells with monocyte colony-stimulating factor (M-CSF) results in sustained Ras activation, ERK phosphorylation, and proliferation associated with impaired Nf1 expression. These M-CSF effects are reversed by ICSBP expression in ICSBP–/– cells. Consistent with this, we find that ICSBP activates the NF1 promoter in myeloid cell line transfectants and identify an ICSBP-binding NF1 cis element. Therefore, the absence of ICSBP leads to Nf1 deficiency, impairing down-regulation of Ras activation by GM-CSF or M-CSF. These results suggest that one mechanism of increased myeloid proliferation, in ICSBP-deficient cells, is decreased NF1 gene transcription. This novel ICSBP function provides insight into regulation of myelopoiesis under normal conditions and in myeloproliferative disorders.

Interferon Consensus Sequence Binding Protein (ICSBP) Decreases β-Catenin Activity in Myeloid Cells by Repressing GAS2 Transcription

ABSTRACT The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor, also referred to as IRF8. ICSBP acts as a suppressor of myeloid leukemia, although few target genes explaining this effect have been identified. In the current studies, we identified the gene encoding growth arrest specific 2 (GAS2) as an ICSBP target gene relevant to leukemia suppression. We find that ICSBP, Tel, and histone deacetylase 3 (HDAC3) bind to a cis element in the GAS2 promoter and repress transcription in myeloid progenitor cells. Gas2 inhibits calpain protease activity, and β-catenin is a calpain substrate in these cells. Consistent with this, ICSBP decreases β-catenin protein and activity in a Gas2- and calpain-dependent manner. Conversely, decreased ICSBP expression increases β-catenin protein and activity by the same mechanism. This is of interest, because decreased ICSBP expression and increased β-catenin activity are associated with poor prognosis and blast crisis in chronic myeloid leukemia (CML). We find that the expression of Bcr/abl (the CML oncoprotein) increases Gas2 expression in an ICSBP-dependent manner. This results in decreased calpain activity and a consequent increase in β-catenin activity in Bcr/abl-positive (Bcr/abl+) cells. Therefore, these studies have identified a Gas2/calpain-dependent mechanism by which ICSBP influences β-catenin activity in myeloid leukemia.

Constitutively Active SHP2 Cooperates with HoxA10 Overexpression to Induce Acute Myeloid Leukemia

The homeodomain transcription factor HoxA10 is maximally expressed in myeloid progenitor cells. Sustained HoxA10 expression during differentiation has been described in poor prognosis human acute myeloid leukemia (AML). Consistent with this, engineered overexpression of HoxA10 in murine bone marrow induces a myeloproliferative disorder that progresses to AML over time. This murine model suggests that HoxA10 overexpression is sufficient for myeloproliferation but that differentiation block, and therefore AML, requires acquisition of additional mutations. In myeloid progenitor cells, HoxA10 represses transcription of genes that encode phagocyte effector proteins such as gp91PHOX and p67PHOX. Tyrosine phosphorylation of HoxA10 during myelopoiesis decreases binding to these target genes. In immature myeloid cells, HoxA10 also activates transcription of the DUSP4 gene that encodes Mkp2, an anti-apoptotic protein. HoxA10 binding to the DUSP4 promoter decreases during myelopoiesis. Therefore, both myeloid-specific gene repression and DUSP4 activation by HoxA10 decrease during myelopoiesis. This results in phenotypic differentiation and facilitates apoptosis as differentiation proceeds. HoxA10 is de-phosphorylated by SHP2 protein-tyrosine phosphatase in myeloid progenitors. This mechanism maintains HoxA10 in a nonphosphorylated state in immature, but not differentiating, myeloid cells. Constitutively active SHP2 mutants have been described in human AML, which dephosphorylate HoxA10 throughout myelopoiesis. In this study, we hypothesize that constitutive SHP2 activation synergizes with HoxA10 overexpression to accelerate progression to AML. Because both HoxA10 overexpression and constitutive SHP2 activation are found in poor prognosis human AML, these studies contribute to understanding biochemical aspects of disease progression in myeloid malignancy.

The Interferon Consensus Sequence Binding Protein (ICSBP/IRF8) Activates Transcription of the FANCF Gene during Myeloid Differentiation

The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor with leukemia-suppressor activity. ICSBP regulates genes that are involved in phagocyte function, proliferation, and apoptosis. In murine models ICSBP deficiency results in a myeloproliferative disorder (MPD) with increased mature neutrophils. Over time this MPD progresses to acute myeloid leukemia (AML), suggesting that ICSBP deficiency is adequate for MPD, but additional genetic lesions are required for AML. The hypothesis of these studies is that dysregulation of key target genes predisposes to disease progression under conditions of decreased ICSBP expression. To investigate this hypothesis, we used chromatin co-immunoprecipitation to identify genes involved the ICSBP-leukemia suppressor effect. In the current studies, we identify the gene encoding Fanconi F (FANCF) as an ICSBP target gene. FancF participates in a repair of cross-linked DNA. We identify a FANCF promoter cis element, which is activated by ICSBP in differentiating myeloid cells. We also determine that DNA cross-link repair is impaired in ICSBP-deficient myeloid cells in a FancF-dependent manner. This effect is observed in differentiating cells, suggesting that ICSBP protects against the genotoxic stress of myelopoiesis. Decreased ICSBP expression is found in human AML and chronic myeloid leukemia during blast crisis (CML-BC). Our studies suggest that ICSBP deficiency may be functionally important for accumulation of chromosomal abnormalities during disease progression in these myeloid malignancies.

Involvement of Cyclin D1 and E2F1 in Intramedullary Apoptosis in Myelodysplastic Syndromes

An unusually high incidence of apoptosis in S-phase cells is characteristically found in the bone marrow (BM) of patients with myelodysplastic syndromes (MDS). Previously, E2F1, c-myc, and Cyclin D1 have been shown to bring about both S-phase changes and/or apoptotic changes. We have already found a stoichiometric imbalance between pRb and E2F1 causing deregulated E2F1 activity in these disorders. In the present study, we investigated the status of Cyclin D1 in relation to E2F1 and apoptosis in 19 patients with a confirmed diagnosis of MDS in comparison with 6 healthy donors. Cyclin D1 was localized immunohistochemically using a specific monoclonal antibody (1:150 dilution) in plastic-embedded BM sections. The nuclear localization of Cyclin D1 graded on a subjective rating scale of 0 (negligible staining) to 8+ (highest), demonstrated negligible levels in normal marrows (median 1+), and in 11/19 evaluable MDS marrows. In contrast, 8/19 MDS biopsies showed an almost four-fold increase in Cyclin D1 localization (p< or =0.001). A western blot analysis of E2F1 in corresponding bone marrow (BM) aspirate mononuclear cells (MNC) demonstrated that the MDS patients with elevated Cyclin D1 expression also had a significant increase in E2F1 protein (p< or =0.03). Additionally, these patients revealed higher levels of mRNA of one of the E2F1 transcriptional target genes, dihydrofolate reductase (DHFR, p=0.01). Subsequently, the relationship of Cyclin D1 with apoptosis was elucidated in a colocalization experiment in BM biopsy sections using immunohistochemistry for Cyclin D1 and in situ end labeling of DNA (ISEL) for apoptosis. The percentage of ISEL-positive apoptotic cells was several fold higher in MDS as compared to normal BMs (p=0.009). Interestingly, 7-41% (median 20%) of the apoptotic cells in different MDS BMs revealed co-localization of Cyclin D1 in their nucleus, whereas in normal BMs co-localization was virtually absent (p=0.008). Thus, it is possible that in a subset of MDS patients, apoptotic death of bone marrow cells may involve Cyclin D1/E2F1 pathway.