Functional interplay of SP family members and nuclear factor Y is essential for transcriptional activation of the human Calreticulin gene

Calreticulin (CALR) is a highly conserved, multifunctional protein involved in a variety of cellular processes including the maintenance of intracellular calcium homeostasis, proper protein folding, differentiation and immunogenic cell death. More recently, a crucial role for CALR in the pathogenesis of certain hematologic malignancies was discovered: in clinical subgroups of acute myeloid leukemia, CALR overexpression mediates a block in differentiation, while somatic mutations have been found in the majority of patients with myeloproliferative neoplasms with nonmutated Janus kinase 2 gene (JAK2) or thrombopoietin receptor gene (MPL). However, the mechanisms underlying CALR promoter activation have insufficiently been investigated so far. By dissecting the core promoter region, we could identify a functional TATA-box relevant for transcriptional activation. In addition, we characterized two evolutionary highly conserved cis-regulatory modules (CRMs) within the proximal promoter each composed of one binding site for the transcription factors SP1 and SP3 as well as for the nuclear transcription factor Y (NFY) and we verified binding of these factors to their cognate sites in vitro and in vivo.

Tyrosine kinase inhibitor-induced CD70 expression mediates drug resistance in leukemia stem cells by activating Wnt signaling.

In chronic myelogenous leukemia (CML), oncogenic BCR-ABL1 activates the Wnt pathway, which is fundamental for leukemia stem cell (LSC) maintenance. Tyrosine kinase inhibitor (TKI) treatment reduces Wnt signaling in LSCs and often results in molecular remission of CML; however, LSCs persist long term despite BCR-ABL1 inhibition, ultimately causing disease relapse. We demonstrate that TKIs induce the expression of the tumor necrosis factor (TNF) family ligand CD70 in LSCs by down-regulating microRNA-29, resulting in reduced CD70 promoter DNA methylation and up-regulation of the transcription factor specificity protein 1. The resulting increase in CD70 triggered CD27 signaling and compensatory Wnt pathway activation. Combining TKIs with CD70 blockade effectively eliminated human CD34(+) CML stem/progenitor cells in xenografts and LSCs in a murine CML model. Therefore, targeting TKI-induced expression of CD70 and compensatory Wnt signaling resulting from the CD70/CD27 interaction is a promising approach to overcoming treatment resistance in CML LSCs.

Immune regulatory and neuroprotective properties of preimplantation factor: From newborn to adult.

Embryonic-maternal interaction from the earliest stages of gestation has a key, sustained role in neurologic development, persisting into adulthood. Early adverse events may be detrimental in adulthood. Protective factors present during gestation could significantly impact post-natal therapy. The role of PreImplantation Factor (PIF) within this context is herein examined. Secreted by viable early embryos, PIF establishes effective embryonic-maternal communication and exerts essential trophic and protective roles by reducing oxidative stress and protein misfolding and by blunting the nocive let-7 microRNA related pathway. PIF's effects on systemic immunity lead to comprehensive immune modulation, not immune suppression. We examine PIF's role in protecting embryos from adverse maternal environment, which can lead to neurological disorders that may only manifest post-nataly: Synthetic PIF successfully translates endogenous PIF features in both pregnant and non-pregnant clinically relevant models. Specifically PIF has neuroprotective effects in neonatal prematurity. In adult relapsing-remitting neuroinflammation, PIF reverses advanced paralysis while promoting neurogenesis. PIF reversed Mycobacterium smegmatis induced brain infection. In graft-vs.-host disease, PIF reduced skin ulceration, liver inflammation and colon ulceration while maintaining beneficial anti-cancer, graft-vs.-leukemia effect. Clinical-grade PIF has high-safety profile even at supraphysiological doses. The FDA awarded Fast-Track designation, and university-sponsored clinical trials for autoimmune disorder are ongoing. Altogether, PIF properties point to its determining regulatory role in immunity, inflammation and transplant acceptance. Specific plans for using PIF for the treatment of complex neurological disorders (ie. traumatic brain injury, progressive paralysis), including neuroprotection from newborn to adult, are presented.

Impact of comorbidities on overall survival in patients with chronic myeloid leukemia: results of the randomized CML study IV.

We studied the influence of comorbidities on remission rate and overall survival (OS) in patients with chronic myeloid leukemia (CML). Participants of the CML Study IV, a randomized 5-arm trial designed to optimize imatinib therapy, were analyzed for comorbidities at diagnosis using the Charlson Comorbidity Index (CCI); 511 indexed comorbidities were reported in 1519 CML patients. Age was an additional risk factor in 863 patients. Resulting CCI scores were as follows: CCI 2, n = 589; CCI 3 or 4, n = 599; CCI 5 or 6, n = 229; and CCI ≥ 7, n = 102. No differences in cumulative incidences of accelerated phase, blast crisis, or remission rates were observed between patients in the different CCI groups. Higher CCI was significantly associated with lower OS probabilities. The 8-year OS probabilities were 93.6%, 89.4%, 77.6%, and 46.4% for patients with CCI 2, 3 to 4, 5 to 6, and ≥7, respectively. In multivariate analysis, CCI was the most powerful predictor of OS, which was still valid after removal of its age-related components. Comorbidities have no impact on treatment success but do have a negative effect on OS, indicating that survival of patients with CML is determined more by comorbidities than by CML itself. OS may therefore be inappropriate as an outcome measure for specific CML treatments. The trial was registered at www.clinicaltrials.gov as #NCT00055874.

A Synthetic Factor XIIa Inhibitor Blocks Selectively Intrinsic Coagulation Initiation.

Coagulation factor XII (FXII) inhibitors are of interest for the study of the protease in the intrinsic coagulation pathway, for the suppression of contact activation in blood coagulation assays, and they have potential application in antithrombotic therapy. However, synthetic FXII inhibitors developed to date have weak binding affinity and/or poor selectivity. Herein, we developed a peptide macrocycle that inhibits activated FXII (FXIIa) with an inhibitory constant Ki of 22 nM and a selectivity of >2000-fold over other proteases. Sequence and structure analysis revealed that one of the two macrocyclic rings of the in vitro evolved peptide mimics the combining loop of corn trypsin inhibitor, a natural protein-based inhibitor of FXIIa. The synthetic inhibitor blocked intrinsic coagulation initiation without affecting extrinsic coagulation. Furthermore, the peptide macrocycle efficiently suppressed plasma coagulation triggered by contact of blood with sample tubes and allowed specific investigation of tissue factor initiated coagulation.

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

Mitochondrial defects in primary leukemia cells: Biological consequences and therapeutic implications

Mitochondria are actively engaged in the production of cellular energy sources, generation of reactive oxygen species (ROS), and regulation of apoptosis. Mitochondrial DNA (mtDNA) mutations/deletions and other mitochondrial abnormalities have been implicated in many diseases, especially cancer. Despite this, the roles that these defects play in cancer development, drug sensitivity, and disease progression still remain to be elucidated. The major objective of this investigation was to evaluate the mechanistic relationship between mitochondrial defects and alterations in free radical generation and chemosensitivity in primary chronic lymphocytic leukemia (CLL) cells. This study revealed that the mtDNA mutation frequency and basal superoxide generation are both significantly higher in primary cells from CLL patients with a history of chemotherapy as compared to cells from their untreated counterparts. CLL cells from refractory patients tended to have high mutation frequencies. The data suggest that chemotherapy with DNA-damaging agents may cause mtDNA mutations, which are associated with increased ROS generation and reduced drug sensitivity. Subsequent analyses demonstrated that CLL cells contain significantly more mitochondria than normal lymphocytes. This abnormal accumulation of mitochondria was linked to increased expression of nuclear respiratory factor-1 and mitochondrial transcription factor A, two key free radical-regulated mitochondrial biogenesis factors. Further analysis showed that mitochondrial content may have therapeutic implications since patient cells with high mitochondrial mass display significantly reduced in vitro sensitivity to fludarabine, a frontline agent in CLL therapy. The reduced in vitro and in vivo sensitivity to fludarabine observed in CLL cells with mitochondrial defects highlights the need for novel therapeutic strategies for the treatment of refractory disease. Brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport that is being developed as an anticancer agent, effectively induces apoptosis in fludarabine-refractory CLL cells through a secretory stress-mediated mechanism involving intracellular sequestration of pro-survival secretory factors. Taken together, these data indicate that mitochondrial defects in CLL cells are associated with alterations in free radical generation, mitochondrial biogenesis activity, and chemosensitivity. Abrogation of survival signaling by blocking ER to Golgi protein transport may be a promising therapeutic strategy for the treatment of CLL patients that respond poorly to conventional chemotherapy. ^

Catalytic antibody response to the Staphylococcus aureus virulence factor Efb

Staphylococcus aureus is a globally prevalent pathogen that can cause a wide variety of acute and chronic diseases in both adults and children, in both immune susceptible populations and healthy individuals. Its ability to cause persistent infections has been linked to multiple immune evasion strategies, including Efb-mediated complement inhibition. As new multi-drug-resistant strains emerge, therapeutic alternatives to traditional antibiotics must be developed. These experiments assessed the ability of healthy patient immunoglobulin to cleave Efb and disable the complement-inhibitory properties of Efb in vitro. Levels of immunoglobulin-mediated Efb catalysis varied both between immunoglobulin isoform/isotype and between individuals. Serum IgG showed the strongest catalytic activity of the immunoglobulin isotypes tested. Additionally, IgG hydrolyzed the virulence factor in a way that enabled only minimal binding to the complement component C3b, effectively blocking Efb-mediated inhibition of complement lysis. Salivary IgA and serum IgM did not block Efb-mediated inhibition of complement. Catalytic IgG selectively cleaved Efb and showed no cleavage of a variety of other proteins tested. Catalytic activity of IgG was inhibited by serine protease inhibitors, but not by other protease inhibitors, suggesting a serine-protease mechanism of catalysis. It is proposed that varying concentrations and activity levels of catalytic IgG between healthy individuals and those with current or recurrent S. aureus infections in both adult and pediatric populations be studied in order to assess the potential effectiveness of passive immunization therapy with catalytic monoclonal IgG. ^

BIOLOGICAL MECHANISMS AND CLINICAL IMPLICATIONS OF BCR-ABL-INDUCED MITOCHONDRIAL OXIDATIVE STRESS AND CELL SURVIVAL IN CHRONIC MYELOID LEUKEMIA

Chronic myeloid leukemia (CML), a myeloproliferative disorder, represents approximately 15-20% of all adult leukemia. The development of CML is clearly linked to the constitutively active protein-tyrosine kinase BCR-ABL, which is encoded by BCR-ABL fusion gene as the result of chromosome 9/22 translocation (Philadelphia chromosome). Previous studies have demonstrated that oxidative stress-associated genetic, metabolic and biological alterations contribute to CML cell survival and drug refractory. Mitochondria and NAD(P)H oxidase (NOX) are the major sources of BCR-ABL-induced cellular reactive oxygen species (ROS) production. However, it is still unknown how CML cells maintain the altered redox status, while escaping from the persistent oxidative stress-induced cell death. Therefore, elucidation of the mechanisms by which CML cells cope with oxidative stress will provide new insights into CML leukemogenesis. The major goal of this study is to identify the survival factors protecting CML cells against oxidative stress and develop novel therapeutic strategies to overcome drug resistance. Several experimental models were used to test CML cell redox status and cellular sensitivity to oxidative stress, including BCR-ABL inducible cell lines, BCR-ABL stably transformed cell lines and BCR-ABL-expressing CML blast crisis cells with differential BCL-XL/BCL-2 expressions. Additionally, an artificial CML cell model with heterogenic BCL-XL/BCL-2 expression was established to assess the correlation between differential survival factor expression patterns and cell sensitivity to Imatinib and oxidative stress. In this study, BCL-XL and GSH have been identified as the major survival factors responsive to BCR-ABL-promoted cellular oxidative stress and play a dominant role in regulating the threshold of oxidative stress-induced apoptosis. Cell survival factors BCL-XL and BCL-2 differentially protect mitochondria under oxidative stress. BCL-XL is an essential survival factor in preventing excessive ROS-induced cell death while BCL-2 seems to play a relatively minor role. Furthermore, the redox modulating reagent β-phenethyl isothiocyanate (PEITC) has been found to efficiently deplete GSH and induce potent cell killing effects in drug-resistant CML cells. Combination of PEITC with BCL-XL/BCL2 inhibitor ABT737 or suppression of BCL-XL by BCR-ABL inhibitor Gleevec dramatically sensitizes CML cells to apoptosis. These results have suggested that elevation of BCL-XL and cellular GSH are important for the development of CML, and that redox-directed therapy is worthy of further clinical investigations in CML.

Seasonal aspects of leukemia

An investigation of (a) month/season-of-birth as a risk factor and (b) month/season-of-treatment initation as a prognostic factor in acute lymphoblastic leukemia (ALL) in children, 0-15 years of age, was conducted. The study population used was that of the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute and included children diagnosed and treated for ALL from 1973-1986. Two separate sets of analyses using different exclusion criteria led to similar results. Specifically, the inability to reject the null hypothesis of no significant difference in the variation of monthly/seasonal incidence rates among children residing within the 10 SEER sites using either cosinor analysis or one-way analysis of variance. No association was established between month/season of treatment initiation and survival in ALL among children using either Kaplan-Meier or cosinor analysis. In separate Kaplan-Meier analyses, age, gender, and treatment type were each found to be significant univariate prognostic factors for survival, however. ^

Leukemia and occupational exposure to electromagnetic fields: An incident case-control study

The existence of an association between leukemia and electromagnetic fields (EMF) is still controversial. The results of epidemiologic studies of leukemia in occupational groups with exposure to EMF are inconsistent. Weak associations have been seen in a few studies. EMF assessment is lacking in precision. Reported dose-response relationships have been based on qualitative levels of exposure to EMF without regard to duration of employment or EMF intensity on the jobs. Furthermore, potential confounding factors in the associations were not often well controlled. The current study is an analysis of the data collected from an incident case-control study. The primary objective was to test the hypothesis that occupational exposure to EMF is associated with leukemia, including total leukemia (TL), myelogenous leukemia (MYELOG) and acute non-lymphoid leukemia (ANLL). Potential confounding factors: occupational exposure to benzene, age, smoking, alcohol consumption, and previous medical radiation exposures were controlled in multivariate logistic regression models. Dose-response relationships were estimated by cumulative occupational exposure to EMF, taking into account duration of employment and EMF intensity on the jobs. In order to overcome weaknesses of most previous studies, special efforts were made to improve the precision of EMF assessment. Two definitions of EMF were used and result discrepancies using the two definitions were observed. These difference raised a question as to whether the workers at jobs with low EMF exposure should be considered as non-exposed in future studies. In addition, the current study suggested use of lifetime cumulative EMF exposure estimates to determine dose-response relationship. The analyses of the current study suggest an association between ANLL and employment at selected jobs with high EMF exposure. The existence of an association between three types of leukemia and broader categories of occupational EMF exposure, is still undetermined. If an association does exist between occupational EMF exposure and leukemia, the results of the current study suggest that EMF might only be a potential factor in the promotion of leukemia, but not its initiation. ^

Inhibition of myogenesis by transforming growth factor β is density-dependent and related to the translocation of transcription factor MEF2 to the cytoplasm

Transforming growth factor β (TGF-β) was found to inhibit differentiation of myogenic cells only when they were grown to high density. Inhibition also occurred when myogenic cells were cocultured with other types of mesenchymal cells but not when they were cocultured with epithelial cells. It is therefore possible that some density-dependent signaling mediates the intracellular response to TGF-β. Within 30 min of treatment, TGF-β induced translocation of MEF2, but not MyoD, myogenin, or p21, to the cytoplasm of myogenic cells grown to high density. Translocation was reversible on withdrawal of TGF-β. By using immune electron microscopy and Western blot analysis on subcellular fractions, MEF2 was shown to be tightly associated with cytoskeleton membrane components. To test whether MEF2 export from the nucleus was causally related to the inhibitory action of TGF-β, we transfected C2C12 myoblasts with MEF2C containing the nuclear localization signal of simian virus 40 large T antigen (nlsSV40). Myogenic cells expressing the chimerical MEF2C/nlsSV40, but not wild-type MEF2C, retained this transcription factor in the nucleus and were resistant to the inhibitory action of TGF-β. We propose a mechanism in which the inhibition of myogenesis by TGF-β is mediated through MEF2 localization to the cytoplasm, thus preventing it from participating in an active transcriptional complex.

SMRT corepressor interacts with PLZF and with the PML-retinoic acid receptor α (RARα) and PLZF-RARα oncoproteins associated with acute promyelocytic leukemia

Retinoic acid receptors (RARs) are hormone-regulated transcription factors that control key aspects of normal differentiation. Aberrant RAR activity may be a causal factor in neoplasia. Human acute promyelocytic leukemia, for example, is tightly linked to chromosomal translocations that fuse novel amino acid sequences (denoted PML, PLZF, and NPM) to the DNA-binding and hormone-binding domains of RARα. The resulting chimeric receptors have unique transcriptional properties that may contribute to leukemogenesis. Normal RARs repress gene transcription by associating with ancillary factors denoted corepressors (also referred to as SMRT, N-CoR, TRAC, or RIP13). We report here that the PML-RARα and PLZF-RARα oncoproteins retain the ability of RARα to associate with corepressors, and that this corepressor association correlates with certain aspects of the leukemic phenotype. Unexpectedly, the PLZF moiety itself can interact with SMRT corepressor. This interaction with corepressor is mediated, in part, by a POZ motif within PLZF. Given the presence of POZ motifs in a number of known transcriptional repressors, similar interactions with SMRT may play a role in transcriptional silencing by a variety of both receptor and nonreceptor transcription factors.

The inv(16) encodes an acute myeloid leukemia 1 transcriptional corepressor

The inv(16) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). The inv(16) fusion protein acts by dominantly interfering with AML-1/core binding factor β-dependent transcriptional regulation. Here we demonstrate that the inv(16) fusion protein cooperates with AML-1B to repress transcription. This cooperativity requires the ability of the translocation fusion protein to bind to AML-1B. Mutational analysis and cell fractionation experiments indicated that the inv(16) fusion protein acts in the nucleus and that repression occurs when the complex is bound to DNA. We also found that the inv(16) fusion protein binds to AML-1B when it is associated with the mSin3A corepressor. An AML-1B mutant that fails to bind mSin3A was impaired in cooperative repression, suggesting that the inv(16) fusion protein acts through mSin3 and possibly other corepressors. Finally, we demonstrate that the C-terminal portion of the inv(16) fusion protein contains a repression domain, suggesting a molecular mechanism for AML-1-mediated repression.

The CXC chemokine stromal cell-derived factor 1 is not responsible for CD8+ T cell suppression of syncytia-inducing strains of HIV-1

Primary CD8+ T cells from HIV+ asymptomatics can suppress virus production from CD4+ T cells acutely infected with either non-syncytia-inducing (NSI) or syncytia-inducing (SI) HIV-1 isolates. NSI strains of HIV-1 predominantly use the CCR5 chemokine receptor as a fusion cofactor, whereas fusion of T cell line-adapted SI isolates is mediated by another chemokine receptor, CXCR4. The CCR5 ligands RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein 1α (MIP-1α), and MIP-1β are HIV-1 suppressive factors secreted by CD8+ cells that inhibit NSI viruses. Recently, the CXC chemokine stromal cell-derived factor 1 (SDF-1) was identified as a ligand for CXCR4 and shown to inhibit SI strains. We speculated that SDF-1 might be an effector molecule for CD8+ suppression of SI isolates and assessed several SDF-1 preparations for inhibition of HIV-1LAI-mediated cell–cell fusion, and examined levels of SDF-1 transcripts in CD8+ T cells. SDF-1 fusion inhibitory activity correlated with the N terminus, and the α and β forms of SDF-1 exhibited equivalent fusion blocking activity. SDF-1 preparations having the N terminus described by Bleul et al. (Bleul, C.C., Fuhlbrigge, R.C., Casasnovas, J.M., Aiuti, A. & Springer, T.A. (1996) J. Exp. Med. 184, 1101–1109) readily blocked HIV-1LAI-mediated fusion, whereas forms containing two or three additional N-terminal amino acids lacked this activity despite their ability to bind and/or signal through CXCR4. Though SDF-1 is constitutively expressed in most tissues, CD8 T cells contained extremely low levels of SDF-1 mRNA transcripts (<1 transcript/5,000 cells), and these levels did not correlate with virus suppressive activity. We conclude that suppression of SI strains of HIV-1 by CD8+ T cells is unlikely to involve SDF-1.