Novel targeted mtLivin nanoparticles treatment for disseminated diffuse large B-cell lymphoma.

We previously showed that Livin, an inhibitor of apoptosis protein, is specifically cleaved to produce a truncated protein, tLivin, and demonstrated its paradoxical proapoptotic activity. We further demonstrated that mini-tLivin (MTV), a 70 amino acids derivative of tLivin, is a proapoptotic protein as potent as tLivin. Based on these findings, in this study we aimed to develop a venue to target MTV for the treatment of diffuse large B-cell lymphoma (DLBCL). MTV was conjugated to poly (lactide-co-glycolic acid) surface-activated nanoparticles (NPs). In order to target MTV-NPs we also conjugated CD40 ligand (CD40L) to the surface of the NPs and evaluated the efficacy of the bifunctional CD40L-MTV-NPs. In vitro, CD40L-MTV-NPs elicited significant apoptosis of DLBCL cells. In a disseminated mouse model of DLBCL, 37.5% of MTV-NPs treated mice survived at the end of the experiment. Targeting MTV-NPs using CD40L greatly improved survival and 71.4% of these mice survived. CD40L-MTV-NPs also greatly reduced CNS involvement of DLBCL. Only 20% of these mice presented infiltration of lymphoma to the brain in comparison to 77% of the MTV-NPs treated mice. In a subcutaneous mouse model, CD40L-MTV-NPs significantly reduced tumor volume in correlation with significant increased caspase-3 activity. Thus, targeted MTV-NPs suggest a novel approach to overcome apoptosis resistance in cancer.

SNP variations in IL10, TNFα and TNFAIP3 genes in patients with dry eye syndrome and Sjogren's syndrome.

BACKGROUND: Cytokines are known to be key players in dry eye syndrome (DES) and Sjogren's syndrome (SS) pathogenesis. In this study we compared single nucleotide polymorphism (SNP) variations in genes encoding cytokine levels among SS and DES patients in Israel. METHODS: We recruited 180 subjects, 82 with SS and 98 with DES. Using a candidate gene approach and allele-specific PCR technique for genotyping, proportions of risk alleles in Tumor Necrosis Factor α (TNFα) (rs1800629), IinterLeukin-10 (IL-10) (rs1800896) and TNFAIP3 (rs2230926) SNPs were compared between study groups. RESULTS: Allelic distribution was found very similar to Caucasian (CEU - Utah residents with Northern and Western European roots) population distributions in these SNPs. While none of the SNPs' variants were significantly associated with SS or DES in a recessive model, in an additive model the TNFα G risk allele was found higher among SS patients compared to DES (Homozygote-G: 84.2% vs. 70.8%; Heterozygote: 26.9% vs. 11.2%, respectively, p = 0.02). After adjustment for age, gender and ethnicity, these variants weren't associated with SS. Genetic scoring reveals that SS patients are more likely to present variants of all three SNPs than DES subjects. CONCLUSIONS: This is the first study evaluating these SNP variations among both patients with DES and patients with SS. We found the allelic distribution in each SNP to be very similar to that found in healthy Caucasian populations presented in the HapMap project. We found the TNFα allele significantly associated with DES for homozygotes, and associated with SS for heterozygotes in the additive model.

Serum organochlorines and non-Hodgkin lymphoma: A case-control study in Israeli Jews and Palestinians.

Associations of organochlorine (OC) pesticides and polychlorinated biphenyls (PCBs) with non-Hodgkin lymphoma are controversial. We compared serum levels of 6 OC pesticides and 38 PCBs in Israeli Jews (IJ) and Palestinian Arabs (PA) and assessed possible associations with B-cell non-Hodgkin lymphoma (B-NHL). Ninety B-NHL cases (50 IJ and 40 PA) and 120 controls (65 IJ and 55 PA) were included. Median concentrations of analytes in controls were compared across ethnic groups using quantile regression, adjusting for age and sex. We used logistic regression to derive odds ratios (OR) and 95% confidence intervals (CI) for detectable analytes and B-NHL, adjusting for age, ethnic group, faming and body mass index. Median values of PCBs and dichlorodiphenyldichloroethylene (DDE) were higher in IJ vs PA controls (P = 0.0007), as were several PCBs (74, 99, 118, 138, 146, 153, 156, 163, 170, and 180). Overall, OC pesticide and PCB exposures were comparable with reports from high-income countries. B-NHL was associated with PCB 146 (OR 1.70, 95% CI: 1.02, 2.83), PCB 156 (OR 1.75, 95% CI: 1.06, 2.89), and high-chlorinated PCBs (OR 1.55, 95% CI: 1.00, 2.40) in all study subjects. These associations were robust in quantile as well as sensitivity analyses. An association of DDE with B-NHL was noted in PA (OR 1.72, 95% CI: 1.07, 2.77), but not in IJ (OR 0.87, 95% CI: 0.59, 1.27). Although high-chlorinated PCB concentrations did not indicate high exposure levels, our findings indicate that B-NHL may be associated with this exposure.

Presence of autoimmune disease affects not only risk but also survival in patients with B-cell non-Hodgkin lymphoma.

Although autoimmune diseases (AIDs) are known to predispose to non-Hodgkin lymphoma (NHL), their association with NHL prognosis has rarely been investigated. We examined associations between autoimmunity and B-cell NHL onset by comparing AID history (determined by self-report and medication review and supplemented by chart review where possible) among 435 adult B-NHL patients in Hadassah-Hebrew University Medical Center, diagnosed 2009-2014, and 414 age-and-sex frequency-matched controls. We examined AIDs as a whole, B- and T-cell-mediated AIDs, and autoimmune thyroid diseases. Among cases, we used Kaplan-Meier and Cox regression models to assess the association of AID with overall survival and relapse-free survival, adjusting for prognostically important patient and disease characteristics such as Ki67% staining, International Prognostic Index, rituximab treatment, and histological subgroup. Autoimmune diseases were associated with B-NHL (odds ratio [OR] = 1.95; 95% confidence interval (CI), 1.31-2.92), especially AIDs mediated by B-cell activation (OR = 5.20; CI, 1.90-14.3), which were particularly associated with marginal zone lymphoma (OR = 19.3; CI, 4.59-80.9). We found that time to relapse for all B-NHL patients with AIDs was significantly shorter (mean of 49.21 mo [±3.22]) than among patients without AID (mean of 59.74 mo [±1.62]), adjusted hazard ratio [HRadj ] = 1.69 (CI, 1.03-2.79). Specifically, in patients with diffuse large B-cell lymphoma, of whom 91.8% had received rituximab, a history of B-cell-mediated AIDs was associated with shorter relapse-free survival and overall survival, HRadj  = 8.34 (CI, 3.01-23.1) and HRadj  = 3.83 (CI, 1.20-12.3), respectively. Beyond confirming the well-known association between AIDs and B-NHL, we found that AID is an adverse prognostic factor in B-cell lymphoma, associated with a shortened time to relapse, suggesting that there are specific therapeutic challenges in the subgroup of patients suffering from both these diseases. Further work is required to address mechanisms of resistance to standard treatment in the setting of AID-associated B-NHL. In the era of immunotherapy, these findings have particular relevance.

Gene and protein analysis reveals that p53 pathway is functionally inactivated in cytogenetically normal Acute Myeloid Leukemia and Acute Promyelocytic Leukemia.

BACKGROUND: Mechanisms that inactivate the p53 pathway in Acute Myeloid Leukemia (AML), other than rare mutations, are still not well understood. METHODS: We performed a bioinformatics study of the p53 pathway function at the gene expression level on our collection of 1153 p53-pathway related genes. Publically available Affymetrix data of 607 de-novo AML patients at diagnosis were analyzed according to the patients cytogenetic, FAB and molecular mutations subtypes. We further investigated the functional status of the p53 pathway in cytogenetically normal AML (CN-AML) and Acute Promyelocytic Leukemia (APL) patients using bioinformatics, Real-Time PCR and immunohistochemistry. RESULTS: We revealed significant and differential alterations of p53 pathway-related gene expression in most of the AML subtypes. We found that p53 pathway-related gene expression was not correlated with the accepted grouping of AML subtypes such as by cytogenetically-based prognosis, morphological stage or by the type of molecular mutation. Our bioinformatic analysis revealed that p53 is not functional in CN-AML and APL blasts at inducing its most important functional outcomes: cell cycle arrest, apoptosis, DNA repair and oxidative stress defense. We revealed transcriptional downregulation of important p53 acetyltransferases in both CN-AML and APL, accompanied by increased Mdmx protein expression and inadequate Chk2 protein activation. CONCLUSIONS: Our bioinformatic analysis demonstrated that p53 pathway is differentially inactivated in different AML subtypes. Focused gene and protein analysis of p53 pathway in CN-AML and APL patients imply that functional inactivation of p53 protein can be attributed to its impaired acetylation. Our analysis indicates the need in further accurate evaluation of p53 pathway functioning and regulation in distinct subtypes of AML.

Ethnic variation in medical and lifestyle risk factors for B cell non-Hodgkin lymphoma: A case-control study among Israelis and Palestinians.

BACKGROUND: Risk factors for B-cell non-Hodgkin lymphoma (B-NHL) have not been assessed among Palestinian Arabs (PA) and Israeli Jews (IJ). METHODS: In a case-control study we investigated self-reported medical and lifestyle exposures, reporting odds ratios (ORs) and 95% confidence intervals [CIs], by ethnicity, for overall B-NHL and subtypes. RESULTS: We recruited 823 cases and 808 healthy controls. Among 307 PA/516 IJ B-NHL cases (mean age at diagnosis = 51 [±17] versus 60 [±15] years, respectively) subtype distributions differed, with diffuse large B-cell lymphoma (DLBCL) being prominent among PA (71%) compared to IJ (41%); follicular lymphoma (FL), was observed in 14% versus 28%, and marginal zone lymphoma, in 2% versus 14%, respectively. Overall B-NHL in both populations was associated with recreational sun exposure OR = 1.43 [CI:1.07-1.91], black hair-dye use OR = 1.70 [CI:1.00-2.87], hospitalization for infection OR = 1.68 [CI:1.34-2.11], and first-degree relative with hematopoietic cancer, OR = 1.69 [CI:1.16-2.48]. An inverse association was noted with alcohol use, OR = 0.46 [CI:0.34-0.62]. Subtype-specific exposures included smoking (FL, OR = 1.46 [CI:1.01-2.11]) and >monthly indoor pesticide use (DLBCL, OR = 2.01 [CI:1.35-3.00]). Associations observed for overall B-NHL in PA only included: gardening OR = 1.93 [CI:1.39-2.70]; history of herpes, mononucleosis, rubella, blood transfusion (OR>2.5, P<0.01 for all); while for IJ risk factors included growing fruits and vegetables, OR = 1.87 [CI:1.11-3.15]; and self-reported autoimmune diseases, OR = 1.99 [CI:1.34-2.95]. CONCLUSIONS: In these geographically proximate populations we found some unique risk factors for B-NHL. Heterogeneity in the observed associations by ethnicity could reflect differences in lifestyle, medical systems, and reporting patterns, while variations by histology infer specific etiologic factors for lymphoma subtypes.

The JAK2V617F mutation in normal individuals takes place in differentiating cells.

The JAK2V617F mutation that results in a hyper-activation of the JAK2 kinase in the erythropoietin pathway is a molecular marker for myeloproliferative neoplasms. Using allele-specific Real-Time PCR, we detected the mutation in the blood of 17.3% (17/98) of normal donors; the mutant allele burden was, however, very low (<0.01% compared to >1% in polycythemia vera). It was much higher in differentiated blood cells in the peripheral blood than in undifferentiated CD34+ cells. Erythropoietin-stimulated differentiation of normal CD34+ cells in liquid culture increased the mutation frequency by 3.34-fold. When progenitors from 9 normal donors were grown in erythropoietin-stimulated semi-solid cultures, the mutation was found in 8.69% of the colonies, but only in <3% of the JAK2 alleles in each positive colony, suggesting that the mutation occurred only in a few cells per colony. In mouse erythroleukemia cells carrying human JAK2 DNA, wild-type or JAK2V617F, the frequencies of mutations from JAK2 wild-type to JAK2V617F and vice versa increased following erythroid differentiation. These results suggest that the mutation occurs and accumulates during differentiation. We hypothesize that genetic stability, which relies on DNA repair, is efficient in normal hematopoietic stem cells but is downgraded in differentiating cells, rendering them susceptible to mutations, including JAK2V617F.

Transmembrane signaling in Saccharomyces cerevisiae as a model for signaling in metazoans: state of the art after 25 years.

In the very first article that appeared in Cellular Signalling, published in its inaugural issue in October 1989, we reviewed signal transduction pathways in Saccharomyces cerevisiae. Although this yeast was already a powerful model organism for the study of cellular processes, it was not yet a valuable instrument for the investigation of signaling cascades. In 1989, therefore, we discussed only two pathways, the Ras/cAMP and the mating (Fus3) signaling cascades. The pivotal findings concerning those pathways undoubtedly contributed to the realization that yeast is a relevant model for understanding signal transduction in higher eukaryotes. Consequently, the last 25 years have witnessed the discovery of many signal transduction pathways in S. cerevisiae, including the high osmotic glycerol (Hog1), Stl2/Mpk1 and Smk1 mitogen-activated protein (MAP) kinase pathways, the TOR, AMPK/Snf1, SPS, PLC1 and Pkr/Gcn2 cascades, and systems that sense and respond to various types of stress. For many cascades, orthologous pathways were identified in mammals following their discovery in yeast. Here we review advances in the understanding of signaling in S. cerevisiae over the last 25 years. When all pathways are analyzed together, some prominent themes emerge. First, wiring of signaling cascades may not be identical in all S. cerevisiae strains, but is probably specific to each genetic background. This situation complicates attempts to decipher and generalize these webs of reactions. Secondly, the Ras/cAMP and the TOR cascades are pivotal pathways that affect all processes of the life of the yeast cell, whereas the yeast MAP kinase pathways are not essential. Yeast cells deficient in all MAP kinases proliferate normally. Another theme is the existence of central molecular hubs, either as single proteins (e.g., Msn2/4, Flo11) or as multisubunit complexes (e.g., TORC1/2), which are controlled by numerous pathways and in turn determine the fate of the cell. It is also apparent that lipid signaling is less developed in yeast than in higher eukaryotes. Finally, feedback regulatory mechanisms seem to be at least as important and powerful as the pathways themselves. In the final chapter of this essay we dare to imagine the essence of our next review on signaling in yeast, to be published on the 50th anniversary of Cellular Signalling in 2039.

tLivin displays flexibility by promoting alternative cell death mechanisms.

Livin is a member of the Inhibitor of Apoptosis (IAP) protein family that inhibits apoptosis triggered by a variety of stimuli. We previously demonstrated that while Livin inhibits caspase activity, caspases can cleave Livin to produce a truncated protein, tLivin and that this newly formed tLivin paradoxically induces cell death. However to date, the mechanism of tLivin-induced cell death is not fully understood. In this study, we set out to characterize the form of cell death mediated by tLivin. Here we demonstrate that, unlike most death-promoting proteins, tLivin is a flexible inducer of cell death capable of promoting necrosis or apoptosis in different cell lines. The unusual flexibility of tLivin is displayed by its ability to activate an alternative form of cell death when apoptosis is inhibited. Thus, tLivin can promote more than one form of cell death in the same cell type. Interestingly, in cells where tLivin induces necrosis, deletion of the caspase binding BIR domain results in tLivin-induced apoptosis, suggesting the BIR domain can potentially hamper the ability of tLivin to induce apoptosis. We further elucidate that tLivin activates the JNK pathway and both tLivin-induced apoptosis and necrosis are partially mediated by JNK activity. Acquired resistance to apoptosis, common in many tumors, impinges on the efficiency of conventional anti-cancer agents that function primarily by inducing apoptosis. The ability of tLivin to induce death of apoptosis-compromised cells makes it an attractive candidate for targeted cancer therapy.

Extracellular matrix constituents interfere with Newcastle disease virus spread in solid tissue and diminish its potential oncolytic activity.

Advanced melanoma cells, characterized by resistance to chemotherapy, have been shown to be highly sensitive to oncolysis by Newcastle disease virus (NDV). In the present study, we investigated the capacity of NDV to specifically infect and spread into solid tissues of human melanoma and lung carcinoma, in vivo and ex vivo. For this purpose a new model of SCID-beige mice implanted with human melanoma was developed. Surprisingly, the replication competent NDV-MTH and the attenuated, single-cycle replication NDV-HUJ strains, demonstrated a similar oncolytic activity in the melanoma-implanted mice. Further, ex vivo analysis, using organ cultures derived from the melanoma tissues indicated a limited spread of the two NDV strains in the tissue. Extracellular matrix (ECM) molecules, notably heparin sulfate and collagen, were found to limit viral spread in the tissue. This observation was validated with yet another solid tumour of human lung carcinoma. Taken together, the results indicate that the ECM acts as a barrier to virus spread within solid tumour tissues and that this restriction must be overcome to achieve effective oncolysis with NDV.

The clinical effect of the inhibitor of apopotosis protein livin in melanoma.

OBJECTIVE: The inhibitor of apoptosis protein (IAP) livin is frequently overexpressed in melanoma. Livin binds caspases and thereby inhibits apoptosis. We found that caspases cleave livin to produce a truncated form with a paradoxical proapoptotic activity. METHODS: We assessed the correlation of livin expression with survival among 114 melanoma patients treated with an autologous melanoma vaccine. In 52 patients, resection resulted in no evidence of disease (NED) and 62 remained with active disease (WAD). Protein levels were assessed using Western blot. RESULTS: We found livin protein expression in 44/114 samples (38.4%). Median overall survival was 1.4 years in NED patients with high levels of livin protein, 8.4 years in those with low-intermediate levels and not reached in patients who did not express livin (p = 0.025). The corresponding overall survival was 2.3 years among WAD patients with high levels of livin protein, 11.3 years in those with low-intermediate levels and, paradoxically, only 4.0 years in patients who did not express livin (p = 0.012). CONCLUSION: Livin protein expression may play a role in the progression of melanoma and correlates with survival. A high level of the protein is associated with a poor prognosis. However, in WAD patients low to intermediate level of livin, rather than absence of the protein, is associated with a favorable prognosis. This is probably due to the paradoxical proapoptotic activity of this important regulator of apoptosis.

Janus Kinase V617F mutation in cigarette smokers.

The JAK2 V617F mutation is responsible for the constitutive activation of the erythropoietin receptor signaling pathway in most cases of polycythemia vera (PV). The mutation has also been described in healthy people. As smoking may result in secondary polycythemia, the goal of this trial was to examine the effect of smoking on the prevalence of the JAK2 mutation and its correlation to erythrocytosis. The study was case-control. Hospitalized smokers (n = 81) and nonsmokers (n = 61) were recruited. Serum was drawn for complete blood count, erythropoietin, ferritin and venous blood gases. JAK2 mutation was analyzed by highly sensitive allele-specific Quantitative Real Time PCR. The JAK2 mutation was found in 29/81 (35.8%) of smokers in comparison to only 9/61 (14.8%) of the control group (P = 0.007). The frequency of the mutation among smokers who were positive for the JAK2 mutation had a mean of 6.78 × 10(-4) ± 1.08 × 10(-3) vs. 1.51 × 10(-4) ± 2.04 × 10(-4) among nonsmokers (P = 0.027). Both frequencies are much lower than those found in PV. There was a medium correlation between older age and mutation frequency in nonsmokers (r= 0.67, P = 0.043). Hematocrit was higher in smokers (47.8 ± 6 vs. 41.7 ± 4.7, P < 0.0001), but no correlation was found to JAK2 mutation. In a cohort of hospitalized smokers and nonsmokers, JAK2 mutation was more prevalent and found in higher frequencies among smokers than nonsmokers. We suggest that accelerated erythropoiesis renders the cells susceptible to JAK2 mutation.

The oncolytic activity of Newcastle disease virus NDV-HUJ on chemoresistant primary melanoma cells is dependent on the proapoptotic activity of the inhibitor of apoptosis protein Livin.

Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. In contrast, melanoma tumor cells that do not express Livin are relatively resistant to NDV-HUJ treatment. Furthermore, we show that NDV-HUJ-induced oncolysis is attributed to the dual function of Livin: although Livin inhibits apoptosis through the inhibition of caspases, under the robust apoptotic stimulation of NDV-HUJ, caspases can cleave Livin to create a truncated protein with a paradoxical proapoptotic activity. Thus, NDV-HUJ is a potent inducer of apoptosis that can overcome the antiapoptotic effect of Livin and allow cleavage of Livin into the proapoptotic tLivin protein. Moreover, the results indicate that the interferon system, which is functional in melanoma, is not involved in NDV-induced oncolysis. Taken together, our data offer the possibility of a new viral oncolytic treatment for chemoresistant melanoma.

The inhibitor of apoptosis protein Livin (ML-IAP) plays a dual role in tumorigenicity.

The inhibitor of apoptosis protein (IAP) family can inhibit apoptosis induced by a variety of stimuli. We and others previously described the IAP Livin (ML-IAP). We found that Livin is unique among the IAP members as, on a strong apoptotic stimulus, it is specifically cleaved by caspases to produce a truncated protein with paradoxical proapoptotic activity (tLivin). We also showed that Livin encodes two splicing variants, termed Livin alpha and beta, with diverse antiapoptotic effects in vitro. In this study, we compared the Livin isoforms in vivo. An animal model was established and the effects of Livin alpha and beta on the initiation and development of tumors were compared. In the animal model, Livin alpha promotes tumor initiation in comparison with control. Interestingly, the growth of tumors originating from cells expressing Livin beta was inhibited. In these tumors, Livin beta was cleaved and produced a high level of the proapoptotic tLivin beta that repressed tumor development. When we eliminated the proapoptotic effect of Livin beta by point mutations, the resulting antiapoptotic Livin beta mutants contributed to tumor progression. In terms of mechanism, we show that Livin beta tumors develop only in mice lacking natural killer (NK) cell activity. Thus, from the animal model, we can conclude that Livin plays a major role in tumorigenicity and that NK cells induce cleavage of Livin to its proapoptotic truncated protein that in turn inhibits tumor growth. Therefore, Livin and tLivin may serve as potential targets for cancer therapy.

Subcellular localization determines the delicate balance between the anti- and pro-apoptotic activity of Livin.

Livin is a member of the Inhibitor of Apoptosis Protein family which inhibits apoptosis induced by a variety of stimuli. We previously identified Livin and demonstrated that following apoptotic stimuli, Livin is cleaved by effector caspases to produce a truncated form with paradoxical pro-apoptotic activity. In the present study, we reveal that while full-length Livin shows diffuse cytoplasmic localization, truncated Livin (tLivin) is found in a peri-nuclear distribution with marked localization to the Golgi apparatus. Using mutation analysis, we identified two domains that are crucial for the pro-apoptotic activity of tLivin: the N-terminal region of tLivin which is exposed by cleavage, and the RING domain. We demonstrate that, of the N-terminal sequence, only the first N-terminal glycine residue dictates the peri-nuclear distribution of tLivin. However, while the perinuclear localization of tLivin is essential, it is not sufficient for tLivin to exert its pro-apoptotic function. Once tLivin is properly localized, an intact RING domain enables its pro-apoptotic function.