bluuebunny/crossref_metadata_2025 huggingface dataset:
Co-Administration of SAHA and 17-AAG Synergistically Induces Apoptosis in Bcr-Abl+ Cells Sensitive and Resistant to STI-571 in Association with Down-Regulation of Bcr-Abl, Abrogation of STAT5 Activity, and Bax Conformational Change.
Authors: Rahmani Mohamed , Reese Erin , Dai Yun , Bauer Cheryl , Kramer Lora , Huang Mei , Jove Richard , Dent Paul , Grant Steven
Published: October 2019

Interactions between the histone deacetylase inhibitors (HDACIs) suberanoylanilide hydroxamic acid (SAHA) and sodium butyrate (SB) and the heat shock protein 90 (Hsp90) antagonist 17-AAG have been examined in Bcr-Abl+ human leukemia cells (K562 and LAMA84), including those sensitive and resistant to STI571. Co-administration of 17-AAG with SAHA or SB for 24 h synergistically induced mitochondrial dysfunction (cytochrome c and AIF release), caspase-3 and -8 activation, apoptosis, and growth inhibition. Similar effects were observed in LAMA84 cells and K562 cells resistant to STI-571 as well as in CD34+ leukemic cells obtained from patients with CML including those who had developed a resistance against STI-571. These events were associated with increased association of Bcr/abl, Raf-1, and Akt with Hsp70, whereas the total level of these proteins markedly decreased. A pronounced inactivation of ERK1/2, and activation of JNK were also observed. In addition, 17-AAG/SAHA abrogated DNA binding and transcriptional activities of STAT5 in K562 cells, including those ectopically expressing a constitutively active STAT5A construct. Co-administration of 17-AAG and SAHA also induced downregulation of Mcl-1, Bcl-xL, B-Raf, and p21CIP1, upregulation of Bak, cleavage of 14-3-3 proteins, and a profound conformational change in Bax accompanied by translocation to the membrane fraction. Together, these findings raise the possibility that combining HDACIs with the Hsp90 antagonist 17-AAG may represent a novel strategy against Bcr-Abl+ leukemias, including those resistant to STI571.
Newly Discovered Polymorphism in the CD34+ Stem Cell Specific AC133-P1 Promoter Linked to Leukemias.
Authors: Shmelkov Sergey V. , St.Clair Ryan , Jun Lin , Calderone Agata , Karajannis Matthias A. , Jensen Kristian K. , Young Lauren M. , Walsh Kathryn , Shido Koji , Jin David , Zhang Fan , Cheng Joseph , Lam George , Tejada Rafael , Avecilla Scott T. , Vincent Loic , Kopp Hans-Georg , Akiri Gal , Lyden David , Rafii Shahin
Published: October 2019

AC133 (CD133, human Prominin-1) is a cell-surface glycoprotein which is expressed on several types of stem cells, including hematopoietic stem cells and endothelial progenitor cells. AC133 is also expressed on acute and chronic myeloid leukemias, as well as lymphoblastic leukemias. AC133 expression is correlated with a poor prognosis in cases of leukemia; in addition, preliminary observations suggest that AC133 may define a population of leukemic stem cells. We previously identified and characterized the AC133 promoter region, and demonstrated that transcription of AC133 is driven by five alternative promoters in a tissue-dependent manner, resulting in the formation of multiple alternatively spliced isoforms. CD34+ hematopoietic stem cells were found to utilize only one of the five promoters, AC133-P1. Furthermore, preliminary data suggest that hematopoietic stem cells express only one AC133 isoform. We identified four allelic variants in the AC133-P1 promoter, 250 bp upstream of the transcription initiation point, which differ by the number of short tandem repeats as well as by a single nucleotide polymorphism (SNP). One allele, (CAA)3CAG, is found in patients with AML, chloroma, and myeloma. Two different alleles, (CAA)4 and (CAAAA)5, are found in cancer cell lines derived from AML, retinoblastoma, colon cancer, and sarcoma. One allele, (CAA)5, is found only in healthy human subjects. Our preliminary results show that these different alleles may affect the activity of the AC133-P1 promoter. These data might lay a foundation for testing a risk of susceptibility to AC133+ leukemias. Additional investigations are required to establish the role of AC133 in emergence, development and survival of leukemias, as well as to explore the viability of AC133 as a primary therapeutic target in patients with aggressive AC133+ leukemia.
Vascular Endothelial Growth Factor 121 (VEGF121) Isoform mRNA Is Predictive of Poor Prognosis in Acute Myeloid Leukemia (AML) .
Authors: Calvo Fabien , Rousselot Philippe , Dombret Herve , Medioni Jacques , Mourah Samia
Published: October 2019

Bone marrow microvascular organisation has been shown to play an important role in Acute Myeloid Leukemia (AML). VEGF has four main isoforms produced by alternative splicing: VEGF121, VEGF165, VEGF189 and VEGF206. The VEGF121 protein which lacks the basic amino acids responsible for heparin binding, is the more soluble and potent isoform. The present work aimed at quantitatively measure the shortest transcript for soluble VEGF121 isoform by quantitative RT-PCR in peripheral blood mononuclear cells (PBMC) from AML patients and to evaluate its value as a prognostic marker for response to therapy and survival. We conducted a single institution prospective study in 67 consecutive AML patients at diagnosis. VEGF121 transcript levels in AML PBMC were significantly higher than in normal controls (25.9 copies/1000 copies of β2 microglobulin (β2m) compared to 1.9 copies in normal participants, p<0.001). No relation was found between VEGF121 level and sex, age, white blood cells counts or the dose intensity of aracytine during induction treatment. Both univariate and multivariate analysis of overall survival showed that high levels of VEGF121 transcripts (VEGF121 in AML patients > 5 copies of VEGF121/1000 copies of β2m; 25th centile, this cut-point were designed after systematic searches) were significantly related with a worse prognosis (p<0.0001 for univariate analysis and OR=11.6 [2.76–48.6, p=0.008 for multivariate analysis). Neither sex (p=0.08) nor age (p=0.90) nor WBC (p=0.96) nor caryotype (p=0.29), nor AraC dose intensity (p=0.29) were related with a bad prognosis in this group of patients. Analysing disease free survival, only high levels of VEGF121 transcripts were significantly related to a worse prognosis (p<0.0001, using univariate analysis). Of note, 94% of the patients who relapsed had an initial high level of VEGF121 transcripts. Our findings support the use of this test as a predictive and prognostic tool, helping the clinician to identify patients who should benefit for alternative therapeutic strategies. This test has advantages over ELISA or RIA monitoring cellular or plasma VEGF protein, since it is more specific of this isoform, independant of the number of circulating cells, of binding to α2 macroglobulin and of contaminating sources of VEGF such as platelets which sequester this cytokine. Monitoring of antiangiogenic treatment through QRT-PCR of VEGF121 could therefore be useful in these patients.
Proteomic Analysis of Acute Promyelocytic Leukemia Reveals That PML-RARalpha Induces Cell Cycle Progression and Mitotic Exit by Increased Expression and Decreased Ser63 Phosphorylation of OP18.
Authors: PeerZada A. , Geletu M. , Pullikan J. , Reddy V. , Hiddemann W. , Behre G.
Published: October 2019

We applied a mass spectrometry based approach to explore the proteins differentially regulated by PML-RARalpha, a translocation characteristic of acute promyelocytic leukemia (APL). Bioinformatic pathway analysis placed the 46 identified PML-RARalpha regulated proteins into three major networks, OP18-MAPK1, HSP-STAT3 and CCT-MYC. Using this approach, we were able to generate a common cell cycle network of the proteins in these pathways. Further analysis indicated that mRNA expression of OP18, which belonged to this network, was elevated in APL patients and the increased OP18 protein expression upon PML-RARalpha induction was overcome by retinoic acid treatment. Here we also report, for the first time a novel role of PML-RARalpha in cell cycle progression and mitotic exit. RNA interference experiments revealed that siRNA against OP18 overcomes PML-RARalpha effects on cell cycle progression. In addition to increased OP18 expression by PML-RARalpha, 2D gel electrophoresis revealed an isomer of OP18, subsequently confirmed by 2D-western as ser63 phosphomer to be downregulated by PML-RARalpha. Based on these findings, point mutation experiments indicated that decreased phosphorylation of ser63 in OP18 is important for PML-RARalpha mediated cell cycle and mitotic index effects since a constitutive phosphorylated mutant (ser63/asp) of OP18 overcame the PML-RARalpha effects in U9/PR cells, NB4 and APL patients. In summary, our results demonstrate that the effect of PML-RARalpha on cell cycle progression and mitotic exit is via two mechanisms: increasing the expression of OP18 and decreasing the phosphorylation of OP18 at ser63.
Identification of RIZ1 Targets Involvedin Erythroid Differentiation of K562 Human Erythroleukemia Cells Using Surface-Enhanced Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (SELDI).
Authors: Takahashi Naoto , Brainbridge Matthew , Scott Stuart A. , Ichinohasama Ryo , Sanche Stephen E. , Dong Wei-Feng , DeCoteau John F. , Geyer C. R.
Published: October 2019

RIZ1 (PRDM2) is a member of the nuclear protein methyltransferase superfamily involved in chromatin remodeling. RIZ1 functions as a tumor suppressor gene in a number of human cancers and is down regulated in some human acute myeloid leukemias. We previously found RIZ-1 to be silenced in K562 erythroleukemia cells by promoter hypermethylation. Furthermore, expression of RIZ1 in K562 promotes erythroid differentiation and also potentiates TGF-β1 mediated differentiation. To investigate similarities between genes altered by RIZ1 expression and the TGF-β1 pathway, we used SELDI to compare the protein profiles of K562 against K562 + RIZ1 and K562 + TGF-β1. Protein extracts for SELDI profiling were separated into six fractions according to their isoelectric points. Proteins from each fraction were then bound to two different protein chip surfaces (H50-hydrophoboic and CM10-cation exhange) and their mass/charge determined using SELDI. We analyzed four replicates from each sample and classified proteins as differentially expressed if their P-values were below 0.05. In total, we observed 104 differentially expressed proteins (60 upregulated and 44 down regulated) between K562 and K562 + RIZ1 and 176 proteins (96 upregulated and 80 down regulated) between K562 and K562 + TGF-β1. We used 2D-PAGE to identify differentially expressed proteins identified by SELDI analysis and located 48 proteins that were over expressed in K562 + RIZ1 and K562 + TGF-β1 relative to K562. To establish whether these proteins were the same proteins observed using SELDI, we determined if the proteins had the same pI and molecular weight and if the gel-eluted proteins bound to the same protein chip surface with the same mass/charge. 15 of 48 proteins passed the above criteria and we determined their identities using Trypsin-based peptide mapping strategies with molecular weight and pI restrictions. We identified two candidate proteins (14-3-3ε and S100/A13) that are similarly over expressed in K562 + RIZ1 and K562 + TGF-β1. These proteins have been shown to be associated with TGF-β1 signaling. Schistosomal 14-3-3ε interacts with SmRK1, a divergent type I transforming growth factor β1 receptor (TR-I) present on the surface of adult parasites and also binds to and activates human TR-I. S100/A13 belongs to a family of low molecular weight proteins characterized by the presence of two calcium-binding EF-hand motifs that includes S100C/A11, a member recently shown to play a key role in a PKCα mediated pathway essential for the growth inhibition of normal human keratinocytes by TGF-β1. In summary, we demonstrate the potential for using SELDI to identify novel proteins involved in regulating and connecting cellular growth and differentiation pathways.
Intra-S-Phase Regulator cdc7 Is a Gene Target of the c-Myb Proto-Oncogene in Human Leukemias.
Authors: Shetzline Susan E. , Hsu June , Thalheim Lindsay , Choi John K. , Gewirtz Alan M.
Published: October 2019

The c-myb proto-oncogene encodes a transcription factor, Myb, which is essential for normal hematopoiesis. Myb may also play a role in leukemogenesis but possible mechanisms remain ill defined. To gain further insights into this issue, we sought to identify Myb regulated genes in human myeloid leukemic cells utilizing a tamoxifen inducible expression system and a microarray approach. Myb function was conditionally abrogated by tamoxifen following infection of K562 cells with a bicistronic retroviral vector MIGR1 which had c-myb’s DNA binding domain (DBD) and the Drosophila engrailed protein transcription repressor domain (MERT) fused to a modified estrogen receptor that binds tamoxifen. MERT was subcloned upstream of an IRES-EGFP cassette in MIGR1 to allow for FACS purification on the basis of GFP expression. To identify Myb regulated genes, purified K562-MERT cells were exposed to 1 μM tamoxifen or ethanol (control) for three days, processed for hybridization to the microarray gene chip and analyzed by software algorithms from Incyte and Arrayex. When endogenous Myb activity was suppressed by MERT, 105 genes out of 10,000 genes on the microarray chip changed > 2-fold in expression. Of these 105 genes, 34 increased their expression >2-fold while 70 decreased their expression >2-fold. Since Myb expression is elevated in leukemic cells, we hypothesized that Myb functions in malignant hematopoietic cells to induce the expression of genes that are essential for their maintenance and survival. Therefore, we focused on those genes that decreased in expression when Myb activity was inhibited by MERT. Among the most repressed was cdc7 (2.8-fold decrease), an intra-S-phase regulator. To verify the microarray data, we utilized real-time PCR to quantitate the expression of cdc7 in our K562-MERT cells. Cdc7 expression decreased 5-fold in tamoxifen treated K562-MERT cells relative to control cells, which is consistent with the microarray data. We then performed chromatin immunoprecipitation (ChIP) experiments to determine whether cdc7 is a direct target of Myb. When the chromatin from untreated K562 and K562-MERT cells was immunoprecipitated with anti-c-Myb, we observed one PCR product using a primer pair that flanked the Myb binding sites in the promoter region of cdc7. This same result was observed in our positive control ChIP experiment in which the chromatin was immunoprecipitated with anti-acetyl histone H4, indicating that the region of the cdc7 promoter containing the Myb binding sites is poised for transcription. When Myb transactivation activity was inhibited by MERT in K562-MERT cells, no PCR product was observed following chromatin immunoprepitation with anti-c-Myb. These results strongly suggest that cdc7 is a direct gene target of c-Myb in malignant hematopoietic cells. Investigation of the transcriptional regulation of cdc7 in hematopoietic cells may yield new clues to Myb’s role in leukemogenesis.
Genes Located within the del(9q) Commonly Deleted Region Are Down-Regualted in Acute Myeloid Leukaemia.
Authors: Zhao Zheng , Daly Sarah , Yin John Liu , Sweetser David , Boultwood Jacqueline , Wainscoat James , Peniket Andrew
Published: October 2019

Deletions of 9q are recurring cytogenetic abnormalities in acute myeloid leukaemia (AML). In approximately one-third of cases del(9q) occurs in association with t(8;21). We have previously identified a 2.4Mb region located on 9q21.32–21.33 which is deleted in cases of del(9q) AML - the del(9q) commonly deleted region (CDR). This region encodes 11 genes which we have also previously shown not to be mutated in del(9q) AML. In order to further investigate the role of these genes in AML and in particular to elucidate the pathogenesis of del(9q) AML we have examined the expression of these genes in AML. RNA was extracted from the bone marrow or peripheral blood of patients with AML at the time of diagnosis. Patient samples from the following cytogenetic subgroups were included in this study:

(1) del(9q) AML (n=8) - this includes 3 patients with associated t(8;21); (2) t(8;21) but no del(9q) (n=15); (3) Normal karyotype (n=6); (4) Complex Karyotype (n=6). Taqman assays were designed for 9 of the 11 genes located within the del(9q) CDR: FRMD3; ENSG00000148057; UBQLN1; GKAP42; Q9UF54; Q8N2B1; Q9H9A7; SLC28A3; NTRK2. For the other 2 genes within the region Taqman assays could not be performed because of uniformly low expression levels (Q8IZ41) and lack of specificity of primer-design (HNRPK). CD34-purified progenitors from normal individuals were used as controls.

It was found that 6 of the 9 genes were significantly down-regulated in del(9q) AML (p<0.05): ENSG00000148057; UBQLN1; Q9UF54; Q8N2B1; Q9H9A7; NTRK2. Since del(9q) is commonly associated with t(8;21), cases of t(8;21) in which del(9q) was not present were also analysed for the expression levels of the del(9q) CDR genes. It was found that 5 of the 9 genes were significantly down regulated in t(8;21) AML (ENSG00000148057; Q9UF54; Q8N2B1; Q9H9A7; SLC28A3) (p<0.05). Only two of these genes were found to be down-regulated in AML of normal karyotype (Q9H9A7 and UBQLN1) (p<0.05) and no significant down-regulation was detected in any of these genes in AML of complex karyotype. Our findings indicate that several genes from within the del(9q) AML CDR are down-regulated in del(9q) AML. A similar pattern of down-regulation is found in cases of t(8;21) even in the absence of del(9q) AML. This suggests that down-regulation of one or more of these genes may be important in the pathogenesis of AML. It may therefore be hypothesized that this pattern of gene down-regulation provides a mechanism common to the development of AML with both del(9q) and t(8;21).
Silencing of Caspase 8 Expression in Leukemia Cells and Patient Samples.
Authors: Disperati Patricia , Suarez-Saiz Fernando , Gronda Marcela , Minden Mark D. , Schimmer Aaron
Published: October 2019

Gene silencing through hypermethylation is common in cancer cells and may contribute to chemoresistance and poor patient outcomes. To investigate this mechanism in leukemia, OCI-AML2 leukemia cells were screened for methylated genes. Among the genes identified using Cpg arrays was caspase-8. Levels of mRNA and protein of caspase-8 were decreased in OCI-AML2 cells compared to control cell lines (Jurkat cells). In contrast, levels of FLIP, caspase-3 and XIAP did not differ between both cell lines. As further evidence of caspase-8 hypermethylation, treatment of OCI-AML2 cells with the demethylating agent 5-Aza-2′-Deoxycytidine (AzaD) increased expression of caspase-8 protein to levels similar to Jurkat cells. To determine whether the silencing of caspase-8 occurs in primary patient samples, levels of caspase-8 were measured by immunoblots in 16 samples from patients with AML. Compared to normal peripheral blood mononuclear stem cells, caspase-8 was reduced in five of 16 (31%) samples. To determine whether the low caspase 8 was a functional consequence, OCI-AML2 cells were treated with CH-11 anti-FAS antibody (100 ng/mL) for 24 hours. Despite the cells expressing the FAS receptor on the surface, less than 5% of apoptosis occurred. In contrast, an equal concentration of CH-11 induced 80% apoptosis in Jurkat cells. To test the status of the death receptor pathway downstream of caspase-8, cytosolic lysates from OCI-AML2 cells were stimulated with recombinant active caspase-8 to directly activate effector caspases. Stimulation of OCI-AML2 lysates with recombinant active caspase-8 increased the maximal rate of effector caspase hydrolysis of Ac-DEVD-AFC 8 fold above buffer treated control, comparable to treatment of Jurkat lysates. These results indicate that OCI-AML2 cells have defects above the level of the effector casapses that renders them resistant to death receptor ligands. To determine if silencing of casp-8 is sufficient to explain these defects, OCI-AML2 cells were cultured with AzaD and CH-11 anti-FAS antibody. Despite restoration of caspase-8 protein levels, AzaD did not restore sensitivity to CH-11. Likewise, transfection of caspase-8 into OCI-AML2 did not restore sensitivity. Therefore, casp-8 is silenced in OCI-AML2 cells and primary patient samples. However, the events indicate the presence of additional defects in the death receptor pathway that cannot be reversed by demethylation. These results suggest that blockade of apoptosis throughout the death receptor pathway may occur at several points and that in some cases, simple reversal of demethylation may not be sufficient.
A Role for IL-1Receptor Antagonist in the Acute Therapeutic Effects of IVIg?.
Authors: Crow Andrew R. , Song Seng , Freedman John , Semple John W. , Lazarus Alan H.
Published: October 2019

It has been suggested that IVIg and anti-D may function through the regulation of various cytokines; in particular, significant increases in the anti-inflammatory compound interleukin-1 receptor antagonist (IL-1Ra) have been reported after exposure of patients to IVIg or anti-D. However, the possible role that IL-1Ra may play in the acute therapeutic effects of IVIg or anti-D is unknown. Using a murine model of ITP in which IVIg and anti-RBC-specific antibodies are therapeutically effective, we observed that mice injected with therapeutic doses of IVIg and one anti-RBC antibody (TER-119, which mimics the effects of anti-D) demonstrated increased serum levels of IL-1Ra, reaching peak levels at a time which coincided with an increase in platelet count. Surprisingly, another RBC-specific antibody which also reverses ITP, failed to increase IL-production above basal levels. Thrombocytopenic mice lacking the IL-1 receptor are known to be completely unresponsive to IL-1Ra. These mice responded to both IVIg and TER-119 as successfully as wild-type mice. Injection of mice with as much as 1 mg of recombinant IL-1Ra did not significantly ameliorate thrombocytopenia. To further study the possible indirect role of the IL-1Ra system in the amelioration of murine ITP, we next looked at nitric oxide (NO), an immunoregulatory compound that can be induced by IL-1, stimulate increases in IL-1Ra levels, and has been previously demonstrated to be modulated by IVIg. We report here that treatment of mice with aminoguanidine, an iNOS-selective inhibitor or with L-NAME, a multi-spectrum NOS inhibitor, did not significantly affect the ability of IVIg to ameliorate ITP. These data suggest that while IVIg and TER-119 may mediate the release of IL-1Ra, it is not required for IVIg or anti-RBC antibodies to exert their acute therapeutic effects.
Thrombocytopenia Is a Strong Predictor of All-Cause and AIDS-Specific Mortality in Women with HIV: The Women’s Interagency HIV Study.
Authors: Pearce C. Leigh , Mack Wendy J. , Levine Alexandra M. , Gravink Jay , Cohen Mardge H. , Machtinger Edward L. , Anastos Kathryn , Silverberg Michael J. , Young Mary A. , Minkoff Howard , Watts D. Heather
Published: October 2019

Background: Thrombocytopenia is a common condition among HIV-infected individuals, however its significance is unclear, particularly among women. Two previous studies, one consisting mostly of men (Sullivan PS, et al. J Acquir Immune Defic Syndr.1997;14:374–379) and one of hemophiliacs (Ehmann WC, et al. Am J Hematol.1997; 54:296–300), have suggested that low platelet count is associated with decreased survival.

Methods: The Women’s Interagency HIV Study (WIHS) is a long-term prospective cohort study of HIV-infected women and HIV-negative women that is being conducted at six urban sites across the United States. 1,990 HIV-infected women and 553 HIV-negative women are included in this report. These women are seen every six months; the median follow-up time is 7.5 years. We conducted extensive multivariate analysis using both generalized estimating equations and Cox proportional hazards models in order to determine the predictors of thrombocytopenia and the role of platelet count in mortality among women being followed as part of this study.

Results: At baseline, 15% of HIV-positive women were thrombocytopenic versus 1.6% of HIV-negative women (p<0.001). Factors associated with increased risk of thrombocytopenia included HIV infection, low CD4 cells, increasing viral load, and smoking. African-American women were significantly protected against thrombocytopenia when compared to Whites, as reported by others (Sloand EM, et al. Eur J Haematol. 1992; 48:168–72; Sullivan PS, et al. J Acquir Immune Defic Syndr.1997;14:374–379 ). Resolution of thrombocytopenia was associated with highly-active antiretroviral therapy (p<0.001), especially that containing zidovudine (<0.0001). On multivariate analysis, thrombocytopenia was a significant predictor of mortality, with women having a platelet count <50,000 cells/mm3 being at more than 5-fold increased risk of dying due to any cause, and at 3-fold increased risk of death due to AIDS compared to women with a platelet count in the normal range. Only CD4+ lymphocyte count <200 cells/mm3 was similar in the magnitude of its effect on mortality. The reasons for decreased survival associated with low platelet count in the context of HIV-infection are unclear and further study is needed.

Conclusions: (1) Thrombocytopenia is associated with HIV infection (p<0.001), and with parameters of more advanced HIV disease in women; (2) African American HIV + women are protected from thrombocytopenia compared to HIV + white women (p<0.0001); (3) HAART is associated with resolution of thrombocytopenia, especially those regimens including AZT (p<0.001); (4) Thrombocytopenia is an independent risk factor for decreased survival in HIV infected women.
Iron Containing Nucleosides as New Cytostatic Substances for Cancer and Leukemia Therapy, Especially for Therapy of Relapsed ALL in Childhoodtransfusion of Red Blood Cells under Shock Conditions in the Rat Microvasculature.
Authors: Prokop Aram , Wieder Thomas , Schlawe Daniel , Lode Holger , Henze Guenter , Daniel Peter T. , Schmalz Hans G.
Published: October 2019

Acute lymphoblastic leukemia (ALL) is the most common malignant disease in childhood. Despite a relatively good prognosis approximately one fourth of the patients suffers from relapse and, consequently, a worse prognosis. Only 40 % of these children with relapsed ALL will survive. Patients who fail to achieve a complete remission in chemotherapy do not survive. This explains the need for new compounds that may overcome resistance against clinically employed cytostatic drugs. Here we describe a new class of iron-containing nucleosides that overcome drug resistance in vitro and ex vivo. Analyses of apoptosis induction in primary lymphoblasts from 30 children with de novo ALL and relapsed ALL showed that these iron-containing nucleoside analogs are far more potent as compared with conventional nucleoside analogs employed in clinical therapy of childhood ALL (cytosinarabinoside: p < 0.0064). Furthermore we demonstrate ex vivo these novel nucleoside analogs overcome drug resistance against fludarabine (p<0.0162) and doxorubicin (p<0.002).

Additional experiments revealed that the prototype of iron-containing nucleoside analogs, N69, specifically induced apoptosis, as evidenced by DNA fragmentation, dissipation of the mitochondrial membrane potential and activation of caspase-3. All those events occurred in the absence of cellular lysis, i. e. in the absence of release of lactate dehydrogenase from the cells, thereby excluding N69-induced necrosis.

N69-induced cell death was functionally characterized by the use of different cellular model systems being devoid of defined molecular parts of the apoptosis machinery. According to those results, the CD95-dependent apoptosis cascade is not involved in N69-induced cell death. Furthermore, N69 triggers apoptosis in a Bax-independent manner in the essentially Bax-free prostata carcinoma cell line DU145. Thus, N69 is able to break the Bax-related drug resistance that plays an important role in the therapy of relapsed ALL in childhood. In addition, we could show that N69-induced apoptosis even proceeds in the absence of caspase-3 expression. We demonstrate here that N69 still exerts a considerable apoptosis-inducing effect in the caspase-3-deficient, multi drug-resistant (i. e. taxol-, epirubicin- and etoposide-resistant) mamma carcinoma cell line MCF-7. Taken together, iron-containing nucleosides comprise a totally new, very promising class of cytostatic agents for cancer and leukemia therapy, especially for the therapy of relapsed ALL in childhood. Future studies will aim to elucidate the target structures of these new compounds and to demonstrate N69-mediated anti-cancer activity in vivo. First experiments in mice show a good tolerability of N69.
In Vivo Bone Marrow Stem Cell Gene Transfer in Mice by In Situ Delivery of a 3rd-Generation Lentiviral Vector Using Intrafemoral Injection.
Authors: Pan Dao , von Kalle Christof , Williams David A. , Worsham D. Nicole , Bohn Kimberly , Lutz Christopher
Published: October 2019

HIV-based lentiviral vectors (LV) have become powerful tools for in vivo gene transfer and gene expression in non-dividing cells after local injection. However, the potential of in vivo bone marrow stem cell gene transfer by “in situ” bone cavity injection has not been well unexplored. This in vivo approach could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo hematopoietic stem cell (HSC) gene transfer. To determine if intra bone marrow (iBM) injection of LV can efficiently transduce BM stem cells, a 3rd-generation self-inactivating LV, containing eGFP regulated transcriptionally by a CMV promoter, was used to inject intra-femorally into adult Boy/J Ly45.1 mice (at 2 x106 IU/mouse). Blood counts were normal in all LV-injected mice (n=4) and buffer-injected mice (n=4). GFP-expressing peripheral blood leukocytes (PBL) were observed in both myeloid (up to 4%) and lymphoid subpopulations (up to 2%) 3-month post injection. To evaluate gene transfer and transgene expression in multi-potential progenitors, the colony-forming cell (CFC) assay was performed using low-density bone marrow 4-months post injection, resulting in 4–5% GFP+ CFU-granulocyte macrophage (CFU-GM) and CFU-granulocyte, erythroid, macrophage and megakaryocyte (CFU-GEMM). This was consistent with the 4-color FACS analysis data demonstrating that up to 3.7% of HSC/progenitors (Lin−c-kit+Sca1+ cells) from LV-injected mice expressed GFP transgene 4-months post injection. To further evaluate HSC gene transfer, BM from injected mice was transplanted into lethally irradiated adult C57/Bl6 (Ly45.2) mice. Four months later, successful engraftment was demonstrated in all BM recipients with 97–99% donor-derived cells in PBL and 91–93.3% in BM. Moreover, significant levels of GFP+ CFU were observed in all recipients ranging from 8.4 ± 2.3% to 17.7 ± 4.2%. To further study the LV-mediated gene transfer profile in HSC, mesenchymal progenitors and systemic distribution by intra-femoral injection, molecular analyses are in progress including real-time QPCR and clonality analysis by LAM-PCR. Our results indicate successful LV-mediated gene transfer into HSC by iBM injection. These data would potentially open a door to a novel approach for treatment of human diseases, but also provide a new tool to study adult stem cell plasticity and the nature of hematopoiesis.
Engineered Oncogene Activation by Somatic Hypermutation Results in a Faithful Mouse Model of MGUS/Multiple Myeloma.
Authors: Robbiani Davide F. , Colon Kaity , Szabo Paul , Affer Maurizio , Nickerson Helen , Qi Chen-Feng , Morse Herbert C. , Chesi Marta , Bergsagel Peter Leif
Published: October 2019

The time in cellular differentiation at which the expression of an oncogene is dysregulated is thought to determine the phenotype of the resulting neoplasia. We tested this assumption in C57BL6/J transgenic mice expressing an HA-tagged c-myc oncogene in B cells under the control of kappa light chain regulatory elements. Two transgenes were engineered that differed by only one nucleotide.

The first set of mice carries a wild type HA-MYC transgene, develops pro-B cell tumors, and succumbs rapidly to pronounced lymphosplenomegaly.

In the second set of mice, a point mutation in the HA-tag portion of the transgene creates a stop codon that abrogates HA-MYC translation. This stop codon was engineered to be a hotspot for somatic hypermutation. Thus sporadically, in a germinal center B cell, somatic hypermutation may revert the stop codon, allowing translation of HA-MYC.

In two independently derived lines, mice spontaneously develop monoclonal gammopathies (50% incidence at 30 weeks (n=35), 80% at 40 weeks (n=35)). Serum protein electrophoresis detects M-spikes that increase in intensity over time, and 6 out of 7 cases tested so far were of IgG1 isotype. Unlike other mouse models of plasma cell neoplasia, no lymphosplenomegaly nor ascites were detected. In analogy to human multiple myeloma, that is a disease of isotype-switched and hypermutated cells homing to the bone, large populations of plasma cells were found in the bone marrow. Bone marrow (but not spleen) lysates reacted for HA and human MYC proteins by western blot and immunohistochemistry, thus indicating that reversion of the stop codon occurred. Remarkably, in two young mice, monoclonal spikes appeared 2 weeks after vaccination with NP-CGG. These spikes were sustained and the monoclonal protein was reactive to the NP antigen.

Together, comparison of the two sets of mice demonstrates that activation of the same transgene at distinct B cell developmental stages results in dramatically different tumor phenotypes. In addition, and opposite to most engineered mice, we have created a system where the oncogene is turned on sporadically, as occurs in the human disease. Furthermore, we have developed a model that faithfully reproduces important clinical aspects of monoclonal gammopathy of undetermined significance (MGUS) and its progression to multiple myeloma. This model will be useful to develop immunologic and chemoterapeutic approaches.
In Vitro Depletion of CD4+CD25+ T Regulatory (Treg) Cells with Denileukin Diftitox (DAB389IL-2) Increases T Cell Alloreactivity.
Authors: Lesnikova Marina , Nikitine Alla , Gass John , Nash Richard A. , Georges George E.
Published: October 2019

We hypothesize that immune tolerance after allogeneic hematopoietic cell transplantation is maintained by an active cellular mechanism that includes Treg CD4+CD25+ cells. In the dog model of mixed hematopoietic chimerism, adoptive immunotherapy with donor lymphocyte infusion (DLI) that successfully converted recipients to all-donor chimerism required the breaking of immune tolerance to target antigens. We reasoned that depletion of Treg cells might break tolerance and could improve the therapeutic efficacy of subsequent DLI. Denileukin diftitox (DAB389IL-2, Ontak) is a fusion protein consisting of the translocation and ADP-ribosylase domains of diphtheria toxin and interleukin 2. DAB389IL-2 binds to the high affinity IL-2 receptor (CD25, CD122 and CD132) expressed on the cell surface of activated T cells. Some Treg cells express the high affinity IL-2 receptor. Although DAB389IL-2 has been shown to deplete alloreactive T cells, we asked if DAB389IL-2 could deplete Treg cells without depleting alloreactive T cells in dog mixed lymphocyte cultures (MLC). First we asked if DAB389IL-2 could induce specific cell death of activated T cells. Five days after initiation of a one-way, dog leukocyte antigen (DLA)-mismatched MLC, DAB389IL-2 was added to the MLC for 24 hours and cells were pulsed with 3H-thymidine. At 10−11 and 10−10 M [DAB389IL-2] there was 34% and 92% inhibition of cell proliferation, respectively. To determine if DAB389IL-2 could deplete Treg cells, we established DLA-mismatched MLC (n=5) with cyclosporine (CSP) 400ng/mL (Martin, PJ et al. BBMT, 2004) and added DAB389IL-2 in half-log increments from 10−11 to 10−9.5 M on day 4 of MLC. On day 5, culture medium was changed to remove DAB389IL-2 and cells were analyzed for expression of CD4 and CD25. Flow cytometric analysis showed that CSP treated MLC had reduced CD4+CD25bright and CD4+CD25dim by 90 ± 3.3% and 64 ± 10%, respectively, compared to no CSP. DAB389IL-2 [10−11 M] treatment of MLC with CSP decreased CD4+CD25bright and dim populations 31 ± 12% and 30 ± 4%, respectively, compared to no DAB389IL-2. On day 10 of the culture, cells were tested in secondary (2°) MLC to determine the effect of depleting CD4+CD25+ cells with DAB389IL-2. 2° MLC was performed without the addition CSP or DAB389IL-2. After 3 days of 2° MLC, cells were assayed for proliferation. Compared to controls with no CSP or DAB389IL-2 treatment during 1° MLC, depletion of CD4+CD25+ cells with DAB389IL-2 increased 2° allo-specific proliferation 36.2 ± 26%. When CSP was present in the 1° MLC, DAB389IL-2 treatment on day 4 increased subsequent 2° MLC allo-specific T cell proliferation 86 ± 22%. These results are consistent with the hypothesis that DAB389IL-2 can efficiently eliminate Treg (CD4+CD25+) cells, particularly when Treg cells are generated in the presence of CSP.
Results of Unrelated Cord Blood Transplants in Fanconi Anemia.
Authors: Gluckman Eliane , Rocha Vanderson , Ionescu Irina , Pasquini Ricardo , Harris Richard , Wagner John , Kurtzberg Joanne , Duval Michel , Darbyshire P.J. , Locatelli Franco , Fernandez M.N.
Published: October 2019

Background: allogeneic hematopoietic stem cell transplant (HSC) is the only chance of cure in patients with Fanconi anemia (FA). Unrelated cord blood transplant is a possible alternative source of HSC. We report here a worldwide experience of unrelated cord blood transplant (UCBT) in a series of 72 patients with FA.

Patients and transplant characteristics : This is a retrospective multicenter analysis of 72 patients (62 children, 12 adults) with FA complicated by severe aplastic anemia or leukemia/MDS. Median age was 8 years ( 1–45), median follow-up after UCBT 23 months. The unrelated CB was HLA identical for A, B DRB1 in 9 cases, had 1 HLA difference in 29 cases and 2 or 3 HLA differences in 33 cases. The median number of nucleated cells (NC) infused was 4.4x107/kg, of CD34+ 2.1x105/kg. Conditioning varied: 24 patients received low dose cyclophosphamide with low dose TBI/TLI, 33 Fludarabine with cyclophosphamide and TBI or Busulfan, 55 patients received ATG. GVH prophylaxis consisted of cyclosporine alone (16) or with prednisone (37).

Results: CI of engraftment was 54%. In multivariate analysis: unfavourable factors were nb of HLA differences>2 (p .007) and NC infused <4.4x107/kg (p.04) CI of GVH> II was 32% and of cGVH 11%. Overall survival was 36%. In multivariate analysis, favourable factors were negative recipient CMV serology ( p.001), nb of NC infused> 4.4x107/kg (p.007) and use of Fludarabine (p.035). Causes of death were non engraftment :10 pts, GVH: 4 pts, infections: 15 pts and toxicity:15 pts. These results are quite similar to our current results of matched unrelated bone marrow transplant.

Conclusion: results of unrelated cord blood transplants could be improved by better patient and cord blood selection with units closely HLA matched and high number of cells, Conditioning with Fludarabine seems to improve results. In FA, use of cord blood might be beneficial because of the reduction of the risk of GVH which might decrease the risk of long term cancers.
Kit PY567- Directed Signals Are Important for Efficient Erythroid Progenitor Cell Proliferation and Survival, and Are Attenuated by Kit PY569.
Authors: Li Ke , Karur Vinit , Vincent Bethany , Curato Jennifer , Kapur Reuben , Besmer Peter , Wojchowski Don M.
Published: October 2019

Anemia incurred by mice with naturally occurring Kit mutations (eg, W41) clearly illustrates important contributions of SCF/Kit signaling to erythropoiesis. Specific cytoplasmic features of Kit and linked signaling events that support such contributions, however, are not well understood. To address this issue, the proliferative and survival signaling activities of a panel of Kit cytoplasmic PY mutants first were studied in SCF- and Epo- dependent G1E2 cells, and also in multi-potential SCF-dependent EML cells. Kit forms studied included PY567, PY569, PY567-plus-569, PY702, PY719, PY728 and PY745 phosphotyrosine add-back forms as well as a biologically inactive baseline mutant, Kit-F6 in which these six PY sites were mutated to phenylalanine. To by-pass endogenous Kit, cytoplasmic PY mutants were expressed from a MIEG3 vector as MCSF-R/Kit “MK” chimeras, and cells expressing matched and physiological levels of M/K chimeras were isolated by FACS. PY567 per se (ie, M/K PY567) selectively supported near wild-type proliferative and anti-apoptotic activities in both G1E2 and EML cells. By direct comparison, PY569 per se has diminished activity in G1E2 cells, and only baseline activity in EML cells. Moreover, the combined restoration of both PY567 and 569 uniformly attenuated the otherwise high proliferative and survival activities of PY567. These findings therefore indicate that Kit PY567 and PY569 do not necessarily exert overlapping roles, but that PY569 instead may preferentially link to negative effects and also act to attenuate key positive signals relayed via Kit PY567 (possibly in a lineage- restricted fashion). In keeping with recent studies in 32D cells, little if any bioactivities were conferred by PY702, 719, 728, or 745 per se. Mechanistically, PY567 also was observed to link with high efficiency to sustained Bcl-xl expression, and to be selectively efficient in Epo receptor co-signaling. While these findings predict that the mutation of Kit Y567 should perturb red cell development, erythropoiesis in Kit-F567 knock-in mice appears near-normal. However, primary erythroid progenitor cells isolated from the spleen of Kit F567 mice at 50hr post- phenylhydrazine injection proved to be markedly deficient in SCF responsiveness. In addition, a >5-fold decrease in SCF and Epo co-signaling was reproducibly observed for Kit-F567 progenitor cell preparations. These novel findings therefore lend new evidence to the case that PY567, in fact, contributes in important ways to Kit’s erythropoietic biofunction.
Signaling by the Granulocyte-Colony Stimulating Factor Receptor Involves Jak2-Dependent Phosphorylation of Gab2.
Authors: Wang Lin , Xue Jia , Corey Seth J. , Robinson Lisa J.
Published: October 2019

Granulocyte colony stimulating factor (G-CSF) is the major cytokine involved in neutrophil production. G-CSF has pleiotropic effects on myeloid cells, initially stimulating proliferation but later promoting differentiation. The specific signaling pathways that mediate the diverse effects of G-CSF remain incompletely understood. Recently, the scaffolding molecule Grb2-associated binder protein 2 (Gab2) was shown to play an important role in G-CSF induced myeloid differentiation (Zhu et al. Blood 2004). Ligand stimulation of the G-CSF receptor results in the rapid phosphorylation of Gab2, but the identity of the responsible kinases and the molecular events dependent on Gab2 phosphorylation remain unclear. Because Janus kinases (Jaks) play a central role in G-CSF signaling, we investigated the involvement of Jaks in G-CSF-stimulated Gab2 phosphorylation using the hematologic DT40 cell line stably transduced with the human G-CSF receptor (DT40GR). Antisense Jak1 and Jak2 constructs expressed in DT40GR cells each produced a marked reduction in their target Jak protein, but only antisense Jak2 reduced G-CSF-stimulated Gab2 phosphorylation. To determine whether Gab2 phosphorylation required Jak2 kinase activity, dominant negative Jak2 mutants lacking catalytic activity were expressed in the DT40GR cells. Expression of dominant negative Jak2 inhibited Gab2 phosphorylation in response to G-CSF. Similarly, treatment with the Jak2-selective kinase inhibitor AG490 markedly reduced G-CSF-dependent Gab2 phosphorylation. Co-immunoprecipitation studies further demonstrated a G-CSF- and Gab2 phosphorylation-dependent association of Jak2 with Gab2 in vivo, which was detectable by 30 seconds after G-CSF stimulation. To determine whether Gab2 was a direct substrate of Jak2, we performed in vitro phosphorylation studies using Gab2-GST fusion protein substrates. Jak2 immunoprecipitated from G-CSF-stimulated cells, but not from control cells, phosphorylated the Gab2 fusion protein. To identify potential Jak2 tyrosine phosphorylation sites in Gab2, we used site-directed mutagenesis to produce three Gab2 tyrosine mutants. Tyrosines 409, 452, and 476 were each replaced by phenylalanine (Y409F, Y452F, and Y476F). The Y452F and Y476F mutations of Gab2 each inhibited G-CSF-stimulated Jak2-dependent phosphorylation of Gab2, both in stably-transfected DT40GR cells and in transiently-transfected 293 cells also transduced with the G-CSF receptor. In contrast, G-CSF-stimulated Gab2 phosphorylation appeared unaffected by the Y409F mutation. We also evaluated downstream events in G-CSF signaling in cells expressing these Gab2 tyrosine- mutants. Akt and Erk phosphorylation following G-CSF stimulation was inhibited by both the Y452F and Y476F Gab2 mutations, but was unaffected by the Y409F mutation. These results suggest that Jak2 may mediate G-CSF differentiation signals through Stat-independent mechanisms.
Pilot Phase Results of the Human Proteome Organization Plasma Proteome Project.
Authors: States David J. , Adamski Marcin , Menon Rajasree , Hanash Samir , Omenn Gilbert S.
Published: October 2019

The protein components of blood play essential roles in coagulation, inflammation, immunity, growth and development. Recognizing the importance of blood proteins in health and disease, the Human Proteome Organization (http://www.hupo.org) chose the Plasma Proteome Project (PPP) as the first tissue specific analysis to be implemented. The PPP is a collaboration of more than 30 laboratories and research groups worldwide. The June 2004 PPP Jamboree held in Ann Arbor, MI marked the completion of the pilot phase of the PPP with more than 50,000 peptide identifications reported leading to a high confidence consensus list of about 2,300 human blood proteins. Important contributions of the pilot phase of the PPP include the identification of over 2 thousands human blood proteins, comparative assessment of proteomic analysis of serum and plasma prepared using citrate, EDTA and heparin anticoagulants samples prepared from a single well defined donor pool; assessment of abundant protein depletion technologies to enhance the dynamic range of proteome analysis; comparative assessment of different protein and peptide fractionation technologies; comparison of MALDI peptide fingerprinting, liquid chromatography electrospray tandem mass spectroscopy, direct MS/SELDI, and accurate mass tag strategies; correlation of protein abundance with success in mass spectroscopy identification; comparison of Sequest, Mascot, SpectrumMill and X!Tandem software for mass spectroscopy data analysis; comparison of NCBI NR, IPI and full 6-frame genome translation databases; establishing quality measures for the different software packages; establishing quality assurance and data validation protocols for large multi-center proteomics studies. These results demonstrate the potential for large scale proteome analysis as well as highlighting important challenges that lie ahead.