bluuebunny/crossref_metadata_2025 huggingface dataset:
The Leukemogenic Properties of MLL Fusion Genes Do Not Require FLT3 Signaling.
Authors: Lavau Catherine , Libura Marta , Morgado Ester , Albouhair Stephanie
Published: April 2020

Leukemias harboring chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene express high levels of the receptor tyrosine kinase FLT3. Activation of FLT3 signaling is very frequent in human acute myeloid leukemias and its role in the transformation of myeloid cells was demonstrated experimentally. To study what role FLT3 activation played in the cellular transformation leading to MLL-rearranged leukemias we used retroviral vectors to express MLL fusion genes in flt3+/+ and flt3−/− bone marrow cells. Cohorts of mice transplanted with flt3+/+ and flt3−/− bone marrow expressing either MLL-ENL or MLL-CBP were established. All mice succumbed to AML within 60 days (MLL-ENL) or 120 days (MLL-CBP) regardless of flt3 status.

Parameters reflecting cellular proliferation (leukocytosis, spleen weights) or maturation (morphology, immunophenotype) were identical in flt3−/− versus flt3+/+ leukemic blasts. Transplantability and clonality of the leukemias were also unchanged. We showed that the FLT3 protein was expressed in flt3+/+ MLL-ENL leukemic blasts and to explore its role in cell growth we studied the sensitivity of the cells to various FLT3 inhibitors ex vivo. We found that PKC412, AG1296 and Herbymicin A had growth inhibitory activity on MLL-ENL leukemic cells in culture. However the IC50 of these drugs was much higher than those determined on cell lines expressing FLT3 mutants that constitutively activate the FLT3 signaling pathway. Furthermore, flt3−/− MLL-ENL leukemic cells were equally sensitive to the growth inhibitory activity of these drugs than flt3+/+ leukemic cells. This demonstrates that PKC412, AG1296 and Herbymicin A impair the growth of MLL cells through the inhibition of tyrosine kinases other than FLT3. Altogether our data question the value of FLT3 as a therapeutic target to fight MLL-rearranged leukemias and suggest that the efficacy of FLT3 inhibitors is in fact mediated by the interference with another pathway.
In AML, Cbl Serves as Nexus for Signaling from Flt3, and Is Required for Coupling JNK1 in a Pathway of Survival and Proliferation Involving c-jun/AP-1.
Authors: Hartman Amy D. , Wilson-Weekes Annique , Suvannasankha Attaya , Burgess Gem S. , Hincher Kathryn J. , Cripe Larry D. , Boswell H. Scott
Published: April 2020

Here we demonstrate by immunoprecipitation and immunoblot, cbl is among the most heavily tyrosine phosphorylated adaptor proteins in primary AML blasts with Flt3 signaling, in the context of either mutation or overexpression/autocrine mechanisms. The human leukemic cell lines MV-4-11 and THP-1 model primary AML blasts in terms of Flt3 signaling by these respective criteria and demonstrate identical coupling between Flt3 and p85, the PI-3-kinase adaptor, by coimmunoprecipitation/blot experiments. Although cbl has no direct binding site on Flt3, it binds tightly to p85 SH2 by virtue of its tyrosine phosphorylation, also demonstrated by co-IP in cell lines and primary cells. Tyrosine phosphorylated cbl is a docking site for CrkII/L SH2’s and this provides a branch point for signals from Flt3 to PI-3-kinase or JNK, respectively, because CrkII(L) is known to bind JNK1 through SH3: polyproline interaction to serve as scaffolding; and interaction of JNK1 and CrkII/L was also observed by co-IP. In a survey of primary AML cases (n=33) there was a strict relationship between expression levels of (active) Flt3 and phospho-c-jun as readout for JNK activity level (p=0.001, r=0.54). To demonstrate the functional relevance of these interactions, siRNA knockdown of components was pursued in the cell lines and in primary AML blasts. JNK1 knockdown, and, to a much lesser degree, JNK2 knockdown, led to loss of phospho-c-jun expression in MV-4-11 and THP-1. Indeed, Flt3 signaling is required for JNK signaling because knockdown of Flt3 led to total loss of p-jun and c-jun expression in MV-4-11 and patient blast. By contrast, cbl knockdown led to selective loss of JNK signaling to p-jun without significantly affecting Flt3 or its downstream activating phosphorylation of AKT. Thus, despite binding by cbl to p85, cbl is not required for PI-3-kinase signaling because of redundancy supplied by the p85-Flt3 interaction. Further, by use of LY294002 to inhibit PI-3-kinase, PI-3-kinase is also not required for JNK signaling. However, selective inhibition of JNK signaling by the small molecule approach in these cell lines and in primary AML blasts leads to loss of proliferation, induction of apoptosis, and synergistic killing with daunorubicin. These observations form the platform for a phase I trial of JNK inhibition in refractory, multidrug-resistant, and Flt3-driven AML.
Leukemic Transformation Induces the Constitutive Expression of Indoleamine 2,3-Dyoxigenase Which Directly Increases CD4+CD25+ T Regulatory Cells.
Authors: Curti Antonio , Pandolfi Simona , Aluigi Michela , Valzasina Barbara , Isidori Alessandro , Ferri Elisa , Rutella Sergio , Durelli Ilaria , Horenstein Alberto L. , Fiore Francesca , Massaia Massimo , Piccioli Milena , Palmerini Emanuela , Pileri Stefano , Colombo Mario P. , Baccarani Michele , Lemoli Roberto M.
Published: April 2020

The expression of the catalytic enzyme of tryptophan, indoleamine 2,3-dyoxigenase (IDO) in different cellular subsets, including solid tumors, has been recently identified as a T-cell inhibitory effector pathway. Here, we show that unlikely normal hematopoietic CD34+ cells expressing IDO only upon stimulation with IFN-γ, acute myeloid leukemia (AML) cells constitutively express IDO and inhibit allogeneic T-cell proliferation. IDO expression correlates with increased CD4+CD25+Foxp3+ T cells in AML patients at diagnosis. In vitro, IDO+ AML cells increase the percentage and the absolute number of CD4+CD25bright T cells, also expressing surface CTLA-4 and Foxp3 mRNA. The addition of the IDO-inhibitor, 1-methyl tryptophan (1-MT), completely abrogates such increase. Purified CD4+CD25+ T cells obtained from coculture with IDO+ AML cells are not proliferating and unable to produce IL-2. They also inhibit naive T-cell proliferation and Th1 skewing. Co-culture with IDO+ AML cells results in the conversion of CD4+CD25− into CD4+CD25+ T cells and the addition of 1-MT completely abrogates this effect. In mice, intrasplenic injection of IDO-expressing leukemia/lymphoma A20 cells induces the increase of CD4+CD25+ T cells, which can be blocked by 1-MT treatment. These data suggest that AML cells have the capacity to directly increase a population of T regulatory cells through the constitutive expression of the functionally active form of IDO. IDO expression can be regarded as a novel mechanism of leukemia escape from immune control and its inhibition may represent a novel anti-leukemia therapeutic strategy.
Gene Expression Profiling of Bulk CD138+ Plasma Cells in Preneoplastic Gammopathy/Asymptomatic Myeloma Identifies Distinct Subsets of Patients.
Authors: Dhodapkar Madhav V. , Zhan Fenghuang , Rasmussen Erik , Burington Bart , Durie Brian , Crowley John , Barlogie Bart , Shaughnessy John
Published: April 2020

Gene expression profiling of plasma cells (GEP-PC) has provided major insights into myeloma pathobiology. However the data about GEP-PC in preneoplastic gammopathy (MGUS) or asymptomatic myeloma (AM) are limited, and gene expression patterns that might predict outcome in these patients have not been defined. We analyzed GEP (using U133Plus Affymetrix microarrays), of plasma cells isolated by immuno-magnetic bead selection with CD138 microbeads, from the bone marrow of patients with MGUS (n=16) and asymptomatic myeloma (AM; n=18) enrolled in a prospective South West Oncology Group (SWOG) observational study. Data from normal plasma cells (PCs) and from 105 myeloma PCs were included as controls. Myeloma PCs were randomly selected to include at least 15 patients from each of the 7 subgroups previously identified based on GEP of myeloma tumor cells (Zhan and Shaughnessy, ASH 2004). After the suppression of immunoglobulin (Ig) genes, there were 1297 genes that significantly differed in expression between MGUS-PCs and MM-PCs, and 1099 genes that differed between MGUS-PCs and normal PCs with a 1% false discovery rate. Hierarchical cluster analysis of all samples was performed using 1000 plasma cell signature genes that were most differentially expressed between normal and myeloma PCs. These data demonstrated that both MGUS and AM samples were distributed between normal and MM samples. A prediction analysis of microarrays (PAM) model (PNAS99:6567, 2002) utilizing 134 genes was then developed to determine if the signature from these genes in MGUS/AM was more similar to normal or to myeloma plasma cells. In this analysis, 11/16 (69%) of the MGUS samples were more similar to normal PC, compared to 6/18 (33%) of the AM samples (p=0.04). At present, there are no reliable phenotypic markers to distinguish between normal and malignant PCs within the bulk CD138+ population. Gene expression spikes for cyclin D1 and MAF/MAF-B were seen in both MGUS and AM cohorts, including in some patients with normal PC signature. These data provide the largest comparison to date, of GEP of PCs in preneoplastic versus malignant gammopathies and suggest that GEP may be a useful tool to prospectively identify subsets of patients within the MGUS/AM population with dominant normal PC or MM PC signatures, and potentially differing prognosis. Further analysis of differentially expressed genes between MGUS/MM PCs identified in this dataset may allow insights into the genomic changes in tumor cells underlying the malignant progression of myeloma.
The Paternally Expressed Gene 10 (PEG10) on Chromosome 7q21 Is Overexpressed and Imprinted in High-Risk B-CLL.
Authors: Kainz Birgit , Bilban Martin , Shehata Medhat , Heintel Daniel , Kienle Dirk , Kroemer Elisabeth , Le Trang , Kroeber Alexander , Schwarzinger Ilse , Gleiss Andreas , Chott Andreas , Doehner Hartmut , Zoechbauer9 Sabine , Heller9 Gerwin , Fonatsch Christa , Gaiger Alexander , Stilgenbauer Stephan , Zielinski Christoph , Wagner Oswald , Jaeger Ulrich ,   German CLL Study Group
Published: April 2020

Introduction: To establish surrogate markers for IgVH mutation status in B-CLL, we performed microarray analysis with mutated and unmutated patient samples. Among the most differentially expressed genes was PEG10, a maternally imprinted gene located on chromosome 7q21. PEG10 is known to be expressed during placental development and in hepatocellular carcinoma. Here we have investigated the association of PEG10 with B-CLL subtypes and risk factors as well as its transcriptional regulation. In addition, we studied its expression in other hematologic malignancies.

Methods and Results: In our microarray experiments, PEG10 was highly expressed (2.1-fold) in unmutated samples. We confirmed these results by real time PCR in 42 B-CLL patients and found a significant correlation between PEG10 expression and unmutated IgVH status (P=0.007). High PEG10 expression was associated with chromosomal del(11q) (P=0.008), a shorter time to treatment (P=0.009) as well as a positive Coombs test (P=0.04). High expression was found in B-CLL and sorted CD19+ B-CLL cells, plasmoblastic DLBCL as well as in the Burkitt’s lymphoma cell line Ramos. In normal tissues, relatively high levels were seen in brain, liver, lung and kidney, but these expression levels were lower than in CD19+ B-CLL cells. Expression in sorted B cells from healthy donors was lower than two orders of magnitude. Intracellular expression of the PEG10 protein in B-CLL cells was confirmed by immunofluorescence studies using antibodies that recognize specifically PEG10-RF1. To address the question whether PEG10 overexpression results from loss of imprinting, we performed allele-specific as well as methylation-specific PCR. Imprinting was maintained in 6 patients with B-CLL. Analysis of a mother and son, both having B-CLL, confirmed that at least in this familial case the maternal PEG10 allele was silenced as expected. Interestingly, similar expression patterns were observed for the neighbouring gene sarcoglycan epsilon indicating a common mechanism of deregulation of the 7q21 locus in high risk B-CLL.

Conclusions: We show that PEG10 is differentially expressed in B-CLL and is associated with del(11q), a shorter time to treatment and unmutated IgVH mutation status, suggesting its role as a marker for high risk B-CLL. PEG10 overexpression is unlikely to result from loss of imprinting. Further studies of the gene locus at 7q21 are in progress.
Aberrant Expression of Ikaros Induces T-Cell Leukemia/Lymphoma in Mice without Wild-Type Allelic Loss.
Authors: Kataoka Itaru , Ishimaru Fumihiko , Ichimura Koichi , Yoshino Tadashi , Tanimoto Mitsune
Published: April 2020

The Ikaros gene consisting of seven exons codes a family member of zinc finger transcription factors essential for establishment of immune systems. A number of isoform proteins are produced by alternative splicing. The activity of Ikaros is strictly regulated by total expression level or mutual dimerization with full-length and dominant-negative isoforms. Findings from Ikaros mutant mice revealed its characteristics as tumor suppressor in addition to functions of hemato-lymphoid development and differentiation. Mutant mice homozygous for lack of dimerization domains evolve T-cell leukemia and lymphoma. Heterozygous mutation of DNA binding domains can also lead to clonal expansion of T-cells through loss of heterozygosity. In both of murine models reported earlier, leukemias arise from the absence of full-length active isoforms. On the other hand, analyses for human diseases conducted by our group elucidated high incidence of over-expression of dominant-negative isoform in adult precursor B-lymphoblastic leukemia. Thus the mechanisms by which Ikaros contribute to leukemogenesis are critical issues to be clarified especially in human. Here, we demonstrate that over-expression of dominant-negative isoform causes leukemia and lymphoma without wild-type allelic loss by murine BMT assay. Retroviral gene transfer of dominant-negative isoform, Ik6, to murine hematopoietic cells developed lethal lympho-proliferative disorders in 3–6 months of latency with complete penetrantion. The diseases involved a variety of organs such as thymus, lymphnode, spleen, liver and bone marrow. Clonality of the infiltrating cells was confirmed by Southern blotting. The leukemic cells showed T-cell phenotype (B220-CD3e+CD4+CD8a+TCRb+Gr1-Mac1-) and express simultaneously full-length Ikaros proteins and transduced dominant-negative isoform. These data suggest that leukemogenesis mediated by decreased activity of Ikaros does not require deletion of full-length isoforms, and disproportional expression of Ikaros directed to dominant-negative isoform is enough to accumulate and immortalize lymphoid blasts in vivo. Silencing dominant-negative Ikaros proteins should be considered as a good candidate of molecular targeted therapy, and quantification of Ikaros isoforms might be useful for monitoring the minimal residual disease.
Potential Genetic Risk Factors for Chronic ITP of Childhood.
Authors: Williams Shirley A. , Imfeld Karen L. , Tai LeiQian , Nakagawa Peggy A. , Chang Mei , Nugent Diane J.
Published: April 2020

While well recognized as an autoantibody mediated disease, the pathophysiology of childhood ITP remains poorly understood. Approximately 20% of children diagnosed with acute ITP will have persistent thrombocytopenia for more than 6 months after the original diagnosis. One hypothesis is that the autoantibody coated platelets are bound by surface Fcγ receptors in the reticuloendothelium system, which then target these platelets for destruction. Although the FcγRIIIA receptor usually shows weak binding, the phenylalanine (F) to valine (V) mutation in the FcγRIIIA receptor results in a higher affinity for IgG1 and IgG3. To test the possibility that the FcγRIIIA receptor genotype is associated with increased autoantibody mediated platelet destruction in ITP, we screened more than 80 ITP patients for this single nucleotide polymorphism (SNP) using an amplicon-down format for a custom Nanochip molecular biology workstation microarray assay.

We observed a large shift in the allele frequencies of the FcγRIIIA receptor SNP (F158V) in ITP patients compared to controls (F =.36, V =.64 vs F =.70, V =.30; P<.001). When we analyzed the ITP patients with regard to chronicity of ITP, we found that chronic ITP patients showed a larger V allele frequency shift (F =.45, V =.55) than acute ITP patients (F =.58, V =.42). The IL-1α-889 SNP genotypes of these ITP patients were also found to have allele frequencies significantly different from controls (C =.67, T =.33 vs C =.84, T =.16, P =.005).

FcγRIIIA SNP Genotype FF FV VV significance ITP patients 12.3 46.5 41 Controls 50.2 39.2 10.4 P<0.001 IL-1 α SNP Genotype CC CT TT significance ITP patients 51.4 33.3 15.2 Controls 71 26.3 2.6 P=0.005

Additionally, we have shown that the VV genotype of FcγRIIIA receptor is associated with 100% response to IVIg treatment in childhood ITP (Tarantino et al J. Thromb. Haemost. 2005). Thus, we propose that the FcγRIIIA receptor and IL-1α-889 genotypes may be useful as potential risk factors for ITP and its response to therapy. Understanding the mechanism whereby these polymorphisms alter the incidence and course of ITP in children will provide critical information in the prevention and treatment of this syndrome.
Cyclosporine Alone for the Treatment of Early Recurrences of TTP.
Authors: Cataland Spero R. , Jin Ming , Ferketich Amy K. , Zheng X. Long , George James N. , Wu Haifeng M.
Published: April 2020

Patients: The 3 patients detailed in this report presented to Ohio State University Hospitals and were enrolled on our prospective study of plasma exchange as initial therapy of TTP. Given the close outpatient follow-up during the first month after the tapering of plasma exchange, 2 patients were found to have an early recurrence of their TTP. The third patient had a recurrence 8 weeks after completing 6 months of CSA after being treated with concurrent CSA and PE for a previous acute episode of TTP. She completed the planned 6 months of cyclosporine therapy but relapsed 8 weeks after discontinuing the drug. Her recurrence was characterized by numbness and tingling in her right hand in addition to laboratory findings consistent with TTP. Complete demographic and clinical details of these 3 patients are shown in Table 1.

Methods: All 3 patients received oral cyclosporine (2–3 mg/kg) daily for six months in a divided dose. None of the patients underwent plasma exchange, infusion, or transfusion of blood products of any kind. Patients were seen daily to monitor for any clinical or laboratory findings that would warrant the re-initiation of plasma exchange therapy. Remissions were defined as a normal platelet count, normalization of LDH for at least 2 consecutive days, and improvement or stabilization of any renal and neurologic abnormalities. Serial measurements of ADAMTS13 activity and inhibitors were performed at regular intervals throughout therapy and follow-up.

Results: All three patients showed clear laboratory and clinical signs of improvement within the first 7 days of therapy and met criteria for clinical remission 14, 24, and 34 days respectively from the time they began treatment with cyclosporine. The third patient that presented with numbness and tingling in her right hand also had complete resolution of her neurologic symptoms within one week of restarting cyclosporine. Pretreatment ADAMTS13 activity was determined to be 33%, 25%, and 10% in all 3 patients respectively with all 3 patients having documented inhibitors of ADAMTS13.

Conclusions: Treatment with cyclosporine alone resulted in the suppression of ADAMTS13 inhibitors and normalization of ADAMTS13 activity.

Figure Figure

All three patients have completed the planned 6 months of cyclosporine and have maintained their remission 4, 3, and 1 month respectively after discontinuing cyclosporine.

Patient Demographic and Clinical Data Laboratory Studies at the TIme CSA Initiated Age Sex Race Therapy Preceeding Recurrence Time to Relapse After Most Recent Therapy Platelet Count (150-450 K/uL) LDH (100-190 U/L) PE=Plasma Exchange, CSA=Cyclosporine, S=Corticosteroids Patient 1 48 F C PE + S 17d 113 212 Patient 2 39 F C PE 6d 135 400 Patient 3 56 F AA CSA 56d 51 418
Mutation in the Zebrafish Homolog of the RNA Helicase DHX8 Result in Hematopoietic Defects.
Authors: English Milton A. , Lei Lin , Blake Trevor , Sood Raman , Liu Paul P.
Published: April 2020

Hematopoiesis is a tightly regulated process that requires the coordinated efforts of many tissue-specific transcription factors. Lateral mesoderm tissue undergoes a series of restricted proliferation and differentiation that result in the production of the hematopoietic stem cells. These cells then differentiate to populate the different cell lineages. While some factors essential for this process have been defined, many remain unknown. Zebrafish offer a powerful model system in which it is possible to conduct forward phenotype-driven screening. We have undertaken a chemical mutagenesis screen using N-nitroso-N-ethylurea (ENU) in the zebrafish to uncover mutants in myelopoiesis and early hematopoiesis. Adult male fish were exposed to ENU in a protocol designed to mutagenize spermatagonia with a mutation frequency of approximately 1:1000. Mutagenized males were crossed to wild-type females to generate F1 females. Haploid embryos from F1 females were screened at 27 hours of development by RNA in situ hybridization using the myeloid-specific marker, leukocyte-specific plastin (l-plastin), and the stem cell marker, core binding factor b (cbfb). One mutant line identified from this screen, mummy, showed reduced cbfb expression and lack of l-plastin expression. Additional in situ staining has confirmed that mummy has a myeloid defect as other myeloid markers such as Lys.C, Mpo, and C/ebp1 were also significantly reduced. Mummy mutant also have a degreased expression of gata1 and bE1 globin (RBC markers) as well as the stem cell marker scl. These results suggested that the defect in mummy is at or up-stream of the HSC level. The mummy mutant embryos also suffer from widespread cell death and die around 36 hours post fertilization. Genetic mapping and positional cloning has identified the mutated gene as the zebrafish homolog of DHX8, which encodes an RNA helicase. The yeast homolog of dhx8, prp22, functions during mRNA splicing and RNA processing. Morpholino injection into wild-type embryos has confirmed that reduced expression of dhx8 can produce a similar phenotype. The wild-type zebrafish dhx8 cDNA but not the mutated dhx8 cDNA identified in mummy was able to partially rescue the mummy phenotype. Preliminary data from RT-PCR and microarray analysis suggest that mummy mutants have defects in the production of spliced transcripts of some genes but not others. Genes with decreased spliced transcripts in the mummy mutant include scl, cbfb, and gata1, which may explain the hematpoietic defects observed. Our results demonstrate that mutations in an RNA helicase involved in splicing could result in specific blockage of hematopoiesis.
Familial Erythrocytosis Associated with a Mutation in the HIF Prolyl Hydroxylase, PHD2.
Authors: Percy Melanie J. , Zhao Q. , Flores A. , Harrison C. N. , Lappin T. R.J. , Maxwell P. H. , McMullin M. F. , Lee F. S.
Published: April 2020

Normal erythropoiesis is precisely regulated by a negative feedback mechanism based on the oxygen status of the body. Whenever a deficit in oxygen supply is detected erythropoietin (Epo) is rapidly synthesised to increase circulating red cells. This process is under the control of the HIF transcription complex, which is composed of one alpha and one beta subunit. Although the alpha subunit is constitutively synthesized, it is maintained at a low level by continual targeting to the proteasome. Prolines 402 and 564 in the oxygen dependent degradation (ODD) domain of HIF-1α are hydroxylated in the presence of oxygen by members of the PHD family of prolyl hydroxylases. Once hydroxylated HIF-1α is captured by the von Hippel Lindau tumour suppressor gene product (VHL), ubiquitylation follows and HIF-1α is then targeted for proteasomal degradation. Defects in the hypoxia response pathway appear to be the most common cause of erythrocytosis associated with inappropriately normal or elevated serum Epo.

Several different mutations in the VHL gene have been detected in erythrocytosis individuals. Although these mutations explain a significant number of cases there remains a large cohort where the molecular defect is undefined. Thus we screened a group of such individuals for base changes in PHD 1–3. Two affected siblings and their deceased father were found to possess a heterozygous C to G change at base 950 in the coding sequence of PHD2, causing loss of proline at codon 317 and replacement with arginine. This base change was not present in their normal mother nor in 200 normal controls. Residue 317 is located in the enzyme’s active site and at a position two residues C-terminal to Asp315, which chelates the active site iron moiety. Functional analysis indicated that the Pro317Arg PHD2 variant bound to HIF-1α and HIF-2α more weakly and displayed substantially less HIF hydroxylase activity than the wild type protein. These results indicate that the Pro317Arg PHD2 mutant protein’s function will be impaired, less hydroxylation of HIF will occur, allowing more HIF to escape proteasomal degradation and to increase transcription of HIF target genes. Consequently, Epo gene expression will be elevated thereby allowing increased erythrocyte production and the development of erythrocytosis.
CD26 Inhibition Enhances Allogeneic Donor Cell Homing and Engraftment after In Utero Bone Marrow Transplantation.
Authors: Peranteau William H. , Endo Masayuki , Adibe Obinna O. , Merchant Aziz , Zoltick Philip , Flake Alan W.
Published: April 2020

In utero bone marrow transplantation (IUBMT) induces donor-specific tolerance for postnatal cellular or organ transplantation. Consistent induction of tolerance requires a threshold of mixed hematopoietic chimerism (>1%). CD26 is a peptidase whose inhibition has been demonstrated to influence granulocyte colony-stimulating factor induced mobilization of hematopoietic stem cells and increase homing and engraftment of BM cells in adult transplantation models. We hypothesized that CD26 inhibition would increase the frequency and levels of allogeneic hematopoietic chimerism after IUBMT.

Methods: B6 GFP BM was injected intravenously into E14 Balb/c fetal mice at a dose of 20e6 cells/fetus with or without CD26 inhibition with diprotin A. Early kinetic analysis was performed to assess donor cell homing to fetal liver (FL). Peripheral blood (PB) was analyzed up to 20 weeks after birth for donor cell chimerism and multilineage engraftment by flow cytometry. PB was also analyzed for donor cell chimerism at the same time points from Balb/c mice receiving 10e6 CD26 inhibited GFP BM cells coinjected with 10e6 noninhibited congenic B6Ly5.2 BM cells at E14 to assess for an in utero competitive advantage provided by CD26 inhibition.

Results: CD26 inhibition increased donor cell homing to the FL at 24, 48 and 96 hours after injection (69.27±8.19 vs 30.21±6.44, 57.20±14.63 vs 36.80±14.20, 46.14±15.79 vs 12.09±7.01; p<0.05 at all time points). The frequency and levels of engraftment at 4 weeks of life were increased in those mice receiving CD26 inhibited BM compared to noninhibited BM (50.0% vs 22.5%; 20.48±14 vs 6.96±8.44, p<0.05). Chimerism was multilineage and maintained at 20 weeks of age (figure 1a) supporting improvement of engraftment at the stem or early progenitor cell level (* p<0.05 comparing chimerism levels between inhibitied and noninhibited cells). The coinjection of CD26 inhibited and noninhibited cells resulted in higher levels of engraftment of inhibited cells at all time points up to 16 weeks of age arguing for a competitive engraftment advantage of early progenitor cells provided by CD26 inhibition (figure 1b).

Conclusion: CD26 inhibition of donor BM prior to IUBMT results in an increased efficiency of donor engraftment and higher levels of chimerism. CD26 inhibition offers a potential mechanism to increase the level of engraftment and the rate of donor specific tolerance and may facilitate combined pre and postnatal strategies for cellular and organ transplantation.

Figure Figure
Immunoregulatory Effects of Allogeneic Mixed Chimerism Induced by T-Cell Depleted, Nonmyeloablative Bone Marrow Transplantation on Chronic Inflammatory Arthritis and Autoimmunity Developed in Interleukin-1 Receptor Antagonist-Deficient Mice.
Authors: Cho Seok-Goo , Park Min-Chung , Min So-Youn , Cho Young-Gyu , Lee Seok , Cho Mi-La , Chang Hong-Seok , Lee Jong-Wook , Min Woo-Sung , Kim Ho-Youn , Kim Chun-Choo
Published: April 2020

Objective: To investigate the immunoregulatory effects of allogeneic mixed chimerism induced by T-cell depleted, nonmyeloablative bone marrow transplantation (TCD-NMT) on chronic inflammatory arthritis and autoimmunity developed in interleukin-1 receptor antagonist-deficient (IL-1Ra−/ −) mice.

Methods: IL-1Ra−/ − mice (H-2kd) were treated with anti-asialoGM1 Ab, TBI 500 cGy, and TCD-NMT from C57BL/6 mice (H-2kb). Engraftment and chimerism were evaluated on peripheral blood (PB), lymph node, and spleen by multi-color flow cytometry. The severity of arthritis was evaluated by clinical score and histopathology. IgG1 and IgG2a subtype of anti-type II collagen (CII) were measured in PB samples. After T cells were stimulated with CII, ovalbumin, and phytohemagglutinin, T-cell proliferation response and cytokines production (INF-g, TNF-a, IL-10, and IL-17) in culture supernatant were assayed.

Results: All the transplanted IL-1Ra mice showed marked improvement of arthritis within 3 weeks after transplantation as well as successful induction of mixed chimerism. Mice in mixed chimerism showed higher level of anti-CII IgG1 and lower level of anti-CII IgG2a and weaker T cell proliferative response than in control groups, such as no-treatment and conditioning only without BM rescue. In mixed chimera, INF-g, TNF-a and IL-17 production from CII-stimulated T cells was significantly suppressed and IL-10 production was significantly increased as compared to the control groups.

Conclusion: These observations indicate that the introduction of allogeneic mixed chimerism has a strong immunoregulatroy potential to correct established chronic inflammatory arthritis and autoimmunity originating from dysregulated proinflammatory cytokine network.
Use of Engineered Autologous BOEC for Gene Therapy of Canine Hemophilia A.
Authors: Hebbel Robert P. , Milbauer Liming , Roney Mark , Lillicrap David , Voorberg Jan , Nichols Timothy C.
Published: April 2020

Hemophilia A is an attractive candidate disease for corrective gene therapy because relatively small amounts of the missing protein, FVIIII, have a significant biological effect. We previously described a novel approach: intravenous administration of ex vivo expanded BOEC (blood outgrowth endothelial cells) engineered to express FVIII. Robust therapeutic results were obtained using NOD/SCID mice and human BOEC stably transfected to express human FVIII (Blood99:457, 2002). We are now conducting scale-up studies in the canine hemophilia A model. Based on the previous mouse data, we estimated that 400 million cells would be required to achieve a therapeutic effect in the dog. First, two normal dogs were infused with autologous unmanipulated BOEC, which revealed removal from the circulation via single-pass kinetics. Then, BOEC cultures were obtained from three hemophilia A dogs, and an onco-retroviral vector (MIRG/cFVIII) was used to obtain transduced BOEC producing B-domainless canine FVIII. Then, for each treatment, autologous engineered BOEC were given IV to the original donor dogs, with dose divided over 3 daily infusions, and whole blood clotting time (WBCT, in minutes) was followed.

Dog Date # cells infused pre Rx WBCT effect on WBCT D28 12/04 274x106 >60 nadir 36 at day 14, Still 39 at day 185 D28 6/05 386x106 39 nadir 24.5 at day 23, now at 30 at day 44, being followed E64 7/04 220x106 >60 nadir 34.5 at day 3, >60 min by day 38 E64 12/04 274x106 >60 still 37.5 at day 189 E64 6/05 185x106 37.5 nadir 34.5 at day 2, at 36 on day 32, being followed H17 6/05 272x106 >60 nadir 29 at day 44, being followed

WBCT (nl = 8-12 min) needs to be <20 min to reflect >0.1% FVIII activity; but the observed WBCT are clearly improved compared to baseline. One dog (E64) was treated for a mouth bleeds on d37 in cycle 2, and d36 in cycle 3. One animal (E64) developed hypotension and tachypnea during one of the infusions (probably because cells were not kept sufficiently in suspension) but recovered. Four of the infusions were associated with mild transient thrombocytopenia. Based on these initial scale-up experiments, it appears that use of engineered BOEC for gene therapy is an approach worthy of continued study, as cell doses can be increased and other improvements in the method are readily envisioned. It has a number of advantages, including use of autologous carrier cells with which any expression vector can be paired, ex vivo exposure to vector rather than in vivo, use of BOEC expansion in culture to simultaneously achieve vector expansion, and the ability to chose a clone of engineered cells having a single, known (and studied) insertion site.
High Dose Cyclophosphamide (CY) for Severe Aplastic Anemia (SAA): Safety and Long Term Follow-Up.
Authors: Brodsky Robert A. , Chen Allan , Dorr Donna M. , Brodsky Isadore , Jones Richard J.
Published: April 2020

Background: Acquired SAA is a potentially life-threatening bone marrow failure disorder that is usually immune-mediated. SAA can be effectively treated with bone marrow transplantation (BMT), immunosuppressive therapy (IST), or CY. BMT can cure the disease but ideally requires a matched sibling donor. IST has a high response rate, but up to 50% of patients relapse, become dependent on cyclosporine, or develop secondary clonal disease such as paroxysmal nocturnal hemoglobinuria (PNH) or myelodysplastic syndromes (MDS). We previously showed that CY induces durable remissions in the majority of SAA patients, but concern has been raised about the safety of this approach.

Methods: Since 1996, we treated 38 previously untreated, and 17 IST failed, SAA patients with CY (50 mg/kg/d x 4) followed by daily G-CSF (5 ug/kg/day) until the neutrophil count (ANC) reached 1000/dl. Response was defined as ANC > 1000/dl and transfusion independence without growth factor support for > 3 months. Relapse was defined as no longer meeting criteria for response. PNH was monitored by flow cytometry.

Results: The median age of the newly diagnosed patients was 40 (range 2–68) years. With a median follow-up of 41 (range, 6 – 111) months, 33/38 patients survive (actuarial survival of 86%, 95% CI 72–95%) with 28 (74%, 95% CI 58–85%) achieving remission, most being complete. Median time to ANC of 500, last platelet and red cell transfusion was 50, 99, and 181 days, respectively. Before treatment, 15 patients met criteria for very (v) SAA (ANC < 200). Mortality within 6 months after CY occurred in 4 (10.5%) patients, all with vSAA; 1 additional patient died from bacterial sepsis 18 months after CY. 22/23 (96%) SAA patients survive (20 in remission) compared to 11/15 (73%) with vSAA (10 in remission). Eight patients had a severe infection at the time of beginning treatment and 5 survive in remission. PNH screening revealed a PNH population ranging from 0.5–40% of granulocytes in 12 patients, and all 12 achieved a durable remission (p = 0.039). No patient in this series has progressed to PNH or MDS, and the PNH clone is regressing in all 12 patients. Two patients have relapsed. One patient, whose first remission lasted 5 years, was retreated with CY into a persisting second complete remission 3 years ago; another patient recently relapsed 3 years after achieving remission. Ten of the 17 patients who failed IST (median age of 31, range 6–58) are alive and nine are in remission.

Conclusions: Cy is safe and highly effective therapy for both untreated and relapsed SAA. Relapses after CY are rare and progression to PNH or MDS in previously untreated patients has not been observed in this series with now 15 patients out beyond 5 years. The presence of a PNH population may be a favorable risk factor, perhaps by excluding non-immune mediated forms of SAA.

Figure Figure
Prevention of Acute Graft-Versus-Host Disease by CD4+CD25+ Regulatory T Cells Is Dependent on the CD30/CD153 Pathway.
Authors: Zeiser Robert , Nguyen Vu , Buess Martin , Karimi Mobin , Bjorck Pia , Baker Jeanette , Negrin Robert S.
Published: April 2020

CD4+CD25+ regulatory T (Treg) cells suppress acute graft versus host disease (aGVHD), prevent autoimmunity and delay allograft rejection. CD30 and other TNF-R family members have been demonstrated to be expressed by Treg and to function as alternative costimulatory pathways for T cell activation. In this study we assessed the significance of the CD30/CD153 pathway in Tregs suppression of aGVHD in a murine major MHC mismatch BMT model. Using bioluminescence imaging proliferation of donor derived luciferase-labeled CD4+ and CD8+ T cells was quantified at serial time points after transplantation. Treg suppressed the early expansion of alloreactive T-cells. Immunofluorescence microscopy revealed a predominant infiltration of donor derived Treg in CD153 positive regions of secondary lymphoid organs, namely parafollicular T cell zones of lymph nodes and the subepithelial dome regions of Peyers Patches. In vivo blockade of the CD30/CD153 pathway with anti CD153 Ab did not alter Treg migration to secondary lymphoid organs but reduced their suppressive effect. Proliferation of donor T cells as measured in photons/second/mouse was significantly higher in animals receiving Treg and CD153 blocking antibodies as compared to recipients of Treg only (p=0.0038). Gene expression profiling of Treg with DNA microarrays indicated a Treg signature that was consistently found in different mouse strains. This Treg signature was altered after CD153 blockade in vitro. Importantly, aGVHD lethality was significantly increased (p=0.021) when CD30-CD153 interaction was blocked during Treg transfer. This study provides direct evidence that the TNF-R family member CD30 is critical for Treg cell function in the regulation of pathological T cell responses that lead to aGVHD.
Inhibition of Graft-Versus-Host Disease (GVHD) by Histone Deacetylase Inhibitor (HDAC) SAHA Leads to Downregulation of STAT1 Activation.
Authors: Leng Corinna , Li Cuiling , Ziegler Judy , Lokshin Anna , Lentzsch Suzanne , Mapara Markus Y.
Published: April 2020

Histone deacetylase (HDAC) inhibitors have been shown to reduce development of graft versus host disease [GVHD] following allogeneic bone marrow transplantation [BMT]. Administration of the HDAC inhibitor suberonylanilide hydroxamic acid [SAHA] resulted in a significantly reduced GVHD-dependent mortality following fully MHC-mismatched allogeneic BMT. Median Survival Time (MST) for vehicle and SAHA-treated mice were 7.5 days and 38 days respectively. However, SAHA treatment did not affect T cell activation nor T cell expansion in vitro and in vivo as determined by MLR assays, phenotypic analysis of donor T cells with regard to expression of the CD25 activation antigen and calculation of donor CD4+ and CD8+ T cell numbers on days +3 and +6 post-BMT. Thus, SAHA treatment was not able to inhibit the strong upregulation of CD25 antigen on CD8+ T cells observed during induction of GVHD on days +3 and +6 post-BMT. We therefore focused on the effects of SAHA treatment on efferent immune effects including cytokine secretion and intracellular signaling events in vitro and in vivo following GVHD induction. SAHA treatment broadly inhibited lipopolysaccharide [LPS] and allo-antigen-induced cytokine/chemokine secretion in vitro like MIP-1-α, IP-10, IFN-γ, TNF-α and IL-6 and led also to a significant decrease in IFN-γ and TNF-α levels in vivo following induction of GVHD. Concomitantly, SAHA treatment inhibited phosphorylation of STAT1 and STAT3 in response to LPS and allo-activation in vitro. Furthermore, analysis of liver tissue and spleens from SAHA-treated animals with GVHD showed a significant decrease in phosphorylated STAT1. In contrast SAHA treatment had only moderate effects on p38 or ERK1,2 Mitogen-activated Protein Kinase (MAPK) pathway underscoring the relevance of the inhibition of the STAT1 pathway. In conclusion, GVHD is associated with a strong induction of phosphorylation of STAT1 in the liver and spleen and SAHA-dependent reduction of GVHD is associated with systemic and local inhibition of pSTAT1 and modulation of the inflammatory cytokine milieu during the efferent immune response.
Immunomodulatory Effects of the Triterpenoid CDDO after Allogeneic Bone Marrow Transplantation in Mice: Reduction of Acute Graft-Versus-Host Disease Lethality.
Authors: Murphy William J. , Frolova Olga , Konopleva Marina , Andreeff Michael , Ma Weihong , Wilkins Danice , Welniak Lisbeth A. , Sun Kai
Published: April 2020

The use of hematopoietic stem cell transplantation (HSCT) in cancer treatment is seriously hampered by the occurrence of graft-versus-host disease (GVHD) and cancer relapse. During acute GVHD, inflammatory cytokines play a pivotal role in the amplification of GVHD. Therefore, assessment of agents with known anti-neoplastic activity that also reduce cytokine production may be useful in both the prevention of GVHD and cancer relapse. The synthetic triterpenoid, CDDO (2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid) is multifunctional molecule which has shown potent anti-cancer activities both in vitro and in vivo through the induction of apoptosis. We first examined the effects of CDDO on both human and murine T cell mitogen responses in vitro. CDDO significantly inhibited mitogen responsiveness of both human and murine T cells in vitro with evidence of cell cycle arrest of the human T cells. We then proceeded to examine the effects of CDDO on acute GVHD induction and progression. In these studies, lethally irradiated C57BL/6 mice received 10 million bone marrow cells (BMC) and 40 million spleen cells from fully MHC-mismatched BALB/c donors. All of the control mice succumbed rapidly due to acute GVHD. In contrast, the mice that received CDDO (120 ug/BID) given from days 0-3 following BMT exhibited significant improvement in survival (P < 0.001). Body weights from the treated mice also were significantly increased compared to untreated controls. We found that the timing of CDDO administration was a critical factor for protection from GVHD as protection only occurred when CDDO was administered early after BMT. Importantly, donor myeloid reconstitution was not adversely affected by CDDO treatment as determined by peripheral blood cell count and donor chimerism assessment on day +14 post-transplant. No adverse toxicities or effects on reconstitution were observed in the mice receiving BMC alone with CDDO being administered continuously. Given the reported direct anti-tumor effects of CDDO, it will be of particular interest in examining the effects of CDDO and allogeneic BMT in tumor-bearing recipients. Our findings suggest that CDDO can enhance the efficacy of allogeneic BMT by decreasing acute GVHD in mice.
Protein-Based Identification of Inflammatory Signaling Pathways in Graft-Versus-Host-Disease (GVHD): Evidence for STAT1 Pathway as a Key Regulator in GVHD.
Authors: Li Cuiling , Ziegler Judy , Leng Corinna , Lokshin Anna , Lentzsch Suzanne , Mapara Markus Y.
Published: April 2020

The pathogenesis of GVHD is influenced by the immunogenetic disparities between donor and recipient, the presence of host-derived antigen-presenting cells, the inflammatory reaction in response to conditioning-induced tissue damage and cytokine secretion. Intestinal damage and subsequent translocation of LPS into the circulation have been shown to be central to the subsequent pathogenetic events occurring in development of GVHD. Interfering with this proinflammatory response by targeted therapy might be an attractive new approach for inhibiting the development of GVHD. We have therefore started to comprehensively analyze the inflammatory signaling pathways, which are activated during the induction phase of GVHD in order to identify key molecular regulators of GVHD, which might serve as targets for future therapy. For this purpose GVHD was in induced in the fully MHC-mismatched BALB/c to B6 strain combination following lethal irradiation with 9.5 Gy. Spleens as well as GVHD target organs liver, skin and small bowel and colon were harvested on days 1, 3, and + 6 post-BMT to study activation of JAK-STAT, MAPK, PI3-Kinase and NF-κB signaling transduction pathways. Studies were performed using western blot, multiplex assays for analysis of phoshotyrosine proteins and transcription factor binding activity and also standard gel shift assays. Furthermore, GVHD liver tissue was subjected to microarray gene expression profiling. Most strikingly, in contrast to syngeneic and allogeneic controls induction of GVHD was associated with a strong activation of JAK-STAT pathway as determined by Tyr701-specific STAT1 phosphorylation and also Tyr705-specific STAT3 phosphorylation in the liver and spleen as early as on day +1 post-BMT and remained elevated until day +6 post-BMT. In line with these results we observed a significant increase of STAT1 dependent gene expression (e.g. CXCL10 5.6-fold, IRF7 4.5-fold, CXCL9 4.2-fold) as studied by microarray gene expression profiling in the liver. Furthermore, multiplex DNA-binding and standard gel shift studies in the liver revealed that GVHD led to an increased DNA binding activity of most prominently NF-κB but also AP-2, EGR1 and NF1. In conclusion we provide evidence that induction of GVHD is associated with strong activation of the STAT1 pathway in the spleen and liver as early as on day +1 post-BMT in additon to activation of the NF-κB pathway. Therefore this study might help to identify candidate molecules to create more specific therapies targeting the inflammatory component of GVHD and thus reducing the development of GVHD without mitigating the GVL response.