bluuebunny/crossref_metadata_2025 huggingface dataset:
Lmo2 Induces T-Cell Leukemia with Epigenetic Deregulation of CD4.
Authors: Cleveland Susan , Dave Utpal P.
Published: October 2019

The LIM domain Only-2 (Lmo2) oncogene is deregulated in the majority of human T-cell acute lymphoblastic leukemias (T-ALL). Enforced, constitutive expression of Lmo2 in transgenic mice using the T-cell-specific Cd2 promoter/enhancer induces T-ALL with median latency of 200 days and 100% penetrance. These transgenic mice have a pre-leukemic differentiation block at the double negative stage (CD4− CD8−) of T-cell differentiation. Despite this, T-ALLs in these mice arise from various stages of T-cell differentiation. To explore the relationship between T-cell development and Lmo2 oncogene function, we established cell lines from four independent T-cell tumors representing the double negative (DN), double positive (DP), and single positive (SP) stages. Two of the lines showed variegated expression of CD4 antigen. One line, 32080, was striking in that CD4 was expressed in 50% of cells whereas CD8 was uniformly present. The cell line expressed markers consistent with the intermediate single positive stage (ISP) to double positive (DP) stage transition. We sorted the 32080 line for highly CD4 positive and negative cells and observed them in culture. After one week, both sorted populations showed variegated CD4 expression. We determined CD4 expression was regulated at the transcriptional level and not due to post-transcriptional effects and that cell replication was necessary to re-establish CD4 variegation. Importantly, variegated CD4 expression was dependent on Lmo2 because its knockdown resulted in decreased CD4 expression. HDAC inhibition by trichostatin A had the reciprocal effect of inducing more CD4 expression suggesting that chromatin modification was playing a role. The CD4 locus was analyzed by chromatin immunoprecipitation and histone H3 methylation patterns consistent with silencing were found in the CD4 negative sorted cells but not in the CD4 positive population. The 32080 cell line is a striking model of ISP to DP T-cell plasticity and invokes a novel mechanism by which Lmo2 functions.
Development of a Preclinical Model for An Erythropoietin Deficient Anemia
Authors: Zeigler Brandon , Qin Wenning , Vajdos Janis , Loverro Linda , Niss Knut
Published: October 2019

The production of red blood cells is tightly controlled by the growth factor erythropoietin (EPO). Mice lacking a functional EPO gene die in-utero due to the complete absence of definitive red blood cells. Thus, studying the role of EPO in adult animals has been limited to the use of artificial anemia models such as phenylhydrazine (PHZ) treatment or phlebotomy. In addition, injections of rHuEPO have been shown to be a valuable tool to study erythropoiesis. However, these approaches have limited use as preclinical models since they do not recapitulate an anemia due to EPO deficiency as it is seen in patients with chronic kidney disease (CKD). These patients develop a chronic anemia due to the lack of a sufficient EPO gene expression response. In order to obtain a preclinical model for an EPO deficient anemia we developed a mouse model in which the EPO gene is flanked by two loxP sites (flox allele). In addition, these animals carry the Cre-ERT2 transgene which allows for a tamoxifen controlled activation of the Cre recombinase. Thus, these mice enable the inactivation of the EPO gene in adult animals by tamoxifen administration.

After induction of Cre activity, mice heterozygous for the EPO allele (EPOko/flox) develop a moderate to severe anemia within 3 weeks. Although anemic, these mice display only low levels of EPO in their serum. However, within 3 months after induction of the knock-out the anemia resolves. This indicates that the Cre mediated excision of the EPO gene is insufficient and some EPO expressing cells remain. This mimics the clinical situation of patients with CKD.

In order to study the response of EPO deficient mice to a severe anemia we treated animals with phenylhydrazine and observed the recovery of these animals. Interestingly, EPO deficient animals, although slightly delayed, fully recover from the induced anemia. Although the measured sEPO levels are significantly lower compared to control animals an increase in splenic erythropoiesis was observed in these animals. This indicates that the hypoxic response triggered by a sudden severe anemia is activating pathways other than EPO that promote erythropoiesis. Taken together, these results show that this novel mouse model will be useful tool as a preclinical model for the anemia associated with CKD. In addition, this model will allow the study of stress erythropoiesis under EPO limiting conditions. The Pfizer Institutional Animal Care and Use Committee reviewed and approved the animal use in these studies. The animal care and use program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International
Chk1 Haploinsufficiency Results in Anemia and Defective Erythropoiesis
Authors: Boles Nathan C , Peddibhotla Sirisha , Chen Alice , Rosen Jeffrey , Goodell Margaret A.
Published: October 2019

Erythropoiesis is a highly regulated and well-characterized developmental process responsible for providing the oxygen transport system of the body. However, besides erythropoietin’s role, few of the mechanisms involved in this process have been elucidated. Checkpoint Kinase 1 (Chk1) is best known for its role in the cell cycle and DNA damage pathways. Chk1 expression in the hematopoietic system has been demonstrated to be restricted to T-cells, erythroid cells and the hematopoietic stem cell. Interestingly, a lack of Chk1 has recently been shown to disrupt T-cell differentiation (Zaugg et al. PNAS 2007) Here, we show that haploinsufficiency of Chk1 leads to a 30% increase in the incidence of anemia in mice. Furthermore, the anemic Chk1 +/− mice exhibit distorted spleen and bone marrow architecture, and an increase in erythroid progenitors (MEP, stages I, and II). In young non-anemic Chk1 +/− mice, a decrease in enucleated cells of the erythroid lineage is observed, suggesting a role for Chk1 in proper erythroid enucleation. In addition, a decrease in Chk1 RNA has been observed in patients with refractory anemia with excess blasts (Pellagatti et al. Blood, 2006), further supporting an important role for Chk1 status in clinical anemia. Finally, Chk1 has recently been show to affect the regulation of Fancd2, one of the gene’s responsible for Fanconi anemia. These results indicate Chk1 plays a part in maintaining the balance between erythroid progenitors and the enucleated erythroid cells during differentiation.
The Impact of Rhg-CSF on the LFA-1 Conformation of CD4+T Cells in Mobilized Hematopoietic Stem Cell Allografts
Authors: Gao Chunji , Chen Weihua , Wang Fei , Li Meng , Zhu Haiyan , Da Wanming
Published: October 2019

In hemotopoietic stem cell transplantation (HSCT), the primary effects of G-CSF on cells of the hemotopoietic system include stimulation of proliferation and differentiation of HSCs, acceleration of neutrophil reconstitution after HSCT and mobilization of bone marrow HSCs into the peripheral blood. In recent years, several investigators have unraveled that G-CSF-mediated immune regulation including the switching T cell cytokine secretion profile to Th2 response, the altering adhesion activity of CD4+T cells to ICAM-1 and so on. Most of these cellular interactions of T cells are dependent on the integrin leukocyte function associated antigen-1 (LFA-1, αLβ2, CD11a/CD18) binding to intercellular adhesion molecule-1 (ICAM-1). Integrin αLβ2 adopts a bent, compact conformation in their low affinity state and an extended conformation in the high affinity state. The epitope of MEM148 is only expressed on the free β2 integrin subunit but is masked on LFA-1. The monoclonal antibody MEM148 reacts with an epitope exposed on free human CD18 chain as well as on high affinity state of LFA-1 that represents a reporter for αLβ2 active conformation. Our previous studies have demonstrated that rhG-CSF mobilization decreased the adhesion activity of CD4+T cells to ICAM-1, but did not find that rhG-CSF mobilization had effect on the level of CD11a expression. rhG-CSF maybe has effect on conformational change of LFA-1 on CD4+T cells. In order to explore the impact of rhG-CSF mobilization on LFA-1 conformation of CD4+ T cells, human CD4+T cells were isolated from peripheral blood mononuclear cells by positive selection with Miltenyi MACS. Isolated CD4+T cells were activated by OKT3+ICAM-1 and PMA+Ion separately. The slides with CD4+T cells were incubated with FITC-CD11a and FITC-MEM148 mAb, then were examined by using fluorescence microscope. The results show that the level of CD25, CD69 expression and MEM148 epitope exposure on activated CD4+T cells were significantly higher compared with unactivated CD4+T cells. rhG-CSF mobilization had no effect on the expression of CD11a, but could decrease expression of CD25, CD69 and exposure of MEM148 epitope on activated CD4+T cells. The percentage of LFA-1 polarized CD4+T cells before and after rhG-CSF mobilization was 85.32%, 61.86% respectively (p <0.01). No MEM148 epitope exposure on unactivated CD4+T cells was detected, but MEM148 epitope exposure on activated CD4+T cells before and after rhGCSF mobilization was 63.63%, 32.79% respectively (p <0.01). Overall, these data suggest that rhG-CSF can affect on the activation of CD4+T cells in mobilized hematopoietic stem cell allografts by altering the conformation of LFA-1.
Induced CD4+CD25+ FOXP-3+ and CD8+CD25+ FOXP-3+t Cells Exhibit a Concurrent Expression of Effector and Selective Suppressive Capacities
Authors: Avivi Irit , Stroopinsky Dina , Rowe Jacob , Avigan David , Katz Tami
Published: October 2019

Naturally occurring regulatory T cells (Tregs), identified as CD4+CD25+FoxP-3+ cells, are known to modulate immunologic tolerance and inhibit primary T cell activation, and therefore, may protect against graft-versus-host disease (GvHD) following allograft. However, having a non-selective suppressive effect, Tregs administration following transplantation may also result in delayed immunological reconstitution and diminished graft-versus-tumor effect, leading to an increased risk of infections and disease relapse. The generation of suppressive T cells that could selectively inhibit GvHD whilst preserving immunity against malignant cells and infection would promote significant contribution to GvHD prevention and therapy. The aim of this study was to investigate the characteristics of T cells undergoing allogeneic stimulation, looking for a subpopulation with a more specific inhibition of host immunity. A negative selection of PBMCs with anti-CD25 magnetic beads resulted in the production of CD25+ cells (-Naturally occurring) and CD25−PBMCs. CD25− PBMCs were than stimulated with allogeneic DC for 5–6. The resultant CD4+CD25+ or CD8+CD25+ populations were isolated with magnetic beads and investigated for immunophenotypical markers, cytokine expression profile (FACS), cell proliferation, inhibitory capacity and anti-viral response (INF-gamma). The current study has shown that stimulation of CD25− T cells with allogeneic peripheral blood mononuclear or dendritic cells (DCs) induces generation of a large fraction of CD4+CD25+ and CD8+CD25+ T-cells. This process was found to be more rapid and prominent following DCs stimulation. The resultant cells exhibit a mixed phenotype, demonstrating expression of the regulatory marker, FoxP3, as well as the inflammatory cytokines, IL-2 and IFNγ. Similar to naturally occurring Tregs, the induced CD4+CD25+ and CD8+CD25+ have markedly inhibited alloreactive T cell expansion. However, in contrast to naturally occurring Tregs, the induced populations, did not suppress proliferation against the cytomegalovirus (CMV) recombinant protein, suggesting their potential for more selective inhibition of host immunity.

Expanded naturally occurring CD4+CD25+T Induced CD4+CD25+T cells Induced CD8+CD25+T cells GITR + ++ ++ Immunophenotype CTLA-4 + ++ ++ FoxP-3 + ++ ++ Self-proliferation − − − Suppression rate Primary allogeneic stimuli + ++ ++ CMV recall antigen stimuli + − − Cytokine expression IL-2 − + + IFN-γ − ++ ++ Cytotoxic capacity (granzyme B) − − +

According to these data, CD25− derived CD4+CD25+FOXP-3+ and CD8+CD25+ FOXP-3, appear to have a dual expression of both effector and selective suppressive capacities, and therefore, might be potentially ideal for the use as post-transplant GvHD prophylaxis.
Only MHC-Identical Donor CD4+CD25+ Regulatory T Cells Convey Full Protection from Lethal Graft-Versus-Host Disease
Authors: Albrecht Julia , Doser Kristina , Andreesen Reinhard , Ermann Joerg , Edinger Matthias , Hoffmann Petra
Published: October 2019

Natural CD4+CD25+ regulatory T cells (Treg) contribute to tolerance induction after transplantation. We previously showed that the adoptive transfer of donor-derived Treg cells prevents lethal graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) in murine disease models. In contrast, host-type Treg cells failed to protect when co-transplanted under identical conditions. We now examined whether MHC compatibility between Treg cells and conventional CD25−CD4+ and CD8+ T cells (Tconv) is required for the suppression of alloresponses, or whether elimination of host-type Treg by allo-aggressive donor Tconv cells occurred. To address this issue, mixed lymphocyte reactions were performed in which CFSE-labelled responder T cells (Tresp), Treg cells and antigen presenting cells (APC) were systematically varied with regard to their MHC haplotype. When BALB/c (H-2d) Tresp cells were stimulated with mixed BALB/c and C57BL/6 (H-2b) APC, cultures contained 26.0 ± 3.1% and 86.2 ± 2.2% proliferating CD4+ and CD8+ T cells, respectively, on 6 d. In the presence of syngeneic BALB/c Treg cells, proliferation was decreased to 9.1 ± 4.7% and 25.1 ± 4.9% for CD4+ and CD8+ Tresp cells, respectively. In contrast, in cultures with allogeneic C57BL/6 Treg cells, proliferation remained at 22.1 ± 1.8% for CD4+ and 89.6 ± 0.4% for CD8+ Tresp cells. Comparable results were obtained with C57BL/6 Tresp cells after stimulation with F1 (C57BL/6 × BALB/c; H-2b/d) or 3rd party (DBA/1; H-2q) APC. Lack of suppression in co-cultures of MHC-mismatched Tresp and Treg cells was not caused by an early elimination of allogeneic Treg cells, as those were still detectable after 6 d of allostimulation. In corresponding in vivo studies, CB6F1 or DBA/1 recipients were protected from lethal GVHD only when Tconv and Treg cells were derived from MHC-identical donors, but not when they were from two MHC-disparate strains. Transplantation of 1 × 106 C57BL/6 Tconv cells resulted in 100% lethality of CB6F1 recipients by d56. When co-transplanted with 1 × 106 C57BL/6 Treg cells, all recipients survived for 100d, whereas only 40% survived after co-transfer of the same number of BALB/c Treg (n = 15; p = 0.004). Similarly, when 1 × 106 BALB/c Tconv cells were transplanted into CB6F1 recipients, all animals died from GVHD by d46. In contrast, all recipients of BALB/c Tconv and Treg cells (ratio1:1) survived for 100d, but only 10% of recipient mice survived after co-transfer of C57BL/6 Treg (n = 10; p < 0.001). Similar results were obtained after BALB/c and C57BL/6 T cell transfer into DBA/1 (3rd party) recipients. In conclusion, these data indicate that MHC-identity between Tconv and Treg cells is required for maximum suppression of an alloresponse and that Treg cells isolated from a 3rd party donor might not be suited for the prevention of GVHD after allogeneic BMT.
Reduction of Inhibitory Anti-FVIII Antibody Titer by Using a B Domain Variant FVIII/N6 cDNA for Nonviral Gene Therapy in Hemophilia a Mice
Authors: Jirovska Dominika , Ye Peiqing , Pipe Steven W. , Miao Carol H.
Published: October 2019

Due to the large size of FVIII, a B-domain deleted FVIII (BDD-FVIII) cDNA is usually used for developing gene therapy protocols for treating hemophilia A. Inefficient transcription of wild- type FVIII cDNA can be overcome by deletion of the heavily glycosylated B-domain encoding portion of the gene. BDD-FVIII is as clinically efficacious and not more immunogenic than full-length recombinant FVIII. More recently, it was demonstrated that a partial deletion of the B-domain leaving an N-terminal 226 amino acid stretch containing 6 putative asparagine-linked glycosylation sites intact (FVIII/N6) was able to increase in vitro and in vivo secretion of FVIII by 10–15 fold. We have inserted this B domain variant FVIII/N6 cDNA into our liver-specific gene expression vector. The resulting construct, FVIII/N6 plasmid was delivered into the hemophilia A mouse liver by the hydrodynamic method. In control mice treated with BDD-FVIII plasmid (n=5/group), FVIII expression dropped to undetectable levels at 2 weeks post injection and high-titer anti-FVIII antibodies were generated in all the plasmid-treated mice. However, in mice treated with FVIII/N6 plasmid (n=5/group), one out of five mice never developed inhibitory antibodies and still had some FVIII gene expression (~10%) at 8 weeks post gene transfer. Three FVIII/N6 plasmid-treated mice developed anti-FVIII antibodies with significantly reduced inhibitor titer and only one mouse developed high-titer inhibitory antibodies. The CD4+ T cells isolated from the spleen of mice injected with FVIII/N6 constructs proliferated less in response to FVIII stimulation than those from mice injected with BDD-FVIII. These results indicate that FVIII/N6 protein is less immunogenic than BDD-FVIII. Interestingly, both BDD-FVIII and FVIII/N6 constructs produced similar levels of FVIII gene expression (100–300%) initially following nonviral gene transfer. However this could be due to saturation of the ER to Golgi transport apparatus for FVIII by the initial high-level gene expression. Gene expression levels produced by using reduced dosages of BDD-FVIII and FVIII/N6 plasmids are currently being evaluated and compared. These findings suggest that use of a FVIII/N6 construct decreases transgene-specific immune responses following nonviral gene transfer and facilitates long-term gene expression.
THP-COP Regimen for the Treatment of Peripheral T Cell Lymphoma and Adult T Cell Leukemia/Lymphoma: A Multicenter Phase II Study
Authors: Takamatsu Yasushi , Suzumiya Junji , Utsunomiya Atae , Maeda Koichi , Matsuoka Hitoshi , Suzushima Hitoshi , Tsukada Junichi , Tamura Kazuo
Published: October 2019

Background: Pirarubicin (THP) is an anthracycline which was introduced to the Japanese market due to less cardiotoxicity compared to doxorubicin. The efficacy of THP-COP regimen (THP, cyclophosphamide (CPM), vincristine (VCR) and prednisolone (PSL)) was compared with CHOP in elderly patients with non- Hodgkin’s lymphoma in the previous Japanese randomized trial [Int J Hematol 81, 246–254, 2005]. The subset analysis showed peripheral T cell lymphoma (PTCL) had significantly better complete response (CR) rate with THP-COP than that of CHOP, whereas no such difference was observed in patients with B cell lymphoma.

Methods: We underwent a multicenter phase II study to investigate the efficacy of THP-COP as a first line treatment for PTCL and adult T cell leukemia/lymphoma (ATLL). THP 50 mg/m2, CPM 750 mg/m2, and VCR 1.4 mg/m2 were given intravenously on day 1. PSL 60 mg/m2 was administered orally on days 1–5. The treatment was repeated at a 21-day interval, and its efficacy and toxicity were investigated.

Results: Fifty-three patients aged from 38 to 83 (median 66) were entered into this study. Twenty-six patients were male and 27 female. Seventeen patients had PTCL including PTCL-U and AILT, and 36 ATLL. Stage IV disease was seen in 33, stage III in 15, and stage II in 5 patients. Thirty-four (64%) patients responded to THP-COP, including 17 (32%) CR and 17 (32%) partial response (PR). The median time to progression and overall survival were 9.5 and 17.5 months, respectively. Grade 3 to 4 neutropenia, anemia and thrombocytopenia occurred in 38 (72%), 18 (34%) and 31 (58%) patients, respectively. Twenty-seven (51%) patients developed febrile neutropenia, while grade 3 of nausea, anorexia, fatigue, arrhythmia, cardiac ischemia, sensory neuropathy, hypoalbuminemia, hyperbilirubinemia, and elevated serum AST/ALT were observed in 1 to 6 (2–11%) patients. No other adverse events greater than grade 3 were encountered.

Conclusion: THP-COP is an effective and well-tolerated treatment regimen in patients with PTCL and ATLL as a first-line therapy.
NR2F6 Initiates Myelodysplasia and Acute Leukemia by Promoting Stem Cell Self-Renewal
Authors: Ichim Christine V. , Reis Marciano D. , Chesney Alden , Knowles Gisele , Iscove Norman N. , Wells Richard A.
Published: October 2019

This abstract was removed at the request of the author.
The Novel Aurora Kinase Inhibitor ENMD-2076 Has Potent Single Agent Activity against Multiple Myeloma (MM) in Vitro and in Vivo, and Shows Synergistic Activity in Combination with Lenalidomide
Authors: Wang Xiaojing , Sinn Anthony L. , Suvannasankha Attaya , Crean Colin D. , Chen Li , Zhang Shuhong , Liang Jing , Zhang Genglin , Pollok Karen E. , Abonour Rafat , Sidor Carolyn , Bray Mark R. , Farag Sherif S.
Published: October 2019

ENMD-2076 is a novel, orally-active molecule that has been shown to have significant activity against Aurora A kinase as well as multiple receptor tyrosine kinases (RTK). We investigated the single agent activity of ENMD-2076 against MM cells in vitro and in vivo, and in combination with lenalidomide. ENMD-2076 free base showed significant cytotoxicity against MM cells with a mean LC50 of 3.84±0.86 μM at 48 hours in vitro. Cytotoxicity was associated with cleavage of caspase 3, 8, 9 and PARP, and loss of mitochondrial membrane potential as early as 6 hours. ENMD-2076 free base inhibited c-kit, FGFR-1, 3 and VEGFR1 and subsequently inhibition of downstream targets phosphorylated (p)-BAD, p-Foxo1a and p-GSK-3β was observed at 6 hours. NOD/SCID mice implanted with H929 human plasmacytoma xenografts and treated for 30 days with 50, 100, 200mg/kg/d ENMD-2076 showed a dose-dependent inhibition of tumor growth (Figure 1), with minimal toxicity as assessed by the stable weight of treated animals. Immunohistochemical staining of tumors from sacrificed animals showed significant reduction in Ki67 at all dose levels of treatment compared to control tumors. An increase in cleaved caspase-3 was observed on Western blot from the lysates of H929 tumors obtained from treated animals. ENMD-2076 free base also showed synergistic cytotoxic activity when combined with lenalidomide against H929, MM1.R and MM1.S cells as assessed by MTT assay and Annexin-V/PI staining. Using the Chou-Talalay method, the combination indices (CI) were < 1 for all three cell lines across a range of concentrations of ENMD-2076 free base (0.25–1.0 μM) plus lenalidomide (2.5–10 μM) indicating synergistic activity (CI=0.362 H929; CI=0.315 MM1.R; CI=0.415 MM1.S). Our results provide rationale for the investigation of ENMD-2076 alone and in combination with lenalidomide in patients with multiple myeloma.

Figure Figure
Results of a Phase I Study of GRN163L, a Direct Inhibitor of Telomerase, in Patients with Relapsed and Refractory Multiple Myeloma (MM)
Authors: Chanan-Khan Asher Alban , Munshi Nikhil C. , Hussein Mohamad A. , Elias Laurence , Benedetti Fabio , Smith Jennifer , Khor Soo-Peang , Huff Carol A.
Published: October 2019

Telomerase over-expression is the predominant mechanism by which cancer cells maintain adequate telomere length to achieve immortalization. Telomere length is often decreased and telomerase activity is often increased in MM. GRN163L is a 13-mer oligonucleotide that directly inhibits telomerase activity and has demonstrated anticancer effects in various preclinical models. We are conducting a phase I dose escalation study to define the maximum tolerated dose (MTD), safety, tolerability, efficacy as well as pharmacokinetics of GRN163L in patients with relapsed or refractory MM. Each treatment cycle consisted of 3 weekly 2 hr i.v. infusions of GRN163L. Dose escalation followed standard “3+3” dose finding rules.

To date, 12 patients, median age 61 years, have been treated in 3 dose cohorts (3.2, 4.8 and 7.2 mg/kg). Patients had received a median of 4.0 prior treatment regimens. All patients had normal baseline neutrophil and 10 had normal baseline platelet counts. Patients completed a median of 2 cycles of GRN163L treatment, with one receiving 4 and another 6 cycles. GRN163L has been generally well-tolerated to date. One patient had Gr 3 anorexia however all other related or possibly related non-hematologic AEs to date were Gr 1 or 2. Treatment related events included thrombocytopenia, neutropenia, aPTT prolongation, anemia, fatigue, nausea, anorexia, and dizziness. No dose limiting toxicity (DLT) occurred among patients in the first 2 cohorts. All patients exhibited transient dose-related aPTT prolongations, which resolved in parallel with decreasing GRN163L plasma levels. There were no bleeding episodes or clinical signs of complement activation. Two of the 5 patients in the 7.2 mg/kg cohort had transient prolongation of aPTT to > 3 fold of the upper limit of normal. One patient in the highest dose group had Gr 4 thrombocytopenia in Cycle 1, which constituted a DLT. Delayed (cycle 2 or later) Gr 3 or 4 neutropenia or thrombocytopenia was noted in 5 additional patients with no episodes of febrile neutropenia. Maximal post-infusion plasma concentrations (Cmax) of GRN163L have been linear with respect to dose. Mean (± SD) plasma concentration of GRN163L declined by 41.1 ±17.6 %, N=9, over the 2 hours following the first infusion, consistent with other previously reported studies. DLTs observed in this study were thrombocytopenia and aPTT prolongation.

The MTD for continuous weekly dosing of GRN163L in this heavily pretreated, relapsed and refractory MM population appears to be ≥ 4.8 and < 7.2 mg/kg. The most marked hematologic toxicity was observed in two patients with prior autologous stem cell transplantation. Exploration of intermediate dose levels in this range is continuing. Additional studies exploring alternative dosing schedules will be initiated.
Phase I Study of IMGN901 in Patients with Relapsed and Relapsed/Refractory CD56-Positive Multiple Myeloma
Authors: Chanan-Khan Asher Alban , Gharibo Mecide , Jagannath Sundar , Munshi Nikhil C. , Anderson Kenneth C , DePaolo Dawn , Lee Kelvin P. , Miller Kena , Guild Robin , Zildjian Sybil , Qin Albert
Published: October 2019

Background: IMGN901 (huN901-DM1) is a novel conjugate of the cytotoxic maytansinoid, DM1, with the humanized CD56-binding monoclonal antibody, N901. Once bound to CD56 on a cancer cell, the conjugate is internalized and releases DM1. About 70% of multiple myeloma (MM) cases have surface expression of CD56. Preclinical investigations demonstrated significant in vitro and in vivo anti-myeloma activity of IMGN901.

Objectives: To determine the maximum tolerated dose (MTD), the dose-limiting toxicities (DLTs), and pharmacokinetics (PK) of increasing doses of IMGN901 given for MM on a weekly schedule.

Methods: Patients with relapsed or relapsed/refractory MM who have failed at least one prior therapy and have CD56-expressing MM received a single IV infusion of IMGN901 on 2 consecutive weeks every 3 weeks. Patients are enrolled in cohorts of 3 at each dose level, with DLT triggering cohort expansion.

Results: Eighteen patients have received IMGN901 to date in this study - 3 patients each at 40, 60, 75, 90, 112, and 140 mg/m2/week. One patient experienced a DLT (grade 3 fatigue) on 140 mg/m2/week, and this cohort is being expanded to enroll up to 6 patients. No patients have experienced serious hypersensitivity reactions or evidence of presence of humoral responses against the huN901 antibody component (HAHA) or against the DM1 component (HADA). Preliminary PK results indicate an approximately linear relationship between dosing and observed maximal serum concentration. A confirmed minor response (MR) was documented in 3 heavily pretreated patients (1 patient each at 60, 90, and 112 mg/m2/week) using the European Bone Marrow Transplant criteria. Durable stable disease was reported at doses of 60, 90, 112, and 140 mg/m2/week. Of the 18 patients treated in the study, eight patients remained on treatment with IMGN901 for at least 15 weeks, five of these 8 patients remained on treatment for at least 24 weeks, and two of these 5 patients remained on treatment for at least 42 weeks. This phase I study provides preliminary evidence of safety as well as clinical activity of IMGN901 in patients with CD56-positive MM who have failed established MM treatments. Updated results of this ongoing study will be presented.
EXEL-8232, a Small Molecule JAK2 Inhibitor, Effectively Treats Thrombocytosis and Extramedullary Hematopoiesis in a Murine Model of Myeloproliferative Disease Induced by MPLW515L
Authors: Wernig Gerlinde , Kharas Michael G. , Leeman Dena S. , Okabe Rachel , Gozo Maricel , Paktinat Mahnaz , Haydu Erika , Ball Brian , Khandan Tulasi , Clary Douglas O. , Gilliland D Gary
Published: October 2019

Approximately 50% of patients with essential thrombocythemia (ET) or myelofibrosis (MF) lack activating mutations in JAK2. Among these patients, ~10% harbor an activating mutation in the thrombopoietin receptor, MPLW515L. We have reported that expression of MPLW515L in a murine bone marrow transplant model recapitulates many features of ET and MF, including severe fibrosis and thrombocytosis, that are not observed in the JAK2V617F model. These observations provide an opportunity to assess the efficacy of small molecule JAK2 inhibitors on a myeloproliferative disease (MPD) induced by MPLW515L in vivo, and to determine whether such inhibitors attenuate thrombocytosis. We have tested EXEL-8232 for efficacy in suppression of thrombocytosis in vivo, and for its ability to attenuate JAK2V617F-negative MPD mediated by MPLW515L. EXEL-8232 is a potent small molecule inhibitor of JAK2 and is structurally similar to XL019, a compound currently in clinical trials for MF and polycythemia vera. EXEL-8232 is selective for JAK2 with a biochemical IC50 of 2 nM, and abolished constitutive phosphorylation of JAK2 and STAT5, as well as cytokine-independent growth, of Ba/F3 cells in vitro. After disease was established 12 days post-bone marrow transplantation, EXEL-8232 was administered for 28 days q12h by oral gavage at doses of 30mg/kg or 100mg/kg respectively. Animals treated with 100mg/kg normalized high platelet counts in excess of2 million/ml and normalized leukocytosis from a median of 134,000/ml in vehicle treated controls. Furthermore, drug treatment eliminated extramedullary hematopoiesis in the spleen, as well as bone marrow fibrosis. Of note, EXEL-8232 had no impact on erythrocytosis in diseased animals or in wild type controls, and wild type animals treated with either dosage of 30mg/kg or 100mg/kg did not develop thrombocytopenia. Consistent with these clinical responses, the surrogate endpoints for response to treatment included a reduction of genomic disease burden in the 100mg/kg treated arm (p<0.05) as assessed by quantitative PCR, a reduction of endogenous colony growth, as well as a inhibition of activation of P-STAT5, P-STAT3 and P-S6K1 kinase as assessed by flow cytometry in immature erythroid and myeloid primary cells both in vitro and upon treatment in vivo. We conclude that EXEL-8232 has efficacy in treatment of thrombocytosis in vivo in a murine model of ET and MF, and may be of therapeutic benefit for patients with JAK2V617F-negative MPD.
NT-ProBNP Is the Strongest Prognostic Factor for Survival in Lymphoma Patients Following Treatment with Curative Intent
Authors: Salar Antonio , Sanchez-Gonzalez Blanca , Alvarez-Larrán Alberto , Comin Josep , Pedro Carmen , Gimeno Eva , Gómez Miquel , Abella Eugenia , Molina Lluis , Garcia Franscesc , Bruguera Jordi , Besses Carlos
Published: October 2019

Introduction: High blood levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), a sensitive indicator of cardiac abnormalities, have been associated with poor outcome in patients with amyloidosis. However, its clinical value in lymphoma is unknown.

Aim: To assess the value of NT-proBNP as a prognostic variable for survival in patients with lymphoma treated with chemotherapy with curative intent.

Patients and methods: Clinical heart examination, evaluation of cardiovascular risk factors, determination of NT-proBNP (ECLIA; Roche) blood levels and high resolution ultrasound echocardiography were done at diagnosis. Clinical variables related with patient characteristics, lymphoma risk and cardiovascular risk were analyzed.

Results: 117 consecutive pts have been analyzed: median age: 63 years (range: 18–88); male: 56%; NHL: 80%; HL: 20%; ECOG/PS ≥2: 29%; III–IV: 56%; B-symptoms: 16%; LDH >1 nv: 34%; b2-microglobulin >1 nv: 57%; albumin <35 g/L: 32%; haemoglobin <105 g/L: 25%; extranodal ≥2: 20%; number of comorbidities ≥2: 51%; previous cardiac disease: 10%; median NT-proBNP: 107 pg/mL (4–9102); median left ventricular ejection fraction (LVEF): 66% (41–83). High blood levels of NT-proBNP were associated with previous cardiologic history but not with LVEF. With a median follow-up of 15.8 moths (0.2–48.9), 16 patients died. Overall survival (OS) at 24 months was 84% (95% CI: 77– 91%). In the univariate analysis, factors associated with worse OS were: NT-proBNP >900, ECOG/PS ≥2, LDH >1 nv, b2-microglobulin >1 nv, albumin < 35 g/L, haemoglobin <105 g/L, comorbidities ≥2, previous cardiologic history, extranodal sites ≥2, age >60. In multivariate analysis, factors predicting for survival were NT-proBNP >900 (HR 15, 95%CI: 3.9–57.8) (p<0.0001) and ECOG/PS ≥2.(HR 4.1, 95%CI: 1.1–16.3) (p=0.04).

Conclusions:

NT-proBNP >900 pg/mL is the strongest prognostic factor for survival in patients with lymphoma following treatment with curative intent. NT-proBNP is an easy, fast, widely available and well-standardized parameter that could be useful in addition to other well-established prognostic indexes.
Use of MicroRNA Expression Profiling to Identify Prognostic Subclasses in Mantle Cell Lymphoma: Mir-29 Family as New Prognostic Markers
Authors: Lin Jianhong , Zhao Jianjun , Lwin Tint , Dessureault Sophie , Moscinski Lynn C. , Dalton William S. , Sotomayor Eduardo , Cheng Jin , Tao Jianguo
Published: October 2019

Patients with mantle cell lymphoma (MCL) are typically older adults with a male predominance and usually present with stage IV disease. Response to chemotherapy usually results in a tumor response but remissions are short. It carries the poorest prognosis among non-Hodgkin’s lymphoma subtypes and the median survival is 3 to 4 years. However, recent clinical observations have identified variable clinical courses of MCL patients: some patients succumb rapidly to the disease, while others have a more chronic course and may not require treatment for prolonged periods of time. Given this clinical heterogeneity, we performed microRNA expression profiling on 34 mantle cell lymphoma (MCL) biopsy specimens to elucidate its pathogenesis and develop prognostic markers for survival of MCL patients. We identified downregulation of 18 miRNAs and upregulation of 22 miRNAs in MCL when compared with normal peripheral blood B lymphocytes, which is closely associated with molecular abnormalities. Furthermore, we found that expression levels of miR-29 family (miR-29a, miR-29b and miR-29c) were inversely correlated with MCL patient survival. A strong correlation between miR-29a, miR-29b and miR-29c values and newly described prognostic index for MCL patients, MIPI is identified. This first microRNA profling in MCL demonstrated that the miR-29a-c could be useful biomarkers for MCL prognosis and aberrant miRNA expression could contribute molecular MCL oncogenesis.
The Stage-Specific Effect of Interleukin-1 Beta (IL-1β) during Human Natural Killer Cell Development
Authors: Hughes Tiffany L , Becknell Michael Brian , Freud Aharon , Schmidt Susan E , Yu Jianhua , Mao Charlene , Gavrilin Mikhail , Wewers Mark , Caligiuri Michael A.
Published: October 2019

Developmental intermediates of human natural killer (NK) cells are found within secondary lymphoid tissue (SLT), and five distinct stages of these intermediates have been identified. While it is well documented that developing NK cells are reliant on interleukin (IL)-15 as a survival factor, it is likely that additional cytokines and growth factors are required for complete NK cell differentiation. Microarray transcriptional profiling of purified stage 1–4 cells from human tonsil and stage 4 and 5 cells from peripheral blood (PB) identified a developmental window of interleukin-1 receptor 1 (IL-1R1) messenger RNA (mRNA) expression restricted to stages 2 and 3. We confirmed this finding by quantitative RT-PCR, and analysis of IL-1R1 surface protein expression revealed that, on average, 81% of stage 3 immature NK cells are IL-1R1(+), whereas the majority of cells from stages 1, 2, and 4 are IL-1R1(−). When cultured in vitro with IL-1β, a physiologic ligand for IL-1R1, cells from all four stages died within 48 hours, consistent with an absolute requirement for IL-15 as a survival factor. However, the combination of IL-1β and IL-15 led to a significant and reproducible 4.64±−0.68–fold increase in stage 3 cell number over that seen with IL-15 alone (p < 0.0005). This phenomenon was completely restricted to stage 3 immature NK cells, and is attributed to increased proliferation. The effects of IL-1β were abrogated by a molar excess of IL-1 receptor antagonist (IL-1RA), a physiologic competitor for IL-1R1 binding. Collectively, our data indicate that IL-1R1 expression fluctuates dramatically during NK cell development, and that unique responses of IL-1R1(+) stage 3 cells to IL-1β and IL-15 govern the expansion of these immature NK cells. Our findings support a model in which IL-1β promotes stage 3 proliferation and survival in vivo, driving stage 3 cells to be the most prevalent NK cell intermediates within SLT.
Two Point Mutations in the MDS-Associated Protein EVI1 Abrogate the Onset of MDS in the Mouse
Authors: Laricchia-Robbio Leopoldo , Fazzina Raffaella , Dickstein Jerome , Nucifora Giuseppina
Published: October 2019

By forcibly expressing EVI1 in murine bone marrow (BM) we previously generated a model for myelodysplastic syndrome (MDS) characterized by megakaryocytic hyperplasia, dysplastic erythropoiesis and fatal anemia. These defects, typical of MDS patients, confirmed the association between EVI1 and MDS in the mouse thus validating our mouse model. We later ascribed the erythroid defects induced by EVI1 to its interaction with GATA1, as shown by the double point mutant EVI1(1+6Mut), unable to bind GATA1, allowing a normal erythroid differentiation of BM progenitors in vitro. We report here that in vivo expression of the mutant EVI1(1+6Mut) abrogates the onset of MDS. We show that whereas EVI1-positive mice invariably die of severe anemia and pancytopenia 11–14 months after BM reconstitution, EVI1(1+6Mut)-positive mice maintained relatively normal erythropoiesis and blood counts until the study was terminated 20 months after BM transplantation. Furthermore, unlike the EVI1-positive mice two GATA1-regulated genes, EpoR and c-Mpl, are not repressed in the BM of the EVI1(1+6Mut)-mice. The normal expression of EpoR restores the response to Epo, in vitro, of the BM cells isolated from the EVI1(1+6Mut)-mice. We thus suggest that the development of drugs interfacing with the first and sixth zinc-finger motifs could be invaluable in the treatment of EVI1-positive MDS patients.
Immunoassay for Human Hepcidin in Blood
Authors: Olbina Gordana , Pastorino Ximena , Girelli Domenico , O’Brien Kimberly O. , Nemeth Elizabeta , Ganz Tomas , Westerman Mark
Published: October 2019

We developed and validated the first ELISA for hepcidin, the principal iron-regulatory hormone that has been very difficult to measure. The competitive ELISA uses a biotin-tagged hepcidin as a tracer to quantify hepcidin in serum, plasma or urine. In healthy volunteers, the 5–95% range of serum hepcidin concentrations was 29–254 ng/ml in men (n=65) and 17–286 ng/ml in women (n=49), with median concentrations 112 vs. 65, p<0.001. The lower limit of detection was 5 ng/ml. Serum, EDTA plasma and heparin plasma hepcidin measurements were in agreement (r> 0.98). Serum and urinary hepcidin concentrations in 24 healthy subjects correlated well (r=0.82). Serum hepcidin appropriately correlated with serum ferritin (r=0.63) reflecting the regulation of both proteins by iron stores. Healthy volunteers showed a diurnal increase of serum hepcidin at noon and 8pm compared to 8am, and a transient rise of serum hepcidin in response to iron ingestion. In 18 young healthy women, serum hepcidin correlated well (p<0.05) with intestinal iron absorption both from a food (r=0.49) and supplemental iron source (r=0.51), as determined by a stable iron isotope technique. In a variety of clinical conditions associated with iron disturbances we observed the appropriate changes in hepcidin concentrations: undetectable or low in patients with iron deficiency anemia (ferritin<10 ng/ml), treated HFE hemochromatosis, and juvenile hemochromatosis, and abnormally high in patients with inflammation (CRP>10 mg/dl), multiple myeloma, or chronic kidney disease. The new blood hepcidin ELISA yields accurate and reproducible measurements that appropriately reflect physiologic, pathologic and genetic influences, and is informative about the etiology of iron disorders.
Hepcidin-Independent Regulation of Dietary Iron Absorption
Authors: Ajioka Richard S. , De Domenico Ivana , Kushner James P.
Published: October 2019

Iron homeostasis in mammals is maintained at the level of iron absorption by the gut. Hepcidin plays a central role in homeostasis by binding to ferroportin and regulating cellular iron export. We found that mice weaned onto diets ranging from 35–350 mg Fe/kg for a period of 4 wk did not change body iron levels as measured by organ iron content and hematological parameters. Direct measure of absorption of 59Fe administered by gavage revealed an inverse correlation between dietary iron content and absorption. Gavage experiments were done following a 4h fast when the stomach and proximal small bowel were free of dietary content. Although iron absorption changed, liver expression of hepcidin mRNA did not. We measured the absorptive response in mice weaned onto diets containing 35 mg Fe/kg for 4 wk and abruptly changed to 350 mg Fe/kg. There was no change in iron absorption at day 1 but by day 3 absorption was reduced nearly 3-fold compared to controls and remained at this level for at least 7d. During this time neither liver nor spleen iron content changed but transferrin saturation increased approximately 1.5-fold. Most importantly, serum hepcidin levels, measured by a competitive binding assay (De Domenico et al. Cell Metab.2008, 8:146–156), were unchanged. Mice were then changed from diets containing 350 mg Fe/kg to diets containing 35 mg Fe/kg. Within 24h mice increased absorption of 59Fe 3-fold. Elevated absorption continued for at least 3d, declined by 7d and at 14d was at a level found in mice maintained on a diet containing 35 mg Fe/kg. During this period there was no change in organ iron content or in transferrin saturation. Serum hepcidin did not change on day 1, but was reduced by approximately 40% on days 3 through 7. Increased iron absorption could be attributed in part to increased expression of Dmt1 but no change in ferroportin message was detected. Enterocyte ferritin levels doubled on day 1 but returned to control levels on days 3 through 7. Finally, mice with a targeted disruption of the hepcidin gene were challenged with an abrupt reduction in dietary iron content and absorption increased approximately 3-fold. These data suggest that iron absorption can respond to changes in dietary iron content independent of hepcidin and that response to changes in luminal iron content are at the level of uptake and storage in a manner intrinsic to the enterocyte.
Identification of hTid1 as a Molecular Partner and a Negative Regulator of the Transcription Factor Stat5B
Authors: Nyga Remy , Dhennin-Duthille Isabelle , Yahiaoui Saliha , Munger Karl , Regnier Aline , Gouilleux-Gruart Valerie , Lassoued Kaiss , Gouilleux Fabrice
Published: October 2019

Stat5A and 5B transcription factors play a major role in the control of hematopoietic cell proliferation and survival and are constitutively activated in number of hematological neoplasia and solid tumors. However, their mechanisms of action remain poorly understood and their molecular partners still need to be identified. Since Stat5A and 5B mainly differ in their carboxy-terminal transactivation domains, we sought to identify molecules that specifically bind to this domain, using a bacteria-match two-hybrid assay to screen a human Jurkat T cells library. This approach enabled us to isolate hTid1, a DNA-J protein homologous to a drosophila tumor suppressor, that interacts with Stat5B but not Stat5A. Specific interaction between Stat5B and hTid1 was confirmed by immunoprecipitation using various mouse and human lymphoïd cell lines and Cos-7 cells transfected with plasmids encoding both molecules. Using various deleted mutants, we showed that the cysteine-repeats containing region (CRR) of hTid1 was required for this interaction. Moreover, we found that the complex formed by hTid1 and STAT5B was dissociated upon cytokines- (IL-3, IL-7) or Tel-Jak2-induced tyrosine phosphorylation of STAT5B. We also brought evidence for a down-regulation of STAT5B transcriptional expression and activity by hTid1 in human hematopoietic cells. Indeed, overexpression of hTid1 but not of its CRR deleted form in Ba/F3 cells, resulted in a sharp reduction of Stat5B expression, a process that probably involves a proteasome-dependent degradation of Stat5B. By contrast, treatment of Ba/F3 cells with siRNA of hTid1 induced an increase of Stat5B expression. Finally, Cos-7 cells cotransfected with the prolactin receptor and Stat5B expression vectors and with a Stat5 specific luciferase constructs enabled us to demonstrate that overexpression of hTid1 inhibits of Stat5B transcription activity in a dose dependent manner. In conclusion, our findings define hTid1 as a novel negative regulator of Stat5B.