bluuebunny/crossref_metadata_2025 huggingface dataset:
p16INK4a Is a Key Downstream Mediator of the Deleterious Effects of FoxO Deficiency on Maintenance of the Hematopoietic Stem Cell Compartment.
Authors: Tothova Zuzana , Sykes Stephen M , Leeman Dena S , Horner James W , Sharpless Norman , DePinho Ronald A , Gilliland D. Gary
Published: April 2020

Regulation of oxidative stress in the hematopoietic stem cell (HSC) compartment is critical for the maintenance of HSC self-renewal. A number of reports have previously implicated p16 in aging of HSCs, pancreatic β-islet cells and subventricular zone progenitors in the brain [1–3]. In the context of the hematopoietic system, p16INK4a expression in HSCs increases with age, and correlates with decreased HSC repopulating ability, decreased self-renewal, and increased apoptosis with stress [1]. We and others have recently reported that FoxO play essential roles in the response to physiologic oxidative stress and thereby mediate quiescence and enhanced survival in the HSC compartment [4, 5]. Young mice deficient in FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system, with striking similarity to aging wild-type mice, show a defect in bone marrow repopulating ability, decrease in self-renewal, myeloid skewing in differentiation and increased levels of apoptosis. Furthermore, young FoxO-deficient HSC show increased levels of p16 when compared to their wildtype counterparts. These collective findings suggested the possibility that FoxO loss could result in accelerated aging of HSC due to increased expression of p16 as a consequence of increased ROS. To test the hypothesis that p16 is one of the key mediators of FoxO loss responsible for accelerated aging of HSC, we deleted FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system of mice deficient in p16INK4a. Young mice deficient in FoxO and p16 shared the same characteristics of their HSC(Lin−Sca1+c-kit+) compartment as mice deficient in FoxO only, including decreased number of HSC, increased percentage of HSC entering S/G2/M and apoptosis, and increased levels of ROS as compared to their wildtype counterparts. However, in a setting of long-term repopulation studies, bone marrow isolated from mice deficient in p16 and FoxO demonstrated a rescue of long-term repopulation for up to 20 weeks, as compared to FoxO deficient bone marrow that showed a severe defect in long-term repopulation. p16 deficiency in the setting of FoxO deficiency did not result in reduction of ROS levels in the HSC compartment. Taken together, these findings indicate that p16 is a critical downstream mediator of FoxO in the maintenance of the HSC compartment, and that it can dissociate the detrimental effects of ROS on HSC self-renewal in a setting of FoxO deficiency.
Mortality in Sickle Cell Patient Transitioning from Pediatric to Adult Program: 10 Years Grady Comprehensive Sickle Cell Center Experience.
Authors: Aduloju Si mbo O , Palmer Sheila , Eckman James R.
Published: April 2020

Over the last three decades there has been improvement in survival in children with sickle cell disease. Overall survival from birth to age 18 of 86% and 95% has been reported in children with sickle cell anemia (HbSS) and sickle hemoglobin C (Hb SC) disease respectively. These encouraging results are secondary to initiation of preventive measures like newborn screening, penicillin prophylaxis, immunization and stroke prevention and other supportive therapies. Ballas (Blood2004;104 supl:Abstr 3743) reported high death rate for young adults with the disease. To determine the death rates and cause of death in our population, we did a retrospective review of our patients who had recently transitioned into adult care. Mortality rate and circumstances of death in a 10 year period of transition from pediatric to adult program in Georgia Comprehensive Sickle Cell Center at Grady Memorial Hospital between 1996 and 2006 were determined. Total number of deaths was obtained from the clinical database. Death, autopsy and medical records were reviewed to determine the cause and circumstances of death. Between 1996 and 2006, 387 young adults with sickle cell disease (HbSS, HbSC, HbS beta thalassemia) transitioned to the adult program at age 18. Twenty two (5.8%) patients died during their first 10 years of transition. Mean age at death was 23.4 (range 21.6–26) and male to female ratio was 1.2 to 1. Ten patients (45%) died due to chronic organ failure all due to chronic iron overload (end stage liver disease in 8 and congestive heart failure in 2). Eight patients (36%) died due to complications of acute vaso-occlusive crisis (sudden death in 4, acute chest/multi-organ failure in 4) while 4 patients (18%) died of non-sickle related causes. Three of four who died of sudden death has documented pulmonary hypertension. Our mortality rate is lower than those reported by Ballas with the most common cause of death being complications of iron overload in chronically transfused patients. Specific interventions targeted at improving management of iron overload need to be developed for the increasing numbers of patients on chronic transfusion transitioning into adult care.
Multi-Modal Intervention for Inpatient Management of Sickle Cell Pain Significantly decreases the rate of Acute Chest Syndrome.
Authors: Reagan Megan , DeBaun Michael R. , Frei-Jones Melissa
Published: April 2020

Painful vaso-occlusive episodes (VOE) account for the majority of hospitalizations and nearly all morbidity in children with sickle cell disease (SCD). Frequently, VOE progresses into acute chest syndrome (ACS), a leading cause of mortality in SCD. Few strategies exist to prevent this potentially fatal complication. We present the results of a multi-modal intervention to improve the inpatient management of SCD. We performed a before-and-after prospective multi-modal intervention. All children with SCD admitted for uncomplicated VOE during the two-year study period were eligible. The multi-modal intervention included standardized VOE admission orders, monthly house staff education, and one-on-one patient and caregiver education. ACS was defined as a new radiologic density on chest radiograph. A total of 332 admissions for uncomplicated VOE occurred during the study period; 159 before the intervention and 173 during the intervention. The ACS rate declined by 50% during the intervention period (25%, 39/159 to 12%, 21/173; p=0.003). No significant difference was found in patient demographics, amount of intravenous fluid administered, frequency of normal saline bolus administration, or cumulative opioid amount delivered in first 24-hours. Patient controlled analgesia (PCA) use was more common after the intervention (52%, 82/159 vs 73%, 126/173; p=0.0001) and fewer patients required changes in analgesic dosing within the first 24-hours after admission (26%, 42/159 vs 16%, 28/173; p=0.015). PCA-use protected against progression to ACS (OR 0.5; 95% CI 0.3–0.0.94) while patients needing escalation of pain medications in the first 24-hours were more likely to develop ACS (OR 2.5; 95% CI 1.3–4.6). A multi-modal intervention to change physician behavior successfully improved patient outcomes by reducing the ACS rate.
Genomic Approaches to Identifying Risk for Pulmonary Artery Hypertension among Individuals with Sickle Cell Disease.
Authors: Silbermins Damian , De Castro Laura M. , Jonassaint Jude C , Hsu Shiaowen David , Telen Marilyn J. , Chi Jen-Tsan
Published: April 2020

Pulmonary artery hypertension (PAH) occurs in 30–50% of adult patients with sickle cell disease (SCD), with mortality ranging from 16 to 50% and a median survival of 25 months. Our objective was to use gene expression profiling to develop a gene signature predictor for PAH through the analysis of gene expression of blood cells from SCD patients with or without PAH. We hypothesized that these gene signatures could allow us to identify patients at risk for PAH, as well as to generate hypotheses as to the pathophysiology of PAH in SCD. We used Affymetrix U133A2 GeneChip to determine the RNA expression of both whole blood and leukocytes using PAXgene and Leukolock methods, respectively. The study population included patients homozygous for HbS or with HbSβ0 thalassemia. Subjects with PAH were ≥18 years old, in steady state, and had PAH either by 2D echo (TR jet ≥ 2.7 m/sec) or right-sided catheterization (mean PA pressure ≥ 30 mmHg). Patients were excluded if they were pregnant, had co-existing rheumatologic conditions or other inflammatory diseases, were on chronic transfusion therapy or had had a vaso-occlusive episode in the previous 4 weeks. The control subjects were patients with SCD but without PAH (TR jet ≤ 1.8 m/sec or mean PA pressure <25 mmHg). Hierarchical clustering based on the gene expression pattern from 7 patients with PAH and 6 controls showed a trend for the clustering of SCD patients with PAH away from SCD patients without PAH. This trend was present for the gene expression in both whole blood and leukocytes. A Bayesian regression analysis was then performed to identify a set of predictor gene signatures for the PAH phenotype (Figure 1) in SCD. Finally, using gene set enrichment analysis, we found that the leukocytes from patients with PAH were highly enriched in the gene sets deriving from hematopoietic stem cells, corroborating the hypothesis of hyperhemolysis and higher blood cell turnover in this population. Other pathways showing upregulation in PAH were PTEN, TGFβ, cyclin D1, WNT and PPAR. Although these data are preliminary, they suggest that PAH in SCD does indeed have a distinct gene signature profile that may become useful in identifying risk for PAH prospectively, as well as in directing further investigation into the pathogenesis of PAH in SCD.

Figure Figure
Inhibition of HIV-1 by Ferroprotin Expression.
Authors: Nekhai Sergei , Foster Altreisha , Xu Min , Niu Xiaomei , Rotimi Jamie , Gordeuk Victor R.
Published: April 2020

HIV-1 transcription is induced by viral Tat protein, which recruits transcriptional co-activators to the HIV-1 promoter. We recently showed that Tat is phosphorylated in the Ser16 and Ser 46 residues by protein kinase CDK2 and that mutations in these residues prevent HIV-1 transcription and viral replication [1]. We also found that iron depletion by iron chelators inhibits cellular activity of CDK2, prevents Tat phosphorylation and inhibits HIV-1 transcription [2]. Thus our previous studies suggest that a decrease in cellular iron might have a protective effect against HIV-1 through inhibition of CDK2 and Tat phosphorylation. Here, we analyzed the effect of the iron exporter, ferroportin, on HIV-1 transcription and viral replication. Increased expression of ferroportin by transfection in iron-treated 293T cells significantly reduced ferritin protein levels compared to increased expression of CD4 or EGFP in iron-treated cells as controls. Treatment with hepcidin increased ferritin levels in 293T cells that expressed wild type ferroportin but not the C326Y mutant of ferroprotin which is not sensitive to hepcidin. Expression of both wild type ferroportin and the C326Y mutant in 293T cells significantly inhibited HIV-1 transcription. Treatment with hepcidin partially restored HIV-1 transcription in the cells expressing wild type ferroportin and not in those expressing the C326Y mutant of ferroportin. Treatment of promonocytic THP-1 cells with iron increased cellular ferritin level. Subsequent treatment with phorbol myristate acetate (PMA) led to increased expression of ferroportin and reduced ferritin level, and this reduction in ferritin was partially alleviated by exposing the cells to hepcidin. Thus, PMA appeared to reduce intracellular iron through increased iron export by ferroportin. HIV-1 replication in iron-supplemented THP-1 cells or primary human monocytes was significantly reduced by treatment with PMA. Subsequent exposure of the PMA-treated monocytes to hepcidin partially restored HIV-1 replication, suggesting that HIV-1 was inhibited in part by the expression of ferroportin and its associated iron-exporting activity. Taken together, our results indicate that expression of ferroportin leads to reduction of cellular iron and also reduced HIV-1 transcription and replication, and that exposure to hepcidin may lead to increased cellular iron content and enhancement of HIV-1 replication. Thus our results suggest that iron depletion of cells that harbor HIV might serve as a strategy to combat this infection, and they point to the need to develop iron chelators specifically designed for HIV-1 therapy.
Novel Heterozygous Mutation 572insA in TACI Identified in a Patient with Selective IgA Deficiency.
Authors: Okada Satoshi , Kajiume Teruyuki , Tsumura Miyuki , Shirao Kenichiro , Ohno Yoshinori , Yasunaga Shin’ichiro , Ohtsubo Motoaki , Sira Mostafa Mofamed , Kanegane Hirokazu , Miyawaki Toshio , Takihara Yoshihiro , Kobayashi Masao
Published: April 2020

Selective IgA deficiency (SIgAD) is relatively common disease in patients with primary immunodeficiency. Recently, mutations in TNFRSF13B, encoding transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), have been identified in 7~15% of patients with common variable immunodeficiency (CVID) and 10% of those with SIgAD. However, the role of TACI mutations in the molecular pathogenesis underlying SIgAD is still controversial and remains unsolved. We identified a novel heterozygous single adenine insertion, 572insA, in exon 4 of TNFRSF13B in a patient with SIgAD. Although the same mutation was detected in her mother and younger brother, their serum immunoglobulin levels were within normal range. This frameshift mutation produces a truncated form of TACI protein lacking majority of the intracellular domain. Peripheral blood B cells from the patient and her siblings with the TACI mutation were able to produce immunoglobulin without inferiority in the presence of dendritic cells, CD40L and IL-10. However, IgD+ or IgM+ B cells from the patient were impaired to produce Cα mature transcripts in response to APRIL, one of the ligands for TACI, reflecting impairment of class-switch recombination required for differentiation into IgA-producing cells. Thus, the T-cell independent B cell activation is impaired in the patient. We overexpressed cDNAs for wild-type (WT), C104R and 572insA mutant TACI in HEK 293 cells, and analyzed the effect on signal transduction through TACI. Immuno-precipitation analysis revealed that either 572insA or C104R mutant associated with WT TACI and formed an oligomeric complex. C104R TACI has been previously shown to exert a dominant-negative effect on ligand-induced TACI signaling, since it forms an oligomeric complex with WT TACI and disrupts ligand-induced receptor signaling. Similarly, since 572insA TACI lacks an intracellular domain that is important for signal transduction, it may exert a dominant-negative effect on ligand-induced TACI signaling. Although the heterozygous 572insA TACI mutation disrupts ligand-induced TACI signaling, not all individuals carrying the mutation display humoral immune deficiency. Thus, an additional genetic modifier and/or an environmental factor may be required for the development of immunological abnormalities due to TACI mutations.
GATA-2 L359V Mutation Is Solely Associated with Cml progression but Not Other Hematological Malignancies.
Authors: Zhang Su-Jiang , Shi Jing-Yi
Published: April 2020

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell disease with distinct biology and clinical features. According to clinical and biology process, CML can be divided as two different phase: the initial chronic phase (CP) and progression phase including accelerated phase (AP) followed by blast crisis (BC). In a previous study, we have identified the transcription factor GATA-2 L359V mutation in CML BC patients but not CP and its pivotal role in CML progression (PNAS 2008 105:2076–2081). Here, we continued to explore the occurrence of GATA-2 L359V mutation in other hematological malignancies using Mass-ARRAY assay and sequence analysis. A total of 652 patient samples were included in our study. These patients were referred to 270 Acute Myeloid Leukemia (AML) including M1–M7, 30 Myelodysplastic Syndrome (MDS), 50 Acute Lymphoblastic Leukemia (ALL), 12 Chronic Lymphocytic Leukemia (CLL), 40 CML CP and 250 BCR/ABL negative Myeloproliferative Disorder (MPD) patients. 5 ml bone marrow sample before therapy was collected with informed consent and genomic DNA was isolated. The diagnosis of AML, ALL, CML, CLL, MDS and MPD was established according to the 2001 WHO diagnostic criteria. In addition, 8 samples of CML BC harboring GATA-2 L359V were supplied into our study as positive control and 100 peripheral blood samples of healthy adult as normal control. As a result, no L359V mutation was detected in AML, ALL, MDS, CLL, BCR/ABL negative MPD and CML CP. Our data strongly suggested that GATA-2 L359V is solely associated with CML progression but not other hematological malignancies. Therefore, we favored this idea, i.e., BCR/ABL underlies the pathogenic basis of CML CP; However, subsequent genomic instabilities contribute to mutation of key transcription factor such as GATA-2 which ultimately trigger the blastic transformation of CML.
Generation of T Cells from Human Embryonic Stem Cells.
Authors: Timmermans Frank , Velghe Imke , Van Walleghem Lieve , De Smedt Magda , Van Coppernolle Stefanie , Leclercq Georges , Taghon Tom , Moore Harry , Langerak Anton W , Kerre Tessa C. C. , Plum Jean , Vandekerckhove Bart
Published: April 2020

Background: Human embryonic stem cells (hESC) are derived from early stage blastocysts and are characterized by the ability to both self-renew and to generate differentiated functional cell types. One of the major challenges in the field of hESC research, is to set up a culture system that drives hESC down a particular lineage fate. To date, studies reporting hematopoietic development have not provided evidence on the differentiation capacity of hESC into T lineage cells in vitro. Material and

Methods: hESC line H1 (National Institutes of Health [NIH] code: WA01), Wisconson, Madison, USA) was used (Passage 30–60) in all experiments. The hESC line was kept in an undifferentiated state on MEFs as previously described. OP9 cells and OP9 cells that express high levels of the Notch ligand Delta-like 1 (OP9-DLL1, a gift from J. C. Zuniga-Pflücker, University of Toronto, Canada) were cultured as previously described in MEM-α with 20 % FCS.

Results: Our data show that T cells can be generated in vitro from hESC in a robust and highly reproducible manner using the sequential exposure of hESC to the murine OP9 cell line and OP9-DLL1. On OP9 stromal layers, a CD34highCD43dim hematopoietic precursor population is generated that is confined to vascular-like structures, reminiscent of blood islands that emerge during in vivo embryonic development. This precursor population becomes T lineage committed when exposed to OP9-DLL1 monolayers, passing sequentially through a CD34+CD7+ phenotype, a CD4+CD8+ double positive intermediate stage and eventually differentiates into a mature T cells. Polyclonal T cells are generated, cell receptor (TCR) alpha-beta and TCRgamma-delta which are functional based on proliferative capacity and production of cytokines after TCR crosslinking.

Conclusion: We show that mature and functional T cells can be generated from hESC using well defined in vitro conditions. This protocol in combination with the recently described induced pluripotent cells may find clinical applicability in tumor immunology.
Tec Kinase Inhibits the Expression of IL-2 Receptor Alpha (CD25) in Human T-Lymphocyte.
Authors: Susaki Kentaro , Kitanaka Akira , Dobashi Hiroaki , Kittaka Katsuharu , Mano Hiroyuki , Kubota Yoshitsugu , Tanaka Terukazu
Published: April 2020

Activation and development of lymphocytes is regulated by the engagement of cell surface immune cell antigen receptors. Following receptor engagement, these receptors transmit signals by the activation of cytoplasmic protein tyrosine kinases (PTKs). The Tec PTK belongs to a group of structurally related nonreceptor PTKs that also includes Btk, Itk (Emt), Rlk (Txk), and Bmx (Etk). We have found the contribution of Tec in the antigen receptor signaling in B-lymphoid cells in the previous studies. Despite the presence of evidences suggesting the involvement of Tec in T-lymphocyte activation pathway via T-cell receptor (TCR) and CD28, the role of Tec in T-lymphocyte still remains unclear because of the lack of apparent defects in T-lymphocyte function in Tec-deficient mice. In this study, we investigated the role of Tec in T-lymphocyte using Jurkat human T-lymphoid cell line stably transfected with a cDNA encoding Tec. We found that the expression of wild type Tec but not kinase-deleted Tec inhibited the expression of IL-2 receptor alpha (CD25) induced by TCR cross-linking. The percentage of CD25 expressing cells after TCR cross-linking was 41.7% in mock-transfected cells, 18.3% in the wild type Tec expressing cells and 41.3% in the kinase-deleted Tec expressing cells. Second, the selective inhibitor of Tec family PTK, LFM-A13, rescued the suppression of TCR-induced CD25 expression observed in the wild type Tec-expressing Jurkat cells. We conclude that Tec PTK mediates signals that negatively regulate CD25 expression induced by TCR cross-linking, implying that this PTK plays a role in the attenuation of the IL-2 activity in human T-lymphocytes.
Results of a Phase 2 NCI Multicenter Study of Romidepsin in Patients with Relapsed Peripheral T-Cell Lymphoma (PTCL).
Authors: Piekarz Richard , Wright John , Frye Robin , Allen Steven L. , Craig Michael , Geskin Larisa , Hutchins Laura , Joske David , Kirschbaum Mark , Leonard John P , Prince Miles , Reeder Craig B. , Jaffe Elaine , Bates Susan
Published: April 2020

Background: The histone deacetylase (HDAC) inhibitors are a class of epigenetic agents undergoing clinical testing. HDAC inhibitors modulate expression of genes involved in cell cycle regulation, leading to induction of differentiation or apoptosis. Romidepsin, a novel, HDAC inhibitor, has previously demonstrated activity as a single agent in patients (pts) with T-cell lymphoma in a phase 1 NCI study.

Aims: To evaluate the efficacy and tolerability of romidepsin in the treatment of advanced PTCL and as an exploratory endpoint, to examine the molecular effects of romidepsin in both PTCL and cutaneous T-cell lymphoma (CTCL).

Methods: This Phase 2, open-label, multi-arm, multicenter study enrolled 43 PTCL pts from the NCI and 9 extramural sites. CTCL pts were also enrolled in this study. The clinical results for pts with PTCL are presented here; the clinical results for the pts with CTCL and the biomarker analyses for both PTCL and CTCL are presented elsewhere. This study is now closed to accrual. Pts with relapsed or refractory PTCL or primary cutaneous anaplastic large cell lymphoma were eligible. Pts received romidepsin 14 mg/m2 as a 4-hr infusion on days 1, 8 and 15 every 28 days. Responses were assessed using elements of the IWG criteria and RECIST, as appropriate, for pts with lymph node involvement and extranodal disease.

Results: 43 pts (23[53%] men and 20 [47%] women) with a mean age of 58.1 (range: 27 to 84) years were treated with romidepsin. 31 pts received ≥ 2 cycles of therapy. Mean number of prior therapies was 3.9 (range 1 to 12). Objective disease response rates (ORR) are summarized in the table. The mean number of cycles of treatment was 6.8 (range 1–37) and the overall median duration of response was 8.3 months (range 1.6 months to 4.8+ yrs) for all pts. The median duration of response for the 7 pts who achieved CR was 8.5 months (range 4.6 months to 4.8+yrs). The most frequent drug-related AEs (all grades, all cycles) were generally mild and included: nausea (86%; 0% ≥grade 3), fatigue (79%; 0% ≥grade 3), decreased platelets (70%; 7% ≥grade 3), and decreased AGC (63%; 5% ≥grade 3). One death, in a pt with significant cardiovascular disease who had achieved a CR, was considered possibly related to treatment.

Conclusions: This study demonstrates tolerability and durable clinical benefit (ORR of 39% and median duration of response of 8.3 months) of romidepsin in pts with recurrent or refractory PTCL. Based on these promising results, a Phase 2B protocol investigating romidepsin in pts with progressive or relapsed PTCL is ongoing at multiple international centers.

All Pts N=43 Pts ≥ 2 cycles N=31 ORR (CR+PR), n (%) 17 (39%) 17 (55%) PR, n (%) 10 (23%) 10 (32%) CR, n (%) 7 (16%) 7 (23%)
Interim Results of the RiPAD+C Regimen Including Velcade in Front Line Therapy for Elderly Patients with Mantle Cell Lymphoma. A Phase II Prospective Study of the GOELAMS Group.
Authors: Gressin Remy , Maugendre Sylvie caulet , Le Gouill Steven , Uribe Mario ojeda , Vigier Magda Alexis , Bouabdallah Krimo , Mounier Christiane , Tournilhac Olivier , Rodon Philippe , Yamani Abderrazak El , Sutton Laurent , Senecal Delphine , Lioure Bruno , Assouline David , Le Mevel Annie , Villemagne Bruno , Dauriac Charles , Gardembas Martine , Park Sophie , Lamy Thierry
Published: April 2020

Elderly patients with mantle cell lymphoma (MCL) do not take benefit from dose-intensive chemotherapy such as HyperCVAD because of its high toxicity (Romaguera JCO 2005). Prior Goelams clinical trials using the VAD plus Chlorambucil regimen with or without Rituximab have demonstrated a good efficacy/toxicity ratio in front line therapy (EHA 2008, a0259). Proteasome inhibitors are among the most promising new agents for relapsing patients (Fisher, JCO 2006). Therefore, the GOELAMS conducted a phase II clinical trial including Velcade plus R-AD and Chlorambucil (RiPAD + Chlorambucil) for elderly MCL patients upfront.

Aims: To evaluate toxicity and overall response rate after 4 cycles of RiPAD + Chlorambucil regimen.

Protocol: RiPAD + C: Rituximab 375 mg/sqm, D1 (and D8 for cycle 1); PS 341, Velcade 1.3 mg/sqm D1, 4, 8 and 11; Adriblastine 9mg/sqm/D in a 4 days continuous infusion; Dexamethasone 20 mgx2/D for D1 to D4; Chlorambucil 12 mg/D, D20 to D29. One cycles every 35 D. The patients received a maximum of 6 cycles.

Inclusion criteria: Untreated MCL patients (from 65 to 80 years) with a stage II to IV disease, and good PS (Ecog < 3). Response criteria is evaluated according to the 1999 Cheson’s criteria, all patients were also evaluated by FDG-PETscan.

Results: since June 2007, 27 patients have been included. Thirteen have been evaluable after 4 cycles. Median age is 71 years (66–80 y). After 4 cycles, disease Status was: 10 patients in response (3 in CR with negative FDG-PETscan and 7 in PR including 3 with a negative PETscan) and 3 patients progressed on therapy. The 3 non responding patients had the highest Ki67 value over 30%. Toxicity evaluation has been evaluated based on 59 cycles. Grade 3–4 toxicities have been reported in 5 cases (2 neurologic, 1 hepatic, 1 cardiac and 1 pulmonary). Only 3 cycles were delayed because of toxic reasons.

Conclusion: this intermediaire analysis shows an ORR of 77% (10/13) including 6 patients with a negative FDG-PET after only 4 cycles. Our study demonstrates that the RiPAD+C regimen has a good efficacy/toxicity ratio and is a promising therapeutic option for MCL elderly patients. The study is ongoing and additional results on response rate and toxicity will be updated for presentation in the ASH meeting.
NPM-ALK Mutants in Kinase Domain Exhibit Altered Kinase Activity and Various Sensitivity to ALK Inhibitors.
Authors: Lu Lihui , Ghose Arup , Quail Matt , Ruggeri Bruce , Cheng Mangeng
Published: April 2020

Abnormal expression of constitutively active anaplastic lymphoma kinase chimeric proteins (NPM-ALK) in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is well defined. Recently, small molecule inhibitors have been reported that provide solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK+ALCL cells. Although robust clinical response of ALCL patients to the ALK inhibitors is expected, some of those patients are also anticipated to develop resistance to an ALK inhibitor, most likely associated with single point mutations in the kinase domain of ALK. In this study, the nonsense mutants of NPM-ALK on the phosphate anchor region and the gatekeeper region were generated by site-directed mutagenesis and their kinase activity was measured in cells. NPM-ALK/L182M, L182V and L256M mutants displayed comparable or higher kinase activity in cells relative to NPM-ALK wt, and were able to render BaF3 cells into growth factor-independent growth, while NPM-ALK/L182R, L256R, L256V, L256P and L256Q displayed much weaker or little kinase activity in cells and failed to transform BaF3 cells. While NPM-ALK/L182M and L182V displayed comparable sensitivity to a fused pyrrolocarbazole (FP)-derived ALK inhibitor as NPM-ALK wt, they were >30-fold less sensitive to a diaminopyrimidine (DAP)-derived ALK inhibitor. On the other hand, NPM-ALK/L256M displayed >30-fold less sensitivity to both the FP-derived and the DAP-derived ALK inhibitors. Consistent with NPM-ALK autophosphorylation inhibition, the FP-ALK inhibitor induced growth inhibition and cytotoxicity of BaF3/NPM-ALK/L182M and L182V cells but not L256M cells, and the DAP ALK inhibitor failed to induce growth inhibition and cytotoxicity of all three BaF3/NPM-ALK mutant cell lines in culture. In the absence of an ALK inhibitor, BaF3 cells harboring NPM-ALK mutants did not display growth advantage in culture with 10% serum and in mice over the NPM-ALK wt cells. However, the BaF3/NPM-ALK mutants displayed significant growth advantage in culture with low serum (2%) over the BaF3/NPM-ALKwt cells, suggesting the BAF3/NPM-ALK mutant cells may proliferate better in the stressed conditions. Analyses of binding of ALK inhibitors to ALK wt and mutants in ALK homology models are in progress and the results will be discussed.
Identification of Core Gene Signature Associated with Synergism Between ABT-869, a FLT3 Inhibitor and SAHA, a HDAC Inhibitor in AML with FLT3-ITD Mutation.
Authors: Zhou Jianbiao , Bi Chonglei , Liu Shaw-Cheng , Chng Wee-Joo , Tay Kian-Ghee , Yu Hanry , Glaser Keith , Albert Daniel , Davidsen Steven K , Chen Chien-Shing
Published: April 2020

Internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) mutation occurs in about 25% of acute myeloid leukemia (AML) patients and is associated with poor prognosis. Various FLT3 inhibitors of different chemical structure are under clinical investigation for the treatment of AML patients with FLT3 mutations. In contrast to their impressive potency in cell culture systems, FLT3 inhibitors as single agents in clinical trials only induce transient reduction of peripheral blasts, but not bone marrow blasts. Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. Small molecule HDAC inhibitors (HDACi) have proven to be a promising new class of anticancer drugs against hematological malignancies, as well as solid tumors. The HDACi, suberoylanilide hydroxamic acid (SAHA, Vorinostat) has been examined in a combinatory fashion with other classes of anticancer agents in acute leukemias, including cyclin-dependent kinase (CDK) inhibitors (flavopiridol), HSP 90 antagonists (17-allylamino- 17-demethoxygeldanamycin, 17-AAG), and estradiol analogs (2-methoxyestradiol, 2-ME). ABT-869, a multi-targeted receptor tyrosine kinase inhibitor, inhibits FLT3 phosphorylation and signaling and now is in active clinical development. In the current study, cell proliferation and apoptosis assays revealed that combining ABT-869 with SAHA led to synergistic killing of AML cells with FLT3 mutations in conventional cell culture and human stromal cell coculture models. To elucidate the molecular mechanism of the synergistic lethality between ABT-869 and SAHA, we compared the gene expression profiles of cells from FLT3 mutated leukemia cell lines, MV4-11 and MOLM-14, treated with DMSO control, ABT-869, SAHA and combination therapy using the Affymetrix microarray platform. Here, we focused on delineating a core gene signature unique to combination therapy and common to both MV4-11 and MOLM-14 cell lines, which could be crucial for the positive cooperation we observed. The core gene signature differentially induced more than two-fold by combination therapy in both cell lines included upregulation of FRY, LMO4, IFI16, ACADSB, and S100A8, and downregulation of PTP4A3 (PRL-3), ORC1L, MND1, ZNF85, DENND3, FAM119A, ACAT2, HMGCS1, DCS2, GSPG4, CBS, TUBE1. Overexpression of PRL-3, a metastasis-associated gene, has been found in different types of solid tumors and multiple myeloma. Modulation of PRL-3 expression level using genetic approaches demonstrated that PRL-3 played an essential role in the synergism in the therapeutic effect of ABT-869 and SAHA.
The CXCR4 Antagonist AMD3100 Impairs Survival of AML Cells and Induces Their Differentiation.
Authors: Tavor Sigal , Eisenbach Manny , Jacob-Hirsch Jasmine , Golan Tammar , Petit Isabelle , Ben-Zion Katz , Kay Sigi , Baron Shoshana , Amariglio Ninette , Deutsch Varda , Naparstek Elizabeth , Rechavi Gideon
Published: April 2020

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, participate in the retention of AML cells within the bone marrow microenvironment and their release into the circulation. We previously showed that the expression of cell surface elastase in AML cells is dependent on the SDF-1/CXCR4 axis, giving a first evidence for the association between the SDF-1 and elastase pathways. In the present study, we hypothesized that inhibition of the SDF-1/CXCR4 axis or elastase may regulate similar pathways and genes in AML cells. In order to test our hypothesis, we compared gene expression profiles of U937 AML cells that express high level of membrane CXCR4, treated with neutralizing CXCR4 mAb or elastase inhibitor (EI), with non-treated cells, by employing gene-array technology. Unsupervised hierarchical clustering analysis demonstrated similar gene expression profiles of anti-CXCR4 antibody or EI treated cells, as compared to control. Analysis of the 364 genes that were differentially expressed in both anti CXCR4 mAb or EI treated cells under both treatment agents identified genes that correspond to the transcriptional profiles of differentiated myeloid cells in a significantly high prevalence. Quantitative Real-time RT-PCR for 4 selected genes C/EBPe, FLT3, HOXA9 and G-CSFR, demonstrated gene expression profiles identical to the microarray expression profiles. Given thus, we further analyzed the effect of CXCR4 inhibition on AML cell growth and differentiation using the antagonist AMD3100. AMD3100 arrested proliferation in five AML cell lines as was suggested by proliferation assay, cell cycle and caspase 3 activity analyses. Moreover, cell differentiation was demonstrated by:

Modifications in cell morphology, Increased expression of myeloid differentiation antigens Acquisition of the ability to produce oxidative bursts An increase in DNA ploidy in megakaryoblasts. Our study defines a new role for the SDF-1/CXCR4 axis in the regulation of leukemic cell survival and differentiation.
Novel Small Molecule Pim Protein Kinase Inhibitors Induce Cell Cycle Arrest and Apoptosis in Human Leukemias: A Potential Therapeutic Approach.
Authors: Lin Yingwei , Song Jin H. , Zhang Zhenhua , Aplan Peter D. , Smith Charles D , Kraft Andrew , Beharry Zanna M
Published: April 2020

The Pim protein kinases, first identified as a proviral integration site in c-Myc overexpressor mice, are increased in multiple human hematopoietic neoplasms including myeloid leukemia, diffuse large B-cell lymphoma, and T-cell lymphoma. In culture these protein kinases have been associated with the signaling pathways that control Flt3-ITD and BCR/ABL driven leukemias. To examine the role of Pim as a target for anticancer chemotherapy, we have screened a 50,000 compound small molecule library and identified and synthesized novel benzylidene-thiazolidine-2,4-diones, D5 and F801, that inhibit the activity of this enzyme with IC50 in the nanomolar range. Screening of 60 serine/threonine and tyrosine kinases demonstrated high specificity of these molecules for Pim kinase. Incubation of these compounds with myeloid leukemias, U937, K562 and MV4;11, inhibited cell growth by inducing a relative cell cycle block in G1. This cell cycle block at G1 phase was associated with a decrease in Cdk2 activity and a translocation of p27Kip1 to the nucleus, suggesting an important role for Pim in controlling the cell cycle. In contrast, we find that in two T-ALL cell lines, Jurkat and 6812, which was established from a double transgenic mouse overexpressing the SCL and LMO1 genes, 70–80% of the cells were killed after 48 hours of treatment with the inhibitors at the concentration of 10 μM. Cell cycle analysis revealed a two-to three-fold increase in sub-G1 population (10.8% in control, 21.2% in D5, and 29.67% in F801), which suggested an increase in apoptosis. Cleavage and activation of the proapopotic protease Caspase 3 was identified by Western blot after 24-hour treatment of exposure to the Pim inhibitors. We further examined the toxicity of D5 and F801 in wild type FVB mice and found that when mice were treated with either 15 mg/kg D5 or 30 mg/kg F801 for 5 days they had no remarkable hematologic toxicity. The antileukemic activity of these agents in vivo is currently under examination. Thus, the Pim protein kinase appears to be a potential target for leukemia therapy using these novel benzylidene-thiazolidine-2,4-diones.
Somatic Expansion of the Frataxin Gene GAA Repeats in MDS Patients.
Authors: Merkel Drorit , Deeg H. Joachim , Simon Amos , Amariglio Ninette , Nagler Arnon , Rechavi Gideon
Published: April 2020

The mitochondria play an important role in both apoptosis and heme synthesis. In patients with Myelodysplastic syndrome (MDS) the marrow is characterized by defective hematopoiesis, increased apoptosis and the presence of iron laden mitochondria. The molecular mechanisms responsible for increased apoptosis remain incompletely understood. Frederic’s ataxia (FRDA), the most common inherited ataxia, is a severe autosomal-recessive disease characterized by neurodegeneration, cardiomyopathy and diabetes, resulting from reduced synthesis of the mitochondrial protein frataxin which is involved in mitochondrial energy production and other cellular functions by providing iron for heme synthesis and iron–sulfur cluster (ISC) assembly and repair, serving as a Fe (II) donor for ferrochelatase. The underlying mutation consists of an unstable expansion of GAA repeats in the first intron of the frataxin gene. Long expansions of a GAA tri-nucleotide in FRDA patients range from 66 to more than 1,700 repeats, whereas the normal range of repeats varies from 7 to 36. Abnormal expansion results in reduced frataxin mRNA levels, leading to reduced function of the respiratory chain. The aim of the present study was to determine if frataxin gene mutations occurred in MDS patients. We analyzed DNA from peripheral blood (PB) of 29 MDS patients and from 22 healthy marrow (BM) donors using repeat-Primed PCR. We also sampled genomic DNA products from buccal smears of the MDS patients. In MDS patients PCR of PB in 9 out of 24 patients (37%) showed short length (2–8 repeats), whereas PB of the remaining 15 patients (62.5%) showed longer PCR products (10–43 repeats, still in the “normal” range for FRDA). The PCR products of the buccal smears from all 14 patient samples were short (2–7 repeats), including those from 9 patients who had longer repeats in PB. In healthy BM donors, PCR of PB detected short length repeats (4–5 repeats) in17 of 20 individuals (85%), whereas 3 samples (15%) had longer PCR products (11–26 repeats). This was statistically significantly different from patients with MDS (P= 0.0014). The results indicate that MDS patients exhibit longer frataxin gene products than healthy individuals in PB, but not in buccal DNA. These data suggest a somatic mutation in the frataxin gene in hematopoetic cells of patients with MDS. Further studies will explore the impact of this mutation on mitochondrial function and on the pathophysiology of MDS.
Trogocytosis in Multiple Myeloma.
Authors: Brown Ross D. , Kabani Karieshma , Yang Shihong , Esther Aklilu , Ho Phoebe Joy , Gibson John , Joshua Douglas E.
Published: April 2020

Trogocytosis is the transfer of cell membrane material from one cell to another during short term cell-cell contact. Recent studies have suggested that the transfer of cell membrane patches across the immunological synapse is at least partly responsible for immune tolerance, tumor escape and the production of adaptive T regulatory cells (Tregs). We sought to identify the extent of tumor-related trogocytosis in patients with multiple myeloma and have developed an in vitro model for further study. Trogocytosis in patients with multiple myeloma was demonstrated by flow cytometry, immunohistochemistry, confocal microscopy, lack of mRNA expression and by the failure to upregulate costimulatory molecules in vitro after stimulation by IL2 and huCD40LT. Of the costimulatory molecules CD80, CD86, B7-H1, B7-H3 and PD-L2, only CD80 and CD86 showed significant transfer to T cells. Increased CD80 expression was found on the T cells of 9% of patients and CD86 on 13% of patients (n= 95). Both CD4 and CD8 memory (CD45RO+) cells were involved but not naïve T cells (CD45RA+). HLA-G expression was found on less than 1% of T cells in 69/70 different myeloma blood samples. Following biotinylation of plasma cells (CD38++) using an in vitro culture model, trogocytosis was demonstrated in up to 36% of T cells. The presence of trogocytosis on CD3- TCRαβ-mutant Jurkat cells (J.RT3-T3.3) and on normal T cells with HLA incompatibility infers that trogocytosis is independent of immune recognition and TCR engagement. In vitro stimulation with IL-2 and huCD40LT and mRNA studies showed that T cells acquire CD80 and CD86 cell surface antigen but unlike B cells do not produce CD80 and CD86 mRNA (n=5) and cannot be stimulated to express CD80 and CD86 (n=6). Trogocytosis was not evident in age-matched control lymphocytes but could be induced in normal lymphocytes in vitro after exposure to malignant plasma cells. Trogocytosis is common in patients with multiple myeloma and involves the transfer of costimulatory molecules and other cell membrane proteins from malignant plasma cells to T cells. Tumor-induced trogocytosis may be a common cause of the failure of cytotoxic T cells and provide a mechanism of tumor escape.
Myeloma and Osteolytic Bone Disease: Is DKK1 All It Takes?.
Authors: Chen Bangzheng , Wu Hong , Zeng Yuxin , Freeman John E , Herman Damir , Shaughnessy John D. , Barlogie Bart , Epstein Joshua
Published: April 2020

The Dickkopf-1 (DKK1) gene product inhibits osteoblast activity by blocking Wnt signaling. Elevated levels of DKK1 in bone marrow plasma and peripheral blood are associated with the presence of osteolytic bone lesions in patients with multiple myeloma (Tian E, et al. N Engl J Med.2003, 349:2483), evidently by inhibiting osteoblast differentiation and, as a result, stimulating osteoclastogenesis driven by osteoblast progenitors’ enhanced production of RANKL and reduced expression of OPG. DKK1-neutralizing antibody suppressed bone resorption induced by inflammatory joint disease and myeloma in murine models (Diarra, Nature Medicine2007, 13:156, Yaccoby, Blood2007, 109:2106), and ectopic Wnt3a expression prevented myeloma induced bone resoption (Qiang, Blood2008;112:374). To further investigate whether DKK1 is essential for producing myeloma bone disease, we used 5 myeloma cell lines with various expression levels of DKK1 in our SCID-hu model. U266 and CAG cells, which express minimal amounts of DKK1 mRNA by qRT-PCR corresponding to 5ng/ml in spent media by ELISA, caused severe bone resorption, whereas ARP1, H929 and OPM2 which express 55, 61, and 320 fold higher levels of DKK1 mRNA (and 80 ng/ml protein for ARP1 cells) had no effects on the bones. OPM2 cells overexpressing DKK1 following transduction with lentiviral vector (127 fold higher than U266 cells), also did not cause bone resorption. To understand if the lack of relationship between DKK1 expression by myeloma cell lines and bone resorption reflected the level of expression, we used the prostate cancer cell line PC3 that expresses 100 times higher DKK1 mRNA than DKK1-transduced OPM2 cells. These cells caused severe bone destruction in the SCID-hu model. DKK1 expression by PC3 was inhibited by 80% by constitutive or conditional (tet-on) shRNA-lentivirus, and resulted in corresponding reduction (75%, p=0.02) in loss of bone mineral density compared with control cells transduced with conditional or constitutive control shRNA lentivirus. PC3 cells recovered from the tumors had 50% reduced levels of DKK1 mRNA. Myeloma associated osteolytic bone disease results from inhibition of osteoblast differentiation, resulting in reduced bone formation and overcharged osteoclastogenesis by osteoblast progenitors. It thus appears that a quantitative threshold exists below which DKK1 inhibition of Wnt is not sufficient to cause osteolytic bone disease. Alternatively, it is possible that the DKK1 produced by the MM cell lines is not funtional, or that factors other than inhibition of Wnt signaling by DKK1 can also be responsible for producing osteolytic bone disease. We are currently investigating whether direct stimulation of osteoclastic activity by the myeloma cell lines that cause osteolysis but express low DKK1 can explain this observed discrepancy, or if they inhibit osteoblast differentiation by other mechanisms. This work was supported in part by NIH grant CA55816.
High-Throughput Microrna Profiling in Patients with Waldenstrom’s Macroglobulinemia.
Authors: Adamia Sophia , Amin Samir B , Li Cheng , Patterson Christopher J , AvetLoiseau Herve , Minvielle Stephane , Moreau Philippe , Anderson Kenneth C , Munshi Nikhil C. , Treon Steven
Published: April 2020

Waldenström’s macroglobulinemia (WM) is an incurable B cell malignancy characterized by the accumulation of IgM secreting clonally related bone marrow lymphoplasmacytic cells (LPC) including CD19+ B-cells and CD138+ plasma cells. Despite intense research efforts, the pathogenetic basis for this disease remains to be clarified. MicroRNAs are small noncoding RNAs that regulate the expression of protein-coding genes by inducing translational inhibition and through cleavage of targeted transcripts by partial or complete base pairing. We therefore evaluated the expression of 384 microRNAs in CD19+ and CD138+ sorted bone marrow lymphoplasmacytic from 13 untreated WM patients, and compared their expression profiling to analogous lymphoplasmacytic cells taken from the bone marrows of 13 healthy donors. Data obtained from microRNA arrays was analyzed using SDS, RQ manager, R and dChip softwares. Relative expression was calculated using the comparative Ct method through RQ manager and dChip softwares. Of the 384 microRNAs evaluated in CD19+ patient cells, miR-192, -125b, -21, -155 demonstrated significant upregulation, whereas miR-181c, -572, and -650 were significantly down regulated compared to healthy donor CD19+ bone marrow cells (p<0.05). Analysis of bone marrow derived CD138+ cells from WM patients demonstrated significant upregulation in miR-192, -193b, -17-3p, -585, -148b, whereas miR-29c, -155, -126, -148a, -125a, -181d, -30a-3p, let-7b, let-7c were downregulated in comparison to healthy donor CD138+ bone marrow cells (p<0.05). Importantly, characterization of the modulated microRNAs found in these studies demonstrated a critical role in growth and survival pathways through modulation of several genes including HOX, BCL-2 and c-myc. Taken together, these studies demonstrate significant differences in microRNA expression between comparable WM and healthy donor lymphoplasmacytic cell populations, and identify aberrancies in microRNAs with a pivotal role in the growth and survival of B-cells.