bluuebunny/crossref_metadata_2025 huggingface dataset:
Randomized, Prospective Clinical Trial of rFVIIa for Secondary Prophylaxis in Hemophilia Patients with Inhibitors.
Authors: Konkle Barbara A. , Ebbesen Liselotte S. , Friedrich Ute , Bianco Raul P. , Lissitchkov Toshko , Rusen Luminita , Serban Margit A.
Published: April 2020

Introduction: Hemophilia patients with inhibitors experience frequent bleeds and reduced quality of life (QoL). This trial evaluated whether once−daily prophylaxis therapy with activated recombinant factor VII (rFVIIa; 90 or 270 μg/kg) for 3 months can effectively reduce bleed frequency compared with on−demand therapy, without compromising safety.

Methods: Thirty−eight hemophilia inhibitor patients with frequent bleeds entered a 3−month observation period where on−demand therapy was continued according to local practice. Twenty−two patients with a stable bleed frequency (≥4 bleeds/month) were randomized 1:1 to once−daily dosing with 90 or 270 μg/kg rFVIIa in a double−blind, parallel−group design. To maintain blinding, the same volume was administered in both groups. Patients then entered a 3−month post−treatment observation period, where on−demand therapy was resumed.

Results: The number of bleeds per month was significantly reduced by 45% and 59% with 90 or 270 μg/kg rFVIIa prophylaxis, respectively, compared with the observation period (Fig.). Similar reductions were seen for all bleeds, irrespective of site or cause. The majority of this reduced bleeding frequency was maintained in the 3 month post−treatment period (27% and 50% reductions, respectively). There were no treatment− or dose−dependent patterns in the number or type of adverse events. No thromboembolic events or withdrawals due to adverse events or ineffective treatment were reported. The benefits of reducing bleeding frequency in this trial translated into improvements in QoL.

Conclusions: These results support the concept of secondary rFVIIa prophylaxis in inhibitor patients with frequent bleeds. Clinically relevant reductions in number of bleeds and improvements in quality of life were observed during prophylaxis compared with conventional on−demand therapy without raising any safety concerns.

Figure Figure
Proteolysis of MLL Family Proteins Is Essential for Taspase1−Orchestrated Cell Cycle Progression.
Authors: Hsieh James J. , Takeda Shugaku , Chen David Y. , Westergard Todd D. , Fisher Jill K. , Rubens Jeffrey A. , Sasagawa Satoru , Kan Jason T. , Korsmeyer Stanley J. , Cheng Emily H.
Published: April 2020

Taspase1 was identified as the threonine endopeptidase that cleaves MLL for proper Hox gene expression in vitro. To investigate its functions in vivo, we generated Taspase1−/− mice. Taspase1 deficiency results in non−cleavage (nc) of MLL and MLL2 and homeotic transformations. Remarkably, our in vivo studies uncover an unexpected role of Taspase1 in cell cycle. Taspase1−/− animals are smaller in size. Taspase1−/− MEFs exhibit impaired proliferation and acute deletion of Taspase1 leads to a marked reduction of thymocytes. Taspase1 deficiency incurs down−regulation of Cyclin Es, As, and Bs and up−regulation of p16Ink4a. We show that MLL and MLL2 directly target E2Fs for Cyclin expression. The un−cleaved precursor MLL displays a reduced histone H3 methyl transferase activity in vitro. Accordingly, CHIP assays demonstrate a markedly decreased histone H3 K4 tri−methylation at Cyclin E1 and E2 genes in Taspase1−/− cells. Furthermore, MLLnc/nc;2nc/nc MEFs are also impaired in proliferation. Our data are consistent with a model in which precursor MLLs, activated by Taspase1, target to Cyclins through E2Fs to methylate histone H3 at K4, leading to activation. Lastly, Taspase1−/− cells are resistant to oncogenic transformation and Taspase1 is over−expressed in many cancer cell lines. Thus, Taspase1 may serve as a target for cancer therapeutics.
Notch1 Expression Is Regulated at Translational Level by 3′UTR Region in Hematopoietic Stem Cell Development.
Authors: Mizuno Shin-ichi , Ozawa Hidetoshi , Shigematsu Hirokazu , Arinobu Yojiro , Yong Chong , Iino Tadafumi , Akashi Koichi
Published: April 2020

In hematopoietic stem cell development, the expression of critical genes is precisely regulated in a stage specific manner, which supports normal hematopoietic development through adequately regulating timing of cell division, self-renewal, and lineage commitment of hematopoietic stem cells. Regulation of gene expression is known to take place at transcriptional level. In addition to the transcriptional regulation, there are growing evidences that translational control of critical genes may play a role, especially in embryogenic development, suggesting an interesting possibility that translational control may also play a role in hematopoiesis. Here, we provide the evidence that the expression of a key transcription factor in lymphoid development, Notch1, is controlled at translational level in hematopoietic stem cell. To examine whether translation of mRNAs of hematopoietic major factors can be regulated at each stage of hematopoietic development, we established a retrovirus sensor vector, in which 3′UTR (untranslated region) of target genes is placed between the GFP coding region and the retrovirus 3′LTR. Since the transcribed mRNA from this vector is a fusion mRNA of GFP and 3′UTR from the gene of interest, this vector allows us to visualize the effect of 3′UTR on translational control of particular genes by GFP as a reporter. We cloned 3′UTR fragments from PU.1, GATA-1, GATA-2, C/EBPα and Notch1 genes into the sensor vector. Then, we introduced these retrovirus vectors into prospectively-purified hematopoietic stem and progenitor cells, including HSC, CMP and GMP, and their GFP intensities were monitored by a flowcytometer. We found that induction of the sensor vector with the 3′UTR sequence from Notch1 showed marked suppression of the GFP intensity at the HSC stage. The 3′UTR sequences from the other 4 transcription factors did not show such intensive inhibitory effect. Suppression of Notch1 transcription by its 3′UTR was further confirmed by using a retrovirus vector which has two distinct markers of YFP and GFP-3′UTR fusion genes under bi-directional EF1α promoter. These data suggest that the expression of Notch1 should be regulated at translational level by its 3′UTR at the HSC stage as well as at transcriptional level. Our data provide the first evidence that the stage-specific translational regulation can play an important role in organization of hematopoietic development.
Hemolysis in Sickle Cell Mice Causes Pulmonary Hypertension Due to Global Impairment in Nitric Oxide Bioavailability.
Authors: Hsu Lewis L. , Champion Hunter C. , Campbell-Lee Sally A. , Bivalacqua Trinity J. , Manci Elizabeth A. , Diwan Bhalchandra A. , Schimel Daniel M. , Cochard Audrey E. , Wang Xunde , Schechter Alan N. , Noguchi Constance Tom , Gladwin Mark T.
Published: April 2020

Pulmonary hypertension is a highly prevalent complication of sickle cell disease and is a strong risk factor for early mortality. However, the pathophysiological mechanisms by which sickle hemoglobin leads to pulmonary vasculopathy remain unclear. Transgenic mice provide opportunities for mechanistic studies of vascular pathophysiology in an animal model of severe sickle cell disease. Using micro-cardiac catheterization we found that all mice expressing exclusively human sickle hemoglobin develop pulmonary hypertension. Recognizing that the NO pathways can have complex abnormalities in other conditions of pulmonary hypertension, the NO axis in sickle mice was assessed by multiple methods. From a mechanistic standpoint the mice exhibit profound pulmonary and systemic endothelial dysfunction and vascular instability characterized by diminished responses to authentic nitric oxide (NO), NO donors and endothelium-dependent pulmonary vasodilators, and enhanced responses to vaso-constrictors. However, endothelium-independent vasodilation in the sickle mice was normal. Mechanisms of vasculopathy in sickle mice involve global dysregulation of the NO-axis: impaired constitutive nitric oxide synthase activity with loss of eNOS coupling (dimerization), increased NO scavenging by plasma hemoglobin and superoxide, increased arginase activity, and depleted intravascular nitrite reserves. Consistent with a functional rather than structural defect, light microscopy and computed tomography of the lungs revealed no plexogenic arterial remodeling, thrombi/emboli, or inflammation. Transplanting sickle marrow into wild-type mice conferred the same phenotype. Similar pathobiology was observed in a non-sickle mouse model of acute alloimmune hemolysis, supporting a major role of hemolysis as a mechanism for this dysregulation of NO and vasculopathy. In this study, alloimmune hemolytic mice were chosen for comparison in order to generalize beyond hemoglobinopathies. Future analogous studies with thalassemic mice may be useful to model pulmonary hypertension in human thalassemia intermedia. In conclusion, this animal model extends the evidence for global impairment in NO responsiveness and NO production in sickle cell disease, and suggests that hemolytic anemia is associated with endothelial dysfunction and pulmonary hypertension.
Regular Blood Transfusion Does Not Prevent Progression of Cerebral Lesions Evidenced by Magnetic Resonance Imaging in Children with Sickle Cell Disease (SCD).
Authors: Brousse Valentine , Hertz-Pannier Lucie , Bresson Jean-Louis , De Montalembert Mariane
Published: April 2020

Purpose: The effects of chronic transfusion on the cerebral vessel abnormalities in SCD patients are unknown. Chronic transfusion has been reported to be effective in preventing recurrence of overt strokes, and occurrence of a first stroke in children with abnormal transcranial Doppler ultrasonography (TCD). However, cerebrovascular events may occur despite adequate prevention, suggesting that transfusion therapy does not cure nor even stabilize vessels disease. We conducted a single center retrospective study to determine the evolution of cerebral lesions in SCD children on regular transfusion therapy using magnetic resonance imaging.

Material and methods: Children with homozygous sickle cell anemia chronically transfused for first or secondary prevention of stroke were included in the study. All patients had been started in a program of monthly transfusions, maintaining constantly hemoglobin S level below 30%, in the month following the stroke or the abnormal TCD. They underwent cerebral magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) every one to two years. An expert neuroradiologist unaware of the date of the imaging and unaware of the identity of the patients reviewed resulting images. Standard MRI criteria were used to identify lacunae, cerebral atrophy, infarcts and leucoencephalopathy, with progressive grading. Standard MRA criteria were used to identify arterial tortuosity, stenosis/occlusion and moya-moya with progressive grading. All MRI and MRA were scored, and these scores were compared longitudinally in each patient using a Paired Rank test.

Results: 18 children (9 males, 9 females) were enrolled. Chronic transfusion therapy was prescribed and initiated for 10 patients with initial strokes (mean age at the stroke 6,8 +/− 2,5 yrs) and 8 patients with abnormal TCD (mean age at the TCD 7,2 +/− 2,9 yrs). Mean follow-up was 6,8 +/− 4,1 yrs in the stroke group, 1,7 +/− 1,0 yrs in the abnormal TCD group. A total of 45 MR images were reviewed (median MR/patient: 3 [2–4]). Initial scores were lower in the abnormal TCD group than in the stroke group (mean score respectively 1 [0–7] versus 12 [2–26]). Comparison of longitudinal scores in the group of patients with initial stroke evidenced progression of lesions (p=0,008). The longitudinal scores were not significantly different in the abnormal TCD group, but this may be explained by the shorter follow-up.

Conclusion: Blood transfusion maintaining permanently HbS level <30% does not prevent progression of cerebral micro or macro vascular lesions in SCD children having had a 1st stroke. A lengthier follow-up is needed to reach a conclusion in the case of children with abnormal TCD. Our results support the case that early bone marrow transplant therapy should be applied to prevent progression of cerebral vasculopathy in SCD children at risk.
Aberrant Expression of the Homeobox Gene CDX2 in Acute Myeloid Leukemia.
Authors: Scholl Claudia , Bansal Dimple , Dohner Konstanze , Eiwen Karina , Lee Benjamin H. , Rucker Frank G. , Schlenk Richard F. , Bullinger Lars , Dohner Hartmut , Gilliland D. Gary , Frohling Stefan
Published: April 2020

The caudal-type homeobox transcription factor 2 (CDX2) plays an important role in embryonic development and regulates the proliferation and differentiation of intestinal epithelial cells in the adult. Ectopic expression of CDX2 in the hematopoietic compartment was previously identified as the key pathogenetic event in a single patient with acute myeloid leukemia (AML) and t(12;13)(p13;q12). Using real-time quantitative PCR, we detected aberrant CDX2 expression in 153 (90%) of 170 patients with AML, in patients with high-risk myelodysplastic syndrome or advanced-stage chronic myeloid leukemia, and in several AML cell lines, but not in bone marrow derived from normal individuals. Expression of CDX2 was monoallelic in the majority of cases with informative single-nucleotide polymorphisms in the CDX2 coding region, but was not related to mutations in the CDX2 coding region or in the predicted CDX2 promoter sequence, gene-specific hypomethylation of the CDX2 promoter, or increased CDX2 gene copy numbers. Stable knockdown of CDX2 expression by lentivirus-mediated RNA interference inhibited the proliferation of various human AML cell lines exhibiting CDX2 transcript levels that were in the range of those observed in most primary AML samples, and strongly reduced their clonogenic potential in vitro. Primary murine hematopoietic progenitor cells transduced with Cdx2 acquired serial replating activity in vitro, could be continuously propagated in liquid culture, generated a fully penetrant and transplantable AML in vivo, and displayed dysregulated expression of Hox family members. Together, these results (i) demonstrate that aberrant expression of CDX2 is a frequent event in myeloid leukemogenesis, (ii) suggest a role for CDX2 as part of a common effector pathway that increases the proliferative capacity and promotes the self-renewal potential of hematopoietic progenitors, and (iii) support the unifying hypothesis that CDX2 is responsible, at least in part, for abnormalities in HOX gene expression in AML.
Inhibition of the NPM-ALK Fusion Tyrosine Kinase in Hematopoietic Neoplasia by the Small Molecule Tyrosine Kinase Antagonist TAE684.
Authors: Sternberg David W. , Ray Arghya , Lee Benjamin H. , Adelsperger Jennifer , Duclos Nicole , McDowell Elizabeth , Haq Naznin , Imbach Patricia , Ohmori Osamu , Honda Toshiyuki , Warmuth Markus , Fabbro Doriano , Griffin James D. , Roesel Johannes
Published: April 2020

Anaplastic large cell lymphoma (ALCL) constitutes approximately 10–30 percent of childhood lymphomas and 3 percent of non-Hodgkin lymphomas in adults. Approximately 50–60 percent of ALCL patients express the ALK tyrosine kinase fused to a variety of partner proteins as a result of chromosomal translocation. The activated ALK fusion kinase drives proliferative and survival signaling in lymphoma cells that carry these translocations, and the native ALK protein is overexpressed in a variety of other hematologic and non-hematologic malignancies. Given the known functional role of NPM-ALK in the pathogenesis of some lymphomas, we developed the small molecule ALK antagonist TAE684 that targets the kinase catalytic domain. In Ba/F3 murine hematopoietic cells and human ALCL cells that require NPM-ALK for growth, TAE684 was selectively cytotoxic and rapidly triggered apoptotic cell death. We have extended these findings to animal models of hematopoietic neoplasia driven by the expression of NPM-ALK. In a murine NPM-ALK-Ba/F3 allograft model, oral administration of TAE684 prolonged the survival to 83 days, compared to 51 days for the control placebo-treated group (p=0.0028). Moreover, we used a murine retroviral bone marrow transplant model of NPM-ALK-driven hematopoietic neoplasia to assess the efficacy of TAE684 treatment. The median survival in the placebo-treated group was 18 days (range 11–18 days), whereas all but one mouse treated with TAE684 oral gavage survived to the study endpoint (range 23–25 days). The prolongation in survival was highly significant (p<0.0001) and was associated with a reduction in peripheral blood leukocytosis, decreased splenomegaly, and resolution of tissue infiltration with atypical granulocytic, B-lymphoid, and histiocytic/macrophage cells. Thus, in two rapidly progressive models of hematopoietic neoplasia, oral administration of the TAE684 compound was able to produce marked control of disease in most recipient mice. The prolonged survival of mice treated with TAE684 can be attributed to the ablation of aggressive hematopoietic malignancy that is driven by expression of the NPM-ALK fusion kinase. We anticipate that TAE684 will be effective in the management of lymphomas that express ALK fusion kinases and in non-hematologic malignancies associated with aberrant expression of the native ALK protein.
Heat Shock Protein 90 (HSP90) Is a Rational Therapeutic Target in Diffuse Large B-Cell Lymphoma.
Authors: Abramson Jeremy S. , Chen Wen , Takahashi Hidenobu , Juszczynski Przemyslaw , Kutok Jeffery L. , Shipp Margaret A.
Published: April 2020

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease that is currently treated with a homogeneous approach based on empiric combination chemotherapy. To elucidate molecular differences in DLBCL subsets and identify more rational treatment targets, coordinate transcriptional profiling was used to define three groups of DLBCLs: “Oxidative Phosphorylation” (OxPhos), “B-cell Receptor/Proliferation” (BCR), and “Host Response” (HR). Of interest, the HSP90 α and β isoforms were differentially expressed in the DLBCL subsets; the stress-induced α isoform was more abundant in OxPhos tumors (p < .0001) whereas the constitutively expressed β isoform was increased in BCR DLBCLs (p < .0001). HSP90 α and β differ in the length of their N-termini but share a common ATP binding domain, the target site for recently developed HSP90 inhibitors, such as IPI-504 (Infinity Pharmaceuticals, Cambridge, MA). Although differential HSP90 isoform expression likely reflects underlying biological differences in the DLBCL subsets, the shared ATP binding domain suggests that both isoforms, and the majority of DLBCLs, may be effectively targeted with HSP90 inhibitors. For these reasons, we determined the IC50 and cytotoxic potential of IPI-504 in an extensive series of DLBCL cell lines. IPI-504 inhibited the proliferation of DLBCL cell lines at IC50s ranging from 0.04 μM in the most sensitive line to 12.67 μM in the most resistant line. Annexin V/PI staining revealed that IPI-504 was cytotoxic to most DLBCL cell lines at low micromolar doses; even cell lines that were relatively resistant to IPI-504 at lower doses (2 μM) underwent apoptosis with prolonged exposure to increased IPI-504 doses (72 hrs. and 8 μM). Cell lines that were most sensitive to IPI-504 expressed high levels of a major HSP90 client protein, pAKT, and exhibited a dose-dependent decrease in pAKT levels following IPI-504 treatment. For these reasons, we assessed the efficacy of IPI-504 in association with a PI3K/AKT pathway inhibitor, LY24009, in the DLBCL panel. Combined treatment with the HSP90 and PI3K inhibitors was highly synergistic (Method of Chou and Talalay; Calcusyn software, Biosoft, Ferguson, MO), inhibiting cellular proliferation by over 95% in evaluated DLBCL cell lines. In contrast, the combination of concurrent IPI-504 and an empiric chemotherapeutic agent, doxorubicin, was antagonistic, underscoring the importance of rational combination therapy. We conclude that: 1) HSP90 isoforms are differentially expressed in primary DLBCL subsets; and 2) inhibition of HSP90, via the conserved ATP binding domain, leads to dose-dependent apoptosis that is associated with depletion of pAKT in DLBCL cell lines. The HSP90 inhibitor IPI-504 possesses both single-agent efficacy as well as synergy when combined with PI3K inhibitors, suggesting that this rational targeted approach warrants further clinical investigation.
Enforced Expression of GATA-2 Confers Quiescence on Human Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.
Authors: Tipping Alex J. , Pina Cristina , Castor Anders , Atzberger Ann , Hong Dengli , Lazzari Lorenza , May Gillian , Jacobsen Sten Eirik , Enver Tariq
Published: April 2020

Hematopoietic stem cells (HSCs) in adults are largely quiescent, periodically entering and exiting cell cycle to replenish the progenitor pool or to self-renew, without exhausting their number. Expression profiling of quiescent HSCs in our and other laboratories suggests that high expression of the zinc finger transcription factor GATA-2 correlates with quiescence. We show here that TGFβ1-induced quiescence of wild-type human cord blood CD34+ cells in vitro correlated with induction of endogenous GATA-2 expression. To directly test if GATA-2 has a causative role in HSC quiescence we constitutively expressed GATA-2 in human cord blood stem and progenitor cells using lentiviral vectors, and assessed the functional output from these cells. In both CD34+ and CD34+ CD38− populations, enforced GATA-2 expression conferred increased quiescence as assessed by Hoechst/Pyronin Y staining. CD34+ cells with enforced GATA-2 expression showed reductions in both colony number and size when assessed in multipotential CFC assays. In CFC assays conducted with more primitive CD34+ CD38− cells, colony number and size were also reduced, with myeloid and mixed colony number more reduced than erythroid colonies. Reduced CFC activity was not due to increased apoptosis, as judged by Annexin V staining of GATA-2-transduced CD34+ or CD34+ CD38− cells. To the contrary, in vitro cultures from GATA-2-transduced CD34+ CD38− cells showed increased protection from apoptosis. In vitro, proliferation of CD34+ CD38− cells was severely impaired by constitutive expression of GATA-2. Real-time PCR analysis showed no upregulation of classic cell cycle inhibitors such as p21, p57 or p16INK4A. However GATA-2 expression did cause repression of cyclin D3, EGR2, E2F4, ANGPT1 and C/EBPα. In stem cell assays, CD34+ CD38− cells constitutively expressing GATA-2 showed little or no LTC-IC activity. In xenografted NOD/SCID mice, transduced CD34+ CD38−cells expressing high levels of GATA-2 did not contribute to hematopoiesis, although cells expressing lower levels of GATA-2 did. This threshold effect is presumably due to DNA binding by GATA-2, as a zinc-finger deletion variant of GATA-2 shows contribution to hematopoiesis from cells irrespective of expression level. These NOD/SCID data suggest that levels of GATA-2 may play a part in the in vivo control of stem and progenitor cell proliferation. Taken together, our data demonstrate that GATA-2 enforces a transcriptional program on stem and progenitor cells which suppresses their responses to proliferative stimuli with the result that they remain quiescent in vitro and in vivo.
Competing Influences of Genetic and Leukaemia-Specific Factors on P-Glycoprotein Expression in Blasts from 817 Patients Entered into the NCRN AML14 and 15 Trials.
Authors: Seedhouse Claire H. , Grundy Martin , White Paul , Li Yun , Fisher Janet , Yakunina Darya , Moorman Anthony , Russell Nigel , Burnett Alan , Pallis Monica
Published: April 2020

Background. P-glycoprotein (pgp), expressed on acute myeloid leukaemia (AML) blasts, is associated with failure to respond to chemotherapy in AML. This study aimed to determine whether expression and function of pgp may be linked to polymorphisms of the encoding gene (MDR1, also known as ABCB1) and whether leukaemia-specific changes in cell biology may override genetic factors in predicting pgp expression. G1199A, G2677T and C3435T polymorphisms in MDR1 (RFLP analysis) as well as pgp protein expression (using MRK-16) and function (modulation of R123 accumulation by PSC 833) were studied in leukaemic blast samples from 817 patients with AML entered into the NCRN AML14 and AML15 clinical trials.

Age, low white blood cell count (WBC), high bcl2, secondary AML and MDS and adverse cytogenetics all correlated with high pgp expression. However, ABCB1 3435TT homozygosity had a negative impact on pgp. Overall, pgp expression was high in 41% of the cohort, but in elderly patients high pgp expression was found in 54% with the 3435C allele versus 32% in the 3435TT group (P=0.009). This is similar to the frequency of pgp in younger patients (34%) and indicates that the 3435TT polymorphism affects pgp expression in elderly patients by preventing the upregulation of pgp protein normally seen in this group. No association was found between genetic factors and pgp function. In a subset of 341 patients, pgp protein levels were analysed on a quantitative scale in relation to C3435T and G2677T gene polymorphisms. The lowest protein expression occurred in the variant TT group for both polymorphisms. Differences were even more marked in the elderly subset (using Mann-Whitney test, P = 0.003 and 0.002 respectively). Linkage disequilibrium occurs between positions 2677 and 3435, and there was a highly significant association between the haplotype and pgp protein expression (P=0.001). ABCB1 mRNA was also measured in 81 patients and was found to be high in patients with the G1199A allele (median expression 0.36 ABCB1/b2M ratio v 0.06 in the GG group, P=0.04), suggesting that the 1199A polymorphism affects ABCB1. However no further correlations were found between the 1199A polymorphism and pgp protein or function. The association between pgp and a low white WBC, (P<0.001) was further examined by cell cycle analysis in 40 patients confirming that pgp is associated with a low proportion of cycling cells - median % cycling cells: pgp negative/low 4.0%, pgp intermediate/high 1.0% (P=0.02).

Conclusions. We conclude that there is an extended MDR phenotype of indolent, pgp positive, bcl2-high cells in AML, particularly in the elderly, and that pgp is affected by an interaction between genetic factors and acquired, leukaemia-specific factors.
Bortezomib Enhances Melphalan Response by Altering Fanconi Anemia (FA)/BRCA Pathway Expression and Function.
Authors: Yarde Danielle N. , Hazlehurst Lori A. , Oliveira Vasco A. , Chen Qing , Dalton William S.
Published: April 2020

The FA/BRCA pathway is involved in DNA damage repair and its importance in oncogenesis has only recently been implicated. Briefly, 8 FA/BRCA pathway family members facilitate the monoubiquitination of FANCD2. Upon monoubiquitination, FANCD2 translocates to the DNA repair foci where it interacts with other proteins to initiate DNA repair. Previously, we reported that the FA/BRCA pathway is upregulated in multiple myeloma cell lines selected for resistance to melphalan (Chen, et al, Blood 2005). Further, reducing FANCF in the melphalan resistant 8226/LR5 myeloma cell line partially reversed resistance, whereas overexpressing FANCF in the drug sensitive 8226/S myeloma line conferred resistance to melphalan. Others have reported, and we have also verified, that bortezomib enhances melphalan response in myeloma cells; however, the mechanism of enhanced melphalan activity in combination with bortezomib has not been reported. Based on our observation that the FA/BRCA pathway confers melphalan resistance, we hypothesized that bortezomib enhances melphalan response by targeting FA/BRCA DNA damage repair pathway genes. To investigate this hypothesis, we first analyzed FA/BRCA gene expression in 8226/S and 8226/LR5 cells treated with bortezomib, using a customized microfluidic card (to detect BRCA1, BRCA2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, RAD51 and RAD51C) and q-PCR. Interestingly, we found that low dose (5nM) bortezomib decreased many FA/BRCA pathway genes as early as 2 hours, with maximal decreases seen at 24 hours. Specifically, 1.5- to 2.5-fold decreases in FANCA, FANCC, FANCD2, FANCE and RAD51C were seen 24 hours post bortezomib exposure. Moreover, pre-treatment of myeloma cells with low dose bortezomib followed by melphalan treatment revealed a greater than 2-fold reduction in FANCD2 gene expression levels. We also found that melphalan treatment alone enhanced FANCD2 protein expression and activation (monoubiquitination), whereas the combination treatment of bortezomib followed by melphalan decreased activation and overall expression of FANCD2 protein. Taken together, these results suggest that bortezomib enhances melphalan response in myeloma by targeting the FA/BRCA pathway. Further understanding of the role of the FA/BRCA pathway in determining melphalan response may allow for more customized and effective treatment of myeloma.
Anti-VEGF Antibody Treatment Markedly Inhibits Tumor Growth in SCID-hu Models of Human Multiple Myeloma.
Authors: Campbell Richard A. , Sanchez Eric , Chen Haiming , Turker Lauren , Trac Olivia , Li Mingjie , Shalitin Dror , Gordon Melinda S. , Pang Shen , Bonavida Benjamin , Said Jonathan , Plowman Gregory D. , Fuh Germaine , Berenson James R.
Published: April 2020

Vascular endothelial growth factor (VEGF) is an important signaling protein that plays a critical role in vasculogenesis and angiogenesis, and serves as one of the contributors to physiological or pathological conditions that can stimulate the formation of new blood vessels. The uncontrolled growth of new blood vessels is an important contributor to a number of pathological conditions, including multiple myeloma (MM). In support of this, bone marrow angiogenesis has been shown to correlate with disease status and poor prognosis in MM. VEGF also directly induces myeloma cell proliferation. Based on these findings, we evaluated a new mouse/human anti-VEGF antibody using our SCID-hu mouse models of human MM. Each immunodeficient (SCID) mouse was implanted with a 2.0 – 4.0 mm3 LAGκ-1A tumor fragment into the left superficial gluteal muscle. The tumors were allowed to grow for 14 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into one of two treatment groups. In one group, anti-VEGF antibody was administered via intraperitoneal injection twice per week at a dose of 5 mg/kg. In the other cohort, control mice were given a control IgG antibody (5 mg/kg) on the same schedule. Mice receiving the anti-VEGF antibody showed marked inhibition of tumor growth (P = 0.0005) and reduction of paraprotein levels (P = 0.0002) compared to mice receiving control antibody. On day 42, LAGκ-1A-bearing mice receiving the anti-VEGF antibody showed a 70% reduction in human paraprotein levels and an 80% decrease in tumor volume compared the control antibody treated animals. Treatment with the anti-VEGF antibody was not associated with any observed toxicity. We are currently evaluating this anti-VEGF antibody in several of our mouse models of human MM and plasma cell leukemia. Based on these data with anti-VEGF monotherapy, we are currently investigating the anti-tumor activity of anti-VEGF antibody plus bortezomib as well as other available anti-MM agents using our in vivo SCID-hu myeloma murine models. Preliminary results are encouraging with single agent anti-VEGF antibody and additional studies may be used to direct the clinical development of anti-VEGF antibody treatment alone and in combination regimens for patients with relapsing or refractory MM.
Cytotoxic CD8 T Cell Immune Responses to Wilms Tumor Protein (WT-1) Characterizes Immunosuppression-Responsive Myelodysplasia (MDS).
Authors: Sloand Elaine M. , Rezvani Katayoun , Yong Agnes , Douek Daniel , Kurlander Roger , Price David , Barrett John , Young Neal S.
Published: April 2020

Clinical observations and experimental evidence link bone marrow failure in myelodysplastic syndrome (MDS) with a T cell-dominated immunologically-mediated pathophysiology in some patients. Indeed, among 129 patients treated with immunosuppression at NIH, trisomy 8 was associated with hematologic response to immunosuppression. We have demonstrated that trisomy 8 patients have oligoclonal CD8 T cell expansion, as determined by increased proportions of one or more T cell receptor (TCR) V beta subfamilies (Sloand et al. Blood106:841; 2005) which had cytotoxic activity toward trisomy 8 cell progenitors. Here we examine the response of cytotoxic T cells to cyclin D1 and Wilms tumor protein (WT-1); these candidate autoantigens had earlier been observed to be over-expressed in trisomy 8 CD34 cell in our microarray analysis (Chen et al. Blood 104:4210, 2004 ). Bone marrow mononuclear cells from 19 trisomy 8 patients (defined by cytogenetics and FISH) had significantly increased levels of WT-1 mRNA and protein expression compared to normal CD34 cells by real time PCR and immunoblot, respectively (p<0.001). When patient and control CD8 cells were cultured in the presence of WT-1- and cyclin D1-loaded HLA-A2-restricted antigen-presenting cells (using three different HLA-A0201- restricted epitopes of WT-1 and four different epitopes for CD1), eight patients’ CD8 cell tested positive for interferon gamma, indicating a cytotoxic response. Staining with labeled MHC class I A2-restricted tetramers used to measure WT-1-specific cells showed concordant results in all eight patients. Tetramer staining was increased in 12 of 14 trisomy 8 patients examined. Cytotoxic T cells specific for WT-1 were present within the expanded V β subfamilies previously found to suppress trisomy 8 cell proliferation in vitro. CD8 cells from another eight MDS patients without trisomy 8 but who had responded to immunosuppression, sampled prior to treatment, also demonstrated WT1-specific cytotoxic activity, but only two of 18 non-responders showed measurable levels of WT-1-specific cytotoxic T cells (entire treated cohort seen in Fig 1). We infer from these data an autoimmune pathophysiology for trisomy 8 MDS with: 1) over-expression of WT-1 by the aneuploid clone; 2) a specific cytotoxic CD8 T cell immune response to WT-1; 3) subsequent cytotoxic T cell marrow suppression by apoptosis, either by cross reactivity or a bystander effect; and 4) improvement in hematopoiesis after immunosuppression. Other MDS which is responsive to immunosuppression may also be mediated by WT1.

Figure Figure
Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.
Authors: Janzen Viktor , Fleming Heather E. , Waring Michael T. , Milne Craig D. , Scadden David T.
Published: April 2020

The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.
Zfx Is an Essential and Specific Regulator of Hematopoietic Stem Cell Self-Renewal.
Authors: Reizis Boris , Harel Sivan , Galan Jose M. , Arenzana Teresita L.
Published: April 2020

Mammalian hematopoiesis is based on the activity of multipotent hematopoietic stem cells (HSC) that undergo continuous self-renewal throughout the adult life. The self-renewal represents a unique property of adult HSC, and appears distinct from the proliferation of hematopoietic progenitors or from the initial specification and expansion of embryonic HSC. Despite recent progress, little is known about the molecular mechanisms governing HSC self-renewal. Zfx is a broadly expressed zinc finger-containing transcriptional activator that is highly conserved in vertebrates. Using conditional gene targeting in mice, we demonstrate that Zfx is an essential and specific regulator of HSC self-renewal. The expression of Zfx was elevated in HSC compared to progenitors and differentiated cells. An inducible deletion of Zfx from the adult bone marrow (BM) resulted in the specific loss of the HSC population. The short-term proliferation and lodging of Zfx-deficient HSC in the BM were normal, suggesting a specific defect of long-term self-renewal. The deletion of Zfx in pre-established BM chimeras completely abrogated HSC maintenance as reflected by the rapid loss of HSC contribution to hematopoiesis. Furthermore, a constitutive pan-hematopoietic deletion of Zfx spared embryonic HSC in the fetal liver, yet resulted in the loss of adult BM HSC. In contrast, adult erythromyeloid progenitors or differentiated cells were not affected by the absence of Zfx. Genome-wide expression analysis identified candidate target genes of Zfx, some of which were controlled by Zfx in HSC but not in their differentiated progeny. Finally, several immediate-early and/or stress-inducible genes were upregulated specifically in Zfx-deficient HSC, suggesting that the latter undergo increased stress-related signaling. Thus, functional and gene expression analysis establishes Zfx as a novel specific regulator of adult HSC self-renewal. Further studies of the target genes and pathways controlled by Zfx should provide novel insights into the molecular basis of HSC function.
Acquisition of FOXP3 Expression by Human Effector CD4+ T Cells Is a Natural Consequence of Antigen Recognition.
Authors: Melenhorst J. Joseph , Scheinberg Phillip , Ambrozak David R. , Hensel Nancy F. , Douek Daniel C. , Price David A. , Barrett A. John
Published: April 2020

Recent data suggest that CD4+CD25+FOXP3+ regulatory T cells (TR) can be generated from CD4+CD25−FOXP3− T cells in the periphery. We studied the induction of CD4+CD25+FOXP3+ T cells during the course of immune responses to cytomegalovirus, tetanus toxoid, purified protein derivative and streptokinase. Peripheral blood mononuclear cells (PBMC) from healthy donors were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA) and stimulated with antigen for 6–7 days. Cells were stained for CD3, CD4, CD25, HLA-DR, CD38, and intracellular FOXP3. Responders to antigens had proliferating (CFDA[dim]) CD4+ T cells expressing CD25 and the activation markers CD38 and HLA-DR. In PBMC from five donors, a median of 37% (range 9–57%) of the proliferating fraction expressed FOXP3 (figure) which mostly co-expressed CD25, HLA-DR and CD38, suggesting that FOXP3 expression is the consequence of cellular activation triggered through the T cell receptor. Since all of these healthy subjects had circulating, pre-existing CD4+CD25+FOXP3+ T cells in the absence of ex vivo antigen stimulation, however, it was possible that these gave rise to the FOXP3+ responder cells. Antigen stimulation of PBMC from three donors aged 69–70 years with no detectable CD4+CD25+FOXP3+ T cells also induced proliferating activated CD4+CD25+FOXP3+ T cells, excluding the possibility that a pre-existing pool of FOXP3+ T cells gave rise to this population. Subsequently, we sorted CD4+ T cells that proliferated in response to antigen by flow cytometry on the basis of FOXP3 expression to sequence the T cell receptor-β CDR3 regions and to establish the T cell clonotype structure of proliferating FOXP3-positive and -negative T cells. These data also indicate that effector CD4+ T cells acquire FOXP3 expression during the course of an immune response. Based on these and previously reported findings, we propose that the acquisition of FOXP3 expression by effector CD4+ T cells is a natural consequence of antigen recognition that serves as a specific regulatory feedback system.

Induction of FOXP3 expression in proliferating CD4+ T cells in response to T cell receptor triggering Induction of FOXP3 expression in proliferating CD4+ T cells in response to T cell receptor triggering
Reversal of Anticoagulation as Assessed by Thrombin Generation Measurement.
Authors: Gatt Alex , van Veen Joost , Cooper Peter , Kitchen Steve , Makris Michael
Published: April 2020

Antidotes to the ever-growing number of anticoagulants are always desirable in order to placate bleeding in emergencies. In general, the older anticoagulants like coumarins and unfractionated heparin (UFH) have proven reversal agents, whereas the newer ones do not. We studied the in vitro effect of 5 different heparinoids (UFH, Tinzaparin, Enoxaparin, Fondaparinux and Danaparoid) on the calibrated automated thrombogram. The assay uses a fluorogenic substrate that is cleaved by the thrombin formed after the addition of plasma to 5pM tissue factor, 4μM phospholipids and calcium chloride. We investigated all the parameters generated by dedicated software (Thrombinoscope™) ie the lag time (LT), the time to peak (ttpeak), the endogenous thrombin potential (ETP) and the peak thrombin. All the five anticoagulants tested inhibited thrombin generation (TG) in a concentration dependent manner.

We subsequently analysed the in vitro effect of different concentrations of six potential reversal agents on correcting TG parameters of maximally inhibited plasma for each anticoagulant. These were protamine sulphate at 2.5, 5 & 8μg/ml, activated FVIIa (Novoseven®) at 5, 10 & 50μg/ml, FEIBA® at 0.5,1 & 2U/ml, Beriplex® at 0.3, 0.6 & 1.2U/ml, Prothromplex® TIM4 at 0.4, 0.8 & 1.8U/ml and fresh frozen plasma (FFP) at 250, 500 & 750μl/ml. The three concentrations reflect the recommended therapeutic doses for each agent together with lower and higher doses than normally used.

As predicted, UFH (final concentration 0.527U/ml) was completely reversed with a standard protamine concentration of 5μg/ml. However, the highest dose of protamine gave slightly lower TG, indicating that higher concentrations of protamine sulphate can have a paradoxical ‘anticoagulant’ effect. High doses of FEIBA (2U/ml) and FVIIa (50μg/ml) restored ~50% of thrombin generation parameters. Tinzaparin (at 1antiXaUnit/ml) was also completely neutralised by protamine. However, a higher concentration of 8μg/ml protamine was required. This effect was not seen with Enoxaparin with this higher concentration of protamine reversing only ~40% of the ETP, 21% of the peak thrombin, 71% of ttpeak and 72% of the LT. There was no positive effect of protamine on Fondaparinux (3μg/ml) and Danaparoid (1antiXa U/ml)-treated plasma. Whereas Danaparoid seemed relatively resistant to all six reversal agents, Fondaparinux effect was completely neutralised by FVIIa at concentrations between 10–50μg/ml.

This study highlights the differences in neutralisation of different low molecular weight heparins and UFH. In particular, Tinzaparin was much more readily reversed with protamine sulphate than Enoxaparin. It also indicates that high doses of FVIIa could completely reverse Fondaparinux anticoagulation.
Evaluation of a VENOus-Return Assist Device (Venowave™) To Treat Severe Post-Thrombotic Syndrome (VENOPTS): A Randomized Controlled Trial.
Authors: O’Donnell Martin , McRae Simon , Kahn Susan , Julian Jim , Kearon Clive , MacKinnon Betsy , Magier Debbie , Strulovitch Carla , Lyons Theresa , Hirsh Jack , Ginsberg Jeffrey
Published: April 2020

Severe post-thrombotic syndrome (PTS) is responsible for considerable disability, reduced quality of life and increased health care costs. Current therapies are limited and often ineffective.

Methods: We performed a two-center, randomized, cross-over controlled trial to evaluate Venowave™, a novel lower-limb venous return assist device, for the treatment of severe PTS. Eligible subjects were allocated to receive, in randomized order, Venowave™ for 8 weeks and control-device for 8 weeks. Participants were crossed-over after a 4-week (wash-out) period. The primary outcome measure was a clinical success, defined as; 1) reported benefit from the device with; 2) moderate (or greater) improvement in symptoms of PTS; and 3) willingness to continue using the device long-term. Secondary outcome measures included venous disease-specific quality of life (QOL) (measured by VEINES-QOL questionnaire; higher scores indicate better QOL) and PTS severity (measured by the Villalta PTS scale; higher scores indicate more severe PTS). The study was registered with ClinicalTrials.gov (NCT00182208).

Results: Thirty-two patients were enrolled. Of these, 26 (80%) were also using graduated compression stockings. Twenty-six participants completed both trial periods. The primary outcome measure occurred in 10 participants receiving Venowave and 4 participants receiving the control-device (P=0.1). Two participants reported a clinical success with both devices. Mean Villalta scale score at the end of treatment period was significantly lower for Venowave™ (12.2; SD 6.3) compared to control (15.0; SD 6.1). (P=0.004) Mean VEINES-QOL score at the end of treatment period was significantly greater for Venowave™ (52.5; SD 5.8) compared to control (50.2; SD 6.2). (P=0.004)

Conclusions: Venowave ™ is a very promising new therapy for patients with severe PTS, which may be used alone or in combination with graduated compression stockings. In our study population of patients with severe PTS, refractory to standard therapies, almost one-third of patients reported clinical improvement with Venowave™.