bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Tumor Suppressor Protein Programmed Cell Death 4 (PDCD4) Suppresses Activity of PI3K/Akt Pathway and Regulates Expression of p27 (Kip1) and c-myc, DAP5 and Willm’s Tumor (WT1) in Acute Myeloid Leukemia.
Authors: Ozpolat Bulent , Akar Ugur , Colburn Nancy H. , Lopez-Berestein Gabriel
Published: April 2020

Programmed cell death 4 (PDCD4) is a recently identied novel tumor suppressor protein that inhibits cap-dependent mRNA translation. PDCD4 inhibits tumor promoter incuced carcinogenesis and transformation by suppressing the helicase activity of eIF4A, leading to translational inhibition. Recently we found that PDCD4 is required for all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of acute promyelocytic leukemia (APL) cells (Ozpolat&Akar et al, Mol Cancer Res, in press), type of acute myeloid leukemia characterized by a t(15;17) and a differentiation block. Here we investigated the downstream mediators or targets of PDCD4 in leukemia cell differentiation. ATRA is currently used as a first line standard treatment in APL. Recently, we reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms that involve suppression of translation initiation, a rate limiting step of protein synthesis (Harris&Ozpolat et al, Blood, 104 (5) 2004). We found that ATRA treatment induced PDCD4 expression in NB4 APL, HL60 AML, primary APL patient leukemia cells and normal human CD34+ bone marrow progenitors cells during granulocytic differentiation. However, ATRA/maturation resistant NB4.R1 and HL60R cells failed to express and translocate PDCD4 into nucleus after ATRA treatment. To identify downstream targets of PDCD4 we knock downed PDCD4 expression by siRNA and examined changes in the expression of target proteins that are known to be regulated by ATRA by Western blot analysis in NB4 cells. We found that PDCD4 represses c-myc, and WT1 expression however, it is required for the expression of DAP5 (death associated protein 5), and cyclin dependent kinase inhibitor p27KIP1, and but not c-jun, p21Cip1, and tissue transglutaminase (TG2). Inhibition of PDCD4 by siRNA resulted in upregulation of phospho-P70S6K, suggesting that PDCD4 inhibits activity of PI3K/Akt pathway. RT-PCR analysis revealed that mRNA of these proteins did not change suggesting that PDCD4 tumor suppressor protein regulates expression of these important cellular proteins at translational level and suppresses PI3K/Akt pathway. Furthermore we rapamycin, a specific mTOR inhibitor currently in clinical trials in AML, induced a marked expression of PDCD4, which regulates c-myc and p27 Kip1, revealing a novel mechanism of action of rapamycin, providing new rationale for targeting translational pathways as a therapeutic intervention in the treatment of AML. Overall, data suggest that PDCD4 regulates expression of critical cellular proteins involved in differentiation of leukemia cells and PDCD4 mediated translational control may be an important regulatory mechanism for regulation of gene expression.
Mice Engineered To Express a Form of Thrombin Favoring Protein C Are Resistant to S. aureus-Induced Sepsis.
Authors: Flick Matthew J. , Chauhan Anil K. , Welch Sara , Shaw Maureen A. , Talmage Kathryn E. , Mullins Eric S. , Palumbo Joseph S. , Thornton Sherry , Wagner Denisa D. , Degen Jay L.
Published: April 2020

Thrombin is central in thrombus formation as both a positive mediator of thrombus formation through the proteolytic activation of PARs, fibrinogen, fXI and other prothrombotic substrates, and a negative modulator of the coagulation cascade through the activation of protein C. Detailed structure-function studies have revealed that thrombin can be redesigned to favor either procoagulant or anticoagulant substrates. The introduction of W215A/E217A substitutions in the murine thrombin active site (fIIWE) results in a pronounced “specificity switch” that reduces catalytic efficiency with fibrinogen by at least 3-orders-magnitude while only modestly reducing activity for protein C activation. To evaluate the effects of fIIWE activity in vivo, we have used a gene-targeting strategy to generate mice carrying the W215A/E217A mutations in the endogenous murine prothrombin gene. The mutant allele was transmitted through the germline and was found to support the expression of normal levels of hepatic fII mRNA and plasma fII in both heterozygous and homozygous neonates. Unlike fII knockout mice, homozygous fIIWE mice were observed at term with the expected Mendelian frequency. Nevertheless, homozygous fIIWE offspring uniformly succumbed to spontaneous bleeding events within days of birth. Heterozygous fIIWE/WT animals generally survived to adulthood, were capable of carrying multiple liters to term, and unchallenged mice displayed a hematological profile similar to wildtype mice. However, consistent with a predicted anticoagulant phenotype, adult fIIWE/WT heterozygotes exhibited significantly delayed thrombus formation following ferric chloride injury of mesenteric arterioles and extended bleeding times following tail tip excision relative to control mice expressing wildtype fII. Given that activated protein C has been shown to be efficacious in the treatment of sepsis, we explored whether the shift in thrombin specificity in heterozygous fIIWE/WT mice would confer the benefit of rendering animals tolerant to acute septic challenges. Kaplan-Meier analyses following intravenous administration of S. aureus revealed that fIIWE/WT mice exhibited a significant survival advantage over littermate wildtype animals challenged in parallel and tracked over a 7-day observation period. Notably, extended thrombus formation and bleeding times as well as resistance to sepsis was not simply a function of half normal wildtype fII expression. When these analyses were performed in animals carrying one wildtype allele and one null mutation allele, results were similar to wiltype. These studies further underscore the interplay between the hemostatic and inflammatory systems in vivo and highlight the possible therapeutic utility of recombinant (pro)thrombin derivatives with selected alterations in substrate specificity.
Subpopulations with Iron Deficiency, Liver Disease, or HFE Mutations Revealed by Statistical Mixture Modeling of Transferrin Saturation and Serum Ferritin Concentration in Asians, African Americans, Hispanics, and Whites.
Authors: McLaren Christine E. , Gordeuk Victor R. , Chen Wen-Pin , Barton James C. , Acton Ronald T. , Speechley Mark , Castro Oswaldo , Adams Paul C. , Snively Beverly M. , Harris Emily L. , Reboussin David M. , McLachlan Geoffrey J. , Bean Richard , McLaren Gordon D.
Published: April 2020

In previous investigations, we modeled the distribution of transferrin saturation (TS) in Caucasians and demonstrated a strong association between HFE genotype and TS subpopulations. Extending this approach, we now have analyzed joint population distributions of TS and serum ferritin concentration (SF) measured in the multi-ethnic Hemochromatosis and Iron Overload Screening (HEIRS) Study and examined the association of these distributions with the presence of HFE C282Y and H63D mutations, self-reported liver disease, and iron deficiency (defined as SF <15 μg/L). Based on separate models for each race/ethnicity by gender, four components with successively increasing age-adjusted means for TS and SF were identified in data from 26,832 African Americans, 12,620 Asians, 12,264 Hispanics, and 43,254 Whites. Fig. 1 illustrates age-adjusted values from 16,662 White men. Superimposed 95% confidence ellipses reflect component probability densities and show separation of the 1st and 4th components that had the lowest and highest means for TS and SF, respectively. Table 1 presents the range of estimates from individual models and indicates that the 2nd (largest) component had TS means of 22–26% for women (29–30% for men) and SF means of 43–82 μg/L for women (165–242 μg/L for men). The 3rd and 4th components had progressively smaller proportions and higher mean values of TS and SF, while the 1st component in each model had mean TS <16% for women (<20% for men), and mean SF <28 μg/L for women (<47 μg/L for men). Compared to the 2nd component:

adjusted odds of iron deficiency were significantly higher in the 1st component (15–48 for women, 61–3530 for men); adjusted odds of self-reported liver disease were significantly higher in the 3rd and 4th components for African-American women and all men; and adjusted odds of any HFE mutation were increased in the 3rd component (1.4–1.8 for women, 1.2–1.9 for men) and in the 4th component for Hispanic and White women (1.5, 5.2, respectively) and men (2.8, 4.7, respectively).

Joint mixture modeling identifies one component with lower mean SF and TS at risk for iron deficiency and two components with higher mean SF and TS at risk for liver disorders and HFE mutations. This approach permits characterization of the aggregate effects of hereditary or acquired factors that influence these serum iron measures in populations, and complements and enhances genetic and phenotypic testing for assessment of disease characteristics.

Table 1 Range of estimates from models.
Screening for Hemochromatosis by Measuring Ferritin Levels: A More Effective Approach.
Authors: Waalen Jill , Beutler Ernest
Published: April 2020

Population screening for hereditary hemochromatosis has traditionally been performed by determining the serum transferrin saturation. With the recognition that the penetrance of HFE hemochromatosis is extremely low, it has become apparent that population screening with transferrin saturation is unlikely to be cost effective as the vast majority of individuals detected neither have clinical disease nor are likely to develop it. Although the factors that cause a subset of patients to develop the severe disease phenotype have not been identified, it is clear from at least three independent studies that only patients with serum ferritin concentrations of over 1000 ng/ml are at risk. We have utilized data from the Scripps-Kaiser hemochromatosis study to model results of a screening program using this ferritin cut-off value. Among 29,699 white subjects screened, only 59 had serum ferritin levels of greater than 1000 ng/ml. Twenty-four of these had homozygous mutant or compound heterozygous mutant HFE genotypes that could account for the ferritin levels. Among the remaining patients, the main causes of elevated ferritin levels were excessive alcohol intake, cancer, or liver disease. Causes for the hyperferritinemia were found in all but 8% of the 59 patients. We conclude that screening for hemochromatosis with serum ferritin levels will detect the vast majority of patients who will be clinically affected, and may detect other clinically significant disease in patients who do not have hemochromatosis phenotypes. Since it is clear from other longitudinal studies that the ferritin level of the vast majority of adult homozygotes for HFE mutations does not rise over long periods of time, excluding subjects with serum ferritin levels <1000 ng/ml should not result in missed opportunities for early treatment of patients who could benefit.
Role of the Direct Interaction between Factor VIII C2 and Factor IXa Gla Domain in the Factor Xase Complex.
Authors: Soeda Tetsuhiro , Nogami Keiji , Takeyama Masahiro , Ogiwara Kenichi , Tomokiyo Kazuhiko , Sakata Yoichi , Yoshioka Akira , Shima Midori
Published: April 2020

Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.
P2 and P2′ Residues Are Important for the Efficient Proteolysis of Factor VIIIa at Arg336 Catalyzed by Activated Protein C.
Authors: Varfaj Fatbardha , DeAngelis Jennifer , Wakabayashi Hironao , Fay Philip J.
Published: April 2020

Activated Protein C (APC) is an anticoagulant serine protease that proteolytically inactivates the coagulation cofactors, factors (F) Va and VIIIa. FVIIIa is a non-covalent heterotrimer consisting of A1, A2 and A3-C1-C2 subunits. APC-catalyzed inactivation of FVIIIa results from proteolysis at the P1 residues Arg336 and Arg562 within the A1 and A2 subunits, respectively, with cleavage at Arg336 representing the dominant reaction. We recently showed that replacement of the P4-P3′ residues surrounding the Arg336 site with the corresponding residues flanking Arg562 resulted in an ∼100-fold reduction in cleavage rate at Arg336 (Varfaj et al, J. Biol. Chem.2007). Comparison of the P4-P3′ residues at the slow-reacting site in FVIIIa (Arg562) with the corresponding residues at the fast-reacting site in FVa (Arg506) revealed that these sequences differ primarily at the P2 and P2′ positions. This observation suggested an important contribution by these residues to cleavage efficiency. The role of the P2 and P2′ residues in the proteolysis of FVIIIa by APC was investigated by preparing recombinant FVIII proteins possessing mutations at Leu335 (P2) and Lys338 (P2′) flanking the P1 Arg336. B-domainless FVIII proteins were stably expressed in BHK cells and purified. Leu335Arg and Leu335Gln mutants were constructed based upon the P2 residues preceding Arg506 in FVa and Arg562 in FVIIIa, respectively. The Lys338Ile mutant was based upon the P2′ residue following Arg506 in FVa. Specific activity values for all FVIII variants were similar to wild type (WT) FVIII. APC-catalyzed inactivation rates for FVIIIa were determined using a FXa generation assay, and rates for proteolysis at the scissile bonds within the A1 and A2 subunits were determined by SDS-PAGE and Western blotting. Rates for APC-catalyzed cleavage of the A1 subunit for the FVIIIa variants Leu335Arg and Leu335Gln were reduced ∼2- and ∼4-fold, respectively, as compared to WT. However, the rate of cleavage of the A1 subunit in the Lys338Ile FVIIIa variant was enhanced ∼3-fold compared to WT. Cleavage rates at Arg562 in all the variants were unaffected by mutation at either residue 335 or 338. These relative values for rates of proteolysis paralleled the observed rates for inactivation of the FVIIIa forms. Overall, these results suggest that both P2 and P2′ residues are important in the efficient proteolysis at Arg336 in FVIIIa. Furthermore, Leu appears more optimal than either Arg or Gln at the P2 position, whereas Ile is preferred over Lys at the P2′ position in this macromolecular substrate for APC.
Inhibition of the Thrombin-Thrombomodulin by a Covalent Antithrombin-Heparin.
Authors: Van Walderveen Maria C. , Berry Leslie R. , Chan Anthony K.C.
Published: April 2020

Endothelial thrombin·thrombomodulin (IIa-TM) complex is capable of activating protein C anticoagulant. In vivo, TM can exist in two forms: one with a chondroitin sulfate (CS) (acidic) and one without (non-acidic). TM binding has been shown to reduce the rate of IIa inhibition by antithrombin (AT) in the presence of unfractionated heparin (UFH), especially when the TM possesses a glycosaminoglycan chain. We have recently developed a covalent antithrombin-heparin (ATH) with high potency for inhibition of IIa bound to surfaces. In the present study we examined the role of the CS chain in the inhibition of IIa-TM complex by the novel anticoagulant, ATH, in comparison to AT + UFH. For this study, discontinuous second order rate constant (k2) inhibition assays of IIa-TM were performed at 37°C. IIa was incubated with TM for 30 min and inhibited using either 20nM AT + varying concentrations (0–6000nM) of UFH or a constant concentration of 10nM ATH. Residual IIa activity was measured at 405nm after stopping the reaction using a solution of polybrene and chromogenic substrate (S-2238). Second order rate constants (mean +/− SEM; n≥5) were calculated from plots of IIa-TM activity vs. time and differences determined using two-sample t-tests. Comparison of the second order rate constants for inhibition of IIa, IIa-TM(−CS), or IIa-TM(+CS) by AT + UFH (2.015 × 108 +/− 7.079 × 106 M−1min−1, 1.633 × 108 +/− 8.922 × 106 M−1min−1, and 5.248 × 107 +/− 5.693 × 106 M−1min−1, respectively) showed that the presence of TM reduced the rate of inhibition (with respect to IIa alone; p=0.012, p<0.001, respectively). Moreover, the presence of a CS on the TM inhibited IIa neutralization to a greater degree than when the CS is absent (p<0.001). Furthermore, ATH was a much faster inhibitor of IIa, IIa-TM(−CS), and TM(+CS) (1.486 × 109 +/− 1.193 × 108 M−1min−1, 1.370 × 109 +/− 1.246 × 108 M−1min−1, and 8.869 × 108 +/− 1.246 × 108 M−1min−1, respectively) compared to AT + UFH (p<0.001). CS removal by chondroitin ABC lyase (ABCase) showed that the rate of IIa-TM(+CS) inhibition significantly increased for both AT + UFH and ATH, while no significant change due to ABCase treatment was found for the rate of inhibition of IIa-TM(−CS) by either AT + UFH or ATH. We speculate that during IIa-TM(+CS) inhibition by ATH, the covalently linked heparin moiety may be able to repel (rotate) the CS chain in order for ATH to interact with the IIa-TM complex. In the case of AT + UFH, lack of permanent UFH linkage to AT may allow the CS to displace the UFH chain from binding both AT and IIa. Steric hindrance by CS chains remains a significant factor since the k2 values were lower for the AT + UFH or ATH reactions with IIa-TM(+CS) compared to those with IIa-TM(−CS). Given the rapid reaction rate with free or bound IIa, ATH significantly limits availability of IIa for coagulant or anticoagulant functions.
Mapping of Allosteric Modulator Effects in the Active Site of Factor IXa.
Authors: Villegas Karla , Baker-Deadmond Kimberly , Neuenschwander Pierre F.
Published: April 2020

Activated factor IX (fIXa) is a vitamin K-dependent blood coagulation serine protease involved in propagation of the coagulant response through activation of fX. Maximal enzymatic and procoagulant activity of fIXa requires the presence of several cofactors; one of which is ionic calcium, which is known to bind to a site in the protease domain of fIXa as well as several sites within the light chain Gla domain region. One of the roles of calcium appears to be allosteric modulation of the fIXa active site as evidenced by an increase in enzymatic activity towards small peptidyl substrates. We and others have additionally found that certain small hygroscopic molecules can also enhance fIXa amidolytic activity. The molecular details involved in either of these effects are not well understood. Previous studies by us have shown that a pentapeptide substrate (AGRSL; the reactive site sequence of antithrombin) is hydrolyzed by fIXa in the absence of cofactors or modulators. This hydrolysis is enhanced in the presence of ionic calcium, ethylene glycol or low molecular weight heparin suggesting effects of these molecules on the immediate active site vicinity of fIXa. In order to gain insight into the potential allosteric modulation that each of these effectors may affect in fIXa, we examined the hydrolysis of four peptide libraries based on the AGRSL pentapeptide sequence, in the presence and absence of various fIXa modulators. The four libraries synthesized were XGRSL, AXRSL, AGRXL and AGRSX; where X denotes any of the possible 20 amino acids. Each of these libraries were screened for hydrolysis by fIXa under various conditions with substrates and products being identified en masse using MALDI-TOF mass spectrometry. The results suggest that ionic calcium enhances fIXa reactivity in part by modulation of the S2 subsite in fIXa. In contrast, ethylene glycol enhances fIXa activity via modulation of the S3 subsite and heparin was found to effect the overall active site region.
Phase II Evaluation of Sunitinib Malate, a Multi-Targeted Inhibitor of Receptor Tyrosine Kinases, in Patients with Myelofibrosis.
Authors: Tam Constantine S. , Thomas Deborah A. , Ravandi Farhad , Richardson Catherine D. , Borthakur Gautam , Burger Jan A. , Estey Elihu , Kantarjian Hagop , Verstovsek Srdan
Published: April 2020

BACKGROUND: Sunitinib Malate is a small molecule inhibitor of receptor tyrosine kinases (TK) with a broad spectrum of activity against VEGFR-1, -2, and -3, PDGFR-α and -β, c-kit, the TK encoded by the ret oncogene, and Flt-3. As TK-mediated angiogenesis may be an integral element in the pathogenesis and progression of myelofibrosis (MF), we investigated the use of sunitinib in MF patients.

METHODS: 14 patients (pt) have been treated with sunitinib 37.5mg orally daily in six-week cycles, with response assessment after 2 and 4 cycles. Median baseline characteristics (range): age 65 years (50–79); 2 previous therapies (0–4); B-symptoms 64%; Hb < 8g/dL or transfusion-dependent 50%; JAK-2 mutated 57%.

RESULTS: Pt received sunitinib for a median of 101 days (23–173). Six stopped study drug prior to the planned four cycles because of toxicity (5) or disease progression (1); four stopped at four cycles because of toxicity (1) or lack of response (3); one stopped at five cycles because of lack of response; and three patients still on therapy had shown no responses at 3, 3 and 5 cycles. No responses were observed. One patient had 50% reduction in splenomegaly but became transfusion-dependent, and was considered a non-response. Grade 1 or 2 hypertension developed in 7 (50%) patients and was more common in patients with a pre-existing history of hypertension (5/7) than patients without a history of hypertension (2/7); in six patients, hypertension led to interruption and/or dose reduction in study medication. Common toxicities were constitutional (fatigue 86%, Grade 3=21%), gastrointestinal (abdominal pain, nausea or diarrhea 79%, Grade 3=14%) or neurologic (pain or numbness in extremity 43%, Grade 3=0%). Grade 3 bleeding (1 epistaxis, 1 upper GI) occurred in 2 patients, and grade 3 optic disc edema in 1. Toxicities were cumulative and dose-related, with dose reductions being required in 7/10 patients who continued beyond 2 cycles.

CONCLUSIONS: At the first planned interim analysis, no responses were recorded in 14 patients treated with sunitinib. This fulfiled the early stopping rule in the Gehan two-stage study design, and the study has been closed to new patient accrual because of inadequate response.
Identification of Critical Phosporylation Sites within NPM-ALK That Regulate JUNB mRNA Translation.
Authors: Fuchs Claudia , Vesely Paul , Bambach Isabella , Schauer Silvia , Linkesch Werner , Sternberg David W. , Hoefler Gerald , Staber Philipp B.
Published: April 2020

Anaplastic large cell lymphoma (ALCL) accounts for approximately 30% of childhood lymphomas and 3% of adult non-Hodgkin lymphomas. The nucleophosmin - anaplastic lymphoma kinase (NPM-ALK) fusion which is the product of a t(2;5)(p23;q35) chromosomal translocation is present in about half of nodal ALCL. Expression of this fusion kinase results in induction of the AP-1 transcription factor JunB and IL-3 independent outgrow of murine hematopoietic Ba/F3 cells. We demonstrated that wild type NPM-ALK increases the amount of ribosomes bound to JUNB mRNA resulting in its more effective translation in large polysomes. The NPM-ALK fusion tyrosine kinase has 20 potential tyrosine residues available for autophosphorylation and phosphorylation by other protein tyrosine kinases. Here we used series of Y-to-F-substituted mutants of NPM-ALK to identify tyrosine residues that are required to regulate the segregation of JUNB mRNAs between polysomes and monosomes as well as ribonucleic particles (RNPs). Neither JUNB transcription nor JunB translation was altered in Ba/F3 cells expressing NPM-ALK mutants Y17F/Y29F/Y67F Y138F/Y152F Y156F/Y191F/Y299F Y378F/Y418F/Y445F and Y646F/Y664F compared to NPM-ALK wild type. Conversely, in NPM-ALK Y567F/Y461F/Y644F mutant cells proliferation was markedly decreased. These cells demonstrated active MEK-ERK pathway, while AKT, mTOR, and rpS6 phosphorylation was impaired. Moreover a shift of JUNB mRNA from the polysomic to the monosomic/mRNP fraction could be observed. In conclusion, we identified specific NPM-ALK phosphorylation sites required to mediate the effect of NPM-ALK on the JUNB translational regulation and therefore provide further insights in the transforming mechanisms of the oncoprotein NPM-ALK.
CMV-Associated Expansion of CD8+CD45RA+CD27− T-Cells in Patients with B-Cell Malignancies.
Authors: Tonino Sanne H. , Van Oers Marinus H.J. , Van Lier Rene A. , Kersten Marie Jose
Published: April 2020

In patients with various malignancies of B-cell origin, changes in the T-cell compartment have been observed. In B-cell chronic lymphocytic leukemia (CLL), we have previously described an expansion of T-cells, largely due to an increase in T-cells exhibiting the CD45RA+CD27- effector phenotype. This was found only in cytomegalovirus (CMV) seropositive patients, suggesting that CMV-antigen drives this expansion. Indeed a considerable fraction of these T-cells were shown to be CMV-specific. At present it is unknown whether this alteration of the T-cell compartment is specific for CLL or rather any B-cell malignancy could induce similar changes. We performed an immunophenotypic analysis of the T-cell compartment in the peripheral blood of 56 patients with a B-cell malignancy (CLL, n=16; indolent lymphoma, n=20; aggressive lymphoma, n=10; multiple myeloma, n=10) and 12 healthy controls. Subjects of the different disease categories and controls were age-matched. We assessed the total CD3 compartment, naïve (CD27+CD45RA+), memory (CD27+CD45RA−) and memory effector (CD27−CD45RA+) CD8+ T cells. Latent CMV infection was assessed both by CMV-IgG titers (ELISA) and CMV viral load (PCR). The absolute number of T-cells was significantly increased (2.5 – 3 fold) in CLL, but not in the other B-cell malignancies. However, in all disease categories the CD4/CD8 ratio was decreased when compared to healthy controls. This was due to a slight decrease in CD4+ T-cells and an increase in CD8+ T-cells. Irrespective of the nature of the malignancy, the expansion of CD8+ T-cells resulted from a significant increase in CD45RA+CD27− effector T-cells. In agreement with our previous observation, CD8+ T-cell changes were only found in CMV-seropositive patients. Based on our earlier studies we expect these T-cells to be CMV-specific. No correlation between CMV-specific antibody titers and effector T-cell numbers was found. We conclude that the, possibly CMV-specific, effector T-cell compartment is expanded in all B-cell maligancies, although these changes seem to be more pronounced in CLL. The expansion of the total T cell compartment is restricted to CLL. The skewing of the T-cell compartment in CMV-seropositive patients could be the result of increased pressure of latent CMV-infection on the cellular immune system due to failure of other aspects of host defence, brought about by the malignancy.
Thalidomide Based Induction Chemotherapy Prior to Stem Cell Transplantation in Myeloma Is Associated with Decreased Response to Retreatment with Thalidomide at First Relapse Post Transplant.
Authors: Simpson Lijo , Gertz Morie , Lacy Martha Q. , Dispenzieri Angela , Hayman Suzanne , Buadi Francis , Rajkumar Vincent S. , Kumar Shaji
Published: April 2020

Background: In patients (pts) with newly diagnosed myeloma (MM) eligible for high dose therapy and autologous stem cell transplantation (ASCT), the standard approach is 4–6 months of induction therapy followed by ASCT. The combination of thalidomide and dexamethasone is one of the commonly used induction regimens, and has superior response rates compared to dexamethasone or VAD. Almost all patients undergoing an ASCT relapse at a median interval of 18–24 months. The choice of treatments at first relapse is variable, but re-application of the original induction regimen, if effective is often considered. It is not clear if the responses obtained at the time of induction therapy can be replicated at the time of relapse. We studied the response of thalidomide based regimens when used as first treatment after relapse from an ASCT in patients receiving thalidomide as induction treatment.

Methods: Pts who received thalidomide and dexamethasone as induction therapy, and thalidomide ± dexamethasone at first relapse after ASCT were identified for the current study. A group of patients receiving dexamethasone as induction therapy and thalidomide ± dexamethasone at first relapse after ASCT was used as the control group.

Results: There were 20 pts in the study group (group 1) and 14 patients in the control group (group 2) identified between 2002 and 2007. There were 15 (75%) males in group 1 and 10 (71%) in group 2. In group 1 there was 1(5%) complete response (CR), 5 (25%) very good partial response (VGPR) and 14 (70%) partial responses (PR) to induction therapy. In group 2 there was 1 (7%)VGPR, 12 (86%) PR and 1 (7%)stable disease following dexamethasone induction. Upon treatment with thalidomide based regimens at first relapse after ASCT, there was a high rate of treatment failures in group 1 as compared to group 2 (no response in 14 out of 20 patients (70%) versus 5 of 14 pts (35%), respectively, p = 0.04). Of the 10 patients with progressive disease in group 1 post transplant, 30% had previously achieved a VGPR and 70% had previously achieved a PR to thalidomide/dexamethasone pre-ASCT.

Conclusions: Our study provides evidence toward the lack of efficacy of thalidomide based regimens on post ASCT relapse in patients who had thalidomide responsive disease prior to ASCT. It is possible that this applies to other induction regimens when used at post ASCT relapse and raises the possibility of selection of drug resistant clones with initial therapy. Response rates seen with drug regimens prior to ASCT may not be reproducible at the time of relapse after ASCT.
Melphalan and Dexamethasone Is an Effective Therapy for Primary Systemic Amyloidosis.
Authors: Tan Tow-Shung , Dispenzieri Angela , Lacy Martha Q. , Hayman Suzanne R. , Buadi Francis K. , Zeldenrust Steven R. , Lust John A. , Russell Stephen J. , Dingli David , Greipp Philip R. , Kyle Robert A. , Rajkumar S. Vincent , Gertz Morie A. , Kumar Shaji
Published: April 2020

Background: Primary systemic amyloidosis (AL) is an incurable plasma cell disorder with a poor survival, especially in patients (pts) with multiple organ involvement. For pts with limited organ involvement, high dose chemotherapy with autologous stem cell (SCT) support is an effective approach. However, the toxicity of SCT limits its widespread use. Melphalan and dexamethasone has been previously shown to be effective in pts ineligible for SCT.

Methods: We reviewed our experience with melphalan and dexamethasone for treatment of AL. Eighty one pts were treated between 2001 and 2003; median age at start of therapy was 63 years (range, 38–80) and 56 pts (69%) were males. Among these, 46 pts (57%) had 2 or more major organs (cardiac, renal, hepatic) involved. Intravenous melphalan (IV) was used in 45 pts (56%), 32 (40%) received oral melphalan (PO) and the rest received cycles of both. The most common doses were 16 mg/m2 for IV administration (every 4–6 weeks) and 0.22 mg/kg for PO administration (every 4 weeks). Dexamethasone was usually given at a dose of 40 mg for the first 4 days of each cycle.

Results: Patients received a median of 5 cycles (range; 1–17). At the time of analysis, 53 (65%) pts were alive with a median follow up of 10.8 months (range, 1.4–47.1).The estimated median overall survival (OS) was 25.6 months for the entire group, with the number or type of organs involved not impacting survival. Hematological response (HR) was defined by a 50% reduction in either serum M-spike, urine M-spike, or the absolute difference between the involved and uninvolved immunoglobulin free light chains. A HR was observed in 43 pts (67%) of the 64 pts measurable for HR by conventional criteria. Achievement of HR was associated with a superior OS with a median post-therapy OS that was not reached, while non-responders had a median OS of 11.1 months (p<0.0001) (Figure 1). Among these pts, an organ response as defined by conventional criteria, was seen in 14 pts (22%), which was significantly correlated with obtaining a hematological response (33% vs. 5%, p=0.01). A 25% reduction in the difference between the involved and uninvolved FLC strongly predicted prolonged OS, with a median post-therapy OS that was not reached for responders vs. 11.3 months for non-responders (p<0.0001) (Figure 2). A decrease in post-therapy brain natriuretic peptide (BNP) also predicted for a survival improvement, with the median OS not reached for responders vs. a median OS of 17 months for non-responders (p<0.016).

Conclusion: Treatment with melphalan and dexamethasone is effective and a significant survival advantage is achieved in patients with a hematological response. FLC and BNP response can help identify patients that are likely to benefit from this therapy.

Figure Figure Figure Figure
Mutation of the Wilms’ Tumor 1 Gene Is a Poor Prognostic Factor Associated with Chemoresistance in Normal Karyotype Acute Myeloid Leukemia.
Authors: Virappane Priya , Gale Rosemary E. , Hills Robert , Kakkas Ioannis , Summers Karin , Stevens Jane , Green Claire , Allen Christopher , Burnett Alan K. , Linch David C. , Bonnet Dominique , Lister T. Andrew , Fitzgibbon Jude
Published: April 2020

The Wilms’ tumour 1 (WT1) gene is highly expressed in various types of leukemia, suggesting a role both as a target for immunotherapy and a means for monitoring minimal residual disease. More recently WT1 has been recognised as an important mutational target in normal karyotype (NK) acute myeloid leukemia (AML), with clustering of mutations to exons 7 and 9. In this study, we set out to determine the clinical relevance of these mutations. Diagnostic DNA samples were obtained from peripheral blood or bone marrow of 470 young adult AML patients entered into the UK Medical Research Council AML 10 and 12 trials. Mutation analysis was performed using standard PCR-based direct sequencing and/or high-resolution capillary electrophoresis of exons 7 and 9. Overall, 51 mutations were observed in 47 cases (10%). Of these, 42 cases had 46 frameshift mutations arising from insertions (n=41) or deletions (n=5) of between 1 and 28 bps that would result in disruption of the C-terminal DNA binding domain of the protein. Median mutant level was 45% (range 8–86%). The remaining 5 cases were non-synonymous amino acid substitutions in exon 9, D396N (n=3) which is implicated in Denys-Drash syndrome, and 1 each of H397Y and H397Q that are of unknown functional significance. There was a borderline inverse relationship between the presence of WT1 mutations and NPM1 exon 12 mutations (p=.05), and an increased proportion of FLT3-ITDs in WT1 mutant cases (p=.08). In order to determine the clinical relevance of WT1 mutations we compared patient characteristics between WT1 mutated and WT1 wild-type cases. There was no difference between the 2 groups in age, gender, presenting WBC, or type of AML (de novo/secondary). Patients with WT1 mutations had an inferior response to therapy compared to WT1 wild-type cases (complete remission [CR] 79% Vs 90% respectively, odds ratio [OR] 3.02, 95% confidence intervals [CI] 1.17–7.82, p=.02) which was due to a higher rate of resistant disease (RD) (15% Vs 4%, OR 9.33, CI 2.38–36.6, p=.001). In univariate analysis mutated cases also had a significantly reduced disease free survival (DFS), 22% Vs 44% at 5 years (OR 2.16, CI 1.32–3.55, p=.005) and overall survival (OS), 26% Vs 47%, OR 1.91, CI 1.23–2.95, p=.007). In multivariate analysis considering age, gender, type of leukemia, WBC, FLT3-ITD and NPM1 mutant status, a WT1 mutation remained an independent adverse prognostic factor for both response to therapy (CR: OR 3.19, CI 1.25–8.13, p=.01; RD: OR 4.93, CI 1.58–15.37, p=.006) and survival (DFS: OR 1.60, CI 1.07–2.38, p=.02; OS: OR 1.52, CI 1.05–2.18, p=.03). These results indicate that WT1 mutations are an additional prognostic marker in NK AML and may be suitable for targeted therapy.
Impact of Prior Lenalidomide Treatment on Peripheral Blood Stem Cell (PBSC) Mobilization in Untreated Multiple Myeloma (MM) Patients.
Authors: Mazumder Amitabha , Kaufman Jonathan , Lonial Sagar , Jagannath Sundar , Vesole David
Published: April 2020

Background: Immunomodulatory drugs (e.g. thalidomide and lenalidomide) are increasingly being used for induction therapy prior to stem cell harvest in MM. We have previously reported that induction therapy with a thalidomide-based regimen leads to a lower yield of stem cells when compared to bortezomib. This resulted in a reduced yield of stem cells per collection and an increased number of phereses required to reach our target goal. Delay in engraftment of platelets by a median of 1 day was also observed. We now report lower PBSC yields in patients receiving lenalidomide induction therapy.

Methods: Data was pooled from 2 centers on patients who received lenalidomide induction therapy followed by PBSC mobilization. Twenty of 28 patients received G-CSF 10 micrograms/kg for four days; 8 patients were mobilized with 7.5 mcg/kg G-CSF plus 7.5 mcg/kg GM-CSF for 5 days.

Results: Eight of 28 patients (40%) failed to collect sufficient cells for even 1 transplant (less than 2 x 106 CD34+ cells/kg. Of note, 2 of these patients also failed to mobilize sufficient stem cells when treated with the CxCR4 inhibitor AMD3100 plus G-CSF on a compassionate use protocol. These patients subsequently had successful mobilization following cyclophosphamide chemotherapy mobilization. Our data mirrored those of Kumar et al (Leukemia 2007 Jun 21; [Epub]) in several aspects. After lenalidomide therapy, there was a decrease in total number of CD34+ cells/kg in day 1 collections and total number of days required to collect sufficient CD34+ cells when compared to chemotherapy or bortezomib induction therapy. However, our data differed from those of Kumar et al regarding the correlation of number of cycles of lenalidomide therapy and subsequent PBSC collection. Kumar et al reported no failures in patients who had less than 6 cycles of lenalidomide. In contrast, 5 of our failures had received only 4 or 5 cycles of lenalidomide.

Conclusions: Patients treated with lenalidomide induction therapy have lower stem cell yields with growth factor mobilization. Biologically, the action of Imids may be different from those of bortezomib on the bone marrow stroma. These lower yields may become clinically important when attempting to obtain stem cells from elderly patients, those with prior radiation therapy or with higher bone marrow plasma cell infiltration. Our group has elected to use cyclophosphamide for mobilization of patients treated with prior lenalidomide and we have been successful in 4/4 patients so far.
Bortezomib [VELCADE™], Pegylated Liposomal Doxorubicin [DOXIL/CAELYX®] and Dexamethasone in the Treatment of Previously Untreated Multiple Myeloma Patients: Impact on Quality-of-Life.
Authors: Belch Andrew , Reece Donna E. , Bahlis Nizar J. , White Darrell , Teixeira Bruno , Camacho Fernando , Plante Richard K.
Published: April 2020

Introduction: Bortezomib in combination with liposomal doxorubicin has shown convincing activity as induction therapy in frontline multiple myeloma (MM) patients with CR rates exceeding those seen with conventional induction therapy. The impact of this treatment on patient Quality-of-Life (QOL), determined by overall health status, cancer and neurotoxicity specific questionnaires, is of clinical interest.

Methods: Newly diagnosed Autologous Stem Cell Transplant (ASCT) eligible MM patients were recruited from nine centres across Canada to this single-arm, open-label, Phase II study. Patients received three to four 21-day cycles of DBd induction therapy (bortezomib 1.3mg/m2 days 1, 4, 8, 11 + pegylated liposomal doxorubicin 30 mg/m2 day 4 + pulsed dexamethasone 40mg days 1–4, 8–11, 15–18 Cycle 1 and days 1–4 cycles 2–4), depending on rapidity of CR achieved, and were then followed post ASCT. Patients completed the European Quality of Life Questionnaire (EQ-5D), the European Organization for Research and Treatment of Cancer’s 30-item Quality of Life Questionnaire (EORTC QLQ-C30) and the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity questionnaire (FACT/GOG-Ntx) prior to each cycle and at study termination.

Results: Fifty patients were enrolled to fulfill the sample size requirement. Mean age was 56.0 (±8.7) years, and 68% of patients were male. Sixty-five percent of the patients had Stage III disease and all but two patients had secretory disease. Mean baseline β2-microglobulin level was 4.2 (1.5–25.4) mg/mL. Overall best response post-induction was 98% (≥ Minimal Response), with a corresponding 26.8% combined complete response (CR) + near CR rate. There was no significant change in the total EQ-5D score throughout the study when compared to baseline. There was a significant improvement at end of study in the mean EQ-5D VAS self-rated health status (Δ+5.14, p<0.05) compared to baseline. Although not significant, there was a trend towards an increase in the proportion of patients reporting no problem in the mobility and anxiety/depression EQ-5D dimensions over the course of induction therapy. There were also no significant changes observed in the EORTC QLQ-C30 scores. Throughout the study, the FACT/GOG-Ntx scores showed no statistically significant changes over time from baseline, in agreement with the overall clinical safety profile observed. The largest mean score change from baseline was at the end-of-study time point (Δ+1.79, SD±6.07, p=0.06).

Conclusion: Although this study was not powered to detect changes in QOL, preliminary results indicate that there is a trend towards improvement in QOL outcomes in multiple myeloma patients being treated with the DBd induction regimen. These results corroborate the safety and tolerability profile observed in the study.
Effects of Soluble Domain 1 of Apolipoprotein ER2′ (BD1 apoER2′) on Pathogenic Effects of a Dimer of β2glycoprotein I (β2GPI) In Vivo.
Authors: Pierangeli Silvia S. , Romay-Penabad Zurina , Urbanus R.T. , Montiel-Manzano Guadalupe , Pennings M.T. , Derksen Ronald , deGroot Philip
Published: April 2020

Background: It has been shown that endothelial cell (EC) activation and tissue factor (TF) upregulation in EC and monocytes and platelet activation by anti-β2glycoprotein I (aβ2GPI) antibodies leads to a prothrombotic state. However, the receptor recognized by aβ2GPI Abs in target cells is not known. It has been demonstrated that dimers of β2GPI mimic the effects of β2GPI-aβ2GPI complexes in that they increased platelet adhesion to collagen and that these dimers bind to apolipoprotein ER2′ (apoER2′) in platelets. Here, we examined the effects of a dimeric form of β2GPI on thrombosis, on platelet activation and TF activity in vivo in mice. The effects of soluble recombinant binding domain 1 (BD1) of apoER2′ were also studied.

Methods: We treated CD1 mice with 50 μg/mL (i.p) recombinant dimeric β2GPI or with recombinant monomeric β2GPI twice at 0 and 48 hours later. Some mice were infused i.p. with 40 μg of BD1. The size of an induced thrombus in the femoral vein of the mice was examined as described elsewhere 72 hours after the first injection. TF activity was determined using a chromogenic assay in homogenates of carotid arteries, and in peritoneal cells of mice. Platelet aggregation was determined by aggregometry in mouse platelet rich plasma.

Results: In vivo, dimers of β2GP1 increased significantly the size of the induced thrombi, TF activity in carotid artery homogenates, in peritoneal macrophages and platelet aggregation, when compared to mice treated with control protein. (Table). These effects were significantly diminished by pre-treatment of the mice with BD1apoER2′.

Conclusions: Thus, when β2GPI is dimerised, it can induce thrombosis, TF and platelet activation in mice independently of the presence of antibodies. The data also indicate that the binding of dimerised β2GPI to target cells may involve apoER2′ since the effects observed were ameliorated by BD1 apoER2′. These findings may be important in understanding the pathogenic mechanisms of thrombosis in Antiphospholipid Syndrome (APS) and in designing new modalities to prevent/ameliorate clinical manifestations of APS.

Treatment Thrombus size (μm2) TF in carotid artery (pM/mg/ml) TF macrophages (pM/mg/ml) Platelet aggregation (%) Dimer 3129±562 346 ± 24 187 ± 36 56 dimer + BD1 533 ± 148 123 ± 15 34 ± 26 23 control monomer 552 ± 79 238 ± 12 42 ± 25 10