bluuebunny/crossref_metadata_2025 huggingface dataset:
The MAML1 Transcriptional Co-Activator Is Required for the Development of Marginal Zone B Cells.
Authors: Wu Lizi , Maillard Ivan , Nakamura Makoto , Pear Warren S. , Griffin James D.
Published: April 2020

Notch1 and Notch2 receptor-mediated signaling appear to have important and unique roles in lymphoid lineage commitment. Notch1 is required for T cell development, while Notch2 is essential for marginal zone B cell development. This specificity is not completely explained by differential expression patterns of Notch1 and 2 or Notch ligands, suggesting that there are other genes that contribute to specifying Notch receptor functions. We have previously shown that the MAML family of transcriptional co-activators is essential for Notch-induced transcriptional events, and functions by forming ternary complexes with Notch and the transcription factor CSL in the nucleus. This MAML family currently consists of three members, MAML1-3, all of which can function as co-activators for Notch receptors in vitro . In this study, we investigated the possibility that MAML1 co-activator contributes to determining Notch receptor function by generating mice deficient in the Maml1 gene. Maml1 -deficient mice fail to thrive and die within 10 days of birth. The morphology of marrow, nodes, and spleen was grossly intact. The ability of Maml1-deficient stem cells to generate different T and B lineages of lymphoid cells was determined by transplanting fetal liver cells isolated from E14.5 embryos into lethally irradiated wild-type recipient mice and analyzing donor-derived lymphoid cells 12 weeks post-transplantation. We found that the deletion of Maml1 results in complete loss of marginal zone B cell lineage (MZB, defined by B220+CD21hiCD23lo). Moreover, the number of MZB cells was reduced to about 50% in Maml1 -heterozygous fetal liver chimeras as compared to wild type controls. However, T cell development was largely unaffected, with only a modest but significant increase in the number of γδ T cells (about 2 fold) in both the thymus and the spleen. Therefore, these results suggest the unexpected finding that targeted deletion of Maml1 in hematopoietic cells is similar to the targeted deletion of either Notch2 or the Notch ligand, Delta-like 1 (Dll1) resulting in the loss of marginal zone B cells and minimal effects on T cell development. Moreover, the number of marginal zone B cells is correlated with Maml1 gene dosage, indicating haploinsufficiency. These data suggest that the Notch ligand Dll1 activates Notch2 signaling resulting in a Notch2/MAML1/CSL complex that is essential for marginal zone B cell development. Further studies with respect to relative expression levels of various MAML genes and interactions of MAML co-activators and Notch receptors may shed additional light into understanding how different Notch receptors regulate cell fate decisions in hematopoiesis.
A Randomized Phase III Trial of Lenalidomide Plus High-Dose Dexamethasone Versus Lenalidomide Plus Low-Dose Dexamethasone in Newly Diagnosed Multiple Myeloma (E4A03): A Trial Coordinated by the Eastern Cooperative Oncology Group.
Authors: Rajkumar S. Vincent , Jacobus Susanna , Callander Natalie , Fonseca Rafael , Vesole David , Greipp Philip
Published: April 2020

Background: Lenalidomide has shown efficacy in patients with relapsed myeloma in phase II and III clinical trials, and is currently being investigated as initial therapy for the disease. We report results of a phase III trial comparing lenalidomide plus high-dose dexamethasone (Dex) versus lenalidomide plus low-dose Dex as first line therapy in newly diagnosed multiple myeloma (MM).

Methods: Pts with newly diagnosed, untreated, symptomatic MM were eligible. Pts in both arms received lenalidomide 25 mg/day PO on days 1–21 every 28 days. In addition, patients in the high-dose Dex arm (Arm A) received Dex 40 mg on days 1–4, 9–12, and 17–20 PO every 28 days, while pts in the low-dose Dex arm (Arm B) received Dex 40 mg on days 1, 8, 15, and 22 PO every 28 days. The primary endpoint was best response at 4 months on intent to treat basis. At 4 months pts could go off study for stem cell transplant or elect to continue therapy until progression. Response was defined as a decrease in serum and urine monoclonal (M) protein by 50% or higher. If the serum M protein was unmeasurable, a 90% or higher decrease in urine M protein was required. Responses need to be confirmed at least 4 weeks apart. Patients with disease progression or not responding to lenalidomide within 4 months switched to thalidomide with the same dose of dexamethasone they were receiving (Arms C and D, respectively). An independent Data Monitoring Committee approved release of these results.

Results: 445 pts were enrolled: 223 randomized to Arm A and 222 to Arm B. Median age was 65 yrs. Serious adverse event data based on expedited reporting (AdEERS) is available on all pts (see table). Common adverse events of Grade 3 or higher were thromboembolism (18.4% in arm A vs 5.4% in Arm B), infection/pneumonia (18.8% vs 9.0%) and hyperglycemia (5.8% vs 1.8%). Incidence of any grade 4 or higher toxicity was 22.0% in Arm A vs 12.6% in Arm B. Response data is being analyzed.

Conclusions: Lenalidomide plus two different schedules of Dex was investigated in this phase III trial. Preliminary results suggest that toxicity rates are higher in the high-dose Dex arm. The differences in the response rates between the two arms will dictate future trials and clinical practice.

Major Toxicties (AdEERS) Toxicity Arm A (n=223) Arm B (n=222) Cardiac ischemia (Grade >=3) 2.7% 0.5% Hyperglycemia (Grade >=3) 5.8% 1.8% Infection/Pneumonitis (Grade >=3) 18.8% 9.0% Neuropathy (Grade >=3) 0.9% 0.9% Thromboembolism (Grade >=3) 18.4% 5.4% Any non-Hem toxicity (Grade >=3) 53.4% 36.0% Any toxicity (Grade >=4) 22.0% 12.6% Death (Grade 5) 4.5% 1.4%
A Novel Fusion between ETV6 and NCOA2 Defines a New Entity of Acute Leukemia with Co-Expression of T-Cell and Myeloid Markers.
Authors: Strehl Sabine , Nebral Karin , König Margit , Harbott Jochen , Bielorai Bella , Struski Stephanie , Izraeli Shai , Haas Oskar A.
Published: April 2020

We have identified a novel recurrent translocation and the genes involved therein, which is associated with both lymphoid and myeloid acute leukemia. Fluorescence in situ hybridization (FISH) and molecular analyses revealed an in-frame fusion of ETV6 (alias TEL) and NCOA2 (alias TIF2) in six cases of childhood acute leukemia. ETV6 encodes an ets transcription repressor and is frequently involved in chromosome rearrangements with a multitude of partners in lymphoid and myeloid leukemia, and to a lesser extent also in solid tumors. NCOA2 is a member of the p160 family of nuclear receptor transcriptional coactivators (NRCoAs), which have a common domain structure with a conserved N-terminal bHLH-PAS domain, a centrally located (nuclear) receptor interaction domain (RID/NID), and a C-terminal transcriptional activation domain (AD2). NRCoAs typically display HAT activity and/or directly interact with CBP. In this context, NCOA2 also contains a C-terminal CBP-interaction domain (CID), and thus is a transcription factor that mediates transcriptional activation in a ligand-dependent fashion by a mechanism involving chromatin remodeling. As a result of the translocation the pointed (PNT) protein interaction domain of ETV6 is fused to the C-terminus of NCOA2 including the CBP and AD2 motifs. The same C-terminal domains of NCOA2 are also involved in the previously identified MOZ-NCOA2 fusion, which is generated by a inv(8)(p11q13). The absence of the reciprocal NCOA2-ETV6 transcript in one of the cases, and the facts that MOZ-NCOA2 is transforming and that the reciprocal NCOA2-MOZ is not expressed, strongly suggest that the ETV6-NCOA2 chimeric protein and not the reciprocal NCOA2-ETV6 is responsible for leukemogenesis. Unlike in cases with a ETV6-RUNX1 fusion, which is frequently associated with a concurrent secondary deletion of the non-rearranged ETV6 allele, in all cases the second ETV6 and also one NCOA2 allele were retained and both genes were readily expressed. With respect to the immunophenotype, according to the European Group for the Immunological Characterization of Leukemias (EGIL), three of the cases were classified as T-ALL (one as T-I My+), two as biphenotypic leukemia (BAL), and one as AML with M1 phenotype. Nevertheless, all leukemias displayed aberrant co-expression of various T-cell and myeloid/NK markers, and were double-negative for CD4/CD8, suggesting transformation of early progenitors. In contrast to the MOZ-NCOA2 fusion, which is exclusively associated with AML, mostly of M4 and M5 subtype, the ETV6-NCOA2 fusion seems to define a new entity of acute leukemia with aberrant co-presence of T lymphoid and myeloid antigens.
Analysis of Loss of Heterozygosity in Adult Patients with De Novo Acute Myeloid Leukemia and Normal Karyotype.
Authors: Schon Christian , Bullinger Lars , Rucker Frank G. , Dohner Konstanze , Dohner Hartmut
Published: April 2020

A large proportion of acute myeloid leukemia (AML) exhibits a normal karyotype in which the underlying pathomechanisms still have to be determined. Novel techniques like arrayCGH or single nucleotide polymorphism (SNP) chip analysis allow the identification and characterization of molecular rearrangements at the sub-megabase level. Recently, the application of genome-wide SNP array technology revealed frequent uniparental disomy (UPD) in approximately 20% of AML suggesting that UPD represents a nonrandom event in leukemogenesis. Uniparental disomy is acquired by somatic recombination and therefore not accessible by conventional cytogenetic methods or arrayCGH. In this study we analyzed DNA from AML patients with normal karyotype for the presence of LOH. SNP analysis was performed on the Mapping 100k GeneChip (Affymetrix, Santa Clara, CA). DNA was extracted from paired samples of 56 de novo AML patients with normal karyotype at diagnosis and in complete remission, respectively. Signal intensity data were analyzed by the GCOS GeneChip analysis software and statistical analysis of SNP call data was performed by the dChipSNP software. In addition, standard mutation screening of the genes encoding NPM1, FLT3, CEBPA, MLL and NRAS was performed in all cases. Using the 100k SNP array, a mean SNP call rate of 98.2% was reached, resulting in > 110,000 SNP genotype calls per sample. Signal intensity data analysis revealed submicroscopic chromosomal deletions resulting in hemizygosity in three patients. Patient 1 had a single 2 Mb deletion in chromosomal band 3p14.1, patient 2 had two small deletions affecting chromosome 12q23 and 12p13, the latter encompassing the ETV6 locus, and patient 3 had two small deletions within the long arm of chromosome 8. Besides these small chromosomal regions of copy number alterations, we found 4 large stretches of somatically acquired homozygosity without numeric alterations, affecting chromosome 6 (6p21 to 6 pter and 6q26 to 6 qter), chromosome 11 (11p12 to 11pter) and chromosome 13 (13q11 to 13qter). Noteworthy, in the case with uniparental disomy of chromosome 13, we could detect a homozygous FLT3-ITD mutation, supporting the findings that acquired isodisomy for chromosome 13 is common in AML, and associated with FLT3-ITD mutations (Griffiths et al., Leukemia, 2005). In summary, high resolution SNP assay technology in AML patients with normal karyotype allowed the identification of distinct chromosomal regions affected by UPD, supporting the postulated nonrandom mechanism of acquired mitotic recombination events in AML. Besides known chromosomal regions known to be affected by genomic aberrations in AML, we found additional submicroscopic chromosomal aberrations in cases with normal karyotype. Analysis of larger patient series will allow the identification of novel regions of interest harboring genes that might be involved in the pathogenesis of AML.
Gene Expression Differences between Low and High Stage Diffuse Large B Cell Lymphoma (DLBCL).
Authors: Roberts Robin A. , Rimsza Lisa M. , Staudt Louis , Rosenwald Andreas , Chan Wing-Chung , Dave Sandeep , Gascoyne Randy D. , Connors Joseph M. , Smeland Erlend B. , Muller-Hermelink H.K. , Campo Elias , Jaffe Elaine S. , Wilson Wyndham , Tan Bruce K. , Fisher Richard I. , Grogan Thomas M. , Miller Thomas P.
Published: April 2020

Low stage DLBCL patients have better prognosis than those with advanced disease. We searched for differences in gene expression between disease stages to find correlations with tumor stage as possible therapeutic targets. We used 2 microarray datasets from the Leukemia/Lymphoma Molecular Profiling Project containing nodal and extra-nodal de novo DLBCL samples on Lymphochip (LC), spotted arrays of ~7400 and Affymetrix arrays (Affy) of ~45,000 elements. The Affy dataset was a subset of the LC samples. LC elements were median-centered, log2 transformed; Affy elements were array-centered to a 500 average. We compared average gene expression in stage I vs. III/IV, looking for elements with >2-fold differences between low and high stage, with p<.05, excluding Affy elements with an average <100. 35 stage I and 131 stage III/IV samples were analyzed with LC and 34 stage I and 118 stage III/IV samples with Affy. Differentially expressed genes were searched by NCBI Entrez and Stratagene PathwayArchitect software to find common pathways of regulation. From the LC data, 6 genes were identified as differentially expressed, 5 were higher in stage I, 1 was lower in stage I. For the Affy data, 29 genes were differentially expressed, 11 were higher in stage I, 18 were lower in stage I (Table 1). Differences were rarely >3-fold. A subset of these genes, CTGF (connective tissue growth factor), FN1 (fibronectin), INHBA (activin), POSTN (periostin), THBS1 (thrombospondin), and BCL2, are coregulated and/or coregulatory. Many are expressed in the microenvironment and associated with fibrosis, wound healing and Th2 immune response. The presence of these genes at higher levels and their known associations implied a pathway of TGF-β signaling present mainly in the low stage DLBCL. Although TGF-β levels were not different between stages, differences in localization/activation of TGF-β could account for observed differences. In conclusion, there are a small number of stage-specific gene expression differences in DLBCL, and many of these relate to TGF-β signaling in the microenvironment. CTGF and FN1 were previously identified as key prognostic molecules in DLBCL (Rosenwald et al, NEJM 2002). These differences may relate to different prognoses between DLBCL stages and constitute therapeutic targets.

Genes Differentially Expressed by Stage higher in stage I higher in stage III/IV LC Affy LC Affy CTGF CTGF IGHM IBRDC2 (2 elements) SERPINA1 MYBPC1 HTR3A POSTN NCR3 BCL2 FN1 INHBA (2 elements) CRYM THBS1 EMP1 SIX1 KLHL14 MGC23911 PP1665 GPM6A (2 elements) FLJ33069 TUBB IgM rheum. factor RF-TT9 CD1C transcribed locus LOC150568 LOC346887 LOC283454 MGC17624 IMAGE:5311619 transcribed locus (2x)
Structural Profiles of p53 Gene Mutations Predict Clinical Outcome in Diffuse Large B-Cell Lymphoma: An International Collaborative Study.
Authors: Young Ken H. , Leory Karen , Møller Michael B. , Sánchez-Beato Margarita , Colleoni Gisele W.B. , Kerbauy Fabio R. , Koduru Prasad P.K. , Haioun Corinne , Gaulard Philippe , Piris Miguel A. , Campo Elias , Delabie Jan , Gascoyne Randy D. , Rosenwald Andreas , Ott German , Huang James , Braziel Rita M. , Jaffe Elaine S. , Staudt Louis M. , Wilson Wyndham H. , Kanehira Kazunori , Rehrauer William M. , Eickhoff Jens C. , Kahl Brad S. , Malter James S. , Chan Wing-Chung , Wisenburger Dennis D. , Greiner Timothy C.
Published: April 2020

Mutations of the p53 tumor suppressor gene have been associated with a poor clinical outcome in some series of diffuse large B-cell lymphoma (DLBCL). However, conflicting results have been reported in other studies. The purpose of this study was to analyze the p53 mutations in DLBCL from twelve centers, and to correlate the structural profiles of the mutations with clinical outcome. The p53 mutations were identified in 102 of 477 cases of DLBCL for a frequency of 21.4%. These included 92 missense mutations, 5 nonsense mutations, 4 deletions, and 1 insertion. The presence of any p53 mutation correlated with poor overall survival (OS; P=0.002). Sixty-two of 102 cases (61%) had mutations in the DNA binding domains of the p53 gene, and the mutations in the DNA-binding domains were found to be the most important predictor of poor OS (P<0.001). In contrast, mutations in the non-DNA binding domains did not correlate with OS (P=0.158). The 5-year survival rate was 24% in patients with any p53 mutation (median survival=1.33 yr) and 19% of patients with the DNA-binding domain mutations (median survival=1.0 yr) compared to 41% for those with wild type p53 (wt-p53, median survival=4.5 yr). The complete remission rate was 57% in patients with any p53 mutation and 54% in patients with the DNA-binding domain mutations compared to 69% for those with wt-p53. Of the mutations in the DNA-binding domains, patients with mutations in the loop-sheet-helix motifs (Loop L1-S10-H2, 20% of all mutations) and DNA minor binding groove motif (Loop L3, 22% of all mutations) had significantly decreased OS (P=0.002). In contrast, OS was not significantly decreased for patients with mutations in Loop L2 (18% of all mutations). Multivariate analysis confirmed that the International Prognostic Index, age, tumor size, serum lactate dehydrogenase, and mutations in the DNA binding motifs were independent predictors of OS. The p53 mutation profile was also found to stratify germinal center B-cell-like DLBCL, but not activated B-cell-like DLBCL, into molecularly distinct subsets with different clinical outcomes. This study demonstrates the importance of the mutational profile in the DNA binding domains of the p53 gene for predicting clinical outcome and refining current prognostic models including gene expression profiling in patients with DLBCL.
Prognostic Indices in Older DLBCL Patients Receiving R-CHOP: An Analysis of the U.S. Intergroup Study (E4494, CALGB 9793).
Authors: Advani R.H. , Chen H. , Habermann T.M. , Morrison V.A. , Weller E. , Fisher R.I. , Peterson B.A. , Gascoyne R.D. , Horning S.J.
Published: April 2020

Background: We reported that addition of rituximab (R) to chemotherapy significantly improves outcome in DLBCL patients (pt) >60 years (JCO24:3121–27, 2006). Although the IPI is a robust clinical prognostic tool in DLBCL, Sehn et al (ASH 2005: abstract 492) reported that a revised (R) IPI more accurately predicted outcome in pt treated with rituximab-chemotherapy.

Methods: We evaluated outcomes of the Intergroup study with respect to the standard IPI, R-IPI, age-adjusted (aa) IPI for evaluable pt treated with R-CHOP alone or with maintenance rituximab. We further assessed a modified IPI (mIPI) using age ≥ 70 y as a cutoff rather than age 60 y.

Results: The 267 pt in this analysis were followed for a median of 4 y. Pt characteristics were: age > 70 (48%) (median=69), male 52%, stage III/IV 75%, >1 EN site 30%, LDH elevated 60%, PS ≥2 15%. On univariate analysis all of these characteristics were significant for 3 y failure-free survival (FFS) and overall survival (OS). The IPI provided additional discrimination of risk compared to the R-IPI with significant differences in FFS and OS for 3 vs 4–5 factors. The aa-IPI defined relatively few pt as low or high risk. The impact of age was studied using a cut-off of 70 years in a modified IPI, yielding 4 risk groups as shown below.

Conclusions: For pt ≥ 60 treated with rituximab-chemotherapy the distinction between 3 vs 4,5 factors in the IPI was significant.The IPI also provided additional discrimination of risk compared to the R-IPI. In this older group of pt, use of an age cutoff ≥70 y placed more patients in the low risk category. It is of interest to apply the mIPI in other datasets with DLBCL pt >60 y.

Group # Factors # Pt % 3y FFS* % 3y OS* *All risk groups significantly different; logrank p < 0.001 **95 % CI: FFS (0.46,0.66), OS (0.58,0.78) ***95 % CI: FFS (0.21,0.45), OS (0.31,0.55) L: Low, LI: Low Intermediate, HI: High Intermediate, H; High IPI L 0–1 12 78 83 LI 2 28 70 80 HI 3 33 56** 68** H 4–5 37 33*** 43*** R-IPI Very Good 0 0 - - Good 1–2 40 72 81 Poor 3–5 60 46 57 aa-IPI L 0 12 78 83 LI 1 35 68 78 HI 2 44 47 59 H 3 9 31 35 mIPI (age ≥ 70) L 0–1 27 77 86 LI 2 28 62 74 HI 3 29 47 58 H 4–5 16 28 36
A Large Scale Evaluation of Genetic Variation in Immune and Inflammation Genes and Risk of Non-Hodgkin Lymphoma.
Authors: Cerhan James R. , Ansell Stephen M. , Fredericksen Zachary S. , Kay Neil E. , Liebow Mark , Call Timothy G. , Dogan Ahmet , Cunningham Julie M. , Liu Wen , Macon William R. , Witzig Thomas E. , Habermann Thomas M. , Slager Susan L.
Published: April 2020

Background: Non-Hodgkin lymphoma (NHL) is a malignancy of lymphocytes, and may develop in the setting of inflammation and immune dysfunction. Small scale evaluations have suggested that common genetic variation in candidate genes related to immune function may predispose to the development of NHL. Here we report a comprehensive analysis of variants within genes associated with immunity and inflammation and risk of NHL.

Methods: We used ParAllele’s Immune and Inflammation panel of 9,412 single nucleotide polymorphisms (SNPs) from 1,450 immune/inflammation genes as a discovery tool in a clinic-based study of 458 NHL cases and 484 frequency matched controls seen at the Mayo Clinic from 2002–2005. The panel included 537 coding non-synonymous SNPs (nsSNPs), with the remainder of the SNPs selected as tags from HapMap (tagSNPs) to provide coverage of the candidate genes (r2 = 0.8 and minor allele frequency >0.05). To assess the association of individual SNPs with risk of NHL, we calculated Odds Ratios (ORs) and 95% confidence intervals, adjusted for age and gender. The most prevalent homozygous genotype was used as the reference group, and each polymorphism was modeled individually as having a log-additive effect in the regression model. Associations between haplotypes from each gene and the risk of NHL were calculated using a score test implemented in HAPLO.SCORE. We also modeled the main effects for all independent (r2 <0.25) SNPs from a gene in a multivariate logistic regression model.

Results: The mean age at diagnosis was 60 years for cases and 58% were male; in controls the mean age at enrollment was 61.6 years and 55% were male. In the gene analyses, the strongest findings (p≤0.001 from multiple SNP logistic regression or haplotype analysis) were for cAMP responsive element binding protein 1 (CREB1; p=0.0004), fibrinogen alpha chain (FGA; p=0.0006), TNF receptor-associated factor 1 (TRAF1; p=0.001), dual specificity phosphatase 2 (DUSP2; p=0.001), and fibrinogen gamma chain (FGG; p=0.001). In the nsSNP analyses, the strongest findings (p≤0.01) were for integrin β3 (ITGB3) L59P (OR=0.64, 0.50–0.83), Beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) H328R (OR=0.72, 0.57–0.91), transporter 2, ATP-binding cassette (TAP2) T665A (OR=1.32, 1.07–1.63), HLA-B associated transcript 2 (BAT2) V1895L (OR=0.60, 0.42–0.85), and complement component 7 (C7) T587P (OR=1.39, 1.07–1.80).

Conclusions. Our results suggest that genetic variability in genes associated with antigen processing (CREB1 and TAP2), lymphocyte trafficking (B3GNT3), immune activation (TRAF1, BAT2), complement and coagulation pathways (FGA, FGG, ITGB3, C7), and MAPK signaling (DUSP2) may be important in the etiology of NHL, and should be pursued in replication studies.
The Focal Adhesion Kinase (FAK) Inhibitor TAE226 Exhibits In Vitro and In Vivo Activity Against Multiple Myeloma.
Authors: Sharkey Janelle , Yeh Sung Lin , Spencer Andrew
Published: April 2020

Multiple Myeloma (MM) is an incurable malignancy of terminally differentiated B-cells characterized by both de novo and acquired resistance to presently available cytotoxic therapeutic agents. TAE226 (Novartis) is a potent and specific inhibitor of focal adhesion kinase (FAK) phosphorylation that also has the capacity to inhibit IGF1-R at sub-micromolar concentrations. FAK inhibition has been shown to abrogate intra-cellular phosphorylation of both ERK and AKT and to have anti-tumor effects in murine solid-tumor xenografts. Based on these data we have undertaken a pre-clinical evaluation of TAE226 as a potential therapeutic for MM. Eight genetically heterogeneous human myeloma cell lines (HMCL) (KMS11, KMS12, JIM1, LP-1, U266, RPMI8226, NCI H929, OPM2) were evaluated by MTS assay following exposure to TAE226 at various concentrations for 24 to 72 hours. All HMCL displayed reductions in viability (45% to 100% at 72 hours with 10μM) that were dose and time-dependent, with the range of effective TAE226 concentration being 1 – 10μM depending on cell type. Under standard culture conditions 5μM TAE226 was markedly cytostatic with complete inhibition of cellular proliferation observed over a 72 hour time period and with cell viability at 72 hours based on tryphan blue exclusion in the range of 40 – 90%. Four HMCL with modest (KMS11/U266) or high (LP-&, OPM2) TAE226 sensitivity were further using 5μM TAE226 for 48 hours. AnnexinV expression confirmed apoptosis induction while PhosFlow measurement of p44/42 MAPK confirmed abrogation of intra-cellular ERK phosphorylation - relative reduction following TAE226 compared to untreated controls of 11% KMS-11, 13% U266, 35% OPM2 and 64% LP-1. The induction of synergistic killing of NCI H929 was observed by combining TAE226 (1μM) with sub-lethal concentrations of either bortezomib (5nM) (synergism quotient (SQ) = 1.5) or adriamycin (10μg/ml) (SQ = 1.4) with the scheduling of TAE226 prior to drug partner superior to the reverse order (SQ both = 1.2) of administration. Primary MM cells from patients (n = 7) with advanced relapsed disease were treated after appropriate informed consent. Cell killing as determined by propidium iodide expression varied widely but with a clear dose-dependency – 5μM median 11%, range 0% – 47%; 10μM median 14%, range 1% – 61%; and, 20μM median 31%, range 9% – 67%. Finally, we tested TAE226 in the 5T33 murine model of systemic myelomatosis. C57BL/KaLwRiJ hosts were treated with TAE226 30mg/kg (n = 9) or vehicle (n = 9) for 14 days from day +7. Median time to hind limb paralysis post-treatment was prolonged in the TAE226 group compared to vehicle treated animals, 16 days vs 9 days, respectively (p = 0.06, Log Rank). We conclude based on these encouraging preliminary data that TAE226 warrants further evaluation as a potential therapeutic for MM.
An International Multi-Center Microarray Study for the Molecular Classification of Leukemia Identifies Novel Sub-Groupings in MDS Overlapping with AML.
Authors: Mills Ken I. , Haferlach Torsten , Hernandez Jesus M. , Hofmann Wolf-Karsten , Kohlmann Alexander , Williams Mickey , Wieczorek Lothar
Published: April 2020

Microarrays can identify robust gene expression signatures associated with distinct sub-classes of paediatric and adult leukemias. Recently, the MILE (Microarray Innovations in LEukemia) study has analysed 1901 expression profiles from retrospective samples in 11 centres (ELN: 7, USA: 3, Singapore: 1). MILE has compared the microarray classification accuracy of 16 acute and chronic leukaemia subclasses, MDS, and non-leukaemia as control group, to routine diagnostic workup. The achieved cross-validation accuracy was very high for the leukaemia subclasses: ~96%.

Included in the study were 175 samples diagnosed as MDS, however, only 49.1% of these samples were correctly called as MDS from their underlying gene expression profiles. The remainder were approximately equally split between a call of “non-leukaemia” (24%) and “AML” (24.6%). A further sub-division of MDS samples called “AML”: 81% called as “AML with normal or other cytogenetics”; and the remainder as “AML with complex cytogenetics”.

MDS is a heterogeneous group of disorders with a wide range in blast cell count, cytogenetics and number of cytopenias. Our analysis showed that neither study centre nor age were a factor in differentiating between “MDS”, “AML” or “non-leukaemia”. However, WHO classification was highly correlated with the microarray classification result; specifically RAEB(I or II) was associated with “AML” call (p < 0.0001). RA/RARS was highly correlated with “MDS” or “non-leukaemia” calls. Furthermore, IPSS was significantly correlated with call (p>0.0001): 65% of patients with an IPSS score of Int-2 or above were classified as “AML”. Examination of the individual components of the IPSS showed that two patients classified as “AML” had a blast count of >20%, under the WHO definition these would be defined as AML and were excluded. Individually, the blast, karyotype and cytopenia contributions were highly significant (p<0.0001, <0.013 and <0.0001 respectively) when comparing “AML”, “MDS” and “non-leukemia” calls. All the “non-leukemia” patients had <10% blasts, with 85% (34/40) having a cytogenetic score of 0 (Normal or Good (1 of: -Y, del(5q), del(20q))) and 82.5% having only 0 or 1 cytopenias. In contrast, 45% of the “AML” samples had between 11 & 20% blasts, 32% with intermediate (0.5) or poor/complex (1) cytogenetic score and 79% had 2 or 3 cytopenias. Furthermore, survival data was available for 122 of the diagnosed MDS patients and showed that MDS patients called “AML” had a trend towards shorter survival (2P=0.2) than those called “MDS” or “non-leukaemia”. These analyses, in combination with gene expression signatures, may contribute to a redefinition of MDS classifications.
The EGFR-Inhibitor Erlotinib Induces Apoptosis in Neoplastic Cells of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), an Effect Determined by the Expression Level of Nucleophosmin (NPM).
Authors: Boehrer Simone , Ades Lionel , Fabre Claire , Fenaux Pierre , Kroemer Guido
Published: April 2020

Background: the epidermal-growth-factor-receptor (EGFR)-inhibitor erlotinib was rationally designed to antagonize the deregulated EGFR-activity in solid tumors. Abundant studies in these entities not only demonstrated clinical efficacy, but also a favorable toxicity profile. In particular the absence of hematopoietic toxicity prompted us to investigate the therapeutic potential of erlotinib in MDS and AML cells.

Methods: We incubated ex vivo cells from patients with MDS (n=4, 2 lower risk and 2 higher risk) and AML (n=6, de novo: 3; post MDS: 3), as well as a broad spectrum of myeloid cell lines (P39, KG-1, HL-60, MV4-11, MOLM-13) with increasing dosages of erlotinib (1μM to 10μM). As controls (n=4) we used non-malignant CD34 + bone marrow cells. Before incubation, all ex vivo cells underwent CD34 + selection. Serial FACS-analyses of parameters determining apoptosis (DIOC/PI and AnnexinV/PI) were carried out over a maximum of 6 days.

Results: We found that erlotinib was able to induce a considerable degree of apoptosis in MDS and AML cells. Although there was a high interindividual difference in sensitivity towards erlotinib, “responders” treated with 10μM erlotinib showed an increase of apoptotic cells between 20–30% after 72h, which reached a maximum of 60% on day 6. This apoptosis-inducing effect was achieved in a dose-dependent manner and not restricted to a specific entity. Noteworthy, erlotinib exhibited no toxicity towards non-neoplastic progenitor cells. Evaluating the molecular mechanisms determining sensitivity we showed that the apoptosis-inducing effect of erlotinib critically depended on the expression level of NPM. Thus erlotinib-resistant myeloid cell lines (i.e. P39) exhibited a higher epression of NPM than sensitive cell lines (i.e. KG-1). In addition, down-regulation of NPM by small-interfering RNA not only increased the apoptosis-inducing effect of erlotinib in sensitive cells, but moreover established sensitivity in otherwise erlotinib-resistant cells. Accordingly, siRNA-induced down-regulation of NPM in P39 cells elevated the percentage of apoptotic cells upon treatment with 10μM erlotinib by about 30% as compared to mock-transfected controls. Conclusion, we showed an off-target effect of erlotinib, as evidenced by its ability to induce apoptosis in EGFR-negative cells. Of particular interest is the observation that erlotinib induced apoptosis exclusively in neoplastic myeloid cells while sparing non-malignant progenitors. To the best of our knowledge, this is the first report providing evidence for the therapeutic potential of erlotinib in MDS and AML.
CD28 Costimulation Induces Transcription of SKP2 and CKS1, the Substrate Recognition Components of the Skp1-Cullin-F-box Ubiquitin Ligase, SCFSkp2.
Authors: Appleman Leonard J. , Chernova Irene , Li Lequn , Boussiotis Vassiliki
Published: April 2020

Cellular immune responses require expansion of antigen-specific T cell clones from the pool of resting T lymphocytes that perform immune surveillance. Regulation of this proliferative potential is critical for defense against pathogens and avoidance of autoimmunity. Costimulation through the T cell receptor and accessory molecule, CD28, triggers replication of T lymphocytes in response to antigen that is mediated via both IL-2-dependent and IL-2-independent mechanisms. This event is associated with decreased abundance of the cyclin-dependent kinase inhibitor p27kip1 and increased cyclin D/cdk4 and cyclin E/cdk2 enzymatic activity. We have previously reported that in contrast to p27kip1 protein, the abundance of P27KIP mRNA transcript is unchanged in response to TCR/CD28 T cell activation. Ubiquitin-targeted degradation by the proteasome regulates the abundance of p27kip1 protein and ubiquitination of p27kip1 occurs in primary human T cells activated through the TCR and CD28. Ubiqutination of p27kip1 is mediated by a Skp1-Cullin-F-box (SCF) family ubiquitin ligase, SCFskp2. The SCFskp2 holoenzyme is comprised of the core components Roc1, Cul1, Skp1, and the substrate recognititon components, an F-box (cyclin F homology) protein, Skp2, that in cooperation with a smaller subunit, Cks1, mediates recognition of p27kip1 substrate. Targeted deletion of the murine Skp2 or Cks1 genes results in accumulation of p27kip1 in multiple cell types. T cell proliferation in response to TCR/CD3-plus-CD28 costimulation is reduced in Skp2−/− mice in comparison to wild-type controls, confirming the role of Skp2 in CD28 costimulation mediated degradation of p27kip1 and T cell proliferation. Here, we examined the mechanisms that regulate Skp2 and Cks1 abundance in primary T lymphocytes in response to TCR/CD3-plus-CD28 costimulation. Using primary human T lymphocytes we observed that the SCFskp2 core components Roc1, Cul1 and Skp1 are constitutively expressed and their levels remain unchanged upon TCR/CD3-plus-CD28 costimulation. In contrast, TCR/CD3-plus-CD28 costimulation lead to striking induction of SKP2 and CKS1 mRNA, and this event is dependent on PI3K/PKB and MEK/ERK signaling pathways. We determined that the SKP2 promoter lies directly upstream of the translation start site and contains binding sites for SP1, Elk-1 and E2F transcription factors. Mutagenesis of SP1 and Elk-1 sites abrogated TCR/CD3-plus-CD28-mediated SKP2 promoter-driven reporter activity, whereas mutagenesis of E2F site enhanced reporter activity, suggesting that SKP2 promoter may act as a node of integration for mitogenic and anti-mitogenic signals. Thus, in primary T lymphocytes CD28 costimulation can directly regulate cell cycle progression by inducing transcription of the substrate recognition components of SCFskp2 ubiquitin ligase that targets p27kip1 for degradation.
Outcomes during and after Pregnancy in Patients with Primary Hypercoagulable States.
Authors: Khalid Aysha , Cohen Alice J.
Published: April 2020

Pregnant women with primary hypercoagulable states have an increased risk of recurrent fetal loss, fetal growth retardation, preclampsia, and placental abruption, as well as thromboembolism in both the antepartum and postpartum periods. Conditions that have been associated with adverse pregnancy outcomes include inherited gene mutation disorders such as Factor V Leiden G1691A (FVL); prothrombin gene mutation G20210A (PGM); hyperhomocysteinemia with C677T mutation (MTHFR); deficiencies of protein S (PS), protein C (PC), antithrombin III (ATIII); and anticardiolipin antibodies/lupus anticoagulants (LA). Screening by obstetricians for these disorders has led to an increase in diagnosis yet there are no established guidelines and therapeutic interventions are variable. A retrospective chart review was conducted on 59 women and 197 pregnancies (1–12 per pt) in whom primary hypercoagulable state was diagnosed: FVL (n=7), PGM (n=11), MTHFR (n=3), ATIII (n=2), PC (n=1), PS (n=8), LA (n=10), and those with more than one thrombophilic risk (TR) (n=12) or lupus (n=2). Of the 197 pregnancies, only 106 (54%) were carried to term in 50 pts (85%). Of the remaining 91 pregnancies, there were 16 terminations, 5 ectopic pregnancies and 4 preterm live births. 66 fetal losses occurred: 45 in the first trimester (26 with single TR/19 with >1 TR), 19 in the 2nd–3rd trimester (14 with single TR/5 with > 1 TR) and 2 unknown. See Table for risk of fetal loss by class of hypercoagulable state. Venous thromboembolism occurred in 8/106 (8%) of term pregnancies, including 5 postpartum. Of women with previous fetal losses, management with anticoagulation (A/C) subsequently was utilized in 20 pregnancies: 9 low molecular weight heparin (LMWH), 6 LMWH+aspirin (ASA), 3 heparin (H), 1 ASA, and 1 H+ASA. 18/20(90%) of these pregnancies resulted in live term births; 1 loss due to intracranial hemorrhage at 27 weeks in a patient on L+ASA. Compared to 146 pregnancies untreated with A/C, successful completion of pregnancy was significantly greater on A/C, 90% vs. 58% (p=0.006).

Conclusions: Fetal losses secondary to TR occurred in both the first and late trimesters in high proportion of pregnancies in women with hypercoagulable states. Outcomes may be improved with the use of A/C therapy.

Table - Risk of Fetal Loss by Class of Hypercoagulable State LA PGM MTHFR PS FVL ATIII FVL/other Trimester1 9/38 (24%) 7/44 (16%) 3/9 (33%) 3/27 (11%) 1/27 (4%) 2/5 (40%) 17/42 (40%) Trimester 2–3 5/38 (13%) 1/36 (3%) 0% 5/16 (31%) 2/27 (7%) 1/5 (20%) 4/44 (9%)
Thrombomodulin-Dependent Protein C Activator Treatment Improves the Short-Term Outcome of Experimental Ischemic Stroke in Mice.
Authors: Gruber Andras , Hurn Patricia D. , Hurst Sawan , Bush Leslie , Marzec Ulla M. , Tucker Erik , Hanson Stephen R. , Di Cera Enrico
Published: April 2020

High dose human or mouse activated protein C (APC) treatment is neuroprotective in the transient intraluminal middle cerebral artery occlusion (MCAO) and reperfusion model of ischemic stroke, in mice. However, the therapeutic potential of systemic APC treatment in stroke might be limited because it can disable hemostasis. Low dose thrombomodulin-dependent protein C activator (PCA) enzyme treatment can generate antithrombotic APC on the intraluminal surface without profound systemic anticoagulation and antihemostatic effects in primates. We thus explored whether PCA treatment could produce short term outcome benefits in comparison to fibrinolysis with tPA in the mouse stroke model. Stroke was induced by MCAO between 0 and 60 min using an intraluminal filament that causes progressive and occlusive secondary thrombosis in the MCA region. Cortical perfusion as a measure of thrombotic occlusion was monitored from start until 75 min, i.e., following removal of the intraluminal filament. Cerebral infarct size (% brain volume, TTC stain), edema (% brain volume; calculated), and neurological damage scores (scale 0 – 5) were assessed in survivors at sacrifice next day. All animals (N=12 to 16 in each treatment group) were evaluated for macroscopic hemorrhage (intracerebral or other sites) on autopsy after death. Treatments with vehicle (saline bolus), recombinant PCA thrombin analog W215A/E217A (0.025 mg/kg IV bolus) or plasminogen activator (2.5 mg/kg Bolus, [tPA-B], or 10 mg/kg Infusion for 45 min, [tPA-I]) were started 15 min after initiation of MCAO. One day mortality rates were similar: 29% in the PCA, 38% in the tPA-B, and 23% in the tPA-I treatment groups vs. 23% in vehicle controls (P>0.05 each). Cerebral infarct size was reduced by 62%, 43%, and 73% in the PCA bolus, tPA-B, and tPA-I treated groups, respectively (p<0.05 for each). Macroscopic hemorrhage (intracranial and/or other sites) was found in 15% (2/13), 14% (2/14), 44% (7/16), and 31% (4/13) in the vehicle, PCA, tPA-B, and tPA-I-treated groups, respectively. One day neurological scores averaged 1.5 in PCA-treated (p<0.05), 3.2 in tPA-B (NS), and 2.2 in tPA-I treated (p<0.05) versus 3.9 in saline vehicle-treated animals. All enzyme treatments significantly reduced secondary (thrombotic) vascular occlusions, as evidenced by 62% (PCA), 66% (tPA-B), and 72% (tPA-I) improvement of early cortical reperfusion vs. vehicle-treated controls (p<0.05 each). The means and SEM are illustrated in the figure below. The apparent efficacy and safety of intermediate dose PCA in this model suggests that PCA treatment might have therapeutic utility in acute ischemic stroke, e.g. when reperfusion of the MCA is achieved by tPA treatment or other modalities.

Figure Figure
Erythrocyte Coupled tPA Improves Outcomes of Percussion Brain Trauma in Rats.
Authors: Ganguly Kumkum , Stein Sherman C. , Groff Robert F. , Zhang Jun , Smith Douglas H. , Cines Douglas B. , Muzykantov Vladimir R.
Published: April 2020

Coupling tissue plasminogen activator (tPA) to carrier red blood cells (RBC): i) restricts tPA’s permeation into tissues and pre-existing hemostatic clots, minimizing hemorrhage; ii) protects it from plasma inhibitors; and iii) prolongs its circulation, permitting incorporation into nascent clots and their lysis from within. These features support the thromboprophylactic utility of RBC coupled tPA (RBC/tPA). In this study we explored the utility of RBC/tPA in traumatic brain injury (TBI), a disorder in which both cerebral thrombosis leading to cerebral ischemia and intracerebral hemorrhage (ICH) have been implicated. Eleven male, Sprague-Dawley rats (340–400g) were subjected to a moderate (avg. peak pressure 2.6atm) lateral fluid percussion injury to the left hemisphere. Rats were given a single intravenous dose of RBC/tPA (0.05mg/Kg, n=5) or vehicle (n=4), 15 min post-injury. Animals were sacrificed 48h later for histopathology and staining for fibrin. The lesions in control animals occupied 8.3 ± 2.8% (mean±SD) of the hemispheric volume. Animals treated with RBC/tPA had a significant decrease in mean lesion volume (1.4±0.7%; p<0.001). Thrombotic burden was reduced from a mean of 10 clots in vehicle-treated to 1 per RBC/tPA-treated rats p<0.001). These data indicate that RBC/tPA attenuates post-traumatic thrombosis without aggravating hemorrhage induced by TBI. These observations support the safety of RBC/tPA in thromboprophylaxis even in the context of brain trauma, due to intravascular containment of RBC/tPA, among other factors. Durable lysis of post-traumatic thrombi by long-circulating RBC/tPA may alleviate secondary brain ischemia and resultant ICH. Correlation between reduction of thrombotic burden and lesion size implicate thrombosis in the pathophysiology of parenchymal brain damage after head trauma. Supported by PENN Research Foundation, HL66442, HL60169 and in part by NS38104.
The Effect of BIBT 986, a Novel Small Molecule Dual Inhibitor of Thrombin and Factor Xa, on Coagulation in a Model of Endotoxin Induced, Tissue Factor Triggered Coagulation Activation in Humans.
Authors: Jilma Bernd , Leitner Judith M. , Cardona Francesco , Mayr Florian B. , Firbas Christa , Rathgen Karin , Staehle Hildegard , Schuehly Uwe , Graefe-Mody Eva U.
Published: April 2020

Background: BIBT 986 is a novel potent anticoagulant that dually inhibits Factors Xa and IIa. We hypothesized that BIBT 986 would dose-dependently decrease endotoxin-induced, tissue factor triggered coagulation activation. Hence it was the aim of the study to compare with placebo the anticoagulant activity of three dosages of BIBT 986 on parameters of coagulation, platelet activation and inflammation and to examine the safety of BIBT 986 in this setting.

Methods: This study was a prospective, randomized, double-blind, placebo-controlled, parallel-group dose escalation trial in 48 healthy male volunteers. Participants were randomised to receive bolus primed continuous infusions of one of the three doses of BIBT 986 or placebo. All of them received a bolus infusion of 2ng/kg body weight lipopolysaccharide (LPS).

Results: BIBT dose-dependently increased anti-Xa activity, activated partial thromboplastin time (APTT), ecarin clotting time (ECT), thrombin time (TT) and the international normalisation ratio (INR). Importantly, BIBT 986 dose-dependently blocked the LPS-induced coagulation as assessed by the in vivo markers of thrombin generation and action: BIBT 986 doses that prolonged APTT by 25% were already effective. The BIBT dose that prolonged APTT by 100%, completely suppressed the increase in prothrombin fragment (F1+2), thrombin-antithrombin complexes (TAT) and D-dimer. BIBT 986 had no influence on activation markers of inflammation, fibrinolysis, endothelial or platelet activation.

Conclusion: Infusion of BIBT 986 was safe and well tolerated. BIBT 986 specifically and dose-dependently blocked LPS-induced, tissue factor trigger coagulation. When compared to different anticoagulants tested previously in this standardized model, BIBT 986 was more effective in suppressing thrombin generation (F1+2 levels) than standard doses of danaparoid, dalteparin or lepirudin. BIBT 986 represents the first drug of a new class of dual FXa and FIIa inhibitors, and displays high potency.