bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Cancer Testis Antigens as Potential Targets for Immunotherapy Is Retained Following Treatment in Multiple Myeloma.
Authors: van Duin Mark , Sahota Surinder S. , de Knegt Yvonne , Broyl Annemiek , Joseph-Pietras Debora , Mulligan George , Bryant Barb , Sonneveld Pieter
Published: April 2020

Background: Multiple myeloma (MM) remains an incurable malignancy, but new treatment modalities can achieve durable remissions in most cases. Remission is generally regarded as a window of opportunity for active immunotherapy, aimed at eradicating residual disease. For immunotherapy, and vaccination in particular, the expression of cancer testis antigens (CTAs) in MM has emerged as a key target that is essentially tumor-specific. These CTAs are expressed at disease presentation, many confirmed also at the protein level. However, the question has remained whether treatment might critically alter the expression profile of CTAs. To address this, we have examined the expression levels of a large panel of known CTAs using gene expression profiles (GEPs) from patient cohorts in clinical trials at defined stages: at disease presentation in HOVON-65 and post-treatment in the APEX, SUMMIT and CREST trials.

Methods and results: The APEX, SUMMIT and CREST trials have been described by Mulligan et al. (Blood, 109, 3177; 2007) and data consist of GEPs obtained from 264 patients with relapse following prior therapies (median number of therapies, 3; min-max, 1–14). In the HOVON-65 study, GEPs of 204 previously untreated myeloma cases were obtained. For comparison of both studies, original raw data files (CEL-files) were obtained and analyzed using Partek software. After normalization and merging, the expression of 62 CTA genes and gene clusters was evaluated. 63 probe sets were used, of which 51 correspond to 50 unique genes and 12 correspond to clusters of cancer antigen genes. Normalized expression levels for 24/62 genes/gene clusters were well above cut-off, and for remainder (38/62) were generally low with medians below 30. High median expression values, in the top 10% of CTA expression values, were found for SSX2, MAGE-A3 and MAGE-C1. Genes in the bottom 10% include MAGE-B1 and SPACA3. Notably, of 62, the fold change in expression level before and after treatment was at most 1.5 in any of the genes studied, indicating that the expression of the majority of CTAs is unchanged by treatment. For 8 genes/gene clusters, significantly decreased expression was found in relapsed cases compared to untreated MM, including GAGE3 and a gene cluster containing the GAGE1 gene.

Conclusion: These clinical trial data indicate that targeting CTAs with immunotherapy during first remission or after relapse of MM remains a viable option.
Treatment of Relapsed and Refractory Multiple Myeloma in Patients with p53 Deletion.
Authors: Reece Donna E. , Trieu Young , Chang Hong , Xu Wei , Anglin Peter , Chen Christine , Kukreti Vishal , Trudel Suzanne , Mikhael Joseph
Published: April 2020

p53 deletion by fluorescence in situ hybridization (FISH) has been reported in about 10% of newly diagnosed patients (pts) with multiple myeloma (MM) and has been associated with a poor prognosis. Previous data from our centre has demonstrated that the progression-free survival (PFS) and overall survival (OS) of these pts after a single autologous stem cell transplant (ASCT) is only 16.9 and 48.2 months, respectively, compared to 24.4 and 76.6 mos, respectively, in all pts transplanted during the same time period (Mikhael et al, Blood2007; 110: abstract 953). Minimal information is available on the treatment (Tx) and course of these pts following disease progression. Therefore, we performed a retrospective review of all MM pts treated at our institution who were found to have p53 deletion. We identified 31 pts with relapsed/refractory (rel/ref) MM with this cytogenetic abnormality. At diagnosis, median age was 54 years (range, 31–70), hemoglobin 97 g/L (range, 67–149), creatinine 96 μmol/L (range, 28–1751), beta 2-microglobulin 314 nmol/L (range, 225–437), CRP 3 mg/L (range, 2–7) and LDH 205 U/L (range, 82–478); 58% were male. Immunoglobulin subtypes included: IgG (14 pts), IgA (8 pts), IgD (2 pts) and light chain only (7 pts). Concomitant cytogenetic abnormalities included 13q deletion in 68% and t(4;14) in 67% of pts evaluable. Thirty-nine percent developed plasma cell leukemia (PCL) at some point in the disease course, which was associated with a poor OS (p=0.005). All but 5 had undergone prior ASCT with a median time from diagnosis to ASCT of 8 (95% CI, 4–145) mos. Txs given for rel/ref MM consisted of the following regimens: thalidomide-based in 15, bortezomib-based in 12, lenalidomide-based in 11, alkylating agents in 9 and steroids only in 5 pts, and other regimens (D-PACE or investigational drugs) in 7 pts. The median follow-up from diagnosis in these pts is 45 (95% CI, 4–145) mos. The overall response rate (≥PR) (ORR), median PFS and median OS from the start of each regimen is shown below.

Agent Median duration of Tx (mos) ORR(%) Median PFS (mos) Median OS (mos) Thalidomide 6.2 20% 5.0 11.9 Bortezomib 5.2 50% 5.6 36.1 Lenalidomide 10.6 60% 4.8 36.1 Alkylating agents 6.0 11% 5.1 30.0 Steroids 1.9 20% 1.9 33.4 Other 2.2 43% 6.3 33.4

We conclude: 1) Median PFS in pts with the p53 deletion is less than 6 months, with an OS of 2.5–3 yrs; 2) novel agents other than thalidomide produce the highest ORR in rel/ref MM with p53 deletion; 3) the short OS in the thalidomide group is likely related to the unavailability of other novel agents for subsequent relapses, as the majority of the pts were treated before 2004; 4) better strategies/drugs are required for these pts.
Development of V617F JAK2 Associated Myeloproliferative Neoplasms Is a Non-Random Event That Is Strongly Dependent on JAK2 Haplotype
Authors: Cross Nicholas C.P. , Jones Amy V. , Silver Richard T. , Oscier David , Metzgeroth Georgia , Wang Y. Lynn , Collins Andrew , Reiter Andreas , Grand Francis , Chase Andrew
Published: April 2020

Epidemiological data and family studies have indicated that inherited factors may predispose to the development of myeloproliferative neoplasms (MPN). It has also been suggested that single nucleotide polymorphisms (SNPs) within JAK2 are associated with specific MPN subtypes. To explore the role of inherited factors in more detail, we initially performed quantitative analysis of a series of JAK2 SNPs in homozygous PV cases (%V617F >50%; n=73). Most mutant haplotypes could be read directly from the distorted allele ratios brought about by expansion of the homozygous clone. In many cases with 50–90% V617F, the residual wild type haplotype could also be read. Strikingly, of the 144 V617F alleles that could be determined, 111 (77%) had an identical core haplotype (subsequently designated 46/1) whereas only 9/76 (12%) residual wild type alleles were 46/1 (P = 1.9e-21, Fisher’s exact test). To explore this observation in more detail we first determined the haplotype structure of JAK2 using 14 SNPs genotyped by the Wellcome Trust Case Control Consortium (WTCCC) in 1500 UK blood donors. Nine haplotypes were inferred using the program PHASE that accounted for 94% of alleles, with a frequency of 0.24 for haplotype 46/1. Haplotype inference and tag SNP analysis revealed that 46/1 was also more frequent in heterozygous V617F positive MPD cases (135/354 alleles) compared to 188 locally sourced healthy controls (92/376 alleles; P = 0.0001) as well as the WTCCC cohort (P = 3.3e-8). Haplotype 46/1 was more frequent in all V617F positive disease entities compared to controls: PV (n=203; P=1.2e-16), ET (n=81; P=1.2e-9) and MF (n=41; P=8.0e-5) however there was no difference in the frequency of 46/1 between controls and V617F negative MPD / idiopathic erythrocytosis (n=123). To determine if heterozygous V617F also preferentially arose on a 46/1 allele as seen for homozygous cases, we developed an allele specific PCR between V617F and a SNP that tags this haplotype. In an analysis of 67 informative heterozygous V617F cases, 50 V617F alleles were 46/1 compared to only 17 residual wild type alleles (P=9.4e-9). We conclude that the 46/1 JAK2 haplotype is a strong predisposition factor for development of V617F associated MPDs (RR=2.6; 95% CI 2.3–2.9). The reason for this predisposition is currently unknown but it is likely that 46/1 is in linkage disequilibrium with an unknown constitutional functional variant that interacts with V617F JAK2.
Imatinib at Weekly Dosage May Induce and Maintain Haematologic and Molecular Remission in Chronic Eosinophilic Leukemia Harbouring FIP1L1-PDGFRA Fusion Gene- An Extended Follow-up Study.
Authors: Helbig Grzegorz , Calbecka Malgorzata , Gajkowska Justyna , Moskwa Andrzej , Urbanowicz Alina , Swiderska Alina , Najda Jacek , Krzemien Slawomira
Published: April 2020

Background. A small proportion of patients with hypereosinophilic syndrome (HES) demonstrate the presence of an interstitial deletion in chromosome 4 leading to the creation of the imatinib-responsive fusion gene- FIP1L1-PDGFRA (F/P). Recently, we showed that a single weekly dose of imatinib is sufficient to induce and maintain remission of chronic eosinophilic leukemia (CEL) with detectable F/P transcript. Here, we present data from 12 patients CEL and HES, 11 of which were F/P positive, who achieved a rapid complete haematologic remission (CHR) with daily imatinib treatment and remission was then maintained with a weekly imatinib schedule.

Design and methods. Twelve patients, 11 out of 12 with detectable F/P were treated with imatinib at the initial doses varies between 100–400mg. There were 10 male and 2 female with a median age of 57 years (19–80). Median time to start imatinib was 23 months (1–204 months). The imatinib dose was de-escalated while patients remained in haematologic remission. As a response maintenance, once weekly imatinib was established in all cases.

Results. All studied patients achieved a complete haematologic remission (CHR) and 100% of cases with detectable F/P fusion gene before imatinib, demonstrated a molecular remission determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The breakpoints occured within exon 12 of PDGFRA whereas breakpoints dispersed across the FIP1L1 locus occuring between exons 10 and 13. Median time to achieve CHR was 13 days (4–90), and median time to molecular remission was 9 months (4–24). As a remission maintenance, imatinib doses were set at 100mg weekly in 9 pts and 200mg weekly in 3. With a median follow-up of 21 months (8–49 months) all pts remain in CHR. The FIP1L1-PDGFRA is undetectable in 11 patients by RT-PCR.

Conclusions. Imatinib at weekly dosage may induce and maintain remission in patient with CEL expressing F/P fusion gene. This strategy appears to be safe and cost savings.
Rac1 GTPase Targeting Inhibits p53-Deficiency Mediated Lymphomagenesis.
Authors: Bosco Emily E , Johnson James F , Ni Wenjun , Zheng Yi
Published: April 2020

Mutation of the p53 tumor suppressor is linked to therapeutic resistance in several tumor types. p53 mutation is generally associated with disease progression and poor prognosis in patients with lymphoid malignancies and can occur in approximately 50% of Burkitt’s lymphomas [1, 2]. Thus, new therapies are needed to specifically target p53-deficient lymphomas with increased efficacy. Rac1 small GTPase signaling is linked to tumorigenesis and Rac1 activity is increased by p53 loss of function. Previous studies in primary cell culture have shown that suppression of Rac1 activity can preferentially inhibit p53-deficiency induced hyperproliferation and cause increase apoptosis [3], indicating that targeting Rac1 may have therapeutic potential for p53-deficient malignancies. In the current study, the specific impact of inhibition of Rac1 on p53-deficient B and T lymphoma cell proliferation, apoptosis, tumor formation, and spontaneous lymphoma development in in vivo mouse lymphoma models was interrogated. p53-deficiency resulted in increased Rac1 activity in both human B and murine T lymphoma lines. Suppression of Rac activity using a dominant negative mutant or the small molecule inhibitor NSC23766 was able to abrogate p53-deficient lymphoma cell proliferation through a G1 checkpoint as determined by cell cycle analysis. Immunoblot analysis revealed that the anti-proliferative effect of Rac1 targeting in lymphoma cells may involve PAK, LIMK, and Akt signaling pathways rather than the MAP kinase pathway. Further, Rac1 targeting by shRNA resulted in an increase in expression of cleaved caspase 3 and cytochrome c by immunoblot concurrent with an elevation in the annexinV/7-AAD positive apoptotic cell population. These data indicate that loss of Rac1 activity can increase apoptosis via a p53-independent mechanism in p53 mutated lymphoma lines. These effects of inhibition of active Rac1 were extended in vivo where Rac1 targeting was able to impair p53-deficient lymphoma cell growth in xenograft models and postpone lymphomagenesis onset in murine transplant models. Taken together, our studies demonstrate that Rac1 activity is inversely regulated by functional p53 and that Rac1 contributes to p53-deficiency induced hyperproliferation by modulating both cell cycle and apoptosis in B and T lymphomas. Because the Rac signaling axis constitutes a critical determinant of cell survival and tumorigenesis associated with p53 defects, it may represent an important target for therapy in the treatment of p53-deficient lymphomas.
Targeting CDK4 in Mantle Cell Lymphoma (MCL) Cell Lines by Specific Lentiviral shRNA Mediated Knockdown Has Profound Effects on Cell Growth and Cell Cycle but Minimal Effects on Apoptosis.
Authors: Klier Margit , Bonzheim Irina , Anastasov Natasa , Angermeier Daniela , Raffeld Mark , Fend Falko , Quintanilla-Martinez Leticia
Published: April 2020

Introduction: The hallmark of MCL is overexpression of cyclin D1 due to the t(11;14). Cyclin D1 accelerates the transition of lymphoma cells from G1 to S phase by binding the cyclin-dependent kinase 4 (CDK4), and inactivating the retinoblastoma protein. Due to the importance of cyclin D1-CDK4 complexes in the control of the cell cycle, CDK4 has become a novel and attractive therapeutic target in MCL. Flavopiridol is a pharmacologically derived CDK inhibitor, which is in phase II trial in relapsed MCL patients. The aim of this study was to investigate the effects of silencing CDK4 expression in MCL using highly specific CDK4-shRNA, and compare the results with known effects of flavopiridol and other CDK inhibitors.

Material and methods: A highly efficient CDK4-shRNA was identified using a LacZ-cyclin D1 fusion gene reporter system in HEK293T cells. This shRNA was cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Four MCL cell lines were analyzed (Granta 519, Jeko-1, Rec-1, Z-138), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantify the knockdown effect. The effect of CDK4 knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis.

Results: In MCL cell lines with high CDK4 levels (Jeko-1, Rec-1, Z-138), profound effects on cell growth and cell cycle were observed with only minor effects in apoptosis. The cells showed 40% growth retardation compared to control cells after seven days of infection. Additionally, a clear shift from the S phase to the G1 phase in the cell cycle was observed as compared to control cells (S phase decreased by 23% and G1 increased by 28%). In contrast, only minimal apoptosis was induced (11% increase compared to control cells). CDK4-shRNA infected cells showed a strong induction of cyclin D2 mRNA (5,6 fold on average). Additionally, Granta 519 and Z-138 cell lines showed an increase in CDK6 mRNA. The expression of cyclin D1 mRNA and protein and cyclin E protein remained unchanged or was slightly induced. No changes were observed in the protein expression of cyclin D3, p27Kip1 and CDK2.

Conclusions: Our data showed that the specific CDK4 downregulation induces important growth retardation but not significant apoptosis. The reported effects after Flavopiridol treatment including downregulation of cyclin D1 and D3, as well as the induction of apoptosis, were not observed after specific CDK4 knockdown, and indicate CDK4-independent effects of this CDK inhibitor. Taken together, targeting specifically CDK4 alone is likely to have less effect on MCL than broader kinase inhibitors like Flavopiridol, used either as a single drug or in combination with conventional chemotherapy.
Genome Wide Analysis of 41 Mycosis Fungoides Tumor Stage Using Array Comparative Genomic Hybridization Technology.
Authors: Salgado Rocio , Servitje Octavio , Gallardo Fernando , Vermeer Maarten H , Romero Pablo Ortiz , Herrera Marta , Karpova Maria , Muniesa Cristina , García-Muret M. Pilar , Estrach Teresa , Sánchez-Schmidt Julia , Romagosa Vicenç , Zipser Marie , Suela Javier , Ferreira Bibiana I , Cigudosa Juan , Barranco Carlos , Dummer Reinhard , Tensen Cornelis P , Serrano Sergi , Pujol Ramon M , Solé Francesc , Espinet Blanca
Published: April 2020

Introduction : Primary cutaneous T cell lymphomas (CTCL) represent 70% of all cutaneous lymphomas. The most frequent is mycosis fungoides/Sézary syndrome (MF/ SS). In this entity, few high resolution cytogenetic studies have been performed. Our aim was to analyze chromosomal abnormalities in MF tumoral stage by array comparative genomic hybridization (ArrayCGH) and to describe potential candidate genes related to this disease.

Patients and methods : Forty-one patients (22 males/19 females) with MF tumor stage were included from centres collaborating in the EORTC Cutaneous Lymphoma Group. DNA was extracted from frozen tissues containing more than 70% of tumor cells. ArrayCGH tecnhology was performed to detect genomic imbalances (gains/losses) using the Humane Genome CGH Microarray Kit 44B (Agilent Techologies). This array consists on 44.000 oligo probes of 60 bp covering all the human genome with a mean resolution of 50–100 Kb. CGH-Analytics 3.2.25 and InSilicoArray CGH (http://isacgh.bioinfo.cipf.es) was used for array analysis and to define SORIs (Smallest Overlapping Region of Imbalance).

Results: Genomic abnormalities were observed in thirty-two cases (76%). Losses (62.36%) were more frequently detected than gains (37.64%). The mean chromosomal imbalances per case were 3.5 gains (0–14) and 5.6 losses (0–30). The minimal common regions altered were gains of 7q33.3 (55%), 17q21.1q21.3 (42.5%) and 8q24.21. Regarding to the losses, 9p21.3 (42,5%), 17p13.1 (42,5%) and 10p11.22 (17,5%) were the most frequent detected. SORIs and potential candidates genes from the most frequently altered regions are summarized in the following table.

Type of change Cytoband First gene start (Kbp) size (Mb) Frequency Candidate genes Loss 9p21.3 MTAP 21795 0,2 42,50% MTAP, CDKN2A, CDKN2B Loss 17p13.1 DULLARD 7094 1,01 27,5% TP53 Loss 10p11.22 chr10:031132968 31132 1,5 17,5% TCF8 Gain 7q33.3 BG495318 135521 14,1 55% BRAF Gain 17q21.1q21.3 SMARCE1 36050 4,7 42,5% STAT5A/STATB Gain 8q24.21 M13930 128816 0,75 32,5% C-MYC

Conclusions : Our results have shown high recurrent chromosomal abnormalities. Moreover, arrayCGH technology has allowed us to define in detail new common regions and to describe potential candidate genes in MF tumor stage as STAT5A/STA5B (17q21.2), BRAF (7q33) and TCF8 (10p11.22). Our findings are similar to those recently published by Vermeer et al in Sézary Syndrome, which lead to confirm the relationship between these two entities. Deletion of 9p21.3 (CDKN2A, CDKN2B, MTAP genes) and 17p13.1 (TP53) are concordance to previous studies.
Proteomic Analysis for the Identification of Disease Biomarkers and Therapeutic Targets in Natural Killer Cell Lymphoma.
Authors: Lee Hojung , Shereck Evan , Cairo Mitchell S. , Seiler Seiler E , Basrur Venkatesha , Fermin Damian , Elenitoba-Johnson Kojo SJ , Lim Megan S
Published: April 2020

Natural killer cell lymphoma (NKL) is a highly aggressive form of hematolymphoid malignancy that arises from natural killer cells, a critical component of the innate immune system. Early diagnosis of the tumor is critical however there are no biomarkers which are useful currently for the diagnosis and monitoring of patients with NKLs. We hypothesized that comprehensive analysis of differentially expressed proteins by NKL and normal NK cells would be a rational strategy for the discovery of NKL biomarkers and lead to better understanding of pathogenetic mechanisms of NKL. We used total cell lysates obtained from highly enriched peripheral blood NK cells (CD3-, CD56dim) and an Epstein-Barr virus negative, IL-2 dependent cell line (NK-92MI) for quantitative proteomic analysis. Isotopecoded affinity tags (ICATTM)-labeling followed by liquid chromatography-tandem mass spectrometry analyses (LC-MS/MS) identified 114 differentially expressed proteins in NKL compared to the normal NK cells. Out of these, 80 were upregulated 1.5-fold or greater and 34 were downregulated 1.5-fold or greater in NKL. Proteins within diverse cellular functional categories including cell signaling, transcriptional regulation, DNA metabolism, and immune response were differentially expression. Furthermore, known proteins relevant to the function of NK cells such as perforin, granzyme and defensins were downregulated in the NKL cell line. We selected eight proteins to validate the changes identified by ICAT-LC-MS/MS using Western blot analysis. HSP90, PCNA, alpha-enolase, GSTP1, galectin-1, and PARK7 were upregulated, and lactoferrin and annexin A1 were downregulated by western blot analysis demonstrating high concordance with the results of ICAT-LC-MS/MS analysis. We validated the functional significance of HSP90 as a potential therapeutic target in NKL by investigating the effect of inhibitors of HSP90 (geldanamycin) on the survival of an NKL cell line. Inhibition of HSP90 led to significant decrease in cell viability and apoptotic cell death as confirmed by western blotting demonstration of PARP-1 induction and detection of its cleavage products. This study represents the first global quantitative proteomic cataloguing of proteins expressed by NKL and normal NK cells, and reveals potential candidate proteins that may be useful for the diagnosis and function as therapeutic targets in patients with NKL.
Alterations of the CxxC Domain Preclude Oncogenic Activation of Mixed Lineage Leukemia 2 (MLL2).
Authors: Slany Robert K. , Bach Christian , Mueller Dorothee , Buhl Sebastian , Garcia-Cuellar Maria-Paz
Published: April 2020

The mixed lineage leukemia (MLL) family of histone methyltransferases has become notorious for the participation of the founding member MLL in fusion proteins that cause acute leukemia. Despite a high overall structural conservation no other MLL homolog has been found involved in leukemogenic fusion proteins. This surprising fact might be either due to a relative genomic stability of the MLL2 locus that prevents chromosomal translocations or MLL2 might be altogether incapable of contributing to oncogenic fusions because of functional constraints. Here we demonstrate the latter to be true for fusion proteins based on MLL2, the closest relative of MLL. A MLL2-ENL protein constructed in analogy to the highly leukemogenic MLL-ENL was incapable of transforming primary hematopoietic cells. Elaborate swap experiments identified the zinc binding “CxxC”- region of MLL2 and an adjacent “post-CxxC” stretch of basic amino acids as the essential determinants for the observed difference between MLL and MLL2. Surprisingly gel shift experiments indicated that the CxxC and post-CxxC domains of MLL and MLL2 conferred almost indistinguishable in vitro DNA binding properties. However, in vivo these motifs guided MLL-ENL and MLL2-ENL to a largely non-overlapping gene repertoire. Divergent nuclear localization, a distinct potency to activate a MLL model promoter in reporter assays, and measurements of homeobox gene levels in primary cells expressing MLL and MLL2 fusion proteins all argued for a separate target site selection of the two fusions. Therefore, the CxxC domain appears to be largely responsible for target finding and as a consequence this domain is a promising object for therapies aimed at MLL fusion proteins without affecting the general function of other MLL family members.
The Essential Role of Stem Cell Factor/KIT Signaling in the Proliferation and Survival of Blast Cells from Transient Leukemia in Neonates with Down Syndrome.
Authors: Toki Tsutomu , Kanezaki Rika , Adachi Souichi , Fujino Hisanori , Xu Gang , Sato Tomohiko , Suzuki Kahori , Tauchi Hisamichi , Endo Mikiya , Ito Etsuro
Published: April 2020

Children with Down syndrome (DS) are predisposed to developing leukemia. A leukemoid reaction occurring uniquely in approximately 10% of newborn infants with DS referred to as transient leukemia (TL). This disorder, in most cases, resolves spontaneously within three months after birth. Of all TL patients approximately 20–30% develop myeloid leukemia (ML-DS) within four years. Although treatment with low dose cytarabine is effective in high-risk TL cases, about 20% of severe patients still suffer early death. Improved treatments for TL are necessary for a better long-term prognosis of DS patients. In this study, we demonstrate abundant KIT expression in all 13 TL patients examined, although no significant difference in expression levels was observed between TL and acute myeloid leukemia (AML). Stem cell factor (SCF) mRNA was expressed at extremely low levels in TL cells and there were no significant differences in SCF expression between TL and AML. SCF stimulated the proliferation of the TL cells from all five patients examined and treatment with the tyrosine kinase inhibitor imatinib suppressed the proliferation and KIT phosphorylation effectively in vitro. To investigate the signal cascade, we established the first SCF-dependent, ML-DS cell line, KPAM1. Withdrawal of SCF or treatment with imatinib induced apoptosis of KPAM1 cells. SCF activated the RAS/MAPK and PI3K/AKT pathways, followed by downregulation of the pro-apoptotic factor BIM and upregulation of the anti-apoptotic factor MCL1. Although we found novel missense mutations of KIT in two of 14 TL patients, functional analysis using KPAM1 cells showed that neither mutation led to KIT activation and neither reduced the cytotoxic effects of imatinib. These results suggest the essential role of SCF/KIT signaling in the proliferation of DS-related leukemia and the possibility of therapeutic benefits of KIT-targeting tyrosine kinase inhibitors for TL patients.
Association of Elevated Soluble P-Selectin Levels and Fetal Loss in Women with a History of Venous Thromboembolism.
Authors: Ay Cihan , Kaider Alexandra , Koder Silvia , Husslein Peter , Pabinger Ingrid
Published: April 2020

Pregnancy is a hypercoagulable state with an increased risk of venous thromboembolism (VTE). Furthermore, accumulating evidence support the hypothesis that pregnancy complications such as fetal loss are associated with both inherited and acquired thrombophilic defects which may predispose to thrombosis of the placental vasculature and lead to subsequent fetal loss. In recent studies the cell adhesion molecule P-selectin has been identified to be a strong risk factor for VTE. Interestingly, soluble P-selectin (sP-selectin) plasma levels were previously reported to increase during pregnancy. Whether P-selectin is also associated with fetal loss in women with a history of venous thromboembolism, is not known yet. Therefore, the aim of our present study was to investigate the significance of elevated sP-selectin levels for fetal loss (e.g. miscarriage or stillbirth) in women with a history of VTE. We retrospectively evaluated data on pregnancy-associated complications in 304 women (mean age [+/-SD]: 45.6 [+/-11.5] yrs) with a history of VTE. sP-selectin plasma levels were measured using a sensitive ELISA (sP-selectin Immunoassay, R&D Systems®, Minneapolis, MN, USA). At the time of measurement of sP-selectin none of the women was pregnant and did not have an acute event of VTE. The mean age (±SD) of patients at the time of the VTE event was 31.3 (+/- 8.4) yrs. The prevalence of miscarriage (defined as intrauterine fetal death before the 24th week of gestation or when fetus weighed <500 g) in our study population was 21.8% and the prevalence of stillbirth (defined as intrauterine fetal death at or after the 24th week of gestation) was 4.3%. Median (interquartile range [IQR]) sP-selectin level of the total study population was 38.0 [21.7-44.4] ng/mL. The cut-off point for elevated sP-selectin was set at 44.4 ng/mL, which represents the 75th percentile of sP-selectin levels of the study population. The prevalence of stillbirth was significantly higher in subjects with elevated sP-selectin levels compared to those with lower levels (10.5% vs. 2.6%, p=0.008), whereas no statistically significant difference in prevalence of miscarriage was observed between women with and without elevated sP-selectin (17.1% vs. 22.9%, p=0.303). The odds ratio [95% CI] of elevated sP-selectin for stillbirth was 4.2 [1.5-12.7] and for miscarriage 0.7 [0.4-1.3]. In summary, elevated sP-selectin plasma levels were associated with a 4-fold risk for stillbirth in women with a history of VTE. Our data support a possible role of P-selectin in the aetiology of late pregnancy complications.
Microparticle-Associated Tissue Factor Activity and Venous Thrombosis in Multiple Myeloma.
Authors: Leebeek Frank , Yuana Yuana , Osanto Suzanne , de Maat Moniek , Auwerda Hajo , Bertina Rogier , Sonneveld Pieter
Published: April 2020

Background: Multiple myeloma (MM) is associated with an increased risk of venous thrombo-embolic complications. The pathogenesis of venous thromboembolism (VTE) in MM is not yet clarified. Recently an association between microparticle-associated tissue factor (MP-TF) activity in plasma and thrombosis in patients with adenocarcinoma has been reported. Aim of this study was to measure MP-TF activity in patients with MM before and after treatment with chemotherapy and to investigate whether MP-TF activity is associated with VTE in these patients.

Patients and results: MP-TF activity was assessed in 123 patients with newly diagnosed multiple myeloma who were eligible for combination chemotherapy and in 20 healthy volunteers. MP-TF activity levels were higher in untreated MM patients compared to the healthy volunteers (17.8 fM Xa/min [8.8–32.8] (median [Inter Quartile Range] versus 4.7 fM Xa/min [2.3–6.6], p<0.001). After three courses of chemotherapy the level of MP-TF activity had decreased significantly to 11.8 fM Xa/min [7.0–18.3], but were still higher than in controls (P<0.001). VTE occurred in 12 patients (10%). In MM patients with VTE the level of MP-TF activity remained elevated after induction chemotherapy (17.9 [9.3– 36.3]), in contrast to MM patients not developing VTE (11.1 [6.9–16.6] ). MP-TF levels after chemotherapy were significantly associated with the risk of VTE, with a 4% increase of VTE risk for every fM Xa/ min MP-TF activity increase (95% CI: 1–8%, p=0.03)

Conclusion: MP-TF activity levels in plasma are increased in patients with multiple myeloma and they decrease significantly after induction chemotherapy. Persistent high MP-TF activity is observed in patients who developed VTE during the induction phase chemotherapy, and is associated with VTE risk. Our findings suggest that MP-TF activity levels may have a pathogenetic role in VTE in multiple myeloma patients.
Protein Kinase Cδ Differentially Regulates Platelet Functional Responses.
Authors: Chari Ramya , Getz Todd , Nagy Bela , Bhavaraju Kamala , Mao Yingying , Bynagari Yamini Saraswathy , Murugappan Swaminathan , Nakayama Keiko , Daniel James , Kunapuli Satya P.
Published: April 2020

Protein Kinase Cδ (PKCδ), a novel PKC isoform is expressed and activated in platelets downstream of PARs and GPVI receptors. In the current study, the role of PKCδ in regulating platelet functional responses was investigated using a pharmacological inhibitor, (δV1-1)TAT (a PKCδ inhibitor) in human platelets. These studies were further confirmed by a knockout approach using PKCδ+/+ and PKCδ−/− mice. In both human and murine platelets, PAR4-mediated dense granule secretions were inhibited, whereas GPVI-mediated dense granule secretions were potentiated. Furthermore, α-granule secretions and thromboxane A2 (TXA2) generation were differentially regulated in murine platelets.. These data suggest a differential role for this isoform in regulating dense granule secretion, α-granule secretion and TXA2 generation. Previous studies have shown that PAR-mediated fibrinogen receptor activation is regulated by a Calcium-dependent and a PKC-dependent pathway. The contribution of PKCδ to PAR-mediated fibrinogen receptor activation was studied by pretreating human and murine platelets with BAPTA. Our results showed a inhibition of AYPGKF-induced aggregation in human and murine platelets in the presence of BAPTA and fibrinogen. These results suggest a small contribution of PKCδ to PAR-4- mediated platelet aggregation and aIIbb3 activation. The in vivo significance of PKCδ was tested using a FeCl3 injury model. While the wildtype mice occluded in 7 minutes, PKCδ −/− mice occluded after 4 minutes of injury with 10 % FeCl3. Therefore, we conclude that PKCδ regulates platelet functional responses such as dense, α-granule secretions, TXA2 generation downstream of both PARs and GPVI receptors, contributes to PAR-4-mediated fibrinogen receptor activation ex vivo and plays a critical role in the thrombus formation in vivo. This study is supported by predoctoral fellowships to Ramya Chari and Swaminathan Murugappan from American Heart Association, Great Rivers affiliate.
The Role of Livin, An Inhibitor of Apoptosis Protein, in Thrombopoiesis.
Authors: Abd-Elrahman Ihab , Deutsch Varda , Hershko Klilah , Noyman Tsahi , Lazar Itay , Nachmias Boaz , Perlman Riki , Ben-Yehuda Dina
Published: April 2020

The exact mechanism of platelet production is poorly understood. A relationship between activation of the apoptotic cell machinery and the formation of pro-platelets has been established. In this study we show that Livin plays a major role in this process. Livin is a member of the Inhibitor of Apoptosis Protein (IAP) family, a novel family of intracellular anti-apoptotic proteins that act by binding and inhibiting caspases. We found that Livin mRNA and protein are expressed in platelets and Livin was also clearly detected by immunohistochemistry staining in normal mature bone marrow megakaryocytes (MK). Overexpression of Livin was also demonstrated in MK of patients with various hematological diseases such as ITP, MDS, Hodgkin’s disease, ET and PV. An in vitro model was established to evaluate the potential role of Livin in thrombopoiesis. The human erythroleukemic cell line, LAMA-84, was induced by a phorbol ester (PMA) to differentiate to MK. Differentiation was characterized by upregulation of the MK marker CD41 from 6% to >60% and downregulation of the erythroid cell marker CD71 from 97% to 70%. Increased cell size, ploidy, and DNA synthesis, all markers of MK differentiation, were detected by flow cytometry. Upon differentiation induced by PMA, LAMA-84 cells formed pro-platelets and produced functional platelets capable of aggregation. This thrombopoiesis was accompanied by Livin overexpression. Moreover, overexpression of Livin by transfection decreased the percentage of the undifferentiated CD71+ cells to 53 % compared to 73 % in control cells (p= 0.007) and increased the production of platelets by two folds compared to control cells. Overexpression of Livin reduced caspase 3 activity and inhibited MK apoptosis. Our results show that anti-apoptotic Livin plays a role in thrombopoiesis possibly by protecting the maturing MK from apoptosis, thus affording more time for efficient platelet production by longer living pro-platelets. We are currently using this new cell line model to further characterize the molecular relationship between apoptosis and thrombopoiesis.
Impaired Activation of Platelets Lacking Protein Kinase C Theta (PKCΘ) Isoform.
Authors: Nagy Bela , Bhavaraju Kamala , Getz Todd , Bynagari Yamini Saraswathy , Kim Soochong , Kunapuli Satya P.
Published: April 2020

Protein kinase C (PKC) has been implicated in platelet functional responses, but the contribution of individual isoforms has not been directly evaluated. PKCΘ is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by ADP. In human platelets, PKCΘ-selective receptor for activated C kinase (RACK) antagonistic peptide inhibited agonist-induced aggregation and secretion. Consistently, in murine platelets lacking PKCΘ, GPVI- or PAR-mediated aggregation and secretion were also impaired. Previously, fibrinogen receptor has been shown to be activated independently by calcium and PKC pathways. In the presence of dimethyl BAPTA, AYPGKF-induced platelet aggregation was inhibited by PKCΘ antagonistic RACK peptides, suggesting a role for this isoform in PKC-dependent fibrinogen receptor activation. In addition, the levels of thromboxane A2 (TXA2) release measured in GPVI and PAR-mediated activation of PKCΘ −/− murine platelets, were significantly lower compared to WT platelets. Moreover, agonist-induced extracellular-signal regulated kinase (ERK) phosphorylation was also significantly decreased in PKCΘ −/− murine platelets, which could be contributing to decreased TXA2 levels. PKCΘ −/− mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl3 in vivo thrombosis model versus WT mice. In conclusion, PKCΘ isoform plays a significant role in platelet functional responses downstream of GPVI and PARs.
Role of Glycoprotein Ibα Mobility in Platelet Function.
Authors: van der Wal Dianne E , Verhoef Sandra , Schutgens Roger EG , Peters Marjolein , Akkerman Jan-Willem
Published: April 2020

For preservation of platelet concentrates, storage at 0°C might be superior to current storage procedures at 22°C since cold storage reduces bacterial growth and might better preserve platelet functions. A major problem is that cold damages the glycosylation of the von Willebrand Factor (VWF) receptor Glycoprotein (GP) Ib&alpha, exposing N-acetyl- D-glucosamine (GlcNAc) residues and inducing receptor clustering. We investigated the effect of dispersed and clustered conformations of GPIb-alpha on the haemostatic functions of the receptor. We deduced the glycosylation state from binding of succinylated wheat germ agglutinin (sWGA) and clustering from binding of AN-51 antibody directed against aa 1–35 which differed between dispersed and clustered GPIb-alpha. We compared different GPIb-alpha conformations with anchorage to the membrane- and cytoskeleton and VWF/ristocetin (VWF*)-induced platelet functions and confirmed specificity for GPIb-alpha and VWF effects by control studies in GPIb-alpha- and VWF-deficient platelets. VWF*- and cold-treatment released GPIb-alpha from the membrane skeleton, inducing sWGA-binding, clustering and anchorage to the cytoskeleton. Addition of GlcNAc restored dispersed GPIb-alpha after cold but not after VWF*-treatment. VWF*- induced aggregation and adhesion to coated VWF under flow were little affected by changes in GPIb-alpha conformation but agglutination, spreading and formation of stress fibers strongly depended on clustered GPIb-alpha. Following cold-induced clustering of GPIb-alpha subsequent incubation at 37°C triggered formation of thromboxane (Tx)A2 in a GPIb-alpha-dependent manner but independent of added or endogenous VWF. GlcNAc strongly inhibited VWF*-induced agglutination, spreading on coated VWF and VWF-independent TxA2 formation. We conclude that incubation at 0°C triggers clustering of GPIb-alpha which activates platelets independent of its ligand VWF.
Genetic Modulators: Prevalence and Their Effect on the Phenotype of HbE/β Thalassemia Patients.
Authors: Sharma Vineeta , Saxena Renu
Published: April 2020

HbE, a β-globin chain variant occurs due to GAG to AAG i.e. Glu-Lys substitution at codon 26.In heterozygous and homozygous state it usually remain asymptomatic but the compound heterozygous state with β thalassemia (HbE/β thalassemia) results in a variable and often severe anemia, with the phenotype ranging from transfusion dependence to a complete lack of symptoms. The basis of the interaction and the cause of the variability remains unexplained. Some of the possible explanations for the observed variable clinical severity are coinheritance of mild β mutations, Effect of alpha gene number and increased production of HbF. Aims of the study are to determine the frequency of primary (β mutations) and secondary modulators (α deletion and α triplication, XmnI polymorphism, HPFH-3 and Asian-Indian Inv/DelGγ(Aγδbeta;)0 deletion) in HbE/β thalassemia patients and to study their effect on the phenotype of the Patients. Subjects were 180 HbE/β thalassemia patients diagnosed by HPLC and confirmed by PCR-RFLP. Patients were divided into three subgroups according to a scoring system based on seven clinical criteria as mild (score 0–3.5)[Gp1], moderate (score 4–7)[Gp2] and severe (score 7.5–10) [Gp3]. β-mutations were studied by ARMS PCR, XmnI polymorphism was studied by PCR-RFLP while α deletions, HPFH-3 and Asian-Indian Inv/DelGγ (Aγ δβ)0 deletions were studied by GAPPCR and alpha triplication by a modified single tube PCR. The frequency of β mutation was almost similar in all the groups (IVS 1–5 G-C (58.8%) was the predominant one). α deletion was found in 19 (10.5%) out of which 13 (12 αα/−α3.7 & 1 αα /--SA) were from Gp1 and 6 (αα/-α3.7) were from Gp2. α triplication was found in 11(6.1%) out of these 11 patients 8 were (αα/αααanti3.7) from Gp3 and 3 were (αα/αααanti3.7) from Gp2. XmnI was found in 112(62.2%), out of which 98 were heterozygous (+/−) and 14 were homozygous (+/+).XmnI +/+ was present in 10 Gp1 & 4 Gp2 patients, while XmnI +/− was present in 36 Gp1 ,43 Gp2 & 33 Gp3 patients.HPFH-3 deletion was present in 8 (4.4%) out of which 5 were from Gp1 and 3 were from Gp2. Asian-Indian Inv/DelGγ (Aγδβ)0 deletion was present in 2(1.1%) patients one from Gp2 and another from GP3. Primary modulator (β mutation) is not the actual factor responsible for the disease heterogeneity. Patients with coexisting α deletion required lesser transfusions and had less severe phenotype while patients with α triplication were on frequent transfusions and had severe phenotype. XmnI polymorphism in homozygous state was observed to alleviate the severity while in heterozygous state it had no effect on the disease severity except the delay of the onset of disease up to some extent. Patients having HPFH3 deletions and Asian-Indian Inv/DelG γ (Aγδβ)0 deletion showed mild to moderate disease severity. Thus it can be concluded that secondary modulators play an important role in the disease heterogeneity of HbE/βthalassemia.
Regulation of the Human α-Globin Genes by GKLF.
Authors: Moi Paolo , Marini Giuseppina Maria , Porcu Loredana , Asunis Isadora , Loi Maria Giuseppina , Cabriolu Lisa Anna , Cao Antonio
Published: April 2020

EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the β- and β-like globin genes. Prompted by the observation that four KLF sites are distributed in the human α-globin promoter, we investigated if any of the β-globin cluster regulating KLFs could also act to modulate the expression of the α-globin genes. We found that, among the globin regulating KLFs (EKLF, LKLF, BKLF, GKLF, KLF6, FKLF and FKLF2), only GKLF and BKLF bound specifically to three out of four KLF sites. In K562 cells, over-expressed GKLF transactivated at high levels a α-globin-luciferase reporter and its action was impaired by point mutations of the KLF sites that disrupted GKLFDNA binding. In K562 cells stably transfected with a Tet-off regulated GKLF expression cassette, GKLF induction stimulated the expression of the endogenous α-globin genes. In a complementary assay in K562 cells, knocking down GKLF expression with small interfering RNAs caused a parallel decrease in the transcription of the α-globin genes. All experiments combined support a main regulatory role of GKLF in the control of α-globin gene expression.
Role of Cjun in Gγ-Globin Gene Trans-Activation.
Authors: Kodeboyina Sirisha , Zein Sima , Lee Moosueng , Balamurugan Parimaladevi , Yao Xiao , Pace Betty
Published: April 2020

Previous studies from our laboratory demonstrated the role of the G-CRE (Gγ-globin cAMP response element) in drug-mediated fetal hemoglobin induction. The G-CRE located at −1222 to −1229 in the promoter of Gγ-globin gene, contains binding site for trans-factors CREB1, ATF-2 and cJun. We previously demonstrated binding of phosphorylated CREB1 and ATF-2 to this element via p38 MAPK signaling triggered by sodium butyrate (NaB) and trichostatin A (TSA). Electrophoretic mobility shift assays with a probe containing the AC → TG mutation in the G-CRE (TGTGGTCA, m2) abolished trans-factor binding to the G-CRE. Furthermore, Gγ promoter activity was abolished in the PGL3 luciferase reporter vector driven by the Gγ promoter (−1500 to +36) carrying the m2 mutation. (Sangerman et al. Blood108:3590–9, 2006). Subsequent studies in our laboratory were aimed at understanding the role of trans-factor cJun, an AP-1 family member, as a regulator of Gγ-globin expression via the G-CRE site. In K562 cells treated with 2mM NaB or 0.3μM TSA for 48 hrs, cJun phosphorylation increased 2.8-fold and 6.4-fold respectively by western blot analysis. Chromatin immunoprecipitation studies showed 16-fold chromatin enrichment in the −1225 Gγ-globin region compared to IgG control studies indicative of significant cJun binding in vivo at steady state. Electrophoretic mobility shift assays using cJun monoclonal antibody demonstrated a supershifted DNA-protein complex confirming binding of cJun to the G-CRE probe. To gain evidence for a functional role of cJun, we performed enforced expression studies using the pLen-cJun vector. In a concentration dependent manner, over-expression of cJun increased luciferase activity up to 350-fold in the luciferase reporter plasmid controlled by the Gγ-promoter (−1500 to +36). As predicted from binding studies, the m2 mutation in this promoter abolished the cJunmediated trans-activation confirming that the G-CRE is required to mediate effects of cJun. We are currently investigating the ability of cJun to trans-activate the endogenous Gγ-globin gene in K562 cells. To achieve this goal, K562 stable lines were established with the expression vectors pLen-cJun and empty vector. A complete analysis of the stable lines is in progress. Future investigations to identify other components of the functional CREB1/ATF2/cJun enhanceosome complex bound to the G-CRE will be performed using affinity chromatography and mass spectrometry. This information will be used to develop strategies for fetal hemoglobin induction.