bluuebunny/crossref_metadata_2025 huggingface dataset:
Grading Follicular Lymphoma: No Difference between 1, 2 and 3a, but 3b Is Something Else.
Authors: Wahlin Björn E. , Sander Birgitta , Christensson Birger , Kimby Eva
Published: April 2020

According to WHO’s current criteria, follicular lymphoma is morphologically divided into grades 1, 2, and 3, and grade 3 is further subdivided into 3a and 3b, depending on the presence of centrocytes and solid sheets of centroblasts. The clinical relevance of this grading system is unclear. Although there is consensus that grades 1 and 2 share clinical characteristics of indolent lymphoma, it is debated whether grade 3 should be treated intensively with anthracycline-containing regimens. Furthermore, the 3a/3b subdivision is not universally implemented. We wanted to investigate the clinical relevance of the grading system with respect to clinical characteristics, treatment and survival. Totally, 186 cases were diagnosed between January 1994 and January 2004 in South Stockholm County with de novo follicular lymphoma and without concomitant transformation. The biopsies were re-graded according to the current WHO criteria by two experienced hematopathologists. 129 cases were grade 1 or 2, 44 grade 3a, and 13 grade 3b. Clinical characteristics in the patients with follicular lymphoma grade 1–2 and 3a are shown in Table 1.

The local policy has been to treat all patients with grade 3 disease aggressively. In grade 3a disease, front-line anthracyclines did not seem to improve overall survival (Figure 1).

Grades 1–2 and 3a show similar overall survival curves of indolent and incurable lymphoma, while the 13 patients with grade 3b disease (of whom 12 received aggressive front-line therapy) seem to reach a plateau after four years (Figure 2).

We conclude that follicular lymphoma grade 3a is not clinically different from grade 1 and 2 disease, and that patients with grade 3a do not seem to benefit from front-line anthracyclines. Although our findings are limited by the few cases of grade 3b, it seems that grade 3b is another disease entity and, when treated aggressively, it is maybe curable with chemotherapy. The histopathological distinction between grade 3a and 3b is clinically of great importance.

Table 1 Grade 1–2 Grade 3a P Clinical characteristics in patients with follicular lymphoma grade 1-2 and 3a. Age >60 years 50% 61% NS Elevated LDH 32% 49% 0.046 Stage III or IV 68% 80% NS Front-line anthracyclines 41% 73% 0.0004 Subsequent transformation 21% 19% NS Figure Figure Figure Figure
Hepatitis B and C Virus in Patients with Non-Hodgkin Lymphoma.
Authors: Anderson Lesley A. , Pfeiffer Ruth , Warren Joan , Landgren Ola , Berndt Sonja , Gadalla Shahinaz , Ricker Winnie , Parsons Ruth , Engels Eric A.
Published: April 2020

Background: Hepatitis C virus (HCV) infection has been implicated in the development of Non-Hodgkin lymphoma (NHL), but subtype specific analyses are limited. Although some studies have reported a higher prevalence of hepatitis B virus (HBV) infection in patients with NHL, results are conflicting.

Methods: From the U.S. SEER-Medicare database, we selected 59,824 patients with NHL (aged 65+ years) and 166,057 population-based controls. Participants were classified as having HCV or HBV infection if they had any Medicare record of infection >1 year prior to diagnosis/selection. Polytomous logistic regression analyses were used to compare HCV and HBV infection in cases and controls with adjustment for gender, age (5-year age bands), calendar year of diagnosis/selection (in tertiles), race (white versus non-white) and the number of physician visits (in quartiles).

Results: Overall, 137 (0.23%) NHL cases and 270 (0.16%) controls had HCV infection and 82 (0.14%) cases and 249 (0.15%) controls had HBV infection. HCV infection was significantly associated with NHL overall (OR 1.3, 95% CI 1.1–1.7) and with diffuse large B-cell lymphoma (n=13,330; OR 1.5, 95% CI 1.1–2.2), Burkitt’s lymphoma (n=221; OR 5.7, 95% CI 1.8–17.9), follicular lymphoma (n=6,142; OR 1.9, 95% CI 1.2–3.0) and marginal zone lymphoma (n=1,989; OR 2.3, 95% CI 1.3–4.1). HCV infection was not associated with T-NHL (n=2,362; OR 0.4, 95% CI 0.1–1.6) or NHL not otherwise specified (n=3330; OR0.9, 95% CI 0.3–2.9). HBV infection was not significantly associated with NHL or NHL subtypes.

Conclusions: HCV, but not HBV, infection was associated with elevated risk of high- and low-grade NHLs. HCV may induce NHL development through chronic immune stimulation or other mechanisms.
PU.1 Cooperates with SOX4 in Myeloid Cells.
Authors: Aue Georg , Du Yang , Jenkins Nancy A. , Dunbar Cynthia E. , Copeland Neal G.
Published: April 2020

Mice that express 20% the normal levels of the Ets transcription factor PU.1 develop AML, unlike mice that express 50% or 80% the normal levels, indicating that PU.1 is a dosage-sensitive tumor suppressor gene. In addition, 3 of 13 AMLs induced by transplanting mice with cells transduced with a Sox4 oncogene-containing retrovirus were found to carry a Sox4 retroviral integration in one PU.1 allele, suggesting that downregulation of PU.1 may cooperate with Sox4 in AML induction. Since the other PU.1 allele remains intact in these AMLs and a 50% decrease in PU.1 expression is not sufficient to induce AML, we hypothesized that Sox4 might further downregulate PU.1 expression in these AMLs. To test this hypothesis, we transfected HL60 cells with an expression vector carrying GFP and Sox4 cDNA or a GFP vector control alone. PU.1 mRNA levels were consistently downregulated 4 to 10 fold in cells transfected with Sox4 cDNA compared to cells transfected with the vector control, confirming that overexpression of Sox4 downregulates PU.1 expression in myeloid cells. The decrease of PU.1 mRNA was observed as early as 8 hours after Sox4 transfection, further suggesting that Sox4 may directly interact with PU.1 in myeloid cells. Consistent with this, analysis of 2 published microarray databases comprising 401 de novo AML patient samples showed that SOX4 expression is significantly negatively correlated with PU.1 expression (coefficient: −0.337, P-value: 1E-07). In order to confirm that downregulation of PU.1 cooperates with Sox4 in AML induction, we infected wild type or PU.1 heterozygous knockout bone marrow cells with the Sox4 retrovirus and then monitored the time of AML development in transplanted mice. Results showed increased penetrance (95%) of myeloid leukemia in mice transplanted with Sox4-infected PU +/– bone marrow compared to mice receiving Sox4-infected wild type marrow (60%). Myeloid leukemia was confirmed by histology in all animals of the Sox4-infected PU +/ cohort while T cell lymphoma was diagnosed in 3 animals of the Sox4 wild type cohort. Together, all experiments support the hypothesis that Sox4 cooperates with the transcription factor PU.1.
SALL4-Dependent Signaling Networks Are Required for Survival of Leukemia Cells.
Authors: Yang Jianchang , Chai Li , Fowles Taylor , Alipio Zaida , Gao Chong , Fink Louis , Ma Yupo
Published: April 2020

Our group and others have recently shown that a murine transcriptional factor, SALL4 plays an essential role in maintaining the properties of embryonic stem cells and in governing the fate of the primitive inner cell mass through regulation of Oct4. Over-expression of SALL4 in mice exhibit myelodysplastic syndrome (MDS) which transforms to acute myeloid leukemia (AML) associated with expansion of leukemic stem cells. Aberrant expression of SALL4 in leukemic blasts is commonly seen in a variety of AML and MDS cases. However, the transcriptional programs downstream of the SALL4 stem cell gene in leukemia are unknown. We mapped its global gene targets using chromatin-immunoprecipitation followed by microarray hybridization (ChIP-on-ChIP). We identified over two thousands high-confidence SALL4 binding sites in the acute leukemic cell line, NB4. Analysis of the SALL4 binding sites revealed that genes involved in cell death, cancer, DNA replication/repair, and cell cycle were highly enriched (p<0.05) when functionally classified. This is consistent with promoter binding of over 280 apoptosis-related genes including important apoptosis and cell-cycle arrest induction genes (DAP3, CARD9, TP53, DAXX, CASP6, CASP9, CYCS, TNF, TAL1, and PTEN) and apoptosis inhibition genes (Bmi-1, BCL2, TEGT and XIAP). This indicates that SALL4 may play a role in leukemogenesis. Reduction of wild type SALL4 mRNA levels by approximately 50 percent resulted in a 21-fold increase in caspase-3 activity from 4.6 percent to 98.3 percent. In addition, suppression of SALL4 expression caused a significant decrease in NB4 cell growth rate. To further study the role of the SALL4 in cell growth, we monitored cell-cycle changes and cellular DNA synthesis in SALL4-suppressed and wild type NB4 cells through flow cytometry analysis and the BrdU incorporation assay. SALL4-reduced NB4 showed about a four-fold decrease in S phase cells and a significant increase in the G1 and G2 phases, which paralleled the drop in DNA synthesis as measurement from the level of BrdU incorporation. Similar results were observed in other cancer cell lines. In addition, SALL4-reduced NB4 cells showed significantly decreased tumorigenicity in immunodeficient mice. Our studies provide a foundation for developing a leukemia stem cell-specific therapy targeting SALL4.
Lack of SSBP2, a Candidate Leukemia Suppressor, Promotes T Cell Lymphomagenesis in p53 Null Mice.
Authors: Wang Yang , Patel Mayur , Ma Xiaoping , Liang Hong , Brandt Brandt , Agulnick Alan , Nagarajan Lalitha
Published: April 2020

Our laboratory positionally cloned a candidate myeloid leukemia suppressor gene, sequence-specific single-stranded DNA binding protein 2 (SSBP2) from chromosome 5q13.3-5q14.1 interval. SSBP2 expression is lost in primary acute myelogenous leukemia (AML) and myelodysplasia (MDS) cells. AML cells induced to express SSBP2 show growth arrest and partial differentiation. SSBP2 directly binds the adaptor protein Ldb1 (LIM domain-binding protein1), which in turn binds LIM domain containing nuclear factors. Although LDB1, SSBP2 containing transcriptional complexes are ubiquitously expressed, they have been best characterized in erythroid differentiation. Stabilization of Ldb1 by its direct interaction with SSBP2 upregulated the transcriptional activity of the Ldb1-Lmo2-E2A/TAL1-containing complex on an erythroid specific gene promoter. Here, we report the generation and characterization of Ssbp2 null mice. Mice with homozygous disruption of Ssbp2 (Ssbp2−/−) are viable and fertile, with no gross hematopoietic abnormalities observed up to six months of age. In order to examine if and how loss of SSBP2 promotes transformation, we crossed Ssbp2−/− mice with p53−/− null mice. Ssbp2−/−, p53−/− mice succumbed to thymic lymphomas with a median survival of 122 days compared to a median survival of 182 days for p53−/− mice (P = 0.0002). Moreover, the spectrum of tumors observed in the Ssbp2−/−, p53−/− mice included 62.5% thymic lymphomas (20/32), whereas only 26.7% of the p53−/− mice developed thymic lymphomas (8/30). In addition, Ldb1 protein levels but not transcript levels were decreased in the thymi of Ssbp2−/−, and Ssbp2−/−, p53−/− mice. Thus, loss of Ssbp2 appears to abrogate Ldb1-Lmo2-mediated T cell differentiation by promoting Ldb1 degradation. Such a block in differentiation cooperates with the loss of cell cycle arrest and apoptosis due to loss of p53. Our findings demonstrate for the first time a functional cooperation between the candidate leukemia suppressor SSBP2 and tumor suppressor p53. Furthermore, these results suggest the Ldb1-Lmo2 complexes to be novel targets for therapeutic intervention in hematologic malignancies.
BCL6 Transcriptionally Reprograms the DLBCL Genome by Forming Distinct Biochemically Active Complexes on Genes Corresponding to Different Pathways.
Authors: Polo Jose M. , Chatzi Katerina , Dell’Oso Tania , Lev Paola , Melnick Ari
Published: April 2020

Aberrant gene expression is a hallmark of cancer, and so it is not surprising that the most common category of oncogenes and tumor suppressors involved in hematologic malignancies are transcription factors. These factors mediate their effects by nucleating biochemically active cofactor complexes to modify the chromatin structure of their respective target genes. BCL6 is a transcriptional repressor and the most commonly involved oncogene in diffuse large B-cell lymphomas. BCL6 represses genes by recruiting several corepressor complexes including SMRT, N-CoR, BCoR; all of which bind to BCL6 through its BTB domain. Each of these complexes has different biochemical functions (e.g. BCoR forms a polycomb complex vs. SMRT which forms an HDAC3 complex). Moreover, our preliminary data suggested that BCL6 uses different sets of corepressors to mediate distinct biological effects, possibly by using different biochemical mechanisms at specific sets of target genes. Therefore, we hypothesized that BCL6 regulates its target genes using different biochemical tools, allowing it to exquisitely fine tune gene expression and provide specific control mechanisms for different biological functions. In order to test this hypothesis we first identified the direct target genes of BCL6 SMRT, N-CoR and BCoR by ChIP-on-chip in DLBCL cells (Ly1 cells) in multiple replicates, and examined whether the overlapping sets of genes corresponded to different gene pathways. We used a 24,000 promoter microarray representing 1.5 KB of sequence for each gene. The results show reproducible binding of BCL6 at 940 promoters, While BCoR bound to 770, SMRT to 545 and N-CoR to 487 promoters respectively. BCL6 and BCoR overlapped at 400 genes, preferentially involved in involve in cell cycle, cell death chromatin structure, ubiquitin dependent process and chemotaxis. BCL6 and SMRT overlapped on 376 genes, involved in immune response, cell motility and also as BCOR cell death, while N-CoR and BCL6 overlapped on 100 genes including transcriptional control and cell death pathways. The overlap between BCoR and SMRT was at 200 genes, BCoR and N-CoR at 60 genes and SMRT and N-CoR at 85 genes. All three overlapped at 50 genes. We also examined whether these corepressors were associated with specific combinations of histone modifications including H3K9 acetylation, H3K9 methylation, H3K4 methylation, H3K27 methylation, H4K16 acetylation and H3K36 acetylation. Taken together, the data indicate that specific subsets of BCL6 target genes are dependent on distinct biochemical mechanisms, suggesting that additional layers of biochemical complexity govern formation of gene repression complexes in DLBCL cells and providing opportunities for highly specific therapeutic targeting of specific gene programs.
Activation of JAZ by Small Molecules in Human Leukemia Cells.
Authors: Yang Mingli , Jia Jinghua , Ostrov David , Qiu Jingxin , May W. Stratford
Published: April 2020

By screening a murine interleukin-3 (IL-3)-dependent myeloid cell cDNA library, we previously identified JAZ (Just Another Zinc Finger Protein), a novel zinc finger protein. JAZ belongs to a new class of evolutionarily conserved C2H2-type ZFPs that feature an unusually long linker sequence and preferentially bind to double-stranded RNA. JAZ localizes in the nucleus and its human gene is localized to the chromosome 5q35-qter, a specific chromosomal region at which deletions and translocations occur in leukemia and lymphoma. We have recently discovered JAZ as a novel direct, positive regulator of p53 transcriptional activity. The mechanism involves direct binding to p53’s C-terminal (negative) regulatory domain to activate “latent” p53 in response to non-genotoxic stress signals (such as interleukin-3 growth factor withdrawal). Thus, we have explored JAZ as a potentially novel molecular target in human leukemia by identifying small molecules to activate JAZ-mediated leukemic cell killing. A structure-based drug design approach was employed to screen small molecules that potentially bind and activate JAZ. Cytotoxicity assays were then carried out with a set of candidate compounds in a dose- and time-dependent manner in various human leukemia cell lines including REH, HL-60, U937, HEL and K562 cells. Several JAZ “activating” compounds were selected, which display differential abilities to induce leukemic cell killing (IC50 = < 1 to 100 μM). Interestingly, biochemical analysis shows that the J-compound(s) can mediate cell death in p53 expressing leukemia cells in association with upregulation of expression of JAZ, p53 and the proapoptotic p53 target gene BAX, indicating activation of p53. Furthermore, the J-compound(s) synergizes induction of leukemic cell death when combined with other known p53 activating agents including cisplatin and nutlin-3, a recently developed small molecule MDM2 antagonist that disrupts the p53-MDM2 interaction. Therefore, the J-compound(s) may mediate leukemic cell death in a mechanism involving activation of “latent”, wild-type p53 by targeting JAZ. However, the J-compound-mediated cell death was also observed in some p53-deficient leukemia cell lines. This suggests that depending on the cell type the J-compounds may also act by a p53-independent mechanism. Since JAZ was recently reported to be a cargo protein for exportin-5, the nuclear export receptor for pre-microRNAs, this dsRNA-binding ZFP may also have an unknown p53-independent function via which the J-compound(s) may possibly act. While these possible mechanisms remain to be further investigated, the J-compound(s) points the way to develop a potentially novel therapeutic strategy targeting JAZ to treat human leukemia.
Modulation of IRP Binding Activity by Oxygen in Primary Erythroid Cells.
Authors: Schranzhofer Matthias , Schifrer Manfred , Ponka Prem , Muellner Ernst W.
Published: April 2020

Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are cytoplasmic RNA-binding proteins that target specific stem-loop RNA structures known as iron responsive elements (IRE). Binding of IRPs to IREs inhibits translation of ferritin mRNA and stabilizes transferrin receptor (TfR) mRNA. Various factors have been reported to regulate binding activity of IRPs, such as iron, phosphorylation, nitric oxide and hypoxia. While there is a consistent agreement on the negative effect of iron on the interaction between IRPs and IREs, reports regarding the influence of hypoxia on the IRE-binding activity of IRPs vary in a species and cell specific manner. It was the aim of this work to study the effect of hypoxic (3% oxygen) and normoxic (20% oxygen) conditions on IRP binding activity in primary erythroid cells. The cells were induced for differentiation and incubated under physiological, low (Desferrioxamine) and high (ferric ammonium citrate) iron conditions. Binding activity of IRPs and protein levels of ferritin and TfR as well as cell proliferation and differentiation parameters were determined to analyze the regulation of iron metabolism during terminal differentiation. The data show, that in developing red blood cells binding activities of IRP1 and IRP2 are reduced at 3% oxygen. This reduction correlates with increased ferritin protein levels and decreased TfR protein levels. Moreover, incubation under hypoxia strongly decreased cell expansion and reduces hemoglobinization. These results suggest that terminal erythroid differentiation in the bone marrow might occur under normoxic rather than hypoxic conditions.
Reducing Iron Stores Lowers Cancer Risk in Patients with Peripheral Arterial Disease.
Authors: Zacharski Leo R. , Chow Bruce K. , Howes Paula S. , Shamayeva Galina , Baron John A. , Dalman Ronald L. , Malenka David J. , Ozaki Keith , Lavori 6 Philip W.
Published: April 2020

Background: Iron accumulation in excess of requirements has been implicated in risk of malignancy through increased iron-catalyzed free radical-mediated oxidative stress. Reduction of body iron stores may lower risk of cancer.

Methods: A multi-center randomized, controlled, single-blinded clinical trial was conducted between May 1, 1999 and April 30, 2005 within the Department of Veterans Affairs Cooperative Studies Program; VA Cooperative Study #410. Its purpose was to test the hypothesis that reducing body iron stores by phlebotomy would influence all cause mortality (the primary endpoint) and combined death plus non-fatal myocardial infarction and stroke (the secondary endpoint) in a cohort of patients with symptomatic peripheral arterial disease. Patients with a diagnosis of visceral malignancy within the preceding five years were excluded. Patients were randomly assigned to control or reduction of iron stores by graded phlebotomy (avoiding iron deficiency) with stratification by hospital, age, entry ferritin, diagnosis of diabetes mellitus, smoking status and HDL/LDL ratio. Information on the occurrence of new visceral malignancies and cause-specific mortality including death due to cancer as collected prospectively.

Results: This study enrolled 1,277 patients: 641 were randomized to control and 636 to iron reduction. Ferritin levels were reduced significantly in patients randomized to iron reduction. A new visceral malignancy was diagnosed during follow-up in 60 control and 38 iron reduction patients, a 37% decrease in risk with iron reduction (p = 0.023). Reduced risk was observed for several common tumor types. Iron reduction patients had lower cancer - specific mortality and lower all - cause mortality in patients diagnosed with cancer (p = 0.003 and 0.009, respectively) than control patients. Neither cancer risk nor mortality was affected by variables used to stratify the randomization or demographic variables at entry. Mean ferritin levels at entry were comparable for all patients that subsequently acquired versus did not acquire cancer (p = 0.314) and for patients randomized to either control or iron reduction that later acquired cancer (p = 0.927). Mean ferritin levels during follow-up in patients randomized to iron reduction that acquired cancer were comparable to levels in control patients (p=0.428) while follow-up ferritin levels were significantly lower in patients not acquiring cancer (p=0.017).

Conclusions: Randomization to iron reduction was associated with a significantly lower cancer risk and mortality during follow-up. Patients randomized to iron reduction that acquired cancer were relatively non-compliant with intervention. These findings invite further study.
Oxidative Stress Contributes to Hemolysis in Hereditary Spherocytosis.
Authors: Ghoti Hussam , Amer Johnny , Shaaban Mohammad Abu , Draz Samir Abu , Issah Abd El Rahman , Fibach Eitan , Rachmilewitz Eliezer A.
Published: April 2020

We have previously shown that oxidative stress is generated in pathological red blood cells (RBC) in congenital (thalassemia, sickle cell anemia) and acquired (myelodysplastic syndrome and paroxysmal nocturnal hemoglobinuria) hemolytic anemias. In the present study we tested whether a similar mechanism occurs in hereditary spherocytosis (HS) which is a heterogeneous group of disorders, where RBC have short survival due to primary deficiency in spectrin, ankyrin-1, band 3 or protein 4.2 in their membranes. Secondary protein deficiencies resulting from oxidative stress are often observed and may be involved in the outcome of the disease. Seventeen patients from 7 families with clinical and laboratory diagnosis of mild to severe HS were examined. All patients were anemic, (Hb ranging from 5.3 to 10.5 g/dl) and 7 were splenectomized. Twelve patients received 5 to 58 units of blood. The diagnosis was based on spherocyte morphology, increased MCHC (33–38 g/dl), decreased MCV (<80 fl in 11 patients) and increased osmotic fragility (>75%) in all of them. Using flow cytometry techniques, we showed that RBC from HS patients generate high amounts (1.65-fold) reactive oxygen species (ROS), as well as membrane lipid peroxides (LP) (3.04-fold), and lower levels (2.05-fold) of the cellular anti-oxidant reduced glutathione (GSH) compared with RBC obtained from normal donors of matched gender and age (Figure). In order to determine whether oxidative stress plays a role in hemolysis in HS, we incubated HS-RBC with their autologous plasma and added antioxidants such as N-acetylcystein, vitamin C and the vitamin E derivative, tocotrinol, each at 1 mM, for 12 hours. The results showed that the antioxidants protected HS-RBC from lysis. Our results provide a direct evidence for oxidative stress in RBC from HS patients similar to findings in other hemolytic anemias which contribute to the magnitude of their lysis, and suggest that treatment with antioxidants may attenuate some of the clinical symptoms of these patients.

Figure Figure
Measurement of Urinary Hepcidin Levels by SELDI-TOF-MS in HFE-Hemochromatosis.
Authors: Bozzini Claudia , Campostrini Natascia , Trombini Paola , Nemeth Elizabetha , Castagna Annalisa , Tenuti Ilaria , Corrocher Roberto , Camaschella Clara , Ganz Tom , Olivieri Oliviero , Piperno Alberto , Girelli Domenico
Published: April 2020

Introduction: An insufficient production of hepcidin, the master regulator of iron metabolism, is recognized as the key pathogenetic feature of HFE-related hereditary hemochromatosis (HH). There is a growing interest in measuring the hepcidin levels, which may improve diagnosis, prognostic evaluation and clinical management of HH. Nevertheless, few investigative tools are available: an immunodot method for urinary hepcidin developed by a single centre (UCLA), not yet ready for large-scale diffusion, and mass spectrometry (MS) based assays, such as surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF-MS). The latter is well suited to small peptides like hepcidin, and can rapidly analyze crude samples with high throughput. Until now, urinary hepcidin has been measured by SELDI-TOF-MS only in small groups of C282Y homozygous patients, the majority of them under phlebotomy treatment. No data are available on C282Y/H63D compound heterozygotes, that can develop a milder clinical form of HH. This study was aimed to measure urinary hepcidin levels by SELDI-TOF-MS in a large group of HH patients.

Methods: We used a protocol based on PBSIIc mass spectromer and Normal Phase chips similar to that recently proven successful for semi-quantitative detection of urinary hepcidin. Urinary samples from 30 control subjects were compared to those obtained from 80 HH patients (57 C282Y homozygotes, 23 C282Y/H63D compound heterozygotes). Eighteen C282Y homozygotes and 11 C282Y/H63D compound heterozygotes were analyzed at diagnosis, the remainder during maintenance phlebotomy (at least 30 days from last phlebotomy).

Results: C282Y homozygotes had significantly lower urinary hepcidin levels vs. controls either at diagnosis, or after phlebotomy (P < 0.05). C282Y/H63D compound heterozygotes had hepcidin levels at diagnosis similar to controls, while the hepcidin:ferritin ratio was significantly decreased (P < 0.001) suggesting a relatively inappropriate hepcidin production. Moreover, also in this group means hepcidin levels after phlebotomy were significantly lower than in controls (P < 0.001). Samples from 12 randomly selected control subjects were sent to UCLA for duplicate measurement by the immunodot method, yielding a good correlation (r= 0.77; P<0.0001).

Conclusions: SELDI-TOF-MS assay is confirmed to be a potentially useful tool for measuring hepcidin levels in HH. The very low hepcidin levels observed in both genotypes after phlebotomy, may suggest that iron depletion as currently achieved by standard protocols may not be the best therapy from a pathophysiological standpoint.
Atiprimod Inhibits the JAK-STAT Pathway and Induces Apoptosis in Human Cells Carrying the JAK2V617F or JAK3 Mutation.
Authors: Manshouri Taghi , Golemovic Mirna , Kantarjian Hagop M.M. , Cortes Jorge E. , Zhang Ying , Priebe Waldemar , Estrov Zeev , Verstovsek Srdan
Published: April 2020

Janus Kinases (JAK), including JAK1, JAK2, TYKa, and JAK3, are a small family of cytoplasmic protein tyrosine kinases, which play an important role in the initiation of cytokine-triggered signaling events by signal transducer and activator of transcription (STAT) proteins, via phosphorylation. The recent reports of an activating somatic mutation in codon 617 of the gene encoding JAK2 (JAK2V617F) in patients with myeloproliferative disorders (MPDs) and 3 mutations in JAK3 (A572V, V722I, P132T) in acute megakaryoblastic leukemia patients, has opened new avenues for the development of targeted therapies for these malignancies. We report here the activity of Atiprimod, a novel compound with anti-inflammatory properties, in retrovirus-transduced JAK2 (JAK2V617F) mutant-expressing murine FDCP-EpoR cells, set-2 cells, CMK cells, and blood cells from patients with polycythemia vera (PV). We compared the growth inhibitory effect of Atiprimod against two mouse FDCP cell lines transfected with erythropoietin receptor (Epo-R), and either wild-type JAK2 (JAK2WT) or mutant JAK2 (JAK2V617F), and human megakaryoblastic leukemia cells with mutated JAK2V617F (set-2 cells) or mutated JAK3 (CMK cells). The growth inhibitory effect was assessed using 3-days MTS assay. Atiprimod was more potent against FDCP cells carrying mutant JAK2 (JAK2V617F) cells (Ic50 0.42 μM) and set-2 cells (Ic50 0.53 μM) than the CMK cells with mutant JAK3 (Ic50 0.79 μM) and FDCP wild-type JAK2 (JAK2WT) cells (Ic50 0.69 μM). Atiprimod, inhibited the phosphorylation of JAK2 and downstream STAT3, STAT5, and AKT proteins in a dose-and time-dependent manner. It exerted its inhibitory activity against cells with mutant JAK2 (JAK2V617F) by affecting the cell cycle progression and induced apoptosis, as evidenced by increase in mitochondrial membrane potential and caspase3 activity. Atiprimod also cleaved PARP protein, increased turnover of the anti-apoptotic X-linked mammalian inhibitor of apoptosis (XIAP) protein, and inhibited the apoptotic protein BCL2 in a time- and dose-dependent manner. In addition, Atiprimod inhibited the proliferation of peripheral blood hematopoietic progenitors from three patients with PV carrying the JAK2 (JAK2V617F) mutation as compared to hematopoietic progenitors from normal individuals, by 2-days MTS assay (p= 0.0013). Our data suggest that Atiprimod is active both in vitro and ex vivo against cells with activated tyrosine kinase of the JAK family.
Analysis of T-Cell Leukemia/Lymphoma (TCL1) Oncoprotein Interacting Proteins by Immunoprecipitation and Tandem Mass Spectrometry.
Authors: Elenitoba-Johnson Kojo S.J. , Basrur Venkatesha , Kronk Adam , Seiler Charles , Fermin Damian , Nesvizhskii Alexey , Lim Megan S.
Published: April 2020

TCL1 is a proto-oncogene whose deregulation has been implicated in the pathogenesis of T- and B-cell lymphoproliferative disorders. TCL1 expression is predominantly observed in developing B lymphocytes, whereas its overexpression in T cells is associated with mature T-cell lymphoproliferation in transgenic mice. Although recent studies indicate that dysregulated expression of TCL1 is important in mature B-cell transformation, very little is currently known about the function or interactions of TCL1, specifically in the B-cell context. In this study, we hypothesized that identification of proteins that interact with TCL1 may facilitate our understanding of the functional properties of TCL1. Proteomic analysis provides an opportunity to carry out functional studies of protein-protein interactions and characterization of functional interactomes. Using a functional proteomic approach, we determined the identity of proteins that interact with TCL1 by co-immunoprecipitation with anti-TCL1 antibody followed by liquid chromatography (LC), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS). Immunoprecipitates of the TCL1 expressing SUDHL-16 B-cell lymphoma derived cell line were compared to that of hyperimmune rabbit immunoglobulin and a cell line that does not express TCL1 (SUDHL-1) by 1-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Silver-stained protein bands from both immunocomplexes were excised and analyzed by ESI-LC-MS/MS. Proteins which were unique to the TCL1 immunocomplex included transcription factors (Jun B), phosphatases (protein phosphatase 2), ubiquitin-associated proteins (ubiquitin activating enzyme), cell adhesion proteins (ADP ribosylation factor 3) and proteins associated with apoptosis regulation (peroxidoxin 1 and scaffold attachment factor). Analysis of subcellular fractions demonstrated that TCL1 and complexes were localized to both the nuclear and cytoplasmic compartments. Importantly, known TCL1 interactors such as histone 3 were uniquely identified in the TCL1 complex. Proteins identified by MS were confirmed by western blotting and reciprocal immunoprecipitation. This study reveals novel TCL1 interactors and indicates that the protein may exert diverse cellular effects impacting apoptosis, cell survival, cytoskeletal organization and cell adhesion. Our interactome analysis provides unique insights into the cellular function of TCL1, a protein whose deregulation is increasingly implicated in the pathogenesis of a variety of hematopoietic neoplasms.
Improving Survival in Multiple Myeloma: Impact of Novel Therapies.
Authors: Kumar Shaji , Rajkumar S. Vincent , Dispenzieri Angela , Lacy Martha Q. , Hayman Suzanne R. , Buadi Francis K. , Zeldenrust Steven R. , Dingli David , Russell Stephen , Lust John A. , Greipp Philip R. , Kyle Robert A. , Gertz Morie A.
Published: April 2020

Background: Recent advances in the therapy of multiple myeloma (MM) have greatly increased the treatment options for this uniformly fatal plasma cell malignancy. It is not clear if introduction of novel therapies and the increased use of high dose therapy (HDT) in the past decade have translated into better outcome for patients (pts) with MM.

Methods: We examined the outcome of two cohorts of pts seen at our institution. The first cohort consisted of 387 pts who relapsed after HDT and was examined for potential improvement in survival following relapse after HDT. These pts were divided into two groups; those who relapsed before or after December 31, 2000. The second cohort consisted of 2981 patients with newly diagnosed MM seen between January 1971 and December 2006 and was used to examine the trends in overall survival (OS).

Results: Among those relapsing after HDT, there were 245 males (63%); median (range) age at HDT was 57 years (32.8–75.4) and the median time to HDT was 8.1 months (1–90 months) from diagnosis. The median time to relapse was 13.2 months (1.1 months-10.3 years) from HDT. In this cohort, a clear improvement in OS from time of relapse was seen over the past decade, with those relapsing after 2000 having a median OS of 23.9 months (95% CI; 19.8, 27.6) compared to 11.8 months (95% CI; 8.7, 14.9) for the rest (P < 0.001) (Figure 1). In a multivariate analysis, the effect of the date of relapse on survival was independent of other prognostic factors such as relapse <12 months after HDT, abnormal cytogenetics, and B2M > 5.5 mg/dL. Pts who were treated with one or more of the newer drugs (thalidomide, lenalidomide, bortezomib) had longer survival from relapse (30.9 months (95% CI; 23.6, 38.2) compared to 14.8 months (95% CI; 11.3, 18.4); P < 0.001) for others. Among the newly diagnosed MM cohort, the median age at diagnosis was 66 years (20.2 – 97 years), and 1,770 (59.4%) were males. The median follow up for the entire group was 27.4 months (0–29.4 years); and at the time of analysis 558 patients (18.7%) were alive with a median follow up of 32.7 months (0–29.4 years). Pts diagnosed in the last decade had an improved OS (44.8 months) compared to those diagnosed before this period (29.9 months; P < 0.001) (Figure 2). The improvement in survival seen in the last decade among newly diagnosed patients was predominantly among those younger than 65 years (60.3 mos vs. 33.3 mos improvement in median survival); compared to those over 65 at diagnosis (26.5 mos vs. 32 mos).

Conclusion: In this study, for the first time, we demonstrate definite proof for improved outcome in patients with myeloma, both in the relapsed setting as well as at diagnosis, a change that is likely to continue with increased use of these drugs.

Figure Figure Figure Figure
The International Prognostic Scoring System for Waldestrom’s Macroglobulinemia (ISSWM) Is Applicable in Patients Treated with Rituximab-Based Regimens.
Authors: Dimopoulos Meletios Athanasios , Kastritis Efstathios , Gika Dimitra , Delimpassi Sossana , Zervas Konstantinos , Simeonidis Argiris , Eleftherakis-Papaiakovou Evangelos , Anagnostopoulos Nicolaos , Michali Evridiki , Kyrtsonis Marie Christine , Terpos Evangelos , Pangalis Gerassimos
Published: April 2020

Introduction: An ISSWM was recently proposed (Morel et al, ASH 2006), which was based on a large number of patients treated primarily with alkylating agents and /or nucleoside analogues. The ISSWM based on 5 adverse covariates wich defined 3 risk groups: low, intermediate and high risk with 5-years survival rates of 87%, 68% and 36% respectively. In our current analysis, we assessed the impact of this system in patients with WM who received primary treatment with rituximab-based regimens.

Patients and methods: Ninety-three previously untreated, symptomatic patients who received treatment either with single agent rituximab (21 patients) or with the combination of dexamethasone, rituximab, and cyclophosphamide (72 patients) were classified according to the ISSWM, which is based on 5 adverse covariates: age> 65 years, hemoglobin ≤11.5 g/dl, platelet count ≤ 100 x 109/L, β2- microglobulin <3mg/L, serum monoclonal protein concentration >70g/L. Low risk is defined by the presence of ≤ 1 adverse characteristics except age, high risk by the presence of >2 adverse characteristics and intermediate risk by the presence of 2 adverse characteristics or age >65 years.

Results: The disease features of the 93 patients were typical of symptomatic WM: age > 65 years in 63%, males 65%, B-symptoms in 22%, splenomegaly in 29%, lymphadenopathy in 34%. 15% of patients were rated as low risk, 65% as intermediate risk and 20% as high risk. Criteria for initiation of therapy included cytopenia, hyperviscosity, constitutional symptoms, organomegaly or IgM-related disorders. Overall, 62% of patients were alive at 6 years. Median survival was not reached for low and intermediate risk and was 38 months for high risk patients (p=0.006). There was a clear separation of the survival curves in the three groups. At the time of last follow-up the percentage of patients alive was 100%, 82% and 58% for patients classified as low, intermediate and high-risk group respectively.

Conclusions: The recently proposed ISSWM is applicable in patients with WM who receive primary treatment with rituximab-based regimens and may serve as a basis to compare outcomes in different studies.
Suppression of Markers of Thrombin Generation and Inflammation in Patients Receiving Low Molecular Weight Heparin Compared to Unfractionated Heparin for TEE-Guided Cardioversion of Atrial Fibrillation.
Authors: Hoppensteadt Debra , Fareed Jawed , Klein Allan , Jasper Susan , Apperson-Hansen Carolyn , Lieber Elizabeth , Katz William , Malouf Joseph , Stoddard Marcus
Published: April 2020

Introduction: Atrial fibrillation (AF) represents a cardiovascular syndrome which can lead to embolic stroke. Several AF clinical trials have shown a decrease risk of stroke with anticoagulation using warfarin. Currently, there are two approaches for anticoagulation in AF patients. For outpatients, warfarin is used with an INR goal 2.0–3.0. For inpatients requiring cardioversion, unfractionated heparin (UFH, IV) is used as a bridge to therapeutic warfarin. More recently, anticoagulation with a low molecular weight heparin, enoxaparin, has been reported to give a more predictable anticoagulant response than UFH in TEEguided cardioversion. The present study compares the markers of inflammation and thrombin generation in patients included in the ACUTE II study.

Methods: This was a randomized multicenter trial of 155 patients from 17 clinical sites, the anticoagulant activity of LMWH (enoxaparin, 1 mg/kg sc bid, Sanofi-Aventis, n=76) was compared to that of UFH (APTT 1.5 – 2.5 x control, n=79). Blood samples were drawn at enrollment (baseline), day 2 (peri cardioversion) and day 4 in both groups. Day 2 and day 4 samples were taken 3–4 hours after the injection of LMWH in patients in the LMWH group. Blood samples were evaluated for inflammation: C-reactive protein (CRP), CD 40 ligand (CD 40L), monocyte chemotactic protein 1 (MCP-1) and anticoagulant effect: thrombin antithrombin complex (TAT) and prothrombin fragment (F 1.2).

Results: The APTT and anti-Xa levels indicated therapeutic anticoagulation was achieved (previously reported). In addition, both TAT and F1.2 levels were increased at baseline and were significantly decreased with LMWH (p<0.01) in comparison to UFH. The CRP, CD40L, MCP-1 were decreased after treatment in both groups of patients. These results suggest that AF patients treated with LMWH demonstrate a stronger anticoagulant effect as measured by a significant reduction in the markers of thrombin generation. Furthermore, anticoagulation with either UFH of LMWH results in a decrease in inflammation which was not different between groups.

Conclusion: In the ACUTE II trial, anticoagulation with LMWH in AF patients may be a better alternate than UFH during TEE-guided cardioversion due to a a stronger anticoagulant.
Prothrombotic Platelet Signaling by the Scavenger Receptor, CD36.
Authors: Chen Kan , Febbraio Maria , Silverstein Roy
Published: April 2020

The scavenger receptor CD36 binds a broad array of ligands, including oxidized low density lipoprotein (oxLDL), thrombospondin-1, fatty acids and apoptotic cells. CD36 was first isolated and characterized structurally from platelets, but the functional role of CD36 on platelets remains relatively obscure. We previously determined that treating platelets with oxLDL activated platelets and that activation was not seen in CD36-null platelets. Using a pharmacological inhibitor we now show that inhibition of JNK MAP kinase abrogated the activation of platelets by oxLDL. This effect was specific to oxLDL-mediated activation because this inhibitor had minimal effect on platelet activation by other classic agonists as exemplified by adenosine diphosphate (ADP). We demonstrated by immunoblotting that JNK2 and its upstream activator MKK4 were phosphorylated in the presence of oxLDL. We also found that the increase of JNK2 phosphorylation by oxLDL was diminished in CD36-null platelets. We showed that a src family kinase inhibitor (AG1879) blocked both platelet activation and JNK2 phosphorylation upon oxLDL treatment. By co-immunoprecipitation we demonstrated that CD36 recruited “active” fyn and lyn in platlets upon oxLDL treatment. These studies suggest that CD36 ligands can activate platelets through a signaling cascade involving src family tyrosine kinases and MAPK signaling molecules such as MKK4 and JNK2. OxLDL forms in the setting of hyperlipidemia and inflammation and plays an important role in atherosclerosis. A common characteristic of atherosclerosis is a prothrombotic state. Our results suggested that a specific signaling cascade activated by CD36 ligands generated in pathological states may contribute to a prothrombotic phenotype in vivo.
Overexpression of the Plasma Lysophospholipase D Autotaxin Reveals a Novel Pathway for Regulation of Hemostasis in Mice.
Authors: Federico Lorenzo , Pamuklar Zehra , Liu Sue , Mills Gordon , Morris Andrew , Smyth Susan
Published: April 2020

The cell-motility factor autotaxin (autotaxin/lysoPLD or Enpp2) is an enzyme with both lysophospholipase D (lysoPLD) and nucleotide pyrophosphatase/phosphodiesterase activities and has recently been identified as a critical component of the major pathway for generation of the bioactive lipid mediator lysophosphatidic acid (LPA) in the blood. Transgenic FVB mice overexpressing autotaxin/lysoPLD under the control of the alpha1-antitrypsin promoter (Enpp2-Tg mice) exhibited elevated plasma autotaxin/lysoPLD protein, lysoPLD activity and LPA levels, with no detectable alteration in plasma levels of ATP, ADP, and adenosine. Ennp2-Tg mice displayed a pronounced bleeding diathesis characterized by markedly prolonged tail bleeding time (>10 min versus 3.2 ± 2.7 min in wild-type mice; p <0.001) and variable protection from thrombosis. Ennp2-Tg mice did not form occlusive thrombus in the carotid artery within 30 min following application of ferric chloride (versus a time to thrombotic occlusion of 9 ± 2 min in wild-type mice; p<0.001), and histologically, only mural thrombus was present along the ferric chloride-treated carotid arteries of the Ennp2-Tg mice. However, intravascular thrombosis in response to intravenous injection of collagen + epinephrine was normal in Ennp2-Tg mice. Enpp2-Tg mice exhibited normal platelet counts (928 ± 184 versus 808 ± 156 x 103/mm3), and normal platelet membrane protein expression, platelet aggregation, and coagulation parameters in ex vivo studies. Although LPA is a weak activator of platelets isolated from the majority of human donors, we found that treatment of isolated murine platelets with physiologically relevant levels of LPA (1μM) attenuated ADP (1μM)-induced and low-dose thrombin (0.01 – 0.05U/ml)-induced aggregation, but did not alter aggregation elicited by higher doses of thrombin or collagen. Treatment of murine platelets with LPA also elevated intraplatelet cAMP levels. Moreover, systemic administration of LPA to mice recapitulated the prolonged bleeding time observed in Enpp2-Tg mice. These results confirm the importance of autotaxin/lysoPLD as the main determinant of circulating LPA levels and suggest a novel pathway for regulation of murine hemostasis and thrombosis involving the autotaxin/lysoPLD-LPA axis.
Lyn and Fyn Kinases Positively Regulate Thromboxane Generation in Platelets.
Authors: Bhavaraju Kamala , Kim Soochong , Kunapuli Satya P.
Published: April 2020

Src family Kinases (SFKs) are important tyrosine kinases in platelets. The family consists of 9 members (viz., Blk, Fgr, Fyn, Hck, Lck, Lyn, Src, Yes and Yrk) and many of the isoforms are expressed in platelets. SFKs are key kinases in glycoprotein (GPVI) mediated platelet activation and we have shown that these kinases play an important role in thromboxane generation. Until now, the role of specific SFKs downstream of G protein signaling in platelets is not well understood. In the present study we characterized functional roles of specific SFK members Fyn, Lyn, Lck and Src Kinases downstream of ADP receptors. Src, Lyn and Fyn are activated downstream of ADP receptors (P2Y) receptors in a time and concentration dependent manner. The presence of fibrinogen receptor antagonist, GR144053, did not affect the activation of Src and Fyn; however, Lyn is not activated in the presence of GR144053, suggesting that Lyn requires outside-in signaling for it’s activation. On the other hand, Lck kinase is not activated downstream of ADP receptors. ADP activates P2Y1 and P2Y12 receptors which in turn couple to Gq and Gi, respectively. In order to further delineate the Src activation pathways we used P2Y1 and P2Y12 receptor antagonists MRS 2179 and AR-C69931MX respectively. Src activation is not inhibited by either P2Y1 or P2Y12 receptor antagonists, suggesting that both receptors independently contribute to the activation of Src kinase. Platelets from mice deficient in Src, Fyn or Lyn were analyzed for thromboxane generation upon stimulation with ADP. Lyn and Fyn KO mice had reduced levels of thromboxane A2 compared to wild type littermates. However, thromboxane generation from platelets lacking Src was unaffected. Hence, we conclude Lyn and Fyn kinases, but not Src, positively regulate thromboxane generation downstream of ADP receptors. We also conclude that Lyn requires outside-in signaling for its activation.