bluuebunny/crossref_metadata_2025 huggingface dataset:
Genistein Promotes Apoptosis, Differentiation and Cell Cycle Arrest in All Trans Retinoic Acid (ATRA) Sensitive and Resistant Acute Promyelocytic Leukemia Cells.
Authors: Khan Matiullah , Ng Angela Ping Ping , Jing Shen , Chen Chien-Shing , Ishii Shunsuke
Published: April 2020

PML, a suppresser of growth and proliferation in many cell types including hematopoetic cells, is localised to nuclear dot like structures known as PML oncogenic domains (POD). PML plays a crucial role in POD dependent and caspase 3 mediated apoptosis. PML-RAR, the causative agent of Acute Promyelocytic Leukaemia (APL), induces the disintegration of PODs, while Retinoid Acid (RA) mediated remission of APL is associated with reorganisation of PODs. These findings led to the concluation that disorganisation of POD is linked with the transforation of APL blasts. We have reported that nuclear hormon receptor co-repressor (N-CoR) is a component of PODs and is essential for PML mediated tumour suppressive function of Mad and Rb. (Khan MM et al. J Biol Chem.279:11814-24). Our most recent findings demonstrate that fusion protein PML-RAR triggers the ER stress and Unfolded Protein Response (UPR) by causing mis-folding and insoluble aggregation of N-CoR protein. (Khan MM et al. J Biol Chem.279:11814-24). These findings suggest that targeting the insoluble aggregation of N-CoR and the ER stress could represent an attractive therapeutic strategy for APL. Based on the hypothesis, we screened compounds known for their inhibitory effect on protein aggregation and ER stress. Among the compounds tested, genistein, a flavonoid, exhibited significant growth inhibitory effect on both RA sensitive and RA resistant APL cell NB4 in dose and time dependant manner (see fig.1 & 2). Genistein inhibited the growth on NB4 cells through collective effects on the cell cycle progression, apoptosis and differentiation. Genistein induced apoptosis in NB4 cells was mediated by the activation of caspase-3 and 9. Genistein also induced differentiation of NB4 cells as marked by appearance of cell surface differentiation marker CD11b. In cell cycle analysis, NB4 cells were accumulated in the G2-M phase after genistein treatment. In Western blotting assay, genistein unregulated and stabilized the level of PML protein in NB4 cells while promoting the degradation of PML-RAR. In protein solubility assay, genistein enhanced the solubility of N-CoR protein and reorganized the micro speckled like distribution of POD to normal dot like pattern in NB4 cells by immunocytochemical assay. In this study, we also identified that genistein abrogates PML-RAR mediated tyrosine phosphorylation of N-CoR protein. These findings, for the first time, have identified the therapeutic potential of genistein in both ATRA sensitive and resistant APL cells.
WP-1034, a Novel Jak-Stat Inhibitor, Has Proapoptotic and Antileukemic Activity in Acute Myeloid Leukemia (AML) Cell Lines and AML Patient Samples.
Authors: Faderl Stefan , Ferrajoli Alessandra , Harris David , Van Quin , Priebe Waldemar , Estrov Zeev
Published: April 2020

Proliferation and growth of AML cells result from stimulation by cytokines and high levels of cytokines are associated with poor prognosis in AML. Cytokines act through cellular receptors that are associated with members of the Jak family of protein tyrosine kinases. Upon phosphorylation and activation of Jak, proteins bound to Jak initiate signaling pathways including those regulated by Stat proteins. Since constitutive activation of Jak-Stat has been associated with leukemogenesis, we hypothesize that inhibition of Jak-Stat inhibits proliferation of AML cells. To do this, we studied the effects of WP-1034, a novel and potent inhibitor of Jak-Stat, in the OCIM2 AML cell line and fresh samples from AML patients. OCIM2 cells were deprived of serum for 2 hours and then incubated with 1 to 5 μM WP-1034 to investigate its effect on OCIM2 cell proliferation. After incubation of the cells without and with 1, 2.5, 5, 7.5, and 10 μM WP-1034 for 1 hour, and at 5 μM for 0, 20 min, 40 min, and 1, 2, 3, and 4 hours, we determined expression of Stat 1, 3, and 5, as well as Phospho-stat 1, 3, and 5 in the cells by Western Immunoblotting. In addition, we analyzed cell cycle status by PI staining and flow cytometry. We further evaluated induction of apoptosis of OCIM2 cells following incubation with WP-1034 at 3, 5, and 6 μM using the annexin V-CY5 assay and analyzed caspase 3 and PARP cleavage using Western Immunoblotting. To demonstrate the effect of WP-1034 on marrow cells from AML patients and healthy volunteers we incubated marrow cells with WP-1034 at increasing concentrations and used the blast colony assay to measure inhibition of proliferation. Our results show that: i) WP-1034 effectively inhibits proliferation of OCIM2 cells and AML blast proliferation from patient samples; ii) WP-1034 blocks activation of Stat 3 and 5 by decreasing the amount of Phospho-stat 3 and 5 in OCIM2 cells; iii) WP-1034 causes cell cycle arrest in sub-G0 phase and is able to induce apoptosis in OCIM2 cells; and iv) WP-1034 induces apoptosis involving cleavage of caspase 3 and PARP. Our data suggest that WP-1034, a potent inhibitor of Jak-Stat, inhibits proliferation of AML cells by inhibition of Stat 3 and 5 and induction of caspase-dependent apoptosis.
Inhibition of p53-MDM2 Interaction by Small-Molecule Antagonist of MDM2 Effectively Induces Apoptosis in Leukemias.
Authors: Kojima Kensuke , Konopleva Marina , Shikami Masato , Cabreira-Hansen Maria , Jackson C. Ellen , Shpall Elizabeth J. , Kornblau Steven M. , Vassilev Lubo T. , Andreeff Michael
Published: April 2020

Alteration of the p53 gene is one of the most frequent events in human tumorigenesis and about 50% of all solid tumors have been reported to carry p53 mutations. The inactivation of p53 in cancer has been associated with poor survival, refractory disease and chemoresistance. p53 mutations rarely occur in hematopoietic malignancies. Instead, MDM2 which is a principal cellular antagonist of p53, is overexpressed in the majority of leukemias. Recently, potent and selective small-molecule antagonists of MDM2, Nutlins, have been identified (Science303:844–888, 2004). Nutlins bind MDM2 in the p53-binding pocket and activate the p53 pathway in human cancer cells with wild-type p53, leading to cell cycle arrest, apoptosis, and growth inhibition of human tumor xenografts in nude mice. In this study, we investigated the potential antileukemic activity of the MDM2 antagonist. Treatment of wild-type p53 OCI-AML-3 cells with 5 μM of an active compound (Nutlin-3a) induced cell cycle arrest and apoptosis as evidenced by flow-cytometric analysis (51% reduction of S-phase at 12 h, 27% sub-G1 DNA content and 57% Annexin V positivity at 48 h). Similar proapoptotic effects were observed in MOLM-13 cells which have wild-type p53, but not in p53-null (HL-60 and U937) or mutant p53 (Raji, Jurkat and NB-4) cells. Nutlin-3a induced apoptosis in a dose- and time-dependent manner, and induced maximal effect on cell cycle arrest at 1 μM. Western blot analysis showed that in OCI-AML-3 cells, wild-type p53 accumulated at 1 h after exposure to Nutlin-3a. Increased levels of MDM2, p21 and Noxa proteins were observed at 1 to 3h. This resulted in cleavage of caspase-9 followed by cleavage of caspase-3. A pharmacologic interaction study between MDM2 inhibitor and Ara-C using a fixed-ratio (1:1) experimental design showed highly synergistic cell growth inhibition (CI = 0.44) and induction of apoptosis (CI = 0.83) in OCI-AML-3 cells. Initial studies conducted in primary leukemia cells demonstrated that Nutlin-3a induced apoptosis in 4 of 5 AML samples tested (68–97% Annexin V induction and 65–93% cell number reduction) and 2 CLL samples (>50% Annexin V induction and 37% and 58% cell number reduction). Since MDM2 protein is overexpressed and p53 is not mutated in the majority of primary leukemia cells, this approach may have therapeutic utility in leukemias.
MLL-AF4 Down-Regulates CDKN1B (P27) Independent of Cell Cycle Progression.
Authors: Xia Zhenbiao , Popovic Relja , Lorenz Tara , Santillan Donna , Erfurth Frank , Zeleznik-Le Nancy J.
Published: April 2020

The MLL gene, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has more than forty known partner genes with which it is able to form in- frame fusions. MLL fusion genes transform hematopoietic cells in vitro, and cause leukemia in mouse models. However, the mechanism is still not clear. Characterizing important downstream target genes may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored potential downstream target genes of the most prevalent MLL fusion protein, MLL-AF4, which is primarily associated with pro-B ALL and is involved in the majority of infant leukemia. To this end, we developed an inducible MLL-AF4 fusion cell line. Overexpression of MLL-AF4 does not lead to increased proliferation in this cell line, but rather, cell growth is slowed compared to similar cell lines inducibly expressing truncated MLL. To try to understand the reason for slower cell growth, we assayed for expression of several CDK inhibitors. We found that in the MLL-AF4 induced cell line, the amount of CDKN1B (cyclin-dependent kinase inhibitor P27) was dramatically decreased both at the RNA and protein levels, in contrast, the levels of CDKN1A (P21) and CDKN2A (P16) were unchanged. Interestingly, we did not observe an increased percentage of cells in S phase of the cell cycle. To explore whether CDKN1B might be a direct target of MLL-AF4, we employed chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. We observed that MLL-AF4 binds to the CDKN1B promoter in vivo and represses CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP assays in the MLL-AF4 leukemia cell line MV4-11. Our results suggest that the CDKN1B may be a downstream target of MLL-AF4, and that MLL-AF4 inhibits CDKN1B expression independent of cell cycle progression.
Genetic Dissection of Cooperating Mutations in BXH-2 Acute Myeloid Leukemia with and without Nf1 Gene Mutation.
Authors: Yin Bin , Walrath Jessica , Shannon Kevin M. , Wallace Margaret R. , Largaespada David A.
Published: April 2020

Loss of the NF1 (Neurofibromatosis Type 1) gene, a tumor suppressor, can cause myeloid diseases juvenile myelomonocytic leukemia (JMML), monosomy 7 syndrome (Mo7), and acute myeloid leukemia (AML). However, using knockout mice, it has been shown that loss of Nf1 expression in hematopoietic cells, by itself, does not lead to aggressive leukemia resulting instead in a relatively indolent myeloproliferative disease. Murine Leukemia Virus (MuLV) insertional mutagenesis in BXH-2 mice provides a model to dissect genetic alterations in AML. We have profiled proviral insertions in BXH-2 AML which do or do not have corresponding loss-of-function of Nf1. 197 PIS (68 from 25 Nf1-wild type AML and 129 from 55 Nf1-defective AML) were isolated. Nf1-defective AML were obtained from BXH-2 AMLs with proviral insertions into the endogenous Nf1 gene and AML that developed in leukemia-prone, heterozygous Nf1+/− BXH-2 mice. These latter AMLs develop faster than wild-type BXH-2 AMLs and show Nf1 gene LOH or proviral insertion into the wild-type Nf1 allele. These analyses led to 37 common proviral insertion sites (CIS), 13 of which have not been reported previously. Several of the CIS (including Lmo2, Cmyb, Meis1, Bcl11a, Spred2, Def8, Edg3, Hoxa9, and a novel Krab domain-zinc finger gene) were found repeatedly among the Nf1-defective group of AML. Expression of most could be detected in human JMML and CMML by RT-PCR, including BCL11A. Importantly, among the CIS we detected, PIS targeting Bcl11a were significantly enriched (p < 0.05) in Nf1-defective leukemia. Retroviral expression vectors for Bcl11a have been constructed and transduced into an immortalized Nf1-/- null myeloblast cell line. Growth assays show that the cumulative cell number of FACS-sorted Bcl11a-Nf1-/- cells increase by ~2.5 fold that of controls. BXH-2 provides a powerful genetic system to dissect Nf1-cooperating genetic events in tumorigenesis. Mutations at several novel common integration sites could be involved in development or progression of leukemia with NF1 gene inactivation. This work was supported by the National Cancer Institute (U01-CA84221-05) and the American Cancer Society (RPG LIB-106632) to DAL and by National Cancer Institute (R01 CA92095) and U.S. Dept. of Defense (DAMD17-97-1-7339) to MRW.
Induction of G1 Arrest and Apoptosis in a Conditional Expression Model of AML1/ETO: P53-Independent Upregulation of P21/WAF/CIP1.
Authors: Berg Tobias , Fliegauf Manfred , Pitako Jurij , Burger Jan , Abdelkarim Mahmoud , Guo Yalin , Staege Martin S. , Burdach Stefan , Lübbert Michael
Published: April 2020

Background: The translocation (8;21) is the most common chromosomal rearrangement in AML, resulting in the expression of the fusion protein AML1/ETO. We have developed an ecdysone-inducible U937 model, in which AML1/ETO is expressed in response to treatment with Ponasterone (Pon) A (Fliegauf et al, Oncogene 2004). This model system was used to determine the cellular effects of AML1/ETO and to identify its target genes in U937 cells.

Methods: Effects of AML1/ETO expression upon cell growth, viability, cell cycle and apoptosis were analyzed by trypan blue exclusion, FACS analysis using propidium iodide and DiOC6 staining, DNA laddering and Western blot for PARP cleavage, respectively. The gene expression profile of U937 with and without conditional AML1/ETO expression was assessed using Affymetrix U133A microarrays. Wild-type U937 cells with and without PonA treatment as well as AML1/ETO-negative and AML1/ETO-positive myeloid cell lines served as controls. Northern and Western Blotting were used for validation of expression changes.

Results: Induction of AML1/ETO expression in U937 resulted in reduced cell growth, G1 arrest and in apoptosis beginning 48–72 hours after PonA treatment. To investigate the underlying mechanisms, microarray analysis was performed. Expression profiles of AML1/ETO-positive and AML1/ETO-negative cell lines formed distinct clusters. Based on stringent criteria, 191 different genes were found upregulated, whereas 37 were downregulated upon expression of AML1/ETO in U937. The identified genes were screened for genes with known functions in cell cycle and apoptosis by automated and manual review and included 13 apoptosis-related genes. Among them, the CDK inhibitor p21/WAF/CIP1 was upregulated 19-fold upon induction of AML1/ETO, whereas the apoptosis regulator MCL-1 was induced 2.5-fold. Based on our criteria, no differential expression of other transcriptionally-controlled apoptosis regulators (such as BCL2, BAX, BAK1, BAD or c-flip) was noted. Northern and Western Blot analysis confirmed the strong induction of p21/WAF/CIP1 that paralleled the expression of AML1/ETO 10 hours after PonA treatment. Induction of p21/WAF/CIP1 was independent of the tumor suppressor protein p53 (Dou et al., Proc. Natl. Acad. Sci. 1995), and by Western blot, p53 was undetectable in U937. Northern Blot analysis revealed a higher expression of p21/WAF/CIP1 in the AML1/ETO-positive cell lines Kasumi-1 and SKNO-1 than in the AML1/ETO-negative cell lines HL-60, KG-1 and U937, supporting our finding that AML1/ETO may induce p21/WAF/CIP1.

Conclusions: AML1/ETO expression resulted in increased expression of p21/WAF/CIP1, which might contribute to the observed growth arrest and induction of apoptosis caused by the conditional expression of AML1/ETO.
High Rate of Deep-Vein Thrombosis in Asia after Major Orthopedic Surgery: The AIDA Study Final Results.
Authors: Piovella Franco , Wang Ching-Jen , Lu Housan , Kenneth Lee , Lee Lai Heng , Lee Won Chul , Turpie Alexander G. , Gallus Alexander , Planes Andre , Passera Raffaella , Rouillon Alain ,   The AIDA Investigators
Published: April 2020

Background: The incidence of post-surgical venous thromboembolism is thought to be low in Asian populations and routine thromboprophylaxis is not implemented.

Objective: In this large multinational, multiethnic study, we evaluated the incidence of deep-vein thrombosis (DVT) patients undergoing major orthopedic surgery of the lower limbs.

Methods: We performed a prospective epidemiological study in 19 centers across Asia (China, Indonesia, South Korea, Malaysia, Philippines, Taiwan and Thailand) in patients undergoing elective total hip replacement (THR), total knee replacement (TKR) or hip fracture surgery (HFS) not receiving pharmacological thromboprophylaxis. The primary endpoint was the rate of DVT of the lower limbs objectively documented by mandatory bilateral ascending venography performed 6 to 10 days after surgery and evaluated by a blinded central adjudication committee.

Results: Overall, 407 Asian patients (20–99 years) undergoing THR (n=175), TKR (n=136) or HFS (n= 96) were recruited in 19 centres. 72.5% of the enrolled patients had adequate venograms. Total DVT was diagnosed in 121 of 295 evaluable patients (41.0%, [95% confidence interval: 35.4–46.7]). Proximal DVT was found in 30 patients (10.2% [7.0–14.2]). Total DVT and proximal DVT rates were the highest in TKR patients (58.1% and 17.1%, respectively), followed by HFS patients (42.0% and 7.2%, respectively), then THR patients (25.6% and 5.8%, respectively). DVT was more frequent in female patients aged at least 65 years. By-country analysis showed that the highest DVT rate was observed in Indonesia (78.6%) and the lowest in China (24.3%). By ethnic group, the lowest DVT rate was observed in Koreans (29.8%), while similar results were found in Chinese (45.0%) and in other non-Korean non-Chinese (46.3%) ethnic groups. Pulmonary embolism was clinically suspected in 10 of 407 patients (2.5%) and objectively confirmed in two patients (0.5%).

Conclusions: Venographically detected thrombosis is well recognized as an appropriate surrogate for symptomatic outcomes and is the basis for the recommendation for routine prophylaxis in Western patients. The high rate of venographic thrombosis in Asian patients is similar to that observed in patients in Western countries. Thrombosis prophylaxis should therefore be considered in Asian patients undergoing major orthopedic procedures.
Lipoprotein (A) and Further Prothrombotic Risk Factors in Women with Unexplained Recurrent Miscarriage- Results of a Multicenter Case-Control Study.
Authors: Nowak-Gottl Ulrike , Sonntag Barbara , Klamroth Robert , Heinecke Achim , Langer Claus , Scharrer Inge , Greb Robert , Krause Manuela
Published: April 2020

Background The present study was performed to assess the association of prothrombotic risk factors and underlying conditions, e.g. smoking and obesity, with unexplained recurrent miscarriage (uRM) in white women.

Methods and Results From 1998 to 2003, 133 Caucasian women aged 18–42 years (median 28 years) suffering from uRM were consecutively enrolled. In patients and 133 age-matched healthy controls prothrombotic risk factors (factor V (FV) G1691A, factor II (FII) G20210A, MTHFR T677T, 4G/5G plasminogen activator inhibitor (PAI)-1, lipoprotein (Lp) (a), protein C (PC), protein S (PS), antithrombin (AT), antiphospholipid/anticardiolipin (APA/ACA) antibodies) as well as associated environmental conditions were investigated. 70 (52.6%) of the patients had at least one prothrombotic risk factor compared with 26 control women (19.5%; p<0.0001). Body mass index (BMI; p=0.78) and smoking habits (p=0.44) did not differ significantly between the groups investigated. Upon univariate analysis the heterozygous FV mutation, Lp(a) > 30 mg/dL, increased APA/ACA and BMI > 25 kg/m2 in combination with a prothrombotic risk factor were found to be significantly associated with uRM. Associations of the heterozygous FII variant, MTHFR or 4G/4G PAI-1 genotypes, deficiency states of PC, PS and AT, as well as the combination of smoking with at least one prothrombotic risk factor did not reach statistical significance. In multivariate analysis, increased Lp(a) (odds ratio (OR): 4.7/95% confidence interval (CI): 2.0–10.7), the FV mutation (OR:3.8/CI:1.4–10.7), and increased APA/ACA (OR: 4.5/CI: 1.1–17,7) had independent associations with uRM.

Conclusion In Caucasian women uRM is associated with the presence of elevated Lp(a) or further prothrombotic risk factors.
Comparison of Compression Ultrasound vs Ascending Contrast Venography for Proximal DVT in Medical-Surgical ICU Patients.
Authors: Crowther Mark A. , Bates Shannon , Schiff David , Dobranowski Julian , Meade Maureen , McDonald Ellen , Geerts Bill , Cook Deborah
Published: April 2020

Background: Ascending contrast venography is the reference standard for diagnosing DVT. Observational studies suggest that compression ultrasound has 97% sensitivity and 94% specificity for proximal DVT in symptomatic patients. However, in asymptomatic patients, such as those in the medical-surgical ICU, the test properties of ultrasound may be substantially worse. Surveys, observational studies and professional documents show that many intensivists, Research Ethics Boards, and the American College of Radiology have rejected the routine use of venography in the ICU setting.

Objective: To compare the results of positive lower limb compression ultrasound with ascending contrast venography in medical-surgical ICU patients.

Design: One year longitudinal cohort study.

Setting: 15 bed medical-surgical closed ICU in Hamilton, Canada.

Methods: We enrolled consecutive patients age >18y with an expected ICU admission >72h. Exclusion criteria were trauma, orthopedic surgery, cardiac surgery, pregnancy or palliative care. Patients underwent bedside lower limb compression ultrasonography by a certified ultrasonographer on ICU admission, twice weekly, and upon clinical suspicion of VTE. Patients with a positive or nondiagnostic ultrasound underwent bilateral lower extremity ascending contrast venography, unless they had pre-specified contraindications: serum creatinine >150 umol/L, pre-existing insulin-requiring diabetes, known or suspected contrast allergy, or contraindications to transport to the Radiology Department. The study radiologist and thrombosis consultant independently interpreted the venograms in duplicate; disagreements were resolved by a third adjudicator.

Results: Of 261 patients, lower limb DVT was identified by compression ultrasound in 32 patients (7, 2.7% on ICU admission and 25, 9.6% during the ICU stay). Using the a priori venogram eligibility criteria, 8 of 32 patients (25.0%) with DVT by screening ultrasound were suitable for venography; 7 of the 8 venograms (87.5%) were positive.

Conclusions: Venogram eligibility criteria (e.g., low oxygen requirements, hemodynamic stability, and low risk for contrast nephropahthy) designed to safely confirm or refute positive or nondiagnostic screening ultrasound findings resulted in only selected, less seriously ill ICU patients undergoing venography. Concerns about the risk: benefit ratio of research interventions not used in current practice means that in multicenter trials, serial compression ultrasound will be the only safe and feasible method of screening for proximal DVT in the medical-surgical critically ill population.
Non-Genomic Effects of Estradiol on Human Platelets: Regulation of Mitogen Activated Protein Kinase Activation and Enhanced Spreading and Aggregation.
Authors: Vijayan Kadekuzhi V. , Liu Yan , Li Tong-Tong , Bray Paul F.
Published: April 2020

17β-estradiol or estrogen (E2) is a sex hormone that modulates platelet function and is widely used in hormone replacement therapy (HRT). We and others have previously demonstrated that human megakaryocytes and platelets posses estrogen receptors ERα and ERβ. HRT treatment augmented the bone marrow megakaryocytes without increasing other bone marrow cells, suggesting that E2 can modulate proliferation of megakaryocytes. Since mitogen activated protein kinases (MAPKs) are critical for 1) cell proliferation, 2) megakarytocyte differentiation and proplatelet formation and estrogen activates MAPK in other cell types, we hypothesized that estrogen regulates the activation of MAPK in human platelets. Signaling was studied using washed platelets from male and female subjects in response to varying concentrations of estrogen. Compared to the ethanol (vehicle) treated platelets, 1 nM E2 treated platelets for 60 seconds resulted in an enhanced activation of extracellular signal-regulated kinase 2 (ERK 2) and P38 but not Jun N-kinase (JNK). These results suggest that E2 can cause a non-genomic signaling in human platelets. The MEK inhibitors PD98059 and U0216 blocked the E2 effect, suggesting that the activation of ERK 2 was mediated through the upstream mitogen activated protein kinase kinase (MAPKK). Because E2 can modulate actin reorganization in other cell types and cell spreading is promoted by ERK 2 activation, we examined the effect of E2 on platelet spreading - a process not dependent on agonist stimulation. Compared to ethanol treated platelets, platelets preincubated with 100 nM E2 for 60 seconds and stained with rhodamine phallodine exhibited a ~60 % greater spreading at 5 and 15 minutes. This observation suggests that that E2 can cause rapid actin cytoskeletal reorganization in platelets. Since inhibition of ERK 2 activation blocks aggregation to low doses of thrombin and collagen, we examined a role for E2 in platelet aggregation. E2 alone did not induce platelet aggregation. However, E2 potentiated aggregation with low but not high doses of thrombin and collagen related peptide (CRP) (P=0.05 for 0.02 μg/ml thrombin and P<0.001 for 0.2 μg/ml CRP). Our data demonstrates that E2 can activate MAPK through a non-genomic mechanism and this activation correlates with greater platelet functions like spreading and aggregation. Our findings support a mechanism whereby a consistent non-genomic enhancement of platelet signaling and reactivity by E2 may underlie the increased cardiovascular events observed in recent randomized clinical trials with HRT.
Evaluation of Baseline Body-Weight Dosing of Yttrium 90 (90Y) Ibritumomab Tiuxetan (Zevalin®) Radioimmunotherapy in Patients with Relapsed or Refractory B-Cell Non-Hodgkin’s Lymphoma (NHL).
Authors: Wiseman Gregory , Conti Peter , Vo Katie , Schilder Russell J. , Foster Paul , Gordon Leo I. , Emmanouilides Christos , Silverman Dan , Witzig Thomas E. , Molina Arturo
Published: April 2020

Background: As the first radiolabled monoclonal antibody approved for the treatment for the treatment of cancer, yttrium 90 (90Y) ibritumomab tiuxetan (Zevalin®) achieves both high response rates and durable remissions in patients with relapsed or refractory, low-grade, follicular, or transformed B-cell NHL. Because of the stability of 90Y ibritumomab tiuxetan and its favorable pharmacokinetic profile (ie, predictable urinary clearance and absence of dehalogenation), dosing is based on patient weight and platelet counts. Yttrium 90 ibritumomab tiuxetan is typically delivered at a dose of 0.4 mCi/kg (in patients with platelets >150,000/mm3) or 0.3 mCi/kg (in patients with platelets ≤150,000/mm3) to a maximum recommended dose (MRD) of 32 mCi. However, patients >80 kg with platelet counts exceeding 150,000/mm3 may receive a lower drug concentration (ie, <0.4 mCi/kg 90Y) as a result of dose capping. We evaluated whether the 32 mCi dose cap influences the safety or efficacy of ibritumomab tiuxetan therapy in patients >80 kg.

Methods: Efficacy and safety data were collected for patients from 3 registrational trials that received either 0.4 mCi/kg 90Y (patients ≤80 kg) or 32 mCi 90Y (patients >80 kg).

Results: Clinical responses in 170 patients were evaluated. Patients ≤80 kg (n = 103) had a median weight of 70 kg (range, 45–80 kg) versus a median weight of 95 kg (range, 81–159) for patients >80 kg (n = 67). Gender (41% M vs 73% M, respectively; P <.01) was the only significantly different baseline characteristic between the ≤80 kg and >80 kg groups. Similar efficacy and safety results were reported for both subsets of patients. Overall response rates of 79% and 70% were observed for patients ≤80 kg and >80 kg, respectively (P =.27); and no significant differences were seen in complete response rates (28% vs 34%, respectively; P =.40). Median TTP (8.9 months vs 9.5 months; P =.53) and median DR (8.5 months vs 11.5 months; P =.34) were equivalent between the 2 weight-based groups. In addition, there were no statistically significant differences in safety measures including grade 3/4 nonhematologic adverse events, neutropenia, thrombocytopenia, or anemia.

Conclusions: As a consequence of dose capping at an MRD of 32 mCi, 39% of patients received a lower dose/kg of 90Y ibritumomab tiuxetan. However, despite the difference in dose administered on a unit of body weight basis, the efficacy and safety results were similar between patients ≤80 kg and >80 kg. We conclude that the 32 mCi dose cap does not influence the efficacy or safety profile of 90Y ibritumomab tiuxetan for patients >80 kg.
Upregulation of the Chemokine Receptor CCR4 in Cutaneous T-Cell Lymphoma and Nodal T-Cell Lymphoma: Implications for Targeted Therapy.
Authors: Jones Dan , Patel Kaushali , Herling Marco , Aguilar Richard J. , Ueda Ryuzo , Inagaki Hiroshi , Fayad Luis , Duvic Madeleine
Published: April 2020

The chemokine receptor CCR4 is expressed by a minor population of non-neoplastic T-cells, particularly Th2 helper T-cells, skin-homing T-cells and CD4+CD8− thymocytes. To assess the suitability of this receptor as a target for immunotherapy, we compared expression of CCR4 in a large series of T-cell malignancies with non-neoplastic T-cells by immunohistochemistry on formalin-fixed, paraffin-embedded material. Using tissue arrays and whole tissue sections, we assessed CCR4 levels in 233 biopsy samples from 182 patients with various T-cell tumors, particularly mycosis fungoides (MF, n = 54) and Sezary syndrome (SS, n = 15). We utilized the anti-CCR4 antibody KM2160 (Kyowa Hakko Kogyo, Tokyo, Japan), whose specificity has been established by immunoprecipitation and flow cytometry on CCR4-expressing transfectants and T-cell lines. Immunostaining was graded on a 4-tier scale (negative, focal, partial and uniform positivity) and compared with staining for markers of tumor progression including CD25, CD30 and the cytotoxic marker granzyme B and with a mediator of lymphocyte homing to skin, cutaneous lymphocyte antigen (CLA). Overall, CCR4 was expressed either partially or uniformly in 49% of T-cell tumors. Among MF/SS cases, partial or uniform CCR4 expression was found in 52% of cases, including in both early patch/plaque stage lesions and disseminated disease. CCR4 was upregulated upon large cell transformation in 39% of 23 MF/SS cases studied in sequential biopsies. There was an 80% concordance rate in CCR4 staining patterns in tumor cells in biopsies taken simultaneously from different anatomic sites. Upregulation of CCR4 upon large cell transformation was independent of other markers of progression, including CD25, CD30 and granzyme B but was significantly correlated with the degree of CLA expression. In contrast to the strong intensity of CCR4 staining in MF/SS cases, dimmer more variable expression was seen in skin homing T-cells in 94% of chronic dermatitis cases (n = 18). Among other T-cell tumors, CCR4 expression was common in AIL (70%), ALCL (53%) and PTCL unspecified (55%) and in the more mature subset of precursor-T lymphoblastic lymphoma/leukemia. CCR4-expressing T-cells were rare in 12 lymph node biopsies with reactive lymphadenitis. In conclusion, the chemokine receptor CCR4 is stably upregulated in cutaneous lymphoma and nodal T-cell lymphoma as compared to levels seen in non-neoplastic T-cells and thus represents a promising antigen for selective targeting of T-cell malignancies by immunotherapy.
Fas Down-Modulation in Epstein Barr Virus (EBV)-Specific Cytotoxic T-Lymphocytes (CTLs) Reduces Their Sensitivity to Fas/Fasl-Induced Apoptosis.
Authors: Dotti Gianpietro , Savoldo Barbara , Pule Martin , Straathof Karin C. , Biagi Ettore , Eric Yvon , Vigoroux Sthephane , Rooney Cliona M. , Brenner Malcolm K.
Published: April 2020

Adoptive immunotherapy with Epstein Barr Virus (EBV)-specific CTL has been successfully used to treat patients with EBV related malignancies including Post Transplant Lymphoproliferative disorder, Hodgkin’s lymphoma and Nasopharyngeal carcinoma. However, these and other potentially immunogenic tumors have evolved evasion strategies that subvert the effectiveness of the immune response. One such strategy involves expression of FasL, which likely impedes the host immune response by accelerating the apoptotic death of Fas-expressing tumor infiltrating T-cells. EBV-specific CTLs, like most effector memory T cells express high levels of Fas and are highly sensitive to cross-linking of the ligand. We therefore determined whether EBV-specific CTLs could be genetically modified to resist FasL-mediated immune evasion. We used retroviral siRNA (pSUPER.retro) directed against the Fas gene product to knockdown receptor expression in tumor-antigen specific CTLs. Transduction of CTLs with siRNA targeting Fas mRNA significantly knocked down Fas expression compared to control cells (Fas MFI 107 ± 47 vs. 402 ± 47, p<0.001). This effect was paralleled by a significant reduction of apoptosis induced by the Fas agonistic antibody (clone CH-11) in modified CTLs compared to control cells (23% ± 3% vs. 44% ± 7% p=0.006). Fas down-modulation was stable over time in CTLs modified, and addition of CH-11 antibody to the culture selected a uniformly Faslow CTL population (Fas MFI 79 ± 29) that was entirely resistant to FasL mediated lysis. However, germ line deletions as well as function-impairing mutations of Fas and FasL can induce T-lymphoproliferation and autoimmune diseases. We therefore compared the growth characteristics and the antigen specificity of unmodified and Fas knockdown CTLs. Survival and proliferation of genetically modified CTLs remain completely dependent on antigen specific stimulation and the presence of other physiological growth signals. Moreover, as assessed by the analysis of the Vb TcR repertoire, IFN-g-release (Elispot) and tetramer staining the modified CTLs remained polyclonal and conserved their antigen specificity. These data suggest that responsiveness to this single death signal may be removed from an effector-memory population of CTLs without adversely affecting their safety. Tumor-antigen-specific CTLs with lower expression of Fas receptor should have a selective functional and survival advantage over unmodified CTLs in the presence of tumors expressing FasL, and may be of value for adoptive cellular therapy of such malignancies.
PLCβ Is Critical for T-Cell Chemotaxis In Vivo.
Authors: Bach Tami L. , Chen Qing-Min , Jordan Martha S. , Choi John K. , Wu Dianqing , Koretzky Gary A. , Zigmond Sally H. , Abrams Charles S.
Published: April 2020

Chemokines acting through G-protein coupled receptors play an essential role in both the immune and inflammatory responses. Phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) are two distinct signaling molecules that have been proposed as potential candidates in the regulation of this process. Studies with knockout mice have demonstrated a critical role for PI3Kγ, but not PLCβ, in Gαi-coupled receptor-mediated neutrophil chemotaxis. We compared the chemotactic response of peripheral T-cells derived from wild type mice with mice containing loss-of-function mutations of either PI3Kγ, or both of the two predominant lymphocyte PLCβ isoforms (PLCβ2 and PLCβ3). In contrast to neutrophils, loss of PI3Kγ did not significantly impair T-cell migration in vitro, although PI3K pharmacologic inhibitor experiments suggest that another isoform of this enzyme might contribute to T-cell migration. However, loss of PLCβ2β3 decreased chemokine-stimulated T-cell migration in vitro. Chelation of intracellular calcium by BAPTA-AM and Quin-2 AM decreased the chemotactic response of wild type lymphocytes, but pharmacologic inhibition of PKC isoforms by GF109203x did not impair T-cell migration. This suggests that the T-cell migration defect seen in the PLCβ2β3-null T-cells may be due to an impaired ability to increase the cytoplasmic calcium concentration, while there appears to be little requirement for PKC activity. Indeed, SDF-1α-induced calcium efflux was not detected in the PLCβ2β3-null lymphocytes. Compared to fluorescently labeled wild type T-cells, labeled PLCβ2β3 knockout T-cells migrated less efficiently into secondary lymphoid organs of recipient mice. This demonstrates that PLCβ is also required for migration in vivo. PLCβ2β3-null mice develop spontaneous skin ulcers starting around 3 months of age. Histological examination of the lesions revealed a dense inflammatory infiltrate composed of neutrophils, macrophages, and plasma cells, consistent with acute and chronic inflammation. Remarkably, lymphocytes, typical of chronic inflammation, were rare to absent by histology and by paraffin immunohistochemistry for CD3, also consistent with an in vivo migratory defect of T-cells.

These results show that phospholipid second messengers generated by PLCβ and isoforms of PI3K, other than PI3Kγ, play a critical role in lymphocyte chemotaxis. Collectively, our data demonstrate that although PLCβ-mediated signaling plays no role in neutrophil chemotaxis, it makes a substantial contribution to this process within T-lymphocytes.
CCL19 and CCL21 Chemokines Induce Endocytosis and Augment Antigen Presentation in Human Mature Dendritic Cells.
Authors: Ogasawara Masahiro , Tanaka Junji , Imamura Masahiro , Kasai Masaharu
Published: April 2020

Dendritic cells (DCs) are potent antigen presenting cells capable of regulating immune responses. DCs lose the ability to capture and process antigens during maturation. In the present study, we examined the effects of CCR7 ligands, CCL19 and CCL21, on endocytosis and antigen presentation in human mature dendritic cells. Immature DCs were generated from peripheral blood monocytes by culturing with GM-CSF and IL4 for 2–3 days. For maturation, immature DCs were cultured with the addition of TNFα, IL1β, IL6 and prostaglandin E2 for another 24 hours. Immature or mature DCs were incubated with FITC-dextran with or without CCL19. Immature DCs internalized FITC-dextran efficiently independent of the presence of CCL19 after 1 hour incubation. On the other hand, mature DCs scarcely internalized FITC-dextran without CCL19. In the presence of CCL19, however, mature DCs internalized FITC-dextran significantly (approximately 60% positive). The effect of CCL19 on the uptake of FITC-dextran in mature DCs was dose and time dependent. CCL21 exerted a similar effect on mature DCs. Next, we examined whether CCL19 facilitates antigen presentation in mature dendritic cells. CD4+ T cells were cultured with irradiated autologous mature DCs which had been incubated with leukemia cell lysate with or without CCL19. Marked proliferation of CD4+ T cells occurred only when these cells were cultured with mature DCs loaded with leukemia cell lysate in the presence of CCL19. This is the first demonstration that chemokines have a pivotal role in endocytosis and antigen presentation by human monocyte-derived dendritic cells to the best of our knowledge. These results demonstrated that generation of potent antigen-loaded mature DCs in relatively short term culture using various cytokines and chemokines may have an important clinical implication to facilitate DC-based immunotherapy.
Reduced Inhibitory KIR Receptors in Patients with NK-Lymphoproliferative Disease of Large Granular Lymphocytes (NK-LDGL) Leads to Dysregulated Killing.
Authors: Epling-Burnette P.K. , Bai Fanqi , Painter Jeffrey S. , Wei Sheng , Djeu Julie Y. , Loughran Thomas P.
Published: April 2020

NK-LDGL is associated with the expansion of CD3−, CD16+ and/or CD56+ lymphocytes, which express a skewed repertoire of NK receptors. (Epling-Burnette et al, Blood103:3431, 2004) Single anti-KIR antibody reactivity was noted in seven patients (54%) and “defective” KIR reactivity in four patients (31%) (n=13). Defective KIR expression was characterized by the loss of reactivity to all available antibodies. These patients expressed a large number of activating KIRs by genotype analysis.

Semi-quantitative RT-PCR demonstrated that lower than normal levels of RNA of the inhibitory KIR was present in some patients in contrast to normal NK cells. Consistent with a high level of activating receptors, we found the NK-LDGL cells have potent cytolytic function in both direct and redirected cytotoxicity assays. Overexpression of activating receptors in the absence of the appropriate inhibitory receptors may contribute to the bone marrow suppression commonly observed in these patients. We first performed Class I typing of the HLA-C locus of five patients with NK-LDGL using PCR-SSP (One Lambda, Inc, Canoga Park, CA). Phenotypes included Cw05/Cw16, Cw15/15, Cw07/12, Cw04/08, and Cw02/03. Each of the five patients had lower then normal levels of inhibitory KIR (KIR2DL1 and KIR2DL2/3) by flow-cytometry and by RT-PCR. In one patient, we examined the functional ability of these inhibitory receptors to block killing of tumor cells expressing cross-reactive Class-I ligands.

Homozygous Cw15/15 was expressed by NK-LDGL5, which is a cross-reactive epitope to Cw3 and recognized by KIR2DL2/3. NK cells from NK-LDGL5 were capable of killing both the class I-negative tumor target 721.221 cells and HLA-Cw3-transfected 721.221 cells suggesting that deficient levels of inhibitory KIR were expressed on the patients NK cells to block activating NK receptor-mediated killing. These results suggest that NK cells from patients with NK-LDGL could mount dysregulated autoimmune-mediated killing, which may impact disease pathogenesis.
Analysis of Clonal Contributions to T Lymphoid and Myeloid Lineages during Early Hematopoiesis Following Autologous Transplantation in the Rhesus Macaque.
Authors: Adler Rima L. , Hematti Peiman , Calmels Boris , Ferguson Cole J. , Keyvanfar Keyvan , Wu Colin , Follmann Dean , Dunbar Cynthia E.
Published: April 2020

Hematopoietic stem cells are defined by their ability both to self-renew and to differentiate into cells that repopulate all hematopoietic lineages. A common lymphoid progenitor (CLP) from mouse marrow has been phenotypically purified and shown to only transiently repopulate all lymphoid lineages (T, B, and NK) but not myeloid lineages when transplanted into lethally irradiated recipients (Kondo et al, 1997). Using the rhesus macaque as a model for human hematopoiesis, we asked whether lineage-restricted repopulating cells contribute to hematopoietic output and for how long following engraftment. In order to follow the progeny of progenitor or stem cell clones in vivo, retroviral vectors were used to mark primitive hematopoietic cells before reinfusion into lethally irradiated autologous recipients. The conditions utilized result in high level of stable retroviral marking in all lineages. We have previously reported use of the sensitive Linear Amplification Mediated - Polymerase Chain Reaction (LAM-PCR) showing that a highly polyclonal set of stable clones accounts for myelopoiesis (Schmidt et al, 2002). In the current study, detailed characterization of the full array of clones contributing to granulocytes, T-lymphocytes, and B-lymphocytes post-engraftment was performed, via sequence analysis of individual clones delineated by individual insertion site sequences at various time points following transplantation of CD34+ cells. Thus far, we have identified 59 granulocyte and 148 T-lymphocyte clones at 1 month following transplantation. Most clones detected at this time point uniquely contribute to only the lineage from which they were identified, only three clones were common to both lineages (1.5% overlap, 95%CI=(0–3.1%)), suggesting independent cells are responsible for hematopoietic reconstitution at this early time point. Six-months following transplantation fewer clones were detected to contribute to hematopoiesis. Interestingly, of the 22 granulocyte and 46 T-lymphocyte clones identified, only two were shared between both lineages (3.0% overlap, 95%CI= (0–7.2%)), further supporting the hypothesis that lineage committed progenitors are responsible for both early and potentially more stable hematopoiesis in this model. To our knowledge, no other study has provided such an extensive characterization of clones contributing to early or more stable in vivo hematopoiesis in the primate model. Preliminary sequence specific tracking experiments reveal that the clones contributing at six-months remain lineage specific over time. Further tracking of clones is being conducted to fully assess the role of such lineage specific progenitor cells in B-lymphopoiesis and long-term hematopoiesis.
Entry Bias: Order of Patient Entry Influences Outcome in Trials of Allogeneic Transplant for Renal Cell Carcinoma.
Authors: Artz Andrew S. , Kocherginsky Masha , Van Besien Koen
Published: April 2020

Early trials evaluating nonmyeloablative allogeneic hematopoietic transplant (NST) for metastatic renal cell carcinoma (RCC) have reported widely discordant results. We performed a meta-analysis of the published literature to identify prognostic factors for NST. However, known RCC prognostic factors are not often detailed in these studies, hindering the identification of patient differences among studies. We explored the order of patient entry as a surrogate marker for patient selection, hypothesizing that patient selection, rather than treatment differences, accounted for early promising results. Patient specific data and adequate follow-up were available for 76 patients from six studies. The rank of patient entry on each individual trial was recorded as well as ≥ grade 2 acute graft-versus-host disease (aGVHD), recipient age, recipient sex, response (partial plus complete), and survival. Multivariable analyses for response and survival were modeled using logistic and cox proportional hazards regression, respectively. The mean overall response rate was 22/76 (29%), with responses across individual studies significantly varying from 0% to 57% P =.009 by Chi-square). Median Kaplan-Meier survival was 263 days. Neither age nor sex was significantly ( associated with response or survival. Acute GVHD occurred in 42% of individuals and correlated with response (OR=9.9, P =0.012) but not survival (HR=1.53, P=0.28). When adjusting by study, later patient entry rank reduced the probability of response (OR=0.30, P =0.007) and survival (HR=1.89, P =0.006). Alternatively, being among the first five patients enrolled in a given study relative to subsequent patients, increased the probability of response (OR=6.69, 95% CI 1.95–39.1, P=0.005), even when adjusting for aGVHD, and afforded a survival benefit (HR= 0.45, 95% CI 0.022 −0.92, P=0.028). The prognostic strength of patient entry rank strongly suggests “entry bias” in patient selection. This bias potentially accounts for the large variation in outcome among studies and for the promising results in early studies. Entry bias analysis offers a novel method to assess early phase trials for selection bias, when detailed individual prognostic information is lacking. Further study is warranted to determine to what extent, if any, entry bias occurs in other clinical trial settings.

Figure Figure