bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Growth Suppression of Philadephia1 Leukemia Cells in Mice by Targeting Both BCR-ABL and VEGF through the Antisense Strategy.
Authors: Han Zhong Chao , Cong Xiu Li , Li Bin , Yang Ren Chi
Published: October 2019

The Philadephia chromosome (Ph1) translocation results in the formation of the BCR-ABL oncogene in over 95% patients with chronic myeloid leukemia (CML). VEGF levels are elevated both in the plasma of CML patients and in conditioned media taken from CML cells. Therefore, simultaneous targeting of BCR-ABL and VEGF might be a rational strategy for attempting treatment of Philadephia1 leukemia. To test this hypothesis, we used an antisense strategy to downregulate BCR-ABL and VEGF expression in K562 cells, a human erythroleukemia cell line. In vitro, combination of bcr/abl and VEGF antisense oligodeoxyribonucleotides (AS-ODNs) exerted a specific synergistic antiproliferative effect on K562 cells and prominently sensitized K562 cells to apoptosis-inducing stimuli. In vivo, nude mice injected with K562 cells were treated systemically with BCR-ABL or VEGF AS-ODNs or with both ODNs in combination. In comparison with the mice treated with individual agents, the mice treated with both ODNs showed a slower growth of leukemia tumors, a reduction of microvessel density and an increased apoptosis in the tumors. These results demonstrate that targeting both BCR-ABL and VEGF may represent an excellent strategy to overcome the resistance to chemotherapeutic agents and ultimately to augment the efficacy of chemotherapy in CML.
Transduction of Primate Cells with Feline Foamy Virus Envelope Pseudotyped Prototype Foamy Virus Vectors.
Authors: Su RuiJun , Rosales Rency L. , Lochelt Martin , Josephson Neil C.
Published: October 2019

Because of their genetic and biological similarity to humans, non-human primates are the best pre-clinical models for testing the efficacy and safety of gene therapy systems. However, the presence of endogenous simian foamy virus infection in nearly all non-human primates kept in captivity complicates foamy virus (FV) vector stem cell transduction studies in these animals. A major concern is that repopulating cells exposed to FV vector stocks will elicit an immune response in non-human primate hosts. Though human serum does not inactivate prototype foamy virus (PFV) vectors, a one hour incubation of PFV vector stock in the presence of serum samples from Papio Cynophalus (baboon), Macaca Mulatta (rhesus macaque), or Macaca Fasicularis (long-tailed macaque) results in a 75–100% drop in titer. To overcome this serum mediated inactivation we sought to pseudotype PFV vectors in the feline foamy virus (FFV) envelope. The wild-type envelope from the FUV strain of FFV does not pseudotype our PFV vectors. Therefore we generated chimeras with regions of both the FFV and PFV envelope. By substituting portions of the FFV envelope leader peptide sequence and membrane spanning domain with corresponding PFV envelope regions we generated chimeric envelopes capable of high titer (105–106 FFU/ml) PFV vector production. Serum samples from Macaca Mulatta produced less inactivation of the FFV pseudotyped than the PFV pseudotyped vectors. Furthermore, both the PFV and FFV pseudotyped vectors demonstrated efficient transduction of baboon mesenchymal stem cells (27–43%) and baboon embryonic stem cells (37–40%). However, the FFV pseudotyped vectors transduced both human and baboon CD34+ cells less efficiently than the PFV pseudotyped vectors. We plan to test PFV vectors pseudotyped by other FV envelopes for inactivation by primate serum, and for their ability to transduce primate hematopoietic cells.
Respiratory Syncytial Virus (RSV) Infections in Immunocompromized Children.
Authors: Chiang Alan Kwok Shing , Chan Godfrey Chi Fung , Ha Shau Yin , Chan Kwok Hung
Published: October 2019

Eleven children who were immunocompromized due to chemotherapy were infected with respiratory syncytial virus (RSV) during an outbreak of RSV infection in the Pediatric Oncology Unit of Queen Mary Hospital, The University of Hong Kong. The clinical symptoms, effect on treatment regimen, outcome of infection and viral clearance had been followed prospectively in all 11 children. Viral clearance was documented by immunofluorescence and PCR studies of nasopharyngeal aspirates or nasal/throat swabs. Eight of the 11 children had upper respiratory symptoms with the remaining 3 patients progressing to lower respiratory tract infections (LRTI). All except one had protracted viral symptoms but eventually recovered without the need for specific anti-viral treatment or intensive support. However, the chemotherapy regimen or scheduled BMT had been significantly interrupted. One patient developed progressively severe pneumonia which was further complicated by adenovirus reactivation causing bone marrow failure and severe sepsis resulting in death of this patient. The viral clearance of RSV was delayed in all patients with median of 20 days (range 9–81 days). One patient had recurrence of RSV detected by PCR 35 days after intial documentation of clearance, after a BMT procedure. The delayed viral clearance correlated with protracted viral symptoms. In conclusion, RSV clearance is delayed in immunocompromized hosts causing protracted viral symptoms, progression to LRTI and interruption to chemotherapy treatment. The delay in viral clearance appears proportional to the degree of immunosuppression of the individual.
Development of a Preference Questionnaire to Compare User Preference for Hemophilia Factor Reconstitution Devices.
Authors: Lloyd Andrew , Mannix Sally , Brewster Jessica , Rentz Anne M. , Butler Regina , Larson Peter J.
Published: October 2019

Introduction: Hemophilia treatment includes reconstitution of lyophilized FVIII concentrates with diluent followed by intravenous injection. Several reconstitution methods are commonly employed during infusion of concentrates. The Bio-Set® (Biodome, Issoire France) is a new needleless device for reconstituting a factor VIII concentrate, Kogenate® FS (Bayer Healthcare, Elkhart IN).

Objective: To develop and evaluate a new preference questionnaire designed to capture nurse, patient, and caregiver preferences for reconstituting factor using the Bio-Set® device, the conventional method for reconstitution and the other available method, the Baxject (Baxter Healthcare, Westlake Village CA).

Methods: A 27-item preference questionnaire was developed based on a previous preference questionnaire. Four hemophilia nurses participated in content validation before pilot-testing the questionnaire. Psychometric properties were tested in a sample of 161 participants (35 hemophilia A patients who infuse themselves, 67 caregivers, 59 infusion nurses). The entire study followed an IRB approved protocol. Method names were blinded to prevent brand recognition by participants. Participants were familiarized with each method by viewing a video, watching an interviewer demonstrate each method, and practicing with each method. After performing a timed round with each method, participants completed the preference questionnaire for each method. After performing the third method, participants ranked methods on 9 attributes.

Results: Item distributions indicated acceptable response spread for most items. Following item analyses and principle components factor analysis (PCA), 2 items were eliminated. The PCA resulted in two factors, worry/safety and ease/confidence. Internal consistency reliability was good for total and subscale scores, ranging from 0.92 to 0.93. Concurrent and discriminant validity was not supported.

Conclusions: This study provides information on the psychometric properties of a new preference questionnaire that will be useful in determining inclination for and differences between patient, caregiver, and nurse preferences for reconstitution devices available for hemophilia factor concentrates.
Dosing Patterns of Erythropoietic Agents and Their Association with Provider Visits in Patients with Cancer and Anemia.
Authors: Bolge Susan C. , Fastenau John M. , Howell Jamie B. , Piech Catherine T. , Donohue Jane A.
Published: October 2019

Background: Understanding current dosing patterns of epoetin alfa (EPO) and darbepoetin alfa (DARB) in clinical practice and their association with provider visits is important to optimize patient management and assess the total costs of anemia care.

Objective: To assess dosing patterns of erythropoietic agents and the frequencies of provider visits in patients with cancer and anemia.

Methods: A cross-sectional study of adults with solid tumor cancers and anemia is on going. 500 planned participants are being recruited through 50 oncologists’ offices across the U.S. Information is collected via a self-administered questionnaire that is divided into two parts. The first, completed by the provider, captures clinical information; the second, completed by patients, records their knowledge, experience and preferences regarding anemia and its treatments, including resource use, quality of life and productivity. This preliminary analysis focuses on the provider-reported dosing section (most recent dosing of the erythropoietic agent) and patient-reported resource utilization section (past month recall of visits to providers).

Results: 276 provider/patient-matched questionnaires have been received. Of these, there were 78 EPO, 172 DARB, 21 Untreated and 5 patients who had received both agents within the past month. 96% of patients were receiving chemotherapy concurrently. The majority of EPO patients were dosed weekly (QW) followed by every 2 week dosing (Q2W) while most DARB patients were dosed Q2W weeks followed by QW dosing (EPO: QW 90%, Q2W 10%; DARB: QW 26%, Q2W 63%, Other 10%). Mean weekly doses for each agent were: EPO 37,574 Units/week; DARB 140 mcg/week; corresponding to an EPO:DARB dose ratio of 267:1. All patients reported a mean of 7.6 visits to providers in the past month, with similar results among the three patient groups (EPO: 8.8, DARB: 7.2, Untreated: 5.9; p=0.316). Most patients described the number of visits during the past month as “typical” (64% overall, range of 60–66% among patient groups; p=0.801). Among patients receiving an erythropoietic agent, the mean number of past monthly visits did not differ by frequency of administration (QW: 8.7, Q2W: 7.4, Other: 4.9; p=0.064).

Conclusion: Based on provider and patient-reported data, dosing patterns showed most EPO patients are dosed QW while most DARB patients are dosed Q2W. The EPO:DARB dose ratio was found to be 267:1 which is consistent with other observational studies using claims data and chart reviews (Proc Am Soc Clin Oncol.2004;23:552 (6144)). Patients with cancer and anemia report similar numbers of visits to their providers each month regardless of their anemia treatment (EPO, DARB or Untreated) or erythropoietic dosing schedule (QW, Q2W, or Other).
The Crystal Structure of Abl Kinase with BMS-354825, a Dual SRC/ABL Kinase Inhibitor.
Authors: Tokarski John S. , Newitt John , Lee Francis Y. , Lombardo Louis , Borzilleri Robert , Kish Kevin , Xie Dianlin , Zhang Yaqun , Cheng Janet D. , Kiefer Susan E. , Chang Chieh Ying J. , Klei Herbert E.
Published: October 2019

Chronic myeloid leukemia (CML) is a stem cell disorder caused by a constitutively activated tyrosine kinase, the BCR-ABL oncoprotein. Imatinib (STI571, Gleevec) is a small-molecule inhibitor of this kinase that produces clinical remissions in CML patients and is now frontline therapy for this disease. While this agent has a high rate of clinical success in early phases of CML, development of resistance to this drug becomes increasingly problematic in later stages of the disease. BMS-354825, a small-molecule dual-function SRC/ABL tyrosine kinase inhibitor, appears to overcome many of the limitations associated with imatinib therapy. BMS-354825 is 500-fold more potent than imatinib against BCR-ABL and more importantly retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants (Shah et al., Science, 2004;305(5682):399). In addition, BMS-354825 proved to be equally effective against several pre-clinically and clinically derived tumor models of imatinib resistance (Lee et al., Proceedings of the AACR, Volume 45, March 2004 abstract number 3937). In order to better understand the molecular basis of the relationship between inhibitor chemistry and biological activity, the three-dimensional structure of the kinase domain of Abl kinase complexed with BMS-354825 was determined by X-ray crystallography. The structure reveals that BMS-354825 binds in the ATP-binding site. A comparison with the imatinib-Abl complex (PDB entry 1IEP) reveals that the central cores of BMS-354825 and imatinib occupy overlapping regions but that these two inhibitors extend in opposite directions. The activation loop is observed to be in the active conformation in the presence of bound BMS-354825 in contrast to bound imatinib. There do not appear to be any steric clashes that would preclude BMS-354825 from also binding to the inactive conformation of the activation loop. This observation suggests that the increased binding affinity of BMS-354825 over imatinib is at least partially due to its apparent ability to recognize multiple states of the enzyme. The P-loop is partially disordered as indicated by high B-factors and broken electron density which suggests that interactions between this part of the protein and BMS-354825 are less critical for binding. Interestingly, several imatinib-resistant mutations occur in the P-loop. The structure was analyzed for the 15 imatinib-resistant BCR-ABL mutants and attempts are made to rationalize the activity of BMS-354825 against these mutants.
FLT3 Mutations Determine the Clinical Outcome in Children with De Novo Acute Myelogenous Leukemia (AML) and Normal Karyotype: Pediatric Oncology Group (POG) Study # 9421.
Authors: Lacayo Norman J. , Meshinchi Soheil , Raimondi Susana C. , Kuo Dennis J. , Yu Ron , Chang Myron N. , Willman Cheryl L. , Tibshirani Robert , Ravindranath Yaddanapudi , Sikic Branimir I. , Weinstein Howard , Dahl Gary V.
Published: October 2019

The karyotype of the leukemia cell at diagnosis is of prognostic importance. The presence of t(8;21), inv(16) and t(15;17) are currently used to make therapeutic decisions. Additionally, specific mutations such as those involving the FLT3 gene are of prognostic importance as they indicate patients likely to relapse early or fail initial induction therapy. Approximately 20% of children with AML have normal karyotypes at diagnosis and no identifiable chromosomal abnormality using standard methods of analysis. In this subgroup there is an increased incidence of FLT3 mutations (internal tandem duplications or point mutations). We observed that patients with normal karyotypes who were enrolled in the Pediatric Oncology Group (POG) study #9421 had two significantly different clinical outcomes that were associated with the expression of FLT3 mutations. We hypothesized that gene expression profiles would identify genes that cooperate with FLT3 mutations in conferring poor clinical outcome.

Bone marrow and/or blood samples from an initial 103 of 622 eligible patients registered on the POG study #9421 were studied. Cytogenetic testing was carried out by clinical laboratories at institutions where AML was diagnosed; all cytogenetic reports underwent centralized review. Samples were analyzed on a 43,760-element spotted array (containing 41,751 unique genes and ESTs) from the Stanford University Microarray Core Facility. Of the 103 diagnostic samples, 20 had normal karyotypes. The FLT3-mutation status was determined by RT-PCR analysis on RNA samples (exon 14 for ITD’s and exon 20 for point mutations). Twelve patients expressed FLT3-mutations and 8 expressed wild-type FLT3 (FLT3-WT). Both groups had a mean WBC of 103x109/L at diagnosis. Probability of event free survival (EFS) was 75% for the FLT3-WT subgroup and 9% for the FLT3-mutant subgroup (P=0.0006). We used Prediction Analysis for Microarrays (PAM) to find the minimum number of genes that could identify samples with FLT3 mutations. PAM identified a 7-gene cluster that differentiated normal cytogenetic cases by clinical outcome. A cluster of 5 overexpressed genes (i.e., HIST1H2AC, HIST1H2AL, HIST1H2BD, HIST1H2BG, and HIST2H2BE) and 2 underexpressed genes (GASP and IVNS1ABP) were associated with poor outcome.

In summary, children with AML, normal karyotype, and absence of FLT3 mutations have a good outcome. In contrast, when these same patients express a FLT3 mutation their outcome is poor despite intensive therapy. This poor outcome is associated with increased expression of several histone genes, which may be downstream targets of the FLT3 signal transduction pathway. We will corroborate these findings in a larger set of POG 9421 diagnostic samples with normal karyotypes.
T Cell Trafficking in Acute GVHD: In Vivo Bioluminescence Evaluation To Elucidate the Role of Different Lymphatic Organs.
Authors: Beilhack Andreas , Schulz Stephan , Baker Jeanette , Beilhack Georg F. , Wieland Courtney B. , Baker Enosh M. , Herman Edward I. , Landan Gilad , Kime Janelle A. , Cao Yu-An , Contag Christopher H. , Negrin Robert S.
Published: October 2019

To study the complex pathophysiology of aGvHD in allogeneic hematopoietic cell transplantation (HCT) we transplanted transgenic luciferase expressing T cell populations into lethally irradiated HCT recipients (murine MHC major mismatch model, H-2q into H-2d). Tracking of light emitting donor T cells in living animals and detailed studies by multi color immunofluorescence microscopy (IFM) and FACS revealed the tight links of spatial and temporal evolution in this complex immune process. Donor derived T cells migrate to T cell areas in lymphoid tissues within a period of 12 hours. In the initial periods donor CD4+ T cells appear first with CD8+ T cell infiltration at later time points. Donor T cells start proliferating in lymphatic tissues on day 2 after transfer, as observed by BrdU stainings. Although alloreactive T cells are similarly activated in all lymphoid organs, they only up-regulate gut homing molecules after more than 5 cell divisions (CFSE proliferation analysis by FACS) in certain lymphoid organs (Peyer’s patches, mesenteric LN and spleen). Abruptly on day 4 after HCT, T cells migrate into intestinal sites. These findings strongly suggested, that specific priming sites are required for alloreactive T cells to induce a distinct type of tissue tropism in GvHD. In contrast to previous reports peformed without host conditioning, depletion of certain lymphoid organs (e.g. Peyer’s patches) before HCT or antibody blocking experiments did not control aGVHD. BLI showed, that anti-L-selectin or anti-MAdCAM-1 antibody treatment alone or in combination was effective in blocking donor T cell migration to lymph nodes and Peyer’s patches, while redirecting these cells to liver and spleen. Subsequently cells proliferated predominantly in the spleen until day 3 after HCT. Surprisingly we observed a full picture of gut infiltration on day 4 and skin involvement on day 5–6, similar in dynamics and strength to the aGvHD isotype control group. These findings demonstrated, that other lymphoid organs can functionally compensate for inducing gut and skin homing of alloreactive T cells. Of importance, we demonstrated that T cells that lacked homing molecules for secondary lymphoid organs had alloreactive properties in vitro, yet did not cause aGVHD in vivo. In summary, the activation of alloreactive T cells in specific sites throughout the body is complex and involves the acquisition of homing molecule expression. Transplantation of T cells with defined homing properties therefore, appears to be a promising alternative in conferring protective immunity early after HCT without the risk of aGvHD.
WIP-Mediated Mechanisms of Recruitment, Activation and Degradation of WASP in T Cells.
Authors: Sasahara Yoji , Ramesh Narayanaswamy , Tsuchiya Shigeru , Geha Raif S.
Published: October 2019

WASP, the product of the gene mutated in Wiskott-Aldrich syndrome (WAS), plays a critical role in T cell activation and actin reorganization. WIP is a WASP-interacting protein that binds to EVH1 domain of WASP, and negatively regulates WASP functions. WASP-mediated actin polymerization and cytoskeletal remodeling are critical for immunological synapse (IS) formation between T cells and antigen presenting cells. To clarify the functions of WIP in T cells, we investigated WIP-mediated mechanisms of recruitment, activation and degradation of WASP following T cell receptor (TCR) ligation and IS formation. WASP translocates to lipid rafts following TCR ligation and localizes at the IS between T cells and B cells presenting superantigen. WASP recruitment is regulated by several TCR signaling pathways mediated by ZAP70-CrkL-WIP and ZAP70-SLP76-Nck complexes. After the recruitment of WASP, WIP is serine-phosphorylated by PKCtheta localized at the IS, and phosphorylated WIP dissociates from WASP, releasing WASP from WIP inhibition. A fraction of WASP is fragmented by Ca-dependent protease calpain and ubiquitinated by Cbl-b, then degraded by the proteasome following TCR ligation. Inhibition of PKCtheta-mediated dissociation of the WASP-WIP complex inhibits WASP degradation. Inhibition of proteasomal degradation results in the accumulation of WASP fragments and enhanced TCR-induced actin polymerization in normal T cells. We demonstrate that WASP protein, but not mRNA, levels are severely diminished in T cells from WIP-deficient mice, but are restored by calpain and proteasome inhibitors. These inhibitors restore WASP protein levels and correct the defect in actin polymerization in response to TCR ligation in T cells from a WAS patient with a missense mutation that disrupts WIP binding. The similarity of the functional defects in WASP- and WIP-deficient mice and the observation that most missense mutations in WAS patients are in the WIP binding EVH1 domain of WASP suggest that WIP may function as a chaperone that controls WASP levels in T cells. These results suggest that WIP is involved in the recruitment and activation of WASP and that degradation of WASP freed from WIP may down-regulate actin polymerization following TCR ligation. More importantly, WIP regulates WASP protein stability in T cells from WAS patients with WASP missense mutations in WIP-binding site and impaired WASP-WIP interaction.
A Novel Synthetic Chemokine Containing D-Amino Acids That Binds to the CXCR4 Receptor and Inhibits HIV-1 Infection.
Authors: Huang Ziwei , Liu Dongxiang , Madani Navid , Kumar Santosh , Choi Won-Tak , Cao Rong , Li Ying , Gao Yigui , Dong Changzhi , Wang Jun , An Jing , Sodroski Joseph G.
Published: October 2019

The viral inflammatory protein II (vMIP-II) is a mammalian chemokine homologue encoded by Kaposi’s Sarcoma-associated herpesvirus. vMIP-II shows a broad spectrum binding activity to CXC, CC and CX3C chemokine receptors. Particularly, vMIP-II binds to CXCR4 and CCR5 which are two major coreceptors for HIV-1 virus entry and infection. In our previous study, we reported an all D-amino acids peptide (DV1) derived from the N-terminus of vMIP-II showing high binding affinity to CXCR4. To study the binding mechanism of vMIP-II with CXCR4, we attached the first 10 D-amino acid residues of this peptide to the N-terminus of vMIP-II, before the first cysteine and synthesized a new chemokine analogue of vMIP-II which we named D10-vMIP-II. Despite of the chiral inverse of the N-terminal residues, D10-vMIP-II shows high CXCR4 binding affinity and inhibits the HIV-1 virus entry as vMIP-II. To explore the structural features of D10-vMIP-II, we determined its crystal structure in high resolution. The crystal structure shows that D10-vMIP-II adopts the same structural folding pattern as vMIP-II except for structural changes at the N-terminus, N-loop and the 30’s loop region, suggesting that the local structures at the N-terminus, N-Loop and the 30’s loop region can be adjusted coordinately. This novel synthetic SMM-Chemokine strongly inhibits the entry and replication of HIV-1 via CXCR4. Our studies demonstrate a new promising lead for the development of therapeutic agents targeted to the HIV-1 entry mechanism.
The Impact of Transplant in AML in 2nd CR: A Prospective Study of 741 in the MRC AML 10 and 12 Trials.
Authors: Burnett Alan K. , Hills Robert K. , Goldstone Anthony H. , Milligan Donald , Prentice Archie G. , Hann Ian , Webb David , Gibson Brenda , Wheatley Keith
Published: October 2019

MRC AML10 and 12 Trials recruited 5425 patients of whom 4557(84%) entered CR and were treated with chemotherapy only. 741 relapsed and achieved a second CR. These patients were recommended for salvage by stem cell transplantation either sibling or MUD allograft or autograft. In a non-randomised comparison, Mantel-Byar and delayed entry proportional hazards regression were used to investigate outcome and which factors were predictive for survival. Of the 741 patients in CR2 480 received a SCT (116 sib Allo: 192 Autograft and 154 MUD 18 other/unknown) at a median of 107 days from second CR. The overall survival at 5 years was significantly better for those who received SCT (39% vs 22% p=0.00001). Of the 116 patients who received a sibling transplant 54% are alive at 5 years; of the 154 patients who received a MUD graft 40% are alive and of 192 who received an autograft 33% are alive and each transplant type was significantly better than chemotherapy alone (p=0.0001, p=0.002, p=0.002). Much of the benefit derives from reduced relapse risk (5yr RR: allograft 32%; autograft 59%; MUD 38%: no transplant 71% (0.0001). Only in MUD transplant was there a greater mortality (allograft 27%; autograft 27%, MUD 42%, no transplant 25% p=0.003). There was, however, no evidence of heterogeneity in the beneficial effects of transplant when analyses were stratified by age, duration of first remission, time taken to achieve second CR, or cytogenetics. Proportional hazards regression shows that duration of 1st CR, cytogenetics and age are important prognostic factors; adjustment for these factors to some extent changes the results of the analyses. After adjustment, there is still strong evidence of the benefits of allograft (p<0.0001), but the evidence for autograft (p=0.09) and MUD (p=0.02) becomes unclear. As a non-randomised comparison, these results cannot provide absolute evidence of benefit. After adjustment for other prognostic factors, the position for autograft and MUD transplants remains unclear especially as not all selection factors can be accounted for. However, the allograft results appear robust to adjustment and imply significant survival benefit for patients receiving allograft in 2nd CR.
Protease-Activator Receptor 4 Is Required for Maximal Thrombus Growth, but Not for Fibrin Generation in Thrombi after Laser Injury.
Authors: Vandendries Erik R. , Hamilton Justin R. , Coughlin Shaun R. , Furie Barbara C. , Furie Bruce
Published: October 2019

The serine protease, thrombin, is necessary for the conversion of fibrinogen to fibrin and is a potent activator of platelets. Thrombin-induced platelet activation, as measured by shape change, calcium mobilization, and ATP secretion, requires the protease-activator receptor 4 (PAR4). Platelets isolated from PAR4 knock-out mice are unresponsive to thrombin, and PAR4 null mice appear to be protected from thrombosis in a ferric chloride-induced injury model of thrombosis and a thromboplastin model of pulmonary embolism. To examine further the role of thrombin-induced platelet activation in developing thrombi, we have examined the in vivo kinetics of thrombus formation in living mice lacking PAR4 using high-speed widefield digital microscopy. In this study, platelets were labeled using anti-CD41 Fab fragments conjugated to Alexa-488. Thrombi were generated by laser-induced injury of the cremaster arteriolar vessel wall, and the total fluorescent antibody accumulation was monitored and quantitated for 5 minutes after injury. In PAR4 null mice, the thrombi generated were significantly smaller with an early arrest of thrombus growth when compared to thrombi generated in wild-type mice. The maximum thrombus platelet accumulation in PAR4 null mice (median of 30 thrombi in 3 mice) was 75% less than that seen in wild-type mice (median of 33 thrombi in 4 mice)(P<0.001). The time to half-maximal and the time to maximal thrombus formation in PAR4 null mice is approximately 5.5 seconds and 16 seconds, respectively, compared to 45 seconds and 74 seconds in wild-type mice (P<0.001). The shortened time to maximal platelet accumulation appears to be secondary to an early termination of thrombus growth. Fibrin generation was monitored using Alexa-647 conjugated to an anti-fibrin antibody that does not recognize fibrinogen in mice simultaneously infused with anti-CD41 Fab conjugated to Alexa-488. No difference in total fibrin accumulation was seen during the first 4 minutes of thrombus formation in PAR4 null mice (median of 23 thrombi in 3 mice) compared to thrombi generated in wild-type mice (median of 26 thrombi in 4 mice) despite a significant decrease in platelet accumulation in PAR4 null thrombi. Most of the fibrin deposition in both wild-type and PAR4 null thrombi was observed at the vascular wall/thrombus interface. In summary, thrombin-induced platelet activation via PAR4 is required for normal thrombus growth. However, in this model of thrombosis, neither PAR4 nor maximal thrombus growth appears to be necessary for fibrin deposition. This suggests that a platelet-independent mechanism of thrombin generation may exist. Alternatively, the amount of platelet accumulation and activation in PAR4 null mice may be sufficient for normal thrombin generation and subsequent fibrin deposition.
Fibroblast Activation Protein (FAP) Is a Critical Microenvironmental Survival Factor for Myeloma Plasma Cells.
Authors: Ge Yun , Zhan Fenghuang , Shaughnessy John , Barlogie Bart , Tricot Guido , Epstein Joshua , Yaccoby Shmuel
Published: October 2019

FAP is one of 32 consistently and significantly upregulated genes in osteoclasts after co-culture with CD138-immunomagnetic bead-selected myeloma plasma cells (MM PCs) from 19 patients and with 3 myeloma cell lines. FAP is a cell surface serine protease with both dipeptidyl peptidase and collagenase activity. This enzyme has been shown to be selectively expressed by tumor stromal fibroblasts in epithelial carcinomas, but not by epithelial carcinoma cells, and to promote tumor metastasis. The aim of the study was to investigate the possible involvement of FAP in myeloma using an ex vivo co-culture system and our established SCID-hu model for primary myeloma (Yaccoby et al., Blood, 1998; 1999). Myeloma-osteoclasts co-cultures were prepared as previously described (Yaccoby et al., Cancer Res., 2004). Mesenchymal stem cells (MSCs) isolated from patient’s bone marrow were cultured on the backside of 1 μM pore-size transwell inserts’ membranes while MM PCs were incubated in the upper chamber of the inserts. Histologic examination demonstrated that the MSC’s cytoplasmic villi pass through the membrane pores, allowing contact with tumor cells. To study the role of FAP in myeloma we initially demonstrated by quantitive real time RT-PCR (qRT-PCR) that FAP was upregulated 3.0 fold (p<0.01, n=5) in osteoclasts and 2.2 fold (p<0.05, n=3) in MSCs after co-culture with MM PCs. In the SCID-hu model, expression of FAP was increased >120 fold in myelomatous vs. nonmyelomatous human bone as determined by a whole bone marrow qRT-PCR. Immunohistochemical staining of myelomatous bone sections from SCID-hu mice revealed expression of FAP by osteoclasts, vascular endothelial cells, osteogenic cells and other stroma elements, but not by myeloma cells. To further study the involvement of FAP in myeloma cell survival we applied the siRNA approach in our myeloma-osteoclasts and myeloma-MSCs co-culture systems. Following preliminary testing of 4 probes, we have chosen a siRNA probe with >75% inhibition of FAP expression in osteoclasts and MSCs as determined by qRT-PCR. Addition of FAP siRNA to co-cultures of CD138-selected MM PCs with osteoclasts (n=5) and MSCs (n=3) for 48 hours, significantly (p<0.05) reduced the number of viable myeloma cells, as determined by trypan blue exclusion and annexin V flow cytometry. To test whether FAP activity is associated with resistance of myeloma cells to drug-induced apoptosis, MM PCs were co-cultured with osteoclasts in the absences and presences of dexamethasone (DEX, 10−6 M) and FAP siRNA. Percent apoptotic MM PCs was significantly higher in co-cultures treated with DEX+siRNA compared with DEX and siRNA alone (p<0.05, n=4). Our results indicate that FAP is critical for the interaction of myeloma cells with the bone marrow microenvironment and promotion of myeloma cell survival. It is therefore a potential therapeutic target in myeloma.
Heparanase Promotes the Spontaneous Metastasis of Myeloma Cells to Bone.
Authors: Yang Yang , MacLeod Veronica , Bendre Manali , Huang Yan , Theus Allison M. , Miao Hua-Quan , Kussie Paul , Yaccoby Shmuel , Epstein Joshua , Suva Larry J. , Kelly Thomas , Sanderson Ralph D.
Published: October 2019

Although widespread skeletal dissemination is a critical step in the progression of myeloma, little is known regarding mechanisms that control this process. High levels of the syndecan-1 heparan sulfate proteoglycan are present in the myeloma microenvironment where they bind numerous growth factors (e.g., HGF, FGF-2) that control myeloma growth, angiogenesis and dissemination. Heparanase-1 (HPSE1) is an enzyme that cleaves heparan sulfate chains of proteoglycans and thus may regulate heparan-binding growth factor activity. We have previously demonstrated that human myeloma cells express heparanase and that active enzyme is present in the plasma harvested from the marrow of myeloma patients (Cancer Res. 63: 8749–56). In the present study, experiments were performed to determine the effects of enhanced expression of heparanase on myeloma tumor growth and dissemination in vivo. The human myeloma cell line, CAG, was transfected with human HPSE1 cDNA (CAGHPSE1) or with corresponding vector only (CAGNeo). The transfected cells were injected either directly into the tibia or subcutaneously into the flank of severe combined immune deficient (SCID) mice and tumor growth rate, microvessel density and metastasis to bone were analyzed. We discovered that expression of heparanase: (i) accelerates the initial growth of the primary tumor, (ii) increases whole body tumor burden as compared to controls, and (iii) enhances both the number and size of microvessels within the primary tumor. In addition, enhanced expression of heparanase dramatically upregulates spontaneous metastasis of subcutaneously-injected myeloma cells to bone (95% in mice bearing CAGHPSE1 tumors, n = 21; as compared to 6% in mice bearing CAGNeo tumors, n=16). When tumor was injected directly into the tibia, 100% of the mice bearing CAGHPSE1 tumor cells had metastases within the contralateral femur (n=10) while only 29% of the mice bearing CAGNeo tumor had metastases (n=7). These studies describe a novel experimental animal model for examining the spontaneous metastasis of bone-homing tumors and indicate that heparanase is a critical determinant of myeloma dissemination and growth in vivo. Thus, therapeutic modulation of heparanase expression or function may be of value in the treatment of myeloma and other cancers that metastasize to bone.
NK4, an Antagonist of Hepatocyte Growth Factor (HGF), Inhibits Growth of Multiple Myeloma Cells In Vitro and In Vivo: A Mouse Model for Molecular Targeting of Angiogenic Growth Factor.
Authors: Hattori Yutaka , Du Wenlin , Yamada Taketo , Matsumoto Kunio , Nakamura Toshikazu , Otsuki Takemi , Niikura Takako , Nukiwa Toshihiro , Ikeda Yasuo
Published: October 2019

Background and rationale: Recent study showed that bone marrow angiogenesis and angiogenic growth factors including hepatocyte growth factor (HGF) are associated with pathogenesis and disease activity of multiple myeloma. In our experience, plasma concentration of HGF significantly elevated in 30 % of patients with clinically active myeloma and declined along with response to therapy. Thus, HGF signaling is considered to play a role in progression of myeloma and is a potential therapeutic target. The aim of this study is to establish a novel therapy targeting HGF and the receptor, c-MET, using NK4 molecule which is an HGF-antagonist consisting of N-terminal kringle domain of HGF.

Results: 1) Among eleven myeloma cell lines established from Japanese patients, two lines, KMS11 and 34, secreted HGF while c-MET is expressed in all eleven cells. 2) 10nM of NK4 protein significantly inhibited growth of the two HGF-producing cells in vitro. NK4 protein showed additive anti-myeloma effect to dexamethasone (Dex) in Dex-sensitive KMS11 cells, and is also effective for growth suppression of Dex-resistant KMS 34 cells. 3) NK4 significantly reduced phosphorylation of ERK1/2 while no significant effect on STAT3 activation. 4) Adenoviral vector carrying NK4 cDNA (Ad-NK4) was injected in femoral muscle of lcr/scid mice. Pharmacokinetic study showed that NK4 proteins were detected in plasma, liver, lung and kidney up to three weeks and attained a maximum level at day 10–14. 5) Ad-NK4 injection to tumor-bearing lcr-SCID mice significantly inhibited growth of subcutaneous tumor derived from KMS11 and 34 cells compared with empty vector injection (n=10, p<. 05). Co-injection of Ad-NK4 and Dex revealed synergistic or additive anti-tumor effect in mice bearing KMS11 tumor. Anti-myeloma effect of Ad-NK4 is also confirmed by histopathological examination.

Conclusion: Above results directly showed that HGF/c-MET signal plays an important role in progression of myeloma cells producing HGF. Molecular targeting of HGF by NK4 is considered as a novel approach for the treatment of myeloma via independent mechanism of glucocorticoid therapy.
Inhibition of Human Plasmacytoma Cell Growth by a Novel JAK Kinase Inhibitor.
Authors: Burger Renate , Legouill Steven , Tai Yu-Tzu , Shringarpure Reshma , Podar Klaus , Catley Laurence , Tassone Pierfrancesco , Neri Paola , Hideshima Teru , Chauhan Dharminder , Fridman Jordan S. , Anderson Kenneth C.
Published: October 2019

Novel strategies in cancer therapy aim at inhibiting distinct signal transduction pathways that are aberrantly activated in malignant cells. Protein tyrosine kinases of the JAK family are associated with a number of cytokine and cytokine-like hormone receptors and regulate important cellular functions such as proliferation, survival, and differentiation. Constitutive or enhanced JAK activation has been implicated in neoplastic transformation and abnormal cell proliferation in various hematological malignancies. In multiple myeloma (MM), JAK kinases play a critical role because of their association with cytokine receptors of the IL-6/gp130 family. A novel small-molecule inhibitor was developed that shows a 100 to 1,000-fold selectivity for JAK1, JAK2, JAK3, and TYK2 relative to other kinases including Abl, Aurora, c-Raf, FGFR3, GSK3b, IGF-1R, Lck, PDGFRa, PKBb, and Zap-70. Growth of MM cell lines and primary patient cells was inhibited by this compound in a dose-dependent manner. The IL-6 dependent cell line INA-6 and derived sublines were sensitive to the drug, with IC50’s of less than 1 mM, in [3H]-thymidine uptake and a colorimetric, tetrazolium compound (MTS) based assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI). Importantly, INA-6 and patient tumor cell growth was also inhibited in the presence of bone marrow stromal cells, which by themselves remained largely unaffected. Growth suppression of INA-6 correlated with a significant and dose-dependent increase in the percentage of apoptotic cells, as evaluated by Apo2.7 staining after 48 hours of drug treatment. In addition, the compound blocked IL-6 induced phosphorylation of STAT3, a direct downstream target of JAK kinases and important transcription factor triggering anti-apoptotic pathways. In other myeloma cell lines, the drug overcame the protective effect of gp130 cytokines on dexamethasone induced apoptosis. In MM1.S cells, it completely blocked IL-6 induced phosphorylation of SHP-2 and AKT, both known to mediate the protective effects of IL-6. In contrast, AKT phosphorylation induced by IGF-1 remained unchanged, demonstrating selectivity of the compound. These studies show that disruption of JAK kinase activity and downstream signaling pathways inhibits myeloma cell growth and survival as well as circumvents drug resistance, thereby providing the conceptual basis for the use of JAK kinase inhibitors as a novel therapeutic approach in MM.
Low-Dose Irradiation Promotes Tissue Revascularization through Matrix Metalloproteinase-9 Mediated VEGF Release from Mast Cells.
Authors: Hattori Koichi , Ohki Yuichi , Sato Yayoi , Rafii Shahin , Werb Zena , Heissig Beate
Published: October 2019

Irradiation is one of the pillars in the treatment of malignancies. The combination of radiotherapy and anti-angiogenic strategies has been shown to increase the tumor response in various tumor models. The more than additive effect of irradiation and anti-angiogenic treatment suggested that irradiation might have a pro-angiogenic effect. However, the mechanism remained unclear.

Bone marrow(BM)-derived progenitor cells contribute to tissue regeneration by promoting angiogenesis/vasculogenesis. We demonstrated that chemokine/cytokine mediated progenitor mobilization is dependent on the activation of matrix metalloproteinase-9 (MMP-9). Here, we show that following irradiation hematopoietic and endothelial progenitors are released into circulation in MMP-9 wild-type, but not MMP-9 deficient (−/−) mice. We have observed that low-dose irradiation fosters vascular regeneration in a limb ischemia model. Vascular regeneration was driven by the upregulation of MMP-9 mediating the release of soluble Kit-ligand (KitL) and increasing plasma vascular endothelial growth factor (VEGF), followed by mobilization of BM-derived hematopoietic and endothelial progenitor cells. Release of sKitL and production of VEGF were impaired in MMP-9-/- mice resulting in failure of mobilization of hematopoietic and endothelial progenitors, and delayed vessel formation in the ischemic limb. The blood vessels forming in the ischemic tissue of MMP-9−/− mice lacked smooth muscle cell coverage, whereas stable vessels were formed in MMP-9 wild-type animals. But which cell type might be responsible for the observed VEGF increase following irradiation? Mast cells are known to harbor a variety of growth factors and angiogenic factors, including VEGF and have been shown to have pro-angiogenic effects. We analyzed various tissues of irradiated and non-irradiated controls. The number of mast cells was increased in the irradiated muscle tissue in MMP-9 wild-type, but not MMP-9−/− mice. VEGF was mainly produced by mast cells in a MMP-9 dependent manner as determined by in situ hybridization. Likewise, the number of mast cells was increased in the ischemic tissue of MMP-9 wild-type, but not in MMP-9 deficient mice. We could show that both VEGF and KitL can promote mast cell migration and that irradiation-induced soluble KitL in collaboration with VEGF promoted migration of mast cells in vitro.

Taken together, low-dose irradiation promoted hematopoietic and endothelial progenitor cell mobilization and activation of mast cells thereby promoting vasculogenesis/angiogenesis in a hind limb model. These data not only show a novel mechanism of neovascularization and tissue regeneration but suggest that low-dose irradiation can be used for therapeutic angiogenesis augmenting collateral vessel growth in ischemic tissues.
P-Selectin Activates Leukocyte Integrin alphaMbeta2 by Recruiting Phosphatidylinositol-3 Kinase to P-Selectin Glycoprotein Ligand-1 Cytoplasmic Domain through Adaptor Protein Naf1.
Authors: Geng Jian-Guo , Wang Hai-Bo , Chen Ming , Xu Tao , Zhang Lei , Zhu Xueliang , Plow Edward F.
Published: October 2019

The interaction of endothelial P-selectin (CD62P) with neutrophil PSGL-1 (P-selectin glycoprotein ligand-1, CD162) mediates neutrophil rolling, which acts in concert with cytokines and chemoattractants for integrin-mediated firm adhesion of neutrophils to vascular endothelial cells. Numerous studies have established that this sequence of events is critical to the extravasation of neutrophils during an inflammatory response. We have recently shown that cross-linking of PSGL-1 with P-selectin acts cooperatively with extracellular stimuli, such as cytokines and chemoattractants released from and displayed on the activated endothelial lining of the vessel walls, for optimal activation of beta2-integrins, an increase in their apparent affinity/avidity for cognate ligands, which in turn supports firm adhesion and transendothelial migration. To identify cellular proteins which interact with human PSGL-1 and trigger this cascade of events, we used the cytoplasmic domain of human PSGL-1 as the bait for screening of human leukocyte yeast two-hybrid library. During this genetic screening, we identified Naf-1 (Nef-associated factor 1; also called ABIN, A20 binding inhibitor of NF-kB, or VAN, virion-associated nuclear shuttle protein) as a binding partner to the cytoplasmic domain of PSGL-1. The specific interaction of Naf1 with PSGL-1 was confirmed by GST-fusion protein pull-down and co-immunoprecipitation experiments. As YPPM at 552–555 of Naf1a amino acid sequence is a known binding motif for p85 subunit of phosphatidylinositol-3 kinase (PI3K; YXXM in which phosphorylated Y is required for p85 binding), we performed co-immunoprecipitation experiments to show that Naf1a actually acted as an adaptor protein for PSGL-1 and p85 subunit of PI3K. In addition, we found that P-selectin activated the enzyme activity of PI3K in human neutrophils and PI3K specific inhibitors, wortmannin and LY294002, inhibited alphaMbeta2 activation and clustering induced by P-selectin. Our data thus delineate a novel PSGL-1 signal transduction pathway essential for transactivation of beta2-integrins.