bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.