Preclinical activity of CPI-0610, a novel small-molecule bromodomain and extra-terminal protein inhibitor in the therapy of multiple myeloma.

Inhibition of the bromodomain and extra-terminal (BET) proteins is a promising therapeutic strategy for various hematologic cancers. Previous studies suggest that BET inhibitors constrain tumor cell proliferation and survival mainly through the suppression of MYC transcription and activity. However, suppression of the transcription of additional genes also contributes to the antitumor activity of BET inhibitors but is less well understood. Here we examined the therapeutic potential of CPI-0610, a potent BET inhibitor currently undergoing phase I clinical testing, in multiple myeloma (MM). CPI-0610 displays potent cytotoxicity against MM cell lines and patient-derived MM cells through G1 cell cycle arrest and caspase-dependent apoptosis. CPI-0610-mediated BET inhibition overcomes the protective effects conferred by cytokines and bone marrow stromal cells. We also confirmed the in vivo efficacy of CPI-0610 in a MM xenograft mouse model. Our study found IKZF1 and IRF4 to be among the primary targets of CPI-0610, along with MYC. Given that immunomodulatory drugs (IMiDs) stabilize cereblon and facilitate Ikaros degradation in MM cells, we combined it with CPI-0610. Combination studies of CPI-0610 with IMiDs show in vitro synergism, in part due to concomitant suppression of IKZF1, IRF4 and MYC, providing a rationale for clinical testing of this drug combination in MM patients.

Polymeric endoaortic paving: Mechanical, thermoforming, and degradation properties of polycaprolactone/polyurethane blends for cardiovascular applications.

Polymeric endoaortic paving (PEAP) is a process by which a polymer is endovascularly delivered and thermoformed to coat or "pave" the lumen of the aorta. This method may offer an improvement to conventional endoaortic therapy in allowing conformal graft application with reduced risk of endoleak and customization to complex patient geometries. Polycaprolactone (PCL)/polyurethane (PU) blends of various blend ratios were assessed as a potential material for PEAP by characterizing their mechanical, thermoforming and degradation properties. Biaxial tension testing revealed that the blends' stiffness is similar to that of aortic tissue, is higher for blends with more PCL content, and may be affected by thermoforming and degradation. Tubes of blends were able to maintain a higher diameter increase after thermoforming at higher PCL content and higher heating temperatures; 50/50 blend tubes heated to 55 °C were able to maintain 90% of the diameter increase applied. Delamination forces of the blends ranged from 41 to 235 N m⁻². In a Pseudomonas lipase solution, the 50/50 blend had a 94% lower degradation rate than pure PCL, and the 10/90 blend exhibited no degradation. These results indicate that PEAP, consisting of a PCL/PU blend, may be useful in developing the next generation of endoaortic therapy.

What retroviruses teach us about the involvement of c-Myc in leukemias and lymphomas.

Overexpression of the cellular oncogene c-Myc frequently occurs during induction of leukemias and lymphomas in many species. Retroviruses have enhanced our understanding of the role of c-Myc in such tumors. Leukemias and lymphomas induced by retroviruses activate c-Myc by: (1) use of virally specified proteins that increase c-Myc transcription, (2) transduction and modification of c-Myc to generate a virally encoded form of the gene, v-Myc, and (3) proviral integration in or near c-Myc. Proviral integrations elevate transcription by insertion of retroviral enhancers found in the long terminal repeat (LTR). Studies of the LTR enhancer elements from these retroviruses have revealed the importance of these elements for c-Mycactivation in several cell types. Retroviruses also have been used to identify genes that collaborate with c-Myc during development and progression of leukemias and lymphomas. In these experiments, animals that are transgenic for c-Mycoverexpression (often in combination with the overexpression or deletion of known proto-oncogenes) have been infected with retroviruses that then insertionally activate novel co-operating cellular genes. The retrovirus then acts as a molecular 'tag' for cloning of these genes. This review covers several aspects of c-Myc involvement in retrovirally induced leukemias and lymphomas.

Type B leukemogenic virus has a T-cell-specific enhancer that binds AML-1.

Type B leukemogenic virus (TBLV) induces rapidly appearing T-cell tumors in mice. TBLV is highly related to mouse mammary tumor virus (MMTV) except that TBLV long terminal repeats (LTRs) have a deletion of negative regulatory elements and a triplication of sequences flanking the deletion. To determine if the LTR triplication represents a viral enhancer element, we inserted the triplication upstream and downstream in either orientation relative to the thymidine kinase promoter linked to the luciferase gene. These experiments showed that upregulation of reporter gene activity by the TBLV triplication was relatively orientation independent, consistent with the activity of eukaryotic enhancer elements. TBLV enhancer activity was observed in T-cell lines but not in fibroblasts, B cells, or mammary cells, suggesting that enhancer function is cell type dependent. To analyze the transcription factor binding sites that are important for TBLV enhancer function, we prepared substitution mutations in a reconstituted C3H MMTV LTR that recapitulates the deletion observed in the TBLV LTR. Transient transfections showed that a single mutation (556M) decreased TBLV enhancer activity at least 20-fold in two different T-cell lines. This mutation greatly diminished AML-1 (recently renamed RUNX1) binding in gel shift assays with a mutant oligonucleotide, whereas AML-1 binding to a wild-type TBLV oligomer was specific, as judged by competition and supershift experiments. The 556 mutation also reduced TBLV enhancer binding of two other protein complexes, called NF-A and NF-B, that did not appear to be related to c-Myb or Ets. AML-1 overexpression in a mammary cell line enhanced expression from the TBLV LTR approximately 30-fold. These data suggest that binding of AML-1 to the TBLV enhancer, likely in combination with other factors, is necessary for optimal enhancer function.

Identification of epitope and surface-exposed domains of Shigella flexneri invasion plasmid antigen D (IpaD).

Transport and surface expression of the invasion plasmid antigens (Ipa proteins) is an essential trait in the pathogenicity of Shigella spp. In addition to the type III protein secretion system encoded by the mxi/spa loci on the large virulence plasmid, transport of IpaB and IpaC into the surrounding medium is modulated by IpaD. To characterize the structural topography of IpaD, the Geysen epitope-mapping system was used to identify epitopes recognized by surface-reactive monoclonal and polyclonal antibodies produced against purified recombinant IpaD or synthetic IpaD peptides. Surface-exposed epitopes of IpaD were confined to the first 180 amino acid residues, whereas epitopes in the carboxyl-terminal half were not exposed on the Shigella surface. By using convalescent-phase sera from 10 Shigella flexneri-infected monkeys, numerous epitopes were mapped within a surface-exposed region of IpaD between amino acid residues 14 and 77. Epitopes were also identified in the carboxyl-terminal half of IpaD with a few convalescent-phase sera. Comparison of IpaD epitope sequences with Salmonella SipD sequences indicated that very similar epitopes may exist in the carboxyl-terminal region of each protein whereas the IpaD epitopes in the surface-exposed amino-terminal region were unique for the Shigella protein. Although the IpaD and SipD homologs may play similar roles in transport, the dominant serum antibody response to IpaD is against the unique region of this protein exposed on the surface of the pathogen.

Conformational changes in cholera toxin B subunit-ganglioside GM1 complexes are elicited by environmental pH and evoke changes in membrane structure.

Fluorescence resonance energy transfer (FRET) was used to monitor pH-dependent structural changes in the cholera toxin B subunit (CTB) and the membranes with which CTB associates. The distance separating the single tryptophan (Trp88) of each CTB monomer and a pyrene probe linked to the membrane-imbedded tail of ganglioside GM1 is not influenced by pH in a range from 3.5 to 7.5, consistent with the position of Trp88 in the GM1 binding site of CTB. In contrast, the distance between the pyrene probe on GM1 and coumarin, stilbene, or fluorescein probes covalently linked to specific sites on CTB appears to increase significantly as the pH is lowered to 5.0 or less. This conformational change is not accompanied by detectable changes in the distance between Trp88 and these extrinsic probe positions in the presence of nonfluorescent GM1. However, when the distance from Trp88 to the extrinsic probes is monitored as a function of pH in the absence of GM1, a conformational change is seen which indicates that receptor binding influences the character of pH-dependent conformational changes that occur within CTB. Interestingly, the observed change in CTB conformation is accompanied by a change in the relative position of GM1 within the membrane as judged by FRET from the pyrene probe on GM1 to a 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) probe linked to the polar head group of phosphatidylethanolamine and positioned at the membrane surface. Taken together, the data imply that low endosomal pH is capable of inducing structural changes in CTB, which, in turn, exert effects on the structure of the membrane to which CTB is bound. These phenomena may have a role in (1) processing of cholera toxin within the endosomal compartments of some target cell types, (2) determining the lag time between cholera toxin binding and the target cell response to cholera intoxication, or (3) the efficiency of CTB and cholera toxin as mucosal adjuvants.

The hysteresis loop as a model for low back motion analysis.

The human low back is highly susceptible to dysfunction. Many diagnostic and treatment modalities for low back problems are used subjectively in clinical practice. Availability of a quantitative reference of back motion could improve practice efficiency. This article describes hysteresis and the hysteresis loop as an objective measurement model of low back motion. The authors constructed an instrument that quantifies force displacement responses of the lower back in passive subjects. Hysteresis loops generated by this instrument lay a scientific foundation on which to base diagnostic procedures and therapeutic measures.

Cloning, expression, and affinity purification of recombinant Shigella flexneri invasion plasmid antigens IpaB and IpaC.

Shigella flexneri and related enteropathogenic bacteria are important agents of bacillary dysentery, a potentially life-threatening illness for children in underdeveloped regions of the world. Onset of shigellosis stems from S. flexneri invasion of colonic epithelial cells, leading to localized cell death and inflammation. Invasion plasmid antigens (Ipa) B, C, and D are three secreted proteins encoded by the large virulence plasmid of S. flexneri that have been implicated as essential effectors of this cell invasion process. These proteins are expressed as part of the ipa operon and are among the major targets of the host immune response to shigellosis. Biochemical characterization of the Ipa invasins has been complicated by the fact they have not been purified in the quantities needed for detailed in vitro analysis. Here we describe the first cloning, expression, and extensive purification of IpaB and IpaC fusion proteins from Escherichia coli for use in dissecting of the protein biochemistry of S. flexneri pathogenesis. A variety of approaches were used to prepare significant quantities of these proteins in their soluble forms, including the use of different host cell lines, modification of bacterial growth conditions, and the use of alternative plasmid expression vectors. Now that these Ipa proteins are available in a highly pure form, it will be possible to initiate studies on their important biological and immunological properties as well as their recruitment into high-molecular-weight protein complexes. Together with IpaD (purified as part of a previous study), these purified proteins will be useful for: (a) exploring properties of the host immune response to S. flexneri invasion, (b) elucidating the specific biochemical properties that lead to pathogen internalization, (c) analyzing the importance of specific Ipa protein complexes in host cell invasions, and (d) monitoring, or perhaps even augmenting, the efficacy of live oral vaccines in human trials.

Fluorescence analysis of galactose, lactose, and fucose interaction with the cholera toxin B subunit.

The cholera toxin B subunit (CTB) recognizes ganglioside GM1 receptors on target cells to facilitate entry of the toxin's A1 polypeptide into the host cytoplasm. GM1 binding to the CTB homopentamer occurs cooperatively with the most prominent interactions involving the terminal galactose residue of the ganglioside. Here, it is shown that association of galactose, lactose, or fucose (6-deoxy-galactose) with CTB is readily monitored using fluorescence spectroscopy. In many respects, however, the formation of CTB complexes with these small sugar analogues of GM1 greatly differs from the formation of complexes with the ganglioside itself. Each of these monosaccharides has a much weaker affinity for CTB than does GM1 and none of the sugars appear to be bound cooperatively. Moreover, GM1 binding conveys a stabilizing effect to CTB which is not seen upon binding of galactose or lactose. These data indicate that CTB-GM1 interactions involving sites other than the terminal galactose of the ganglioside serve prominently in the proper placement of CT on the target cell surface.