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.

Dual-modality endomicroscopy with co-registered fluorescence and phase contrast.

We describe a dual-modality laser scanning endomicroscope that provides simultaneous fluorescence contrast based on confocal laser endomicroscopy (CLE) and phase-gradient contrast based on scanning oblique back-scattering microscopy (sOBM). The probe consists of a 2.6mm-diameter micro-objective attached to a 30,000-core flexible fiber bundle. The dual contrasts are inherently co-registered, providing complementary information on labeled and un-labeled sample structure. Proof of principle demonstrations are presented with ex-vivo mouse colon tissue.

Phase-gradient contrast in thick tissue with a scanning microscope.

It is well known that the principle of reciprocity is valid for light traveling even through scattering or absorptive media. This principle has been used to establish an equivalence between conventional widefield microscopes and scanning microscopes. We make use of this principle to introduce a scanning version of oblique back-illumination microscopy, or sOBM. This technique provides sub-surface phase-gradient and amplitude images from unlabeled tissue, in an epi-detection geometry. That is, it may be applied to arbitrarily thick tissue. sOBM may be implemented as a simple, cost-effective add-on with any scanning microscope, requiring only the availability of an extra input channel in the microscope electronics. We demonstrate here its implementation in combination with two-photon excited fluorescence (TPEF) microscopy and with coherent anti-Stokes Raman scattering (CARS) microscopy, applied to brain or spinal cord tissue imaging. In both cases, sOBM provides information on tissue morphology complementary to TPEF or CARS contrast. This information is obtained simultaneously and is automatically co-registered. Finally, we show that sOBM can be operated at video rate.

View synthesis with a partitioned-aperture microscope.

We present a simple and fast algorithm for view synthesis based on the acquisition of four high-resolution oblique images with a conventional widefield microscope. The images are acquired simultaneously using a partitioned aperture add-on. The technique provides physically valid views of thin samples that are transmitting or fluorescent, as demonstrated with biopsied tissue or green fluorescent protein-labeled brain slices. The goal of this technique is to facilitate image interpretation by conferring impressions of depth that are otherwise absent in standard microscope images.

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.

Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy.

When a two-photon excited fluorescence (TPEF) microscope is used to image deep inside tissue, out-of-focus background can arise from both ballistic and nonballistic excitation. We propose a solution to largely reject TPEF background in thick tissue. Our technique is based on differential-aberration imaging with a deformable mirror. By introducing extraneous aberrations in the excitation beam path, we preferentially quench in-focus TPEF signal while leaving out-of-focus TPEF background largely unchanged. A simple subtraction of an aberrated, from an unaberrated, TPEF image then removes background while preserving signal. Our differential aberration (DA) technique is simple, robust, and can readily be implemented with standard TPEF microscopes with essentially no loss in temporal resolution when using a line-by-line DA protocol. We analyze the performance of various induced aberration patterns, and demonstrate the effectiveness of DA-TPEF by imaging GFP-labeled sensory neurons in a mouse olfactory bulb and CA1 pyramidal cells in a hippocampus slice.

Electro-optic response of second-harmonic generation membrane potential sensors.

We quantify and ascertain the nature of the second-harmonic generation (SHG) response of amphiphilic push-pull chromophores to a transmembrane electric field. Our technique is based on the application of an alternating field across labeled giant unilamelar vesicles. For chromophore responses that are purely electro-optic, our technique provides an estimate of photoinduced charge shifts based on the observed dispersion of the field response, in accord with a two-level perturbation theory. These results are applicable to the optimization of membrane potential sensors for SHG microscopy.

Transmission confocal laser scanning microscopy with a virtual pinhole based on nonlinear detection.

We present a transmission-mode confocal laser scanning microscope system based on the use of second-harmonic generation (SHG) for signal detection. Our method exploits the quadratic intensity dependence of SHG to preferentially reveal unscattered signal light and reject out-of-focus scattered background. The SHG crystal acts as a virtual pinhole that remains self-aligned without the need for descanning.

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.

Critical roles of nuclear receptor response elements in replication of hepatitis B virus.

Functional analysis of the roles of the nuclear receptor response elements (NRREs) in the transcription and replication of hepatitis B virus (HBV) in the context of its whole genome has been hampered by the extensive overlapping of the NRREs with the regions encoding viral proteins. We introduced point mutations that inactivate the NRREs individually without altering the open reading frames of viral proteins. These mutations in the context of a plasmid containing 1.2 copies of the HBV genome were transiently transfected into the human hepatoma cell line Huh7. Inactivation of the NRRE in either the preC promoter (NRRE(preC)) or enhancer I (NRRE(enhI)) led to moderate reductions in synthesis of viral RNAs. Concurrent inactivation of both NRREs led to 7- to 8-fold reductions in synthesis of the preC, pregenomic, and preS RNAs and a 15-fold reduction in synthesis of the S RNA. The accumulation of viral DNA in the cytoplasmic nucleocapsids and virion particles in the culture medium was also reduced seven- to eightfold. These results suggest that these NRREs are critical for the efficient propagation of HBV in hepatocytes. In cotransfection experiments we also found that overexpression of PPARalpha-RXRalpha in the presence of their respective ligands led to a fourfold increase in pregenomic RNA synthesis and a four- to fivefold increase in viral DNA synthesis, while it had little or no effect on synthesis of the other viral RNAs. Similar effects were observed with overexpression of PPARgamma-RXRalpha in the presence of their respective ligands. This activation was dependent on NRRE(preC), because the increase in synthesis of viral RNA and DNA was not observed when this site was mutated. Likewise, no activation of synthesis of pregenomic RNA and viral DNA by PPARalpha-RXRalpha was observed in a naturally occurring NRRE(preC)(-) mutant of HBV. Our results suggest that interactions between nuclear receptors and NRREs present in the HBV genome may play critical roles in regulating its transcription and replication during HBV infection of hepatocytes.

Two-photon microscopy in brain tissue: parameters influencing the imaging depth.

Light scattering by tissue limits the imaging depth of two-photon microscopy and its use for functional brain imaging in vivo. We investigate the influence of scattering on both fluorescence excitation and collection, and identify tissue and instrument parameters that limit the imaging depth in the brain. (i) In brain slices, we measured that the scattering length at lambda=800 nm is a factor 2 higher in juvenile cortical tissue (P14-P18) than in adult tissue (P90). (ii) In a detection geometry typical for in vivo imaging, we show that the collected fraction of fluorescence drops at large depths, and that it is proportional to the square of the effective angular acceptance of the detection optics. Matching the angular acceptance of the microscope to that of the objective lens can result in a gain of approximately 3 in collection efficiency at large depths (>500 microm). A low-magnification (20x), high-numerical aperture objective (0.95) further increases fluorescence collection by a factor of approximately 10 compared with a standard 60x-63x objective without compromising the resolution. This improvement should allow fluorescence measurements related to neuronal or vascular brain activity at >100 microm deeper than with standard objectives.

Nuclear factor 1 family members mediate repression of the BK virus late promoter.

BK virus (BKV) is a member of the polyoma virus family that is ubiquitous in humans. Its 5-kb DNA genome consists of a bidirectional promoter region situated between two temporally regulated coding regions. We mapped the transcription initiation site of the major late promoter (MLP) of the archetype strain BKV(WW) to nt 185. We found that it lies within the sequence TGGN6GCCA, a binding site for members of the nuclear factor 1 (NF1) family of transcription factors. Competition electrophoretic mobility shift and immunoshift assays confirmed that NF1 factors present in nuclear extracts of HeLa and CV-1 cells bind to the BKV-MLP. Because BKV(WW) grew poorly in tissue culture and failed to express detectable levels of RNA in vitro, SV40-BKV chimeric viruses were constructed to investigate the transcriptional function of this NF-1 binding site. These sequence-specific factors repressed transcription in a cell-free system when template copy number was low. This repression could be relieved by the addition in trans of oligonucleotides containing wild-type, but not mutated, NF1-binding site sequences. SV40-BKV chimeric viruses defective in this NF1-binding site overproduced late RNA at early, but not late, times after transfection of CV-1 cells. Finally, transient expression in 293 cells of cDNAs encoding the family members NF1-A4, NF1-C2, and NF1-X2 specifically repressed transcription from the BKV late promoter approximately 3-, 10-, and 10-fold, respectively, in a DNA binding-dependent manner. We conclude that some members of the NF1 family of transcription factors can act as sequence-specific cellular repressors of the BKV-MLP. We propose that titration of these and other cellular repressors by viral genome amplification may be responsible in part for the replication-dependent component of the early-to-late switch in BKV gene expression.

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.

Coherent scattering in multi-harmonic light microscopy.

By focusing a pulsed laser beam into a sample, harmonic up-conversion can be generated as well as multi-photon excited fluorescence. Whereas multi-photon excited fluorescence microscopy is well established, the use of multi-harmonic generation for three-dimensional image contrast is very recent. Both techniques can provide similar resolution and, for adequate radiating source density, comparable signal levels, allowing them to be combined in a single versatile instrument. However, harmonic generation differs fundamentally from fluorescence generation in that it is coherent and produces radiation patterns that are highly sensitive to phase. As such, multi-harmonic generation microscopy provides a unique window into molecular spatial organization that is inaccessible to fluorescence.

Odor-evoked calcium signals in dendrites of rat mitral cells.

Mitral cell dendrites do more than passively integrate and convey synaptic potentials to the soma, they release transmitter onto local interneurones to mediate recurrent and lateral inhibition. Several mechanisms may control the level of dendritic intracellular calcium ([Ca(2+)]) and define timing for dendritic release. Here we investigated in vivo, how odor controls calcium dynamics in mitral cell dendrites by combining intracellular recording and two-photon microscopy imaging of [Ca(2+)]. During odor stimulation, two types of [Ca(2+)] changes accompany membrane potential oscillations that are phase-locked with the respiratory cycle: (i) one is graded and parallels the membrane potential, even below the threshold for action potential firing; (ii) a second is transient, triggered by sodium action potentials that invade the entire dendritic tree. These results indicate that mitral cell dendritic compartments are synchronized by action potentials and suggest that the efficacy of dendritic synapses is finely tuned by odor-evoked graded changes in [Ca(2+)].

Identification of a novel element involved in regulation of the lytic switch BZLF1 gene promoter of Epstein-Barr virus.

Epstein-Barr virus (EBV) is a human herpesvirus capable of establishing a latent state in B lymphocytes. EBV's BZLF1 gene product plays a central role in regulating the switch from latency to productive infection. Here, we identify a sequence element, 5'-CAGGTA-3', called ZV, located at nucleotides -17 to -12 relative to the transcription initiation site of the BZLF1 promoter. ZV sequence-specifically binds a cellular nuclear factor(s), ZVR. ZVR DNA-binding activity was present in the EBV-negative B-lymphocytic cell line DG75, the EBV-positive B-lymphocytic cell lines GG68 and 721, the cervical cell line C33A, and the kidney cell line CV-1 but not in the breast carcinoma cell line MCF-7. Mutations in ZV that relieve binding of ZVR lead to a two- to fourfold increase in basal expression of the BZLF1 promoter in DG75, C33A, and CV-1 cells. The same mutants exhibited a 40- to 180-fold increase in tetradecanoyl phorbol acetate-ionomycin-induced expression in DG75 cells and a 22-fold increase in C33A cells. Thus, ZVR functions as a regulator of the BZLF1 promoter, repressing transcription when bound to the ZV site in the absence of inducers. No differences in basal or induced transcription between wild-type and ZV mutant BZLF1 promoters were observed in ZVR-negative MCF-7 cells. ZVR failed to bind any of the previously identified negative regulatory elements within the BZLF1 promoter. We conclude that ZV functions as an important regulatory element of the BZLF1 promoter, with ZVR likely playing important roles in the maintenance of latency and reactivation of EBV.