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Table S1: Crystallographic data collection and refinement statistics Table S2: Inhibitory activity against other BET bromodomains Table S3: Bromodomain profiling data Fig. S1: Detailed binding interactions of compounds 1 - 5 in BRD4-1 Fig. S2: Comparison of compound 2 and JQ1 in Brd4-1 Fig. S3: Chemical structures of other compounds used in this work. Fig. S4: Statistical evaluation of kinome profiling data using the Gini coefficient Fig. S5: Original flow cytometry data of MM1.S cells Fig. S6: MM1.S cells require stimulation with IL-6 to detect phospho-STAT3 levels Fig. S7: Effect of dual BRD4-kinase inhibitors on MV-4-11 cells. Fig. S8: Time dependent alteration of c-Myc and p21Cip1 levels in MM1.S cells. Fig. S9: Effect of dual BET-kinase inhibitors on ruxolitinib sensitive MPN cell lines Fig. S10: Dose-effect analysis of UKE-1 cells treated with drug combinations Fig. S11: Cell line sensitivity by tissue type

Synergistic action of kinase and BET bromodomain inhibitors in cell killing has been reported for a variety of cancers. Using the chemical scaffold of the JAK2 inhibitor TG101348, we developed and characterized single agents which potently and simultaneously inhibit BRD4 and a specific set of oncogenic tyrosine kinases including JAK2, FLT3, RET, and ROS1. Lead compounds showed on-target inhibition in several blood cancer cell lines and were highly efficacious at inhibiting the growth of hematopoietic progenitor cells from patients with myeloproliferative neoplasm. Screening across 931 cancer cell lines revealed differential growth inhibitory potential with highest activity against bone and blood cancers and greatly enhanced activity over the single BET inhibitor JQ1. Gene drug sensitivity analyses and drug combination studies indicate synergism of BRD4 and kinase inhibition as a plausible reason for the superior potency in cell killing. Combined, our findings indicate promising potential of these agents as novel chemical probes and cancer therapeutics. Mol Cancer Ther; 16(6); 1054–67. ©2017 AACR.

Table S4: Kinome profiling data (Microsoft Excel sheet) Table S5: Cell line screening data (Microsoft Excel sheet)

A method for non-invasive visualization of genetically labelled cells in animal disease models with micron-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the “optical window” above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune, previously the brightest monomeric FP when excited beyond 600 nm. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence, while the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts and stem cells into myocytes in living mice with high anatomical detail.

Synergistic action of kinase and BET bromodomain inhibitors in cell killing has been reported for a variety of cancers. Using the chemical scaffold of the JAK2 inhibitor TG101348, we developed and characterized single agents which potently and simultaneously inhibit BRD4 and a specific set of oncogenic tyrosine kinases including JAK2, FLT3, RET, and ROS1. Lead compounds showed on-target inhibition in several blood cancer cell lines and were highly efficacious at inhibiting the growth of hematopoietic progenitor cells from patients with myeloproliferative neoplasm. Screening across 931 cancer cell lines revealed differential growth inhibitory potential with highest activity against bone and blood cancers and greatly enhanced activity over the single BET inhibitor JQ1. Gene drug sensitivity analyses and drug combination studies indicate synergism of BRD4 and kinase inhibition as a plausible reason for the superior potency in cell killing. Combined, our findings indicate promising potential of these agents as novel chemical probes and cancer therapeutics. Mol Cancer Ther; 16(6); 1054–67. ©2017 AACR.

Screening for effects of small molecules on cells grown in culture is a well-established method for drug discovery and testing, and faster throughput at lower cost is needed. Small-molecule arrays and microfluidics are promising approaches. Here we introduce a simple method of surface-mediated delivery of drugs to cells from a microarray of phospholipid multilayers (layers thicker than a bilayer) encapsulating small molecules. The multilayer patterns are of sub-cellular dimensions and controllable thickness and were formed by dip-pen nanolithography. The patterns successfully delivered a rhodamine-tagged lipid and drugs only to the cells directly over them, indicating successful encapsulation and no cross-contamination to cells grown next to the patterns. We also demonstrated multilayer thickness-dependant uptake of the lipids from spots with sub-cellular lateral dimensions, and therefore the possibility of delivering different dosages from different areas of the array. The efficacies of two drugs were assayed on the same surface, and we were able to deliver dosages comparable to those of solution based delivery (up to the equivalent of 30 μg/mL). We expect our method to be a promising first step toward producing a single high-throughput liposome-based screening microarray plate that can be used in the same way as a standard well plate.

A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones. © 2012 Nature America, Inc. All rights reserved.

Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins.

Multicolour labelling with fluorescent proteins is frequently used to differentially highlight specific structures in living systems. Labelling with fusion proteins is particularly demanding and is still problematic with the currently available palette of fluorescent proteins that emit in the red range due to unsuitable subcellular localization, protein-induced toxicity and low levels of labelling efficiency. Here we report a new monomeric red fluorescent protein, called FusionRed, which demonstrates both high efficiency in fusions and low toxicity in living cells and tissues.

Commonly used monomeric blue fluorescent proteins suffer from moderate brightness. The brightest of them, mTagBFP, has a notably low chemical stability over time. Prolonged incubation of mTagBFP leads to its transition from a blue fluorescent state with absorbance at 401 nm to a non-fluorescent state with absorbance at 330 nm. Here, we have determined the chemical structure of the degraded product of the blue mTagBFP-like chromophore. On the basis of mTagBFP we have developed an improved variant, named mTagBFP2. mTagBFP2 exhibits 2-fold greater chemical stability and substantially higher brightness in live cells than mTagBFP. mTagBFP2 is also 1.2-fold and 1.7-fold more photostable than mTagBFP in widefield and confocal microscopy setups, respectively. mTagBFP2 maintains all other beneficial properties of the parental mTagBFP including the high pH stability and fast chromophore formation. The enhanced photostability and chromophore chemical stability of mTagBFP2 make it a superior protein tag. mTagBFP2 performs well in the numerous protein fusions and surpasses mTagBFP as a donor in Forster resonance energy transfer with several green fluorescent protein acceptors.

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

The original green fluorescent protein (GFP) was discovered back in the early 1960s when researchers studying the bioluminescent properties of the Aequorea victoria jellyfish isolated a blue-light-emitting bioluminescent protein called aequorin together with another protein that was eventually named

The advent of fluorescent proteins (FP) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning the blue to red spectral regions. Common to autofluorescent FPs is their tight β-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has its own unique photophysical properties. Thus, there is no single “best” fluorescent protein for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for any given application, we have characterized quantitatively over 40 different FPs for their brightness, photostability, pH stability, and monomeric properties, which permits easy apples-to-apples comparisons between these FPs. We report the values for all of the FPs measured, but focus the discussion on the more popular and/or best performing FPs in each spectral region.