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In a seismic PSA, dependency among seismic failures of components has not been explicitly modeled in the fault tree or event tree. This dependency is separately identified and assigned with numbers that range from zero to unity that reflect the level of the mutual correlation among seismic failures. Because of complexity and difficulty in calculating combination probabilities of correlated seismic failures in complex seismic event tree and fault tree, there has been a great need of development to explicitly model seismic correlation in terms of seismic common cause failures (CCFs). If seismic correlations are converted into seismic CCFs, it is possible to calculate an accurate value of a top event probability or frequency of a complex seismic fault tree by using the same procedure as for internal, fire, and flooding PSA. This study first proposes a methodology to explicitly model seismic dependency by converting correlated seismic failures into seismic CCFs. As a result, this methodology will allow systems analysts to quantify seismic risk as what they have done with the CCF method in internal, fire, and flooding PSA.

DNA–nanoparticle conjugates have found widespread use in sensing, imaging, and as components of devices. However, their synthesis remains relatively complicated and empirically based, often requiring specialized protocols for conjugates of different size, valence, and elemental composition. Here we report a novel, bottom-up approach for the synthesis of DNA–nanoparticle conjugates, based on ring-opening metathesis polymerization (ROMP), intramolecular crosslinking, and template synthesis. Using size, valence, and elemental composition as three independent synthetic parameters, various conjugates can be obtained using a facile and universal procedure. Examples are given to show the usefulness of these conjugates as sensing probes, building blocks for self-assembly, and as model particles for structure–property relationship studies., DNA–nanoparticle conjugates can be synthesized with independent control over size, valence and elemental composition using a template-based strategy.

Pinpointing subcellular protein localizations from microscopy images is easy to the trained eye, but challenging to automate. Based on the Human Protein Atlas image collection, we held a competition to identify deep learning solutions to solve this task. Challenges included training on highly imbalanced classes and predicting multiple labels per image. Over 3 months, 2,172 teams participated. Despite convergence on popular networks and training techniques, there was considerable variety among the solutions. Participants applied strategies for modifying neural networks and loss functions, augmenting data and using pretrained networks. The winning models far outperformed our previous effort at multi-label classification of protein localization patterns by ~20%. These models can be used as classifiers to annotate new images, feature extractors to measure pattern similarity or pretrained networks for a wide range of biological applications., The 2018 Human Protein Atlas Image Classification competition sought to improve automated classification of protein subcellular localizations from fluorescence images. The winning strategies involved innovative deep learning approaches for multi-label classification.

Many different metal ions are involved in various biological functions including metallomics and trafficking, and yet there are currently effective sensors for only a few metal ions, despite the first report of metal sensors for calcium more than 40 years ago. To expand upon the number of metal ions that can be probed in biological systems, we and other laboratories employ the in vitro selection method to obtain metal-specific DNAzymes with high specificity for a metal ion and then convert these DNAzymes into fluorescent sensors for these metal ions using a catalytic beacon approach. In this Forum Article, we summarize recent progress made in developing these DNAzyme sensors to probe metal ions in living cells and in vivo, including several challenges that we were able to overcome for this application, such as DNAzyme delivery, spatiotemporal control, and signal amplification. Furthermore, we have identified a key remaining challenge for the quantitative detection of metal ions in living cells and present a new design and the results of a Forster resonance energy transfer (FRET)-based DNAzyme sensor for the ratiometric quantification of Zn2+ in HeLa cells. By converting existing DNAzyme sensors into a ratiometric readout without compromising the fundamental catalytic function of the DNAzymes, this FRET-based ratiometric DNAzyme design can readily be applied to other DNAzyme sensors as a major advance in the field to develop much more quantitative metal-ion probes for biological systems.

Spatial and temporal distributions of metal ions in vitro and in vivo are crucial in our understanding of the roles of metal ions in biological systems, and yet there is a very limited number of methods to probe metal ions with high space and time resolution, especially in vivo. To overcome this limitation, we report a Zn(2+)-specific near infrared (NIR) DNAzyme nanoprobe for real-time metal ion tracking with spatiotemporal control in early embryos and larvae of zebrafish. By conjugating photocaged DNAzymes onto lanthanide-doped upconversion nanoparticles (UCNPs), we have achieved upconversion of a deep tissue penetrating NIR 980 nm light into 365 nm emission. The UV photon then efficiently photo-decages a substrate strand containing a nitrobenzyl group at the 2’-OH of adenosine ribonucleotide, allowing enzymatic cleavage by a complementary DNA strand containing a Zn(2+)-selective DNAzyme. The product containing a visible FAM fluorophore that is initially quenched by BHQ1 and Dabcyl quenchers, is released after cleavage, resulting in higher fluorescent signals. The DNAzyme-UCNP probe enables Zn(2+) sensing by exciting in the NIR biological imaging window in both living cells and zebrafish embryos, and detecting in the visible region. This report introduces a platform that can be used to understand the Zn(2+) distribution with spatiotemporal control, thereby giving insights into the dynamical Zn(2+) ion distribution in intracellular and in vivo models.

There has been increasing interest on the use of natural organic coagulants to replace synthetic inorganic coagulants due to environment and health concerns and its failure to comply with discharge regulations. In this study, chickpea (Cicer arietinum) was utilized as a natural and novel coagulant and flocculant in the treatment of palm oil mill effluent (POME) produced from crude palm oil extraction. The standard jar test method was utilized to investigate the effects of pH, dosage and rapid mixing speed on the removal of total suspended solids (TSS), turbidity and chemical oxygen demand (COD). By applying response surface methodology with a central composite design, the optimum condition was established at pH of 6.69, chickpea dosage of 2.6 g/L, and rapid mixing speed of 140 rpm. At the optimum condition, the removal percentages of turbidity, COD and TSS were determined to be 86%, 56% and 87% respectively. Characterization of chickpea with Fourier transform infrared spectroscopy revealed the presence of OH, CH, NH, CC, CO, CN bonds which is most likely to be found in proteins and polysaccharides; while results from scanning electron microscopy showed the rough and porous structure of chickpea and confirms the interaction between the suspended solid pollutants of POME and chickpea. On top of that, analytical results from energy dispersive x-ray spectroscopy and bomb calorimeter discovered that the dewatered sludge recovered post-treatment at optimum condition have a high potential to be used as a clean solid biomass fuel.

This review summarizes research on the metal-binding properties of catalytic DNAzymes, towards the goal of understanding the structural properties leading to metal ion specificity. Progress made and insight gained from a range of biochemical and biophysical techniques are covered, and promising directions for future investigations are discussed.

Aptamer-based targeted drug delivery systems have shown significant promise for clinical applications. Although much progress has been made in this area, it remains unclear how PEG coating would affect the selective binding of DNA aptamers and thus influence the overall targeting efficiency. To answer this question, we herein report a systematic investigation of the interactions between PEG and DNA aptamers on the surface of liposomes by using a series of nanoscale liposomal doxorubicin formulations with different DNA aptamer and PEG modifications. We investigated how the spatial size and composition of the spacer molecules affected the targeting ability of the liposome delivery system. We showed that a spacer of appropriate length was critical to overcome the shielding from surrounding PEG molecules in order to achieve the best targeting effect, regardless of the spacer composition. Our findings provide important guidelines for the design of aptamer-based targeted drug delivery systems.

Nanomaterials which can respond to external stimuli have attracted considerable attention due to their potential applications in sensing and biomedicine. One of the most promising classes of such materials is the stimuli-responsive liposome that can release its contents in response to a specific target. Despite recent progress, development of liposomes responsive to small molecular targets remains a challenge, due to the difficulty in designing the transduction process to link between target binding and triggered release, even though small molecular metabolites play important roles in many biological processes. Herein, we demonstrate a combination of an aptamer (apt) for target recognition and enzyme phosphatidylcholine 2-acetylhydrolase (PLA2) for rupture of liposome. As a proof-of-concept, cocaine molecules were used to trigger the release of the enzyme. The exposure to DNA-PLA2 conjugates induced the rupture of liposome containing uranin and gadopentetic acid (Gd-DTPA), allowing multimodal fluorescent and MRI detection of cocaine. The strategy demonstrated in this work can be generally applied to other imaging modalities by loading different imaging agents, as well as other targets by using different functional DNAs.

Liposomes are nanocarriers comprised of lipid bilayers encapsulating an aqueous core. The ability of liposomes to encapsulate a wide variety of diagnostic and therapeutic agents has led to significant interest in utilizing liposomes as nanocarriers for theranostic applications. In this review, we highlight recent progress in developing liposomes as nanocarriers for a) diagnostic applications to detect proteins, DNA, and small molecule targets using fluorescence, magnetic resonance, ultrasound, and nuclear imaging; b) therapeutic applications based on small molecule-based therapy, gene therapy and immunotherapy; and c) theranostic applications for simultaneous detection and treatment of heavy metal toxicity and cancers. In addition, we summarize recent studies towards understanding of interactions between liposomes and biological components. Finally, perspectives on future directions in advancing the field for clinical translations are also discussed.

Pinpointing subcellular protein localizations from microscopy images is easy to the trained eye, but challenging to automate. Based on the Human Protein Atlas image collection, we held a competition to identify deep learning solutions to solve this task. Challenges included training on highly imbalanced classes and predicting multiple labels per image. Over 3 months, 2,172 teams participated. Despite convergence on popular networks and training techniques, there was considerable variety among the solutions. Participants applied strategies for modifying neural networks and loss functions, augmenting data and using pretrained networks. The winning models far outperformed our previous effort at multi-label classification of protein localization patterns by ~20%. These models can be used as classifiers to annotate new images, feature extractors to measure pattern similarity or pretrained networks for a wide range of biological applications.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Over the past two decades, enormous progress has been made in designing fluorescent sensors or probes for divalent metal ions. In contrast, the development of fluorescent sensors for monovalent metal ions, such as sodium (Na + ), has remained underdeveloped, even though Na + is one the most abundant metal ions in biological systems and plays a critical role in many biological processes. Here, we report the in vitro selection of the first (to our knowledge) Na + -specific, RNA-cleaving deoxyribozyme (DNAzyme) with a fast catalytic rate [observed rate constant ( k obs ) ∼0.1 min −1 ], and the transformation of this DNAzyme into a fluorescent sensor for Na + by labeling the enzyme strand with a quencher at the 3′ end, and the DNA substrate strand with a fluorophore and a quencher at the 5′ and 3′ ends, respectively. The presence of Na + catalyzed cleavage of the substrate strand at an internal ribonucleotide adenosine (rA) site, resulting in release of the fluorophore from its quenchers and thus a significant increase in fluorescence signal. The sensor displays a remarkable selectivity (>10,000-fold) for Na + over competing metal ions and has a detection limit of 135 µM (3.1 ppm). Furthermore, we demonstrate that this DNAzyme-based sensor can readily enter cells with the aid of α-helical cationic polypeptides. Finally, by protecting the cleavage site of the Na + -specific DNAzyme with a photolabile o -nitrobenzyl group, we achieved controlled activation of the sensor after DNAzyme delivery into cells. Together, these results demonstrate that such a DNAzyme-based sensor provides a promising platform for detection and quantification of Na + in living cells.

Active surveillance has been increasingly utilized as a strategy for the management of favorable-risk, localized prostate cancer. In this review, we describe contemporary management strategies of active surveillance, with a focus on traditional stratification schemes, new prognostic tools, and patient outcomes. Patient selection, follow-up strategy, and indication for delayed intervention for active surveillance remain centered around PSA, digital rectal exam, and biopsy findings. Novel tools which include imaging, biomarkers, and genetic assays have been investigated as potential prognostic adjuncts; however, their role in active surveillance remains institutionally dependent. Although 30–50% of patients on active surveillance ultimately undergo delayed treatment, the vast majority will remain free of metastasis with a low risk of dying from prostate cancer. The optimal method for patient selection into active surveillance is unknown; however, cancer-specific mortality rates remain excellent. New prognostication tools are promising, and long-term prospective, randomized data regarding their use in active surveillance will be beneficial.

A systematic investigation of the effects of different DNA sequences on the morphologies of silver nanoparticles (AgNPs) grown from Ag nanocube seeds is reported. The presence of 10-mer oligo-A, -T, and -C directed AgNPs growth from cubic seeds into edge-truncated octahedra of different truncation extents and truncated tetrahedral AgNPs, while AgNPs in the presence of oligo-G remained cubic. The shape and morphological evolution of the nanoparticle growth for each system is investigated using SEM and TEM and correlated with UV-vis absorption kinetic studies. In addition, the roles of oligo-C and oligo-G secondary structures in modulating the morphologies of AgNPs are elucidated, and the morphological evolution for each condition of AgNPs growth is proposed. The shapes were found to be highly dependent on the binding affinity of each of the bases and the DNA secondary structures, favoring the stabilization of the Ag{111} facet. The AgNPs synthesized through this method have morphologies and optical properties that can be varied by using different DNA sequences, while the DNA molecules on these AgNPs are also stable against glutathione. The AgNP functionalization can be realized in a one-step synthesis while retaining the biorecognition ability of the DNA, which allows for programmable assembly.

DNAzymes, which are sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. Despite their significant promise, cellular sensing using DNAzymes has however been difficult, mainly because of the “always-on” mode of first-generation DNAzyme sensors. In this paper, to overcome this limitation, a photoactivatable (or photocaged) DNAzyme was designed and synthesized, and its application in sensing ZnlI in living cells was demonstrated. In this design, the adenosine ribonucleotide at the scissile position of the 8–17 DNAzyme was replaced by 2'-O-nitrobenzyl adenosine, rendering the DNAzyme inactive and thus allowing its delivery into cells intact, protected from nonspecific degradation within cells. Irradiation at 365 nm restored DNAzyme activity, thus allowing the temporal control over the sensing activity of the DNAzyme for metal ions. Finally, the same strategy was also applied to the GR-5 DNAzyme for the detection of PbII, thus demonstrating the possible scope of the method.

This review highlights recent progress in developing DNA aptamers for personalized medicine, with more focus on in vivo studies for potential clinical applications. Examples include design of aptamers in combination with DNA nanostructures, nanomaterials, or microfluidic devices as diagnostic probes or therapeutic agents for cancers and other diseases. The use of aptamers as targeting agents in drug delivery is also covered. The advantages and future directions of such DNA aptamer-based technology for the continued development of personalized medicine are discussed.

Background: Comprehensive spatial phenotyping is an increasingly important pathology research tool for investigating the highly complex biology of tumors and their micro-environment. High multiplex immunohistochemical (IHC) strategies enable spatial profiling of tumor samples and expand the depth of information obtained from a single tissue section. Conventional IHC technologies overcome the limitations of low multiplexing by testing serial sections; however, this method does not provide evidence of true marker colocalization and results in inaccurate phenotyping. More recent multiplex methods involving iterative staining and stripping steps have been developed but suffer from low-throughput workflow and can potentially damage samples. Alternative strategies using localized release of mass- or oligonucleotide-tags are not only low-throughput but are also high-cost and require specialized instruments. In this study, we developed a highly streamlined 15-plex fluorescence IHC assay to identify and subtype lymphocytes, macrophages, dendritic cells, antigen presenting cells, and tumor cells on single FFPE tissue sections in a single work day. Methods: InSituPlex® technology was used to perform high-multiplex immune profiling (15 targets) of different FFPE tumor sections. Slides were stained with a Leica® Bond®RX autostainer using a cocktail of 15 primary antibodies. Whole slide images were obtained using commercially available multi-color fluorescence slide scanners. The resulting images were then segmented and analyzed for different phenotypes and their spatial relationships using the Indica Labs HALOTM software. Tissue sections processed with single-antibody staining were used as references to characterize the performance of the 15-plex assay. Results: We demonstrate a 15-plex immuno-fluorescence assay on whole FFPE tumor tissue sections in a single work day using the InSituPlex technology. Equivalence of assay performance between the 15-plex and individually stained reference samples confirmed assay specificity and sensitivity in the high multiplexed format. High-dimensional data obtained from the 15-plex assay allowed for a detailed sub-classification of various cell types and their spatial relationship on each single section. Conclusions: InSituPlex technology empowers immuno-oncology research through the staining of 15 different immune and tumor markers on single FFPE tumor sections, with a fast, automated workflow and reproducible assay. The resulting high-dimensional images are then used to spatially profile various sub-populations of lymphocytes, macrophages, and tumor cells. This study demonstrates the promises of the InSituPlex technology for translational research and discovery applications where rapid imaging of a large number of biomarkers over the entire tissue is highly desired. Citation Format: Abdul Majeed Mohammed, Zhixin Xia, Gourab Chatterjee, Kevin Hwang, Mael Manesse. High-plex spatial profiling of whole FFPE tissue sections using InSituPlex® technology for discovery applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1183.

DNAzymes have shown great promise as a general platform for detecting metal ions, as many metal-specific DNAzymes can be obtained using in vitro selection. While DNAzyme-based metal sensors have found many applications in the extracellular environment, no intracellular application of DNAzyme sensors has yet been reported. Here, we demonstrate a novel type of metal ion sensor for intracellular metal ion detection. The probe consists of a 13 nm gold nanoparticle (AuNP) core functionalized with a shell consisting of a uranyl-specific 39E DNAzyme whose enzyme strand contains a thiol at the 3' end for conjugation to the AuNP, and whose substrate strand is modified with a Cy3 fluorophore at the 5' end and a molecular quencher at the 3' end. In the absence of uranyl, the fluorescence of the Cy3 is quenched by both AuNP and the molecular quencher. In the presence of uranyl, the DNAzyme cleaves the fluorophore-labeled substrate strand, resulting in release of the shorter product strand containing the Cy3 and increased fluorescence. We demonstrate that this DNAzyme-AuNP probe can readily enter cells and can serve as a metal ion sensor within a cellular environment, making it the first demonstration of DNAzymes as intracellular metal ion sensors. Such a method can be generally applied to the detection of other metal ions using other DNAzymes selected through in vitro selection.

Selective targeting of cancer cells is a critical step in cancer diagnosis and therapy. To address this need, DNA aptamers have attracted significant attention as possible targeting ligands. However, while their use in targeting cancer cells in vitro has been reported, their effectiveness has rarely been established in vivo. Here we report the development of a liposomal drug delivery system for targeted anticancer chemotherapy. Liposomes were prepared containing doxorubicin as a payload, and functionalized with AS1411, a DNA aptamer with strong binding affinity for nucleolin. AS1411 aptamer-functionalized liposomes increased cellular internalization and cytotoxicity to MCF-7 breast cancer cells as compared to non-targeting liposomes. Furthermore, targeted liposomal doxorubicin improved antitumor efficacy against xenograft MCF-7 breast tumors in athymic nude mice, attributable to their enhanced tumor tissue penetration. This study suggests that AS1411 aptamer-functionalized liposomes can recognize nucleolin overexpressed on MCF-7 cell surface, and therefore enable drug delivery with high specificity and selectivity.

Lanthanide-doped upconversion nanoparticles (UCNPs) have shown promise in biomedical applications. However, as the UCNPs are normally capped with hydrophobic ligands, it remains challenging to prepare biocompatible UCNPs with specific molecular recognition capabilities. We herein report an exceptionally simple strategy to prepare uniform DNA-modified upconversion nanoparticles (DNA-UCNPs) as versatile bioprobes. The approach can directly convert as-prepared hydrophobic UCNPs into water-soluble DNA-UCNPs without any chemical modification of UCNPs or oligonucleotides. Furthermore, DNA molecules on the DNA-UCNPs retain their biorecognition ability, allowing programmable assembly of hybrid nanostructures. More importantly, we show that these DNA-UCNPs are capable of crossing cell membranes without the need of transfection agents, and their use as agents for bioimaging and DNA delivery are also demonstrated. Finally, DNA aptamer-conjugated UCNPs can be readily used for targeted imaging of cancer cells.

A paramagnetic manganese complex of a fluorescein-based probe affords a dual-modality zinc sensor featuring an improved fluorescence dynamic range and an MRI readout., National Institute of General Medical Sciences (U.S.) ((Grant GM065519), SBS Foundation, National Institutes of Health (U.S.) (NIH Grant DP2-OD2441), Raymond and Beverley Sackler Foundation

AP23464 is a potent Src and Abl inhibitor (in vitro IC50 < 1 nM), which has been shown to effectively inhibit growth of imatinib-resistant Bcr-Abl+ cells, and thus, is a promising compound for treating patients with imatinib-resistant leukemia. We conducted a study to evaluate growth inhibition and inhibition of Src versus Abl protein tyrosine kinases in human myeloid cell lines: MV4-11 expressing an internal tandem duplication of Flt3 (Flt3-ITD), the murine pro-B cell line Ba/F3 that expresses the Flt3-ITD, the GM-CSF dependent Mo7e, and the G-CSF-responsive BaF3-GR (Ba/F3 cells expressing the human G-CSF receptor). We compared AP23464 with the PP1, a previously described Src kinase inhibitor (IC50 < 1 uM). We sought to correlate growth inhibition with Src or Abl inhibition. Methods: Growth inhibition was assessed by Trypan blue exclusion and MTT assay using drug concentrations 0.1 uM – 10 uM. Drugs were added daily to the cell suspension during the 3-day experiment. After a 60 min incubation at concentrations 0.1 nM – 1 uM, Src or Abl kinase inhibition was analyzed by blotting with a polyclonal phospho-Src (Tyr416) antibody or a polyclonal phospho-Abl (Tyr245) antibody. Results: In MV4-11 cells AP23464 was more potent than PP1 in causing growth inhibition with IC50 at 50% growth inhibition with 70% knock-down of Lyn. The IC50 for phosphoSrc was 1 nM for both AP23464 and PP1. Abl was present in Mo7e cells, but no phosphoAbl was demonstrated (K562 cells served as positive control). In BaF3-GR cells grown in G-CSF, the IC50 was 1 uM for AP23464 vs. 10 uM for PP1. In western blotting, the IC50 for phospho-Src 416 was detected at 10 nM AP23464. Abl was present in Ba/F3GR cells, but no phosphoAbl was demonstrated (K562 cells served as positive control). Conclusions: AP23464 is more potent than PP1 in causing growth inhibition and Src kinase inhibition in these cell lines that serve as models for acute myeloid leukemia. It is unlikely that Abl is the drug target, because MV4-11 cells do not express detectable Abl, and phospho-Abl was not detected in Mo7e or Ba/F3 cells. These results suggest that inhibition of Src tyrosine kinases contributes predominantly to growth inhibition caused by the Src/Abl kinase inhibitor AP23464 in these cell types.

BMS-354825 is a dual SRC and ABL inhibitor, which has been shown effective in imatinib-resistant BCR-ABL+ cells, and it is in phase I trials for patients with imatinib-resistant leukemia. We conducted a study to evaluate growth inhibition and inhibition of Src v. Abl protein tyrosine kinases in human myeloid cell lines: MV4-11 expressing an internal tandem duplication of Flt3 (Flt3-ITD), the murine pro-B cell line Ba/F3 that expresses the Flt3-ITD, the GM-CSF dependent Mo7e, and the G-CSF-responsive BaF3-GR (Ba/F3 cells expressing the human G-CSF receptor). We compared BMS-354825 with PP1, a SRC kinase inhibitor with in vitro IC50 at sub-micromolar concentrations. We sought to correlate growth inhibition with SRC or ABL inhibition. In these myeloid cell lines, LYN is the predominant SRC kinase. Methods: Growth inhibition was assessed by Trypan blue exclusion and MTT assay using drug concentrations 0.1 uM – 10 uM. Drugs were added daily to the cell suspension during the 3-day experiment. After a 60 min incubation at concentrations 0.1 nM – 1 uM, SRC or ABL kinase inhibition was analyzed by blotting with a polyclonal phospho-SRC (Tyr416) antibody or a polyclonal phospho-ABL (Tyr245) antibody. Results: In MV4-11 cells BMS-354825 and PP1 caused similar growth inhibition IC50 at 5 uM. By western blotting, inhibition of phospho-Src 416 occurred at 1 nM concentrations of both compounds. Protein expression of ABL was not detected in MV4-11 cells. In Ba/F3-ITD cells, the IC50 for BMS-354825 was 1–10 uM (grown in IL-3) and 0.01 uM (without IL-3). The IC50 for PP1 was 1–10 uM (grown in IL-3) and 0.1 uM (without IL-3). Inhibition of phospho-SRC occurred at 10 nM. In Mo7e cells, grown in the presence of GM-CSF, the IC50 was 5 uM for BMS-354825 v. 10 uM for PP1 by MTT assay. By western blotting, inhibition of phospho-SRC 416 occurred at 1 nM for both BMS-354825 and PP1. To determine specific contribution of LYN to Mo7e growth, we treated Mo7e cells with LYN siRNA. With 70% knock-down of LYN, there was 50% growth inhibition. ABL was present in Mo7e cells, but no phosphoAbl was demonstrated (K562 cells served as positive control). In BaF3-GR cells grown in G-CSF, the IC50 was 5 uM for BMS-354825 vs. 10 uM for PP1 by MTT assay. In western blotting, inhibition of phospho-Src 416 was detected at 10 nM BMS-354825. ABL was present in Ba/F3GR cells, but no phospho-ABL was demonstrated (K562 cells served as positive control). Conclusions: BMS-354825 is more potent than PP1 in causing growth inhibition and SRC kinase inhibition in Mo7e and Ba/F3GR cells that serve as models for acute myeloid leukemia. It is unlikely that ABL is the drug target, because MV4-11 cells do not express ABL and phospho-ABL was not found in Mo7e or Ba/F3 cells. These results suggest that inhibition of SRC tyrosine kinases contributes predominantly to growth inhibition caused by the dual SRC/ABL kinase inhibitor BMS-354825 in myeloid cell lines expressing Flt3-ITD and cytokine-driven proliferation and survival via the IL-3/GM-CSF Receptor or G-CSF Receptor.