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Understanding how a subset of expressed genes dictates cellular phenotype is a considerable challenge owing to the large numbers of molecules involved, their combinatorics and the plethora of cellular behaviours that they determine1,2. Here we reduced this complexity by focusing on cellular organization—a key readout and driver of cell behaviour3,4—at the level of major cellular structures that represent distinct organelles and functional machines, and generated the WTC-11 hiPSC Single-Cell Image Dataset v1, which contains more than 200,000 live cells in 3D, spanning 25 key cellular structures. The scale and quality of this dataset permitted the creation of a generalizable analysis framework to convert raw image data of cells and their structures into dimensionally reduced, quantitative measurements that can be interpreted by humans, and to facilitate data exploration. This framework embraces the vast cell-to-cell variability that is observed within a normal population, facilitates the integration of cell-by-cell structural data and allows quantitative analyses of distinct, separable aspects of organization within and across different cell populations. We found that the integrated intracellular organization of interphase cells was robust to the wide range of variation in cell shape in the population; that the average locations of some structures became polarized in cells at the edges of colonies while maintaining the ‘wiring’ of their interactions with other structures; and that, by contrast, changes in the location of structures during early mitotic reorganization were accompanied by changes in their wiring.

PDF file - 279K, Immunoblots of MV4-11, MOLM14, and SEM-K2 with quizartinib DR

PDF file - 116K, Elevation of pro-apoptotic BimEL mirrors loss of anti-apoptotic Mcl-1

PDF file - 46K, Durability of quizartinib inhibition translates to loss of viability after brief exposure

PDF file - 54K, DMSO and DR to quizartinib, percent apoptotic cells is percent of cells with

PDF file - 42K, Quizartinib inhibition of MV4-11 tumor grwoth in vivo

PDF file - 74K, CEP-32496 Supplemental Data.

PDF file - 54K, Inhibition of FLT3 phosphorylation and cell viability by FLT3 inhibitors

Summary: We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis. : Gunawardane and colleagues use CRISPR/Cas9 to deliver an excisable cassette to transcriptionally silent loci in hiPSCs, then accomplish excision of the cassette in a second step utilizing Cas9/CRISPR and the MMEJ and NHEJ DNA-repair pathways. Excision results in mEGFP tagging of the targeted loci. Upon differentiation, each of five tagged cell lines appropriately expresses a unique fluorescent fusion protein localized to the sarcomere in live cardiomyocytes. Keywords: CRISPR/Cas9, genome editing, cardiomyocyte differentiation, stem cells, iPSCs, MMEJ, live imaging, endogenous fluorescent tagging, mEGFP, HDR

To address health disparities and facilitate increasingly personalized treatments, we need to develop new models for basic and disease research that reflect diverse ancestral backgrounds and sex, and ensure that diverse populations are included among donors and research participants.

We performed a comprehensive analysis of the transcriptional changes within and across cell populations during human induced pluripotent stem cell (hiPSC) differentiation to cardiomyocytes. Using the single cell RNA-seq combinatorial barcoding method SPLiT-seq, we sequenced >20,000 single cells from 55 independent samples representing two differentiation protocols and multiple hiPSC lines. Samples included experimental replicates ranging from undifferentiated hiPSCs to mixed populations of cells at D90 post-differentiation. As expected, differentiated cell populations clustered by time point, with differential expression analysis revealing markers of cardiomyocyte differentiation and maturation changing from D12 to D90. We next performed a complementary cluster-independent sparse regression analysis to identify and rank genes that best assigned cells to differentiation time points. The two highest ranked genes between D12 and D24 (MYH7 and MYH6) resulted in an accuracy of 0.84, and the three highest ranked genes between D24 and D90 (A2M, H19, IGF2) resulted in an accuracy of 0.94, revealing that low dimensional gene features can identify differentiation or maturation stages in differentiating cardiomyocytes. Expression levels of select genes were validated using RNA FISH. Finally, we interrogated differences in differentiation population composition and cardiac gene expression resulting from two differentiation protocols, experimental replicates, and three hiPSC lines in the WTC-11 background to identify sources of variation across these experimental variables.

Our goal is to identify and understand cellular behaviors using 3D live imaging of cell organization. To do this, we image human inducible pluripotent stem cell (hiPSC) lines expressing fluorescently tagged protein representing specific cellular organelles and structures. To produce large numbers of standardized cell images, we developed an automated hiPSC culture procedure, to maintain, passage and Matrigel coat 6-well plastic plates and 96-well glass plates compatible with high-resolution 3D microscopy. Here we describe this system including optimization procedures and specific values for plate movement, angle of tips, speed of aspiration and dispense, seeding strategies and timing of every step. We validated this approach through a side-by-side comparison of quality control results obtained from manual and automated methods. Additionally, we developed an automated image-based colony segmentation and feature extraction pipeline to predict cell count and select wells with consistent morphology for high resolution 3D microscopy.

SummaryWe present a quantitative co-analysis of RNA abundance and sarcomere organization in single cells and an integrated framework to predict subcellular organization states from gene expression. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes expressing mEGFP-tagged alpha-actinin-2 to develop quantitative image analysis tools for systematic and automated classification of subcellular organization. This captured a wide range of sarcomeric organization states within cell populations that were previously difficult to quantify. We performed RNA FISH targeting genes identified by single cell RNA sequencing to simultaneously assess the relationship between transcript abundance and structural states in single cells. Co-analysis of gene expression and sarcomeric patterns in the same cells revealed biologically meaningful correlations that could be used to predict organizational states. This study establishes a framework for multi-dimensional analysis of single cells to study the relationships between gene expression and subcellular organization and to develop a more nuanced description of cell states.Graphical AbstractTranscriptional profiling and structural classification was performed on human induced pluripotent stem cell-derived cardiomyocytes to characterize the relationship between transcript abundance and subcellular organization.

Although some cell types may be defined anatomically or by physiological function, a rigorous definition of cell state remains elusive. Here, we develop a quantitative, imaging-based platform for the systematic and automated classification of subcellular organization in single cells. We use this platform to quantify subcellular organization and gene expression in >30,000 individual human induced pluripotent stem cell-derived cardiomyocytes, producing a publicly available dataset that describes the population distributions of local and global sarcomere organization, mRNA abundance, and correlations between these traits. While the mRNA abundance of some phenotypically important genes correlates with subcellular organization (e.g., the beta-myosin heavy chain, MYH7), these two cellular metrics are heterogeneous and often uncorrelated, which suggests that gene expression alone is not sufficient to classify cell states. Instead, we posit that cell state should be defined by observing full distributions of quantitative, multidimensional traits in single cells that also account for space, time, and function.

The methods described below were adapted from the small molecule protocol described in Lian et al.1, and have been optimized for the differentiation of cardiomyocytes in a glass 96-well plate format using the Allen Cell Collection, derived from the hiPSC WTC parental line released by the Conklin Laboratory2 at the J. David Gladstone Institute. Differentiation on glass allows high resolution imaging without replating steps. It also allows observationsat the earliest transitions and cell states during differentiation.

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouse(TM) platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.

We describe a multi-step CRISPR/Cas9 gene editing method to create endogenously tagged GFP-fusions of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs), allowing visualization of proteins that are only expressed upon differentiation. To do this, we designed a donor template containing the monomeric EGFP (mEGFP) fusion tag and an mCherry selection cassette delivered in tandem to a target locus via homology directed repair (HDR). mCherry expression was driven by a constitutive promoter and served as a drug-free, excisable selection marker. Following selection, the mCherry cassette was excised with Cas9, creating an mEGFP-fusion with the target gene. We achieved scarless excision by using repetitive sequences to guide microhomology-mediated end joining (MMEJ) and introduce linker sequences between the mEGFP tag and the target gene. Using this strategy, we successfully tagged genes encoding the cardiomyocyte sarcomeric proteins troponin I (TNNI1), alpha-actinin (ACTN2), titin (TTN), myosin light chain 2a (MYL7), and myosin light chain 2v (MYL2) with mEGFP in undifferentiated hiPSCs. This methodology provides a general strategy for scarlessly introducing tags to transcriptionally silent loci in hiPSCs.

LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholesterol and corneal opacity. Here we report the 2.65 A crystal structure of the human LCAT protein. Crystallization required enzymatic removal of N-linked glycans and complex formation with a Fab fragment from a tool antibody. The crystal structure reveals that LCAT has an α/β hydrolase core with two additional subdomains that play important roles in LCAT function. Subdomain 1 contains the region of LCAT shown to be required for interfacial activation, while subdomain 2 contains the lid and amino acids that shape the substrate binding pocket. Mapping the naturally occurring mutations onto the structure provides insight into how they may affect LCAT enzymatic activity.

The generation of a collection of human induced pluripotent stem cell (hiPSC) lines expressing endogenously GFP-tagged proteins using CRISPR/Cas9 methods is described. The methods used and the genomic and cell biological data validating the GFP-tagged hiPSC lines are also presented., We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in

SummaryBreast cancer is a heterogeneous disease and can be classified based on gene expression profiles that reflect distinct epithelial subtypes. We identify prostate-derived ETS factor (PDEF) as a mediator of mammary luminal epithelial lineage-specific gene expression and as a factor required for tumorigenesis in a subset of breast cancers. PDEF levels strongly correlate with estrogen receptor (ER)-positive luminal breast cancer, and PDEF transcription is inversely regulated by ER and GATA3. Furthermore, PDEF is essential for luminal breast cancer cell survival and is required in models of endocrine resistance. These results offer insights into the function of this ETS factor that are clinically relevant and may be of therapeutic value for patients with breast cancer treated with endocrine therapy.

Fms-like tyrosine kinase 3 (FLT3) is implicated in the pathogenesis of acute myeloid leukemia (AML). FLT3-activating internal tandem duplication (ITD) mutations are found in approximately 30% of patients with AML and are associated with poor outcome in this patient population. Quizartinib (AC220) has previously been shown to be a potent and selective FLT3 inhibitor. In the current study, we expand on previous observations by showing that quizartinib potently inhibits the phosphorylation of FLT3 and downstream signaling molecules independent of FLT3 genotype, yet induces loss of viability only in cells expressing constitutively activated FLT3. We further show that transient exposure to quizartinib, whether in vitro or in vivo, leads to prolonged inhibition of FLT3 signaling, induction of apoptosis, and drastic reductions in tumor volume and pharmacodynamic endpoints. In vitro experiments suggest that these prolonged effects are mediated by slow binding kinetics that provide for durable inhibition of the kinase following drug removal/clearance. Together these data suggest quizartinib, with its unique combination of selectivity and potent/sustained inhibition of FLT3, may provide a safe and effective treatment against FLT3-driven leukemia. Mol Cancer Ther; 12(4); 438–47. ©2013 AACR.

Serine/threonine protein kinases Aurora A, B, and C play essential roles in cell mitosis and cytokinesis. Currently a number of Aurora kinase inhibitors with different isoform selectivities are being evaluated in the clinic. Herein we report the discovery and characterization of 21c (AC014) and 21i (AC081), two structurally novel, potent, kinome-selective pan-Aurora inhibitors. In the human colon cancer cell line HCT-116, both compounds potently inhibit histone H3 phosphorylation and cell proliferation while inducing 8N polyploidy. Both compounds administered intravenously on intermittent schedules displayed potent and durable antitumor activity in a nude rat HCT-116 tumor xenograft model and exhibited good in vivo tolerability. Taken together, these data support further development of both 21c and 21i as potential therapeutic agents for the treatment of solid tumors and hematological malignancies.

Activating mutations in the receptor tyrosine kinase FLT3 are present in up to approximately 30% of acute myeloid leukemia (AML) patients, implicating FLT3 as a driver of the disease and therefore as a target for therapy. We report the characterization of AC220, a second-generation FLT3 inhibitor, and a comparison of AC220 with the first-generation FLT3 inhibitors CEP-701, MLN-518, PKC-412, sorafenib, and sunitinib. AC220 exhibits low nanomolar potency in biochemical and cellular assays and exceptional kinase selectivity, and in animal models is efficacious at doses as low as 1 mg/kg given orally once daily. The data reveal that the combination of excellent potency, selectivity, and pharmacokinetic properties is unique to AC220, which therefore is the first drug candidate with a profile that matches the characteristics desirable for a clinical FLT3 inhibitor.

Treatment of AML patients with small molecule inhibitors of FLT3 kinase has been explored as a viable therapy. However, these agents are found to be less than optimal for the treatment of AML because of lack of sufficient potency or suboptimal oral pharmacokinetics (PK) or lack of adequate tolerability at efficacious doses. We have developed a series of extremely potent and highly selective FLT3 inhibitors with good oral PK properties. The first series of compounds represented by 1 (AB530) was found to be a potent and selective FLT3 kinase inhibitor with good PK properties. The aqueous solubility and oral PK properties at higher doses in rodents were found to be less than optimal for clinical development. A novel series of compounds were designed lacking the carboxamide group of 1 with an added water solubilizing group. Compound 7 (AC220) was identified from this series to be the most potent and selective FLT3 inhibitor with good pharmaceutical properties, excellent PK profile, and superior efficacy and t...

Cell migration and invasion are two critical cellular processes that are often deregulated during tumorigenesis. To identify factors that contribute to oncogenic progression by stimulating cell migration, we conducted a powerful retroviral based migration screen using an MCF7 cDNA library and the immortalized human breast epithelial cell line MCF-10A. We identified prostate derived Ets factor (PDEF), an Ets transcription factor that is overexpressed in both prostate and breast carcinoma, as a candidate promigratory gene from this screen. Whereas PDEF induced limited motility of MCF-10A cells, coexpression of PDEF with the receptor tyrosine kinases (RTK) ErbB2 and colony-stimulating factor receptor (CSF-1R)/CSF-1 significantly enhanced MCF-10A motility. Furthermore, cells coexpressing PDEF with either ErbB2 or CSF-1R/CSF-1 induced a dramatic invasive phenotype in three-dimensional cultures. Constitutive activation of the extracellular signal–regulated kinase (ERK) pathway also enhanced PDEF-induced motility and invasion, suggesting that activation of the ERK/mitogen-activated protein kinase by ErbB2 and CSF-1R/CSF-1 can cooperate with PDEF to promote motility and invasion. Furthermore, PDEF promoted anchorage-independent growth of ErbB2 and CSF-1R/CSF-1–expressing cells. Using laser capture microdissection, we also found that PDEF mRNA is overexpressed in breast tumor epithelia throughout tumor progression. Taken together, these findings suggest that the transcription factor PDEF may play an important role in breast tumorigenesis and that PDEF overexpression may be particularly significant in tumors that exhibit activation of oncogenic RTKs such as ErbB2 and CSF-1R. (Cancer Res 2005; 65(24): 11572-80)

The gamma-tubulin ring complex (gammaTuRC) is important for microtubule nucleation from the centrosome. In addition to gamma-tubulin, the Drosophila gammaTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small gamma-tubulin complex (gammaTuSC) and the gammaTuRC, while the remaining subunits are found only in the gammaTuRC. To study gammaTuRC assembly and function, we first reconstituted gammaTuSC using the baculovirus expression system. Using the reconstituted gammaTuSC, we showed for the first time that this subcomplex of the gammaTuRC has microtubule binding and capping activities. Next, we characterized two new gammaTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known gammaTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with gammaTuSC. However, this interaction is insufficient for gammaTuRC assembly.

Mutations in the BRAF gene have been identified in approximately 7% of cancers, including 60% to 70% of melanomas, 29% to 83% of papillary thyroid carcinomas, 4% to 16% colorectal cancers, and a lesser extent in serous ovarian and non–small cell lung cancers. The V600E mutation is found in the vast majority of cases and is an activating mutation, conferring transforming and immortalization potential to cells. CEP-32496 is a potent BRAF inhibitor in an in vitro binding assay for mutated BRAFV600E (Kd BRAFV600E = 14 nmol/L) and in a mitogen-activated protein (MAP)/extracellular signal–regulated (ER) kinase (MEK) phosphorylation (pMEK) inhibition assay in human melanoma (A375) and colorectal cancer (Colo-205) cell lines (IC50 = 78 and 60 nmol/L). In vitro, CEP-32496 has multikinase binding activity at other cancer targets of interest; however, it exhibits selective cellular cytotoxicity for BRAFV600E versus wild-type cells. CEP-32496 is orally bioavailable in multiple preclinical species (>95% in rats, dogs, and monkeys) and has single oral dose pharmacodynamic inhibition (10–55 mg/kg) of both pMEK and pERK in BRAFV600E colon carcinoma xenografts in nude mice. Sustained tumor stasis and regressions are observed with oral administration (30–100 mg/kg twice daily) against BRAFV600E melanoma and colon carcinoma xenografts, with no adverse effects. Little or no epithelial hyperplasia was observed in rodents and primates with prolonged oral administration and sustained exposure. CEP-32496 benchmarks favorably with respect to other kinase inhibitors, including RAF-265 (phase I), sorafenib, (approved), and vemurafenib (PLX4032/RG7204, approved). CEP-32496 represents a novel and pharmacologically active BRAF inhibitor with a favorable side effect profile currently in clinical development. Mol Cancer Ther; 11(4); 930–41. ©2012 AACR.

The Ras/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway plays a central role in the regulation of cell growth, differentiation, and survival. Expression of mutant BRAF(V600E) results in constitutive activation of the MAPK pathway, which can lead to uncontrolled cellular growth. Herein, we describe an SAR optimization campaign around a series of quinazoline derived BRAF(V600E) inhibitors. In particular, the bioisosteric replacement of a metabolically sensitive tert-butyl group with fluorinated alkyl moieties is described. This effort led directly to the identification of a clinical candidate, compound 40 (CEP-32496). Compound 40 exhibits high potency against several BRAF(V600E)-dependent cell lines and selective cytotoxicity for tumor cell lines expressing mutant BRAF(V600E) versus those containing wild-type BRAF. Compound 40 also exhibits an excellent PK profile across multiple preclinical species. In addition, significant oral efficacy was observed in a 14-day BRAF(V600E)-dependent human Colo-205 tumor xenograft mouse model, upon dosing at 30 and 100 mg/kg BID.

Aryl phenyl ureas with a 4-quinazolinoxy substituent at the meta-position of the phenyl ring are potent inhibitors of mutant and wild type BRAF kinase. Compound 7 (1-(5-tert-butylisoxazol-3-yl)-3-(3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)urea hydrochloride) exhibits good pharmacokinetic properties in rat and mouse and is efficacious in a mouse tumor xenograft model following oral dosing.

Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different “poor-outcome” cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by “poor-prognosis” signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.

Treatment of AML patients with small molecule inhibitors of FLT3 kinase has been explored as a viable therapy. However, these agents are found to be less than optimal for the treatment of AML because of lack of sufficient potency or suboptimal oral pharmacokinetics (PK) or lack of adequate tolerability at efficacious doses. We have developed a series of extremely potent and highly selective FLT3 inhibitors with good oral PK properties. The first series of compounds represented by 1 (AB530) was found to be a potent and selective FLT3 kinase inhibitor with good PK properties. The aqueous solubility and oral PK properties at higher doses in rodents were found to be less than optimal for clinical development. A novel series of compounds were designed lacking the carboxamide group of 1 with an added water solubilizing group. Compound 7 (AC220) was identified from this series to be the most potent and selective FLT3 inhibitor with good pharmaceutical properties, excellent PK profile, and superior efficacy and tolerability in tumor xenograft models. Compound 7 has demonstrated a desirable safety and PK profile in humans and is currently in phase II clinical trials.

Functional proteomics approaches that comprehensively evaluate the biological activities of human cDNAs may provide novel insights into disease pathogenesis. To systematically investigate the functional activity of cDNAs that have been implicated in breast carcinogenesis, we generated a collection of cDNAs relevant to breast cancer, the Breast Cancer 1000 (BC1000), and conducted screens to identify proteins that induce phenotypic changes that resemble events which occur during tumor initiation and progression. Genes were selected for this set using bioinformatics and data mining tools that identify genes associated with breast cancer. Greater than 1000 cDNAs were assembled and sequence verified with high-throughput recombination-based cloning. To our knowledge, the BC1000 represents the first publicly available sequence-validated human disease gene collection. The functional activity of a subset of the BC1000 collection was evaluated in cell-based assays that monitor changes in cell proliferation, migration, and morphogenesis in MCF-10A mammary epithelial cells expressing a variant of ErbB2 that can be inducibly activated through dimerization. Using this approach, we identified many cDNAs, encoding diverse classes of cellular proteins, that displayed activity in one or more of the assays, thus providing insights into a large set of cellular proteins capable of inducing functional alterations associated with breast cancer development.

The gamma-tubulin ring complex (gammaTuRC), consisting of multiple protein subunits, can nucleate microtubule assembly. Although many subunits of the gammaTuRC have been identified, a complete set remains to be defined in any organism. In addition, how the subunits interact with each other to assemble into gammaTuRC remains largely unknown. Here, we report the characterization of a novel gammaTuRC subunit, Drosophila gamma ring protein with WD repeats (Dgp71WD). With the exception of gamma-tubulin, Dgp71WD is the only gammaTuRC component identified to date that does not contain the grip motifs, which are signature sequences conserved in gammaTuRC components. By performing immunoprecipitations after pair-wise coexpression in Sf9 cells, we show that Dgp71WD directly interacts with the grip motif-containing gammaTuRC subunits, Dgrips84, 91, 128, and 163, suggesting that Dgp71WD may play a scaffolding role in gammaTuRC organization. We also show that Dgrips128 and 163, like Dgrips84 and 91, can interact directly with gamma-tubulin. Coexpression of any of these grip motif-containing proteins with gamma-tubulin promotes gamma-tubulin binding to guanine nucleotide. In contrast, in the same assay Dgp71WD interacts with gamma-tubulin but does not facilitate nucleotide binding.

Publisher Summary This chapter discusses the purification and reconstitution of Drosophila γ-tubulin complexes. To clone the individual Xenopus and Drosophila γTuRC subunits there are two approaches: (1) raising mouse polyclonal antibodies against purified γTuRCs and (2) obtaining protein sequences for individual γTuRC subunits. To date, four of the Drosophila γTuRC subunits are cloned, sequenced, and characterized. The isolation of γTuRC from Drosophila embryos involves an affinity purification step, using antipeptide antibodies specific for the C-terminal 17 amino acids of Drosophila γ-tubulin. An advantage of using Drosophila extracts is that once fly population cages are established, embryos are readily available in large quantities and flies do not require hormone injections. Another advantage is that Drosophila embryo extracts are less variable in quality than Xenopus egg extracts. Although the γ-tubulin-containing complexes—namely, γTuRC and γTuSC—can be purified from Drosophila embryos with relative ease, a system is developed that would allow to express the Dgrip proteins individually and in various combinations to study the organization and function of the γ-tubulin complexes biochemically. The chapter describes methods used to culture and infect the Sf9 cells and focuses on the general methods used to construct and assay the baculovirus.

Publisher Summary This chapter discusses the purification and characterization of γ-tubulin ring complex (γTuRC) and γ-tubulin small complex (γTuSC) and the function of γTuRC at the centrosome. The proposed mechanism of γTuRC-mediated microtubule nucleation is also reviewed. The identification of γ-tubulin and its essential role in microtubule nucleation is a major breakthrough in the study of microtubules and centrosomes. The subsequent purification of γTuRC provides the much-needed tool to investigate the role of γ-tubulin in microtubule nucleation and centrosome assembly. γTuRC is essential for both microtubule nucleation and formation of a functional centrosome. Further characterization of the γTuRC components helps to study the assembly of γTuRC and its recruitment to the centrosome. It provides an understanding of centrosome assembly, which is an essential process for proper cell division. The mechanism of microtubule nucleation by γ-tubulin comes from both biochemical and structural studies. γ-Tubulin exists as a complex with other proteins inside the cell; in certain cell types, multiple γ-tubulin complexes of different sizes co-exist. Antibody affinity production of γ-tubulin complexes from Drosophila embryos and Xenopus egg extracts is diagrammatically represented in the chapter.

Abstract 2607 Fms-like tyrosine kinase 3 (Flt3) plays a critical role in the leukemogenesis of a significant percentage of acute myeloid leukemias (AML) and acute lymphoblastic leukemias making it an attractive target for therapeutic intervention with small molecule inhibitors. To evaluate the pharmacodynamic (PD) effects of AC220, a highly selective and potent Flt3 inhibitor, in phase I and II clinical trials, we developed Flt3 immunoassays using the Meso Scale Discovery (MSD) 96-well platform. Several antibodies were screened to identify capture and detection pairs for both total (t) and phospho (p) Flt3 that were optimized for selectivity, sensitivity, and reproducibility. To validate the assays, cell lysates from SEM-K2 cells that express constitutively phosphorylated Flt3, the MSD assays reproducibly detected tFlt3 with high signal to background ratios, and consistently tracked changes in Flt3 phosphorylation when cells were subjected to Flt3 inhibitors. The functionality of these assays was minimally affected in the context of a whole blood matrix, as p/tFlt3 levels were similarly detected when SEM-K2 cells were spiked into human blood before lysis. To determine the PD effects of AC220 in humans, we tested whole blood samples from a phase I trial in predominantly relapsed or refractory AML patients (pts). AC220 was dosed once daily with escalations from 12 to 450 mg/day. Blood samples were initially examined in 6 pts taken pre-drug administration, at 2, 9, and 24 hrs, and 8 and 15 days post-administration. AC220 markedly reduced pFlt3 levels in the blood lysates at the 2 hr time point and this reduction was maintained over the 5 subsequent time points. Interestingly, the tFlt3 levels increased in all 6 pts at 2 and 9 hrs after treatment, began decreasing by 24 hrs (albeit still elevated from pre-dose levels), and was severely reduced by day 8 and day 15, consistent with elimination of Flt3-expressing peripheral blasts. Expanding the analysis to 45 patients from this phase I study, statistically significant reductions were observed in pFlt3 levels at 2 hrs, 24 hrs, and 15 days after dosing. Furthermore, pts receiving >90 mg demonstrated significantly greater reductions in pFlt3 compared to those receiving More recently, the PD effects of AC220 were assessed for pts from the exploratory part of an ongoing phase II trial in relapsed/refractory Flt3 ITD+ AML pts. Blood was taken at day 1 just before the first dose, and 24 hrs and 8 days following the initial dose. Phosphorylated STAT5, a marker closely associated with the Flt3 ITD mutation, was also monitored using a commercially available MSD assay. Blood samples from 52 pts were tested in both MSD assays. For Flt3, 13 pts were excluded from the analysis due to signals below the level of quantitation. Of the 39 pts remaining, a median pFlt3 inhibition of 75% (mean 70%) was observed at 24 hrs following the first dose, consistent with a direct effect of AC220 on its intended target. Similar to what was observed in the phase I trial, 52% of the pts had an increase in tFlt3 levels at 24 hrs. Importantly, 47% of pts had >90% reduction in tFlt3 levels after one week on AC220, signifying a major inhibitory effect on peripheral blast counts. Consistent with the strong linkage between Flt3 ITD autoactivation and STAT5 phosphorylation, there was a significant (p Disclosures: Nepomuceno: Ambit Biosciences: Employment, Equity Ownership. Gunawardane:Ambit Biosciences: Employment, Equity Ownership. Rooks:Ambit Biosciences: Employment, Equity Ownership. Armstrong:Ambit Biosciences: Employment, Equity Ownership.

Gene expression signatures are used clinically as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. However, we lack a clear understanding of whether these correlative biomarkers link to a common biological network that regulates metastasis. METHODS: In order to address this question, we analyzed thirteen different well-described (including two in clinical use) “poor outcome” cancer signatures in a cell line model of estrogen receptor positive breast cancer (MCF7) manipulated by diverse oncogenic stimuli. RESULTS: We find that the c-MYC oncoprotein coordinately regulates the expression of these thirteen different “poor outcome” cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits their distant metastasis in vivo and invasive behavior in vitro. CONCLUSION: These results suggest that MYC oncogene activity, as reflected by “poor prognosis” signature expression, may be necessary for the translocation of “poor outcome” human breast tumors to distant sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3300.

Fms-like tyrosine kinase 3 (FLT3) is thought to be a major driver in the pathogenesis of acute myeloid leukemia (AML). FLT3-activating mutations are found in ∼ 30% of AML patients and are associated with poor outcome in this patient population. AC220 is a small molecule kinase inhibitor with potent and selective FLT3 inhibitory activity. Here we characterize the effect of AC220 on FLT3 autophosphorylation, activation of downstream signaling pathways (STAT5, ERK and AKT), cell cycle distribution, and apoptosis endpoints in multiple leukemia cell lines expressing either wild type FLT3 (FLT3-WT, SEM-K2 cells (FLT3-WT overexpression) and RS4;11 cells (FLT3-WT)) and/or ITD-mutated FLT3 (FLT3-ITD, MV4-11 (FLT3-ITD), MOLM-14 (FLT3-ITD/FLT3-WT)). Across each of these parameters we also compare the effects of AC220 to those of other known FLT3 inhibitors including, sorafenib, sunitinib, lestaurtinib, and midostaurin. AC220 inhibited FLT3 autophosphorylation in both FLT3-WT and FLT3-ITD cells with roughly equal potency, while other FLT3 inhibitors showed differential pFLT3 inhibition across several cell lines. Although AC220-mediated inhibition of FLT3 autophosphorylation was observed in all cell lines, subsequent cell death was only detected in the cell lines with activated FLT3. This contrasts with other FLT3 inhibitors, where potent inhibition of cell proliferation in the non-FLT3 activated RS4;11 cells was observed, highlighting the lack of FLT3 selectivity of these compounds. Consistent with FLT3 inhibition, AC220 also inhibited constitutively activated STAT5, ERK and AKT in these cell lines, suggesting that one or more of these pathways play a role in FLT3-mediated cell growth and survival. Furthermore, the inhibition of these downstream markers correlated with the inhibition of FLT3 across each of the FLT3 inhibitors tested. Lastly, in an MV4-11 murine xenograft model, AC220 administration resulted in the reduction of phosphorylated FLT3 and downstream signaling molecules concomitant with tumor reduction. Together, these data suggest that selective FLT3 inhibition is sufficient to inhibit the growth and induce cell death of FLT3-driven leukemia cells. AC220 is currently being evaluated in a phase II clinical trials in relapsed or refractory AML patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3619.

Abstract 636 Activating mutations in the FLT3 RTK are present in ∼30% of AML patients (pts), who have a significantly worse prognosis than pts with wild type (WT) FLT3. AC220 is a novel 2nd generation RTK inhibitor with potent in vitro and in vivo activity in FLT3- and KIT-dependent tumor cell lines. It is highly selective for both WT and mutant FLT3 with significant activity against KIT. A first-in-human Phase 1 study investigating AC220 in predominantly relapsed or refractory AML pts, unselected for FLT3 mutations, was recently completed, using a standard 3+3 dose escalation with 50% dose increments. AC220 was administered once daily as an oral solution initially with an intermittent dosing (ID) regimen: 14 days on and 14 days off (1 cycle). Starting dose was escalated from 12 to 450 mg/day ID. Additional cohorts were investigated on a continuous dosing (CD) regimen: 200 and 300 mg/day for 28 days (1 cycle). A total of 76 pts (46 male, 30 female) were dosed with AC220. Median age was 60 yrs (23-86), median number of prior therapies was 4 (0-9), 12 pts had prior allogeneic transplant and 3 elderly pts (≥72 yrs) unfit for induction chemotherapy were previously untreated. Eighteen pts (24%) had FLT3 ITD mutations, 47 (62%) were WT, and 11 (14%) were undetermined. Pts were also evaluated for PK, phosphorylated (p) FLT3, pKIT, pSTAT5, and ex vivo plasma inhibitory activity. AC220 plasma exposure was sustained between dose intervals and continued to increase in a dose-proportional manner from 12 to 450 mg with a half-life of ∼1.5 days. An active metabolite, AC886, was detected that has similar potency and activity to AC220. Patient plasma at '12 mg potently inhibited pFLT3 in ex vivo FLT3-ITD cell lines and complete inhibition of pFLT3 in WT cell lines was observed at higher doses. Target inhibition was also observed, with suppression of pFLT3, pSTAT5 and pKIT in peripheral blasts. The most commonly reported possibly drug-related AEs were GI events, peripheral edema, and dysguesia, which were Grade ≤2. DLT was observed at 300 mg CD and 200 mg CD was declared as the MTD. Two pts at 300 mg CD had possibly study drug-related DLTs with grade 3 QTc prolongation, but had confounding factors including concomitant medications known to prolong QTc. Responses (IWG criteria) were observed in 23 (30%) pts. PR and CR were observed as low as the 18 and 40 mg cohorts, respectively. Most responses occurred within cycle 1. Overall, 9 (12%) pts had a complete response (CR) with 2 CR, 5 CRi, and 2 CRp. One of these pts also had complete resolution of leukemia cutis. In addition, 14 (18%) pts achieved PR. Overall median duration of response (MDOR) was 14 (4-62+) weeks and overall median survival (MS) was 14 (1-68+) weeks. In FLT3-ITD pts the MDOR was 12 (4-27+) weeks and the MS was 18 (3-42) weeks. In FLT3-WT pts the MDOR was 32 (8-62+) weeks and the MS was 11 (1-68+) weeks. 10 (56%) of 18 FLT3-ITD pts were responders (1 CR, 3 CRi, 6 PR), 9 (19%) of 47 FLT3-WT pts (1 CRi, 2 CRp, 6 PR), and 4 (36%) of 11 undetermined pts (1 CR, 1 CRi, 2 PR). At 200 mg CD (MTD expansion), 4 of 6 FLT3-ITD pts responded (1 CR, 1 CRp, 1 CRi, 1 PR). Of the 4 responders, 2 failed prior treatment with sorafenib and 2 previously refractory pts went onto transplant. The 2 FLT3-ITD non-responders had 6 and 8 prior lines of therapy, respectively. These encouraging efficacy results and an acceptable safety profile warrant continued evaluation of AC220 as monotherapy and in combination therapy for the treatment of AML. Phase 2 studies in FLT3-ITD positive and WT pts are in progress. Disclosures: Cortes: Ambit Biosciences: Research Funding. Foran:Ambit Biosciences: Research Funding. Ghirdaladze:Ambit Biosciences: Research Funding. DeVetten:Ambit Biosciences: Research Funding. Zodelava:Ambit Biosciences: Research Funding. Holman:Ambit Biosciences: Research Funding. Levis:Ambit Biosciences: Consultancy, Research Funding. James:Ambit Biosciences: Employment. Zarringkar:Ambit Biosciences: Employment. Gunawardane:Ambit Biosciences: Employment. Armstrong:Ambit Biosciences: Employment. Padre:Ambit Biosciences: Employment. Wierenga:Ambit Biosciences: Employment. Corringham:Ambit Biosciences: Employment. Trikha:Ambit Biosciences: Employment.

Activating mutations in the receptor tyrosine kinase FLT3 are present in up to approximately 30 % of acute myeloid leukemia (AML) patients, implicating FLT3 as a potential target for kinase inhibitor therapy. While several compounds have been evaluated in the clinic as FLT3 inhibitors, including CEP-701 (lestaurtinib), PKC-412 (midostaurin), MLN-518 (tandutinib) and most recently sorafenib, none of these was originally developed as a FLT3 inhibitor. We report here the characterization of AC220, a novel compound that has been expressly optimized as a selective FLT3 inhibitor. We demonstrate that this second generation FLT3 inhibitor has a unique combination of high potency, exceptional selectivity, bioavailability, and pharmacokinetic properties compatible with once a day oral dosing. In an in vitro binding assay AC220 interacted with FLT3 with high affinity (Kd = 1.6 nM). In the FLT3 dependent human leukemia cell line MV4-11, which harbors a homozygous activating FLT3 internal tandem duplication (ITD) mutation, AC220 inhibited FLT3 autophosphorylation and cellular proliferation with subnanomolar potency. Inhibition of proliferation of a FLT3 independent cell line was several hundred-fold less potent, demonstrating cellular selectivity for FLT3. A biochemical screen against a panel of 402 kinase assays representing almost 80 % of human protein kinases revealed a highly focused and selective interaction pattern. The only targets with affinity for AC220 within 10-fold that for FLT3 were closely related class III receptor tyrosine kinases (KIT, PDGFR, RET, CSF1R), and the only targets with affinity within 100-fold that for FLT3 were four additional receptor tyrosine kinases (FLT1, FLT4, DDR1, VEGFR2). When orally administered to mice at a dose of 10 mg/kg, AC220 achieved a peak plasma concentration (Cmax) of 3.8 μM (2,100 ng/mL) within two hours of dosing. When corrected for plasma protein binding, the concentration of AC220 in plasma remained above the cellular IC50 for FLT3 inhibition 24 hours after dosing. Total exposure (AUC0-24 h) as well as Cmax increased proportionally with the administered dose from 0.1 to approximately 30 mg/kg. At higher doses, both Cmax and AUC0-24 h continued to increase, approaching a plateau above 100 mg/kg. In a FLT3-ITD-dependent MV4-11 tumor xenograft model, AC220 showed substantial, dose dependent efficacy when dosed at 1, 3 and 10 mg/kg orally once a day for 28 days. Tumors regressed at 3 and 10 mg/kg, and remained static at 1 mg/kg. In a follow-on study at the 10 mg/kg oral dose, tumor size was monitored for an additional 60 days after dosing was discontinued. By the end of the study eight complete responses and two partial responses were observed in the ten animals treated with AC220. AC220 also had activity in a leukemia tumor model at doses as low as 1 mg/kg given orally once a day. A direct comparison of AC220 with the first generation FLT3 inhibitors CEP-701, MLN-518, PKC-412, sorafenib and sunitinib revealed that the combination of potency, selectivity and pharmacokinetic properties is unique to AC220. AC220 is a second generation FLT3 inhibitor that has been explicitly optimized for the combination of properties believed to be required for the successful treatment of FLT3-dependent AML, and specifically to test the hypothesis that selective FLT3 inhibition will result in clinical benefit. AC220 is currently being evaluated in a phase I clinical trial in relapsed or refractory AML patients.