Screening Of Small-molecule Inhibitors Research Articles

Computational discovery of pathway-level genetic vulnerabilities in non-small-cell lung cancer.

Motivation: Novel approaches are needed for discovery of targeted therapies for non-small-cell lung cancer (NSCLC) that are specific to certain patients. Whole genome RNAi screening of lung cancer cell lines provides an ideal source for determining candidate drug targets.Results: Unsupervised learning algorithms uncovered patterns of differential vulnerability across lung cancer cell lines to loss of functionally related genes. Such genetic vulnerabilities represent candidate targets for therapy and are found to be involved in splicing, translation and protein folding. In particular, many NSCLC cell lines were especially sensitive to the loss of components of the LSm2-8 protein complex or the CCT/TRiC chaperonin. Different vulnerabilities were also found for different cell line subgroups. Furthermore, the predicted vulnerability of a single adenocarcinoma cell line to loss of the Wnt pathway was experimentally validated with screening of small-molecule Wnt inhibitors against an extensive cell line panel.Availability and implementation: The clustering algorithm is implemented in Python and is freely available at https://bitbucket.org/youngjh/nsclc_paper.Contact: marcotte@icmb.utexas.edu or jon.young@utexas.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Phenotypic Screening of Small-Molecule Inhibitors: Implications for Therapeutic Discovery and Drug Target Development in Traumatic Brain Injury.

The inability of central nervous system (CNS) neurons to regenerate damaged axons and dendrites following traumatic brain injury (TBI) creates a substantial obstacle for functional recovery. Apoptotic cell death, deposition of scar tissue, and growth-repressive molecules produced by glia further complicate the problem and make it challenging for re-growing axons to extend across injury sites. To date, there are no approved drugs for the treatment of TBI, accentuating the need for relevant leads. Cell-based and organotypic bioassays can better mimic outcomes within the native CNS microenvironment than target-based screening methods and thus should speed the discovery of therapeutic agents that induce axon or dendrite regeneration. Additionally, when used to screen focused chemical libraries such as small-molecule protein kinase inhibitors, these assays can help elucidate molecular mechanisms involved in neurite outgrowth and regeneration as well as identify novel drug targets. Here, we describe a phenotypic cellular (high content) screening assay that utilizes brain-derived primary neurons for screening small-molecule chemical libraries.

A Novel, Oncogenic CSF2RB Mutation Found in a Primary Leukemia Sample Results in Constitutive Signaling, Increased Receptor Stability, and Formation of Intermolecular Complexes

BackgroundColony stimulating factor 2 receptor beta (CSF2RB) is the shared beta-chain receptor and an essential component for IL-3, IL-5 and GM-CSF receptor activation. In the context of GM-CSF signaling, the ligand-specific alpha chain (CSF2RA) complexes with CSF2RB and GM-CSF ligand, forming a dodecameric complex in which the proximity of CSF2RB subunits allows associated JAK2 kinases to trans-phosphorylate. CSF2RB signals through several pathways, including JAK2/STAT5, PI3K/AKT and MEK/ERK to promote survival, proliferation and differentiation. Several studies have demonstrated that mutations in CSF2RB can lead to cellular transformation in vitro, but no oncogenic mutations have yet been identified in primary leukemia samples. Here we report the first such mutation, found in a pediatric patient with T-cell acute lymphoblastic leukemia (T-ALL), resulting in the formation of intermolecular disulfide bonds, prolonged protein half-life, and ligand-independent receptor activation that is sensitive to JAK2 kinase inhibition.Methods and ResultsThrough an ongoing effort to identify oncogenic mutations in primary leukemias, we performed whole-exome sequencing on 18 T-ALL patient samples. CSF2RB R461C was identified in a pediatric T-ALL and stably expressed in Ba/F3 cells to test its capacity to confer factor-independent growth. R461C consistently and rapidly transformed Ba/F3 cells, while wild-type (WT) and vector controls did not. R461C expression resulted in a substantial accumulation of CSF2RB protein relative to WT, both prior to and following Ba/F3 transformation. Cycloheximide time-course experiments demonstrated that the R461C mutant possessed a prolonged surface half-life relative to WT CSF2RB (3.6 vs. 6.5 hours; p<0.01). Given the presence of a novel cysteine in the R461C mutant, we hypothesized that the increased surface stability could result from abnormal, disulfide-linked receptor oligomerization. To test this, we compared the WT and R461C receptors using a non-reducing PAGE immunoblot and observed higher molecular weight, CSF2RB-containing complexes of 500 KDa only in R461C-expressing cells. The size of this complex approximately corresponds to a CSF2RB tetramer and could account for receptor activation and accumulation.The UniProt-listed and commonly used transmembrane domain for CSF2RB (444-460) indicates R461C is a cytoplasmic residue and therefore unlikely to form a disulfide bond in the reducing conditions of the cytoplasm. To resolve this discrepancy, we modeled the transmembrane domain of CSF2RB using several advanced prediction platforms and concluded that residue 461 lies well within the membrane spanning region for both the WT and mutant receptors. While the CSF2RB transmembrane domain has not been empirically studied, modern models delineate that the domain spans residues 443-465, thereby creating the potential for cysteines 461 and 463 to form intermolecular disulfide bonds with homologous or heterogeneous receptor chains.Using immunoblot analysis, we showed that transformed Ba/F3 cells expressing CSF2RB R461C exhibited constitutive receptor phosphorylation and signaled through several pathways, including STAT-1, -3, and -5, AKT/mTOR1, MEK/ERK, and SRC. In a small-molecule inhibitor screen, several JAK2 inhibitors (ruxolitinib, tofacitinib, AZD1480) achieved significantly lower IC50 concentrations in CSF2RB R461C cells relative to WT (p<0.05). These results indicate that R461C primarily signals through JAK2, which is consistent with ligand-independent activation of CSF2RB.ConclusionsWe have demonstrated for the first time that a novel and actionable primary CSF2RB mutation confers factor-independent growth, receptor accumulation, and constitutive JAK/STAT pathway activation. Furthermore, we identified a novel mechanism of constitutive CSF2RB activation where incorporation of a transmembrane cysteine results in novel intermolecular disulfide bonds that could account for constitutive signaling. JAK inhibitors blocked the growth of R461C-transformed cells, thereby providing a potential rationale for using JAK inhibitors to treat patients with CSF2RB-mutated leukemia. Future research could also utilize R461C to study the impact of the transmembrane domain on CSF2RB receptor complex formation and recycling. DisclosuresTyner:Aptose Biosciences: Research Funding; Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding. Druker:Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; McGraw Hill: Patents & Royalties; Oncotide Pharmaceuticals: Research Funding; Millipore: Patents & Royalties; Henry Stewart Talks: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Sage Bionetworks: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding. Agarwal:CTI BioPharma: Research Funding.

A Role for IL1RAP in Acute Myelogenous Leukemia Stem Cells Following Treatment and Progression

BackgroundAcute Myelogenous Leukemia (AML) evolves as many patients who are responsive to therapy upfront are resistant to the same agents when applied at relapse. We previously reported the results of our prospective efforts to formally assess the evolution of the leukemia stem cell (LSC) population(s) during patients' clinical courses. We identified a 9-90 fold increase in LSC activity and greatly increased phenotypic diversity of the LSC population. To identify the potential mechanisms underlying these changes we further characterized functionally-defined LSC populations from paired diagnosis and relapse samples.MethodsPrimary bone marrow and peripheral blood samples were collected on IRB approved protocols from patients with newly diagnosed AML undergoing induction therapy as well as normal donors. Twenty-five patients who relapsed after achieving a complete remission were selected for further study. Screening studies identified seven patients whose pre-therapy samples demonstrated sustained engraftment of NSG mice following transplantation. Transcriptional profiling of highly enriched LSC populations from seven patients was performed using ABI TaqMan® Low Density Array (TLDA) qPCR analyses following pre-amplification using a novel 153 gene expression platform. Protein expression levels of interleukin-1 receptor accessory protein (IL1RAP) on bulk leukemia cells and LSC populations from 25 patients were assessed by flow cytometry. The impact of loss of IL1RAP was assessed using lentiviral based shRNA targeting all IL1RAP isoforms followed by assessment of proliferation, apoptosis, colony forming unit (CFU) activity and NSG engraftment capacity in human cell lines as well as in primary patient samples. Downstream signaling events for IL1RAP were probed using a small molecule inhibitor approach.ResultsWhile the majority of the LSC populations' gene expression profile remained stable, twelve genes were differentially expressed between pre-treatment and relapsed LSC populations including IL1RAP. Flow cytometric analyses confirmed that IL1RAP is overexpressed on both bulk leukemia populations as well as LSC populations at diagnosis and relapse in comparison to normal hematopoietic stem cell (HSC) populations. Targeting ILRAP1 using shRNA in both cell lines and primary AML samples resulted in impaired proliferation, increased apoptosis, a marked loss of CFU capacity and impaired NSG engraftment. IL1 signaling is known to involve both the MAPkinase and NFKappB pathways. To determine which pathways are involved in IL1RAP mediated LSC survival, we performed a small molecule inhibitor screen targeting elements in both signaling cascades. Established inhibitors of the NFKappaB pathway resulted in loss in loss of leukemic cell function while MAPK signaling inhibition had minimal to no effect.ConclusionsWe identified IL1RAP as being overexpressed in both bulk leukemia and functionally defined LSC populations from pre-treatment and relapsed AML samples. Loss of IL1RAP was associated with a marked decline in LSC function. Preliminary studies support a primary role for the NF Kappa B pathway in LSC function. Our findings support a critical role for IL1RAP in LSC function and support its development as a target for AML therapy in both the upfront and relapse setting. DisclosuresWang:Immunogen: Research Funding. Calvi:Fate Therapeutics: Patents & Royalties. Becker:Millenium: Research Funding.

Novel Method to Perform Functional Drug Screens on Frozen Primary Leukemia Samples

The ability to assess anti-leukemic activity of drugs on patient samples is a powerful tool in determining potential drug targets and may enhance selection of therapeutic agents with a biologic and functional rationale for individual patients. We have previously optimized small-molecule screens performed on freshly isolated leukemia cells for this purpose. Here we describe a novel method we have recently developed that produces reliable functional testing results from previously frozen specimens. This method was established to take advantage of bio-repositories containing archival primary leukemia specimens and to aid in the validation of potential drug target analysis and pathway activation.Three different culture media comprising various cocktails of cytokines were tested for their ability to maintain cell viability after thawing and to produce inhibitor results similar to those obtained using freshly isolated acute myeloid leukemia (AML) cells. We identified a method of thawing cells by first culturing for 24-48 hours in cytokine-enriched medium (RPMI-1640, 10% fetal bovine serum (FBS), and low concentrations of GM-CSF, G-CSF, SCF, and IL-3) prior to plating in RPMI-1640 and 10% FBS (R10). This method maintained high cell viability and produced inhibitor results comparable to those of freshly isolated cells plated in R10. Half-maximal inhibitory concentration (IC50) analysis was used to determine the relative success of the thaw process and reliability of results. Comparison of data produced from frozen cells initially cultured in cytokine-enriched medium to data obtained from freshly isolated cells showed few differences in effective drug hits.To examine the effects of freeze-thawing and cytokines on cell subpopulations, we performed FACS analysis on 8 markers to examine the level of cell differentiation under each condition. FACS sorting revealed increases in expression of myeloid differentiation markers (CD11b and CD14) after thaw in all media tested compared to fresh cells in cultured in R10. Thawed cells grown in cytokine-enriched medium remained closest to fresh controls as measured by antigen expression, including CD11b and CD14.Cell viability post-thaw was a strong indicator of samples that would fail to produce reliable inhibitor results. We observed that post-thaw viability <80% served as a strong indicator of unreliable inhibitor data, even if dead cells were removed prior to plating. Cells cultured in cytokine-enriched medium for 24-48 hours resulted in 11-33% higher viability prior to plating in R10. Percent median IC50 for specific inhibitors assayed on fresh and frozen samples showed similar trends in effective drugs (Fig. 1A). Discrepancies observed in fresh and frozen sample data may be due to inconsistent fresh sample data as we demonstrated that frozen samples were highly consistent compared to other frozen samples (Fig. 1B). Lower R2 values could be attributed to variability observed with non-effective drugs. Taken together, our data indicate that thawing leukemia specimens in cytokine-enriched media can allow for the generation of informative small-molecule inhibitor screening data and provide a novel method for making use of archived primary human leukemia specimens for further downstream functional analyses. [Display omitted] DisclosuresAgarwal:CTI BioPharma: Research Funding. Druker:Oregon Health & Science University: Patents & Royalties; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Sage Bionetworks: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncotide Pharmaceuticals: Research Funding; ARIAD: Research Funding; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; McGraw Hill: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

A Novel Inhibitor of AKT1-PDPK1 Interaction Efficiently Suppresses the Activity of AKT Pathway and Restricts Tumor Growth In Vivo.

The serine/threonine kinase AKT/PKB has a critical role in the regulation of cell proliferation. Because AKT signaling is deregulated in numerous human malignancies, it has become an attractive anticancer drug target. A number of small molecule AKT kinase inhibitors have been developed; however, severe side effects have prevented their use in clinical trials. To find inhibitors of AKT1 signaling with principally novel mechanism of action, we carried out a live cell-based screen for small molecule inhibitors of physical interaction between AKT1 and its primary activator PDPK1. The screen revealed one molecule-NSC156529, which downregulated AKT1 signaling, efficiently decreased the proliferation of human cancer cells in vitro, and substantially inhibited the growth of prostate tumor xenografts in vivo. Interestingly, the treated tumor xenografts exhibited higher expression level of normal prostate differentiation markers but did not show augmented cell death, suggesting that the identified compound primarily enhances the differentiation of malignant cells toward normal prostate epithelium and thus poses as an attractive lead compound for developing novel antitumor agents with less cytotoxic side effects.

From GFP to β-lactamase: advancing intact cell imaging for toxins and effectors.

Canonical reporters such as green fluorescent protein (GFP) and luciferase have assisted researchers in probing cellular pathways and processes. Prior research in pathogenesis depended on sensitivity of biochemical and biophysical techniques to identify effectors and elucidate entry mechanisms. Recently, the β-lactamase (βlac) reporter system has advanced toxin and effector reporting by permitting measurement of βlac delivery into the cytosol or host βlac expression in intact cells. βlac measurement in cells was facilitated by the development of the fluorogenic substrate, CCF2-AM, to identify novel effectors, target cells, and domains involved in bacterial pathogenesis. The assay is also adaptable for high-throughput screening of small molecule inhibitors against toxins, providing information on mechanism and potential therapeutic agents. The versatility and limitations of the βlac reporter system as applied to toxins and effectors are discussed in this review.

Abstract POSTER-THER-1405: Targeting proteasome-associated deubiquitinating enzymes for ovarian cancer treatment

Abstract Purpose of the study: Recent studies have shown that genetic events in cancer activate signaling pathways that ultimately alter cell metabolism, thereby rendering cancer cells more dependent upon “metabolic pathways” as compared to normal cells. These findings have resulted in refocused efforts in the strategy for the selective design of anticancer therapies to target the metabolic dependencies of cancer cells. Deubiquitinating enzymes (DUBs) are an essential component of the ubiquitin-dependent protein degradation system and, owing to their recently described role during cancer progression and chemoresistance, DUBs are an attractive new target for cancer treatment. The purpose of our study is to develop novel small molecule inhibitors of proteasome-associated DUBs as an alternative therapeutic approach for treatment of resistant forms of ovarian cancer. The rationale driving this study is that up-regulation of DUBs (at protein and enzymatic activity levels) is a feature of cancer and of chemoresistant versus chemosensitive ovarian cancer. Experimental Procedure: We conducted a high-throughput screening for small molecule inhibitors of DUBs, resulting in the identification of a lead compound, RA-9. Potency, selectivity, cell permeability and potential off-target effects of RA-9 were evaluated in vitro in primary ovarian cancer cells derived from clinical specimens and in vivo in a preclinical mouse xenograft model of human ovarian cancer. Preclinical efficacy and drug safety were also evaluated in a mouse model of human ovarian cancer. Summary of the data: RA-9 was shown to be a potent, selective and cell permeable inhibitor of a specific subset of DUBs associated with the 19S subunit of proteasomes. RA-9 selectively killed primary ovarian cancer cells derived from patients resistant to chemotherapy at clinically achievable concentrations. Mechanistically, RA-9 caused the onset of apoptosis in ovarian cancer cells via a mechanism involving exacerbation of endoplasmic-reticulum stress responses. In vivo, RA-9 was well tolerated, significantly decreased tumor burden, and increases overall survival in a preclinical model of human ovarian cancer. Conclusion: Our findings indicate that RA-9 has significant in vitro and in vivo anti-cancer activity at doses well tolerated in a mouse xenograft model. Based on these promising data, our laboratory is committed to the further development of this class of inhibitors. This includes medicinal chemistry study to optimize selectivity and potency of our current lead compound and pharmacology efforts to optimize pharmacokinetic and pharmacological properties of RA-9 and its most promising derivatives. Our immediate goal is to rapidly translate our findings from the benchside by performing a phase 0 clinical trial with our new investigational drugs. Importantly, Phase 0 studies are highly feasible in that they require: GLP grade drugs, single doses of drugs that are about 1% of the amount designed for clinical trials and a relatively low number of subjects. This work was supported by the Department of Defense Ovarian Cancer Research Program (OCRP) OC093424 and Minnesota Ovarian Cancer Alliance to MB. Citation Format: Kathleen Coughlin, Thanasak Sueblinvong, Rachel Isaksson Vogel, Ravi Anchoori, Martina Bazzaro. Targeting proteasome-associated deubiquitinating enzymes for ovarian cancer treatment [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1405.

Characterization of a Steroid Receptor Coactivator Small Molecule Stimulator that Overstimulates Cancer Cells and Leads to Cell Stress and Death

By integrating growth pathways on which cancer cells rely, steroid receptor coactivators (SRC-1, SRC-2, and SRC-3) represent emerging targets in cancer therapeutics. High-throughput screening for SRC small molecule inhibitors (SMIs) uncovered MCB-613 as a potent SRC small molecule "stimulator" (SMS). We demonstrate that MCB-613 can super-stimulate SRCs' transcriptional activity. Further investigation revealed that MCB-613 increases SRCs' interactions with other coactivators and markedly induces ER stress coupled to the generation of reactive oxygen species (ROS). Because cancer cells overexpress SRCs and rely on them for growth, we show that we can exploit MCB-613 to selectively induce excessive stress in cancer cells. This suggests that over-stimulating the SRC oncogenic program can be an effective strategy to kill cancer cells.

Abstract 266: Screening and Validation of Small Molecule Inhibitors of Serum and Glucocorticoid Regulated Kinase-1 as Novel Therapy for Cardiac Arrhythmia Disorders

Alterations in sodium flux (INa) play an important role in the pathogenesis of cardiac arrhythmias and may also contribute to the development of cardiomyopathies. Recent data demonstrates a critical role for the serum and glucocorticoid regulated kinase-1 (SGK1) by modulation of INa in the heart, by regulating the voltage-gated sodium channel NaV1.5. To better understand and pharmacologically probe the significance of SGK1 in cardiac dysrhythmias, we have used computer aided drug discovery (CADD) to identify small molecule inhibitors of SGK1. Expression of a constitutively active form of SGK1 (SGK1-CA) increased INa (1.7 fold, p &lt;0.005) in a stable line of HEK cells expressing NaV1.5. Conversely, expression of a dominant negative form (SGK-DN) decreased NaV1.5 channel activity (2.8 fold, p &lt;0.005). We examined the effects of SGK1 inhibition in a LQT model, by quantifying the ability of SGK1 inhibition to rescue the 2:1 AV block phenotype of the potassium channel zebrafish mutant, breakdance (bkd). Morpholino injection or expression of SGK1-DN significantly rescued the 2:1 AV block phenotype as compared to controls (p &lt; 0.05). Using CADD partnered with iterative empirical screens we identified several hit chemical scaffolds. Our lead compound inhibits the phosphorylation of the SGK1 target gene, GSK3-β in a dose dependent manner in cardiomyocytes (CMs) expressing SGK1-CA at the lowest effective concentration of 0.5μM. There was no significant inhibition of AKT dependent phosphorylation of GSK3-β up to a concentration of 50μM, demonstrating specificity of the inhibitor for SGK1. Incubation of bkd zebrafish mutants with the inhibitor rescued the 2:1 AV block in a dose dependent manner (60% rescue with 45μM, p &lt; 0.05). Acute application of the inhibitor dramatically inhibited INa with either expression of SGK1-CA (90.8% reduction, p &lt;0.05 ) or with RFP only (77.5% reduction p &lt; 0.005). The half-time of inhibition was 200s with resulting current densities that were not statistically different than those observed with genetic inhibition by expression of SGK1-DN. We conclude SGK1 activity regulates INa and speculate that structure activity relationship (SAR) derivatives of our lead compound might have a role in treatment of human cardiac arrhythmias.

Small molecule inhibitor screen identifies synergistic activity of the bromodomain inhibitor CPI203 and bortezomib in drug resistant myeloma.

Despite significant therapeutic progress in multiple myeloma, drug resistance is uniformly inevitable and new treatments are needed. Our aim was to identify novel, efficacious small-molecule combinations for use in drug resistant multiple myeloma. A panel of 116 small molecule inhibitors was used to screen resistant myeloma cell lines for potential therapeutic targets. Agents found to have enhanced activity in the bortezomib or melphalan resistant myeloma cell lines were investigated further in combination. Synergistic combinations of interest were evaluated in primary patient cells. The overall single-agent drug sensitivity profiles were dramatically different between melphalan and bortezomib resistant cells, however, the bromodomain inhibitor, CPI203, was observed to have enhanced activity in both the bortezomib and melphalan resistant lines compared to their wild-type counterparts. The combination of bortezomib and CPI203 was found to be synergistic in both the bortezomib and melphalan resistant cell lines as well as in a primary multiple myeloma sample from a patient refractory to recent proteasome inhibitor treatment. The CPI203-bortezomib combination led to enhanced apoptosis and anti-proliferative effects. Finally, in contrast to prior reports of synergy between bortezomib and other epigenetic modifying agents, which implicated MYC downregulation or NOXA induction, our analyses suggest that CPI203-bortezomib synergy is independent of these events. Our preclinical data supports a role for the clinical investigation of the bromodomain inhibitor CPI203 combined with bortezomib or alkylating agents in resistant multiple myeloma.

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis.

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.

Effective Quenchers Are Required to Eliminate the Interference of Substrate: Cofactor Binding in the HAT Scintillation Proximity Assay.

Histone acetyltransferases (HATs) mediate the transfer of an acetyl group from the cofactor, acetyl-CoA, to the side chain amino group of specific lysines in diverse protein substrates, most notably nuclear histones. The deregulation of HATs is connected to a number of disease states. Reliable and rapid biochemical assays for HATs are critical for understanding biological functions of protein acetylation, as well as for screening small-molecule inhibitors of HAT enzymes. In this report, we present a scintillation proximity assay (SPA) for the measurement of HAT enzymatic activities. The acetyl donor was [(3)H]Ac-CoA, and a biotin-modified histone peptide served as the HAT substrate. After the HAT reaction, streptavidin-coated beads were added to induce proximity of acetylated substrate to the scintillant molecules. However, we observed strong nonspecific binding between the cofactor and the histone peptide substrates, which adversely complicated the SPA performance. To prevent this problem, a set of chemical agents were evaluated to eliminate the cofactor-substrate interaction, thus providing reliable SPA readings. With optimization, the SPA showed consistent and robust performance for HAT activity measurement and HAT inhibitor evaluation. Overall, this mix-and-measure assay does not require any washing procedure, can be utilized in the microplate format, and is well suited for high-throughput screening of HAT chemical modulators.

PDK1 is a potential therapeutic target against angiosarcoma cells

BackgroundAngiosarcoma is a rare and aggressive malignant neoplasm of endothelial cells. Recent studies have shown that the mTOR pathway is also aberrantly activated in cutaneous angiosarcoma. New therapeutic strategies are required because the prognosis of this disease is still poor. ObjectiveThe aim of the present study was to determine the driver gene of angiosarcoma useful for the screening of small molecule inhibitors. MethodsWe investigated the sensitivity of inhibitors for the PI3K/AKT/mTOR pathway in ISOS-1 and ISO-HAS cutaneous angiosarcoma cell lines. Flow cytometric analysis was conducted to evaluate cell-cycle progression and apoptosis. Western blot analyses were performed to elucidate the possible underlying mechanisms of growth inhibition. The colony formation assay was conducted to evaluate the clonogenic potential. We used the siRNA for PDK1 to examine the role of PDK1 on the growth of angiosarcoma cells. ResultsThe PI3K inhibitor and mTOR inhibitor inhibited the growth of both cell lines in a dose-dependent manner. The PI3K inhibitor more effectively induced cell-cycle arrest at the G1 phase with the downregulated expression of cyclin D in ISOS-1 cells than the mTOR inhibitor. The PI3K inhibitor and mTOR inhibitor weakly but significantly induced G1 cell cycle arrest at the same degree in ISO-HAS cells. The expression of cyclin D was downregulated by the treatment with siRNA for PDK1, but not by the AKT inhibitor in ISOS-1 and ISO-HAS cells. The knock down of PDK1 with siRNA was more effective at reducing colony numbers than the mTOR inhibitor in ISOS-1 cells. ConclusionThese data showed that PDK1 played a pivotal role in the growth of angiosarcoma cells. Therefore, inhibition of PDK1, but not AKT, may be a more appropriate strategy than that of mTORC1 for the treatment of cutaneous angiosarcoma; the PDK1 inhibitor is promising as a therapeutic agent.

Quantification of Histone H3 Lys27 Trimethylation (H3K27me3) by High-Throughput Microscopy Enables Cellular Large-Scale Screening for Small-Molecule EZH2 Inhibitors

EZH2 inhibition can decrease global histone H3 lysine 27 trimethylation (H3K27me3) and thereby reactivates silenced tumor suppressor genes. Inhibition of EZH2 is regarded as an option for therapeutic cancer intervention. To identify novel small-molecule (SMOL) inhibitors of EZH2 in drug discovery, trustworthy cellular assays amenable for phenotypic high-throughput screening (HTS) are crucial. We describe a reliable approach that quantifies changes in global levels of histone modification marks using high-content analysis (HCA). The approach was validated in different cell lines by using small interfering RNA and SMOL inhibitors. By automation and miniaturization from a 384-well to 1536-well plate, we demonstrated its utility in conducting phenotypic HTS campaigns and assessing structure-activity relationships (SAR). This assay enables screening of SMOL EZH2 inhibitors and can advance the mechanistic understanding of H3K27me3 suppression, which is crucial with regard to epigenetic therapy. We observed that a decrease in global H3K27me3, induced by EZH2 inhibition, comprises two distinct mechanisms: (1) inhibition of de novo DNA methylation and (II) inhibition of dynamic, replication-independent H3K27me3 turnover. This report describes an HCA assay for primary HTS to identify, profile, and optimize cellular active SMOL inhibitors targeting histone methyltransferases, which could benefit epigenetic drug discovery.

FL118 induces p53-dependent senescence in colorectal cancer cells by promoting degradation of MdmX.

Anticancer agent FL118 was recently identified in screening of small-molecule inhibitors of human survivin expression. Although FL118 is a camptothecin analogue, its antitumor potency is much superior to other FDA-approved camptothecin analogues (irinotecan and topotecan). The mechanism of action (MOA) underlying the antitumor effects of FL118 remains to be fully elucidated. Here, we report that FL118 activates tumor suppressor p53 as a novel MOA in p53 wild-type cancer cells. Our studies show that this MOA involves an induction of proteasomal degradation of MdmX, a critical negative regulator of p53, in a manner largely independent of ATM-dependent DNA damage signaling pathway but dependent on E3-competent Mdm2. FL118 inhibits p53 polyubiquitination and monoubiquitination by Mdm2-MdmX E3 complex in cells and in cell-free systems. In contrast, FL118 stimulates Mdm2-mediated MdmX ubiquitination. Coimmunoprecipitation revealed that FL118 slightly decreases Mdm2-p53 interactions and moderately increases Mdm2-MdmX interactions, suggesting a change of targeting specificity of Mdm2-MdmX E3 complex from p53 to MdmX, resulting in accelerated MdmX degradation. As a result, p53 ubiquitination by Mdm2-MdmX E3 complex is reduced, which in turn activates p53 signaling. Activation of the p53 pathway by FL118 induces p53-dependent senescence in colorectal cancer cells. However, in the absence of p53 or in the presence of MdmX overexpression, FL118 promotes p53-independent apoptosis. These two distinct cellular consequences collectively contribute to the potent effects of FL118 to inhibit clonogenic potential of colon cancer cells. This study identifies a potential application of FL118 as an MdmX inhibitor for targeted therapies.

Identification and characterization of small-molecule inhibitors of lysine acetyltransferases.

Lysine acetyltransferases (KATs) acetylate various proteins including histones, transcription factors, metabolic enzymes, and other cellular substrates. Protein acetylation significantly impacts protein stability and function. Certain KATs such as p300 (KAT3B) are overexpressed in cancer cells and are linked to tumor progression and drug resistance. Thus, pharmacologic inhibition of KATs represents a new strategy for cancer therapy. Quantitative biochemical assays of KAT enzymatic activity have been developed and adapted for high-throughput screens of small-molecule compounds to discover specific KAT inhibitors. Such compounds are useful probes for understanding the cellular functions of these critical enzymes and importantly, they may be further developed as anticancer therapeutics. Here we describe a fluorescence-based KAT activity assay and cell-based validation of KAT inhibition by small-molecule compounds.

Characterization of Early-Stage Amyloid Aggregates by Incorporating Extrinsic Fluorescence and Atomic Force Microscopy

Amyloid-β (Aβ) oligomers are nanosized bio-assemblies that cause Alzheimer's disease. Characterizing early-stage Aβ aggregates becomes an important issue because it is a prerequisite in exploring small molecule inhibitors that bind to Aβ oligomers. Of special interest are efficient screening systems that characterize the Aβ oligomer size with respect to the aging time. In this work, highly sensitive fluorescence techniques and atomic force microscopy (AFM) were employed to investigate the size determination of Aβ and screening of small molecule inhibitors. A solvatochromic dye, 1-anilinonaphthalene-8-sulfonic acid (ANS), was used as an extrinsic fluorophore to monitor the growth mechanism of the Aβ aggregates. Then, the time-resolved fluorescence anisotropy method was employed to estimate the hydrodynamic size of Aβ oligomers. Finally, AFM was used to characterize the Aβ oligomer size in the absence and presence of potential inhibitors. We present that the combination of such three experimental techniques is an excellent way to detect the early stage of Aβ aggregation and to screen small molecule inhibitors.

AXL inhibition sensitizes mesenchymal cancer cells to antimitotic drugs.

Molecularly targeted drug therapies have revolutionized cancer treatment; however, resistance remains a major limitation to their overall efficacy. Epithelial-to-mesenchymal transition (EMT) has been linked to acquired resistance to tyrosine kinase inhibitors (TKI), independent of mutational resistance mechanisms. AXL is a receptor tyrosine kinase associated with EMT that has been implicated in drug resistance and has emerged as a candidate therapeutic target. Across 643 human cancer cell lines that were analyzed, elevated AXL was strongly associated with a mesenchymal phenotype, particularly in triple-negative breast cancer and non-small cell lung cancer. In an unbiased screen of small-molecule inhibitors of cancer-relevant processes, we discovered that AXL inhibition was specifically synergistic with antimitotic agents in killing cancer cells that had undergone EMT and demonstrated associated TKI resistance. However, we did not find that AXL inhibition alone could overcome acquired resistance to EGFR TKIs in the EMT setting, as previously reported. These findings reveal a novel cotreatment strategy for tumors displaying mesenchymal features that otherwise render them treatment refractory.

Abstract 693: AXL tyrosine kinase inhibition selectively sensitizes mesenchymal cancer cells to antimitotic agents

Abstract Molecularly-targeted drug therapies have revolutionized cancer treatment, however, resistance remains a major limitation to their overall efficacy. Epithelial to mesenchymal transition (EMT) has been linked to acquired resistance to tyrosine kinase inhibitors (TKIs), independent of mutational resistance mechanisms. AXL is a receptor tyrosine kinase (RTK) associated with EMT that has been implicated in drug resistance, and has emerged as a candidate therapeutic target. Across 643 human cancer cell lines that were analyzed, we found that elevated AXL was strongly associated with a mesenchymal phenotype, particularly in triple negative breast cancer and non-small cell lung cancer. Here we experimentally established a model system to induced EMT by exposure to TGF-β and we observed that the resulting TGF-β-induced mesenchymal cells exhibit substantially increased AXL expression, become relatively drug-resistant to their appropriate TKI and cross-resistant to a variety of anti-cancer agents. We tested the ability of AXL inhibition to restore erlotinib sensitivity, in both a TGF-β-induced resistance model and in two different models of acquired resistance to erlotinib. Here, our observations do not support the previously reported role for AXL inhibition in overcoming acquired resistance to TKIs in the EMT context. However, in an unbiased screen of small molecule inhibitors of cancer-relevant processes, we discovered that AXL inhibition was specifically synergistic with anti-mitotic agents in killing cancer cells that had undergone EMT and demonstrated associated TKI resistance. Inhibition of AXL in combination with anti-mitotic agents enhances death in mitosis by promoting cells to enter mitosis through the down-regulation of CDK1, resulting in G2M cell cycle arrest. In summary, our findings highlight the utility of combination drug screening to reveal novel potentially useful anti-cancer drug combinations to improve clinical responses. While we did not identify a functional role for AXL in the context of acquired resistance to TKI therapies, we did observe that AXL inhibition may be an effective therapy in the context of mesenchymal tumors, specifically when used in combination with anti-mitotic agents. These findings reveal a novel therapeutic strategy for tumors displaying mesenchymal features that otherwise render them treatment-refractory. Citation Format: Catherine Wilson, Thinh Pham, Xiaofen Ye, Eva Lin, Sara Chan, Erin McNamara, Richard M. Neve, Lisa Belmont, Hartmut Koeppen, Robert L. Yauch, Avi Ashkenazi, Jeff Settleman. AXL tyrosine kinase inhibition selectively sensitizes mesenchymal cancer cells to antimitotic agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 693. doi:10.1158/1538-7445.AM2014-693