Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Scaffolding Protein Connector Enhancer of Kinase Suppressor of Ras 1 (CNKSR1) Regulates MAPK Inhibition Responsiveness in Pancreas Cancer via Crosstalk with AKT Signaling.

Combinatorial molecular therapy in pancreatic ductal adenocarcinoma (PDAC) has yielded largely disappointing results in clinical testing to-date as a multitude of adaptive resistance mechanisms is making selection of patients via molecular markers that capture essential, intersecting signaling routes challenging. Here, we report the scaffolding protein connector enhancer of kinase suppressor of Ras 1 (CNKSR1) as mediator of resistance to MAPK (MEK) inhibition. MEK inhibition in CNKSR1high cancer cells induces translocation of CNKSR1 to the plasma membrane where the scaffolding protein interacts with and stabilizes the phosphorylated form of AKT. CNKSR1-mediated AKT activation following MEK inhibition was associated with increased cellular p-PRAS40 levels and reduced nuclear translocation and cellular levels of FoxO1, a negative regulator of AKT signaling. In clinical PDAC specimens, high cytoplasmatic CNKSR1 levels correlated with increased cellular phospho-AKT and mTOR levels. Pharmacological co-blockade of AKT and MEK ranked top in induced synergies with MEK inhibition in CNKSR1high pancreas cancer cells among other inhibitor combinations targeting known CNKSR1 signaling. In vivo, CNKSR1high pancreatic tumors treated with AKT and MEK inhibitors showed improved outcome in the combination arm compared with single-agent treatment, an effect not observed in CNKSR1low models.Our results identify CNKSR1 as regulator of adaptive resistance to MEK inhibition by promoting crosstalk to AKT signaling via a scaffolding function for the phosphorylated form of AKT. CNSKR1 expression might be a possible molecular marker to enrich patients for future AKT-MEK inhibitor precision medicine studies. IMPLICATIONS: The CNKSR1 scaffold, identified within an RNAi screen as a novel mediator of resistance to MEK inhibition in pancreas cancer, connects the MAPK pathway and AKT signaling and may be adopted as a biomarker to select patients for combined MEK AKT blockade.

Discovery and Optimization of Pyrrolopyrimidine Derivatives as Selective Disruptors of the Perinucleolar Compartment, a Marker of Tumor Progression toward Metastasis.

The perinucleolar compartment (PNC) is a dynamic subnuclear body found at the periphery of the nucleolus. The PNC is enriched with RNA transcripts and RNA-binding proteins, reflecting different states of genome organization. PNC prevalence positively correlates with cancer progression and metastatic capacity, making it a useful marker for metastatic cancer progression. A high-throughput, high-content assay was developed to identify novel small molecules that selectively reduce PNC prevalence in cancer cells. We identified and further optimized a pyrrolopyrimidine series able to reduce PNC prevalence in PC3M cancer cells at submicromolar concentrations without affecting cell viability. Structure-activity relationship exploration of the structural elements necessary for activity resulted in the discovery of several potent compounds. Analysis of in vitro drug-like properties led to the discovery of the bioavailable analogue, metarrestin, which has shown potent antimetastatic activity with improved survival in rodent models and is currently being evaluated in a first-in-human phase 1 clinical trial.

LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors.

PURPOSE: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254. EXPERIMENTAL DESIGN: LXH254 was profiled in biochemical, in vitro, and in vivo assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF. RESULTS: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive. CONCLUSIONS: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.

Identification of Small Molecule Enhancers of Immunotherapy for Melanoma.

Small molecule based targeted therapies for the treatment of metastatic melanoma hold promise but responses are often not durable, and tumors frequently relapse. Response to adoptive cell transfer (ACT)-based immunotherapy in melanoma patients are durable but patients develop resistance primarily due to loss of antigen expression. The combination of small molecules that sustain T cell effector function with ACT could lead to long lasting responses. Here, we have developed a novel co-culture cell-based high throughput assay system to identify compounds that could potentially synergize or enhance ACT-based immunotherapy of melanoma. A BRAFV600E mutant melanoma cell line, SB-3123p which is resistant to Pmel-1-directed ACT due to low gp100 expression levels was used to develop a homogenous time resolve fluorescence (HTRF), screening assay. This high throughput screening assay quantitates IFNγ released upon recognition of the SB-3123p melanoma cells by Pmel-1 CD8+ T-cells. A focused collection of approximately 500 small molecules targeting a broad range of cellular mechanisms was screened, and four active compounds that increased melanoma antigen expression leading to enhanced IFNγ production were identified and their in vitro activity was validated. These four compounds may provide a basis for enhanced immune recognition and design of novel therapeutic approaches for patients with BRAF mutant melanoma resistant to ACT due to antigen downregulation.

Identification of a Small-Molecule Inhibitor That Disrupts the SIX1/EYA2 Complex, EMT, and Metastasis.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFß signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFß signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis in vivo without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.

Design and Discovery of N-(3-(2-(2-Hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide, a Selective, Efficacious, and Well-Tolerated RAF Inhibitor Targeting RAS Mutant Cancers: The Path to the Clinic.

Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, 3 (RAF709; Nishiguchi, G. A.; et al. J. Med. Chem. 2017, 60, 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.

Identification of Schlafen-11 as a Target of CD47 Signaling That Regulates Sensitivity to Ionizing Radiation and Topoisomerase Inhibitors.

Knockdown or gene disruption of the ubiquitously expressed cell surface receptor CD47 protects non-malignant cells from genotoxic stress caused by ionizing radiation or cytotoxic chemotherapy but sensitizes tumors in an immune competent host to genotoxic stress. The selective radioprotection of non-malignant cells is mediated in part by enhanced autophagy and protection of anabolic metabolism pathways, but differential H2AX activation kinetics suggested that the DNA damage response is also CD47-dependent. A high throughput screen of drug sensitivities indicated that CD47 expression selectively sensitizes Jurkat T cells to inhibitors of topoisomerases, which are known targets of Schlafen-11 (SLFN11). CD47 mRNA expression positively correlated with schlafen-11 mRNA expression in a subset of human cancers but not the corresponding non-malignant tissues. CD47 mRNA expression was also negatively correlated with SLFN11 promoter methylation in some cancers. CD47 knockdown, gene disruption, or treatment with a CD47 function-blocking antibody decreased SLFN11 expression in Jurkat cells. The CD47 signaling ligand thrombospondin-1 also suppressed schlafen-11 expression in wild type but not CD47-deficient T cells. Re-expressing SLFN11 restored radiosensitivity to a CD47-deficient Jurkat cells. Disruption of CD47 in PC3 prostate cancer cells similarly decreased schlafen-11 expression and was associated with a CD47-dependent decrease in acetylation and increased methylation of histone H3 in the SLFN11 promoter region. The ability of histone deacetylase or topoisomerase inhibitors to induce SLFN11 expression in PC3 cells was lost when CD47 was targeted in these cells. Disrupting CD47 in PC3 cells increased resistance to etoposide but, in contrast to Jurkat cells, not to ionizing radiation. These data identify CD47 as a context-dependent regulator of SLFN11 expression and suggest an approach to improve radiotherapy and chemotherapy responses by combining with CD47-targeted therapeutics.

Target Deconvolution of a Multikinase Inhibitor with Antimetastatic Properties Identifies TAOK3 as a Key Contributor to a Cancer Stem Cell-Like Phenotype.

Pancreatic cancer remains an incurable condition. Its progression is driven, in part, by subsets of cancer cells that evade the cytotoxic effects of conventional chemotherapies. These cells are often low-cycling, multidrug resistant, and adopt a stem cell-like phenotype consistent with the concept of cancer stem cells (CSC). To identify drugs impacting on tumor-promoting CSCs, we performed a differential high-throughput drug screen in pancreatic cancer cells cultured in traditional (2D) monolayers versus three-dimensional (3D) spheroids which replicate key elements of the CSC model. Among the agents capable of killing cells cultured in both formats was a 1H-benzo[d]imidazol-2-amine-based inhibitor of IL2-inducible T-cell kinase (ITK; NCGC00188382, inhibitor #1) that effectively mediated growth inhibition and induction of apoptosis in vitro, and suppressed cancer progression and metastasis formation in vivo An examination of this agent's polypharmacology via in vitro and in situ phosphoproteomic profiling demonstrated an activity profile enriched for mediators involved in DNA damage repair. Included was a strong inhibitory potential versus the thousand-and-one amino acid kinase 3 (TAOK3), CDK7, and aurora B kinases. We found that cells grown under CSC-enriching spheroid conditions are selectively dependent on TAOK3 signaling. Loss of TAOK3 decreases colony formation, expression of stem cell markers, and sensitizes spheroids to the genotoxic effect of gemcitabine, whereas overexpression of TAOK3 increases stem cell traits including tumor initiation and metastasis formation. By inactivating multiple components of the cell-cycle machinery in concert with the downregulation of key CSC signatures, inhibitor #1 defines a distinctive strategy for targeting pancreatic cancer cell populations.

Sunitinib promotes myogenic regeneration and mitigates disease progression in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal, muscle degenerative disease causing premature death of affected children. DMD is characterized by mutations in the dystrophin gene that result in a loss of the dystrophin protein. Loss of dystrophin causes an associated reduction in proteins of the dystrophin glycoprotein complex, leading to contraction-induced sarcolemmal weakening, muscle tearing, fibrotic infiltration and rounds of degeneration and failed regeneration affecting satellite cell populations. The α7ß1 integrin has been implicated in increasing myogenic capacity of satellite cells, therefore restoring muscle viability, increasing muscle force and preserving muscle function in dystrophic mouse models. In this study, we show that a Food and Drug Administration (FDA)-approved small molecule, Sunitinib, is a potent α7 integrin enhancer capable of promoting myogenic regeneration by stimulating satellite cell activation and increasing myofiber fusion. Sunitinib exerts its regenerative effects via transient inhibition of SHP-2 and subsequent activation of the STAT3 pathway. Treatment of mdx mice with Sunitinib demonstrated decreased membrane leakiness and damage owing to myofiber regeneration and enhanced support at the extracellular matrix. The decreased myofiber damage translated into a significant increase in muscle force production. This study identifies an already FDA-approved compound, Sunitinib, as a possible DMD therapeutic with the potential to treat other muscular dystrophies in which there is defective muscle repair.

Mutation Profiles in Glioblastoma 3D Oncospheres Modulate Drug Efficacy.

Glioblastoma (GBM) is a lethal brain cancer with a median survival time of approximately 15 months following treatment. Common in vitro GBM models for drug screening are adherent and do not recapitulate the features of human GBM in vivo. Here we report the genomic characterization of nine patient-derived, spheroid GBM cell lines that recapitulate human GBM characteristics in orthotopic xenograft models. Genomic sequencing revealed that the spheroid lines contain alterations in GBM driver genes such as PTEN, CDKN2A, and NF1. Two spheroid cell lines, JHH-136 and JHH-520, were utilized in a high-throughput drug screen for cell viability using a 1912-member compound library. Drug mechanisms that were cytotoxic in both cell lines were Hsp90 and proteasome inhibitors. JHH-136 was uniquely sensitive to topoisomerase 1 inhibitors, while JHH-520 was uniquely sensitive to Mek inhibitors. Drug combination screening revealed that PI3 kinase inhibitors combined with Mek or proteasome inhibitors were synergistic. However, animal studies to test these drug combinations in vivo revealed that Mek inhibition alone was superior to the combination treatments. These data show that these GBM spheroid lines are amenable to high-throughput drug screening and that this dataset may deliver promising therapeutic leads for future GBM preclinical studies.

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin. In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Combination of anthracyclines and anti-CD47 therapy inhibit invasive breast cancer growth while preventing cardiac toxicity by regulation of autophagy.

BACKGROUND: A perennial challenge in systemic cytotoxic cancer therapy is to eradicate primary tumors and metastatic disease while sparing normal tissue from off-target effects of chemotherapy. Anthracyclines such as doxorubicin are effective chemotherapeutic agents for which dosing is limited by development of cardiotoxicity. Our published evidence shows that targeting CD47 enhances radiation-induced growth delay of tumors while remarkably protecting soft tissues. The protection of cell viability observed with CD47 is mediated autonomously by activation of protective autophagy. However, whether CD47 protects cancer cells from cytotoxic chemotherapy is unknown. METHODS: We tested the effect of CD47 blockade on cancer cell survival using a 2-dimensional high-throughput cell proliferation assay in 4T1 breast cancer cell lines. To evaluate blockade of CD47 in combination with chemotherapy in vivo, we employed the 4T1 breast cancer model and examined tumor and cardiac tissue viability as well as autophagic flux. RESULTS: Our high-throughput screen revealed that blockade of CD47 does not interfere with the cytotoxic activity of anthracyclines against 4T1 breast cancer cells. Targeting CD47 enhanced the effect of doxorubicin chemotherapy in vivo by reducing tumor growth and metastatic spread by activation of an anti-tumor innate immune response. Moreover, systemic suppression of CD47 protected cardiac tissue viability and function in mice treated with doxorubicin. CONCLUSIONS: Our experiments indicate that the protective effects observed with CD47 blockade are mediated through upregulation of autophagic flux. However, the absence of CD47 in did not elicit a protective effect in cancer cells, but it enhanced macrophage-mediated cancer cell cytolysis. Therefore, the differential responses observed with CD47 blockade are due to autonomous activation of protective autophagy in normal tissue and enhancement immune cytotoxicity against cancer cells.

Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis.

Metastasis remains a leading cause of cancer mortality due to the lack of specific inhibitors against this complex process. To identify compounds selectively targeting the metastatic state, we used the perinucleolar compartment (PNC), a complex nuclear structure associated with metastatic behaviors of cancer cells, as a phenotypic marker for a high-content screen of over 140,000 structurally diverse compounds. Metarrestin, obtained through optimization of a screening hit, disassembles PNCs in multiple cancer cell lines, inhibits invasion in vitro, suppresses metastatic development in three mouse models of human cancer, and extends survival of mice in a metastatic pancreatic cancer xenograft model with no organ toxicity or discernable adverse effects. Metarrestin disrupts the nucleolar structure and inhibits RNA polymerase (Pol) I transcription, at least in part by interacting with the translation elongation factor eEF1A2. Thus, metarrestin represents a potential therapeutic approach for the treatment of metastatic cancer.

Antitumor Properties of RAF709, a Highly Selective and Potent Inhibitor of RAF Kinase Dimers, in Tumors Driven by Mutant RAS or BRAF.

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537-48. ©2018 AACR.

Design and Discovery of N-(2-Methyl-5'-morpholino-6'-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A Potent, Selective, and Efficacious RAF Inhibitor Targeting RAS Mutant Cancers.

RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

SU9516 Increases α7ß1 Integrin and Ameliorates Disease Progression in the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7ß1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7ß1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7ß1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD.

Large-scale pharmacological profiling of 3D tumor models of cancer cells.

The discovery of chemotherapeutic agents for the treatment of cancer commonly uses cell proliferation assays in which cells grow as two-dimensional (2D) monolayers. Compounds identified using 2D monolayer assays often fail to advance during clinical development, most likely because these assays do not reproduce the cellular complexity of tumors and their microenvironment in vivo. The use of three-dimensional (3D) cellular systems have been explored as enabling more predictive in vitro tumor models for drug discovery. To date, small-scale screens have demonstrated that pharmacological responses tend to differ between 2D and 3D cancer cell growth models. However, the limited scope of screens using 3D models has not provided a clear delineation of the cellular pathways and processes that differentially regulate cell survival and death in the different in vitro tumor models. Here we sought to further understand the differences in pharmacological responses between cancer tumor cells grown in different conditions by profiling a large collection of 1912 chemotherapeutic agents. We compared pharmacological responses obtained from cells cultured in traditional 2D monolayer conditions with those responses obtained from cells forming spheres versus cells already in 3D spheres. The target annotation of the compound library screened enabled the identification of those key cellular pathways and processes that when modulated by drugs induced cell death in all growth conditions or selectively in the different cell growth models. In addition, we also show that many of the compounds targeting these key cellular functions can be combined to produce synergistic cytotoxic effects, which in many cases differ in the magnitude of their synergism depending on the cellular model and cell type. The results from this work provide a high-throughput screening framework to profile the responses of drugs both as single agents and in pairwise combinations in 3D sphere models of cancer cells.

Chemical Screens Identify Drugs that Enhance or Mitigate Cellular Responses to Antibody-Toxin Fusion Proteins.

The intersection of small molecular weight drugs and antibody-based therapeutics is rarely studied in large scale. Both types of agents are currently part of the cancer armamentarium. However, very little is known about how to combine them in optimal ways. Immunotoxins are antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. For fusion proteins derived from Pseudomonas exotoxin (PE), potency relies on the enzymatic domain of the toxin which catalyzes the ADP-ribosylation of EF2 causing inhibition of protein synthesis leading to cell death. Candidate immunotoxins have demonstrated clear value in clinical trials but generally have not been curative as single agents. Therefore we undertook three screens to discover effective combinations that could act synergistically. From the MIPE-3 library of compounds we identified various enhancers of immunotoxin action and at least one major class of inhibitor. Follow-up experiments confirmed the screening data and suggested that immunotoxins when administered with everolimus or nilotinib exhibit favorable combinatory activity and would be candidates for preclinical development. Mechanistic studies revealed that everolimus-immunotoxin combinations acted synergistically on elements of the protein synthetic machinery, including S61 kinase and 4E-BP1 of the mTORC1 pathway. Conversely, PARP inhibitors antagonized immunotoxins and also blocked the toxicity due to native ADP-ribosylating toxins. Thus, our goal of investigating a chemical library was justified based on the identification of several approved compounds that could be developed preclinically as 'enhancers' and at least one class of mitigator to be avoided.