Purpose: Although many cancers are showing remarkable responses to targeted therapies, pediatric sarcomas, including Ewing sarcoma, remain recalcitrant. To broaden the therapeutic landscape, we explored the in vitro response of Ewing sarcoma cell lines against a large collection of investigational and approved drugs to identify candidate combinations.Experimental Design: Drugs displaying activity as single agents were evaluated in combinatorial (matrix) format to identify highly active, synergistic drug combinations, and combinations were subsequently validated in multiple cell lines using various agents from each class. Comprehensive metabolomic and proteomic profiling was performed to better understand the mechanism underlying the synergy. Xenograft experiments were performed to determine efficacy and in vivo mechanism.Results: Several promising candidates emerged, including the combination of small-molecule PARP and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, a rational combination as NAMPTis block the rate-limiting enzyme in the production of nicotinamide adenine dinucleotide (NAD(+)), a necessary substrate of PARP. Mechanistic drivers of the synergistic cell killing phenotype of these combined drugs included depletion of NMN and NAD(+), diminished PAR activity, increased DNA damage, and apoptosis. Combination PARPis and NAMPTis in vivo resulted in tumor regression, delayed disease progression, and increased survival.Conclusions: These studies highlight the potential of these drugs as a possible therapeutic option in treating patients with Ewing sarcoma. Clin Cancer Res; 23(23); 1-11. ©2017 AACR.

Ketamine is rapidly metabolized in the human body to a variety of metabolites, including the hydroxynorketamines. At least two hydroxynorketamines have significant antidepressant action in rodent models, with limited action against the N-methyl-d-aspartate (NMDA) receptor. The synthesis of 12 hydroxynorketamines and their binding affinity to the NMDA receptor is presented here.

The Plasmodium falciparum malarial parasite genome appears to encode one and only one phosphatidylinositol 3'-kinase (PI3K), and sequence analysis suggests that the enzyme is a "class III"- or "Vps34"-type PI3K. PfVps34 has generated excitement as a possible druggable target and potentially a key target of artemisinin-based antimalarials. In this study, we optimize the PfVps34 gene for heterologous expression in yeast, purify the protein to homogeneity, use a recently validated quantitative assay for phosphatidylinositol 3'-phosphate production from phosphatidylinositol ( Hassett et al., companion paper; DOI 10.1021/acs.biochem.7b00416 ) to quantify activity and drug inhibition of that activity, and investigate the importance of key residues in the enzyme's catalytic and "N-lobe" domains. Data suggest that PfVps34 is indeed inhibited by artemisinin and related drugs but only under conditions that cleave the drugs' endoperoxide bridge to generate reactive alkylating agents.

Primary CNS lymphoma (PCNSL) harbors mutations that reinforce B cell receptor (BCR) signaling. Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, targets BCR signaling and is particularly active in lymphomas with mutations altering the BCR subunit CD79B and MYD88. We performed a proof-of-concept phase Ib study of ibrutinib monotherapy followed by ibrutinib plus chemotherapy (DA-TEDDi-R). In 18 PCNSL patients, 94% showed tumor reductions with ibrutinib alone, including patients having PCNSL with CD79B and/or MYD88 mutations, and 86% of evaluable patients achieved complete remission with DA-TEDDi-R. Increased aspergillosis was observed with ibrutinib monotherapy and DA-TEDDi-R. Aspergillosis was linked to BTK-dependent fungal immunity in a murine model. PCNSL is highly dependent on BCR signaling, and ibrutinib appears to enhance the efficacy of chemotherapy.

BACKGROUND: Patients with hereditary diffuse gastric cancer (HDGC), a cancer predisposition syndrome associated with germline mutations of the CDH1 (E-cadherin) gene, have few effective treatment options. Despite marked differences in natural history, histopathology, and genetic profile to patients afflicted by sporadic gastric cancer, patients with HDGC receive, in large, identical systemic regimens. The lack of a robust preclinical in vitro system suitable for effective drug screening has been one of the obstacles to date which has hampered therapeutic advances in this rare disease. METHODS: In order to identify therapeutic leads selective for the HDGC subtype of gastric cancer, we compared gene expression profiles and drug phenotype derived from an oncology library of 1912 compounds between gastric cancer cells established from a patient with metastatic HDGC harboring a c.1380delA CDH1 germline variant and sporadic gastric cancer cells. RESULTS: Unsupervised hierarchical cluster analysis shows select gene expression alterations in c.1380delA CDH1 SB.mhdgc-1 cells compared to a panel of sporadic gastric cancer cell lines with enrichment of ERK1-ERK2 (extracellular signal regulated kinase) and IP3 (inositol trisphosphate)/DAG (diacylglycerol) signaling as the top networks in c.1380delA SB.mhdgc-1 cells. Intracellular phosphatidylinositol intermediaries were increased upon direct measure in c.1380delA CDH1 SB.mhdgc-1 cells. Differential high-throughput drug screening of c.1380delA CDH1 SB.mhdgc-1 versus sporadic gastric cancer cells identified several compound classes with enriched activity in c.1380 CDH1 SB.mhdgc-1 cells including mTOR (Mammalian Target Of Rapamycin), MEK (Mitogen-Activated Protein Kinase), c-Src kinase, FAK (Focal Adhesion Kinase), PKC (Protein Kinase C), or TOPO2 (Topoisomerase II) inhibitors. Upon additional drug response testing, dual PI3K (Phosphatidylinositol 3-Kinase)/mTOR and topoisomerase 2A inhibitors displayed up to >100-fold increased activity in hereditary c.1380delA CDH1 gastric cancer cells inducing apoptosis most effectively in cells with deficient CDH1 function. CONCLUSION: Integrated pharmacological and transcriptomic profiling of hereditary diffuse gastric cancer cells with a loss-of-function c.1380delA CDH1 mutation implies various pharmacological vulnerabilities selective to CDH1-deficient familial gastric cancer cells and suggests novel treatment leads for future preclinical and clinical treatment studies of familial gastric cancer.

Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1 (P3F). The mechanisms by which P3F dysregulates chromatin are unknown. We find P3F reprograms the cis-regulatory landscape by inducing (de novo) super enhancers (SEs). P3F uses SEs to setup auto-regulatory loops in collaboration with master transcription factors MYOG, MYOD and MYCN. This myogenic SE circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1 occupied SEs. Furthermore, P3F recruits and requires BET bromodomain protein BRD4 to function at SEs, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield novel insights into the epigenetic functions of P3F, and reveal a specific vulnerability that can be exploited for precision therapy.

Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is a lipid kinase involved in endosome maturation that emerged from a haploid genetic screen as being required for Ebola virus (EBOV) infection. Here we analyzed the effects of apilimod, a PIKfyve inhibitor that was reported to be well tolerated in humans in phase 2 clinical trials, for its effects on entry and infection of EBOV and Marburg virus (MARV). We first found that apilimod blocks infections by EBOV and MARV in Huh 7, Vero E6 and primary human macrophage cells, with notable potency in the macrophages (IC50, 10 nM). We next observed that similar doses of apilimod block EBOV-glycoprotein-virus like particle (VLP) entry and transcription-replication competent VLP infection, suggesting that the primary mode of action of apilimod is as an entry inhibitor, preventing release of the viral genome into the cytoplasm to initiate replication. After providing evidence that the anti-EBOV action of apilimod is via PIKfyve, we showed that it blocks trafficking of EBOV VLPs to endolysosomes containing Niemann-Pick C1 (NPC1), the intracellular receptor for EBOV. Concurrently apilimod caused VLPs to accumulate in early endosome antigen 1-positive endosomes. We did not detect any effects of apilimod on bulk endosome acidification, on the activity of cathepsins B and L, or on cholesterol export from endolysosomes. Hence by antagonizing PIKfyve, apilimod appears to block EBOV trafficking to its site of fusion and entry into the cytoplasm. Given the drug's observed anti-filoviral activity, relatively unexplored mechanism of entry inhibition, and reported tolerability in humans, we propose that apilimod be further explored as part of a therapeutic regimen to treat filoviral infections.

Aortic valve disease (AVD) is a common condition with a progressive natural history, and presently, there are no pharmacologic treatment strategies. Elastic fiber fragmentation (EFF) is a hallmark of AVD, and increasing evidence implicates developmental elastic fiber assembly defects. Emilin1 is a glycoprotein necessary for elastic fiber assembly that is present in both developing and mature human and mouse aortic valves. The Emilin1-deficient mouse (Emilin1(-/-) ) is a model of latent AVD, characterized by activated TGFβ/MEK/p-Erk signaling and upregulated elastase activity. Emilin1(-/-) aortic valves demonstrate early EFF and aberrant angiogenesis followed by late neovascularization and fibrosis. The objective of this study was to test the effectiveness of three different targeted therapies. Aged (12-14 months) Emilin1(-/-) mice were treated with refametinib (RDEA-119, MEK1/2 inhibitor), doxycycline (elastase inhibitor), or G6-31 (anti-VEGF-A mouse antibody) for 4 weeks. Refametinib- and doxycycline-treated Emilin1(-/-) mice markedly reduced MEK/p-Erk activation in valve tissue. Furthermore, both refametinib and doxycycline attenuated elastolytic cathepsin K, L, MMP-2, and MMP-9 activation, and abrogated macrophage and neutrophil infiltration in Emilin1(-/-) aortic valves. RNAseq analysis was performed in aortic valve tissue from adult (4 months) and aged (14 months) Emilin1(-/-) and age-matched wild-type control mice, and demonstrated upregulation of genes associated with MAPK/MEK/p-Erk signaling and elastases at the adult stage and inflammatory pathways at the aged stage controlling for age. These results suggest that Erk1/2 signaling is an important modulator of early elastase activation, and pharmacological inhibition using refametinib may be a promising treatment to halt AVD progression.

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.

In the fall of 2014, an international news agency reported that patients suffering from Ebola virus disease (EVD) in Liberia were treated successfully with lamivudine, an antiviral drug used to treat human immunodeficiency virus-1 and hepatitis B virus infections. According to the report, 13 out of 15 patients treated with lamivudine survived and were declared free from Ebola virus disease. In this study, the anti-Ebola virus (EBOV) activity of lamivudine and another antiretroviral, zidovudine, were evaluated in a diverse set of cell lines against two variants of wild-type EBOV. Variable assay parameters were assessed to include different multiplicities of infection, lengths of inoculation times, and durations of dosing. At a multiplicity of infection of 1, lamivudine and zidovudine had no effect on EBOV propagation in Vero E6, Hep G2, or HeLa cells, or in primary human monocyte-derived macrophages. At a multiplicity of infection of 0.1, zidovudine demonstrated limited anti-EBOV activity in Huh 7 cells. Under certain conditions, lamivudine had low anti-EBOV activity at the maximum concentration tested (320 μM). However, lamivudine never achieved greater than 30% viral inhibition, and the activity was not consistently reproducible. Combination of lamivudine and zidovudine showed no synergistic antiviral activity. Independently, a set of in vitro experiments testing lamivudine and zidovudine for antiviral activity against an Ebola-enhanced green fluorescent protein reporter virus was performed at the Centers for Disease Control and Prevention. No antiviral activity was observed for either compound. A study evaluating the efficacy of lamivudine in a guinea pig model of EVD found no survival benefit. This lack of benefit was observed despite plasma lamivudine concentrations in guinea pig of about 4 μg/ml obtained in a separately conducted pharmacokinetics study. These studies found no evidence to support the therapeutic use of lamivudine for the treatment of EVD.