Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration-and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers. Mol Cancer Ther; 13(4); 842-54. Ó2014 AACR.

Background:Eribulin mesilate (eribulin), a non-taxane microtubule dynamics inhibitor, has shown trends towards greater overall survival (OS) compared with progression-free survival in late-stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional, previously unrecognised antitumour mechanisms beyond its established antimitotic activity. To investigate this possibility, eribulin's effects on the balance between epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) in human breast cancer cells were investigated.Methods:Triple negative breast cancer (TNBC) cells, which are oestrogen receptor (ER−)/progesterone receptor (PR−)/human epithelial growth receptor 2 (HER2−) and have a mesenchymal phenotype, were treated with eribulin for 7 days, followed by measurement of EMT-related gene and protein expression changes in the surviving cells by quantitative real-time PCR (qPCR) and immunoblot, respectively. In addition, proliferation, migration, and invasion assays were also conducted in eribulin-treated cells. To investigate the effects of eribulin on TGF-β/Smad signalling, the phosphorylation status of Smad proteins was analysed. In vivo, the EMT/MET status of TNBC xenografts in mice treated with eribulin was examined by qPCR, immunoblot, and immunohistochemical analysis. Finally, an experimental lung metastasis model was utilised to gauge the metastatic activity of eribulin-treated TNBC in the in vivo setting.Results:Treatment of TNBC cells with eribulin in vitro led to morphological changes consistent with transition from a mesenchymal to an epithelial phenotype. Expression analyses of EMT markers showed that eribulin treatment led to decreased expression of several mesenchymal marker genes, together with increased expression of several epithelial markers. In the TGF-β induced EMT model, eribulin treatment reversed EMT, coincident with inhibition of Smad2 and Smad3 phosphorylation. Consistent with these changes, TNBC cells treated with eribulin for 7 days showed decreased capacity for in vitro migration and invasiveness. In in vivo xenograft models, eribulin treatment reversed EMT and induced MET as assessed by qPCR, immunoblot, and immunohistochemical analyses of epithelial and mesenchymal marker proteins. Finally, surviving TNBC cells pretreated in vitro with eribulin for 7 days led to decreased numbers of lung metastasis when assessed in an in vivo experimental metastasis model.Conclusions:Eribulin exerted significant effects on EMT/MET-related pathway components in human breast cancer cells in vitro and in vivo, consistent with a phenotypic switch from mesenchymal to epithelial states, and corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo. These preclinical findings may provide a plausible scientific basis for clinical observations of prolonged OS by suppression of further spread of metastasis in breast cancer patients treated with eribulin.

E7389, a macrocyclic ketone analog of the marine natural product halichondrin B, currently is undergoing clinical trials for cancer. This fully synthetic agent exerts its highly potent in vitro and in vivo anticancer effects via tubulin-based antimitotic mechanisms, which are similar or identical to those of parental halichondrin B. In an attempt to understand the impressive potency of E7389 in animal models of human cancer, its ability to induce apoptosis following prolonged mitotic blockage was evaluated. Treatment of U937 human histiocytic lymphoma cells with E7389 led to time-dependent collection of cells in the G 2 -M phase of the cell cycle, beginning as early as 2 h and becoming maximal by 12 h. Increased numbers of hypodiploid events were seen beginning at 12 h, suggesting initiation of apoptosis after prolonged E7389-induced mitotic blockage. The identity of hypodiploid events as apoptotic cells under these conditions was confirmed by two additional morphologic criteria: green to orange/yellow shifts on acridine orange/ethidium bromide staining, and cell surface annexin V binding as assessed by flow cytometry. Several biochemical correlates of apoptosis also were seen following E7389 treatment, including phosphorylation of the antiapoptotic protein Bcl-2, cytochrome c release from mitochondria, proteolytic activation of caspase-3 and -9, and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). In LNCaP human prostate cancer cells, treatment with E7389 also led to generation of hypodiploid cells, activation of caspase-3 and -9, and appearance of cleaved PARP, indicating that E7389 can activate cellular apoptosis pathways under anchorage-independent and -dependent cell culture conditions. These results show that prolonged mitotic blockage by E7389 can lead to apoptotic cell death of human cancer cells in vitro and can provide a mechanistic basis for the significant in vivo anticancer efficacy of E7389.

Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. Although immunotherapies are approved for MIBC, the majority of patients fail to respond, suggesting existence of complementary immune evasion mechanisms. Here, we report that the PPARγ/RXRα pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC. Recurrent mutations in RXRα at serine 427 (S427F/Y), through conformational activation of the PPARγ/RXRα heterodimer, and focal amplification/overexpression of PPARγ converge to modulate PPARγ/RXRα-dependent transcription programs. Immune cell-infiltration is controlled by activated PPARγ/RXRα that inhibits expression/secretion of inflammatory cytokines. Clinical data sets and an in vivo tumor model indicate that PPARγHigh/RXRαS427F/Y impairs CD8+ T-cell infiltration and confers partial resistance to immunotherapies. Knockdown of PPARγ or RXRα and pharmacological inhibition of PPARγ significantly increase cytokine expression suggesting therapeutic approaches to reviving immunosurveillance and sensitivity to immunotherapies. Our study reveals a class of tumor cell-intrinsic “immuno-oncogenes” that modulate the immune microenvironment of cancer.

Thapsigargin, a tumour-promoting sesquiterpene lactone, selectively inhibits the Ca(2+)-ATPase responsible for Ca2+ accumulation by the endoplasmic reticulum (ER). Mobilization of ER-sequestered Ca2+ to the cytosol and to the extracellular fluid subsequently ensues, with concomitant alteration of cellular functions. Thapsigargin was found to serve as a rapid, potent and efficacious inhibitor of amino acid incorporation in cultured mammalian cells. At concentrations mobilizing cell-associated Ca2+ to the extracellular fluid, thapsigargin provoked extensive inhibition of protein synthesis within 10 min. The inhibition in GH3 pituitary cells involved the synthesis of almost all polypeptides, was not associated with increased cytosolic free Ca2+ concentration ([Ca2+]i), and was not reversed at high extracellular Ca2+. The transient rise in [Ca2+]i triggered by ionomycin was diminished by thapsigargin. Polysomes failed to accumulate in the presence of the drug, indicative of impaired translational initiation. With longer (1-3 h) exposures to thapsigargin, recovery of translational activity was observed accompanied by increased synthesis of the ER protein glucose-regulated stress protein 78 or immunoglobulin heavy-chain binding protein ('GRP78/BiP') and its mRNA. Such inductions were comparable with those observed previously with Ca2+ ionophores which mobilize the cation from all intracellular sequestered sites. Actin mRNA concentrations declined significantly during such treatments. In HepG2 cells processing and secretion of the glycoprotein alpha 1-antitrypsin were rapidly suppressed by thapsigargin. Ca2+ sequestered specifically by the ER is concluded to be essential for optimal protein synthesis and processing. These rapid effects of thapsigargin on mRNA translation, protein processing and gene expression should be considered when evaluating potential mechanisms by which this tumour promoter influences cellular events.

We report here that des-methyl, des-amino pateamine A (DMDA-PatA), a structurally simplified analogue of the marine natural product pateamine A, has potent antiproliferative activity against a wide variety of human cancer cell lines while showing relatively low cytotoxicity against nonproliferating, quiescent human fibroblasts. DMDAPatA retains almost full in vitro potency in P-glycoprotein-overexpressing MES-SA/Dx5-Rx1 human uterine sarcoma cells that are significantly resistant to paclitaxel, suggesting that DMDA-PatA is not a substrate for P-glycoprotein-mediated drug efflux. Treatment of proliferating cells with DMDA-PatA leads to rapid shutdown of DNA synthesis in the S phase of the cell cycle. Cell-free studies show that DMDA-PatA directly inhibits DNA polymerases α and γ in vitro albeit at concentrations considerably higher than those that inhibit cell proliferation. DMDA-PatA shows potent anticancer activity in several human cancer xenograft models in nude mice, including significant regressions observed in the LOX and MDA-MB-435 melanoma models. DMDA-PatA thus represents a promising natural product-based anticancer agent that warrants further investigation.

Thyroglobulin (Tg), the major protein secreted by thyroid epithelial cells and precursor of thyroid hormones, is a large dimeric glycoprotein with multiple disulfide bonds. The folding and assembly of this complex molecule begins in the endoplasmic reticulum (ER) and is likely to involve a variety of reactions catalyzed by molecular chaperones (Kuznetsov, G., Chen, L. B., and Nigam, S. K. (1994) J. Biol. Chem. 269, 22990 -22995). By coimmunoprecipitation in rat thyroid cells, we were able to demonstrate that BiP, grp94, ERp72, and grp170, four proteins believed to function as specific molecular chaperones, complex with Tg during its maturation. The same complex of the four putative chaperones with Tg was observed in cells treated with tunicamycin, indicating that these four ER chaperones stably associate with Tg when it is misfolded/misassembled due to inhibition of its glycosylation. BiP, grp94, and ERp72 were also found to associate with Tg in cells in which misfolding was induced by perturbing ER calcium stores. To determine if the assembly of a complex between the four chaperones and Tg under conditions of misglycosylation was unique to the maturation of this particular secretory protein or a more general phenomenon, adenovirus-transformed rat thyroid cells that do not synthesize Tg were analyzed. In these transformed cells, the only protein these same four chaperones were found to complex with was a protein of approximately 200 kDa. This protein was subsequently identified as thrombospondin, which, like Tg, is a large oligomeric secreted glycoprotein with multiple disulfide bonds. We therefore propose that these ER chaperones complex together with a variety of large oligomeric secretory glycoproteins as they fold and assemble in the ER.

Mutations in estrogen receptor alpha (ER) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Since a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ER signaling, there remains a critical need to develop the next generation of ER antagonists that can overcome aberrant ER activity. Through our drug discovery efforts, we identified H3B-5942 which covalently inactivates both wild-type and mutant ER by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ER antagonist referred to as Selective Estrogen Receptor Covalent Antagonists (SERCAs).In vitro comparisons of H3B-5942 with standard of care (SoC) 10, 2018; DOI: 10.1158/2159-8290.CD-17-1229 3Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July SignificanceNearly 30% of endocrine-therapy resistant breast cancer metastases harbor constitutively activating mutations in ER. Selective Estrogen Receptor Covalent Antagonist (SERCA) H3B-5942 engages C530 of both ER WT and ER MUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over standard of care (SoC) agents.Importantly, single agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors.