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Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient’s 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

Clancy et al. develop a specific chemical inhibitor of USP9X and characterize its effects upon the cellular proteome. This analysis reveals a central role in the regulation of ribosomal stalling through control of critical E3 ligases., When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.

Inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) is associated with robust antitumor activity. Ribociclib (LEE011) is an orally bioavailable CDK4/6 inhibitor that is approved for the treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer, in combination with an aromatase inhibitor, and is currently being evaluated in several additional trials. Here, we report the preclinical profile of ribociclib. When tested across a large panel of kinase active site binding assays, ribociclib and palbociclib were highly selective for CDK4, while abemaciclib showed affinity to several other kinases. Both ribociclib and abemaciclib showed slightly higher potency in CDK4-dependent cells than in CDK6-dependent cells, while palbociclib did not show such a difference. Profiling CDK4/6 inhibitors in large-scale cancer cell line screens in vitro confirmed that RB1 loss of function is a negative predictor of sensitivity. We also found that routinely used cellular viability assays measuring adenosine triphosphate levels as a proxy for cell numbers underestimated the effects of CDK4/6 inhibition, which contrasts with assays that assess cell number more directly. Robust antitumor efficacy and combination benefit was detected when ribociclib was added to encorafenib, nazartinib, or endocrine therapies in patient-derived xenografts.

In breast cancer, estrogen receptor alpha (ERα) positive cancer accounts for approximately 74% of all diagnoses, and in these settings, it is a primary driver of cell proliferation. Treatment of ERα positive breast cancer has long relied on endocrine therapies such as selective estrogen receptor modulators, aromatase inhibitors, and selective estrogen receptor degraders (SERDs). The steroid-based anti-estrogen fulvestrant (5), the only approved SERD, is effective in patients who have not previously been treated with endocrine therapy as well as in patients who have progressed after receiving other endocrine therapies. Its efficacy, however, may be limited due to its poor physicochemical properties. We describe the design and synthesis of a series of potent benzothiophene-containing compounds that exhibit oral bioavailability and preclinical activity as SERDs. This article culminates in the identification of LSZ102 (10), a compound in clinical development for the treatment of ERα positive breast cancer.

In the tumor microenvironment, ATP released by dying cells is converted to adenosine, a well-established potent suppressor of immune cell activity. The immune suppressive function of extracellular adenosine is mediated through two G-protein coupled receptors known as A2A and A2B. Both receptors are expressed on many types of immune cells. While A2B has traditionally been considered of less relevance compared to A2A due to lower affinity to its ligand adenosine, recent evidence suggests a specific role of A2B in immune suppressive myeloid cells in cancer. To determine whether a small molecule inhibitor which blocks both adenosine receptors on immune cells can be used as an immunotherapy to enhance anti-tumor immune responses, we initiated a discovery campaign to identify dual antagonist inhibitors of A2A and A2B. INCB106385 is a potent, selective and orally bioavailable dual antagonist of A2A and A2B receptors. In vitro, INCB106385 potently binds to both A2A and A2B receptors in the single-digit nanomolar range and antagonizes downstream signaling as measured by cAMP production in A2A and A2B expressing cells. In functional studies, INCB106385, in the presence of 50μM AMP, restores effector T cell activity as measured by interferon gamma (IFNγ) production. In vivo, INCB106385 inhibited both the downstream effector pCREB and tumor growth in CT26 and B16-F10 syngeneic tumor models. Furthermore, in in vitro and in vivo combination studies, INCB106385 and anti-PD-1/PD-L1 CPIs demonstrated an increase in T cell function and anti-tumor activity compared to the single agent treatment groups. These data suggests that inhibition of the adenosine pathway is non-redundant to current checkpoint inhibitor therapies and can be an effective combination strategy to enhance anti-tumor immune activities. In summary, the data presented in this study demonstrate that INCB106385 is a potent, selective and orally bioavailable A2A/A2B dual antagonist that can overcome the immune-suppressive effects of high levels of adenosine in the tumor microenvironment. INCB106385 can promote anti-tumor immunity as a monotherapy and in combination with anti-PD-1/PD-L1 treatment. Citation Format: Hui Wang, Alexandra Alexandra, Michael Hansbury, Jennifer Mason, Jennifer Harris, Christina Stevens, Christopher Maddage, Xiaodi Ren, Mingming Gao, Kerri Kurzeja-Lipinski, Gengjie Yang, Patricia Conlen, Kristine Stump, Patricia Feldman, Pramod Thekkat, Luping Lin, Maryanne Covington, Swamy Yeleswaram, Chao Qi, Xiaozhao Wang, Wenqing Yao, Sunkyu Kim, Susan Wee, Yingnan Chen, Holly Koblish. Discovery and characterization of INCB106385: a novel A2A/A2B adenosine receptor antagonist, as a cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB157.

A uniformly colored TiO 2 , on which the surface is functionalized with nanotubes to control wettability, was prepared by a two-step anodization; the first anodization was carried out to prepare nanotubes for a super-hydrophilic or -hydrophobic surface and the second anodization was performed to fabricate a thin film barrier oxide to ensure uniform coloring. The effect of the nanotubes on barrier oxide coloring was examined by spectrophotometry and UV-vis-IR spectroscopy. We found four different regimes governing the color changes in terms of anodization voltage, indicating that the color of the duplex TiO 2 was primarily determined by the thickness of the barrier oxide layer formed during the second anodization step. The surface wettability, as confirmed by the water contact angle, revealed that the single barrier TiO 2 yielded 74.6° ± 2.1, whereas the nanotubes on the barrier oxide imparted super-hydrophilic properties as a result of increasing surface roughness as well as imparting a higher hydrophobicity after organic acid treatment.

Purpose: Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition. Experimental Design: We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies. Results: Sensitivity to binimetinib and ribociclib was inversely related (r = −0.58, P = 0.009). MYCN amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs. Conclusions: Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS–MAPK signaling. Clin Cancer Res; 23(7); 1785–96. ©2016 AACR.

MnO 2 was homogenously doped into anodic nanotubular TiO 2 by a potential shock method, in which a high potential was imposed on the anodic nanotubes immediately after anodization process. We found that the potential shock process is inapplicable in a high-conductivity aqueous electrolyte, e.g. a MnO 2 precursor solution (herein, 0.006 M KMnO 4 : 852–933 μS/m). To avoid exceeding the output compliance of the current source, the potential shock voltage was optimized in ethylene glycol for the application of water oxidation. We found an optimal potential shock voltage of 140 V, which led to the doping of 0.7 at. % MnO 2 into the high-aspect-ratio nanotubular TiO 2 within 10 s. The TiO 2 nanotubes doped with MnO 2 were successfully employed as electrodes for the non-noble catalysis of water oxidation. Although the doping concentration of Mn was found to be linearly proportional to the applied potential shock voltages, potential shocks greater than 140 V significantly increased the thickness of the barrier oxide layer, which increased the overpotential in the water oxidation process.

Antigen recognition and T-cell receptor (TCR) activation are fundamental processes that drive anti-tumor T cell responses. HPK1 has been identified as a negative regulator of TCR activation, as well as BCR activation, and is a potential anticancer target for immuno-oncology. We show that genetic ablation of HPK1 in human T cells resulted in increased cytokine production upon cell stimulation. Jurkat cells with genetic HPK1 knockout were unable to phosphorylate SLP76, a direct downstream target of HPK1, upon cell stimulation which was associated with increased IL-2 production. Genetic deletion of HPK1 in human RAMOS B cells reduced phosphorylation of BLNK upon IgM stimulation and led to increased TNF alpha and TNF beta production. Treatment of HPK1 knockout primary human T cells with pembrolizumab enhanced IFN gamma secretion compared to the knockout cells alone. Based on these genetic data, HPK1 may be an attractive target for immuno-oncology. We describe herein the in vitro and in vivo profile of several small molecule HPK1 inhibitors and report a surprising disparity between in vitro and in vivo findings. In vitro, the HPK1 inhibitors tested phenocopied the genetic data by potently inhibiting pSLP76 and enhancing IL-2 production in Jurkat cells following stimulation. Similarly, HPK1 inhibitors enhanced IL-2 production in human PBMCs, and increased IFN gamma production in combination with atezolizumab in a primary T cell co-culture assay. In vivo, the HPK1 small molecule inhibitors inhibited tumor growth in the MC38 model, which was further enhanced in combination with anti-PD-L1 in 3 different syngeneic models (MC38, CT26 and MBT-2). Despite these results, tumor growth inhibition was not observed in the GL261 glioma syngeneic model whose growth was recently shown to be inhibited when implanted into HPK1 kinase-dead mice. Further, although several in vitro assays demonstrated increased functional cytokine production with HPK1 inhibitor treatment, the compounds did not increase in vivo cytokine production in tumors. Moreover, combining HPK1 inhibitors with a PD-L1 antibody in vivo abolished the anti-PD-L1-induced production of IFN gamma in the CT26 model. In summary, while our genetic data support the role of HPK1 as a negative regulator of T and B cells, the in vitro activity of HPK1 kinase activity inhibitors was not correlated with functional effects in in vivo syngeneic tumor models. These results underline the complexity of interpreting HPK1 biology and also highlight challenges for the development of clinically active compounds targeting this pathway. Citation Format: Yaoyu Chen, Jonathan Rios-Doria, Michelle Pusey, Kerri Lasky, Min Ye, Pramod Thekkat, Karen Gallagher, Kristine Stump, Patricia Conlen, Christine Gardiner, Hui Wang, Alexander Sokolsky, Mark Rupar, Luping Lin, Elham Behshad, Maryanne Covington, Holly Koblish, Oleg Vechorkin, Wenqing Yao, Sunkyu Kim, Yingnan Chen. The role of HPK1 in the regulation of T cell function and anti-tumor immune activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4513.

Tri-reforming of methane (TRM) produces syngas by directly utilizing flue gas from a fossil fuel-fired power plant without requiring post-combustion CO2 separation. In this work, different yolk sizes of a NiCe@SiO2 multi–yolk–shell nanotube catalyst were prepared and their catalytic properties were evaluated at different oxidizer (CO2 + H2O + O2) to methane (O/M) feed ratios for TRM. The NiCe@SiO2 multi–yolk–shell nanotube catalyst can exhibit longer stability than the conventional NiCe/SiO2Imp catalyst synthesized by impregnation method due to its controlled morphology and synergetic interactions of Ni–Ce and Ni–Si species. At a low O/M feed ratio of 1.0, NiCe@SiO2 with smaller yolks ( 30 nm) presents stable TRM activity at a high O/M feed ratio of 1.1, whereas NiCe@SiO2 consisting of smaller yolks deactivates. Deactivation of NiCe@SiO2 with smaller yolks can be explained by the re-oxidation of active Ni species, in which carbon formation and oxidation rates, and Ce3+/Ce4+ redox properties play a crucial role. Our results indicate that the NiCe@SiO2 multi–yolk–shell nanotube structures can provide high TRM activity, yet their structure should be tuned for stable performance by considering the yolk sizes and interaction of Ni–Ce species.

Motivation Traditional drug discovery approaches identify a target for a disease and find a compound that binds to the target. In this approach, structures of compounds are considered as the most important features because it is assumed that similar structures will bind to the same target. Therefore, structural analogs of the drugs that bind to the target are selected as drug candidates. However, even though compounds are not structural analogs, they may achieve the desired response. A new drug discovery method based on drug response, which can complement the structure-based methods, is needed. Results We implemented Siamese neural networks called ReSimNet that take as input two chemical compounds and predicts the CMap score of the two compounds, which we use to measure the transcriptional response similarity of the two compounds. ReSimNet learns the embedding vector of a chemical compound in a transcriptional response space. ReSimNet is trained to minimize the difference between the cosine similarity of the embedding vectors of the two compounds and the CMap score of the two compounds. ReSimNet can find pairs of compounds that are similar in response even though they may have dissimilar structures. In our quantitative evaluation, ReSimNet outperformed the baseline machine learning models. The ReSimNet ensemble model achieves a Pearson correlation of 0.518 and a precision@1% of 0.989. In addition, in the qualitative analysis, we tested ReSimNet on the ZINC15 database and showed that ReSimNet successfully identifies chemical compounds that are relevant to a prototype drug whose mechanism of action is known. Availability and implementation The source code and the pre-trained weights of ReSimNet are available at https://github.com/dmis-lab/ReSimNet. Supplementary information Supplementary data are available at Bioinformatics online.

For the preparation of a highly stable and active electrode for hydrogen evolution reaction (HER) and oxygen evolution reaction (OER), Pt was doped into TiO2 nanotubes by means of an underpotential shock method, in which positively biased voltage lower than the anodizing voltage was applied to anodic TiO2 for short seconds immediately after anodization, to reduce the overpotential of the catalyzed hydrogen and oxygen evolution reactions of water splitting. Because aqueous acidic H2PtCl6 solution was used as a doping precursor, Cl– ions were generated during imposing high positive voltage (so-called potential shock), which would aggressively damage the anodic oxide if a normal potential shock were applied. Instead, the novel underpotential shock method allowed Pt to be homogeneously doped into TiO2 films without destroying the structures. Pt could not thoroughly penetrate the barrier oxide into the interface between the oxide and Ti metal because the voltage of the potential shock was lower than the anodiz...

Tungsten trioxide (WO 3 ) prepared by anodization of a W foil was doped with NH 4 through NH 4 OH treatment at 450 °C. Since aqueous NH 4 OH was used during doping instead of NH 3 gas, the treatment step does not require complicated annealing facilities. Moreover, the state of doped N is a form of NH 3 -W instead of W 2 N, which lowers the bandgap but increases photocorrosion. We found that incorporation of NH 4 into WO 3 leads to reduction of the bandgap from 2.9 eV to 2.2 eV, regardless of the amount of NH 4 OH treatment, lowering the onset potential and increasing the current density at fixed potential for oxygen evolution reaction under illumination. Scanning electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy were employed to investigate the surface morphologies, crystallinities of tungsten oxides and existence of NH 4 doping, respectively. The bandgap energy was determined by UV–Vis spectroscopy to measure the transmittance and refraction. The water splitting performance of each sample was measured by electrochemical linear sweep voltammetry in a 3-electrode configuration under illumination.

Tetrahydroisoquinoline 40 has been identified as a potent ERα antagonist and selective estrogen receptor degrader (SERD), exhibiting good oral bioavailability, antitumor efficacy, and SERD activity in vivo. We outline the discovery and chemical optimization of the THIQ scaffold leading to THIQ 40 and showcase the racemization of the scaffold, pharmacokinetic studies in preclinical species, and the in vivo efficacy of THIQ 40 in a MCF-7 human breast cancer xenograft model.

SummaryActivation of the phosphoinositide 3-kinase (PI3K) pathway occurs frequently in breast cancer. However, clinical results of single-agent PI3K inhibitors have been modest to date. A combinatorial drug screen on multiple PIK3CA mutant cancers with decreased sensitivity to PI3K inhibitors revealed that combined CDK 4/6-PI3K inhibition synergistically reduces cell viability. Laboratory studies revealed that sensitive cancers suppress RB phosphorylation upon treatment with single-agent PI3K inhibitors but cancers with reduced sensitivity fail to do so. Similarly, patients’ tumors that responded to the PI3K inhibitor BYL719 demonstrated suppression of pRB, while nonresponding tumors showed sustained or increased levels of pRB. Importantly, the combination of PI3K and CDK 4/6 inhibitors overcomes intrinsic and adaptive resistance leading to tumor regressions in PIK3CA mutant xenografts.

Castration-resistant prostate cancer (CRPC) is the most aggressive, incurable form of prostate cancer. MDV3100 (enzalutamide), an antagonist of the androgen receptor (AR), was approved for clinical use in men with metastatic CRPC. Although this compound showed clinical efficacy, many initial responders later developed resistance. To uncover relevant resistant mechanisms, we developed a model of spontaneous resistance to MDV3100 in LNCaP prostate cancer cells. Detailed characterization revealed that emergence of an F876L mutation in AR correlated with blunted AR response to MDV3100 and sustained proliferation during treatment. Functional studies confirmed that ARF876L confers an antagonist-to-agonist switch that drives phenotypic resistance. Finally, treatment with distinct antiandrogens or cyclin-dependent kinase (CDK)4/6 inhibitors effectively antagonized ARF876L function. Together, these findings suggest that emergence of F876L may (i) serve as a novel biomarker for prediction of drug sensitivity, (ii) predict a “withdrawal” response to MDV3100, and (iii) be suitably targeted with other antiandrogens or CDK4/6 inhibitors. Significance: We uncovered an F876L agonist-switch mutation in AR that confers genetic and phenotypic resistance to the antiandrogen drug MDV3100. On the basis of this finding, we propose new therapeutic strategies to treat patients with prostate cancer presenting with this AR mutation. Cancer Discov; 3(9); 1030–43. ©2013 AACR. See related commentary by Nelson and Yegnasubramanian, p. 971 This article is highlighted in the In This Issue feature, p. 953

Purpose: Although significant progress has been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), many patients will require additional therapy for relapsed/refractory disease. Cyclin D3 (CCND3) and CDK6 are highly expressed in T-ALL and have been effectively targeted in mutant NOTCH1-driven mouse models of this disease with a CDK4/6 small-molecule inhibitor. Combination therapy, however, will be needed for the successful treatment of human disease. Experimental Design: We performed preclinical drug testing using a panel of T-ALL cell lines first with LEE011, a CDK4/6 inhibitor, and next with the combination of LEE011 with a panel of drugs relevant to T-ALL treatment. We then tested the combination of LEE011 with dexamethasone or everolimus in three orthotopic mouse models and measured on-target drug activity. Results: We first determined that both NOTCH1-mutant and wild-type T-ALL are highly sensitive to pharmacologic inhibition of CDK4/6 when wild-type RB is expressed. Next, we determined that CDK4/6 inhibitors are antagonistic when used either concurrently or in sequence with many of the drugs used to treat relapsed T-ALL (methotrexate, mercaptopurine, asparaginase, and doxorubicin) but are synergistic with glucocorticoids, an mTOR inhibitor, and gamma secretase inhibitor. The combinations of LEE011 with the glucocorticoid dexamethasone or the mTOR inhibitor everolimus were tested in vivo and prolonged survival in three orthotopic mouse models of T-ALL. On-target activity was measured in peripheral blood and tissue of treated mice. Conclusions: We conclude that LEE011 is active in T-ALL and that combination therapy with corticosteroids and/or mTOR inhibitors warrants further investigation. Clin Cancer Res; 23(4); 1012–24. ©2016 AACR. See related commentary by Carroll et al., p. 873

Herein, we describe the discovery of potent and highly selective inhibitors of both CDK4 and CDK6 via structure-guided optimization of a fragment-based screening hit. CDK6 X-ray crystallography and pharmacokinetic data steered efforts in identifying compound 6, which showed >1000-fold selectivity for CDK4 over CDKs 1 and 2 in an enzymatic assay. Furthermore, 6 demonstrated in vivo inhibition of pRb-phosphorylation and oral efficacy in a Jeko-1 mouse xenograft model.

Identification and structure-guided optimization of a series of 4-(pyrazol-4-yl)-pyrimidines as selective CDK4/6 inhibitors is reported herein. Several potency and selectivity determinants were established based on the X-ray crystallographic analysis of representative compounds bound to monomeric CDK6. Significant selectivity for CDK4/6 over CDK1 and CDK2 was demonstrated with several compounds in both enzymatic and cellular assays.

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3γ, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3γ. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.

The Drosophila norpA gene encodes at least two subtypes of phospholipase C (PLC), one of which is essential for phototransduction and the other is utilized in signalling pathways other than phototransduction. The two subtypes of norpA-PLC differ by 14 amino acids that have been proposed as important for the function of PLC in different signalling pathways. The present study aimed to determine whether norpA subtype II enzyme can functionally substitute for the subtype I enzyme in the phototransduction pathway. We found that the non-retinal norpA-PLC enzyme can substitute for its retinal counterpart, but that there is a reduced rate of repolarization of photoreceptors following intense light stimuli. This reduced repolarization might be due to the inability of a regulatory component being able to interact with the non-retinal norpA-PLC enzyme.

Although chlorhexidine is one of the most efficacious antimicrobial agents used for the prevention of dental caries, side effects limit its application. The effects of gaegurin 6 (GGN6), an animal-derived cationic peptide, and its derivatives PTP6 and PTP12 on the growth of oral streptococci were investigated to assess the potential of these agents for use in the prevention of dental caries. The minimal inhibitory concentrations of the peptides for inhibition of the growth of oral streptococci (Streptococcus mutans, S. sobrinus, S. sanguis and S. gordonii) ranged from 1.2 to 8.2 µM. The peptides also exhibited marked synergistic antibacterial effects with chlorhexidine or xylitol. The most effective combinations (fractional inhibitory concentration index of 0.5) were xylitol with GGN6 against S. gordonii 10558 and chlorhexidine with either GGN6 or PTP6 against S. sobrinus OMZ-175. These results indicate that cationic peptides alone or in combination with chlorhexidine or xylitol might prove effective for the inhibition of the growth of cariogenic oral streptococci in situ.

In this study, partially crystalline anodic TiO2 with SiO2 well-distributed througout the entire oxide film is prepared using plasma electrolytic oxidation (PEO) to obtain a high-capacity anode with an excellent cycling stability for Li-ion batteries. The micropore sizes in the anodic film become inhomogeneous as the SiO2 content is increased from 0% to 25%. The X-ray diffraction peaks show that the formed oxide contains the anatase and rutile phases of TiO2. In addition, X-ray photoelectron spectroscopy and energy-dispersive X-ray analyses confirm that TiO2 contains amorphous SiO2. Anodic oxides of the SiO2/TiO2 composite prepared by PEO in 0.2 m H2SO4 and 0.4 m Na2SiO3 electrolyte deliver the best performance in Li-ion batteries, exhibiting a capacity of 240 µAh cm−2 at a fairly high current density of 500 µA cm–2. The composite film shows the typical Li–TiO2 and Li–SiO2 redox peaks in the cyclic voltammogram and a corresponding plateau in the galvanostatic charge/discharge curves. The as-prepared SiO2/TiO2 composite anode shows at least twice the capacity of other types of binder-free TiO2 and TiO2 composites and very stable cycling stability for more than 250 cycles despite the severe mechanical stress.

Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure-activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an alpha-helical structure in trifluoroethanol (TFE)/H(2)O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H(2)O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic alpha-helix spanning residues 3-18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic alpha-helix that potentially disrupts membrane integrity, which would result in cell death.

Mantle cell lymphoma (MCL) is an aggressive malignancy that is characterized by poor prognosis. Large-scale pharmacological profiling across more than 100 hematological cell line models identified a subset of MCL cell lines that are highly sensitive to the B cell receptor (BCR) signaling inhibitors ibrutinib and sotrastaurin. Sensitive MCL models exhibited chronic activation of the BCR-driven classical nuclear factor-κB (NF-κB) pathway, whereas insensitive cell lines displayed activation of the alternative NF-κB pathway. Transcriptome sequencing revealed genetic lesions in alternative NF-κB pathway signaling components in ibrutinib-insensitive cell lines, and sequencing of 165 samples from patients with MCL identified recurrent mutations in TRAF2 or BIRC3 in 15% of these individuals. Although they are associated with insensitivity to ibrutinib, lesions in the alternative NF-κB pathway conferred dependence on the protein kinase NIK (also called mitogen-activated protein 3 kinase 14 or MAP3K14) both in vitro and in vivo. Thus, NIK is a new therapeutic target for MCL treatment, particularly for lymphomas that are refractory to BCR pathway inhibitors. Our findings reveal a pattern of mutually exclusive activation of the BCR-NF-κB or NIK-NF-κB pathways in MCL and provide critical insights into patient stratification strategies for NF-κB pathway-targeted agents.

PTP7 is a 13-amino acid residue peptide designed from gaegurin 6, an antimicrobial peptide isolated from skin secretions of Rana rugosa. In order to examine the effect of hydrophobicity on antimicrobial activity, a series of PTP7 derivatives were constructed and analyzed the activity against bacteria and artificial membrane. We found that the mean hydrophobicity by simple summation of hydrophobicity of each constituent amino acid did not necessarily describe the hydrophobic property of antimicrobial peptides. The mean hydrophobicity did not show close correlation with the observed hydrophobicity by measuring reverse phase high performance liquid chromatography (RP HPLC) retention time. The observed hydrophobicity represented as RP HPLC retention time correlated well with the activity against artificial membrane and Gram positive bacterial species, such as Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus, rather than mean hydrophobicity. However, antimicrobial activity against Gram negative bacteria, such as Escherichia coli, did not show correlation with RP HPLC retention time. These data indicate that the RP HPLC retention time should be exploited rather than the mean hydrophobicity in the analysis of the relationship between hydrophobicity and antimicrobial activity.

In order to develop peptide agents with reduced length and enhanced tumoricidal activity, we have designed gaegurin 6 (GGN6) derivatives through deletions and/or substitutions of amino acids. The deletion of hydrophobic amino terminal region completely abolished antitumor activity whereas the deletion of carboxy terminal region had little influence on antitumor activity. Antitumor activity of the PTP peptides did not correlate with antibacterial activity. PTP7, the most potent derivative, was found to have comparable antitumor activity to GGN6 in spite of reduced number of amino acids which is about half the size of gaegurin 6; furthermore, it showed little cytotoxicity on PBMCs and RBCs. GGN6 and PTP7 also showed equivalent cytotoxicity against drug sensitive (MCF-7) and multidrug-resistant cell lines (MCF-7/DOX). Plasma membrane blebbing and DNA fragmentation of peptide-treated tumor cells indicated that the peptides could induce apoptosis in tumor cells. These results suggest that GGN6 and its derivatives can be developed as new anticancer agents and may provide a new strategy for overcoming MDR which is a major problem in cancer therapy.

The tumor suppressor Retinoblastoma protein (Rb) is often inactivated in cancer. In many tumors, the Rb protein itself is retained but functionally inactivated by increased CDK4/6 kinase activity. A number of key oncogenic aberrations can result in this increased activity, including inactivation of CDKN2A (p16), translocation or amplification of D-cyclins, amplification of CDK4/6 and mutations/deletions upstream of cyclin D, such as activating mutations of BRAF/PIK3CA and PTEN deletion. Abolishing CDK4/6 kinase activity and subsequent reactivation of Rb in these tumors has been demonstrated to inhibit tumor initiation and growth. Here we will describe LEE011- an orally bioavailable, selective small molecule inhibitor of CDK4/6 kinases. LEE011 inhibits CDK4/6 kinase activity with nM IC50 and is highly selective for these targets. In a number of preclinical tumor models, LEE011 demonstrates a dose dependent anti-tumor activity that tracks well with CDK4/6 inhibition. The primary mechanism for growth inhibitory effect appears to be G1 arrest in vitro, although, in some sensitive in vivo models, regressions are observed. Importantly, given the role of CDK4/6 downstream of other oncogenic driver mutations, LEE011 shows single agent activity in melanomas with activating mutations of BRAF or NRAS, and in breast cancers with intact estrogen receptor and/or activating aberrations of PIK3CA/Her-2. Combining LEE011 with LGX818, a V600E BRAF specific inhibitor, leads to robust anti-tumor activity in melanoma models that are both sensitive and, importantly, those that are resistant to LGX818. Furthermore, combining LEE011 with BYL719, a PIK3CA specific inhibitor, also leads to significant anti-tumor activity in breast cancer models both sensitive and resistant to BYL719. Several clinical studies evaluating LEE011 as single agent and in combinations are underway. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR02. Citation Format: Sunkyu Kim, Alice Loo, Rajiv Chopra, Giordano Caponigro, Alan Huang, Sadhna Vora, Sudha Parasuraman, Steve Howard, Nicholas Keen, William Sellers, Christopher Brain. LEE011: An orally bioavailable, selective small molecule inhibitor of CDK4/6– Reactivating Rb in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR02.

Background: Neuroblastoma is a pediatric embryonal cancer for which the survival of patients with high-risk disease is less than 50% and has not dramatically changed over the last several years. Recently, a number of cell cycle genes_particularly those within the Cyclin D/CDK4/CDK6/RB network_have been identified as oncogenic vulnerabilities in neuroblastoma, suggesting that their therapeutic exploitation might improve survivability. Indeed, genomic amplifications of CDK4, CDK6, and CCND1 have been reported in primary neuroblastomas, and we have previously shown via an unbiased loss of function screen that CDK4 depletion is associated with potent anti-tumor activity (Cole, PNAS 2011). Here, we sought to translate these findings into novel therapies for children with neuroblastoma by evaluating the effect of pharmacologic Cdk4/Cdk6 inhibition on neuroblastoma viability. Methods: We analyzed the effect of combined Cdk4/6 inhibition in a comprehensive panel of human-derived neuroblastoma cell lines using LEE011, a highly specific Cdk4/6 small molecule inhibitor. Anti-tumor activity was also determined in vivo in three neuroblastoma xenograft models, and integrative genomics was used to identify biomarkers of drug sensitivity. Results: Treatment with LEE011 significantly inhibited proliferation in 10 of 15 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 361 ± 97 nM, considering sensitive lines only), as evidenced by significant cell cycle arrest and senescence that were likely attributed to dose-dependent decreases in phosphorylated RB and FOXM1. In addition, responsiveness of neuroblastoma xenografts to LEE011 was reflective of in vitro data in that there was a direct correlation of IC50 values with degree of subcutaneous xenograft growth delay, with the most sensitive lines in vitro showing profound growth inhibition in vivo. While our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (p= 0.04, student's t test), a supervised hierarchical clustering of gene expression data identified several potential gene signatures that could explain the observed differential sensitivity to Cdk4/6 inhibition. Conclusions: Our data show that LEE011 is highly active in a large subset of neuroblastoma cell lines and xenograft models, and therefore support the clinical development of LEE011 as a therapy for neuroblastoma as well as efforts to validate biomarkers of drug activity. Citation Format: JulieAnn Rader, Lori Hart, Mike Russell, Michael Nakazawa, Lili Belcastro, Daniel Martinez, Erica Carpenter, Sunkyu Kim, Sudha Parasuraman, Giordano Caponigro, Robert Schnepp, Andrew Wood, Bruce Pawel, Deborah Watson, Patrick Warren, Kristina Cole, John Maris. CDK4/CDK6 inhibition is potently active in a definable subset of human neuroblastomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2744. doi:10.1158/1538-7445.AM2013-2744

Background: Well-differentiated and de-differentiated liposarcomas (WD/DDLPS) are characterized by high-level chromosomal amplification of 12q13–15, a region including the cyclin-dependent kinase 4 (CDK4) oncogene. In this study, we explored the effects of LEE011, a novel selective inhibitor of CDK4/CDK6 on human liposarcoma cell lines and primary tumor xenografts. Materials and Methods: We examined the expression of cell cycle regulatory proteins in human liposarcoma cell lines and human normal preadipocytes/adipocytes. CDK4 small interfering RNA (siRNA) or LEE011 was applied to liposarcoma cell lines and the biological consequences were determined by phospho-RB immunoblot, cell cycle assay and cell enumeration. In addition, liposarcoma cells were transfected with RB siRNA to determine the specificity of the effects of LEE011. Nude mice implanted with human liposarcoma cell lines or primary tumors were treated with LEE011 or vehicle by oral gavage. After 3 daily doses, in vivo BrdU incorporation assay, immunostains for phospho-RB, and 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET) scan were performed. Tumor size and animal weight were serially measured during 3 weeks of dosing. Results: CDK4 and p-Rb are highly expressed in human liposarcoma cell lines in comparison with normal preadipocytes/adipocytes. Both CDK4 siRNA and LEE011 inhibited RB phosphorylation at the CDK4/6-specific sites Ser780 and Ser807/811 in a dose-dependent manner and dramatically inhibited liposarcoma cell growth. Cell cycle analysis demonstrated arrest at G0/G1. siRNA-mediated knockdown of RB1 rescued the inhibitory effects of LEE011, suggesting that LEE011 inhibited proliferation as expected through the RB pathway. Oral administration of LEE011 to mice bearing human liposarcoma xenografts reduced tumor 18F-FDG uptakes by approximately 50% and dramatically decreased biomarkers including RB phosphorylation and BrdU incorporation in vivo. Continued LEE011 treatment inhibited growth of established cell line xenografts as well as primary human liposarcoma tumor xenografts without detrimental effects on mouse weight. Conclusions: LEE011 decreases cell cycle progression, hypermetabolism, and proliferation of human liposarcomas in an RB-dependent manner, consistent with inhibition of CDK4. Clinical trials of LEE011 in patients with liposarcoma are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A236.