Discovery of a highly potent, selective, orally bioavailable inhibitor of KAT6A/B histone acetyltransferases with efficacy against KAT6A-high ER+ breast cancer.

KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.

Proton exchange rate of chemical exchange saturation transfer MRI constructed from direct saturation-removed omega plots to improve the assessment of patients with ischemic stroke.

Background: Proton exchange rate (k ex) magnetic resonance imaging (MRI) has recently been developed, with preliminary results demonstrating its potential for evaluating reactive oxygen species. This prospective cohort study investigated the k ex in different stroke stages and its correlation with stroke severity and prognosis. Methods: In all, 96 ischemic stroke patients were included in the study. Patients were divided into 3 groups based on stroke phase (acute, subacute, and chronic). A spin echo-echo planar imaging sequence with presaturation powers of 1.5, 2.5, and 3.5 µT was implemented to obtain Z-spectra, and k ex maps were constructed from direct saturation-removed omega plots. Relative k ex (rk ex) and the relative apparent diffusion coefficient (rADC) were calculated as the ratio of k ex or ADC in the infarcts to values in contralateral tissue, respectively. Correlations between both k ex and rk ex and National Institute of Health Stroke Scale (NIHSS) scores were evaluated. Receiver operating characteristic (ROC) analysis was used to evaluate the performance of k ex, rk ex, rADC, and lesion volume for predicting acute stroke outcome. Results: The k ex was significantly higher in ischemic lesions than in contralateral tissue at all stages. In addition, the k ex of acute lesions was higher than that of subacute and chronic lesions [mean (± SD) 935.1±81.5 vs. 881.4±55.7 and 866.9±76.7 s-1, respectively; P<0.05 and P<0.01, respectively]. The difference in k ex between subacute and chronic lesions was not significant. In acute stroke, there was a limited correlation between a lesion's k ex and NIHSS score (R2=0.16; P=0.01) and between rk ex and NIHSS score (R2=0.28; P=0.001). Acute stroke patients with poor prognosis had significantly higher lesion k ex and rk ex than did those with good prognosis (k ex: 991.1±78.2 vs. 893.1±55.1 s-1, P<0.001; rk ex: 1.28±0.09 vs. 1.15±0.06, P<0.001). In ROC analyses, k ex and rk ex showed favorable predictive performance for acute stroke outcome, with areas under the curve (AUC) of 0.837 and 0.880, respectively, which were slightly but not significantly higher than the AUCs for lesion volume (0.730) and rADC (0.673). Conclusions: This study indicates that k ex MRI is promising for the diagnosis and management of ischemic stroke because it can reflect the oxidative stress of lesions and predict prognosis.

Bone metastasis of hepatocellular carcinoma: facts and hopes from clinical and translational perspectives.

Patients with hepatocellular carcinoma (HCC) and bone metastasis (BM) suffer from greatly reduced life quality and a dismal prognosis. However, BM in HCC has long been overlooked possibly due to its relatively low prevalence in previous decades. To date, no consensus or guidelines have been reached or formulated for the prevention and management of HCC BM. Our narrative review manifests the increasing incidence of HCC BM to sound the alarm for additional attention. The risk factors, diagnosis, prognosis, and therapeutic approaches of HCC BM are detailed to provide a panoramic view of this disease to clinicians and specialists. We further delineate an informative cancer bone metastatic cascade based on evidence from recent studies and point out the main factors responsible for the tumor-associated disruption of bone homeostasis and the formation of skeletal cancer lesions. We also present the advances in the pathological and molecular mechanisms of HCC BM to shed light on translational opportunities. Dilemmas and challenges in the treatment and investigation of HCC BM are outlined and discussed to encourage further endeavors in the exploration of underlying pathogenic and molecular mechanisms, as well as the development of novel effective therapies for HCC patients with BM.

Structure-Based Drug Design and Synthesis of PI3Kα-Selective Inhibitor (PF-06843195).

The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway is a frequently dysregulated pathway in human cancer, and PI3Kα is one of the most frequently mutated kinases in human cancer. A PI3Kα-selective inhibitor may provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family. Here, we describe our efforts to discover a PI3Kα-selective inhibitor by applying structure-based drug design (SBDD) and computational analysis. A novel series of compounds, exemplified by 2,2-difluoroethyl (3S)-3-{[2'-amino-5-fluoro-2-(morpholin-4-yl)-4,5'-bipyrimidin-6-yl]amino}-3-(hydroxymethyl)pyrrolidine-1-carboxylate (1) (PF-06843195), with high PI3Kα potency and unique PI3K isoform and mTOR selectivity were discovered. We describe here the details of the design and synthesis program that lead to the discovery of 1.

Joint construction of micro-vibration stimulation and BCP scaffolds for enhanced bioactivity and self-adaptability tissue engineered bone grafts.

The bone defects caused by trauma and disease have become a major difficulty in the treatment of clinical bone defects, and bone tissue engineering has become a promising treatment strategy. It was found that mechanical stimulation regulated the development of bone constructs by affecting the distribution and differentiation of cells on them. In this study, tissue-engineered bone grafts with enhanced bioactivity and self-adaptability were constructed by BMSCs and biphasic calcium phosphate (BCP) scaffolds under periodic micro-vibration stimulation (MVS) with a frequency of 40 Hz and a magnitude of 0.3 g. The results of the material characterization indicated that the BCP scaffolds created a more favourable osteogenic micro-environment with promoted calcium ion release, protein adsorption and mineralization deposition under the micro-vibration stimulation. The in vitro results showed that the apoptosis of BMSCs increased significantly on day 1, but from day 3 on, the proliferation increased and apoptosis decreased. Cells were evenly distributed on the scaffolds, exhibiting tight adhesion in a flat-shape and distinct matrix mineralization. F-actin and ALP expression significantly increased and meanwhile osteogenesis-related genes including Runx2, Col-I, ALP, and OCN were significantly up-regulated. Western blotting results suggested that the ERK1/2 and Wnt/ß-catenin signalling pathways were involved in the osteogenic behaviour of BMSCs induced by MVS. In vivo experiments showed that grafts had stronger osteoinduction and mechanical adaptability. Taken together, this study suggested that micro-vibration stimulation combined with BCP scaffolds with good osteoinduction could be a promising approach for constructing tissue engineered bone grafts with enhanced bioactivity, mechanical adaptability, and bone regeneration repair capability.

Grading and proliferation assessment of diffuse astrocytic tumors with monoexponential, biexponential, and stretched-exponential diffusion-weighted imaging and diffusion kurtosis imaging.

PURPOSE: To compare the main parameters derived from monoexponential, biexponential and stretched-exponential diffusion-weighted imaging (DWI) and diffusion kurtosis imaging (DKI) with respect to diagnostic performance for tumor grading and proliferation assessment in diffuse astrocytic tumors (DATs). MATERIALS AND METHODS: Fifty-eight pathologically confirmed DAT patients who underwent DWI and DKI on a 3-T scanner were prospectively collected and retrospectively reviewed. Measurements including the apparent diffusion coefficient (ADC), true diffusion coefficient (D), pseudodiffusion coefficient (D*), perfusion fraction (f), distributed diffusion coefficient (DDC), heterogeneity index (α), mean diffusivity (MD), fractional anisotropy (FA), and mean kurtosis (MK) were compared between tumor grades (Ⅱ, Ⅲ, and Ⅳ) by using a Jonckheere-Terpstra test. Receiver operating characteristic (ROC) curves were used to assess the diagnostic efficacy of these parameters. Spearman's rho with the Ki-67 labeling index (LI) was calculated for each parameter. RESULTS: MK values differed significantly between all DAT subtypes and increased with grade. The ADC, D, f, DDC, α and MD values were significantly higher in grade Ⅱ tumors than in grade Ⅲ/Ⅳ tumors. D* values were significantly lower in grade Ⅱ tumors than in grade Ⅳ tumors (all P < 0.05). In discriminating between grade Ⅱ and Ⅲ tumors, α, MK, MD, D and f had significantly greater area under the ROC curve (AUC) values than D* and FA (0.927, 0.901, 0.896, 0.895, and 0.889, respectively vs 0.659 and 0.598, respectively, P < 0.05). In discriminating between grade Ⅲ and Ⅳ tumors, only MK demonstrated acceptable discrimination (AUC = 0.711). MK and D showed a strong correlation with the Ki-67 LI (ρ = 0.791 and -0.789, respectively, P < 0.001). D*, f, MD, ADC, DDC and α showed a moderate correlation (|ρ| ranged from 0.415 to 0.698, P < 0.05). CONCLUSION: MK and D have considerable potential to predict the degree of proliferation of DATs. MK could effectively characterize microstructural changes throughout the malignant transformation of DATs and provided useful complementary information for grading.

Anisotropy of anomalous diffusion improves the accuracy of differentiating low- and high-grade cerebral gliomas.

BACKGROUND: Anomalous diffusion model has been introduced and shown to be beneficial in clinical applications. However, only the directionally averaged values of anomalous diffusion parameters were investigated, and the anisotropy of anomalous diffusion remains unexplored. The aim of this study was to demonstrate the feasibility of using anisotropy of anomalous diffusion for differentiating low- and high-grade cerebral gliomas. METHODS: Diffusion MRI images were acquired from brain tumor patients and analyzed using the fractional motion (FM) model. Twenty-two patients with histopathologically confirmed gliomas were selected. An anisotropy metric for the FM-related parameters, including the Noah exponent (α) and the Hurst exponent (H), was introduced and their values were statistically compared between the low- and high-grade gliomas. Additionally, multivariate logistic regression analysis was performed to assess the combination of the anisotropy metric and the directionally averaged value for each parameter. The diagnostic performances for grading gliomas were evaluated using a receiver operating characteristic (ROC) analysis. RESULTS: The Hurst exponent H was more anisotropic in high-grade than in low-grade gliomas (P = 0.015), while no significant difference was observed for the anisotropy of α. The ROC analysis revealed that larger areas under the ROC curves were produced for the combination of α (1) and the combination of H (0.813) compared with the directionally averaged α (0.979) and H (0.594), indicating an improved performance for tumor differentiation. CONCLUSION: The anisotropy of anomalous diffusion can provide distinctive information and benefit the differentiation of low- and high-grade gliomas. The utility of anisotropic anomalous diffusion may have an improved effect for investigating pathological changes in tissues.

Xylanilyticolides A-C, Three New Compounds from Cultures of the Actinomycete Promicromonospora xylanilytica YIM 61515.

Three new lactones, xylanilyticolides A-C (1-3), were isolated from cultures of the actinomycete Promicromonospora xylanilytica YIM 61515. Their structures were elucidated by 1D and 2D NMR spectroscopic data in conjunction with HRESIMS analysis. Compound 1 exhibited potent cytotoxicities against five human cancer cell lines HL-60, A-549, SMMC-7721, MCF-7 and SW480 with the IC50 values of 3.9, 15.2, 11.2, 5.9, and 4.7 µM, respectively.

Anemhupehins A-C, Podocarpane Diterpenoids from Anemone hupehensis.

Three new podocarpane diterpenoids, namely anemhupehins A-C (1-3), together with four known analogues (4-7), have been isolated from aerial parts of Anemone hupehensis. Their structures were characterized based on extensive spectroscopic data. Compounds 1 and 4 showed certain cytotoxicities against human cancer cell lines.

Anomalous diffusion in cerebral glioma assessed using a fractional motion model.

PURPOSE: To demonstrate the capability of the fractional motion (FM) model for describing anomalous diffusion in cerebral gliomas and to assess the potential feasibility of FM for grading these tumors. METHODS: Diffusion MRI images were acquired from brain tumor patients using a special Stejskal-Tanner diffusion sequence with variable diffusion gradient amplitudes and separation times. Patients with histopathologically confirmed gliomas, including astrocytic and oligoastrocytic tumors, were selected. The FM-related parameters, including the Noah exponent ( α), the Hurst exponent ( H), and the memory parameter ( µ=H-1/α), were calculated and compared between low- and high-grade gliomas using a two-sample t-test. The grading performance was evaluated using the receiver operating characteristic analysis. RESULTS: Twenty-two patients were included in the present study. The calculated α, H, and µ permitted the separation of tumor lesions from surrounding normal tissues in parameter maps and helped differentiate glioma grades. Moreover, α showed greater sensitivity and specificity in distinguishing low- and high-grade gliomas compared with the apparent diffusion coefficient. CONCLUSION: The FM model could improve the diagnostic accuracy in differentiating low- and high-grade gliomas. This improved diffusion model may facilitate future studies of neuro-pathological changes in clinical populations. Magn Reson Med 78:1944-1949, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

Pulmonary sequestration: a case report and literature review.

PURPOSE: Pulmonary sequestration (PS) is a rare congenital lung malformation. It is characterized by an abnormal mass of dysplastic lung tissue supplied by an anomalous systemic artery and separated from normal bronchopulmonary tree. Misdiagnosis and inadequate treatment can lead to recurrent pneumonia and fatal hemoptysis. METHODS: We report a 45 years female was diagnosed PS, and performed a brief review about the clinical features, diagnostic strategies, and management options of the PS. RESULTS: Her remarkable symptoms were cough and hemoptysis, the contrast- enhanced computed tomography of the chest revealed a multiloculated cystic solid mass filled with low density lesions and a feeding artery from the descending abdominal aorta to the cystic solid mass was visualized, then the patient suffered a right lower- lobe resection, and the surgery and pathological examination all supported the diagnosis of intralobar sequestration. CONCLUSIONS: Symptomatic patients of the pulmonary sequestration should be treated by surgery to avoid the risk of death due to massive hemoptysis.

HIP1-ALK, a novel ALK fusion variant that responds to crizotinib.

INTRODUCTION: The aim of this study was to identify anaplastic lymphoma kinase (ALK) rearrangements in lung cancer patient-derived xenograft (PDX) models and to explore their responses to crizotinib. METHODS: Screening of 99 lung cancer PDX models by the NanoString ALK fusion assay identified two ALK-rearranged non-small-cell lung cancer (NSCLC) tumors, including one harboring a previously known echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion and another containing an unknown ALK fusion variant. Expression array, RNA-Seq, reverse transcription polymerase chain reaction, and direct sequencing were then conducted to confirm the rearrangements and to identify the novel fusion partner in the xenograft and/or the primary patient tumor. Finally, pharmacological studies were performed in PDX models to evaluate their responses to ALK inhibitor crizotinib. RESULTS: Two ALK-rearranged NSCLC PDX models were identified: one carried a well-known EML4-ALK variant 3a/b and the other harbored a novel huntingtin interacting protein 1 (HIP1)-ALK fusion gene. Exon 28 of the HIP1 gene located on chromosome 7 was fused to exon 20 of the ALK gene located on chromosome 2. Both cases were clinically diagnosed as squamous cell carcinoma. Compared with the other lung cancer PDX models, both ALK-rearranged models displayed elevated ALK mRNA expression. Furthermore, in vivo efficacy studies demonstrated that, similar to the EML4-ALK-positive model, the HIP1-ALK-containing PDX model was sensitive to treatment with crizotinib. CONCLUSIONS: Discovery of HIP1 as a fusion partner of ALK in NSCLC is a novel finding. In addition, the HIP1-ALK-rearranged tumor is sensitive to treatment with crizotinib in vivo, implicating HIP1-ALKas an oncogenic driver of lung tumorigenesis. Collectively, our results indicate that HIP1-ALK-positive NSCLC may benefit from clinical applications of crizotinib.

SAR and evaluation of novel 5H-benzo[c][1,8]naphthyridin-6-one analogs as Aurora kinase inhibitors.

Several potent Aurora kinase inhibitors derived from 5H-benzo[c][1,8]naphthyridin-6-one scaffold were identified. A crystal structure of Aurora kinase A in complex with an initial hit revealed a binding mode of the inhibitor within the ATP binding site and provided insight for structure-guided compound optimization. Subsequent SAR campaign provided a potent and selective pan Aurora inhibitor, which demonstrated potent target modulation and antiproliferative effects in the pancreatic cell line, MIAPaCa-2. Furthermore, this compound inhibited phosphorylation of histone H3 (pHH3) in mouse bone morrow upon oral administration, which is consistent with inhibition of Aurora kinase B activity.

BRAF activation initiates but does not maintain invasive prostate adenocarcinoma.

Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAF(V600E)--a mutation found in approximately 10% of human prostate tumors--was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAF(V600E) in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAF(V600E) is not required for its maintenance.

Mutations in BRAF and KRAS converge on activation of the mitogen-activated protein kinase pathway in lung cancer mouse models.

Mutations in the BRAF and KRAS genes occur in approximately 1% to 2% and 20% to 30% of non-small-cell lung cancer patients, respectively, suggesting that the mitogen-activated protein kinase (MAPK) pathway is preferentially activated in lung cancers. Here, we show that lung-specific expression of the BRAF V600E mutant induces the activation of extracellular signal-regulated kinase (ERK)-1/2 (MAPK) pathway and the development of lung adenocarcinoma with bronchioloalveolar carcinoma features in vivo. Deinduction of transgene expression led to dramatic tumor regression, paralleled by dramatic dephosphorylation of ERK1/2, implying a dependency of BRAF-mutant lung tumors on the MAPK pathway. Accordingly, in vivo pharmacologic inhibition of MAPK/ERK kinase (MEK; MAPKK) using a specific MEK inhibitor, CI-1040, induced tumor regression associated with inhibition of cell proliferation and induction of apoptosis in these de novo lung tumors. CI-1040 treatment also led to dramatic tumor shrinkage in murine lung tumors driven by a mutant KRas allele. Thus, somatic mutations in different signaling intermediates of the same pathway induce exquisite dependency on a shared downstream effector. These results unveil a potential common vulnerability of BRAF and KRas mutant lung tumors that potentially affects rational deployment of MEK targeted therapies to non-small-cell lung cancer patients.

Characterization of melanocyte-specific inducible Cre recombinase transgenic mice.

Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.

Replacement of amino acid sequence features of a- and c-subunits of ATP synthases of Alkaliphilic Bacillus with the Bacillus consensus sequence results in defective oxidative phosphorylation and non-fermentative growth at pH 10.5.

Mitchell's (Mitchell, P. (1961) Nature 191, 144-148) chemiosmotic model of energy coupling posits a bulk electrochemical proton gradient (Deltap) as the sole driving force for proton-coupled ATP synthesis via oxidative phosphorylation (OXPHOS) and for other bioenergetic work. Two properties of proton-coupled OXPHOS by alkaliphilic Bacillus species pose a challenge to this tenet: robust ATP synthesis at pH 10.5 that does not correlate with the magnitude of the Deltap and the failure of artificially imposed potentials to substitute for respiration-generated potentials in energizing ATP synthesis at high pH (Krulwich, T. (1995) Mol. Microbiol. 15, 403-410). Here we show that these properties, in alkaliphilic Bacillus pseudofirmus OF4, depend upon alkaliphile-specific features in the proton pathway through the a- and c-subunits of ATP synthase. Site-directed changes were made in six such features to the corresponding sequence in Bacillus megaterium, which reflects the consensus sequence for non-alkaliphilic Bacillus. Five of the six single mutants assembled an active ATPase/ATP synthase, and four of these mutants exhibited a specific defect in non-fermentative growth at high pH. Most of these mutants lost the ability to generate the high phosphorylation potentials at low bulk Deltap that are characteristic of alkaliphiles. The aLys(180) and aGly(212) residues that are predicted to be in the proton uptake pathway of the a-subunit were specifically implicated in pH-dependent restriction of proton flux through the ATP synthase to and from the bulk phase. The evidence included greatly enhanced ATP synthesis in response to an artificially imposed potential at high pH. The findings demonstrate that the ATP synthase of extreme alkaliphiles has special features that are required for non-fermentative growth and OXPHOS at high pH.

A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg2+ uptake.

The atp operon of alkaliphilic Bacillus pseudofirmus OF4, as in most prokaryotes, contains the eight structural genes for the F-ATPase (ATP synthase), which are preceded by an atpI gene that encodes a membrane protein of unknown function. A tenth gene, atpZ, has been found in this operon, which is upstream of and overlapping with atpI. Most Bacillus species, and some other bacteria, possess atpZ homologues. AtpZ is predicted to be a membrane protein with a hairpin topology, and was detected by Western analyses. Deletion of atpZ, atpI, or atpZI from B. pseudofirmus OF4 led to a requirement for a greatly increased concentration of Mg2+ for growth at pH 7.5. Either atpZ, atpI, or atpZI complemented the similar phenotype of a triple mutant of Salmonella typhimurium (MM281), which is deficient in Mg2+ uptake. atpZ and atpI, separately and together, increased the Mg2+-sensitive 45Ca2+ uptake by vesicles of an Escherichia coli mutant that is defective in Ca2+ and Na+ efflux. We hypothesize that AtpZ and AtpI, as homooligomers, and perhaps as heterooligomers, are Mg2+ transporter, Ca2+ transporter, or channel proteins. Such proteins could provide Mg2+, which is required by ATP synthase, and also support charge compensation, when the enzyme is functioning in the hydrolytic direction; e.g., during cytoplasmic pH regulation.