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Lipopolysaccharide (LPS) destroys intestinal mechanical barrier and causes apoptosis by triggering oxidative stress and inflammatory responses. Glutamine (Gln) can maintain normal intestinal function under various stressed or pathological conditions. Thereby, this study aims to evaluate the protection of glutamine on intestinal health of snakehead (Channa argus), specifically regarding the NF-κB/MLCK/MLC2 signaling pathway mediated by tight junction affecting oxidative stress, inflammation and apoptosis. In this work, a model of intestinal tight junction injury in intestine of snakehead was constructed by injecting 4 mg/mL LPS into anus for 96 h. Before constructing the model, fish were treated with different levels of alanyl-glutamine (Ala-Gln) (0 %, 0.3 %, 0.6 %, 0.9 %, 1.2 % and 1.5 %) for 56 days. Microstructure and ultra microstructure showed that LPS-induced obvious intestinal damage and tight connection destruction, while Gln effectively alleviated these phenomena. In addition, results also showed that Gln can effectively inhibit LPS-induced damage to intestinal tight junction (zo-1, occludin, claudin5, claudin1, nf-κb p65, mlck and mlc2), alleviate oxidative stress (nrf2, sod, gsh, gpx and cat), ameliorate intestinal inflammation (tnf-α, il-1β, il-8, tlr5 and tlr2), thereby reduce apoptosis (p38mapk, caspase9, caspase8, caspase3 and bax). Crucially, the above results were related to NF-κB/MLCK/MLC2 signaling pathway mediated by tight junction. In conclusion, Gln has a good protective effect on LPS-induced intestinal injury in northern snakehead, providing a new perspective for regulating fish intestinal health., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Targeting of the estrogen receptor (ER) by anti-estrogens is the standard-of-care for patients with ER+ HER2- advanced/metastatic breast cancer. While anti-estrogens that degrade ERα (fulvestrant) or block estrogen production (aromatase inhibitors) have improved patient outcomes, clinically important challenges remain related to drug administration, limited bioavailability, lack of brain exposure, and acquired resistance due to ESR1 mutations. These limitations indicate a need for more robust ER-targeted therapies. Here, we discovered and characterized imlunestrant, a next-generation potent, brain-penetrant oral selective estrogen receptor degrader (SERD). Imlunestrant degraded ERα and decreased ERα-mediated gene expression both in vitro and in vivo. Cell proliferation and tumor growth in ESR1 wildtype and mutant models were significantly inhibited by imlunestrant. Combining imlunestrant with abemaciclib (CDK4/6 inhibitor), alpelisib (PI3K inhibitor), or everolimus (mTOR inhibitor) further enhanced tumor growth inhibition, regardless of ESR1 mutational status. In an ER+ breast cancer intracranial tumor model, imlunestrant prolonged survival compared to vehicle or alternative SERD therapies. Together, these finding support the potential of imlunestrant to degrade ERα and suppress the growth of ESR1 wildtype and mutant breast cancer, including brain metastatic tumors.

The cortical bone trajectory (CBT) is a novel technique in lumbar fixation and fusion. The unique caudocephalad and medial-lateral screw trajectories endow it with excellent screw purchase for vertebral fixation via a minimally invasive method. The combined use of CBT screws with transforaminal or posterior lumbar interbody fusion can treat a variety of lumbar diseases, including spondylolisthesis or stenosis, and can also be used as a remedy for revision surgery when the pedicle screw fails. CBT has obvious advantages in terms of surgical trauma, postoperative recovery, prevention and treatment of adjacent vertebral disease, and the surgical treatment of obese and osteoporosis patients. However, the concept of CBT internal fixation technology appeared relatively recently; consequently, there are few relevant clinical studies, and the long-term clinical efficacy and related complications have not been reported. Therefore, large sample and prospective studies are needed to further reveal the long-term complications and fusion rate. As a supplement to the traditional pedicle trajectory fixation technique, the CBT technique is a good choice for the treatment of lumbar diseases with accurate screw placement and strict indications and is thus deserving of clinical recommendation., Competing Interests: Conflict-of-interest statement: The authors declare that no competing interest exists., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

In nature, plants are often confronted with wide variations in light intensity, which may cause a massive carbon loss and water waste. Here, we investigated the response of photosynthetic rate and stomatal conductance to fluctuating light among ten rice genotypes and their influence on plant acclimation and intrinsic water-use efficiency (WUE i ). Significant differences were observed in photosynthetic induction and stomatal kinetics across rice genotypes. However, no significant correlation was observed between steady-state and non-steady-state gas exchange. Genotypes with a greater range of steady-state and faster response rate of the gas exchange showed stronger adaptability to fluctuating light. Higher stomatal conductance during the initial phase of induction had little effect on the photosynthetic rate but markedly decreased the plant WUE i . Clarification of the mechanism influencing the dynamic gas exchange and synchronization between photosynthesis and stomatal conductance under fluctuating light may contribute to the improvement of photosynthesis and water-use efficiency in the future., Competing Interests: The authors declare that they have no conflict of interest., (Copyright: © 2022 Xiong et al.)

KRAS mutant lung adenocarcinomas remain intractable for targeted therapies. Genetic interrogation of KRAS downstream effectors, including the MAPK pathway and the interphase CDKs, identified CDK4 and RAF1 as the only targets whose genetic inactivation induces therapeutic responses without causing unacceptable toxicities. Concomitant CDK4 inactivation and RAF1 ablation prevented tumor progression and induced complete regression in 25% of KRAS/p53-driven advanced lung tumors, yet a significant percentage of those tumors that underwent partial regression retained a population of CDK4/RAF1-resistant cells. Characterization of these cells revealed two independent resistance mechanisms implicating hypermethylation of several tumor suppressors and increased PI3K activity. Importantly, these CDK4/RAF1-resistant cells can be pharmacologically controlled. These studies open the door to new therapeutic strategies to treat KRAS mutant lung cancer, including resistant tumors., Competing Interests: Competing interest statement: S.-B.P. is an employee at the Oncology Research at Eli Lilly and Company. M.B. reports a research contract with Oncology Research, Eli Lilly and Company.

Colorectal cancer (CRC) is prevalent with high mortality, with liver metastasis contributing as a major factor that worsens the survival of patients. The roles of miRNAs in CRC have been elucidated, subsequent to recent studies that suggest the involvement of miRNAs in cancer biology. In this study, we compare the miRNA and gene expression profiles of primary tumors between two groups of patients (with and without liver metastasis) to identify the metastasis-initiating microRNA-target gene regulations. Analysis from 33 patients with metastasis and 14 patients without metastasis revealed that 17 miRNAs and their 198 predicted target genes are differentially expressed, where the target genes showed association with cancer progression and metastasis with statistical significance. In order to evaluate the clinical implications of the findings, we classified CRC patients of independent data into two groups based on the identified miRNA-target regulations, where one group was closer to primary tumors with metastasis than the other group. The comparison of survival showed statistically significant difference, thereby implying the roles of the identified miRNA-target regulations in cancer progression and metastasis. The identification of metastasis-initiating miRNA-target regulations in this study will lead to better understanding of the roles of miRNAs in CRC progression.

Metastasis is the primary cause of cancer mortality. The primary tumors of colorectal cancer (CRC) often metastasize to the liver. In this study, we have collected 122 samples from 45 CRC patients. Among them, 32 patients have primary tumors, adjacent normal tissues, and matched liver metastases. Thirteen patients have primary tumors without distant metastasis and matched normal tissues. Characterization of these samples was conducted by whole-exome and RNA sequencing and SNP6.0 analysis. Our results revealed no significant difference in genetic alterations including common oncogenic mutations, whole genome mutations and copy number variations between primary and metastatic tumors. We then assembled gene co-expression networks and identified metastasis-correlated gene networks of immune-suppression, epithelial-mesenchymal transition (EMT) and angiogenesis as the key events and potentially synergistic drivers associated with CRC metastasis. Further independent cohort validation using published datasets has verified that these specific gene networks are up regulated throughout the tumor progression. The gene networks of EMT, angiogenesis, immune-suppression and T cell exhaustion are closely correlated with the poor patient outcome and intrinsic anti-PD-1 resistance. These results offer insights of combinational strategy for the treatment of metastatic CRC.

We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC., Competing Interests: Declaration of Interests C.J.D. is a consultant/advisory board member for Mirati Therapeutics and Deciphera Pharmaceuticals. C.J.D. has received research funding support from Mirati Therapeutics and Deciphera Pharmaceuticals and has consulted for Ribometrix, Jazz Therapeutics, SVB Leerink, Axon Advisors LLC, Third Bridge, SmartAnalyst, Turning Point Therapeutics, and Eli Lilly. A.D.C. has consulted for Eli Lilly and Mirati Therapeutics. E.F.P. and M.P. receive royalties from Avant Diagnostics. E.F.P. is a consultant to and shareholder of Avant Diagnostics, Inc. and Perthera, Inc. and received funding support from Mirati Therapeutics, Genentech, Inc., and Abbvie, Inc. R.V.T. was previously employed at Eli Lilly and Company and is now employed at Astellas Pharma US. S.V.B., S.-B.P., and R.V.T. are shareholders of Eli Lilly., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: This was an open-label phase 1a study assessing the maximum tolerated dose (MTD), safety, and tolerability of CXCR4 peptide antagonist, LY2510924, administered in combination with durvalumab in patients with advanced refractory solid tumors. Methods: Patients received LY2510924 at 20, 30, or 40 mg subcutaneous (SC) once daily in combination with durvalumab at 1500 mg intravenously (IV) on day 1 of each 28-day cycle. The primary objective was to assess the MTD and safety of LY2510924 SC daily in combination with durvalumab in patients with advanced (metastatic and/or unresectable) solid tumors. Secondary objectives included pharmacokinetics (PK) and the antitumor activity of LY2510924 in combination with durvalumab. Exploratory objectives were biomarker analysis, including pharmacodynamic markers, relevant to LY2510924 and durvalumab, including immune functioning, drug targets, cancer-related pathways, and the disease state. Results: Nine patients (three each at 20, 30, and 40 mg) were enrolled in the study (eight patients with pancreatic cancer and one patient with rectal cancer). The majority of patients completed one or two cycles (100.0% ≥ 1 cycle; 88.9% ≥ 2 cycles) of LY2510924 and durvalumab. No dose limiting toxicities were reported. Most common (>10%) treatment-emergent adverse events were injection-site reaction (44.4%), fatigue (33.3%), and increased white blood cell count (33.3%). PK parameters for combination were similar to those reported in previous studies when given as monotherapy. Best overall response of stable disease was observed in four (44.4%) patients and one patient had unconfirmed partial response. Conclusion: The recommended phase 2 dose is 40 mg SC once-daily LY2510924 in combination with durvalumab 1500 mg IV and showed acceptable safety and tolerability in patients with advanced refractory tumors., Competing Interests: M.H.O. received research funding support in the form of salary from Eli Lilly and Company; is a consultant advisor for Karyopharm and Exelixis; and received grant/research support from Bristol Meyers Squibb, Eli Lilly, Celldex, and Astra Zeneca. W.M. received research support from Eli Lilly and Company for this study. H.K. is a consultant advisor for AstraZeneca, Merck, BMS, Boehringer Ingelheim, Ipsen, Erytech, Five Prime, Paredox, Aldeyra, Kyowa, and Astellas; received grants/research support from Aduro, AstraZeneca, Bayer, GSK, Merck, MedImmune, Verastem, BMS, Eli Lilly, Polaris, Deciphera, Inhibrx, and Roche/Genentech. W.Z., C.P., A.M.S., S.-B.P., and B.V. are employees and shareholders of Eli Lilly and Company. A.A.K. is a full-time employee and stock shareholder of AstraZeneca. No potential conflicts of interests were reported by D.W. and M.K. A.W.-G. served on the advisory board for Ipsen, Merrimack, BMS, Vicus, Tyme, Jacobio, and Repugene., (© Mark H. O'Hara et al. 2020; Published by Mary Ann Liebert, Inc.)

The ERK pathway is critical in oncogenesis; aberrations in components of this pathway are common in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of the pathway. BRAF- and MEK-targeted therapies are effective in BRAF V600E/K metastatic melanoma and lung cancers; however, responses are short-lived due to emergence of resistance. Reactivation of ERK signaling is central to the mechanisms of acquired resistance. Therefore, ERK inhibition provides an opportunity to overcome resistance and leads to improved efficacy. In addition, KRAS -mutant cancers remain an unmet medical need in which ERK inhibitors may provide treatment options alone or in combination with other agents. Here, we report identification and activity of LY3214996, a potent, selective, ATP-competitive ERK inhibitor. LY3214996 treatment inhibited the pharmacodynamic biomarker, phospho-p90RSK1, in cells and tumors, and correlated with LY3214996 exposures and antitumor activities. In in vitro cell proliferation assays, sensitivity to LY3214996 correlated with ERK pathway aberrations. LY3214996 showed dose-dependent tumor growth inhibition and regression in xenograft models harboring ERK pathway alterations. Importantly, more than 50% target inhibition for up to 8 to 16 hours was sufficient for significant tumor growth inhibition as single agent in BRAF - and KRAS -mutant models. LY3214996 also exhibited synergistic combination benefit with a pan-RAF inhibitor in a KRAS -mutant colorectal cancer xenograft model. Furthermore, LY3214996 demonstrated antitumor activity in BRAF -mutant models with acquired resistance in vitro and in vivo . Based on these preclinical data, LY3214996 has advanced to an ongoing phase I clinical trial (NCT02857270)., (©2019 American Association for Cancer Research.)

Mutations in ERK signaling drive a significant percentage of malignancies. LY3009120, a pan-RAF and dimer inhibitor, has preclinical activity in RAS - and BRAF -mutated cell lines including BRAF -mutant melanoma resistant to BRAF inhibitors. This multicenter, open-label, phase I clinical trial (NCT02014116) consisted of part A (dose escalation) and part B (dose confirmation) in patients with advanced/metastatic cancer. In part A, oral LY3009120 was dose escalated from 50 to 700 mg twice a day on a 28-day cycle. In part B, 300 mg LY3009120 was given twice a day. The primary objective was to identify a recommended phase II dose (RP2D). Secondary objectives were to evaluate safety, pharmacokinetics, and preliminary efficacy. Identification of pharmacodynamic biomarkers was exploratory. In parts A and B, 35 and 16 patients were treated, respectively ( N = 51). In part A, 6 patients experienced eight dose-limiting toxicities. The RP2D was 300 mg twice a day. Common (>10%) any-grade drug-related treatment-emergent adverse events were fatigue ( n = 15), nausea ( n = 12), dermatitis acneiform ( n = 10), decreased appetite ( n = 7), and maculopapular rash ( n = 7). The median duration of treatment was 4 weeks; 84% of patients completed one or two cycles of treatment. Exposures observed at 300 mg twice a day were above the preclinical concentration associated with tumor regression. Eight patients had a best overall response of stable disease; there were no complete or partial clinical responses. Despite adequate plasma exposure levels, predicted pharmacodynamic effects were not observed., (©2019 American Association for Cancer Research.)

The KRASG12C protein product is an attractive, yet challenging, target for small molecule inhibition. One option for therapeutic intervention is to design small molecule ligands capable of binding to and inactivating KRASG12C via formation of a covalent bond to the sulfhydryl group of cysteine 12. In order to better understand the cellular off-target interactions of Compound 1 , a covalent KRASG12C inhibitor, we have completed a series of complementary chemical proteomics experiments in H358 cells. A new thiol reactive probe (TRP) was designed and used to construct a cellular target occupancy assay for KRASG12C. In addition, the thiol reactive probes allowed us to profile potential off-target interactions of Compound 1 with over 3200 cysteine residues. In order to complement the TRP data we designed Compound 2 , an alkyne containing version of Compound 1 , to serve as bait in competitive chemical proteomics experiments. Herein, we describe and compare data from both the TRP and the click chemistry probe pull down experiments., Competing Interests: The authors declare no competing financial interest.

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, such as melanoma, lung, colorectal and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor advanced to clinical study has been shown to inhibit both RAS and BRAF mutant cell proliferation in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4/6-selective inhibitor, is currently in phase III studies for ER-positive breast cancer and KRAS mutant lung cancer. In this study, we found that combinatory treatment with LY3009120 and abemaciclib synergistically inhibited proliferation of tumor cells in vitro and led to tumor growth regression in xenograft models with a KRAS, NRAS or BRAF mutation at the doses of two drugs that were well tolerated in combination. Further in vitro screen in 328 tumor cell lines revealed that tumor cells with KRAS, NRAS or BRAF mutation, or cyclin D activation are more sensitive, whereas tumor cells with PTEN, PIK3CA, PIK3R1 or retinoblastoma (Rb) mutation are more resistant to this combination treatment. Molecular analysis revealed that abemaciclib alone inhibited Rb phosphorylation partially and caused an increase of cyclin D1. The combinatory treatment cooperatively demonstrated more complete inhibition of Rb phosphorylation, and LY3009120 suppressed the cyclin D1 upregulation mediated by abemaciclib. These results were further verified by CDK4/6 siRNA knockdown. Importantly, the more complete phospho-Rb inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combination led to more significant cell cycle G 0 /G 1 arrest of tumor cells. These preclinical findings suggest that combined inhibition of RAF and d-cyclin-dependent kinases might provide an effective approach to treat patients with tumors harboring mutations in RAS or RAF genes.

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a critical role in mobilization and redistribution of immune cells and hematopoietic stem cells (HSCs). We evaluated effects of two CXCR4-targeting agents, peptide antagonist LY2510924 and monoclonal antibody LY2624587, on mobilizing HSCs and white blood cells (WBCs) in humans, monkeys, and mice. Biochemical analysis showed LY2510924 peptide blocked SDF-1/CXCR4 binding in all three species; LY2624587 antibody blocked binding in human and monkey, with minimal activity in mouse. Cellular analysis showed LY2624587 antibody, but not LY2510924 peptide, down-regulated cell surface CXCR4 and induced hematological tumor cell death; both agents have been shown to inhibit SDF-1/CXCR4 interaction and downstream signaling. In animal models, LY2510924 peptide induced robust, prolonged, dose- and time-dependent WBC and HSC increases in mice and monkeys, whereas LY2624587 antibody induced only moderate, transient increases in monkeys. In clinical trials, similar pharmacodynamic effects were observed in patients with advanced cancer: LY2510924 peptide induced sustained WBC and HSC increases, while LY2624587 antibody induced only minimal, transient WBC changes. These distinct pharmacodynamic effects in two different classes of CXCR4 inhibitors are clinically important and should be carefully considered when designing combination studies with immune checkpoint inhibitors or other agents for cancer therapy., Competing Interests: CONFLICTS OF INTEREST All authors are full-time employees/shareholders of Eli Lilly and Company.

Purpose: To evaluate the antitumor efficacy of cetuximab in combination with LSN3074753, an analog of LY3009120 and pan-RAF inhibitor in 79 colorectal cancer patient-derived xenograft (PDX) models. Experimental Design: Seventy-nine well-characterized colorectal cancer PDX models were employed to conduct a single mouse per treatment group ( n = 1) trial. Results: Consistent with clinical results, cetuximab was efficacious in wild-type KRAS and BRAF PDX models, with an overall response rate of 6.3% and disease control rate (DCR) of 20.3%. LSN3074753 was active in a small subset of PDX models that harbored KRAS or BRAF mutations. However, the combination treatment displayed the enhanced antitumor activity with DCR of 35.4%. Statistical analysis revealed that BRAF and KRAS mutations were the best predictors of the combinatorial activity and were significantly associated with synergistic effect with a P value of 0.01 compared with cetuximab alone. In 12 models with BRAF mutations, the combination therapy resulted in a DCR of 41.7%, whereas either monotherapy had a DCR of 8.3%. Among 44 KRAS mutation models, cetuximab or LSN3074753 monotherapy resulted in a DCR of 13.6% or 11.4%, respectively, and the combination therapy increased DCR to 34.1%. Molecular analysis suggests that EGFR activation is a potential feedback and resistant mechanism of pan-RAF inhibition. Conclusions: MAPK and EGFR pathway activations are two major molecular hallmarks of colorectal cancer. This mouse PDX trial recapitulated clinical results of cetuximab. Concurrent EGFR and RAF inhibition demonstrated synergistic antitumor activity for colorectal cancer PDX models with a KRAS or BRAF mutation. Clin Cancer Res; 23(18); 5547-60. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Activating mutations in the KRAS and BRAF genes, leading to hyperactivation of the RAS/RAF/MAPK oncogenic signaling cascade, are common in patients with colorectal cancer (CRC). While selective BRAF inhibitors are efficacious in BRAFmut melanoma, they have limited efficacy in BRAFmut CRC patients. In a RASmut background, selective BRAF inhibitors are contraindicated due to paradoxical activation of the MAPK pathway through potentiation of CRAF kinase activity. A way to overcome such paradoxical activation is through concurrent inhibition of the kinase activity of both RAF isoforms. Here, we further examined the effects of LY3009120, a panRAF and RAF dimer inhibitor, in human models of CRC with various mutational backgrounds. We demonstrate that LY3009120 induced anti-proliferative effects in BRAFmut and KRASmut CRC cell lines through G1-cell cycle arrest. The anti-proliferative effects of LY3009120 in KRASmut CRC cell lines phenocopied molecular inhibition of RAF isoforms by simultaneous siRNA-mediated knockdown of ARAF, BRAF and CRAF. Additionally, LY3009120 displayed significant activity in in vivo BRAFmut and KRASmut CRC xenograft models. Examination of potential resistance to LY3009120 demonstrated RAF-independent ERK and AKT activation in the KRASmut CRC cell line HCT 116. These findings describe the preclinical activity of a panRAF inhibitor in a BRAFmut and KRASmut CRC setting.

It is known that the identification performance of a multi-aircraft formation (MAF) of narrowband radar mainly depends on the time on target (TOT). To realize the identification task in one rotated scan with limited TOT, the paper proposes a novel identification-while-scanning (IWS) method based on sparse recovery to maintain high rotating speed and super-resolution for MAF identification, simultaneously. First, a multiple chirp signal model is established for MAF in a single scan, where different aircraft may have different Doppler centers and Doppler rates. Second, based on the sparsity of MAF in the Doppler parameter space, a novel hierarchical basis pursuit (HBP) method is proposed to obtain satisfactory sparse recovery performance as well as high computational efficiency. Furthermore, the parameter estimation performance of the proposed IWS identification method is analyzed with respect to recovery condition, signal-to-noise ratio and TOT. It is shown that an MAF can be effectively identified via HBP with a TOT of only about one hundred microseconds for IWS applications. Finally, some numerical experiment results are provided to demonstrate the effectiveness of the proposed method based on both simulated and real measured data., Competing Interests: The authors declare no conflict of interest.

SDF-1 and CXCR4 are a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of CXCR4 is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin's lymphoma, and generally correlates with a poor prognosis. In this study, we developed a humanized anti-CXCR4 monoclonal antibody, LY2624587 as a potent CXCR4 antagonist that was advanced into clinical study for cancer. LY2624587 blocked SDF-1 binding to CXCR4 with an IC50 of 0.26 nM, and inhibited SDF-1-induced GTP binding with a Kb of 0.66 nM. In human lymphoma U937 and leukemia CCRF-CEM cells expressing endogenous CXCR4, LY2624587 inhibited SDF-1-induced cell migration with IC50 values of 3.7 and 0.26 nM, respectively. This antibody also inhibited CXCR4 and SDF-1 mediated cell signaling including activation of MAPK and AKT in tumor cells expressing CXCR4. Bifocal microscopic and flow cytometry analyses revealed that LY2624587 mediated receptor internalization and caused CXCR4 down-regulation on the cell surface. In human hematologic cancer cells, LY2624587 caused dose dependent apoptosis in vitro and in vivo. In mouse xenograft models developed with human leukemia and lymphoma cells expressing high levels of CXCR4, LY2624587 exhibited dose-dependent tumor growth inhibition and provided significant survival benefit in a disseminated lymphoma model. Collectively, we have demonstrated that CXCR4 inhibition by LY2624587 has the potential for the treatment of human hematological malignancies.

Unlabelled: We have identified previously undiscovered BRAF in-frame deletions near the αC-helix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers. These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the β3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors dimer formation. Expression of L485-P490-deleted BRAF is able to transform NIH/3T3 cells in a BRAF dimer-dependent manner. BRAF homodimer is confirmed to be the dominant RAF dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions, LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive., Significance: This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer., (©2016 American Association for Cancer Research.)

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Targeting the stromal cell-derived factor 1α (SDF-1α)/C-X-C chemokine receptor type 4 (CXCR4) axis has been shown to be a promising therapeutic approach to overcome chemoresistance in acute myeloid leukemia (AML). We investigated the antileukemia efficacy of a novel peptidic CXCR4 antagonist, LY2510924, in preclinical models of AML. LY2510924 rapidly and durably blocked surface CXCR4 and inhibited stromal cell-derived factor 1 (SDF-1)α-induced chemotaxis and prosurvival signals of AML cells at nanomolar concentrations more effectively than the small-molecule CXCR4 antagonist AMD3100. In vitro, LY2510924 chiefly inhibited the proliferation of AML cells with little induction of cell death and reduced protection against chemotherapy by stromal cells. In mice with established AML, LY2510924 caused initial mobilization of leukemic cells into the circulation followed by reduction in total tumor burden. LY2510924 had antileukemia effects as monotherapy as well as in combination with chemotherapy. Gene expression profiling of AML cells isolated from LY2510924-treated mice demonstrated changes consistent with loss of SDF-1α/CXCR4 signaling and suggested reduced proliferation and induction of differentiation, which was proved by showing the attenuation of multiple prosurvival pathways such as PI3K/AKT, MAPK, and β-catenin and myeloid differentiation in vivo. Effective disruption of the SDF-1α/CXCR4 axis by LY2510924 may translate into effective antileukemia therapy in future clinical applications., (© 2015 by The American Society of Hematology.)

The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.

Selective BRAF inhibitors have demonstrated significant clinical benefit in melanoma patients harboring oncogenic BRAF mutations. However, the majority of such patients either exhibit de novo resistance from the beginning of the treatment or acquire resistance and eventually relapse. Despite tremendous progress in understanding the underlying mechanisms of resistance, overcoming resistance to BRAF inhibitors remains an unmet medical need. Constitutive activation of cyclin-dependent kinases (CDK) 4/6 as a result of genetic aberrations including CDKN2A inactivation and CCND1 amplification is common across many cancer types and frequently co-occurs with oncogenic BRAF mutations. Also, cyclin D1 overexpression is a common feature of resistance to BRAF inhibitors. Here we review CDK4/6 as a therapeutic target in BRAF mutant cancers and discuss emerging evidence supporting a critical role of cyclin D1/CDK4/6 axis in de novo and acquired resistance to BRAF inhibitors. Co-targeting CDK4/6 and BRAF could be a more effective therapy to augment clinical response of BRAF inhibitors and overcome resistance in BRAF mutant cancers., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: To observe the effect of removable denture with metal stent of baked plastic occlusal pad in the reconstruction of dentition defects accompanied with severe residual teeth attrition., Methods: Twenty one patients were selected with dentition defects accompanied with severe residual teeth attrition, who needed occlusal reconstruction. Their removable dentures with metal stent of baked plastic occlusal pad were reconstructed, and their occlusal vertical dimensions were restored. The restoration effect was evaluated., Results: Twenty-one patients were treated and followed up for 12-24 months. Excellent results were achieved in 18 patients, and fair results were obtained in 3 patients., Conclusions: Using the baked plastic occlusal pad can not only recover patients' occlusal vertical dimension, but also restore the shapes and function of their residual teeth to a better extent. It is a good method for occlusal reconstruction.

Emerging evidence demonstrates that stromal cell-derived factor 1 (SDF-1) and CXCR4, a chemokine and chemokine receptor pair, play important roles in tumorigenesis. In this report, we describe a small cyclic peptide, LY2510924, which is a potent and selective CXCR4 antagonist currently in phase II clinical studies for cancer. LY2510924 specifically blocked SDF-1 binding to CXCR4 with IC50 value of 0.079 nmol/L, and inhibited SDF-1-induced GTP binding with Kb value of 0.38 nmol/L. In human lymphoma U937 cells expressing endogenous CXCR4, LY2510924 inhibited SDF-1-induced cell migration with IC50 value of 0.26 nmol/L and inhibited SDF-1/CXCR4-mediated intracellular signaling. LY2510924 exhibited a concentration-dependent inhibition of SDF-1-stimulated phospho-ERK and phospho-Akt in tumor cells. Biochemical and cellular analyses revealed that LY2510924 had no apparent agonist activity. Pharmacokinetic analyses suggested that LY2510924 had acceptable in vivo stability and a pharmacokinetic profile similar to a typical small-molecular inhibitor in preclinical species. LY2510924 showed dose-dependent inhibition of tumor growth in human xenograft models developed with non-Hodgkin lymphoma, renal cell carcinoma, lung, and colon cancer cells that express functional CXCR4. In MDA-MB-231, a breast cancer metastatic model, LY2510924 inhibited tumor metastasis by blocking migration/homing process of tumor cells to the lung and by inhibiting cell proliferation after tumor cell homing. Collectively, the preclinical data support further investigation of LY2510924 in clinical studies for cancer., (©2014 American Association for Cancer Research.)

B-RAF selective inhibitors, including vemurafenib, were recently developed as effective therapies for melanoma patients with B-RAF V600E mutation. However, most patients treated with vemurafenib eventually develop resistance largely due to reactivation of MAPK signaling. Inhibitors of MAPK signaling, including MEK1/2 inhibitor trametinib, failed to show significant clinical benefit in patients with acquired resistance to vemurafenib. Here, we describe that cell lines with acquired resistance to vemurafenib show reactivation of MAPK signaling and upregulation of cyclin D1 and are sensitive to inhibition of LY2835219, a selective inhibitor of cyclin-dependent kinase (CDK) 4/6. LY2835219 was demonstrated to inhibit growth of melanoma A375 tumor xenografts and delay tumor recurrence in combination with vemurafenib. Furthermore, we developed an in vivo vemurafenib-resistant model by continuous administration of vemurafenib in A375 xenografts. Consistently, we found that MAPK is reactivated and cyclin D1 is elevated in vemurafenib-resistant tumors, as well as in the resistant cell lines derived from these tumors. Importantly, LY2835219 exhibited tumor growth regression in a vemurafenib-resistant model. Mechanistic analysis revealed that LY2835219 induced apoptotic cell death in a concentration-dependent manner in vemurafenib-resistant cells whereas it primarily mediated cell-cycle G1 arrest in the parental cells. Similarly, RNAi-mediated knockdown of cyclin D1 induced significantly higher rate of apoptosis in the resistant cells than in parental cells, suggesting that elevated cyclin D1 activity is important for the survival of vemurafenib-resistant cells. Altogether, we propose that targeting cyclin D1-CDK4/6 signaling by LY2835219 is an effective strategy to overcome MAPK-mediated resistance to B-RAF inhibitors in B-RAF V600E melanoma., (©2014 American Association for Cancer Research.)

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 16 cancer subtypes and identified 486 genes that were amplified in two or more datasets. The list was narrowed to 75 cancer-associated genes with potential "druggable" properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 42 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 42 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapters GRB2 and GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.

In an on-going effort to discover metallotherapeutic alternatives to the chemotherapy drug cisplatin, neutral distorted square pyramidal gold(III) coordination complexes possessing 2,9-disubstituted-1,10-phenanthroline ligands {[((R)phen)AuCl3]; R = n-butyl, sec-butyl} have been previously synthesized and characterized. A structurally analogous gold(III) complex bearing a 6,6'-di-methylbipyridine ligand ([((methyl)bipy)AuCl3]) has been synthesized and fully characterized to probe the effect of differing aromatic character of the ligand on solution stability and tumor cell cytotoxicity. The two compounds [((sec-butyl)phen)AuCl3] and [((methyl)bipy)AuCl3]) were subsequently assessed for their stability against the biological reductant glutathione, and it was found that the [((sec-butyl)phen)AuCl3] complex exhibits slightly enhanced stability compared to the [((methyl)bipy)AuCl3] complex and significantly higher stability than previously reported square planar gold(III) complex ions. Furthermore, these complexes were tested for cytotoxic effects against existing lung and head and neck cancer cell lines in vitro. The [((sec-butyl)phen)AuCl3] complex was found to be more cytotoxic than cisplatin against five different tumor cell lines, whereas [((methyl)bipy)AuCl3] had more limited in vitro antitumor activity. Given that [((sec-butyl)phen)AuCl3] had significantly higher antitumor activity, it was tested against an in vivo tumor model. It was found that this complex did not significantly reduce the growth of xenograft tumors in mice and initial model binding studies with bovine serum albumin indicate that interactions with serum albumin proteins may be the cause for the limited in vivo activity of this potential metallotherapeutic., (© 2013.)

Breast cancer resistance protein (BCRP), also known as ABCG2, is a member of the ATP-binding cassette (ABC) transporter superfamily, and is known to play important roles in cancer multidrug resistance. The BCRP promoter lacks a TATA-box and contains a CAAT-box, lots of AP1, AP2 sites and several putative Sp1 sites which are downstream of a putative CpG island. Several transcription factors, such as progesterone receptor (PR), estrogen receptor (ER), nuclear factor-κB (NF-κB), hypoxia-inducible factors (HIFs), nuclear factor erythroid 2-related factor 2 (Nrf2), aryl hydrocarbon receptor (AhR), peroxisome proliferator-activated receptors (PPARs) and Krüppel-like factor 5 (KLF5), have been recently shown to bind to their response elements in the promoter/enhancer to activate the transcription of BCRP. BCRP transcription can be influenced by proinflammatory cytokines, growth factors, and homeobox protein MSX2. Signaling pathways, such as Sonic hedgehog (Shh), Notch and RAR/RXR pathways, may also involve in the transcriptional regulation of BCRP. In addition, promoter methylation and histone acetylation are essential for the BCRP transcription, especially for the drug-induced BCRP expression. This paper reviews the recent research progresses in this field with an emphasis on the roles of transcription factors and epigenetics in the transcriptional regulation of BCRP.

Oncogenic B-RAF V600E mutation is found in 50% of melanomas and drives MEK/ERK pathway and cancer progression. Recently, a selective B-RAF inhibitor, vemurafenib (PLX4032), received clinical approval for treatment of melanoma with B-RAF V600E mutation. However, patients on vemurafenib eventually develop resistance to the drug and demonstrate tumor progression within an average of 7 months. Recent reports indicated that multiple complex and context-dependent mechanisms may confer resistance to B-RAF inhibition. In the study described herein, we generated B-RAF V600E melanoma cell lines of acquired-resistance to vemurafenib, and investigated the underlying mechanism(s) of resistance. Biochemical analysis revealed that MEK/ERK reactivation through Ras is the key resistance mechanism in these cells. Further analysis of total gene expression by microarray confirmed a significant increase of Ras and RTK gene signatures in the vemurafenib-resistant cells. Mechanistically, we found that the enhanced activation of fibroblast growth factor receptor 3 (FGFR3) is linked to Ras and MAPK activation, therefore conferring vemurafenib resistance. Pharmacological or genetic inhibition of the FGFR3/Ras axis restored the sensitivity of vemurafenib-resistant cells to vemurafenib. Additionally, activation of FGFR3 sufficiently reactivated Ras/MAPK signaling and conferred resistance to vemurafenib in the parental B-RAF V600E melanoma cells. Finally, we demonstrated that vemurafenib-resistant cells maintain their addiction to the MAPK pathway, and inhibition of MEK or pan-RAF activities is an effective therapeutic strategy to overcome acquired-resistance to vemurafenib. Together, we describe a novel FGFR3/Ras mediated mechanism for acquired-resistance to B-RAF inhibition. Our results have implications for the development of new therapeutic strategies to improve the outcome of patients with B-RAF V600E melanoma.

The fibroblast growth factor receptors (FGFR) are tyrosine kinases that are present in many types of endothelial and tumor cells and play an important role in tumor cell growth, survival, and migration as well as in maintaining tumor angiogenesis. Overexpression of FGFRs or aberrant regulation of their activities has been implicated in many forms of human malignancies. Therefore, targeting FGFRs represents an attractive strategy for development of cancer treatment options by simultaneously inhibiting tumor cell growth, survival, and migration as well as tumor angiogenesis. Here, we describe a potent, selective, small-molecule FGFR inhibitor, (R)-(E)-2-(4-(2-(5-(1-(3,5-Dichloropyridin-4-yl)ethoxy)-1H-indazol-3yl)vinyl)-1H-pyrazol-1-yl)ethanol, designated as LY2874455. This molecule is active against all 4 FGFRs, with a similar potency in biochemical assays. It exhibits a potent activity against FGF/FGFR-mediated signaling in several cancer cell lines and shows an excellent broad spectrum of antitumor activity in several tumor xenograft models representing the major FGF/FGFR relevant tumor histologies including lung, gastric, and bladder cancers and multiple myeloma, and with a well-defined pharmacokinetic/pharmacodynamic relationship. LY2874455 also exhibits a 6- to 9-fold in vitro and in vivo selectivity on inhibition of FGF- over VEGF-mediated target signaling in mice. Furthermore, LY2874455 did not show VEGF receptor 2-mediated toxicities such as hypertension at efficacious doses. Currently, this molecule is being evaluated for its potential use in the clinic.

A gold(III) complex possessing 5,6-dimethyl-1,10-phenanthroline (5,6DMP) was synthesized and fully characterized using standard spectroscopic techniques, as well as X-ray crystallography and elemental analysis. The complex [(5,6DMP)AuCl(2)][BF(4)] (2) was found to possess a distorted square planar geometry about the gold(III) center, commonplace for d(8) Au(III) cations possessing sterically un-hindered polypyridyl ligands. Compound 2 was evaluated for its potential use as an anticancer therapeutic. It was determined that the complex is stable in phosphate buffer over a 24-hour period, thought it does undergo rapid reduction in the presence of equimolar amounts of reduced glutathione (GSH) and ascorbic acid. The DNA binding and in vitro tumor cytotoxicity of the title compound 2 were also determined. It was found to undergo weak and reversible binding to calf thymus DNA, and was more cytotoxic towards a panel of human cancer cell lines than the commonly used chemotherapy agent cisplatin. Cytotoxicity experiments with the free 5,6DMP ligand indicate that the ligand has IC(50) values that are slightly lower than those observed for the gold complex (2), and coupled with the fact that the ligand appears to be released from the gold(III) metal center in reducing environments, this suggests the ligand itself may play an important role in the antitumor activity of the parent gold complex., (Copyright © 2011 Elsevier Inc. All rights reserved.)

DNA ligases are the enzymes essential for DNA replication, repair and recombination in all organisms. The bacterial DNA ligases involved in DNA replication require NAD(+) for activity, but eukaryotic and viral DNA ligases require ATP. Because of their essential nature, unique structures and widespread existence in nature, bacterial DNA ligases represent a class of valuable targets for identifying novel and selective antibacterial agents. In this study, we cloned and expressed the ligA gene from Streptococcus pneumoniae, and characterized this ligA-encoded NAD(+)-dependent DNA ligase. We then screened small molecule chemical libraries using a biochemical assay and identified a new small molecule with a structure of 2,4-diamino-7-dimethylamino-pyrimido[4,5-d]pyrimidine. We show that this small molecule is a specific inhibitor of bacterial NAD(+)-dependent DNA ligases. Biochemical studies show that this molecule inhibits NAD(+)-dependent DNA ligases, but not ATP-dependent enzymes. The molecule inhibits NAD(+)-dependent DNA ligases competitively with respect to NAD(+) and specifically inhibits enzyme adenylation, but not DNA adenylation or ligation. Labeling studies establish that this molecule inhibits the incorporation of thymidine into DNA and that overexpression of DNA ligase in the cell abolishes this inhibition. Finally, microbiological studies show that this molecule exhibits a broad spectrum of antibacterial activity. Together, this study shows that this small molecule inhibitor identified is specific to bacterial NAD(+)-dependent DNA ligases, exhibits a broad spectrum of antibacterial activities, and has the potential to be developed into an antibacterial agent.

Background: Numerous studies have used microarrays to identify gene signatures for predicting cancer patient clinical outcome and responses to chemotherapy. However, the potential impact of gene expression profiling in cancer diagnosis, prognosis and development of personalized treatment may not be fully exploited due to the lack of consensus gene signatures and poor understanding of the underlying molecular mechanisms., Methods: We developed a novel approach to derive gene signatures for breast cancer prognosis in the context of known biological pathways. Using unsupervised methods, cancer patients were separated into distinct groups based on gene expression patterns in one of the following pathways: apoptosis, cell cycle, angiogenesis, metastasis, p53, DNA repair, and several receptor-mediated signaling pathways including chemokines, EGF, FGF, HIF, MAP kinase, JAK and NF-kappaB. The survival probabilities were then compared between the patient groups to determine if differential gene expression in a specific pathway is correlated with differential survival., Results: Our results revealed expression of cell cycle genes is strongly predictive of breast cancer outcomes. We further confirmed this observation by building a cell cycle gene signature model using supervised methods. Validated in multiple independent datasets, the cell cycle gene signature is a more accurate predictor for breast cancer clinical outcome than the previously identified Amsterdam 70-gene signature that has been developed into a FDA approved clinical test MammaPrint., Conclusion: Taken together, the gene expression signature model we developed from well defined pathways is not only a consistently powerful prognosticator but also mechanistically linked to cancer biology. Our approach provides an alternative to the current methodology of identifying gene expression markers for cancer prognosis and drug responses using the whole genome gene expression data.

The CXCR4/stromal cell-derived factor-1 (SDF-1) axis plays important roles in development, leukocyte trafficking, HIV infection, and tumorigenesis. Its critical function in bone marrow stem cell and hematopoietic progenitor cell retention, homing and release has been well-characterized by genetic and pharmacological analyses. However, its role in neutrophil retention and release is still poorly understood. In this study, we demonstrated that T134, a peptide antagonist of human CXCR4, is also a potent antagonist of mouse CXCR4. Treatment of C57BL/6 mice with T134 resulted in a rapid and time-dependent increase of white blood cells (WBC) and neutrophils, as well as hematopoietic stem and progenitor cells in peripheral blood. Interestingly, recurrent WBC and neutrophil mobilization was achieved by repeated T134 treatment, and the T134-mediated increase and subsequent retreat of WBC and neutrophils correlated with T134 activity in the peripheral blood. Kinetic analysis revealed that T134 binding to CXCR4 did not induce any significant cell-surface receptor downregulation, indicating that T134-induced WBC and neutrophil mobilization is likely due to direct blockage of the CXCR4/SDF-1 interaction. The results from this study support an important role of CXCR4/SDF-1 axis in neutrophil retention and release in the marrow.

To investigate the association between single nucleotide polymorphisms (SNPs) rs2970847(Thr394Thr) and rs8192678(Gly482Ser) of peroxisome proliferators activated receptor gamma coactivator-1(PPARGC1A)gene and type 2 diabetes in Chinese Han population in Hubei. The Thr394Thr and Gly482Ser polymorphisms of the PPARGC1A gene were genotyped in 307 controls and 344 type 2 diabetes patients. Single marker/haplotype association analyses and logistic re-gression analysis were performed. Significant differences in genotypic and allelic frequencies were observed between cases and controls for the Thr394Thr polymorphism (genotype, P=0.006; allele, Plt;0.001). The genotype AA of the Thr394Thr polymorphism and the haplotype Thr394(ACA)- Ser482 were significantly associated with type 2 diabetes. No differences in the genotypic and allelic frequencies between cases and controls were found for the Gly482Ser polymorphism. PPARGC1A is a susceptibility gene for type 2 diabetes in Chinese Han population in Hubei.

Stromal Cell-derived factor 1 (SDF-1) is a CXC chemokine that binds to the CXCR4 receptor. Recent publication indicates that the SDF-1/CXCR4 signaling pathway plays a pivotal role during development and in many patho-physiological conditions including hematopoiesis, blood vessel formation, cancer metastasis, angiogenesis and HIV infection. Two human SDF-1 isoforms, SDF-1alpha and SDF-1beta, have been reported to date. Here we report the identification of four additional human SDF-1 isoforms derived from alternative splicing events, SDF-1gamma, SDF-1delta, SDF-1epsilon and SDF-1phi. These SDF-1 splice variants all share the same first three exons but contain different fourth exons. The human SDF-1 gene spans over 88 kilobase-pairs on chromosome 10. Using the semi-quantitative RT-PCR method, we determined the tissue distribution of these SDF-1 isoforms. SDF-1alpha and SDF-1beta share similar expression patterns and the highest expression were detected in liver, pancreas and spleen. SDF-1gamma seems to be the human orthologue of recently isolated rat SDF-1gamma, and its expression was only detected in the heart. SDF-1delta expression can be detected in several adult tissues but the highest expression was detected in fetal liver. When transfected into HEK293 cells, all the SDF-1 isoforms can be detected as secreted proteins in the cell culture media. The conditioned media from transfected cells can stimulate cell migration in a CXCR4-dependent manner. These data suggest that the novel SDF-1 splice variants encode functional proteins.

G protein-coupled receptors (GPCRs) play important roles in a variety of biological and pathological processes. They are considered among the most desirable targets for drug development. Recent studies have demonstrated that many GPCRs, such as endothelin receptors, chemokine receptors and lysophosphatidic acid receptors have been implicated in the tumorigenesis and metastasis of multiple human cancers. In this study, we conducted an in silico analysis of GPCR gene expression in primary human tumors by analyzing some publicly available gene expression profiling data. Statistical analysis was performed on eight microarray data sets of non-small cell lung cancer, breast cancer, prostate cancer, melanoma, gastric cancer and diffused large B cell lymphoma to identify GPCRs that are up-regulated in primary or metastatic cancer cells. Our analysis has demonstrated overexpression of several GPCRs in primary tumor cells, including chemokine receptors and protease-activated receptors that were shown to be important for tumorigenesis by previous studies. In addition, we have uncovered several GPCRs, such as neuropeptide receptors, adenosine A2B receptor, P2Y purinoceptor, calcium-sensing receptor and metabotropic glutamate receptors, that are expressed at a significantly higher level in some cancer tissue and may play a role in cancer progression. Analysis of cancer samples in different disease stages also suggests that some GPCRs, such as endothelin receptor A, may be involved in early tumor progression and others, such as CXCR4, may play a critical role in tumor invasion and metastasis. The present study demonstrates the value of publicly available microarray data as a resource to gain more understanding of cancer biology, to validate previous findings from in vitro experiments, and to identify potential novel anticancer targets and biomarkers.

Emerging evidence shows that the stromal cell-derived factor 1 (SDF-1)/CXCR4 interaction regulates multiple cell signaling pathways and a variety of cellular functions such as cell migration, proliferation, and survival. There is little information linking the cellular functions and individual signaling pathways mediated by SDF-1 and CXCR4 in human cancer cells. In this study, we have shown that human epitheloid carcinoma HeLa cells express functional CXCR4 by reverse transcription-PCR, immunofluorescent staining, and 125I-SDF-1alpha ligand binding analyses. The treatment of HeLa cells with recombinant SDF-1alpha results in time-dependent Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) activations. The SDF-1alpha-induced Akt and ERK1/2 activations are CXCR4 dependent as confirmed by their total inhibition by T134, a CXCR4-specific peptide antagonist. Cell signaling analysis with pathway-specific inhibitors reveals that SDF-1alpha-induced Akt activation is not required for ERK1/2 activation and vice versa, indicating that activations of Akt and ERK1/2 occur independently. Functional analysis shows that SDF-1alpha induces a CXCR4-dependent migration of HeLa cells. The migration can be totally blocked by phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, whereas mitogen-activated protein/ERK kinase inhibitors, PD98059 and U0126, have no significant effect on SDF-1alpha-induced migration, suggesting that Akt activation, but not ERK1/2 activation, is required for SDF-1alpha-induced migration of epitheloid carcinoma cells.

Transforming growth factor beta (TGF-beta) signaling pathways regulate a wide variety of cellular processes including cell proliferation, differentiation, extracellular matrix deposition, development, and apoptosis. TGF-beta type-I receptor (TbetaRI) is the major receptor that triggers several signaling events by activating downstream targets such as the Smad proteins. The intracellular kinase domain of TbetaRI is essential for its function. In this study, we have identified a short phospho-Smad peptide, pSmad3(-3), KVLTQMGSPSIRCSS(PO4)VS as a substrate of TbetaRI kinase for in vitro kinase assays. This peptide is uniquely phosphorylated by TbetaRI kinase at the C-terminal serine residue, the phosphorylation site of its parent Smad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylated by only TbetaRI and not TGF-beta type-II receptor kinase, indicating that the peptide is a physiologically relevant substrate suitable for kinetic analysis and screening of TbetaRI kinase inhibitors. Utilizing pSmad3(-3) as a substrate, we have shown that novel pyrazole compounds are potent inhibitors of TbetaRI kinase with K(i) value as low as 15 nM. Kinetic analysis revealed that these pyrazoles act through the ATP-binding site and are typical ATP competitive inhibitors with tight binding kinetics. More importantly, these compounds were shown to inhibit TGF-beta-induced Smad2 phosphorylation in vivo in NMuMg mammary epithelial cells with potency equivalent to the inhibitory activity in the in vitro kinase assay. Cellular selectivity analysis demonstrated that these pyrazoles are capable of inhibiting activin signaling but not bone morphogenic protein or platelet-derived growth factor signal transduction pathways. Further functional analysis revealed that pyrazoles are capable of blocking the TGF-beta-induced epithelial-mesenchymal transition in NMuMg cells, a process involved in the progression of cancer, fibrosis, and other human diseases. These pyrazoles provide a foundation for future development of potent and selective TbetaRI kinase inhibitors to treat human disease.

Signal peptidase (SPase) I is responsible for the cleavage of signal peptides of many secreted proteins in bacteria. Because of its unique physiological and biochemical properties, it serves as a potential target for development of novel antibacterial agents. In this study, we report the production, isolation, and structure determination of a family of structurally related novel lipoglycopeptides from a Streptomyces sp. as inhibitors of SPase I. Detailed spectroscopic analyses, including MS and NMR, revealed that these lipoglycopeptides share a common 14-membered cyclic peptide core, an acyclic tripeptide chain, and a deoxy-alpha-mannose sugar, but differ in the degree of oxidation of the N-methylphenylglycine residue and the length and branching of the fatty acyl chain. Biochemical analysis demonstrated that these peptides are potent and competitive inhibitors of SPase I with K(i) 50 to 158 nm. In addition, they showed modest antibacterial activity against a panel of pathogenic Gram-positive and Gram-negative bacteria with minimal inhibitory concentration of 8-64 microm against Streptococcus pneumonniae and 4-8 microm against Escherichia coli. Notably, they mechanistically blocked the protein secretion in whole cells as demonstrated by inhibiting beta-lactamase release from Staphylococcus aureus. Taken together, the present discovery of a family of novel lipoglycopeptides as potent inhibitors of bacterial SPase I may lead to the development of a novel class of broad-spectrum antibiotics.

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.

Source, sink, and translocation capacity of assimilates play important roles during the formation of grain yield. The present study was conducted to characterize the genetic bases of traits representing source, sink and transport tissue, and their relationships with yield traits in rice, by analyzing QTLs for these traits and various ratios among them. The genetic materials were a recombinant inbred population derived from a cross between two indica cultivars Zhenshan 97 and Minghui 63, the parents of the most-widely grown hybrid rice in China. Using a linkage map that covers a total of 1,796 cM based on 221 molecular marker loci, a total of 81 QTLs were identified for the 15 traits studied (three leaf areas as the source, total spikelets per panicle as the sink, the number of large vascular bundles in the stem as transport tissue, three source to sink ratios, three transport tissue to source ratios, one transport tissue to sink ratio and three yield traits). The amount of variation explained by individual QTLs ranged from 1.12% to 24.14%. Five QTLs were identified to show interaction effects with the environment, which explained from 3.19% to 9.15% of the variation. The results showed that close linkage or pleiotropy is the genetic basis for the correlations of grain yield traits with source, sink, transport tissue and the various ratios among them. Of the 25 QTLs identified for source-sink-transport tissue trait, and 43 for various ratios, 8 and 22 QTLs, respectively, were mapped to the similar genomic blocks harboring QTLs for yield traits, especially for grain weight. Co-location of QTLs for yield traits with those for ratios among source, sink and transport tissue may provide a genetic explanation for the physiological expression of yield traits, and also suggest that improvement in ratios among source, sink and transport tissue may result in improvement in yield potential.

We have previously demonstrated that Streptococcus pneumoniae signal peptidase (SPase) I catalyzes a self-cleavage to result in a truncated product, SPase37-204 [Peng, S.B., Wang, L., Moomaw, J., Peery, R.B., Sun, P.M., Johnson, R.B., Lu, J., Treadway, P., Skatrud, P.L. & Wang, Q.M. (2001) J. Bacteriol.183, 621-627]. In this study, we investigated the effect of phospholipid on invitro self-cleavage of S. pneumoniae SPase I. In the presence of phospholipid, the self-cleavage predominantly occurred at one cleavage site between Gly36-His37, whereas the self-cleavage occurred at multiple sites in the absence of phospholipid, and two additional self-cleavage sites, Ala65-His66 and Ala143-Phe144, were identified. All three self-cleavage sites strongly resemble the signal peptide cleavage site and follow the (-1, -3) rule for SPase I recognition. Kinetic analysis demonstrated that self-cleavage is a concentration dependent and intermolecular event, and the activity in the presence of phospholipid is 25-fold higher than that in the absence of phospholipid. Biochemical analysis demonstrated that SPase37-204, the major product of the self-cleavage totally lost activity to cleave its substrates, indicating that the self-cleavage resulted in the inactivation of the enzyme. More importantly, the self-cleavage was demonstrated to be happening in vivo in all the growth phases of S. pneumoniae cells. The bacterial cells keep the active SPase I at the highest level in exponential growth phase, suggesting that the self-cleavage may play an important role in regulating the activity of the enzyme under different conditions.

Recent studies have demonstrated that bacteria possess an essential protein translocation system similar to mammalian signal recognition particle (SRP). Here we have identified the Ffh, a homologue of the mammalian SRP54 subunit from S. pneumoniae. Ffh is a 58-kDa protein with three distinct domains: an N-terminal hydrophilic domain (N-domain), a G-domain containing GTP/GDP binding motifs, and a C-terminal methionine-rich domain (M-domain). The full-length Ffh and a truncated protein containing N and G domains (Ffh-NG) were overexpressed in E. coli and purified to homogeneity. The full-length Ffh has an intrinsic GTPase activity with k(cat) of 0.144 min(-1), and the K(m) for GTP is 10.9 microM. It is able to bind to 4.5S RNA specifically as demonstrated by gel retardation assay. The truncated Ffh-NG has approximately the same intrinsic GTPase activity to the full-length Ffh, but is unable to bind to 4.5S RNA, indicating that the NG domain is sufficient for supporting intrinsic GTP hydrolysis, and that the M domain is required for RNA binding. The interaction of S. pneumoniae Ffh with its receptor, FtsY, resulted in a 20-fold stimulation in GTP hydrolysis. The stimulation was further demonstrated to be independent of the 4.5S RNA. In addition, a similar GTPase stimulation is also observed between Ffh-NG and FtsY, suggesting that the NG domain is sufficient and the M domain is not required for GTPase stimulation between Ffh and FtsY.

In the study, ten panicle traits associated with yield sink size were measured in a recombinant inbred population derived from Zhenshan 97 x Minghui 63. Generally, spikelets per panicle were more closely correlated with number of secondary branch per panicle, spikelets on secondary branch per secondary branch, and spikelet density. A total of 53 QTL were detected for ten traits in two years. Approximately 43.4% QTLs were detected in both two years, suggesting environmental effects on traits. Five chromosomal regions (G359-RG532 and C567-C86-RG236 on chromosome 1, R712-RM29 on chromosome 2, P-RG424 on chromosome 6, C148-RM258 on chromosome 10) were detected to have effects on multiple panicle traits. QTLs for traits, which were correlated, were generally localized in similar chromosomal regions, suggesting that pleiotropy and (or) linkage are the molecular basis of relationship between them. A large number of digenic interactions were detected, 18.2% of which were detected simultaneously in both two years. The proportion of common interactions was trait-depended, ranging 8.7% for spikelets on secondary branch per secondary branch to 32.6% for panicle length. Approximately 26.7% of common two-locus combinations had pleiotropic effects by simultaneously influencing two or more traits. Overall, the results indicate that each panicle trait is controlled by several QTLs, genotype x environment interaction, and a large number of epistatic interactions.

Signal peptidase (SPase) I is responsible for the cleavage of signal peptides of many secreted proteins in bacteria and serves as a potential target for the development of novel antibacterial agents due to its unique physiological and biochemical properties. In this paper, we describe a novel fluorogenic substrate, KLTFGTVK(Abz)PVQAIAGY(NO2)EWL, in which 2-aminobenzoic acid (Abz) and 3-nitrotyrosine (Y(NO2)) were used as the fluorescent donor and acceptor, respectively. The substrate can be cleaved by both Streptococcus pneumoniae and Escherichia coli SPase I. Upon cleavage of the fluorogenic substrate by SPase I, the fluorescent intensity increases and can be monitored continuously by spectrofluorometer. Kinetic analysis with S. pneumoniae SPase I demonstrated that the K(m) value for the substrate is 118.1 microM, and the k(cat) value is 0.032 s(-1). Mass spectrometric analysis and peptide sequencing of the two cleaved products confirmed that the cleavage occurs specifically at the predicted site. More interestingly, the positively charged lysine in the N-terminus of the substrate was demonstrated to be important for effective cleavage. Phospholipids were found to stimulate the cleavage reaction. This stimulation by phospholipids is dependent upon the N-terminal charge of the substrate, indicating that the interaction of the positively charged substrate with anionic phospholipids is important for maintaining the substrate in certain conformation for cleavage. The substrate and assay described here can be readily automated and utilized for the identification of potential antibacterial agents., (Copyright 2001 Academic Press.)

Bacterial signal peptidase I is responsible for proteolytic processing of the precursors of secreted proteins. The enzymes from gram-negative and -positive bacteria are different in structure and specificity. In this study, we have cloned, expressed, and purified the signal peptidase I of gram-positive Streptococcus pneumoniae. The precursor of streptokinase, an extracellular protein produced in pathogenic streptococci, was identified as a substrate of S. pneumoniae signal peptidase I. Phospholipids were found to stimulate the enzymatic activity. Mutagenetic analysis demonstrated that residues serine 38 and lysine 76 of S. pneumoniae signal peptidase I are critical for enzyme activity and involved in the active site to form a serine-lysine catalytic dyad, which is similar to LexA-like proteases and Escherichia coli signal peptidase I. Similar to LexA-like proteases, S. pneumoniae signal peptidase I catalyzes an intermolecular self-cleavage in vitro, and an internal cleavage site has been identified between glycine 36 and histidine 37. Sequence analysis revealed that the signal peptidase I and LexA-like proteases show sequence homology around the active sites and some common properties around the self-cleavage sites. All these data suggest that signal peptidase I and LexA-like proteases are closely related and belong to a novel class of serine proteases.

Vacuolar H+-ATPases (V-ATPases) are involved in a wide variety of essential cellular processes. An unresolved question is how the cell regulates the activity of these proton pumps and their targeting to distinct cellular compartments. There is growing evidence for the presence of subunit diversity amongst V-pumps, particularly regarding the 116-kDa subunit (called the a subunit). We have cloned and characterized three isoforms (a1, a2 and a3) of this subunit from chicken. The amino-acid sequences of these homologues are approximately 50% similar and their nucleotide differences indicate that they are products of distinct genes. The levels of mRNA expression of these isoforms was quantified by ribonuclease protection analysis. The a1 and a2 isoforms have a similar tissue distribution, with the highest level of mRNA expression in brain, an intermediate level in kidney and relatively low levels in liver and bone. In contrast, the highest level of expression of the a3 isoform is in bone and liver, with a moderate level in kidney, and the lowest level in brain. An antibody against the a1 isoform reacted with a 116 kDa protein in a brain V-ATPase preparation that was not detected in bone or liver V-ATPase preparations, whereas an antibody against the a3 isoform reacted with a 116-kDa peptide in bone and liver, but not brain V-ATPases preparations. The bone and brain V-ATPases showed differential sensitivity to the inhibitors bafilomycin and (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-[4-(2, 2,6,6-tetramethyl)piperidinyl]-2,4-pentadienamide. Thus, this work demonstrates the presence of structurally and functionally distinct V-ATPases in a single vertebrate species.

The vacuolar proton pump of clathrin-coated vesicles is composed of two general sectors, a cytosolic, ATP hydrolytic domain (V1) and an intramembranous proton channel, V0. V1 is comprised of 8-9 subunits including polypeptides of 50 and 57 kDa, termed SFD (Sub Fifty-eight-kDa Doublet). Although SFD is essential to the activation of ATPase and proton pumping activities catalyzed by holoenzyme, its constituent polypeptides have not been separated to determine their respective roles in ATPase functions. Recent molecular characterization of these subunits revealed that they are isoforms that arise through an alternative splicing mechanism (Zhou, Z., Peng, S.-B., Crider, B.P., Slaughter, C., Xie, X.S., and Stone, D.K. (1998) J. Biol. Chem. 273, 5878-5884). To determine the functional characteristics of the 57-kDa (SFDalpha)1 and 50-kDa (SFDbeta) isoforms, we expressed these proteins in Escherichia coli. We determined that purified recombinant proteins, rSFDalpha and rSFDbeta, when reassembled with SFD-depleted holoenzyme, are functionally interchangeable in restoration of ATPase and proton pumping activities. In addition, we determined that the V-pump of chromaffin granules has only the SFDalpha isoform in its native state and that rSFDalpha and rSFDbeta are equally effective in restoring ATPase and proton pumping activities to SFD-depleted enzyme. Finally, we found that SFDalpha and SFDbeta structurally interact not only with V1, but also withV0, indicating that these activator subunits may play both structural and functional roles in coupling ATP hydrolysis to proton flow.