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Autosomal-recessive mutations inSPEG(striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy. Loss of SPEG is associated with defective triad formation, abnormal excitation-contraction coupling, and calcium mishandling in skeletal muscles. To elucidate the underlying molecular pathways, we have utilized multi-omics tools and analysis to obtain a comprehensive view of the complex biological processes. We identified that SPEG interacts with myospryn complex proteins (CMYA5, FSD2, RyR1), and SPEG deficiency results in myospryn complex abnormalities. In addition, transcriptional and protein profiles of SPEG-deficient muscle revealed defective mitochondrial function including aberrant accumulation of enlarged mitochondria on electron microscopy. Furthermore, SPEG regulates RyR1 phosphorylation at S2902, and its loss affects JPH2 phosphorylation at multiple sites. On analyzing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix-receptor interaction and peroxisome proliferator-activated receptors signaling, which may be due to defective triad and mitochondrial abnormalities. In summary, we have elucidated the critical role of SPEG in triad as it works closely with myospryn complex, phosphorylates JPH2 and RyR1, and demonstrated that its deficiency is associated with mitochondrial abnormalities. This study emphasizes the importance of using multi-omics techniques to comprehensively analyze the molecular anomalies of rare diseases.SynopsisWe have previously linked mutations inSPEG(striated preferentially expressed protein) with a recessive form of centronuclear myopathy and/or dilated cardiomyopathy and have characterized a striated muscle-specific SPEG-deficient mouse model that recapitulates human disease with disruption of the triad structure and calcium homeostasis in skeletal muscles. In this study, we applied multi-omics approaches (interactomic, proteomic, phosphoproteomic, and transcriptomic analyses) in the skeletal muscles of SPEG-deficient mice to assess the underlying pathways associated with the pathological and molecular abnormalities.SPEG interacts with myospryn complex proteins (CMYA5, FSD2, RyR1), and its deficiency results in myospryn complex abnormalities.SPEG regulates RyR1 phosphorylation at S2902, and its loss affects JPH2 phosphorylation at multiple sites.SPEGα and SPEGβ have different interacting partners suggestive of differential function.Transcriptome analysis indicates dysregulated pathways of ECM-receptor interaction and peroxisome proliferator-activated receptor signaling.Mitochondrial defects on the transcriptome, proteome, and electron microscopy, may be a consequence of defective calcium signaling.

Background and aimsWNT2B is a canonical Wnt ligand previously thought to be fully redundant with other Wnts in the intestinal epithelium. However, humans with WNT2B deficiency have severe intestinal disease, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis.MethodsWe investigated the intestinal health ofWnt2bknock out (KO) mice. We assessed the impact of inflammatory challenge to the small intestine, using anti-CD3χ antibody, and to the colon, using dextran sodium sulfate (DSS). In addition, we generated human intestinal organoids (HIOs) from WNT2B-deficient human iPSCs for transcriptional and histological analyses.ResultsMice with WNT2B deficiency had significantly decreasedLgr5expression in the small intestine and profoundly decreased expression in the colon, but normal baseline histology. The small intestinal response to anti-CD3χ antibody was similar inWnt2bKO and wild type (WT) mice. In contrast, the colonic response to DSS inWnt2bKO mice showed an accelerated rate of injury, featuring earlier immune cell infiltration and loss of differentiated epithelium compared to WT. WNT2B-deficient HIOs showed abnormal epithelial organization and an increased mesenchymal gene signature.ConclusionWNT2B contributes to maintenance of the intestinal stem cell pool in mice and humans. WNT2B deficient mice, which do not have a developmental phenotype, show increased susceptibility to colonic injury but not small intestinal injury, potentially due to a higher reliance on WNT2B in the colon compared to the small intestine.WNT2B deficiency causes a developmental phenotype in human intestine with HIOs showing a decrease in their mesenchymal component and WNT2B-deficient patients showing epithelial disorganization.Data Transparency StatementAll RNA-Seq data will be available through online repository as indicated in Transcript profiling. Any other data will be made available upon request by emailing the study authors.

Supplementary Figure 2 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

Supplementary Figure 1 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

Recepteur d'origine nantais (RON) is a receptor tyrosine kinase closely related to c-Met. Both receptors are involved in cell proliferation, migration, and invasion, and there is evidence that both are deregulated in cancer. Receptor overexpression has been most frequently described, but other mechanisms can lead to the oncogenic activation of RON and c-Met. They include activating mutations or gene amplification for c-Met and constitutively active splicing variants for RON. We identified a novel inhibitor of RON and c-Met, compound I, and characterized its in vitro and in vivo activities. Compound I selectively and potently inhibited the kinase activity of RON and c-Met with IC50s of 9 and 4 nmol/L, respectively. Compound I inhibited hepatocyte growth factor–mediated and macrophage-stimulating protein–mediated signaling and cell migration in a dose-dependent manner. Compound I was tested in vivo in xenograft models that either were dependent on c-Met or expressed a constitutively active form of RON (RONΔ160 in HT-29). Compound I caused complete tumor growth inhibition in NIH3T3 TPR-Met and U-87 MG xenografts but showed only partial inhibition in HT-29 xenografts. The effect of compound I in HT-29 xenografts is consistent with the expression of the activating b-Raf V600E mutation, which activates the mitogen-activated protein kinase pathway downstream of RON. Importantly, tumor growth inhibition correlated with the inhibition of c-Met–dependent and RON-dependent signaling in tumors. Taken together, our results suggest that a small-molecule dual inhibitor of RON/c-Met has the potential to inhibit tumor growth and could therefore be useful for the treatment of patients with cancers where RON and/or c-Met are activated. [Cancer Res 2008;68(16):6680–7]

Supplementary Figure Legends 1-3 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

Supplementary Figure 3 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

Purpose To develop nomograms that predict the detection of clinically significant prostate cancer at diagnostic biopsy based on multiparametric prostate MRI (mpMRI), serum biomarkers, and patient clinicodemographic features. Materials and Methods Nomograms were developed from a cohort of biopsy-naïve men presenting to our 11-hospital system with a PSA of 2-20ng/mL who underwent pre-biopsy mpMRI from March 2018-June 2021 (n = 1494). The outcomes were the presence of clinically significant and high-grade prostate cancer (defined as ≥ GG2 [Grade Group 2] and ≥ GG3 prostate cancer, respectively). Using significant variables on multivariable logistic regression, individual nomograms were developed for men with PSA, % free PSA, or prostate health index (PHI) when available. The nomograms were both internally validated and evaluated in an independent cohort of 366 men presenting to our hospital system from July 2021-February 2022. Results 1031 of 1494 men (69%) underwent biopsy after initial evaluation with mpMRI, 493 (47.8%) of whom were found to have ≥ GG2 PCa, and 271 (26.3%) were found to have ≥ GG3 PCa. Age, race, highest PIRADS score, prostate health index (PHI) when available, % free PSA when available, and PSA density were significant predictors of ≥ GG2 and ≥ GG3 PCa on multivariable analysis and were used for nomogram generation. Accuracy of nomograms in both the training cohort and independent cohort were high, with areas under the curves (AUC) of ≥ 0.885 in the training cohort and ≥ 0.896 in the independent validation cohort. In our independent validation cohort, our model for ≥ GG2 prostate cancer with PHI saved 39.1% of biopsies (143/366) while only missing 0.8% of csPCa (1/124) with a biopsy threshold of 20% probability of csPCa. Conclusions Here we developed nomograms combining serum testing and mpMRI to help clinicians risk stratify patients with elevated PSA of 2-20ng/mL who are being considered for biopsy. Our nomograms are available at https://rossnm1.shinyapps.io/MynMRIskCalculator/ to aid with biopsy decisions.

Objectives: To quantify the volume and characterize the accuracy of men’s health-related content on TikTok and Instagram Methods: We conducted a search on both TikTok and Instagram for the terms: testosterone, erectile dysfunction, male infertility, semen retention, Peyronie’s disease, and vasectomy. The top 10 hashtags for each term were identified and used to estimate the total number of impressions for each term on each platform. We evaluated the top 40 posts for each topic on each platform and characterized the post by creator type, content type, and accuracy. Content accuracy was scored on a scale from one to five, with one being completely inaccurate information and five being completely accurate.Results: Overall, TikTok had more than 2.3 billion impressions and Instagran had more than 3 million posts across the men’s health topics. Semen retention had the most impressions on TikTok (1.2 billion) and posts on Instagram (1 million). Physicians created only a small portion of content comprising only 10.3% and 12.9% of total TikTok and Instagram posts, respectfully. Across all men’s health topics, the accuracy of content was poor with a score of 2.6 +/- 1.7 but physician posts were significantly more accurate than non-physician posts (4.2 vs 2.3, p < 0.001 respectfully).Conclusions: Men’s health content is popular on TikTok and Instagram but has a low level of overall accuracy. Given the higher accuracy of physician-created content, we recommend that physicians actively engage, on both individual and organizational levels, in social media to address misinformation

Striated preferentially expressed gene (SPEG), a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle-specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here, we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amount of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1 and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and β1 integrin, both of which are critical components of the focal adhesion complex. Further, β1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.

Centronuclear myopathies (CNMs) are a subtype of congenital myopathies characterized by skeletal muscle weakness and an increase in the number of central myonuclei. SPEG (striated preferentially expressed protein kinase) has been identified as the sixth gene associated with CNM, and it has been shown that striated muscle-specific Speg-knockout (KO) mice have defective triad formation, abnormal excitation-contraction coupling, and calcium mishandling. The impact of SPEG deficiency on the survival and function of myogenic cells remains to be deciphered. In this study, the authors examined the overall population, proliferation, and differentiation of myogenic cells obtained from striated muscle-specific Speg-KO mice and compared them with wild-type (WT) controls. SPEG-deficient skeletal muscles contained fewer myogenic cells, which on further study demonstrated reduced proliferation and delayed differentiation compared with those from WT muscles. Regenerative response to skeletal muscle injury in Speg-KO mice was compared with that of WT mice, leading to the identification of similar abnormalities including fewer satellite cells, fewer dividing cells, and an increase in apoptotic cells in KO mice. Overall, these results reveal specific abnormalities in myogenic cell number and behavior associated with SPEG deficiency. Similar satellite cell defects have been reported in mouse models of MTM1- and DNM2-associated CNM, suggestive of shared underlying pathways.

Pathogenic variants in the SRCAP (SNF2-related CREBBP activator protein) gene, which encodes a chromatin-remodeling ATPase, cause neurodevelopmental disorders including Floating Harbor syndrome (FLHS). Here, we report the discovery of a de novo transposon insertion in SRCAP exon 13 from trio genome sequencing in a 28-year-old female with failure to thrive, developmental delay, mood disorder and seizure disorder. The insertion was a full-length (~2.8 kb), antisense-oriented SVA insertion relative to the SRCAP transcript, bearing a 5' transduction and hallmarks of target-primed reverse transcription. The 20-bp 5' transduction allowed us to trace the source SVA element to an intron of a long non-coding RNA on chromosome 12, which is highly expressed in testis. RNA sequencing and qRT-PCR confirmed significant depletion of SRCAP expression and low-level exon skipping in the proband. This case highlights a novel disease-causing structural variant and the importance of transposon analysis in a clinical diagnostic setting.

Striated preferentially expressed protein kinase (SPEG), a myosin light chain kinase, is mutated in centronuclear myopathy (CNM) and/or dilated cardiomyopathy. No precise therapies are available for this disorder, and gene replacement therapy is not a feasible option due to the large size of SPEG. We evaluated the potential of dynamin-2 (DNM2) reduction as a potential therapeutic strategy because it has been shown to revert muscle phenotypes in mouse models of CNM caused by MTM1, DNM2, and BIN1 mutations. We determined that SPEG-β interacted with DNM2, and SPEG deficiency caused an increase in DNM2 levels. The DNM2 reduction strategy in Speg-KO mice was associated with an increase in life span, body weight, and motor performance. Additionally, it normalized the distribution of triadic proteins, triad ultrastructure, and triad number and restored phosphatidylinositol-3-phosphate levels in SPEG-deficient skeletal muscles. Although DNM2 reduction rescued the myopathy phenotype, it did not improve cardiac dysfunction, indicating a differential tissue-specific function. Combining DNM2 reduction with other strategies may be needed to target both the cardiac and skeletal defects associated with SPEG deficiency. DNM2 reduction should be explored as a therapeutic strategy against other genetic myopathies (and dystrophies) associated with a high level of DNM2.

Inhibitors of the voltage-gated sodium channel NaV1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified NaV1.7-inhibitory peptides GpTx-1 and JzTx-V from tarantula venom screens. Potency and selectivity were modulated through attribute-based positional scans of native residues via chemical synthesis. Herein, we report JzTx-V lead optimization to identify a pharmacodynamically active peptide variant. Molecular docking of peptide ensembles from NMR into a homology model-derived NaV1.7 structure supported prioritization of key residues clustered on a hydrophobic face of the disulfide-rich folded peptide for derivatization. Replacing Trp24 with 5-Br-Trp24 identified lead peptides with activity in electrophysiology assays in engineered and neuronal cells. 5-Br-Trp24 containing peptide AM-6120 was characterized in X-ray crystallography and pharmacokinetic studies and blocked histamine-induced pruritis in mice after subcutaneous administration, demonstrating systemic NaV1.7-dependent pharmacodynamics. Our data suggests a need for high target coverage based on plasma exposure for impacting in vivo end points with selectivity-optimized peptidic NaV1.7 inhibitors.

SLC25A46 (solute carrier family 25 member 46) mutations have been linked to various neurological diseases with recessive inheritance, including Leigh syndrome, optic atrophy, and lethal congenital pontocerebellar hypoplasia. SLC25A46 is expressed in the outer membrane of mitochondria, where it plays a critical role in mitochondrial dynamics. A deceased 7-month-old female infant was suspected to have Leigh syndrome. Clinical exome sequencing was non-diagnostic, but research reanalysis of the sequencing data identified two novel variants in SLC25A46: a missense (c.1039C>T, p.Arg347Cys; NM_138773, hg19) and a donor splice region variant (c.283+5G>A) in intron 1. Both variants were predicted to be damaging. Sanger sequencing of cDNA detected a single missense allele in the patient compared to control, and the SLC25A46 transcript levels were also reduced due to the splice region variant. Additionally, Western blot analysis of whole-cell lysate showed a decrease of SLC25A46 expression in proband fibroblasts, relative to control cells. Further, analysis of mitochondrial morphology revealed evidence of increased fragmentation of the mitochondrial network in proband fibroblasts, compared to control cells. Collectively, our findings suggest that these novel variants in SLC24A46, the donor splice one and the missense variant, are the cause of the neurological phenotype in this proband.

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

Paid crowdsourcing platforms suffer from low-quality work and unfair rejections, but paradoxically, most workers and requesters have high reputation scores. These inflated scores, which make high-quality work and workers difficult to find, stem from social pressure to avoid giving negative feedback. We introduce Boomerang, a reputation system for crowdsourcing that elicits more accurate feedback by rebounding the consequences of feedback directly back onto the person who gave it. With Boomerang, requesters find that their highly-rated workers gain earliest access to their future tasks, and workers find tasks from their highly-rated requesters at the top of their task feed. Field experiments verify that Boomerang causes both workers and requesters to provide feedback that is more closely aligned with their private opinions. Inspired by a game-theoretic notion of incentive-compatibility, Boomerang opens opportunities for interaction design to incentivize honest reporting over strategic dishonesty.

Efforts to improve upon the physical properties and metabolic stability of Aurora kinase inhibitor 14a revealed that potency against multidrug-resistant cell lines was compromised by increased polarity. Despite its high in vitro metabolic intrinsic clearance, 23r (AMG 900) showed acceptable pharmacokinetic properties and robust pharmacodynamic activity. Projecting from in vitro data to in vivo target coverage was not practical due to disjunctions between enzyme and cell data, complex and apparently contradictory indicators of binding kinetics, and unmeasurable free fraction in plasma. In contrast, it was straightforward to relate pharmacokinetics to pharmacodynamics and efficacy by following the time above a threshold concentration. On the basis of its oral route of administration, a selectivity profile that favors Aurora-driven pharmacology and its activity against multidrug-resistant cell lines, 23r was identified as a potential best-in-class Aurora kinase inhibitor. In phase 1 dose expansion studies with G-CSF support, 23r has shown promising single agent activity.

Human genetic evidence has identified the voltage-gated sodium channel Na

Human genetic evidence has identified the voltage-gated sodium channel NaV1.7 as an attractive target for the treatment of pain. We initially identified naphthalene sulfonamide 3 as a potent and selective inhibitor of NaV1.7. Optimization to reduce biliary clearance by balancing hydrophilicity and hydrophobicity (Log D) while maintaining NaV1.7 potency led to the identification of quinazoline 16 (AM-2099). Compound 16 demonstrated a favorable pharmacokinetic profile in rat and dog and demonstrated dose-dependent reduction of histamine-induced scratching bouts in a mouse behavioral model following oral dosing.

Recepteur d'origine nantais (RON) is a receptor tyrosine kinase closely related to c-Met. Both receptors are involved in cell proliferation, migration, and invasion, and there is evidence that both are deregulated in cancer. Receptor overexpression has been most frequently described, but other mechanisms can lead to the oncogenic activation of RON and c-Met. They include activating mutations or gene amplification for c-Met and constitutively active splicing variants for RON. We identified a novel inhibitor of RON and c-Met, compound I, and characterized its in vitro and in vivo activities. Compound I selectively and potently inhibited the kinase activity of RON and c-Met with IC50s of 9 and 4 nmol/L, respectively. Compound I inhibited hepatocyte growth factor–mediated and macrophage-stimulating protein–mediated signaling and cell migration in a dose-dependent manner. Compound I was tested in vivo in xenograft models that either were dependent on c-Met or expressed a constitutively active form of RON (RONΔ160 in HT-29). Compound I caused complete tumor growth inhibition in NIH3T3 TPR-Met and U-87 MG xenografts but showed only partial inhibition in HT-29 xenografts. The effect of compound I in HT-29 xenografts is consistent with the expression of the activating b-Raf V600E mutation, which activates the mitogen-activated protein kinase pathway downstream of RON. Importantly, tumor growth inhibition correlated with the inhibition of c-Met–dependent and RON-dependent signaling in tumors. Taken together, our results suggest that a small-molecule dual inhibitor of RON/c-Met has the potential to inhibit tumor growth and could therefore be useful for the treatment of patients with cancers where RON and/or c-Met are activated. [Cancer Res 2008;68(16):6680–7]

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinasein vitroat 0.06 μM and was inactive against 26 other kinases. GW2580 at 1 μM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophagesin vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cellsin vivomakes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.

c-Met is a receptor tyrosine kinase with oncogenic potential for which several small molecule inhibitors are currently being tested in clinical trials. There are suggestions in the literature that c-Met inhibitors could have anti-tumor effects under a variety of contexts, including in tumors in which the MET gene is amplified, contains a gain-of-function mutation or in tumors with receptor overexpression. We have developed ATP-competitive c-Met inhibitors that possess different selectivity profiles and fall into two categories. Class I molecules are highly selective for c-Met while class II molecules are multi-kinase inhibitors. These molecules were profiled in vitro and in vivo to identify biomarkers of c-Met dependence. We show that both classes of molecules inhibited c-Met kinase activity in vitro and in vivo. However, class I and class II molecules differed extensively in their spectrum of anti-tumor activity. The anti-proliferative effects of the different c-Met inhibitors were tested in 359 cancer cell lines in vitro. The activity of class I molecules was exclusively restricted to cell lines that harbor MET amplification. Western blotting in sensitive or resistant cell lines showed that c-Met inhibitors effectively block signaling downstream of c-Met only in MET amplified cell lines. In vivo, class I molecules inhibited only the growth of tumor models that are highly dependent on c-Met activity due to MET amplification or due to a hepatocyte growth factor (HGF)-driven autocrine loop. The same models that were sensitive to class I inhibitors were also sensitive to class II inhibitors. However, class II inhibitors had additional anti-tumor activities in vitro and in vivo in models that did not respond to class I inhibitors. The majority of cell lines that responded only to a class II molecule in vitro required much higher drug concentrations than those required to inhibit the growth of MET amplified cell lines. Finally, the growth inhibitory effects observed in vitro and in vivo were consistent with the selectivity profiles of the different c-Met inhibitors. Together our data demonstrate that MET amplification is a clinically identifiable, potential sensitivity biomarker for selective c-Met kinase inhibitors. c-Met expression alone is not a sensitivity biomarker in preclinical models. The spectrum of c-Met inhibitor sensitive models defined in this work could be used to understand whether other small molecule c-Met inhibitors are exquisitely selective for c-Met or harbor additional activities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3558. doi:10.1158/1538-7445.AM2011-3558

Introduction: AMG 900 is a small molecule being developed as an orally administered, highly potent and selective pan-aurora kinase inhibitor. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. The objectives of the studies were to characterize single dose pharmacokinetics (PK) of AMG 900 in preclinical species and use the preclinical PK data to predict its human PK. Methods: Following a single intravenous and oral administration of AMG 900 to mice, rats, dogs, and monkeys, blood samples were collected and processed for plasma by centrifugation. AMG 900 plasma concentrations were determined by LC-MS/MS. Non-compartmental analysis of the concentration - time data was performed to calculate PK parameters of AMG 900. Human PK were predicted based on data from preclinical species using the simple allometric scaling method assuming a human body weight of 60 kg. Results: AMG 900 exhibited low (mouse, monkey and dog) to moderate (rat) clearance in the species evaluated. The volume of distribution at steady state was less than 0.5 L/kg across species. Following intravenous administration, the compound had terminal elimination half-life ranging from 0.6 to 2.4 hours. AMG 900 was well absorbed in fasted animals across species with an oral bioavailability ranging from 31% to 104%. The clearance and volume of distribution at steady state in humans were predicted to be 0.03 L/hr/kg and 0.1 L/kg, respectively, with the predicted half-life to be approximately 2.3 hours. Bioavailability after oral administration of AMG 900 was predicted to be 75%, calculated from the mean values observed in non-clinical species. Conclusion: AMG 900 exhibits good PK properties in preclinical species and it was predicted to have low clearance and good oral bioavailability in human. Available PK results from the on-going phase 1 trial are consistent with the allometric scaling predictions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2663.

Background: The aurora family of serine-threonine kinases (aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora-A and aurora-B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora-C function appears restricted to male meiosis. Aurora-B is responsible for the direct phosphorylation of histone H3 on serine-10. Aurora-A and aurora-B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. The emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis has focused substantial interest in developing selective small molecule inhibitors for the treatment of human cancers. Objective: The aim of this study was to determine the in vitro effects of AMG 900 on a panel of tumor cell lines and profiling its antitumor activity across multiple human xenograft models. Results: AMG 900 is a novel ATP competitive small molecule inhibitor that is potent and highly selective for aurora kinases A, B, and C. In cells, AMG 900 inhibits autophosphorylation of aurora-A and aurora-B as well as the phosphorylation of histone H3. The predominant cellular response of tumor cells to AMG 900 treatment is aborted cell division, which leads to endoreduplication and cell death. The effect of AMG 900 was tested in a panel of 26 cell lines to evaluate its potential to inhibit cell proliferation across multiple tumor types. AMG 900 inhibited the proliferation of tumor cell lines at low nanomolar concentrations (EC50 values 1-6 nM), including a number of multidrug resistant (MDR) cell lines expressing ATP-binding cassette transporters (P-gp and BCRP1). In contrast, paclitaxel and three aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358) showed a loss of potency in these MDR cell lines compared to matched parental cell lines. In nude mice, oral administration of AMG 900 inhibited phosphorylation of histone H3 in tumors in a concentration- and dose-dependent manner. The effect of AMG 900 was tested in a panel of nine human xenografts representing five tumor types (breast, colon, lung, pancreatic, and uterine). Oral administration of AMG 900 at 15 mg/kg BID for two consecutive days per week or 3 mg/kg BID every day inhibited tumor growth (50-97%, P < 0.005) in all nine of the xenograft models. Importantly, three of these xenograft models are resistant to taxanes. Mice treated with efficacious doses of AMG 900 showed a transient loss of body weight and bone marrow cellularity, consistent with its on-mechanism effects on normal proliferating cells. Conclusion: Based on these preclinical activities, AMG 900 has the potential to treat advanced cancers, including tumors resistant to chemotherapeutic agents such as paclitaxel and docetaxel. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4437.

The aurora family of serine/threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Whereas aurora kinase C function appears restricted to meiosis in males, aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis. Aurora kinases A and B have been implicated in tumorigenesis, with overexpression levels correlating to clinical staging of cancers and poor prognosis. Thus, these mitotic kinases have become the subject of much interest as targets for anticancer therapy. N-(4-((3-(2-amino-4-pyrimidinyl)-2-pyridinyl)oxy)phenyl)-4-phenyl-1-phthalazinamine was a key aurora kinase inhibitor lead, possessing oral bioavailability in rats that was lacking in the anthranilamide compounds from which it was derived. This phthalazine compound possessed a key feature that was deemed important to maintain in a clinical candidate: potency against a model multidrug resistant (MDR) cell line (MES-SA Dx5) commensurate with its activity against a cell line that does not overexpress P-gp (HeLa). Improved in vivo potency was desired, as measured by suppression of the phosphorylation of the aurora kinase B substrate Histone H3 on Ser10 six hours after dosing. SAR from targeting this improvement in in vivo activity uncovered a delicate balance between protein binding, pharmacokinetic parameters, and cell potency in MES-SA Dx5 cells. AMG 900 was identified as a suitable candidate for clinical development based on its low single digit nanomolar potency against MDR cell lines, robust PD response (with complete suppression of Histone H3 phosphorylation at six hours), and high selectivity against other kinases. Oral administration of AMG 900 at a well-tolerated dose of 4 mg/kg BID inhibited tumor growth (83% TGI; p < 0.0001) in an HCT116 xenograft model. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5776.

Background: The aurora family of serine-threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora kinase C function appears restricted to male meiosis. Aurora kinase B is responsible for the direct phosphorylation of histone H3 on serine-10 (p-histone H3). Aurora kinase A and B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. With the emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis, we developed AMG 900, a novel ATP competitive small molecule inhibitor, that is highly potent and selective for aurora kinases A, B, and C. In vivo, AMG 900 inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models. Objective: The aim of this study was to establish a pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 in human tumor xenografts and surrogate tissues. Results: In nude mice, oral administration of AMG 900 inhibited p-histone H3 in tumors and bone marrow in a concentration- and dose-dependent manner. To further refine the AMG 900 PK-PD relationship, p-histone H3 IC50 values for bone marrow and dissociated COLO 205 tumors from mice were associated with plasma concentrations of 294 and 273 ng/mL, respectively. The duration of target coverage required for anti-tumor efficacy was determined to be > 6 hours per day above the in vivo p-histone H3 IC50 using different dosing schedules. To explore whether skin could be a suitable surrogate tissue for measuring p-histone H3 inhibition, tumor-bearing mice were administered AMG 900 at 3.75, 7.5, and 15 mg/kg for 3 hours. Significant suppression of p-histone H3 in skin was only observed at 15 mg/kg (p < 0.0001), whereas in tumor, a dose-dependent inhibition was achieved at all doses (p < 0.0015). To determine the feasibility of measuring p-histone H3 using fine-needle aspirate (FNA) tumor biopsies, tumor-bearing mice were administered AMG 900 at 15 mg/kg for 3 hours. Treatment with AMG 900 significantly inhibited p-histone H3 (> 99%, p < 0.0001) in COLO 205 tumors, suggesting that FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic. Conclusion: These studies demonstrate that AMG 900 inhibits p-histone H3 in tumors and surrogate tissues, although tissues such as skin may be less sensitive to assess PD effects. The use of p-histone H3 as a biomarker to determine plasma levels of AMG 900 required to inhibit aurora B activity may be useful in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3600.