Wild-Type KRAS Is Required for Panitumumab Efficacy in Patients With Metastatic Colorectal Cancer.

PURPOSE: Panitumumab, a fully human antibody against the epidermal growth factor receptor (EGFR), has activity in a subset of patients with metastatic colorectal cancer (mCRC). Although activating mutations in KRAS, a small G-protein downstream of EGFR, correlate with poor response to anti-EGFR antibodies in mCRC, their role as a selection marker has not been established in randomized trials. PATIENTS AND METHODS: KRAS mutations were detected using polymerase chain reaction on DNA from tumor sections collected in a phase III mCRC trial comparing panitumumab monotherapy to best supportive care (BSC). We tested whether the effect of panitumumab on progression-free survival (PFS) differed by KRAS status. RESULTS: KRAS status was ascertained in 427 (92%) of 463 patients (208 panitumumab, 219 BSC). KRAS mutations were found in 43% of patients. The treatment effect on PFS in the wild-type (WT) KRAS group (hazard ratio [HR], 0.45; 95% CI: 0.34 to 0.59) was significantly greater (P < .0001) than in the mutant group (HR, 0.99; 95% CI, 0.73 to 1.36). Median PFS in the WT KRAS group was 12.3 weeks for panitumumab and 7.3 weeks for BSC. Response rates to panitumumab were 17% and 0%, for the WT and mutant groups, respectively. WT KRAS patients had longer overall survival (HR, 0.67; 95% CI, 0.55 to 0.82; treatment arms combined). Consistent with longer exposure, more grade III treatment-related toxicities occurred in the WT KRAS group. No significant differences in toxicity were observed between the WT KRAS group and the overall population. CONCLUSION: Panitumumab monotherapy efficacy in mCRC is confined to patients with WT KRAS tumors. KRAS status should be considered in selecting patients with mCRC as candidates for panitumumab monotherapy.

Limited differential expression of miRNAs and other small RNAs in LPS-stimulated human monocytes.

Monocytes are a distinct subset of myeloid cells with diverse functions in early inflammatory immune modulation. While previous studies have surveyed the role of miRNA regulation on different myeloid cell lines and primary cultures, the time-dependent kinetics of inflammatory stimulation on miRNA expression and the relationship between miRNA-to-target RNA expression have not been comprehensively profiled in monocytes. In this study, we use next-generation sequencing and RT-PCR assays to analyze the non-coding small RNA transcriptome of unstimulated and lipopolysaccharide (LPS)-stimulated monocytes at 6 and 24 hours. We identified a miRNA signature consisting of five mature miRNAs (hsa-mir-146a, hsa-mir-155, hsa-mir-9, hsa-mir-147b, and hsa-mir-193a) upregulated by LPS-stimulated monocytes after 6 hours and found that most miRNAs were also upregulated after 24 hours of stimulation. Only one miRNA gene was down-regulated and no other small RNAs were found dysregulated in monocytes after LPS treatment. In addition, novel tRNA-derived fragments were also discovered in monocytes although none showed significant changes upon LPS stimulation. Interrogation of validated miRNA targets by transcriptomic analysis revealed that absolute expression of most miRNA targets implicating in innate immune response decreased over time in LPS-stimulated monocytes although their expression patterns along the treatment were heterogeneous. Our findings reveal a potential role by which selective miRNA upregulation and stable expression of other small RNAs enable monocytes to develop finely tuned cellular responses during acute inflammation.

Haploinsufficiency of the Insulin Receptor in the Presence of a Splice-Site Mutation in Ppp2r2a Results in a Novel Digenic Mouse Model of Type 2 Diabetes.

Insulin resistance in mice typically does not manifest as diabetes due to multiple compensatory mechanisms. Here, we present a novel digenic model of type 2 diabetes in mice heterozygous for a null allele of the insulin receptor and an N-ethyl-N-nitrosourea-induced alternative splice mutation in the regulatory protein phosphatase 2A (PP2A) subunit PPP2R2A. Inheritance of either allele independently results in insulin resistance but not overt diabetes. Doubly heterozygous mice exhibit progressive hyperglycemia, hyperinsulinemia, and impaired glucose tolerance from 12 weeks of age without significant increase in body weight. Alternative splicing of Ppp2r2a decreased PPP2R2A protein levels. This reduction in PPP2R2A containing PP2A phosphatase holoenzyme was associated with decreased serine/threonine protein kinase AKT protein levels. Ultimately, reduced insulin-stimulated phosphorylated AKT levels were observed, a result that was confirmed in Hepa1-6, C2C12, and differentiated 3T3-L1 cells knocked down using Ppp2r2a small interfering RNAs. Altered AKT signaling and expression of gluconeogenic genes in the fed state contributed to an insulin resistance and hyperglycemia phenotype. This model demonstrates how genetic changes with individually small phenotypic effects interact to cause diabetes and how differences in expression of hypomorphic alleles of PPP2R2A and potentially other regulatory proteins have deleterious effects and may therefore be relevant in determining diabetes risk.

A mutation in Ampd2 is associated with nephrotic syndrome and hypercholesterolemia in mice.

BACKGROUND: Previously, we identified three loci affecting HDL-cholesterol levels in a screen for ENU-induced mutations in mice and discovered two mutated genes. We sought to identify the third mutated gene and further characterize the mouse phenotype. METHODS: We engaged, DNA sequencing, gene expression profiling, western blotting, lipoprotein characterization, metabolomics assessment, histology and electron microscopy in mouse tissues. RESULTS: We identify the third gene as Ampd2, a liver isoform of AMP Deaminase (Ampd), a central component of energy and purine metabolism pathways. The causative mutation was a guanine-to-thymine transversion resulting in an A341S conversion in Ampd2. Ampd2 homozygous mutant mice exhibit a labile hypercholesterolemia phenotype, peaking around 9 weeks of age (251 mg/dL vs. wildtype control at 138 mg/dL), and was evidenced by marked increases in HDL, VLDL and LDL. In an attempt to determine the molecular connection between Ampd2 dysfunction and hypercholesterolemia, we analyzed hepatic gene expression and found the downregulation of Ldlr, Hmgcs and Insig1 and upregulation of Cyp7A1 genes. Metabolomic analysis confirmed an increase in hepatic AMP levels and a decrease in allantoin levels consistent with Ampd2 deficiency, and increases in campesterol and ß-sitosterol. Additionally, nephrotic syndrome was observed in the mutant mice, through proteinuria, kidney histology and effacement and blebbing of podocyte foot processes by electron microscopy. CONCLUSION: In summary we describe the discovery of a novel genetic mouse model of combined transient nephrotic syndrome and hypercholesterolemia, resembling the human disorder.

Sclerostin expression is induced by BMPs in human Saos-2 osteosarcoma cells but not via direct effects on the sclerostin gene promoter or ECR5 element.

Sclerostin is a secreted inhibitor of Wnt signaling and plays an essential role in the regulation of bone mass. The expression of sclerostin is largely restricted to osteocytes although its mode of transcriptional regulation is not well understood. We observed regulated expression of sclerostin mRNA and protein that was directly correlated with the mineralization response in cultured human Saos-2 osteosarcoma cells and rat primary calvarial cells. Sclerostin mRNA and protein levels were increased following treatment of cells with BMP2, BMP4 and BMP7. Analysis of deletion mutants from the -7.4 kb upstream region of the human sclerostin promoter did not reveal any specific regions that were responsive to BMPs, Wnt3a, PTH, TGFß1 or Activin A in Saos-2 cells. The downstream ECR5 element did not show enhancer activity in Saos-2 cells and also was not affected when Saos-2 cells were treated with BMPs or PTH. Genome-wide microarray analysis of Saos-2 cells treated with BMP2 showed significant changes in expression of several transcription factors with putative consensus DNA binding sites in the region of the sclerostin promoter. However, whereas most factors tested showed either a range of inhibitory activity (DLX family, MSX2, HEY1, SMAD6/7) or lack of activity on the sclerostin promoter including SMAD9, only MEF2B showed a positive effect on both the promoter and ECR5 element. These results suggest that the dramatic induction of sclerostin gene expression by BMPs in Saos-2 cells occurs indirectly and is associated with late stage differentiation of osteoblasts and the mineralization process.

FusionMap: detecting fusion genes from next-generation sequencing data at base-pair resolution.

MOTIVATION: Next generation sequencing technology generates high-throughput data, which allows us to detect fusion genes at both transcript and genomic levels. To detect fusion genes, the current bioinformatics tools heavily rely on paired-end approaches and overlook the importance of reads that span fusion junctions. Thus there is a need to develop an efficient aligner to detect fusion events by accurate mapping of these junction-spanning single reads, particularly when the read gets longer with the improvement in sequencing technology. RESULTS: We present a novel method, FusionMap, which aligns fusion reads directly to the genome without prior knowledge of potential fusion regions. FusionMap can detect fusion events in both single- and paired-end datasets from either RNA-Seq or gDNA-Seq studies and characterize fusion junctions at base-pair resolution. We showed that FusionMap achieved high sensitivity and specificity in fusion detection on two simulated RNA-Seq datasets, which contained 75 nt paired-end reads. FusionMap achieved substantially higher sensitivity and specificity than the paired-end approach when the inner distance between read pairs was small. Using FusionMap to characterize fusion genes in K562 chronic myeloid leukemia cell line, we further demonstrated its accuracy in fusion detection in both single-end RNA-Seq and gDNA-Seq datasets. These combined results show that FusionMap provides an accurate and systematic solution to detecting fusion events through junction-spanning reads. AVAILABILITY: FusionMap includes reference indexing, read filtering, fusion alignment and reporting in one package. The software is free for noncommercial use at (http://www.omicsoft.com/fusionmap).

Motesanib inhibits Kit mutations associated with gastrointestinal stromal tumors.

BACKGROUND: Activating mutations in Kit receptor tyrosine kinase or the related platelet-derived growth factor receptor (PDGFR) play an important role in the pathogenesis of gastrointestinal stromal tumors (GIST). METHODS: This study investigated the activity of motesanib, an inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3; PDGFR; and Kit, against primary activating Kit mutants and mutants associated with secondary resistance to imatinib. Single- and double-mutant isoforms of Kit were evaluated for their sensitivity to motesanib or imatinib in autophosphorylation assays and in Ba/F3 cell proliferation assays. RESULTS: Motesanib inhibited Kit autophosphorylation in CHO cell lines expressing primary activating mutations in exon 9 (AYins503-504, IC50 = 18 nM) and exon 11 (V560 D, IC50 = 5 nM; Delta552-559, IC50 = 1 nM). Motesanib also demonstrated activity against kinase domain mutations conferring imatinib resistance (V560D/V654A, IC50 = 77 nM; V560D/T670I, IC50 = 277 nM; Y823 D, IC50 = 64 nM) but failed to inhibit the imatinib-resistant D816V mutant (IC50 > 3000 nM). Motesanib suppressed the proliferation of Ba/F3 cells expressing Kit mutants with IC50 values in good agreement with those observed in the autophosphorylation assays. CONCLUSIONS: In conclusion, our data suggest that motesanib possesses inhibitory activity against primary Kit mutations and some imatinib-resistant secondary mutations.

A comparability study of 5 commercial KRAS tests.

BACKGROUND: Activating mutations in the KRAS gene occur frequently in human tumors, including colorectal carcinomas; most mutations occur in codons 12 and 13. Mutations in KRAS have been associated with poor response to anti-epidermal growth factor receptor antibodies. Therefore, an accurate and readily available analysis of KRAS mutational status is needed. The aim of this study was to evaluate concordance between KRAS assays performed by 6 different laboratories. METHODS: Forty formalin-fixed paraffin-embedded colorectal cancer tumor samples were obtained. Sample sections were submitted for KRAS mutation analysis to 5 independent commercial laboratories (Agencourt, Gentris, Genzyme, HistoGeneX, and Invitek) and to the Amgen DNA Sequencing Laboratory for direct polymerase chain reaction sequencing. The assay used by Invitek is no longer commercially available and has been replaced by an alternative technique. Results from the commercial services were compared with those from Amgen direct sequencing by kappa statistics. RESULTS: KRAS mutations were observed in codon 12 and/or 13 in 20 of 40 (50%) samples in Amgen direct sequencing assays. Results from HistoGeneX (kappa = 0.95), Genzyme (kappa = 0.94), and Agencourt (kappa = 0.94) were in almost perfect agreement with these results, and the results from Gentris were in substantial agreement with the results from Amgen (kappa = 0.75). The Invitek allele-specific assay demonstrated slight agreement (kappa = 0.13). CONCLUSIONS: This study provides data on the comparability of KRAS mutational analyses. The results suggest that most (but not all) commercial services provide analysis that is accurate and comparable with direct sequencing.

Biochemical characterization of AMG 102: a neutralizing, fully human monoclonal antibody to human and nonhuman primate hepatocyte growth factor.

AMG 102 is a fully human monoclonal antibody that selectively targets and neutralizes hepatocyte growth factor/scatter factor (HGF/SF). A detailed biochemical and functional characterization of AMG 102 was done to support its clinical development for the treatment of cancers dependent on signaling through the HGF/SF:c-Met pathway. In competitive equilibrium binding experiments, AMG 102 bound to human and cynomolgus monkey HGF with affinities of approximately 19 pmol/L and 41 pmol/L, respectively. However, AMG 102 did not detect mouse or rabbit HGF on immunoblots. Immunoprecipitation experiments showed that AMG 102 preferentially bound to the mature, active form of HGF, and incubation of AMG 102/HGF complexes with kallikrein protease indicated that AMG 102 had no apparent effect on proteolytic processing of the inactive HGF precursor. AMG 102 inhibited human and cynomolgus monkey HGF-induced c-Met autophosphorylation in PC3 cells with IC(50) values of 0.12 nmol/L and 0.24 nmol/L, respectively. AMG 102 also inhibited cynomolgus monkey HGF-induced migration of human MDA-MB-435 cells but not rat HGF-induced migration of mouse 4T1 cells. Epitope-mapping studies of recombinant HGF molecules comprising human/mouse chimeras and human-to-mouse amino acid substitutions showed that amino acid residues near the NH(2)-terminus of the beta-chain are critical for AMG 102 binding. Bound AMG 102 protected one trypsin protease cleavage site near the NH(2)-terminus of the beta-chain of human HGF, further substantiating the importance of this region for AMG 102 binding. Currently, AMG 102 is in phase II clinical trials in a variety of solid tumor indications. Mol Cancer Ther; 9(2); 400-9.

Pharmacological effects of nonselective and subtype-selective nicotinic acetylcholine receptor agonists in animal models of persistent pain.

Nicotinic acetylcholine receptors (nAChRs) are longstanding targets for a next generation of pain therapeutics, but the nAChR subtypes that govern analgesia remain unknown. We tested a series of nicotinic agonists, including many molecules used or tried clinically, on a panel of cloned neuronal nAChRs for potency and selectivity using patch-clamp electrophysiology and a live cell-based fluorescence assay. Nonselective nicotinic agonists as well as compounds selective either for alpha4beta2 or for alpha7 nAChRs were then tested in the formalin and complete Freund's adjuvant models of pain. Nonselective nAChR agonists ABT-594 and varenicline were effective analgesics. By contrast, the selective alpha4beta2 agonist ispronicline and a novel alpha4beta2-selective potentiator did not appear to produce analgesia in either model. alpha7-selective agonists reduced the pain-related endpoint, but the effect could be ascribed to nonspecific reduction of movement rather than to analgesia. Neither selective nor nonselective alpha7 nicotinic agonists affected the release of pro-inflammatory cytokines in response to antigen challenge. Electrophysiological recordings from spinal cord slice showed a strong nicotine-induced increase in inhibitory synaptic transmission that was mediated partially by alpha4beta2 and only minimally by alpha7 subtypes. Taken with previous studies, the results suggest that agonism of alpha4beta2 nAChRs is necessary but not sufficient to produce analgesia, and that the spinal cord is a key site where the molecular action of nAChRs produces analgesia.

Antihepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammation-induced anemia.

Iron maldistribution has been implicated in multiple diseases, including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino acid peptide. Hepcidin is induced by inflammation, causes iron to be sequestered, and thus, potentially contributes to AI. Human hepcidin (hHepc) overexpression in mice caused an iron-deficient phenotype, including stunted growth, hair loss, and iron-deficient erythropoiesis. It also caused resistance to supraphysiologic levels of erythropoiesis-stimulating agent, supporting the hypothesis that hepcidin may influence response to treatment in AI. To explore the role of hepcidin in inflammatory anemia, a mouse AI model was developed with heat-killed Brucella abortus treatment. Suppression of hepcidin mRNA was a successful anemia treatment in this model. High-affinity antibodies specific for hHepc were generated, and hHepc knock-in mice were produced to enable antibody testing. Antibody treatment neutralized hHepc in vitro and in vivo and facilitated anemia treatment in hHepc knock-in mice with AI. These data indicate that antihepcidin antibodies may be an effective treatment for patients with inflammatory anemia. The ability to manipulate iron metabolism in vivo may also allow investigation of the role of iron in a number of other pathologic conditions.

Phase II study of safety and efficacy of motesanib in patients with progressive or symptomatic, advanced or metastatic medullary thyroid cancer.

PURPOSE: This phase II study investigated the efficacy and tolerability of motesanib, an investigational, highly selective inhibitor of vascular endothelial growth factor receptors 1, 2, and 3; platelet-derived growth factor receptor; and Kit in advanced medullary thyroid cancer (MTC). PATIENTS AND METHODS: Patients with locally advanced or metastatic, progressive or symptomatic MTC received motesanib 125 mg/d orally for up to 48 weeks or until unacceptable toxicity or disease progression. The primary end point was objective response by independent review. Other end points included duration of response, progression-free survival, safety, pharmacokinetics, and changes in tumor markers. RESULTS: Of 91 enrolled patients who received motesanib, two (2%) achieved objective response (95% CI, 0.3% to 7.7%); their duration of response was 32 weeks (censored) and 21 weeks (disease progressed). Eighty-one percent of patients had stable disease (48% had durable stable disease > or = 24 weeks), 8% had disease progression as best response, and 9% were not evaluated; 76% experienced a decrease from baseline in target lesion measurement. Median progression-free survival was 48 weeks (95% CI, 43 to 56 weeks). Among patients with tumor marker analysis, 69 (83%) of 83 and 63 (75%) of 84 had decreased serum calcitonin and carcinoembryonic antigen during treatment, respectively, compared with baseline. The most common treatment-related adverse events were diarrhea (41%), fatigue (41%), hypothyroidism (29%), hypertension (27%), and anorexia (27%). In pharmacokinetic analyses, motesanib trough concentrations were lower compared with differentiated thyroid cancer patients from the same study. CONCLUSION: Although the objective response rate was low, a significant proportion of MTC patients (81%) achieved stable disease while receiving motesanib.

Activity of panitumumab alone or with chemotherapy in non-small cell lung carcinoma cell lines expressing mutant epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) kinase domain mutations cause hyperresponsiveness to ligand and hypersensitivity to small-molecule tyrosine kinase inhibitors. However, little is known about how these mutations respond to antibodies against EGFR. We investigated the activity of panitumumab, a fully human anti-EGFR monoclonal antibody, in vitro in mutant EGFR-expressing non-small cell lung carcinoma (NSCLC) cells and in vivo with chemotherapy in xenograft models. Mutant EGFR-expressing NSCLC cells (NCI-H1975 [L858R+T790M] and NCI-H1650 [Delta746-750]) and CHO cells were treated with panitumumab before EGF stimulation to assess the inhibition of EGFR autophosphorylation. Established tumors were treated with panitumumab (25, 100, or 500 mug/mouse twice a week) alone or with docetaxel (10 or 20 mg/kg once a week) or cisplatin (7.5 mg/kg once a week). Antitumor activity and levels of proliferation markers were analyzed. Treatment of mutant EGFR-expressing CHO and NSCLC cells with panitumumab inhibited ligand-dependent autophosphorylation. In NCI-H1975 and NCI-H1650 xenografts, treatment with panitumumab alone or with cisplatin inhibited tumor growth compared with control (P < 0.0003). With panitumumab plus docetaxel, enhanced antitumor activity was seen in both xenografts versus panitumumab alone. Panitumumab treatment alone decreased Ki-67 and phospho- mitogen-activated protein kinase (pMAPK) staining in both xenografts compared with control. Docetaxel enhanced panitumumab activity in NCI-H1650 xenografts (decreased Ki-67 and pMAPK staining by >60%) when compared with either agent alone. Panitumumab inhibits ligand-induced EGFR phosphorylation, tumor growth, and markers of proliferation alone or with docetaxel in NSCLC cell lines that express clinically observed EGFR kinase domain mutations, including the small-molecule tyrosine kinase inhibitor-resistant T790M mutation.

New variants in the Enpp1 and Ptpn6 genes cause low BMD, crystal-related arthropathy, and vascular calcification.

A large genome-wide, recessive, N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen was performed on a mixed C57BL/6J and C3H.SW-H2/SnJ mouse background to identify genes regulating bone mass. Approximately 6500 male and female G(3) hybrid mice were phenotyped at 8 and 10 wk of age by DXA analysis for evidence of changes in unadjusted or body weight-adjusted BMD or BMC. Phenodeviant lines were identified based on statistical criteria that included a false discovery rate (FDR) <20% and Z-score >2.8. Genome-wide mapping scans were initiated on 22 lines, with evidence of high or low BMD or BMC that deviated by approximately -30% to +50% from the means. Several lines were discontinued as showing lack of heritability, but two heritable lines were identified with narrow chromosomal regions that allowed sequencing of potential mutant candidate genes. Novel mutations were identified in the Enpp1 (C397S) gene on chromosome 10 (line 4482) and the Ptpn6 (I482F) gene on chromosome 6 (line 4489) that were both associated with low bone mass. In addition, the phenotype of the Enpp1 mice showed a striking joint disease and calcification of blood vessels including the aorta, myocardium, and renal arteries and capillaries. These results support a role for the Enpp1 gene in the pathogenesis associated with mineralization of articular cartilage and vascular calcification. This work confirms the utility of the chemical mutagenesis approach for identification of potential disease genes and confirms the role of Enpp1 and Ptpn6 in regulating mineralization and skeletal bone mass.

An antibody to IP-10 is a potent antagonist of cell migration in vitro and in vivo and does not affect disease in several animal models of inflammation.

IP-10 secretion is induced by pro-inflammatory cytokines and mediates the migration of CXCR3+ cells. Its elevation in clinical samples has been associated with multiple inflammatory diseases and its antagonism has been reported to be effective in several animal models of inflammatory disease. We generated a mouse anti-mouse IP-10 monoclonal antibody (mAb; Clone 20A9) that specifically bound murine IP-10 with high affinity and inhibited in vitro IP-10 induced BaF3/mCXCR3 cell migration with an IC(50) of approximately 4 nM. The 20A9 mAb was completely absorbed in vivo and had dose proportional pharmacokinetic exposure with a serum half life of 2.4-6 days. The 20A9 mAb inhibited IP-10 mediated T-cell recruitment to the airways, indicating that it is effective in vivo. However, administration of the 20A9 mAb had no significant effect on disease in mouse models of delayed type hypersensitivity, collagen induced arthritis, cardiac allograft transplantation tolerance, EAE or CD4+ CD45RBHi T-cell transfer-induced IBD. These data suggest that the 20A9 mAb can antagonize IP-10 mediated chemotaxis in vitro and in vivo and that this is insufficient to cause a therapeutic benefit in multiple mouse models of inflammatory disease.

Targeted deletion of Nm23/nucleoside diphosphate kinase A and B reveals their requirement for definitive erythropoiesis in the mouse embryo.

The ubiquitously expressed nucleoside diphosphate kinases (Nm23/NDPK/Awd) are a large family of multifunctional enzymes implicated in nucleic acid metabolism and in normal and abnormal development. Here, we describe the generation and characterization of NDPK A- and B-deficient (Nme1(-/-)/Nme2(-/-)) mice in which >95% of the enzyme activity is eliminated. These mice are undersized, die perinatally, and exhibit a spectrum of hematological phenotypes including severe anemia, impaired maturation of erythrocytes, and abnormal hematopoiesis in the liver and bone marrow. Flow cytometric analysis of developing Nme1(-/-)/Nme2(-/-) erythroid cells indicated that the major iron transport receptor molecule TfR1 is attenuated concomitant with a reduction of intracellular iron, suggesting that TfR1 is a downstream target of NDPKs and that reduced iron in Nme1(-/-)/Nme2(-/-) erythroblasts is inhibiting their development. We conclude that Nm23/NDPKs play critical roles in definitive erythroid development. Our novel mouse model also links erythropoiesis and nucleotide metabolism.

Pancreatic islet expression profiling in diabetes-prone C57BLKS/J mice reveals transcriptional differences contributed by DBA loci, including Plagl1 and Nnt.

BACKGROUND: C57BLKS/J (BLKS) mice are susceptible to islet exhaustion in insulin-resistant states as compared with C57BL6/J (B6) mice, as observed by the presence of the leptin receptor (Lepr) allele, Leprdb/db. Furthermore, DBA2/J (DBA) mice are also susceptible to beta-cell failure and share 25% of their genome with BLKS; thus the DBA genome may contribute to beta-cell dysfunction in BLKS mice. RESULTS: Here we show that BLKS mice exhibit elevated insulin secretion, as evidenced by improved glucose tolerance and increased islet insulin secretion compared with B6 mice, and describe interstrain transcriptional differences in glucose response. Transcriptional differences between BLKS and B6 mice were identified by expression profiling of isolated islets from both strains. Genomic mapping of gene expression differences demonstrated a significant association of expression differences with DBA loci in BLKS mice (P = 4x10-27). CONCLUSION: Two genes, Nicotinamide nucleotide transhydrogenase (Nnt) and Pleiomorphic adenoma gene like 1 (Plagl1), were 4 and 7.2-fold higher respectively in BLKS islets, and may be major contributors to increased insulin secretion by BLKS islets. Contrary to reports for B6 mice, BLKS mice do not harbor a mutant Nnt gene. We detected 16 synonymous polymorphisms and a two-amino acid deletion in the Plagl1 gene in BLKS mice. Several inflammatory glucose-responsive genes are expressed at a higher level in BLKS, suggesting an inflammatory component to BLKS islet dysfunction. This study describes physiological differences between BLKS and B6 mice, and provides evidence for a causative role of the DBA genome in beta-cell dysfunction in BLKS mice.

Identification of three loci affecting HDL-cholesterol levels in a screen for chemically induced recessive mutations in mice.

We conducted a genome-wide screen using the mutagen N-ethyl-N-nitrosourea to identify recessive mutations in genes that lead to altered lipid traits in mice. We screened 7,546 G3 mice that were of mixed C57BL/6J (B6) x C3.SW-H2(b)/SnJ (C3) genomes and identified three pedigrees with differences in plasma HDL-cholesterol. Genome scan analyses mapped three distinct loci to chromosomes 3, 4, and 7. An S1748L missense mutation was identified in ABCA1 in one pedigree with undetectable levels of HDL-cholesterol and resulted in reduced protein levels. This phenotype was completely penetrant, semi-dominant, and cosegregated with high plasma triglycerides. Mice in a second pedigree had very high levels of plasma total cholesterol and HDL-cholesterol (up to 800 mg/dl total cholesterol). Despite a high degree of phenotype lability and reduced penetrance, an I68N missense mutation was identified in the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). Finally, a second high HDL-cholesterol pedigree of mice, again with a highly labile phenotype and reduced penetrance, was mapped to a 7 Mb locus on chromosome 3. These results illustrate the use of a hybrid background for simultaneous screening and mapping of mutagenized pedigrees of mice and identification of three novel alleles of HDL-cholesterol phenotypes.

Motesanib diphosphate in progressive differentiated thyroid cancer.

BACKGROUND: The expression of vascular endothelial growth factor (VEGF) is characteristic of differentiated thyroid cancer and is associated with aggressive tumor behavior and a poor clinical outcome. Motesanib diphosphate (AMG 706) is a novel oral inhibitor of VEGF receptors, platelet-derived growth-factor receptor, and KIT. METHODS: In an open-label, single-group, phase 2 study, we treated 93 patients who had progressive, locally advanced or metastatic, radioiodine-resistant differentiated thyroid cancer with 125 mg of motesanib diphosphate, administered orally once daily. The primary end point was an objective response as assessed by an independent radiographic review. Additional end points included the duration of the response, progression-free survival, safety, and changes in serum thyroglobulin concentration. RESULTS: Of the 93 patients, 57 (61%) had papillary thyroid carcinoma. The objective response rate was 14%. Stable disease was achieved in 67% of the patients, and stable disease was maintained for 24 weeks or longer in 35%; 8% had progressive disease as the best response. The Kaplan-Meier estimate of the median duration of the response was 32 weeks (the lower limit of the 95% confidence interval [CI] was 24; the upper limit could not be estimated because of an insufficient number of events); the estimate of median progression-free survival was 40 weeks (95% CI, 32 to 50). Among the 75 patients in whom thyroglobulin analysis was performed, 81% had decreased serum thyroglobulin concentrations during treatment, as compared with baseline levels. The most common treatment-related adverse events were diarrhea (in 59% of the patients), hypertension (56%), fatigue (46%), and weight loss (40%). CONCLUSIONS: Motesanib diphosphate can induce partial responses in patients with advanced or metastatic differentiated thyroid cancer that is progressive. (ClinicalTrials.gov number, NCT00121628.)

Association of K-ras mutational status and clinical outcomes in patients with metastatic colorectal cancer receiving panitumumab alone.

BACKGROUND: Identifying predictive biomarkers is important to optimally treat patients. This analysis evaluated the association of K-ras, BRAF, and PIK3CA gene mutations with tumor resistance to panitumumab alone. PATIENTS AND METHODS: From 3 phase II panitumumab metastatic colorectal cancer (mCRC) studies, 62 of 533 patient samples were available. Mutations were identified from genomic DNA by sequencing. RESULTS: Of the 62 samples, 24 (38.7%) harbored a K-ras mutation, and 38 (61.3%) were wild type. In the wild-type K-ras group, 11% of patients had a partial response (PR), 53% had stable disease (SD), and 37% had progressive disease (PD). In the mutant K-ras group, 21% of patients had SD, and 79% of patients had PD; there were no responses. The absence of a K-ras mutation was associated with response to panitumumab (PR vs. SD vs. PD; P = .0028). The hazard ratio for wild-type versus mutant K-ras was 0.4 (95% CI, 0.2-0.7) for progression-free survival and 0.5 (95% CI, 0.3-0.9) for overall survival. Four patients had a V600E BRAF mutation, and 2 patients had a PIK3CA mutation. CONCLUSION: These data suggest that patients with mCRC with activating K-ras mutations are less likely to respond to panitumumab alone. The small sample size limits us from defining a predictive role of PIK3CA and BRAF mutations for panitumumab treatment.