GAS1 is required for NOTCH-dependent facilitation of SHH signaling in the ventral forebrain neuroepithelium.

Growth arrest-specific 1 (GAS1) acts as a co-receptor to patched 1, promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in induced pluripotent stem cell-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating NOTCH signaling, which is essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives NOTCH pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating NOTCH and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.

Tyrosine Kinase Inhibitors. 20. Optimization of Substituted Quinazoline and Pyrido[3,4-d]pyrimidine Derivatives as Orally Active, Irreversible Inhibitors of the Epidermal Growth Factor Receptor Family.

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of quinazoline- and pyrido[3,4-d]pyrimidine-based analogues of the irreversible pan-erbB inhibitor, canertinib. Cyclic amine bearing crotonamides were determined to provide rapid inhibition of cellular erbB1 autophosphorylation and good metabolic stability in liver microsome and hepatocyte assays. The influence of 4-anilino substitution on pan-erbB inhibitory potency was investigated. Several anilines were identified as providing potent, reversible pan-erbB inhibition. Optimum 4- and 6-substituents with known 7-substituents provided preferred irreversible inhibitors for pharmacodynamic testing in vivo. Quinazoline 54 and pyrido[3,4-d]pyrimidine 71 were identified as clearly superior to canertinib. Both compounds possess a piperidinyl crotonamide Michael acceptor and a 3-chloro-4-fluoroaniline, indicating these as optimized 6- and 4-substituents, respectively. Pharmacokinetic comparison of compounds 54 and 71 across three species selected compound 54 as the preferred candidate. Compound 54 (PF-00299804) has been assigned the nomenclature of dacomitinib and is currently under clinical evaluation.

Ihog and Boi elicit Hh signaling via Ptc but do not aid Ptc in sequestering the Hh ligand.

Hedgehog (Hh) proteins are secreted molecules essential for tissue development in vertebrates and invertebrates. Hh reception via the 12-pass transmembrane protein Patched (Ptc) elicits intracellular signaling through Smoothened (Smo). Hh binding to Ptc is also proposed to sequester the ligand, limiting its spatial range of activity. In Drosophila, Interference hedgehog (Ihog) and Brother of ihog (Boi) are two conserved and redundant transmembrane proteins that are essential for Hh pathway activation. How Ihog and Boi activate signaling in response to Hh remains unknown; each can bind both Hh and Ptc and so it has been proposed that they are essential for both Hh reception and sequestration. Using genetic epistasis we established here that Ihog and Boi, and their orthologs in mice, act upstream or at the level of Ptc to allow Hh signal transduction. In the Drosophila developing wing model we found that it is through Hh pathway activation that Ihog and Boi maintain the boundary between the anterior and posterior compartments. We dissociated the contributions of Ptc from those of Ihog/Boi and, surprisingly, found that cells expressing Ptc can retain and sequester the Hh ligand without Ihog and Boi, but that Ihog and Boi cannot do so without Ptc. Together, these results reinforce the central role for Ptc in Hh binding in vivo and demonstrate that, although Ihog and Boi are dispensable for Hh sequestration, they are essential for pathway activation because they allow Hh to inhibit Ptc and thereby relieve its repression of Smo.

Contribution of a single host genetic locus to mouse adenovirus type 1 infection and encephalitis.

UNLABELLED: Susceptibility to mouse adenovirus type 1 (MAV-1) is mouse strain dependent; susceptible mice die from hemorrhagic encephalomyelitis. The MAV-1 susceptibility quantitative trait locus Msq1 accounts for ~40% of the phenotypic (brain viral load) variance that occurs between resistant BALB/c and susceptible SJL mice after MAV-1 infection. Using an interval-specific congenic mouse strain (C.SJL-Msq1(SJL)), in which the SJL-derived allele Msq1(SJL) is present in a BALB/c background, we demonstrate that Msq1(SJL) controls the development of high brain viral titers in response to MAV-1 infection, yet does not account for the total extent of brain pathology or mortality in SJL mice. C.SJL-Msq1(SJL) mice had disruption of the blood-brain barrier and increased brain water content after MAV-1 infection, but these effects occurred later and were not as severe, respectively, as those noted in infected SJL mice. As expected, BALB/c mice showed minimal pathology in these assays. Infection of SJL- and C.SJL-Msq1(SJL)-derived primary mouse brain endothelial cells resulted in loss of barrier properties, whereas BALB/c-derived cells retained their barrier properties despite being equally capable of supporting MAV-1 infection. Finally, we provide evidence that organ pathology and inflammatory cell recruitment to the brain following MAV-1 infection were both influenced by Msq1. These results validate Msq1 as an important host factor in MAV-1 infection and refine the major role of the locus in development of MAV-1 encephalitis. They further suggest that additional host factors or gene interactions are involved in the mechanism of pathogenesis in MAV-1-infected SJL mice. IMPORTANCE: A successful viral infection requires both host and viral factors; identification of host components involved in viral replication and pathogenesis is important for development of therapeutic interventions. A genetic locus (Msq1) controlling mouse adenovirus type 1 (MAV-1) brain infection was previously identified. Genes in Msq1 belong to the same family of genes associated with susceptibility to other encephalitic viruses, HIV-1 and West Nile virus. We constructed an interval-specific congenic mouse strain to examine the contribution of Msq1 to MAV-1 susceptibility and brain morbidity. We compared infected resistant, susceptible, and congenic mice regarding known MAV-1 disease manifestations in the brain (survival, viral loads, blood-brain barrier disruption, edema, mouse brain endothelial cell barrier properties, pathology, and inflammatory cell recruitment) to determine the extent to which Msq1 influences MAV-1 infection outcome. Our results showed that Msq1 is a critical host genetic factor that controls many aspects of MAV-1 infection.

Overlapping roles and collective requirement for the coreceptors GAS1, CDO, and BOC in SHH pathway function.

Secreted Hedgehog (HH) ligands signal through the canonical receptor Patched (PTCH1). However, recent studies implicate three additional HH-binding, cell-surface proteins, GAS1, CDO, and BOC, as putative coreceptors for HH ligands. A central question is to what degree these coreceptors function similarly and what their collective requirement in HH signal transduction is. Here we provide evidence that GAS1, CDO, and BOC play overlapping and essential roles during HH-mediated ventral neural patterning of the mammalian neural tube. Specifically, we demonstrate two important roles for these molecules: an early role in cell fate specification of multiple neural progenitors and a later role in motor neuron progenitor maintenance. Most strikingly, genetic loss-of-function experiments indicate an obligatory requirement for GAS1, CDO, and BOC in HH pathway activity in multiple tissues.

The major locus for mouse adenovirus susceptibility maps to genes of the hematopoietic cell surface-expressed LY6 family.

Susceptibility to mouse adenovirus type 1 is associated with the major quantitative trait locus Msq1. Msq1 was originally mapped to a 13-Mb region of mouse chromosome (Chr) 15 in crosses between SJL/J and BALB/cJ inbred mice. We have now narrowed Msq1 to a 0.75-Mb interval from 74.68 to 75.43 Mb, defined by two anonymous markers, rs8259436 and D15Spn14, using data from 1396 backcross mice. The critical interval includes 14 Ly6 or Ly6-related genes, including Ly6a (encoding Sca-1/TAP), Ly6e (Sca-2/Tsa1), Ly6g (Gr-1), and gpihbp1 (GPI-anchored high-density lipoprotein-binding protein 1), as well as the gene encoding an aldosterone synthase (Cyp11b2). The Ly6 family members are attractive candidates for virus susceptibility genes because their products are GPI-anchored membrane proteins expressed on lymphoid and myeloid cells, with proposed functions in cell adhesion and cell signaling. To determine interstrain variation in susceptibility and produce additional resources for cloning Msq1, we assayed the susceptibility phenotype of four previously untested inbred mouse strains. Susceptibility of strain 129S6/SvEvTac was subsequently localized to the Ly6 complex region, using polymorphic genetic markers on Chr 15 in a population of 271 (129S6/SvEvTac x BALB/cJ)F(1) x BALB/cJ backcross mice. We identified a major 129S6/SvEvTac susceptibility allele, Msq1(129S6), on Chr 15 in the same region as Msq1(SJL). The results indicate that a major host factor in mouse adenovirus type 1 susceptibility is likely to be a member of the Ly6 gene family.

Antitumor activity and pharmacokinetic properties of PF-00299804, a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor.

Signaling through the erbB receptor family of tyrosine kinases contributes to the proliferation, differentiation, migration, and survival of a variety of cell types. Abnormalities in members of this receptor family have been shown to play a role in oncogenesis, thus making them attractive targets for anticancer treatments. PF-00299804 is a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor currently in phase I clinical trials. PF-00299804 is believed to irreversibly inhibit erbB tyrosine kinase activity through binding at the ATP site and covalent modification of nucleophilic cysteine residues in the catalytic domains of erbB family members. Oral administration of PF-00299804 causes significant antitumor activity, including marked tumor regressions in a variety of human tumor xenograft models that express and/or overexpress erbB family members or contain the double mutation (L858R/T790M) in erbB1 (EGFR) associated with resistance to gefitinib and erlotinib. Furthermore, PF-00299804 shows exceptional distribution to human tumor xenografts and excellent pharmacokinetic properties across species.

PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to gefitinib.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib are effective treatments for a subset of non-small cell lung cancers. In particular, cancers with specific EGFR-activating mutations seem to be the most sensitive to these agents. However, despite their initial response, such cancers almost invariably develop resistance. In 50% of such cancers, a secondary EGFR mutation, T790M, has been identified that renders gefitinib and erlotinib ineffective inhibitors of EGFR kinase activity. Thus, there is a clinical need to develop novel EGFR inhibitors that can effectively inactivate T790M-containing EGFR proteins. In this study, we evaluate the effectiveness of a novel compound, PF00299804, an irreversible pan-ERBB inhibitor. The results from these studies show that PF00299804 is a potent inhibitor of EGFR-activating mutations as well as the EGFR T790M resistance mutation both in vitro and in vivo. Additionally, PF00299804 is a highly effective inhibitor of both the wild-type ERBB2 and the gefitinib-resistant oncogenic ERBB2 mutation identified in lung cancers. These preclinical evaluations support further clinical development of PF00299804 for cancers with mutations and/or amplifications of ERBB family members.

Tyrosine kinase inhibitors. 19. 6-Alkynamides of 4-anilinoquinazolines and 4-anilinopyrido[3,4-d]pyrimidines as irreversible inhibitors of the erbB family of tyrosine kinase receptors.

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of alkynamide analogues of quinazoline- and pyrido[3,4-d]pyrimidine-based compounds. The compounds were prepared by coupling the appropriate 6-aminoquinazolines or 6-aminopyrido[3,4-d]pyrimidines with alkynoic acids, using EDCI.HCl in pyridine. The compounds showed pan-erbB enzyme inhibition but were on average about 10-fold more potent against erbB1 than against erbB2 and erbB4. For cellular inhibition, the nature of the alkylating side chains was an important determinant, with 5-dialkylamino-2-pentynamide type Michael acceptors providing the highest potency. This is suggested to be due to an improved ability of the amine to participate in an autocatalysis of the Michael reaction with enzyme cysteine residues. Pyrido[3,4-d]pyrimidine analogue 39 was selected for in vivo evaluation and achieved tumor regressions at 10 mg/kg in the A431 human epidermoid carcinoma and at 40 mg/kg for the SF767 human glioblastoma and the SKOV3 human ovarian carcinoma. Complete stasis was observed at 40 mg/kg in the BXPC3 human pancreatic carcinoma as well as in the H125 human non-small-cell lung carcinoma.