Avapritinib-based SAR studies unveil a binding pocket in KIT and PDGFRA.

Avapritinib is the only potent and selective inhibitor approved for the treatment of D842V-mutant gastrointestinal stromal tumors (GIST), the most common primary mutation of the platelet-derived growth factor receptor α (PDGFRA). The approval was based on the NAVIGATOR trial, which revealed overall response rates of more than 90%. Despite this transformational activity, patients eventually progress, mostly due to acquired resistance mutations or following discontinuation due to neuro-cognitive side effects. These patients have no therapeutic alternative and face a dismal prognosis. Notable, little is known about this drug's binding mode and its medicinal chemistry development, which is instrumental for the development of the next generation of drugs. Against this background, we solve the crystal structures of avapritinib in complex with wild-type and mutant PDGFRA and stem cell factor receptor (KIT), which provide evidence and understanding of inhibitor binding and lead to the identification of a sub-pocket (Gα-pocket). We utilize this information to design, synthesize and characterize avapritinib derivatives for the determination of key pharmacophoric features to overcome drug resistance and limit potential blood-brain barrier penetration.

Indoleamine 2,3-dioxygenase attenuates inhibitor development in gene-therapy-treated hemophilia A mice.

A serious impediment to gene and protein replacement therapy in hemophilia A is the development of inhibitors. Mechanisms responsible for inhibitor development include T-cell-dependent adaptive immune responses and the CD28-B7 signaling pathway that eventually leads to the formation of antibodies directed against factor VIII (FVIII). Indoleamine 2,3-dioxygenase (IDO) is a potent immunosuppressive enzyme that can inhibit T-cell responses and induce T-cell apoptosis by regulation of tryptophan metabolism. Kynurenine, one of the metabolites of tryptophan, has been implicated as an immune modulator. Here we hypothesize that co-delivery of the genes for FVIII and IDO can attenuate inhibitor formation. Using transposon-based gene delivery, we observed long-term therapeutic FVIII expression and significantly reduced inhibitor titers when the genes were co-delivered. Co-expression of FVIII and IDO in the liver was associated with increased plasma kynurenine levels, an inhibition of T-cell infiltration and increased apoptosis of T cells within the liver. These experiments suggest that modulation of tryptophan catabolism through IDO expression provides a novel strategy to reduce inhibitor development in hemophilia gene/protein therapy.

Seasonal phenology of Bactrocera minax (Diptera: Tephritidae) in western Bhutan.

The Chinese citrus fruit fly, Bactrocera (Tetradacus) minax (Enderlein), is one of the major citrus pests in Bhutan and can cause >50% mandarin (Citrus reticulata Blanco) fruit drop. As part of the development of a management strategy for the fly in mandarin orchards, population monitoring and experimental manipulations were carried out to determine: (i) adult emergence period; (ii) adult phenology patterns; (iii) period of crop susceptibility; and (iv) period from fruit drop to pupation. In western Bhutan, adult flies emerge from the overwintering pupal stage in late April/early May. Most flies are mature by the end of May and it is inferred that mating occurs at this time: from the beginning of June males rapidly disappear from the population and by mid- to late June are rare or absent from traps. Mature females are present in the mandarin crop at the beginning of June, but very little oviposition occurs until mid-June, while most damage has occurred by mid-July. Initiation of oviposition into mandarins is almost certainly linked to crop phenology. Adult flies disappear from the orchard system during August. After fruit drop, larvae were recorded leaving the fruit to pupate within 13 days. The use of early to mid-season protein bait sprays and/or targeted use of systemic insecticides during the one month oviposition period, plus the removal of fallen fruit once every 10 days, are recommended as control strategies.

Molecular alterations resulting from frameshift mutations in peripheral myelin protein 22: implications for neuropathy severity.

Alterations in peripheral myelin protein 22 (PMP22) expression are associated with a heterogeneous group of hereditary demyelinating peripheral neuropathies. Two mutations at glycine 94, a single guanine insertion or deletion in PMP22, result in different reading frameshifts and, consequently, an extended G94fsX222 or a truncated G94fsX110 protein, respectively. Both of these autosomal dominant mutations alter the second half of PMP22 and yet are linked to clinical phenotypes with distinct severities. The G94fsX222 is associated with hereditary neuropathy with liability to pressure palsies, whereas G94fsX110 causes severe neuropathy diagnosed as Dejerine-Sottas disease or Charcot-Marie-Tooth disease type IA. To investigate the subcellular changes associated with the G94 frameshift mutations, we expressed epitope-tagged forms in primary rat Schwann cells. Biochemical and immunolabeling studies indicate that, unlike the wild-type protein, which is targeted for the plasma membrane, frameshift PMP22s are retained in the cell, prior to reaching the medial Golgi compartment. Similar to Wt-PMP22, both frameshift mutants are targeted for proteasomal degradation and accumulate in detergent-insoluble, ubiquitin-containing aggregates upon inhibition of this pathway. The extended frameshift PMP22 shows the ability to form spontaneous aggregates in the absence of proteasome inhibition. On the other hand, Schwann cells expressing the truncated protein proliferate at a significantly higher rate than Schwann cells expressing the wild-type or the extended PMP22. In summary, these results suggest that a greater potential for PMP22 aggregation is associated with a less severe phenotype, whereas dysregulation of Schwann cell proliferation is linked to severe neuropathy.

Functional cloning of SPIN-2, a nuclear anti-apoptotic protein with roles in cell cycle progression.

The balance between hematopoietic cell viability and apoptosis is regulated by exogenous growth factors, however, the molecular mechanisms by which these trophic factors exert their effects remain obscure. A functional retroviral cDNA library-based screen was employed to identify genes that prevent growth factor withdrawal-mediated apoptosis in the myeloid progenitor cell 32Dcl3. This approach identified three classes of genes: those with known roles in apoptosis (bcl-X(L) and ornithine decarboxylase); genes previously identified but not linked directly to apoptotic signaling (O-linked N-acetylglucosamine transferase); and a previously uncharacterized gene we termed SPIN-2. In 32Dcl3 cells, expression of exogenous SPIN-2 provides 25% protection from apoptosis following growth factor withdrawal compared to controls which show approximately 1-2% survival. SPIN-2 overexpression slows cell growth rates and increases the percentage of cells in G(2)/M (32% vs control cells at 12%). Immunolocalization studies indicate that myc-epitope tagged SPIN-2 proteins, which retain their anti-apoptotic function, reside in the nucleus, whereas a C-terminal deletion mutant that loses its anti-apoptotic activity is located in the cytoplasm. These studies suggest that SPIN-2 is a novel nuclear protein that functions to regulate cell cycle progression and this activity is related to the inhibition of apoptosis following the removal of essential growth factors.

Peripheral myelin protein 22 is a constituent of intercellular junctions in epithelia.

Alterations in peripheral myelin protein 22 (PMP22) gene expression are associated with a host of heritable demyelinating peripheral neuropathies, yet the function of the protein remains unknown. PMP22 expression is highest in myelinating Schwann cells of peripheral nerves; however, significant levels of PMP22 mRNAs can be detected in a variety of non-neural tissue, including epithelia. To date, PMP22 protein expression and localization in non-neural tissues have not been studied in detail. In adult rat liver and intestine, and cultured epithelial cells, we detected PMP22-like immunoreactivity associated with markers of the tight junctional complex, including zonula occludens 1 (ZO-1) and occludin. Upon disruption of intercellular contacts, PMP22 was internalized into vesicles that were immunoreactive for both anti-occludin and anti-PMP22 antibodies. Nonionic detergent extraction of cultured epithelial cells did not solubilize PMP22, as the majority of the protein remained in the detergent insoluble fraction, as did ZO-1 and occludin. We also observed the targeting of exogenous myc-tagged PMP22 to apical cell junctions in polarized epithelia and to anti-ZO-1 antibody immunoreactive cell contacts of L fibroblasts. These studies support a role for PMP22 at intercellular junctions of epithelia and may indicate a similar function in myelinating Schwann cells. Furthermore, our findings could provide an explanation for certain phenotypes of PMP22 neuropathy mice that cannot be accounted for by dysmyelination.

Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells.

Childhood acute lymphoblastic leukaemia (ALL) is treated by combination chemotherapy with a number of drugs, always including the enzyme L-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance are a significant problem. In vitro studies have demonstrated increased asparagine synthetase (AS) expression in ASNase-resistant cells, which has led to the hypothesis that elevated AS activity permits drug-resistant survival. The data presented show that not only is elevated AS expression a property of ASNase-resistant MOLT-4 human leukaemia cells, but that short-term (12 h) treatment of the cells with ASNase causes a relatively rapid induction of AS expression. The results also document that the elevated expression of AS in ASNase-resistant cells is not fully reversible, even 6 weeks after ASNase removal from the culture medium. Furthermore, ASNase resistance, assessed as both drug-insensitive cell growth rates and decreased drug-induced apoptosis, parallels this irreversible AS expression. Mimicking the elevated AS activity in ASNase-resistant cells by overexpression of the human AS protein by stable retroviral transformation of parental MOLT4 cells is sufficient to induce the ASNase-resistance phenotype. These data document that ASNase resistance in ALL cells is a consequence of elevated AS expression and that although other drug-induced metabolic changes occur, they are secondary to the increased asparagine biosynthetic rate.

Transcriptional induction of prostaglandin G/H synthase-2 by basic fibroblast growth factor.

In serum-free mouse osteoblastic MC3T3-E1 cells, basic fibroblastic growth factor (bFGF) induced mRNA and protein for prostaglandin G/H synthase-2 (PGHS-2), the major enzyme in arachidonic acid (AA) conversion to prostaglandins. mRNA accumulation peaked at 1 h with bFGF 1 nM. In cells stably transfected with a 371-bp PGHS-2 promoter-luciferase reporter, bFGF stimulated luciferase activity, which peaked at 2-3 h with bFGF 1-10 nM. In the presence of exogenous AA, bFGF stimulated PGE2 production, which paralleled luciferase activity. In serum-free neonatal mouse calvarial cultures, bFGF stimulated PGE2 production in the absence of exogenous AA. bFGF stimulated PGHS-2 mRNA accumulation, which peaked at 2-4 h and then decreased; there were later mRNA elevations at 48 and 96 h that were inhibited by indomethacin. In both MC3T3-E1 cells and neonatal calvariae, bFGF produced smaller and slower increases in PGHS-1 mRNA levels than for PGHS-2. bFGF stimulated bone resorption in mouse calvariae with a maximal increase of 80% at 1 nM. Stimulation was partially inhibited by nonsteroidal anti-inflammatory drugs. We conclude that bFGF rapidly stimulates PGE2 production in osteoblasts, largely through transcriptional regulation of PGHS-2, and that prostaglandins mediate some of bFGF's effects on bone resorption.

v-src induction of the TIS10/PGS2 prostaglandin synthase gene is mediated by an ATF/CRE transcription response element.

We recently reported the cloning of a mitogen-inducible prostaglandin synthase gene, TIS10/PGS2. In addition to growth factors and tumor promoters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region between -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60v-src induction in 3T3 cells. This region contains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGTCA) and E-box (CACGTG) sequences. Gel shift-oligonucleotide competition experiments with nuclear extracts from cells stably transfected with a temperature-sensitive v-src gene demonstrate that the CGTCACGTG sequence can bind proteins at both the ATF/CRE and E-box sequences. Dominant-negative CREB and Myc proteins that bind DNA, but do not transactivate, block v-src induction of a luciferase reporter driven by the first 80 nucleotides of the TIS10/PGS2 promoter. Mutational analysis distinguishes which TIS10/PGS2 cis-acting element mediates pp60v-src induction. E-box mutation has no effect on the fold induction in response to pp60v-src. In contrast, ATF/CRE mutation attenuates the pp60v-src response. Antibody supershift and methylation interference experiments demonstrate that CREB and at least one other ATF transcription factor in these extracts bind to the TIS10/PGS2 ATF/CRE element. Expression of a dominant-negative ras gene also blocks TIS10/PGS2 induction by v-src. Our data suggest that Ras mediates pp60v-src activation of an ATF transcription factor, leading to induced TIS10/PGS2 expression via the ATF/CRE element of the TIS10/PGS2 promoter. This is the first description of v-src activation of gene expression via an ATF/CRE element.

Platelet-activating factor and retinoic acid synergistically activate the inducible prostaglandin synthase gene.

Platelet-activating factor (PAF), a potent lipid mediator generated in cell injury and in the inflammatory and immune responses, promotes transcriptional activation of several primary response genes. TIS10/PGS-2 is a primary response gene encoding the inducible form of prostaglandin synthase. The inductive effects of PAF and retinoic acid (RA), alone and in combination, were studied with the regulatory region of TIS10/PGS-2 transfected into an exponentially growing glioblastoma-neuroblastoma NG108-15 hybrid in the human SH-SY5Y neuroblastoma or in the NIH 3T3 cell. RA alone exhibited only a small inductive effect. However, in the presence of RA (100 nM), a PAF-dependent (1-50 nM) synergistic activation of luciferase reporter constructs driven by regulatory regions of the TIS10/PGS-2 gene was found. The hetrazepine BN-50730, an antagonist selective for intracellular PAF binding sites, inhibited PAF and RA induction of luciferase from the TIS10/PGS-2 promoter. Thus, the intracellular PAF binding site is involved in TIS10/PGS-2 expression. Induction is rapid, suggesting that the combination of PAF and RA activates a preexisting latent transcription factor(s). Deletion studies restrict the major PAF and RA cis-acting response element of the TIS10/PGS-2 gene to a 70-nucleotide sequence as an intracellular inducer of TIS10/PGS-2 expression. The synergistic effect of RA and PAF represents an unusual convergence of nuclear signaling pathways by which, through the modulation of preexisting transcription factors, specific gene expression can be upregulated. PAF-dependent induction of TIS10/PGS-2 expression may play a role in cell injury, differentiation, inflammation, and immune responses.

Expression of the protein product of the prostaglandin synthase-2/TIS10 gene in mitogen-stimulated Swiss 3T3 cells.

TIS10/PGS-2 encodes a prostaglandin synthase (PGS) distinct from the previously described enzyme PGS-1 (EC 1.14.99.1). We have now generated antipeptide antisera, directed to an amino acid sequence unique to the murine TIS10/PGS-2 protein, which specifically recognize the TIS10/PGS-2 antigen. TIS10/PGS-2 protein was undetectable in quiescent Swiss 3T3 cells. The level of TIS10/PGS-2 protein peaked between 6 and 8 h following phorbol ester stimulation of cells, then declined to basal levels after 18-24 h. Synthesis of TIS10/PGS-2 protein was dramatically increased in the second hour following mitogen stimulation and remained elevated for several hours. The half-life of the TIS10/PGS-2 protein was 4 h. Immunofluorescence studies demonstrated a perinuclear and cytoplasmic localization of the TIS10/PGS-2 antigen. As expected, detection of induced TIS10/PGS-2 antigen was dependent on protein synthesis. Metabolically labeled TIS10/PGS-2 protein migrated as a 71/73-kDa doublet following immunoprecipitation. Dexamethasone blocked both the TPA- and serum-induced appearance of TIS10/PGS-2 antigen. These studies demonstrate the existence of a mitogen-inducible, glucocorticoid-inhibitable, immunologically distinct prostaglandin synthase protein.

Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase.

The TIS10 cDNA was cloned as a primary response gene transcript whose mRNA rapidly accumulates in 3T3 cells treated with serum, polypeptide growth factors, or phorbol esters. The sequence of the TIS10 cDNA suggested that the gene encodes a protein with strong similarities to prostaglandin G/H synthase/cyclooxygenase (EC 1.14.99.1). Transient transfection into COS-1 cells of an expression vector driving the TIS10 cDNA leads to production and secretion of prostaglandin E2. Microsomes prepared from COS-1 cells transfected with this construct demonstrate both hydroperoxidase and cyclooxygenase activities similar to that demonstrated by cells transfected with a vector encoding the ovine prostaglandin G/H synthase. These data demonstrate that the TIS10 gene encodes a functional prostaglandin synthase/cyclooxygenase distinct from the prostaglandin synthase/cyclooxygenase whose cDNAs and/or genes have previously been cloned from sheep, mouse, and man. The structure of the TIS10 gene, determined by a combination of sequencing of genomic clones and polymerase chain reactions from genomic clones, demonstrates remarkable exon-intron conservation with the human prostaglandin synthase/cyclooxygenase gene. A 1-kilobase sequence located immediately proximal to the start site of transcription of the TIS10 gene can confer phorbol ester and serum inducibility to a luciferase reporter gene following transient transfection into NIH 3T3 cells, suggesting that this region of the gene is responsible for transcriptional regulation of the TIS10 gene by mitogens in fibroblasts.

Structure and expression of TIS21, a primary response gene induced by growth factors and tumor promoters.

The TIS21 gene is a primary response gene that is induced rapidly and transiently in 3T3 cells by the tumor promoter and mitogen tetradecanoyl phorbol acetate. The predicted open reading frame of the TIS21 cDNA encodes a protein of 158 amino acids with no obvious similarity to any known protein. Antiserum prepared to TIS21 recombinant protein produced in Escherichia coli precipitates a 17-kDa protein from Swiss 3T3 cells. The 2040-nucleotide 3'-untranslated region of the cDNA includes an unusual T18 sequence. The TIS21 gene has a single 1.4-kilobase intron which interrupts the open reading frame and is otherwise identical to the cDNA sequence. The 5'-flanking sequence of the TIS21 gene contains TATA and CAAT box-type sequences, three potential Sp1 sites, two putative cyclic AMP response elements, two potential AP1 binding elements, and an AP2 element. A possible Z-DNA structure of 29 AC repeats is present 660 nucleotides from the start of transcription. Expression from a luciferase reporter construct containing a 460-nucleotide fragment of the TIS21 promoter is induced by tetradecanoyl phorbol acetate, forskolin, epidermal growth factor, and serum, despite the absence of a consensus serum response element.

TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue.

TIS10 is a primary response gene whose cDNA was cloned as a result of its rapid, superinducible expression in Swiss 3T3 cells in response to 12-O-Tetradecanoylphorbol-13-acetate. The 5'-untranslated region of the 3.9-kilobase TIS10 message contains only 124 nucleotides, whereas the 3'-untranslated region is almost 2 kilobases in length. Within this long 3' region, there are multiple repeats of the sequence ATTTA, a sequence often present in rapidly degraded mRNA species. Primer extension revealed that the TIS10 cDNA begins 16 base pairs downstream of the transcription start site for the TIS10 gene. The TIS10 cDNA encodes a predicted protein of 604 amino acids. A computer search identified striking similarities between the predicted TIS10 protein product and the murine, sheep, and human prostaglandin synthase/cyclooxygenase proteins. The TIS10 protein has many of the same conserved amino acids that are thought to be important for cyclooxygenase function. TIS10 mRNA is undetectable by Northern analysis in quiescent 3T3 cells. The TIS10 gene is rapidly and transiently induced by forskolin and serum, as well as by 12-O-tetradecanoylphorbol-13-acetate, in Swiss 3T3 cells. These agents elicit far more dramatic changes in TIS10 mRNA levels than in cyclooxygenase mRNA levels. The expression of the TIS10 gene appears to be highly cell type-restricted in cultured cell lines; of 12 cell lines tested under superinducing conditions, only the rodent embryonic Swiss 3T3 and Rat1 cell lines expressed TIS10 gene.