Hyperactive Mutant Research Articles

Construction of Efficient Recombinant Strain Through Genome Shuffling in Marine Endophytic Fusarium sp. ALAA-20 for Improvement Lovastatin Production Using Agro-Industrial Wastes

During searching for marine endophytic fungi that have the potential to effectively produce lovastatin using agro-industrial residues, the endophytic Fusarium sp. ALAA-20 was isolated from marine sponge Aplysina fistularis with lovastatin production of 4.51 mg/gds in the solid-state fermentation of rice straw. Lovastatin yield was improved by enhancing approaches including mutation induction and three consecutive cycles of genome shuffling of the producing strain ALAA-20 to obtain the hyperactive recombinant strain FR3/1 with a higher yield of lovastatin (52.1 mg/gds), which is 11.55- and 3.1-fold of the parent strain ALAA-20 and the hyperactive mutant NE21 (16.8 mg/gds), respectively. Moreover, optimization of the solid-state fermentation of oil cakes by the recombinant FR3/1 significantly increased the lovastatin production by 64.19-, 17.23- and 5.56-fold over the selected wild strain (ALAA-20), mutant (NE21) and fusant (FR3/1) prior to the optimization process. Lovastatin obtained from the recombinant FR3/1 strain showed pleiotropic effects such as antibacterial activity against drug-resistant Staphylococcus, Pseudomonas and Klebsiella species and antifungal activity against Candida, Aspergillus, Fusarium and Trichophyton species. New recombinant strain FR3/1 lovastatin showed potent antitumor activity against liver (HepG-2), colon (HCT-116), breast (MCF-7) and lung (A-549) cancer cell lines with IC50 equal to 8.0, 7.2, 4.8 and 9.1 μM, respectively.

Phosphatidylinositol 3,5-bisphosphate regulates Ca2+ transport during yeast vacuolar fusion through the Ca2+ ATPase Pmc1.

The transport of Ca2+ across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles under hyperosmotic stress become fragmented through fission events that requires the release of Ca2+ stores through the TRP channel Yvc1. This requires the phosphorylation of phosphatidylinositol-3-phosphate (PI3P) by the PI3P-5-kinase Fab1 to produce transient PI(3,5)P2 pools. Ca2+ is also released during vacuole fusion upon trans-SNARE complex assembly, however, its role remains unclear. The effect of PI(3,5)P2 on Ca2+ flux during fusion was independent of Yvc1. Here, we show that while low levels of PI(3,5)P2 were required for Ca2+ uptake into the vacuole, increased concentrations abolished Ca2+ efflux. This was as shown by the addition of exogenous dioctanoyl PI(3,5)P2 or increased endogenous production of by the hyperactive fab1T2250A mutant. In contrast, the lack of PI(3,5)P2 on vacuoles from the kinase dead fab1EEE mutant showed delayed and decreased Ca2+ uptake. The effects of PI(3,5)P2 were linked to the Ca2+ pump Pmc1, as its deletion rendered vacuoles resistant to the effects of excess PI(3,5)P2 . Experiments with Verapamil inhibited Ca2+ uptake when added at the start of the assay, while adding it after Ca2+ had been taken up resulted in the rapid expulsion of Ca2+ . Vacuoles lacking both Pmc1 and the H+ /Ca2+ exchanger Vcx1 lacked the ability to take up Ca2+ and instead expelled it upon the addition of ATP. Together these data suggest that a balance of efflux and uptake compete during the fusion pathway and that the levels of PI(3,5)P2 can modulate which path predominates.

An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian Applications.

As non-viral transgenic vectors, the piggyBac transposon system represents an attractive tool for gene delivery to achieve a long-term gene expression in immunotherapy applications due to its large cargo capacity, its lack of a trace of transposon and of genotoxic potential, and its highly engineered structure. However, further improvements in transpose activity are required for industrialization and clinical applications. Herein, we established a one-plasmid effective screening system and a two-step high-throughput screening process in yeast to isolate hyperactive mutants for mammalian cell applications. By applying this screening system, 15 hyperactive piggyBac transposases that exhibited higher transpose activity compared with optimized hyPBase in yeast and four mutants that showed higher transpose activity in mammalian cells were selected among 3000 hyPBase mutants. The most hyperactive transposase, bz-hyPBase, with four mutation sites showed an ability to yield high-efficiency editing in Chinese hamster ovarian carcinoma (CHO) cells and T cells, indicating that they could be expanded for gene therapy approaches. Finally, we tested the potential of this screening system in other versions of piggyBac transposase.

The neonatal microenvironment programs innate γδ T cells through the transcription factor STAT5.

IL-17-producing RORγt+ γδ T cells (γδT17 cells) are innate lymphocytes that participate in type 3 immune responses during infection and inflammation. Herein, we show that γδT17 cells rapidly proliferate within neonatal lymph nodes and gut, where, upon entry, they upregulate T-bet and coexpress IL-17, IL-22, and IFN-γ in a STAT3- and retinoic acid-dependent manner. Neonatal expansion was halted in mice conditionally deficient in STAT5, and its loss resulted in γδT17 cell depletion from all adult organs. Hyperactive STAT5 mutant mice showed that the STAT5A homolog had a dominant role over STAT5B in promoting γδT17 cell expansion and downregulating gut-associated T-bet. In contrast, STAT5B preferentially expanded IFN-γ-producing γδ populations, implying a previously unknown differential role of STAT5 gene products in lymphocyte lineage regulation. Importantly, mice lacking γδT17 cells as a result of STAT5 deficiency displayed a profound resistance to experimental autoimmune encephalomyelitis. Our data identify that the neonatal microenvironment in combination with STAT5 is critical for post-thymic γδT17 development and tissue-specific imprinting, which is essential for infection and autoimmunity.

Identification of novel regulators of STAT3 activity.

STAT3 mediates signalling downstream of cytokine and growth factor receptors where it acts as a transcription factor for its target genes, including oncogenes and cell survival regulating genes. STAT3 has been found to be persistently activated in many types of cancers, primarily through its tyrosine phosphorylation (Y705). Here, we show that constitutive STAT3 activation protects cells from cytotoxic drug responses of several drug classes. To find novel and potentially targetable STAT3 regulators we performed a kinase and phosphatase siRNA screen with cells expressing either a hyperactive STAT3 mutant or IL6-induced wild type STAT3. The screen identified cell division cycle 7-related protein kinase (CDC7), casein kinase 2, alpha 1 (CSNK2), discoidin domain-containing receptor 2 (DDR2), cyclin-dependent kinase 8 (CDK8), phosphatidylinositol 4-kinase 2-alpha (PI4KII), C-terminal Src kinase (CSK) and receptor-type tyrosine-protein phosphatase H (PTPRH) as potential STAT3 regulators. Using small molecule inhibitors targeting these proteins, we confirmed dose and time dependent inhibition of STAT3-mediated transcription, suggesting that inhibition of these kinases may provide strategies for dampening STAT3 activity in cancers.

Activated by interaction

Signal Transduction Cytokines trigger immune responses when they bind to their cognate receptors. Class I cytokine receptors rely on the associated Janus kinase 2 (JAK2) to initiate signal transduction. There has been debate over whether activation involves ligand-induced dimerization of these receptors or ligand-induced conformational change of preformed dimers. Wilmes et al. imaged cytokine receptors in the plasma membranes of live human cells by single-molecule fluorescence microscopy and observed ligand-induced dimerization. They found that the JAK2 pseudokinase domains contribute to dimerization and that hyperactive JAK2 mutants promote dimerization, consistent with the model that dimerization triggers activation. Science , this issue p. [643][1] [1]: /lookup/doi/10.1126/science.aaw3242

Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations.

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.

Efficient tissue-type specific expression of target genes in a tetracycline-controlled manner from the ubiquitously active Eef1a1 locus

Using an efficient gene targeting approach, we developed a novel mouse line that expresses the tetracycline-controlled transactivator (tTA) from the constitutively active Eef1a1 locus in a Cre recombinase-inducible manner. The temporally and spatially controlled expression of the EF1-LSL-tTA knockin and activation of tTA-driven responder transgenes was tested using four transgenic lines that express Cre under tissue-specific promoters of the pancreas, mammary gland and other secretory tissues, as well as an interferon-inducible promoter. In all models, the endogenous Eef1a1 promoter facilitated a cell-type-specific activation of target genes at high levels without exogenous enhancer elements. The applicability of the EF1-LSL-tTA strain for biological experiments was tested in two studies related to mammary gland development and tumorigenesis. First, we validated the crucial role of active STAT5 as a survival factor for functionally differentiated epithelial cells by expressing a hyperactive STAT5 mutant in the mammary gland during postlactational remodeling. In a second experiment, we assessed the ability of the EF1-tTA to initiate tumor formation through upregulation of mutant KRAS. The collective results show that the EF1-LSL-tTA knockin line is a versatile genetic tool that can be applied to constitutively express transgenes in specific cell types to examine their biological functions at defined developmental stages.

Gene-expression profile reveals the genetic and acquired phenotypes of hyperactive mutant SPORTS rat.

Spontaneously Running Tokushima Shikoku (SPORTS) rat is a hyperactive rat strain. However, the causative mutation of this phenotype has not yet been identified. To investigate the molecular basis for the unique phenotype of SPORTS rats, we examined gene-expression profiles by microarray analyses. Among adenylate kinase isozymes that maintain the homeostasis of cellular adenine nucleotide composition in the cell, only adenylate kinase 1 is highly up-regulated in both exercised and sedentary SPORTS rats compared with wild-type (WT) rats, 5.5-fold and 3.3-fold, respectively. Further comparative analyses revealed that genes involved in glucose metabolism were up-regulated in skeletal muscle tissue of exercised SPORTS rats compared with sedentary mutants, whereas genes related to extracellular matrix or region were down-regulated compared with WT rats. In brain tissue of sedentary SPORTS rats, genes associated with defense and catecholamine metabolism were highly expressed compared with WT rats. These findings suggest that genetic mutation(s) in SPORTS rat remodels metabolic demands through differentially regulating gene expression regardless of exercise. Therefore, the SPORTS rats are useful animal model not only for further examining the effects of exercise on metabolism but also for deeply studying the molecular basis how mutation affect the psychological motivation with spontaneous voluntary exercise phenotype. J. Med. Invest. 67 : 51-61, February, 2020.

The Arabidopsis cryptochrome 2 I404F mutant is hypersensitive and shows flavin reduction even in the absence of light.

The cryptochrome photoreceptor mutant cry2I404F exhibits hyperactivity in the dark, hypersensitivity in different light conditions, and in contrast to the wild-type protein, its flavin chromophore is reducible even in the absence of light. Plant cryptochromes (cry) are blue-light photoreceptors involved in multiple signaling pathways and various photomorphogenic responses. One biologically hyperactive mutant of a plant cryptochrome that was previously characterized is Arabidopsis cry1L407F (Exner et al. in Plant Physiol 154:1633-1645, 2010). Protein sequence alignments of different cryptochromes revealed that L407 in cry1 corresponds to I404 in cry2. Point mutation of Ile to Phe in cry2 in this position created a novel mutant. The present study provided a baseline data on the elucidation of the properties of cry2I404F. This mutant was still able to bind ATP-triggering conformational changes, as confirmed by partial tryptic digestion and thermo-FAD assays. Surprisingly, the FAD cofactor of cry2I404F was reduced by the addition of reductant even in the absence of light. In vivo, cry2I404F exhibited a cop phenotype in the dark and hypersensitivity to various light conditions compared to cry2 wild type. Overall, these data suggest that the hypersensitivity to red and blue light and hyperactivity of this novel mutant in the dark can be mostly accounted to structural alterations brought forth by the Ile to Phe mutation at position 404 that allows reduction of the flavin chromophore even in the absence of light.

Regulation of plant ER oxidoreductin 1 (ERO1) activity for efficient oxidative protein folding

In the endoplasmic reticulum (ER), ER oxidoreductin 1 (ERO1) catalyzes intramolecular disulfide-bond formation within its substrates in coordination with protein-disulfide isomerase (PDI) and related enzymes. However, the molecular mechanisms that regulate the ERO1-PDI system in plants are unknown. Reduction of the regulatory disulfide bonds of the ERO1 from soybean, GmERO1a, is catalyzed by enzymes in five classes of PDI family proteins. Here, using recombinant proteins, vacuum-ultraviolet circular dichroism spectroscopy, biochemical and protein refolding assays, and quantitative immunoblotting, we found that GmERO1a activity is regulated by reduction of intramolecular disulfide bonds involving Cys-121 and Cys-146, which are located in a disordered region, similarly to their locations in human ERO1. Moreover, a GmERO1a variant in which Cys-121 and Cys-146 were replaced with Ala residues exhibited hyperactive oxidation. Soybean PDI family proteins differed in their ability to regulate GmERO1a. Unlike yeast and human ERO1s, for which PDI is the preferred substrate, GmERO1a directly transferred disulfide bonds to the specific active center of members of five classes of PDI family proteins. Of these proteins, GmPDIS-1, GmPDIS-2, GmPDIM, and GmPDIL7 (which are group II PDI family proteins) failed to catalyze effective oxidative folding of substrate RNase A when there was an unregulated supply of disulfide bonds from the C121A/C146A hyperactive mutant GmERO1a, because of its low disulfide-bond isomerization activity. We conclude that regulation of plant ERO1 activity is particularly important for effective oxidative protein folding by group II PDI family proteins.

Skin Associated Staphylococcus Aureus Contributes to Disease Progression in CTCL

It has been proposed that bacteria play a direct role in progression of cutaneous T cell lymphoma (CTCL), although definitive evidence is missing, and the underlying mechanism of how microbes contribute to disease progression remains unknown. The skin of CTCL patients is frequently colonized with Staphylococcus aureus (S. aureus) strains and infections with hospital and community associated strains of S. aureus are a frequent cause of morbidity and mortality among patients with advanced CTCL. Here we provide a comprehensive analysis of the association between CTCL and S. aureus colonization, and use our unique pre-clinical animal model of CTCL to determine the cause-effect relationship between skin-associated S. aureus and CTCL progression. To understand the relationship between bacterial colonization and CTCL we collected skin swabs from active lesions, unaffected skin and nares of CTCL patients to perform S. aureus cultures and 16S rRNA gene sequencing. Skin swabs of psoriasis patients and healthy donors served as controls. The frequency of S. aureus colonization determined by culture based techniques revealed that >65% of advanced stage patients had S. aureus present at lesional/tumor sites, while corresponding sites in patients with psoriasis and in healthy controls rarely had detectable S. aureus. Colonization rates correlated positively with the disease stage. Unbiased, 16s sequencing based analysis of the skin microbiome from advanced CTCL patients revealed that the overall skin microbiome of these patients is distinct from that of healthy individuals and patients with psoriasis. A lower phylogenetic diversity and significantly higher relative abundance of Staphylococcus species was found in CTCL patients. To determine the causal relationship between skin flora and progression of CTCL we used our mouse model of CTCL and assessed disease progression in both conventionally housed specific-pathogen-free (SPF) conditions and in germ free (GF) isolators using a standardized clinical score. The CD4CreSTAT3stopfl/+ mice express a hyper-active STAT3C mutant protein selectively in T lymphocytes and virtually all mutant mice develop T cell infiltration in the epidermis causing skin lesions resembling CTCL, by eight months of age. In contrast to the SPF housed animals, GF mice remained disease free or developed only a mild phenotype (clinical score 1 out of 5) after 11 months of follow-up. Notably, when GF CD4CreSTAT3stopfl/+ mice were transitioned to SPF conditions they all developed advanced disease. Finally, we examined the role of T cell antigen receptor (TCR) signaling in mediating the transformation of T lymphocytes. R26STAT3Cstopfl/+CD4Cre Rag2KO OTII mice express only OVA-specific TCRs. T cells from R26STAT3Cstopfl/+ CD4Cre Stim1fl/fl mice express a normal TCR repertoire, but exhibit defective T cell receptor signaling due to compromised calcium influx. Both strains failed to develop typical skin lesions, suggesting an essential role for TCR interaction with tumor microenvironment and microbial antigens in the pathogenesis of CTCL. In conclusion, we demonstrate a strong correlation between CTCL staging and rates of S. aureus colonization. Our study supports a cause-effect relationship between skin flora and CTCL oncogenesis. We propose that CTCL represents an antigen driven malignancy. Further studies using mono-colonization with single bacterial strains are needed to further interrogate the role of specific bacteria. Disclosures Hymes: Celgene: Consultancy. Odum:Micreos human Health B.V: Consultancy. Geskin:Merck: Other: Supported/Contracted Research; UpToDate: Patents & Royalties: Royalty, Receipt of Intellectual Property Rights / Patent Holder; Mallinckrodt: Consultancy, Honoraria, Other: Supported/Contracted Research; Helsinn: Consultancy, Honoraria, Other: Supported/Contracted Research; Stratpharma: Other: Supported/Contracted Research; Medivir: Consultancy, Honoraria; Medscape: Speakers Bureau; Actelion: Other: Supported/Contracted Research.

Abstract 201: Roles of PIKfyve in Cardiac Fibroblast Migration

Cell migration is fundamental to proper development of the heart as well as other organs. Our characterization of a hypomorphic PIKfyve β-geo/β-geo mouse mutant, revealed that has an abnormally small heart, and raised the possibility that this could be due in part to a defect in cell migration. Moreover, a few studies suggested that PIKfyve, the sole lipid kinase that generates phosphatidylinositol 3,5-bisphosphate (PI3,5P2) and provides most of the PI5P, may have a role in migration of cultured cells. However, the localization of PIKfyve and the molecular mechanisms whereby PIKfyve regulates cell movement remain elusive. Here I show that inhibition of PIKfyve significantly impairs cell migration in cultured cell lines, as well as in primary cardiac fibroblasts, which are essential for proper heart development. Moreover, expression of a hyperactive PIKfyve mutant promotes cell migration. In order to detect the localization of endogenous PIKfyve, we used CRISPR-Cas9 genome editing to tag endogenous PIKfyve with an HA peptide at its N-terminus. Interestingly, in addition to PIKfyve localization on endosomes, a pool of PIKfyve was located at the cell leading edge. We observed PIKfyve in both lamellipodia and filopodia. Notably, we found that acute addition of PI3,5P2 or PI5P for 5 min altered filopodia dynamics. Our preliminary results suggest that PI3,5P2 and PI5P alter different properties of filopodia. These findings suggest that cell migration may be controlled in part by PI3,5P2 and PI5P which arise due to PIKfyve localization to the cell leading edge. Together, these studies suggest that mechanistic insights into how PIKfyve regulates cell migration may reveal new tools or concepts that can be utilized to study heart development in mammals.

RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and Integration.

CRISPR-based technologies have become central to genome engineering. However, CRISPR-based editing strategies are dependent on the repair of DNA breaks via endogenous DNA repair mechanisms, which increases susceptibility to unwanted mutations. Here we complement Cas9 with a recombinase's functionality by fusing a hyperactive mutant resolvase from transposon Tn3, a member of serine recombinases, to a catalytically inactive Cas9, which we term integrase Cas9 (iCas9). We demonstrate iCas9 targets DNA deletion and integration. First, we validate iCas9's function in Saccharomyces cerevisiae using a genome-integrated reporter. Cooperative targeting by CRISPR RNAs at spacings of 22 or 40 bp enables iCas9-mediated recombination. Next, iCas9's ability to target DNA deletion and integration in human HEK293 cells is demonstrated using dual GFP-mCherry fluorescent reporter plasmid systems. Finally, we show that iCas9 is capable of targeting integration into a genomic reporter locus. We envision targeting and design concepts of iCas9 will contribute to genome engineering and synthetic biology.

An Essential Regulator of Bacterial Division Links FtsZ to Cell Wall Synthase Activation

Bacterial growth and division require insertion of new peptidoglycan (PG) into the existing cell wall by PG synthase enzymes. Emerging evidence suggests that many PG synthases require activation to function; however, it is unclear how activation of division-specific PG synthases occurs. The FtsZ cytoskeleton has been implicated as a regulator of PG synthesis during division, but the mechanisms through which it acts are unknown. Here, we show that FzlA, an FtsZ-binding protein and essential regulator of constriction in Caulobacter crescentus, helps link FtsZ to PG synthesis to promote division. We find that hyperactive mutants of the PG synthases FtsW and FtsI specifically render fzlA, but not other division genes, non-essential. However, FzlA is still required to maintain proper constriction rate and efficiency in a hyperactive PG synthase background. Intriguingly, loss of fzlA in the presence of hyperactivated FtsWI causes cells to rotate about the division plane during constriction and sensitizes cells to cell-wall-specific antibiotics. We demonstrate that FzlA-dependent signaling to division-specific PG synthesis is conserved in another α-proteobacterium, Agrobacterium tumefaciens. These data establish that FzlA helps link FtsZ to cell wall remodeling and is required for signaling to both activate and spatially orient PG synthesis during division. Overall, our findings support the paradigm that activation of SEDS-PBP PG synthases is a broadly conserved requirement for bacterial morphogenesis.

A Novel High-Throughput Screening Assay for State-Dependent and Subunit-Dependent BK Channel Modulators

BK potassium channels are attractive drug targets for a wide variety of human disorders affecting almost every organ system. However, the therapeutic potential of many BK channel modulators is limited by lack of tissue specificity, reflecting that the pore forming a-subunit (Slo1) is encoded by a single gene. Properties of modulators that can potentially enhance tissue specificity include (a) sensitivity to regulatory subunits that are expressed in a tissue-dependent manner and (b) state-dependent action, conferring sensitivity to tissue-specific differences in membrane voltage and Ca2+ that activate BK channels. We have developed a novel 384-well fluorescent thallium-flux kinetic (FLIPR) assay to identify modulators with these properties. The screen includes assays on a panel of BK channel variants composed of hyperactive human Slo1 mutants (F380Y or R275C) in the presence or absence of different regulatory subunits (β1, β2, β2αFIW, β4, γ1), expressed in U2OS cells with Bacmam virus. Hyperactive Slo1 mutants allow both inhibitors and activators to be reliably detected under resting cellular conditions. Comparison of R275C and F380Y identifies state-dependent modulators since the voltage-sensors of R275C are constitutively activated while F380Y are not. A test screen of small molecules and arachnid venoms revealed a surprising degree of variant-selectivity and identified novel modulators with activities that depend on voltage-sensor activation and/or regulatory subunit expression. Thus, the screen detects modulators that would fail to be detected by conventional screens while also providing information about mechanism of action that should facilitate SAR analysis and development of potent tissue-selective modulators.

Interaction between the transmembrane domains of Sho1 and Opy2 enhances the signaling efficiency of the Hog1 MAP kinase cascade in Saccharomyces cerevisiae.

To cope with increased extracellular osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK), which controls a variety of adaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of a core MAPK cascade and two independent upstream branches (SHO1 and SLN1 branches) containing distinct osmosensing machineries. In the SHO1 branch, a homo-oligomer of Sho1, the four-transmembrane (TM) osmosensor, interacts with the transmembrane co-osmosensors, Hkr1 and Msb2, and the membrane anchor protein Opy2, through their TM domains, and activates the Ste20-Ste11-Pbs2-Hog1 kinase cascade. In this study, we isolated and analyzed hyperactive mutants of Sho1 and Opy2 that harbor mutations within their TM domains. Several hyperactive mutations enhanced the interaction between Sho1 and Opy2, indicating the importance of the TM-mediated interaction between Sho1 and Opy2 for facilitating effective signaling. The interaction between the TM domains of Sho1 and Opy2 will place their respective cytoplasmic binding partners Pbs2 and Ste11 in close proximity. Indeed, genetic analyses of the mutants showed that the Sho1-Opy2 interaction enhances the activation of Pbs2 by Ste11, but not Hog1 by Pbs2. Some of the hyperactive mutants had mutations at the extracellular ends of either Sho1 TM4 or Opy2 TM, and defined the Sho1-Opy2 binding site 1 (BS1). Chemical crosslinking and mutational analyses revealed that the cytoplasmic ends of Sho1 TM1 and Opy2 TM also interact with each other, defining the Sho1-Opy2 binding site 2 (BS2). A geometric consideration constrains that one Opy2 molecule must interact with two adjacent Sho1 molecules in Sho1 oligomer. These results raise a possibility that an alteration of the conformation of the Sho1-Opy2 complex might contributes to the osmotic activation of the Hog1 MAPK cascade.

A Genetic Screen for the Isolation of Mutants with Increased Flux in the Isoprenoid Pathway of Yeast.

The yeast Saccharomyces cerevisiae is one of the preferred hosts for the production of terpenoids through metabolic engineering. A genetic screen to identify novel mutants that can increase the flux in the isoprenoid pathway has been lacking. We present here the method that has led to the development of a carotenoid based visual screen by exploiting the carotenogenic genes from the red yeast Rhodosporidium toruloides, an organism known to have high levels of carotenoids. We also discuss the methods to use this screen for the identification of mutants that can lead to higher isoprenoid flux. The carotenoid based screen was developed in S. cerevisiae using phytoene synthase RtPSY1 and a hyperactive mutant of the enzyme phytoene dehydrogenase, RtCRTI(A393T) from Rhodosporidium toruloides. As validation of the genetic screen is critical at all stages, we describe the method to validate the screen using a known flux increasing gene, a truncated HMG1 (tHMG1). To demonstrate how this screen can be exploited to isolate mutants, we described how targeted mutagenesis of candidate gene, SPT15 a TATA binding protein involved in the global transcription machinery can be carried out to yield novel mutants with increased metabolic flux. Since it is also important to ensure that the isolated mutants are enhancing general isoprenoid flux, we describe how this can be established using an alternate isoprenoid, α-farnesene.

Abstract A40: Specific mutations in the D1-D2 linker region of VCP/p97 enhance ATPase activity and confer resistance to VCP inhibitors

Abstract Valosin-containing protein (VCP) has been shown to be involved in various protein quality control pathways including ubiquitin proteasome system, endoplasmic reticulum-associated degradation, chromatin-associated degradation, protein aggregate processing, and autophagy. Recent studies also identified VCP as a lineage-specific essential gene in ovarian cancer. An orally bioavailable VCP inhibitor, CB-5083, is currently in phase I clinical trials because it shows therapeutic effects in multiple tumor xenograft models. However, the mechanism of resistance to CB-5083 is unknown. Here, we characterized molecular mechanism of resistance to CB-5083. Using incremental exposure to CB-5083, we established CB-5083-resistant ovarian cancer cells that showed 5-6 fold resistance in vitro compared to parental cells. Genomic and complementary DNA sequencing of the VCP coding region reveal a pattern of coselected mutations: (1) missense mutations at codon 470 in one copy resulting in increased ATPase activity and (2) nonsense or frameshift mutations at codon 606 or codon 616 in another copy causing the loss of allele-specific expression. Unbiased molecular docking studies show codon 470 as a putative binding site for CB-5083. Analysis of somatic mutations in cancer genomes from The Cancer Genome Atlas indicates that codon 616 contains hotspot mutations in VCP. Thus, identification of these mutations associated with in vitro resistance to VCP inhibitors may be useful as potential theranostic markers while screening for patients to enroll in clinical trials. VCP has emerged as a viable therapeutic target for several cancer types, and therefore targeting such hyperactive VCP mutants should aid in improving the therapeutic outcome in cancer patients. Citation Format: Prabhakar Bastola, Bret Freudenthal, Matthew Schaich, Tsui-Fen Chou, Taiping Gan, Feng Wang, Jeremy Chien. Specific mutations in the D1-D2 linker region of VCP/p97 enhance ATPase activity and confer resistance to VCP inhibitors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A40.

Abstract 2366: The tyrosine phosphatase, SHP-2, participates in regulating PD-L1 expression in non-small cell lung cancer

Abstract Immune checkpoint inhibitors, specifically those which target T cell surface receptor Programmed Cell Death-1 (PD-1) and its ligand, Programmed cell death ligand-1 (PD-L1), have demonstrated clinical benefit in patients with non-small cell lung cancer (NSCLC). Currently, tumor expression of PD-L1 serves as the FDA-approved companion diagnostic for response to anti-PD-1/PD-L1 therapy. However, a fraction of patients who express PD-L1 exhibit a response to treatment, and in some cases, patient tumors that do not express PD-L1 demonstrate response to therapy, making this a poor predictive biomarker. Further, the mechanism by which PD-L1 expression is controlled remains under-studied. In this study, we seek to determine the impact of the protein tyrosine phosphatase, SHP-2, on PD-L1 expression through Jak/STAT- mediated signaling in NSCLC cell lines. SHP-2 can harbor activating mutations which prevent auto-inhibition of the catalytic phosphatase domain, and that may be pro-tumorigenic in some tumors. To understand the impact of SHP-2 activity on PD-L1 expression, we first assessed activation of STAT3 in NSCLC tumor cells after treatment with IFNγ and other mitogens. We then transfected NSCLC cells with wildtype and hyperactive SHP-2 mutants or ablated SHP-2 with siRNA specific for SHP-2 and measured both STAT3 and PD-L1 expression. Cell lines A549 and H661 exhibit low endogenous expression of both SHP-2 and PD-L1, and thus were chosen for transfection with hyperactive SHP-2 mutants (E76K and G503V), which also altered PD-L1 expression by western blot analysis. Quantitative real-time PCR (qPCR) and western blot analysis revealed that siRNA ablation of SHP-2 led to increased PD-L1 expression in the H460 cell line. Cells transfected with the SHP-2 activating mutants will also be assessed alteration of cell proliferation. Finally, using data obtained from The Cancer Genome Atlas project, STAT3 and PD-L1 expression levels are being evaluated in NSCLC tumor samples in which SHP-2 is mutated (E76K or G503V). With these data, we expect to gain a more complete understanding of the role of SHP-2 within the tumor microenvironment and the extent to which it controls PD-L1 expression, leading to new drug targets or combinations of drugs and better predictive biomarkers of response to PD-1 and PD-L1 inhibitors. Citation Format: Keller Toral, Brent Harris, Jarrod Creameans, Penni Black. The tyrosine phosphatase, SHP-2, participates in regulating PD-L1 expression in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2366.