Synthetic Lethal Combinations Research Articles

L04.05 - Targeted Drug Development in Pediatric Cancer Despite a Low Frequency of Tumor Driving Mutations

Neuroblastoma are a childhood tumors of the peripheral sympathetic nervous system. The pathogenesis has for a long time been quite enigmatic, as only very few gene defects were identified in this often lethal tumor. Frequently detected gene alterations were limited to MYCN amplification and ALK activations. Our recent studies on the genomic landscape of primary neuroblastoma revealed two novel molecular defects. The analysis of structural defects identified a local shredding of chromosomes, known as chromothripsis in 18% of high stage neuroblastoma. These tumors are associated with a poor outcome. In addition, an integrated analysis of mutation data and structural aberration data showed frequent alterations in genes involved in neuritogenesis (1). Despite these newly identified genomic aberrations the absolute number of genomic aberrations in neuroblastoma is much lower compared to adult tumors. Whole genome sequencing of multiple subclones of the same primary tumor showed that in some neuroblastoma tumors we could not detect any early tumor driving mutations. Therefore these tumors are most likely depending on copy number aberrations and structural variations for tumor initiation. Also sequence efforts in other pediatric cancers showed a low frequency of early tumor driving mutations. This urges an approach in which individualized therapeutic strategies in pediatric cancer not only use strong biomarkers as activating gene mutations. Also synthetic lethal combinations and milder aberrations as copy number gains and epigenetic aberrations should be explored as biomarkers for targeted therapy. We have now validated a series of target genes and corresponding targeted compounds through an effective pipeline. Integrated analysis of highthroughput molecular biological data using the R2 bioinformatic platform (http://r2.amc.nl) allowed identification of neuroblastoma driving oncogenes as BCL2, BIRC5, CDK4, CDK2, DNA-PK, EZH2, LIN28B and FOXR1 (2). Compounds against several of these target genes were further validated in vitro and in vivo. We conclude that a personalized treatment approach in pediatric cancer should include a broad biological typing.

Why Cancer & Metabolism? Why now?

Why Cancer & Metabolism? Why now?

Targeting BRCA1 localization to augment breast tumor sensitivity to poly(ADP-Ribose) polymerase inhibition.

PARP inhibitors have gained recent attention due to their highly selective killing of BRCA1/2-mutated and DNA double-strand break (DSB) repair-deficient tumors. Unfortunately, the majority of sporadic breast cancers carry wild-type BRCA1/2 and are proficient in DSB repair. We and others have shown that BRCA1 is a nuclear/cytoplasm shuttling protein that is transiently exported from the nucleus to the cytosol upon various stimuli. Thus, we hypothesized that depletion of nuclear BRCA1 would compromise DSB repair and subsequently render sporadic tumors susceptible to PARP inhibition. Indeed, in human sporadic breast cancer cells with functional BRCA1 and proficient DSB repair, a transient nuclear depletion of BRCA1 and subsequent homologous recombination repair deficit was induced with either truncated BRCA1 or irradiation. This rendered these human sporadic breast cancer cells susceptible to PARP inhibition. These observations were confirmed genetically using mislocated BRCA1 mutants as well as in vivo in mice bearing breast tumor xenografts. These data support the potential strategy of targeting BRCA1 location to convert BRCA1-proficient sporadic tumors to be susceptible to the synthetic lethal combination with PARP inhibitors.

Abstract 1459: Combined inhibition of Wee1 and PARP1 sensitizes pancreatic cancer cells to radiation

Abstract PARP1 [poly (ADP-ribose) polymerase-1] inhibitors are maximally effective in cells with HRR (homologous recombination repair) defects, a concept known as synthetic lethality. We have previously shown that inhibition of HRR and the G2 checkpoint by small molecule inhibitors of Chk1 (checkpoint kinase 1), can induce synthetic lethality by increasing PARP1 inhibitor-mediated radiosensitization selectively in tumor cells and preferentially in tumor cells with p53 mutations. In order to begin to determine whether other inhibitors of the DNA damage response might induce synthetic lethality in combination with PARP1 inhibition and radiation, we evaluated MK-1775, a Wee1 kinase inhibitor. Wee1 regulates the G2 checkpoint by phosphorylation of Cdk1, thus preventing entry into mitosis in the presence of DNA damage, while its influence on HRR is not clear. To determine whether inhibition of Wee1 might synergistically radiosensitize in combination with PARP1 inhibition, we treated pancreatic cancer cells with MK-1775 (100-200nM) and the PARP1 inhibitor, olaparib (1uM) and assessed radiosensitization by clonogenic survival. We found that the combination of MK-1775 with olaparib induced marked radiosensitization in MiaPaCa-2 (RER: 1.9) and AsPC-1 (RER: 1.7) pancreatic cancer cells, which was greater than the radiosensitization produced by either agent alone. In order to determine the mechanism(s) by which Wee1 and PARP1 inhibitors interact to produce radiosensitization, we investigated the G2 checkpoint as well as HRR. In response to radiation, MK-1775 abrogated the G2 checkpoint while olaparib treatment resulted in increased G2 accumulation which was associated with increased γH2AX, suggesting the presence of unrepaired DNA double strand breaks. Abrogation of the olaparib+radiation-mediated G2 checkpoint, by MK-1775 resulted in even greater DNA damage as assessed by γH2AX expression. Given the efficacy of PARP1 inhibitors in HRR defective cells, we also investigated the effect of MK-1775 on HRR. We found that MK-1775 inhibited homology-directed repair of an I-SceI endonuclease-induced DNA double strand break in MiaPaCa-2 cells (Control 5.2% versus MK-1775 3.3%, P<0.05) as well as impaired Rad51 focus formation in response to radiation. Taken together, these results suggest that inhibition of HRR and the G2 checkpoint by inhibition of Wee1 can induce synthetic lethality with PARP1 inhibitors in combination with radiation by increasing unrepaired DNA double strand breaks. These studies support the continued investigation of combined molecularly targeted agents such as Wee1 and PARP1 inhibitors with radiation, to induce synthetic lethality selectively in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1459. doi:1538-7445.AM2012-1459

Mouse Genetics Suggests Cell-Context Dependency for Myc-Regulated Metabolic Enzymes during Tumorigenesis

c-Myc (hereafter called Myc) belongs to a family of transcription factors that regulates cell growth, cell proliferation, and differentiation. Myc initiates the transcription of a large cast of genes involved in cell growth by stimulating metabolism and protein synthesis. Some of these, like those involved in glycolysis, may be part of the Warburg effect, which is defined as increased glucose uptake and lactate production in the presence of adequate oxygen supply. In this study, we have taken a mouse-genetics approach to challenge the role of select Myc-regulated metabolic enzymes in tumorigenesis in vivo. By breeding λ-Myc transgenic mice, Apc Min mice, and p53 knockout mice with mouse models carrying inactivating alleles of Lactate dehydrogenase A (Ldha), 3-Phosphoglycerate dehydrogenase (Phgdh) and Serine hydroxymethyltransferase 1 (Shmt1), we obtained offspring that were monitored for tumor development. Very surprisingly, we found that these genes are dispensable for tumorigenesis in these genetic settings. However, experiments in fibroblasts and colon carcinoma cells expressing oncogenic Ras show that these cells are sensitive to Ldha knockdown. Our genetic models reveal cell context dependency and a remarkable ability of tumor cells to adapt to alterations in critical metabolic pathways. Thus, to achieve clinical success, it will be of importance to correctly stratify patients and to find synthetic lethal combinations of inhibitors targeting metabolic enzymes.

Histone Chaperone Asf1 Plays an Essential Role in Maintaining Genomic Stability in Fission Yeast

The histone H3-H4 chaperone Asf1 is involved in chromatin assembly (or disassembly), histone exchange, regulation of transcription, and chromatin silencing in several organisms. To investigate the essential functions of Asf1 in Schizosaccharomyces pombe, asf1-ts mutants were constructed by random mutagenesis using PCR. One mutant (asf1-33(ts)) was mated with mutants in 77 different kinase genes to identify synthetic lethal combinations. The asf1-33 mutant required the DNA damage checkpoint factors Chk1 and Rad3 for its survival at the restrictive temperature. Chk1, but not Cds1, was phosphorylated in the asf1-33 mutant at the restrictive temperature, indicating that the DNA damage checkpoint was activated in the asf1-33 mutant. DNA damage occured in the asf1-33 mutant, with degradation of the chromosomal DNA observed through pulse-field gel electrophoresis and the formation of Rad22 foci. Sensitivity to micrococcal nuclease in the asf1-33 mutant was increased compared to the asf1+ strain at the restrictive temperature, suggesting that asf1 mutations also caused a defect in overall chromatin structure. The Asf1-33 mutant protein was mislocalized and incapable of binding histones. Furthermore, histone H3 levels at the centromeric outer repeat region were decreased in the asf1-33 mutant and heterochromatin structure was impaired. Finally, sim3, which encodes a CenH3 histone chaperone, was identified as a strong suppressor of the asf1-33 mutant. Taken together, these results clearly indicate that Asf1 plays an essential role in maintaining genomic stability in S. pombe.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Synthetic Lethal Compound Combinations Reveal a Fundamental Connection between Wall Teichoic Acid and Peptidoglycan Biosyntheses in Staphylococcus aureus

Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase (TP) with reduced susceptibility to β-lactam antibiotics. PBP2A cross-links nascent peptidoglycan when the native TPs are inhibited by β-lactams. Although mecA expression is essential for β-lactam resistance, it is not sufficient. Here we show that blocking the expression of wall teichoic acids (WTAs) by inhibiting the first enzyme in the pathway, TarO, sensitizes methicillin-resistant S. aureus (MRSA) strains to β-lactams even though the β-lactam-resistant transpeptidase, PBP2A, is still expressed. The dramatic synergy between TarO inhibitors and β-lactams is noteworthy not simply because strategies to overcome MRSA are desperately needed but because neither TarO nor the activities of the native TPs are essential in MRSA strains. The "synthetic lethality" of inhibiting TarO and the native TPs suggests a functional connection between ongoing WTA expression and peptidoglycan assembly in S. aureus. Indeed, transmission electron microscopy shows that S. aureus cells blocked in WTA synthesis have extensive defects in septation and cell separation, indicating dysregulated cell wall assembly and degradation. Our studies imply that WTAs play a fundamental role in S. aureus cell division and raise the possibility that synthetic lethal compound combinations may have therapeutic utility for overcoming antibiotic-resistant bacterial infections.

Isolation of synthetic lethal mutations in the rsm1-null mutant of fission yeast

To identify mutations in genes that are genetically linked to rsm1, we performed a synthetic lethal genetic screen in the fission yeast, Schizosaccharomyces pombe. Four mutations that showed synthetic lethality in combination with the rsm1null allele were isolated from approximately 320,000 colonies and defined in three complementation groups. One mutant (SLrsm1) exhibited a significant accumulation of poly(A)(+) RNA in the nucleus under synthetic lethal conditions, while the rest had no mRNA export defects. In addition, some genes (spmex67, rae1, or mlo3) required for mRNA export complemented the growth defects of the identified mutants. These results suggest that the isolated mutants contain mutations in genes that are involved in mRNA export and/or pre-mRNA retention.

The Effect of the IGF1R Antibody R1507 on ER Positive Breast Cancer Cell Lines Growth and Survival.

Abstract Background: The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of IGF-1 and IGF-2 and is expressed in most breast cancer cells, where receptor activation induces proliferative, cell-survival, and transforming activities. Because crosstalk exists between ER and IGF1R blockade of both pathways could increase antitumor activity. Further, IGF signaling may be enhanced in ER+ cells that have become refractory to long term estrogen deprivation (LTED) explaining resistance to aromatase inhibitor therapy. The effect of the selective IGF1R antibody (R1507) was therefore assessed in a series of breast cancer cell lines in both estrogen-dependent and estrogen-independent growth phases.Methods: The ER positive breast cancer cells lines MCF7, T47D and HCC712 were kept in phenol red-free medium containing 5% charcoal-stripped serum. Several doses of R1507 were used to evaluate effects on cell proliferation, signaling and apoptosis. Western blotting with phospho-specific antibodies was used to observe effects on cell signaling, cell growth was measured using resazurin reduction and apoptosis was quantified by flow cytometry using the TUNEL assay. STED (short-term estrogen deprived) cells were treated with R1507 between 2 and 3 weeks after estrogen depletion and LTED were treated with R1507 after 6+ months of estrogen depletion.Results: R1507 suppressed IGF1R expression and inhibited IGF-1 stimulated IGF1R and AKT phosphorylation in all cell lines. R1507 had anti-proliferative effects in MCF7 cells in both estrogen-dependent and independent growth phases and the magnitude of the effect was similar to fulvestrant. R1507 had a smaller effect on cell proliferation in HCC712 cells and no effects on proliferation were observed in T47D cells. Antibody treatment resulted in significant induction of apoptosis in MCF7 STED but MCF7 LTED cells were resistant to the apoptotic effects of IGF1R blockade.Conclusions: R1507 had an inhibitory effect on IGF1R signaling and an anti-proliferative effect in two of the three cell lines tested. In addition R1507 induced synthetic lethality in combination with acute estrogen deprivation in MCF7 cells, an effect we have recently reported for direct PI3 kinase inhibitors under the same circumstances (1). While the proapoptotic effects of R1507 were lost in the estrogen-independent growth phase of MCF7 cells, the anti-proliferative effect was not, indicating that R1507 might have efficacy in patients with endocrine therapy resistant tumors. In other cell lines the efficacy of R1507 was variable and synthetic lethality was not observed, suggesting that the development of predictive biomarkers for R1507 should be a priority. We are currently addressing the activity of R1507 in a clinical trial with two cohorts, one with acquired resistance to letrozole, where the antibody is added to continued letrozole treatment and a second group of patients with stable disease on letrozole to determine if R1507 can induce tumor regression under these circumstances.1. Crowder et al Cancer Research 69:3955-62, 2009 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3126.

Functional Analysis of saxophone, the Drosophila Gene Encoding the BMP Type I Receptor Ortholog of Human ALK1/ACVRL1 and ACVR1/ALK2

In metazoans, bone morphogenetic proteins (BMPs) direct a myriad of developmental and adult homeostatic events through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like Kinase1 and -2 (ALK1/ACVRL1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp+. In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sax mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by saxP, a viable allele previously reported to be null, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVRl-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-beta signaling.

Metabolite-centric approaches for the discovery of antibacterials using genome-scale metabolic networks

Development of genome-scale metabolic models and various constraints-based flux analyses have enabled more sophisticated examination of metabolism. Recently reported metabolite essentiality studies are also based on the constraints-based modeling, but approaches metabolism from a metabolite-centric perspective, providing synthetic lethal combination of reactions and clues for the rational discovery of antibacterials. In this study, metabolite essentiality analysis was applied to the genome-scale metabolic models of four microorganisms: Escherichia coli, Helicobacter pylori, Mycobacterium tuberculosis and Staphylococcus aureus. Furthermore, chokepoints, metabolites surrounded by enzymes that uniquely consume and/or produce them, were also calculated based on the network properties of the above organisms. A systematic drug targeting strategy was developed by combining information from these two methods. Final drug target metabolites are presented and examined with knowledge from the literature.

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

The absence of Tsa1, a key peroxiredoxin that functions to scavenge H(2)O(2) in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations including gross chromosomal rearrangements (GCRs). Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD6 and several key genes involved in DNA double-strand break repair. In the present study we investigated the causes of GCRs and cell death in these mutants. tsa1-associated GCRs were independent of the activity of the translesion DNA polymerases zeta, eta, and Rev1. Anaerobic growth reduced substantially GCR rates of WT and tsa1 mutants and restored the viability of tsa1 rad6, tsa1 rad51, and tsa1 mre11 double mutants. Anaerobic growth also reduced the GCR rate of rad27, pif1, and rad52 mutants, indicating a role of reactive oxygen species in GCR formation in these mutants. In addition, deletion of TSA1 or H(2)O(2) treatment of WT cells resulted in increased formation of Rad52 foci, sites of repair of multiple DNA lesions. H(2)O(2) treatment also induced the GCRs. Our results provide in vivo evidence that oxygen metabolism and reactive oxygen species are important sources of DNA damages that can lead to GCRs and lethal effects in S. cerevisiae.

Genome-wide RNAi to identify genes that confer synthetic lethality with BRCA1

We have previously demonstrated that a functional orthologue of the breast cancer tumour suppressor gene BRCA1 exists in C. elegans (brc-1). Deletion mutants in C. elegans brc-1 or its heterodimeric partner, brd-1, share many of the phenotypic hallmarks of BRCA1-deficient human cells, yet are homozygous viable thus permitting extensive reverse genetic analysis. Using a rapid and inexpensive genome-wide screen in C. elegans we set out to identify genes that could be targeted in human patients to selectively kill tumours defective in the BRCA pathway. To this end we have utilized the complete C. elegans RNA-mediated interference library to systematically inactivate all 19,500 C. elegans genes and have identified those genes whose depletion confers synthetic lethality in combination with brc-1 and brd-1 mutations. In total, this screen identified 20 genes including pme-1 and pme-2, the C. elegans counterparts of PARP, a gene whose inhibition selectively kills BRCA defective tumour cells. We are currently using siRNA to knockdown all human homologues to identify those genes whose inactivation specifically kills mammalian cells harbouring mutations in BRCA1. These results and our current progress will be presented.

Synthetic Lethal and Biochemical Analyses of NAD and NADH Kinases in Saccharomyces cerevisiae Establish Separation of Cellular Functions

Production of NADP and NADPH depends on activity of NAD and NADH kinases. Here we characterized all combinations of mutants in yeast NAD and NADH kinases to determine their physiological roles. We constructed a diploid strain heterozygous for disruption of POS5, encoding mitochondrial NADH kinase, UTR1, cytosolic NAD kinase, and YEF1, a UTR1-homologous gene we characterized as encoding a low specific activity cytosolic NAD kinase. pos5 utr1 is a synthetic lethal combination rescued by plasmid-borne copies of the POS5 or UTR1 genes or by YEF1 driven by the ADH1 promoter. Respiratory-deficient and oxidative damage-sensitive defects in pos5 mutants were not made more deleterious by yef1 deletion, and a quantitative growth phenotype of pos5 and its arginine auxotrophy were repaired by plasmid-borne POS5 but not UTR1 or ADH1-driven YEF1. utr1 haploids have a slow growth phenotype on glucose not exacerbated by yef1 deletion but reversed by either plasmid-borne UTR1 or ADH1-driven YEF1. The defect in fermentative growth of utr1 mutants renders POS5 but not POS5-dependent mitochondrial genome maintenance essential because rho-utr1 derivatives are viable. Purified Yef1 has similar nucleoside triphosphate specificity but substantially lower specific activity and less discrimination in favor of NAD versus NADH phosphorylation than Utr1. Low expression and low intrinsic NAD kinase activity of Yef1 and the lack of phenotype associated with yef1 suggest that Utr1 and Pos5 are responsible for essentially all NAD/NADH kinase activity in vivo. The data are compatible with a model in which there is no exchange of NADP, NADPH, or cytoplasmic NAD/NADH kinase between nucleocytoplasmic and mitochondrial compartments, but the cytoplasm is exposed to mitochondrial NAD/NADH kinase during the transit of the molecule.

Role of the spindle-pole-body protein ApsB and the cortex protein ApsA in microtubule organization and nuclear migration in Aspergillus nidulans

Nuclear migration and positioning in Aspergillus nidulans depend on microtubules, the microtubule-dependent motor protein dynein, and auxiliary proteins, two of which are ApsA and ApsB. In apsA and apsB mutants nuclei are clustered and show various kinds of nuclear navigation defects, although nuclear migration itself is still possible. We studied the role of several components involved in nuclear migration through in vivo fluorescence microscopy using fluorescent-protein tagging. Because ApsA localizes to the cell cortex and mitotic spindles were immobile in apsA mutants, we suggest that astral microtubule-cortex interactions are necessary for oscillation and movement of mitotic spindles along hyphae, but not for post-mitotic nuclear migration. Mutation of apsA resulted in longer and curved microtubules and displayed synthetic lethality in combination with the conventional kinesin mutation DeltakinA. By contrast, ApsB localized to spindle-pole bodies (the fungal centrosome), to septa and to spots moving rapidly along microtubules. The number of cytoplasmic microtubules was reduced in apsB mutants in comparison to the wild type, indicating that cytoplasmic microtubule nucleation was affected, whereas mitotic spindle formation appeared normal. Mutation of apsB suppressed dynein null mutants, whereas apsA mutation had no effect. We suggest that nuclear positioning defects in the apsA and apsB mutants are due to different effects on microtbule organisation. A model of spindle-pole body led nuclear migration and the roles of dynein and microtubules are discussed.

Cooperative function of the CHD5-like protein Mdm39p with a P-type ATPase Spf1p in the maintenance of ER homeostasis in Saccharomyces cerevisiae

Spf1p is a P-type ATPase that is mainly localized to the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. The protein is involved in the maintenance of ion homeostasis in the ER. To investigate the intracellular role of Spf1p in more detail, we performed a genetic screen for mutations that lead to synthetic lethality in combination with a disruption of SPF1; the mutations identified have been termed lws (for lethal with spf1) mutations. Mutant alleles of five LWS genes (MDM39, RIC1, LAS21, TUP1 and BTS1) were recovered. The identification of these genes provides clues to the physiological relationships between Spf1p function and the secretory pathway. Among the five genes identified, MDM39 encodes a membrane protein that is similar to the protein CHD5/WRB, which is involved in the pathogenesis of Down syndrome-associated congenital heart disease in humans. We localized Mdm39p to the ER. The Deltamdm39 mutant exhibited defects in glycosylation, cell wall organization and the unfolded protein response. It also showed calcium-related phenotypes and synthetic lethal interactions with deletion mutations in other LWS genes. Our findings imply a homeostatic role for Mdm39p, which may be related to the regulation of calcium ion fluxes in the ER, and is indispensable for mutants that lack Spf1p.

Genetic, biochemical, and morphological evidence for the involvement of N-glycosylation in biosynthesis of the cell wall beta1,6-glucan of Saccharomyces cerevisiae.

Recent evidence indicates that Stt3p plays a central role in the recognition and/or catalytic step in N-glycosylation (asparagine-linked glycosylation) in the lumen of the endoplasmic reticulum. It is known that stt3 mutants exhibit certain phenotypic features that are suggestive of a cell wall defect. To understand the basis of these phenotypes, we devised a genetic screen to isolate strains bearing mutations that lead to synthetic lethality in combination with the stt3-1 mutation. Using this screen, we were surprised to identify two KRE genes (KRE5 and KRE9) that are involved in the biosynthesis of the cell wall beta1,6-glucan. This finding led us to propose that the N-glycosylation process is essential in the biosynthesis of cell wall beta1,6-glucan. This proposal was supported by the observation that several stt3 mutants exhibited a 60-70% reduction in the content of cell wall beta1,6-glucan as compared with WT cells. Transmission electron microscopy revealed that the stt3 mutant strains exhibit a diffused cell wall with loss of the outer mannoprotein layer as compared with the WT cells. Thus, we provide genetic, morphological, and biochemical evidence for the critical involvement of N-glycosylation in some step in assembly of the cell wall beta1,6-glucan in Saccharomyces cerevisiae.

TINA interacts with the NIMA kinase in Aspergillus nidulans and negatively regulates astral microtubules during metaphase arrest.

The tinA gene of Aspergillus nidulans encodes a protein that interacts with the NIMA mitotic protein kinase in a cell cycle-specific manner. Highly similar proteins are encoded in Neurospora crassa and Aspergillus fumigatus. TINA and NIMA preferentially interact in interphase and larger forms of TINA are generated during mitosis. Localization studies indicate that TINA is specifically localized to the spindle pole bodies only during mitosis in a microtubule-dependent manner. Deletion of tinA alone is not lethal but displays synthetic lethality in combination with the anaphase-promoting complex/cyclosome mutation bimE7. At the bimE7 metaphase arrest point, lack of TINA enhanced the nucleation of bundles of cytoplasmic microtubules from the spindle pole bodies. These microtubules interacted to form spindles joined in series via astral microtubules as revealed by live cell imaging. Because TINA is modified and localizes to the spindle pole bodies at mitosis, and lack of TINA causes enhanced production of cytoplasmic microtubules at metaphase arrest, we suggest TINA is involved in negative regulation of the astral microtubule organizing capacity of the spindle pole bodies during metaphase.