Tiffany T. Huang

Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome

Mutations in the rpoB gene of Escherichia coli result in resistance to the antibiotic rifampicin (Rif(r)) by altering the beta subunit of RNA polymerase. Previous studies have identified 39 single base substitutions in the rpoB gene that lead to Rif(r) at 37 degrees C and an additional two mutations that result in temperature sensitive cells. We have extended this work and identified an additional 30 single base substitutions that result in the Rif(r) phenotype. With these mutations the rpoB/Rif(r) system now allows the monitoring of 69 base substitutions at 37 degrees at 37 sites (base pairs) distributed among 24 coding positions. Each of the six possible base substitutions is represented by 8-17 mutations. More than 90% of the mutations are within a small enough region of the rpoB gene to allow PCR amplification with a single pair of oligonucleotide primers, followed by sequencing with a single primer, leading to rapid analysis of numerous mutations. The remaining mutations can be monitored using an additional primer pair. To calibrate this system we sequenced over 500 mutations in rpoB occurring spontaneously or generated by different mutagens and mutators with known specificity. These results show that rpoB/Rif(r) is an accurate and easy to employ detection system, and offers the advantage of allowing analysis of mutations occurring on the chromosome rather than on an extrachromosomal element. The mutS, mutT, mutY, M mutators, as well as the mutagenic agents ethyl methanesulfonate (EMS), ultraviolet (UV) irradiation, 2-aminopurine (2AP), 5-azacytidine (5AZ), and cisplatin (CPT) gave results predicted by their characterized specificities. The number of different sequence contexts is sufficient to reveal significant hotspots among the spontaneous mutS, 2-aminopurine, ultraviolet light, 5-azacytidine, and cisplatin mutational spectra. The cisplatin distribution is particularly striking, with 68% of the mutations resulting from an A:T-->T:A transversion at a single site. Because of the conservation of key regions of RNA polymerase among many microorganisms, using the Rif(r)/rpoB system may be a general method for studying mutational processes in microorganisms without well developed genetic systems.

EGFR Signaling Through an Akt-SREBP-1–Dependent, Rapamycin-Resistant Pathway Sensitizes Glioblastomas to Antilipogenic Therapy

Inhibitors of fatty acid signaling promote apoptosis in glioblastoma cells with highly active EGFR signaling. Inhibiting Lipid Metabolism to Combat Glioblastoma Glioblastoma, the most common form of brain cancer, is frequently lethal. Glioblastoma is often associated with increased signaling through the epidermal growth factor receptor (EGFR); however, therapeutic efforts focused on inhibiting EGFR signaling have been disappointing. Guo et al. analyzed tumor tissue removed from glioblastoma patients before and during treatment with the EGFR inhibitor lapatinib and found that EGFR signaling activated sterol regulatory element–binding protein 1 (SREBP-1), a key regulator of lipid metabolism, and increased the cellular concentrations of fatty acids. Intriguingly, inhibiting fatty acid synthesis promoted apoptosis in glioblastoma cells with substantial EGFR signaling both in vitro and when transplanted into immunodeficient mice, but not in glioblastoma cells with little EGFR signaling. Thus, inhibition of fatty acid synthesis may represent a new avenue toward treating glioblastomas driven by EGFR signaling. Glioblastoma, the most common malignant brain tumor, is among the most lethal and difficult cancers to treat. Although epidermal growth factor receptor (EGFR) mutations are frequent in glioblastoma, their clinical relevance is poorly understood. Studies of tumors from patients treated with the EGFR inhibitor lapatinib revealed that EGFR induces the cleavage and nuclear translocation of the master transcriptional regulator of fatty acid synthesis, sterol regulatory element–binding protein 1 (SREBP-1). This response was mediated by Akt; however, clinical data from rapamycin-treated patients showed that SREBP-1 activation was independent of the mammalian target of rapamycin complex 1, possibly explaining rapamycin’s poor efficacy in the treatment of such tumors. Glioblastomas without constitutively active EGFR signaling were resistant to inhibition of fatty acid synthesis, whereas introduction of a constitutively active mutant form of EGFR, EGFRvIII, sensitized tumor xenografts in mice to cell death, which was augmented by the hydroxymethylglutaryl coenzyme A reductase inhibitor atorvastatin. These results identify a previously undescribed EGFR-mediated prosurvival metabolic pathway and suggest new therapeutic approaches to treating EGFR-activated glioblastomas.

Fyn and SRC are effectors of oncogenic epidermal growth factor receptor signaling in glioblastoma patients

Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR- and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies.

Pseudomonas fluorescens Encodes the Crohn's Disease-Associated I2 Sequence and T-Cell Superantigen

ABSTRACT Commensal bacteria have emerged as an important disease factor in human Crohns disease (CD) and murine inflammatory bowel disease (IBD) models. We recently isolated I2, a novel gene segment of microbial origin that is associated with human CD and that encodes a T-cell superantigen. To identify the I2 microorganism, BLAST analysis was used to identify a microbial homologue, PA2885, a novel open reading frame (ORF) in the Pseudomonas aeruginosa genome. PCR and Southern analysis identified Pseudomonas fluorescens as the originating species of I2, with homologues detectable in 3 of 13 other Pseudomonas species. Genomic cloning disclosed a locus containing the full-length I2 gene (pfiT) and three other orthologous genes, including a homologue of the pbrA/pvdS iron response gene. CD4+ T-cell responses to recombinant proteins were potent for I2 and pfiT, but modest for PA2885. pfiT has several features of a virulence factor: association with an iron-response locus, restricted species distribution, and T-cell superantigen bioactivity. These findings suggest roles for pfiT and P. fluorescens in the pathogenesis of Crohns disease.

CD44v6 Regulates Growth of Brain Tumor Stem Cells Partially through the AKT-Mediated Pathway

Identification of stem cell-like brain tumor cells (brain tumor stem-like cells; BTSC) has gained substantial attention by scientists and physicians. However, the mechanism of tumor initiation and proliferation is still poorly understood. CD44 is a cell surface protein linked to tumorigenesis in various cancers. In particular, one of its variant isoforms, CD44v6, is associated with several cancer types. To date its expression and function in BTSC is yet to be identified. Here, we demonstrate the presence and function of the variant form 6 of CD44 (CD44v6) in BTSC of a subset of glioblastoma multiforme (GBM). Patients with CD44high GBM exhibited significantly poorer prognoses. Among various variant forms, CD44v6 was the only isoform that was detected in BTSC and its knockdown inhibited in vitro growth of BTSC from CD44high GBM but not from CD44low GBM. In contrast, this siRNA-mediated growth inhibition was not apparent in the matched GBM sample that does not possess stem-like properties. Stimulation with a CD44v6 ligand, osteopontin (OPN), increased expression of phosphorylated AKT in CD44high GBM, but not in CD44low GBM. Lastly, in a mouse spontaneous intracranial tumor model, CD44v6 was abundantly expressed by tumor precursors, in contrast to no detectable CD44v6 expression in normal neural precursors. Furthermore, overexpression of mouse CD44v6 or OPN, but not its dominant negative form, resulted in enhanced growth of the mouse tumor stem-like cells in vitro. Collectively, these data indicate that a subset of GBM expresses high CD44 in BTSC, and its growth may depend on CD44v6/AKTpathway.

Development of a Real-time RT-PCR Assay for Detecting EGFRvIII in Glioblastoma Samples

Purpose: Epidermal growth factor receptor variant III (EGFRvIII) is an oncogenic, constitutively active mutant form of the EGFR that is commonly expressed in glioblastoma and is also detected in a number of epithelial cancers. EGFRvIII presents a unique antigenic target for anti-EGFRvIII vaccines and it has been shown to modulate response to EGFR kinase inhibitor therapy. Thus, detection in clinical samples may be warranted. Existing patents preclude the use of anti-EGFRvIII antibodies for clinical detection. Further, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffin-embedded (FFPE) clinical samples is a major challenge. Experimental Design: We developed a real-time reverse transcription-PCR (RT-PCR) assay for detecting EGFRvIII in FFPE samples and analyzed 59 FFPE glioblastoma clinical samples with paired frozen tissue from the same surgical resection. We assessed EGFRvIII protein expression by immunohistochemistry using two distinct specific anti-EGFRvIII antibodies and examined EGFR gene amplification by fluorescence in situ hybridization. Results: The FFPE RT-PCR assay detected EGFRvIII in 16 of 59 (27%) samples, exclusively in cases with EGFR amplification, consistent with the expected frequency of this alteration. The FFPE RT-PCR assay was more sensitive and specific for detecting EGFRvIII than either of the two antibodies alone, or in combination, with a sensitivity of 93% (95% confidence interval, 0.78-1.00) and a specificity of 98% (95% confidence interval, 0.93-1.00). Conclusion: This assay will facilitate accurate assessment of EGFRvIII in clinical samples and may aid in the development of strategies for stratifying patients for EGFRvIII-directed therapies.

Targeted Therapy for Malignant Glioma Patients: Lessons Learned and the Road Ahead

SummaryMolecularly targeted therapies are transforming the care of patients with malignant gliomas, including glioblastoma, the most common malignant primary brain tumor of adults. With an arsenal of small molecule inhibitors and antibodies that target key components of the signal transduction machinery that are commonly activated in gliomas, neurooncologists and neurosurgeons are poised to transform the care of these patients. Nonetheless, successful application of targeted therapies remains a challenge. Strategies are lacking for directing kinase inhibitor or other pathway-specific therapies to individual patients most likely to benefit. In addition, response to targeted agents is determined not only by the presence of the key mutant kinases, but also by other critical changes in the molecular circuitry of cancer cells, such as loss of key tumor suppressor proteins, the selection for kinase-resistant mutants, and the deregulation of feedback loops. Understanding these signaling networks, and studying them in patients, will be critical for developing rational combination therapies to suppress resistance for malignant glioma patients. Here we review the current status of molecular targeted therapies for malignant gliomas. We focus initially on identifying some of the insights gained to date from targeting the EGFR/PI3K/Akt/mTOR signaling pathway in patients and on how this has led toward a reconceptualization of some of the challenges and directions for targeted treatment. We describe how advances from the world of genomics have the potential to transform our approaches toward targeted therapy, and describe how a deeper understanding of the complex nature of cancer, and its adeptness at rewiring molecular circuitry to evade targeted agents, has raised new challenges and identified new leads.

Resident enteric microbiota and CD8+ T cells shape the abundance of marginal zone B cells

Since enteric microbial composition is a distinctive and stable individual trait, microbial heterogeneity may confer lifelong, non‐genetic differences between individuals. Here we report that C57BL/6 mice bearing restricted flora microbiota, a distinct but diverse resident enteric microbial community, are numerically and functionally deficient in marginal zone (MZ) B cells. Surprisingly, MZ B‐cell levels are minimally affected by germ‐free conditions or null mutations of various TLR signaling molecules. In contrast, MZ B‐cell depletion is exquisitely dependent on cytolytic CD8+ T cells, and includes targeting of a cross‐reactive microbial/endogenous MHC class 1B antigen. Thus, members of certain enteric microbial communities link with CD8+ T cells as a previously unappreciated mechanism that shapes innate immunity dependent on innate‐like B cells.

Competition between MutY and Mismatch Repair at A · C Mispairs In Vivo

We show that the MutY protein competes with the MutS-dependent mismatch repair system to process at least some A. C mispairs in vivo, converting them to G. C pairs. In the presence of an increased dCTP pool resulting from the loss of nucleotide diphosphate kinase, the frequency of A. T-->G. C transitions at a hot spot in the rpoB gene is 30-fold lower in a MutY-deficient derivative than in the wild type.

Intravenous Administration of Retroviral Replicating Vector, Toca 511, Demonstrates Therapeutic Efficacy in Orthotopic Immune-Competent Mouse Glioma Model

Toca 511 (vocimagene amiretrorepvec), a nonlytic, amphotropic retroviral replicating vector (RRV), encodes and delivers a functionally optimized yeast cytosine deaminase (CD) gene to tumors. In orthotopic glioma models treated with Toca 511 and 5-fluorocytosine (5-FC) the CD enzyme within infected cells converts 5-FC to 5-fluorouracil (5-FU), resulting in tumor killing. Toca 511, delivered locally either by intratumoral injection or by injection into the resection bed, in combination with subsequent oral extended-release 5-FC (Toca FC), is under clinical investigation in patients with recurrent high-grade glioma (HGG). If feasible, intravenous administration of vectors is less invasive, can easily be repeated if desired, and may be applicable to other tumor types. Here, we present preclinical data that support the development of an intravenous administration protocol. First we show that intravenous administration of Toca 511 in a preclinical model did not lead to widespread or uncontrolled replication of the RVV. No, or low, viral DNA was found in the blood and most of the tissues examined 180 days after Toca 511 administration. We also show that RRV administered intravenously leads to efficient infection and spread of the vector carrying the green fluorescent protein (GFP)-encoding gene (Toca GFP) through tumors in both immune-competent and immune-compromised animal models. However, initial vector localization within the tumor appeared to depend on the mode of administration. Long-term survival was observed in immune-competent mice when Toca 511 was administered intravenously or intracranially in combination with 5-FC treatment, and this combination was well tolerated in the preclinical models. Enhanced survival could also be achieved in animals with preexisting immune response to vector, supporting the potential for repeated administration. On the basis of these and other supporting data, a clinical trial investigating intravenous administration of Toca 511 in patients with recurrent HGG is currently open and enrolling.