Genome-wide screens were performed in yeast to identify genes that enhance the toxicity of a mutant huntingtin fragment or of α-synuclein. Of 4850 haploid mutants containing deletions of nonessential ...genes, 52 were identified that were sensitive to a mutant huntingtin fragment, 86 that were sensitive to α-synuclein, and only one mutant that was sensitive to both. Genes that enhanced toxicity of the mutant huntingtin fragment clustered in the functionally related cellular processes of response to stress, protein folding, and ubiquitin-dependent protein catabolism, whereas genes that modified α-synuclein toxicity clustered in the processes of lipid metabolism and vesicle-mediated transport. Genes with human orthologs were overrepresented in our screens, suggesting that we may have discovered conserved and nonoverlapping sets of cell-autonomous genes and pathways that are relevant to Huntington's disease and Parkinson's disease.
After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with ...additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement
characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing
, was effective
in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.
Target selection for antibody-drug conjugates (ADC) frequently focuses on identifying antigens with differential expression in tumor and normal tissue, to mitigate the risk of on-target toxicity. ...However, this strategy restricts the possible target space. SLC34A2/NaPi2b is a sodium phosphate transporter expressed in a variety of human tumors including lung and ovarian carcinoma, as well as the normal tissues from which these tumors arise. Previous clinical trials with a NaPi2b targeting MMAE-ADCs have shown objective durable responses. However, the protein-based biomarker assay developed for use in that study was unable to discern a statistically significant relationship between NaPi2b protein expression and the probability of response. XMT-1536 is a NaPi2b targeting ADC comprised of a unique humanized antibody conjugated with 10-15 auristatin F- hydroxypropylamide (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is a cell-permeable, antimitotic compound that is slowly metabolized intratumorally to an active, very low-permeable metabolite, auristatin F (AF), resulting in controlled bystander killing. We describe the preclinical
and
antitumor effects of XMT-1536 in models of ovarian and lung adenocarcinoma. Pharmacokinetic analysis showed approximately proportional increases in exposure in rat and monkey. Systemic free AF-HPA and AF concentrations were observed to be low in all animal species. Finally, we describe a unique IHC reagent, generated from a chimeric construct of the therapeutic antibody, that was used to derive a target expression and efficacy relationship in a series of ovarian primary xenograft cancer models.
Background: Mig1 is a transcriptional repressor responsible for glucose repression of many genes in the budding yeast Saccharomyces cerevisiae. Glucose regulates Mig1 function by affecting its ...phosphorylation, which is catalyzed by the Snf1 protein kinase. Phosphorylation alters the subcellular localization of Mig1, causing it to be nuclear when glucose is present, and cytoplasmic when glucose is absent.
Results: Here, we report that Msn5, a member of the importin β family of nuclear transport receptors, is required to export Mig1 from the nucleus when glucose is removed. Mig1 and Msn5 interacted in a yeast two-hybrid assay. Within the portion of Mig1 that regulates its nuclear transport, we found a region that directed its nuclear export. Within this region, two leucine-rich sequences similar to known nuclear export signals were not required for Mig1 export. The corresponding domain of the yeast Kluyveromyces lactis Mig1 conferred glucose-regulated Msn5-dependent protein export from the nucleus in S. cerevisiae. Sequence comparison with S. cerevisiae Mig1 revealed short patches of homology in K. lactis and K. marxianus Mig1 that might be Msn5-interaction domains. These regions overlapped with the serine residues predicted to be Snf1 phosphorylation sites, suggesting that Msn5 and Snf1 recognize similar sequences in Mig1. Altering these serines abolished glucose-dependent phosphorylation of Mig1 and caused it to be a constitutive repressor that was retained in the nucleus.
Conclusions: Mig1 contains a new nuclear export signal that is phosphorylated by Snf1 upon glucose removal, causing it to be recognized by the nuclear exportin Msn5 and carried out of the nucleus into the cytoplasm where it contributes to derepression of glucose-repressed genes.
Mig1 and Mig2 are proteins with similar zinc fingers that are required for glucose repression of SUC2 expression. Mig1, but not Mig2, is required for repression of some other glucose-repressed genes, ...including the GAL genes. A second homolog of Mig1, Yer028, appears to be a glucose-dependent transcriptional repressor that binds to the Mig1-binding sites in the SUC2 promoter, but is not involved in glucose repression of SUC2 expression. Despite their functional redundancy, we found several significant differences between Mig1 and Mig2: (1) in the absence of glucose, Mig1, but not Mig2, is inactivated by the Snf1 protein kinase; (2) nuclear localization of Mig1, but not Mig2, is regulated by glucose; (3) expression of MIG1, but not MIG2, is repressed by glucose; and (4) Mig1 and Mig2 bind to similar sites but with different relative affinities. By two approaches, we have identified many genes regulated by Mig1 and Mig2, and confirmed a role for Mig1 and Mig2 in repression of several of them. We found no genes repressed by Yer028. Also, we identified no genes repressed by only Mig1 or Mig2. Thus, Mig1 and Mig2 are redundant glucose repressors of many genes.
Abstract
XMT-1522 is an anti-HER2 ADC that incorporates HT-19, a novel, human anti-HER2 antibody optimized for cytotoxic payload delivery. Several parameters such as cell binding, internalization ...rate, cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) downstream signaling, affinity maturation, NHP cross-reactivity, normal human tissue cross-reactivity and in vivo efficacy were used to screen a wide range of antibodies to select a lead candidate optimized for use in ADC applications. In addition HT-19 was selected to be non-competitive for HER2 binding with existing therapies - trastuzumab or pertuzumab, to allow for potential combinability. In vivo efficacy as an ADC did not directly correlate with typical parameters used in antibody screening cascade such as in vitro cytotoxicity, internalization or binding affinity. The lead antibody underwent affinity maturation, and despite increases in affinity, affinity maturation significantly decreased the in vivo efficacy of the ADC in vivo xenograft models. This phenomenon was observed in all the antibody hits. HT-19 showed antibody-dependent cell-mediated cytotoxicity (ADCC) activity. When administered as a single agent in NCI-N87 gastric cancer xenograft model it had biological activity at 20 mg/kg as well as at 3 mg/kg. Consistent with the hypothesis that a non-competitive ADC is combinable with current anti-HER2 regimens, the combination of XMT-1522 with trastuzumab and/or pertuzumab showed more rapid internalization, more complete HER2 degradation, and significantly great anti-tumor activity in the NCI-N87 gastric cancer xenograft model relative to XMT-1522 alone or the combination of pertuzumab + trastuzumab.
Citation Format: Natasha Bodyak, Alex Yurkovetskiy, Dmitry R. Gumerov, Dongmei Xiao, Joshua D. Thomas D. Thomas, Laura L. Poling, LiuLiang Qin, Mao Yin, Michael J. DeVit, Peter U. Park, Winnie Lee, Bianka Prinz, Donald A. Bergstrom, Timothy B. Lowinger. Optimization of lead antibody selection for XMT-1522, a novel, highly potent HER2-targeted antibody-drug conjugate (ADC). abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 596.
Glucose represses the transcription of many genes in bakers yeast (Saccharomyces cerevisiae). Mig1 is a Cys2-His2 zinc finger protein that mediates glucose repression of several genes by binding to ...their promoters and recruiting the general repression complex Ssn6-Tup1. We have found that the subcellular localization of Mig1 is regulated by glucose. Mig1 is imported into the nucleus within minutes after the addition of glucose and is just as rapidly transported back to the cytoplasm when glucose is removed. This regulated nuclear localization requires components of the glucose repression signal transduction pathway. An internal region of the protein separate from the DNA binding and repression domains is necessary and sufficient for glucose-regulated nuclear import and export. Changes in the phosphorylation status of Mig1 are coincident with the changes in its localization, suggesting a possible regulatory role for phosphorylation. Our results suggest that a glucose-regulated nuclear import and/or export mechanism controls the activity of Mig1.
Foreign genes were expressed in liver and skin cells of live mice by using a new apparatus to accelerate DNA-coated microprojectiles into tissues. After introduction of a plasmid in which the firefly ...luciferase gene was controlled by the human β-actin promoter, luciferase activity was detectable for up to 14 days in mouse tissues (skin and liver). In situ hybridization histochemistry revealed that microprojectiles penetrated through multiple cell layers without evidence of tissue injury and that 10-20% of the cells in the bombarded area expressed the foreign gene. An advantage of the new design is that internal organs, such as liver, can be transfected without subjecting the tissue to a vacuum. This procedure potentially is applicable to a wide variety of tissues and cell types for studies of transcriptional control elements and for expression of foreign proteins in intact animals.
This study investigated the recycling of solid waste residues from rock asphalt extraction via 5-h calcination at 900 °C and indirect carbon mineralization. The calcined powder was first dissolved at ...60 °C in 2 M CH3COOH, carbonized in NH4HCO3, and agitated at 1200 rpm in 2 M NaOH for 30 minutes at 30–60 °C and pH 12. The precipitated calcium carbonate (PCC) results are calcite (24.4–56.3 wt%) and vaterite (75.6–3.7 wt%), as confirmed by XRD analysis, carbonate FTIR spectra, and SEM images of prismatic calcite and spherical vaterite. This high-value PCC product has potential industrial applications, such as paper and plastics.
Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (∼45%) of the ...1,101 essential yeast genes, with ∼30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.
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