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CIViC (Clinical Interpretation of Variants in Cancer; civicdb.org) is a crowd-sourced, public domain knowledgebase composed of literature-derived evidence characterizing the clinical utility of cancer variants. As clinical sequencing becomes more prevalent in cancer management, the need for cancer variant interpretation has grown beyond the capability of any single institution. CIViC contains peer-reviewed, published literature curated and expertly-moderated into structured data units (Evidence Items) that can be accessed globally and in real time, reducing barriers to clinical variant knowledge sharing. We have extended CIViC's functionality to support emergent variant interpretation guidelines, increase interoperability with other variant resources, and promote widespread dissemination of structured curated data. To support the full breadth of variant interpretation from basic to translational, including integration of somatic and germline variant knowledge and inference of drug response, we have enabled curation of three new Evidence Types (Predisposing, Oncogenic and Functional). The growing CIViC knowledgebase has over 300 contributors and distributes clinically-relevant cancer variant data currently representing >3200 variants in >470 genes from >3100 publications., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To evaluate the clinical impact of molecular tumor profiling (MTP) with targeted sequencing panel tests, pediatric patients with extracranial solid tumors were enrolled in a prospective observational cohort study at 12 institutions. In the 345-patient analytical population, median age at diagnosis was 12 years (range 0-27.5); 298 patients (86%) had 1 or more alterations with potential for impact on care. Genomic alterations with diagnostic, prognostic or therapeutic significance were present in 61, 16 and 65% of patients, respectively. After return of the results, impact on care included 17 patients with a clarified diagnostic classification and 240 patients with an MTP result that could be used to select molecularly targeted therapy matched to identified alterations (MTT). Of the 29 patients who received MTT, 24% had an objective response or experienced durable clinical benefit; all but 1 of these patients received targeted therapy matched to a gene fusion. Of the diagnostic variants identified in 209 patients, 77% were gene fusions. MTP with targeted panel tests that includes fusion detection has a substantial clinical impact for young patients with solid tumors., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: Molecular tumor profiling is becoming a routine part of clinical cancer care, typically involving tumor-only panel testing without matched germline. We hypothesized that integrated germline sequencing could improve clinical interpretation and enhance the identification of germline variants with significant hereditary risks., Materials and Methods: Tumors from pediatric patients with high-risk, extracranial solid malignancies were sequenced with a targeted panel of cancer-associated genes. Later, germline DNA was analyzed for a subset of these genes. We performed a post hoc analysis to identify how an integrated analysis of tumor and germline data would improve clinical interpretation., Results: One hundred sixty participants with both tumor-only and germline sequencing reports were eligible for this analysis. Germline sequencing identified 38 pathogenic or likely pathogenic variants among 35 (22%) patients. Twenty-five (66%) of these were included in the tumor sequencing report. The remaining germline pathogenic or likely pathogenic variants were single-nucleotide variants filtered out of tumor-only analysis because of population frequency or copy-number variation masked by additional copy-number changes in the tumor. In tumor-only sequencing, 308 of 434 (71%) single-nucleotide variants reported were present in the germline, including 31% with suggested clinical utility. Finally, we provide further evidence that the variant allele fraction from tumor-only sequencing is insufficient to differentiate somatic from germline events., Conclusion: A paired approach to analyzing tumor and germline sequencing data would be expected to improve the efficiency and accuracy of distinguishing somatic mutations and germline variants, thereby facilitating the process of variant curation and therapeutic interpretation for somatic reports, as well as the identification of variants associated with germline cancer predisposition., Competing Interests: Alanna J. Church Honoraria: The Jackson Laboratory Consulting or Advisory Role: AlphaSights, The Jackson Laboratory, Bayer Travel, Accommodations, Expenses: Bayer Laura B. Corson Employment: Sema4 Stock and Other Ownership Interests: Alnylam, Inovio Pharmaceuticals, Agios Consulting or Advisory Role: Biomatics Patents, Royalties, Other Intellectual Property: Patent from work done at H3 Biomedicine before 2016 Brennan Decker Employment: Foundation Medicine Stock and Other Ownership Interests: Avidea Technologies, Roche Consulting or Advisory Role: Foundation Medicine, Avidea Technologies Catherine M. Clinton Employment: Helix OpCo LLC Mark A. Applebaum Consulting or Advisory Role: Fennec Pharma Rochelle Bagatell Uncompensated Relationships: Ymabs Therapeutics Inc Steven G. DuBois Consulting or Advisory Role: Bayer, Amgen Research Funding: Merck (Inst), Roche/Genentech (Inst), Lilly (Inst), Curis (Inst), Loxo (Inst), BMS (Inst), Eisai (Inst), Pfizer (Inst), Turning Point Therapeutics (Inst), Bayer (Inst), Salarius Pharmaceuticals (Inst) Travel, Accommodations, Expenses: Roche/Genentech, Salarius Pharmaceuticals Uncompensated Relationships: Ymabs Therapeutics Inc Julia L. Glade-Bender Research Funding: Eisai (Inst), Lilly (Inst), Loxo (Inst), Roche/Genentech (Inst), Bayer (Inst) Patents, Royalties, Other Intellectual Property: Patent on a T lympboblastic lymphoma cell line, CUTLL1 Travel, Accommodations, Expenses: Amgen (Inst) Uncompensated Relationships: SpringWorks Therapeutics, Bristol Myers Squibb, Merck, Eisai Open Payments Link: https://openpaymentsdata.cms.gov/physician/708514 AeRang Kim Research Funding: Esai (Inst); AstraZeneca (Inst); Oncternal (Inst); Ascentage (Inst); BioAlta (Inst). Theodore W. Laetsch Consulting or Advisory Role: Novartis, Bayer, Cellectis, Aptitude Health, Clinical Education Alliance, Deciphera, Jumo Health, Massive Bio, Med Learning Group, Medscape, Physicans' Education Resource, Y-mAbs Therapeutics Research Funding: Pfizer (Inst), Novartis (Inst), Bayer (Inst), AbbVie (Inst), Amgen (Inst), Atara Biotherapeutics (Inst), Bristol Myers Squibb (Inst), Lilly (Inst), Epizyme (Inst), GlaxoSmithKline (Inst), Janssen (Inst), Jubilant Pharmaceuticals (Inst), Novella Clinical (Inst), Servier (Inst), Foundation Medicine (Inst), Merck Sharp & Dohme (Inst) Margaret E. Macy Stock and Other Ownership Interests: Johnson & Johnson, GE Healthcare, Varian Medical Systems Consulting or Advisory Role: Ymabs Therapeutics Inc Research Funding: Bayer (Inst), Ignyta (Inst), Roche (Inst), Lilly (Inst), Merck (Inst), Oncternal Therapeutics Inc (Inst), AbbVie (Inst), Jubilant DraxImage (Inst), Actuate Therapeutics (Inst) Patents, Royalties, Other Intellectual Property: Patent for non-invasive methods of leukemia cell detection with MRI/MRS—Patent No. 8894975 Luke Maese Honoraria: Jazz Pharmaceuticals Consulting or Advisory Role: Jazz Pharmaceuticals Amit J. Sabnis Consulting or Advisory Role: Cardinal Health Joshua D. Schiffman Employment: PEEL Therapeutics Leadership: PEEL Therapeutics Stock and Other Ownership Interests: ItRunsInMyFamily.com, PEEL Therapeutics Honoraria: Affymetrix Consulting or Advisory Role: N-of-One, Fabric Genomics Samuel L. Volchenboum Stock and Other Ownership Interests: Litmus Health Consulting or Advisory Role: Accordant Travel, Accommodations, Expenses: Sanford Health Daniel A. Weiser Consulting or Advisory Role: Illumina Radiopharmaceuticals Jonathan A. Nowak Research Funding: NanoString Technologies, Illumina Katherine A. Janeway Honoraria: Foundation Medicine, Takeda Consulting or Advisory Role: Bayer, Ipsen Travel, Accommodations, Expenses: Bayer Brian D. Crompton Employment: Acceleron Pharma (I) Stock and Other Ownership Interests: Acceleron Pharma (I) Research Funding: Gradalis Junne Kamihara Stock and Other Ownership Interests: PanTher Therapeutics (I), ROME Therapeutics (I), TellBio (I) Honoraria: Pfizer (I), NanoString Technologies (I), Foundation Medicine (I), Ikena Oncology (I) Consulting or Advisory Role: ROME Therapeutics (I), Third Rock Ventures (I), Tekla Capital Management (I) Research Funding: PureTech (I), Ribon Therapeutics (I), ACD Biotechne (I) Patents, Royalties, Other Intellectual Property: Patent on drug delivery device licensed to PanTher Therapeutics (I), Patents on Repeat RNA biomarkers and therapeutics licensed to Rome Therapeutics (I), Patents on Circulating Tumor Cell Biomarkers Licensed to TellBio Inc (I) No other potential conflicts of interest were reported. Alanna J. Church Honoraria: The Jackson Laboratory Consulting or Advisory Role: AlphaSights, The Jackson Laboratory, Bayer Travel, Accommodations, Expenses: Bayer Laura B. Corson Employment: Sema4 Stock and Other Ownership Interests: Alnylam, Inovio Pharmaceuticals, Agios Consulting or Advisory Role: Biomatics Patents, Royalties, Other Intellectual Property: Patent from work done at H3 Biomedicine before 2016 Brennan Decker Employment: Foundation Medicine Stock and Other Ownership Interests: Avidea Technologies, Roche Consulting or Advisory Role: Foundation Medicine, Avidea Technologies Catherine M. Clinton Employment: Helix OpCo LLC Mark A. Applebaum Consulting or Advisory Role: Fennec Pharma Rochelle Bagatell Uncompensated Relationships: Ymabs Therapeutics Inc Steven G. DuBois Consulting or Advisory Role: Bayer, Amgen Research Funding: Merck (Inst), Roche/Genentech (Inst), Lilly (Inst), Curis (Inst), Loxo (Inst), BMS (Inst), Eisai (Inst), Pfizer (Inst), Turning Point Therapeutics (Inst), Bayer (Inst), Salarius Pharmaceuticals (Inst) Travel, Accommodations, Expenses: Roche/Genentech, Salarius Pharmaceuticals Uncompensated Relationships: Ymabs Therapeutics Inc Julia L. Glade-Bender Research Funding: Eisai (Inst), Lilly (Inst), Loxo (Inst), Roche/Genentech (Inst), Bayer (Inst) Patents, Royalties, Other Intellectual Property: Patent on a T lympboblastic lymphoma cell line, CUTLL1 Travel, Accommodations, Expenses: Amgen (Inst) Uncompensated Relationships: SpringWorks Therapeutics, Bristol Myers Squibb, Merck, Eisai Open Payments Link: https://openpaymentsdata.cms.gov/physician/708514 AeRang Kim Research Funding: Esai (Inst); AstraZeneca (Inst); Oncternal (Inst); Ascentage (Inst); BioAlta (Inst). Theodore W. Laetsch Consulting or Advisory Role: Novartis, Bayer, Cellectis, Aptitude Health, Clinical Education Alliance, Deciphera, Jumo Health, Massive Bio, Med Learning Group, Medscape, Physicans' Education Resource, Y-mAbs Therapeutics Research Funding: Pfizer (Inst), Novartis (Inst), Bayer (Inst), AbbVie (Inst), Amgen (Inst), Atara Biotherapeutics (Inst), Bristol Myers Squibb (Inst), Lilly (Inst), Epizyme (Inst), GlaxoSmithKline (Inst), Janssen (Inst), Jubilant Pharmaceuticals (Inst), Novella Clinical (Inst), Servier (Inst), Foundation Medicine (Inst), Merck Sharp & Dohme (Inst) Margaret E. Macy Stock and Other Ownership Interests: Johnson & Johnson, GE Healthcare, Varian Medical Systems Consulting or Advisory Role: Ymabs Therapeutics Inc Research Funding: Bayer (Inst), Ignyta (Inst), Roche (Inst), Lilly (Inst), Merck (Inst), Oncternal Therapeutics Inc (Inst), AbbVie (Inst), Jubilant DraxImage (Inst), Actuate Therapeutics (Inst) Patents, Royalties, Other Intellectual Property: Patent for non-invasive methods of leukemia cell detection with MRI/MRS—Patent No. 8894975 Luke Maese Honoraria: Jazz Pharmaceuticals Consulting or Advisory Role: Jazz Pharmaceuticals Amit J. Sabnis Consulting or Advisory Role: Cardinal Health Joshua D. Schiffman Employment: PEEL Therapeutics Leadership: PEEL Therapeutics Stock and Other Ownership Interests: ItRunsInMyFamily.com, PEEL Therapeutics Honoraria: Affymetrix Consulting or Advisory Role: N-of-One, Fabric Genomics Samuel L. Volchenboum Stock and Other Ownership Interests: Litmus Health Consulting or Advisory Role: Accordant Travel, Accommodations, Expenses: Sanford Health Daniel A. Weiser Consulting or Advisory Role: Illumina Radiopharmaceuticals Jonathan A. Nowak Research Funding: NanoString Technologies, Illumina Katherine A. Janeway Honoraria: Foundation Medicine, Takeda Consulting or Advisory Role: Bayer, Ipsen Travel, Accommodations, Expenses: Bayer Brian D. Crompton Employment: Acceleron Pharma (I) Stock and Other Ownership Interests: Acceleron Pharma (I) Research Funding: Gradalis Junne Kamihara Stock and Other Ownership Interests: PanTher Therapeutics (I), ROME Therapeutics (I), TellBio (I) Honoraria: Pfizer (I), NanoString Technologies (I), Foundation Medicine (I), Ikena Oncology (I) Consulting or Advisory Role: ROME Therapeutics (I), Third Rock Ventures (I), Tekla Capital Management (I) Research Funding: PureTech (I), Ribon Therapeutics (I), ACD Biotechne (I) Patents, Royalties, Other Intellectual Property: Patent on drug delivery device licensed to PanTher Therapeutics (I), Patents on Repeat RNA biomarkers and therapeutics licensed to Rome Therapeutics (I), Patents on Circulating Tumor Cell Biomarkers Licensed to TellBio Inc (I) No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Cancer treatment decisions are increasingly based on the genomic profile of the patient's tumor, a strategy called "precision oncology." Over the past few years, a growing number of clinical trials and case reports have provided evidence that precision oncology is an effective approach for at least some children with cancer. Here, we review key factors influencing pediatric drug development in the era of precision oncology. We describe an emerging regulatory framework that is accelerating the pace of clinical trials in children as well as design challenges that are specific to trials that involve young cancer patients. Last, we discuss new drug development approaches for pediatric cancers whose growth relies on proteins that are difficult to target therapeutically, such as transcription factors., (Copyright © 2019, American Association for the Advancement of Science.)

A series of trisubstituted hydroxylactams was identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Utilizing structure-based design and physical property optimization, multiple inhibitors were discovered with <10 μM lactate IC 50 in a MiaPaca2 cell line. Optimization of the series led to 29 , a potent cell active molecule (MiaPaca2 IC 50 = 0.67 μM) that also possessed good exposure when dosed orally to mice.

Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enone using structure-based design strategies resulted in inhibitors with considerable improvement in biochemical potency against human lactate dehydrogenase A (LDHA). These potent inhibitors were typically selective for LDHA over LDHB isoform (4–10 fold) and other structurally related malate dehydrogenases, MDH1 and MDH2 (>500 fold). An X-ray crystal structure of enzymatically most potent molecule bound to LDHA revealed two additional interactions associated with enhanced biochemical potency.

A series of 3,6-disubstituted dihydropyrones were identified as inhibitors of human lactate dehydrogenase (LDH)-A. Structure activity relationships were explored and a series of 6,6-spiro analogs led to improvements in LDHA potency (IC50 <350 nM). An X-ray crystal structure of an improved compound bound to human LDHA was obtained and it illustrated additional opportunities to enhance the potency of these compounds, resulting in the identification of 51 (IC50=30 nM)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50=1.7 μM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.18 μM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure-activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F=45%)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A 2-amino-5-aryl-pyrazine was identified as an inhibitor of human lactate dehydrogenase A (LDHA) via a biochemical screening campaign. Biochemical and biophysical experiments demonstrated that the compound specifically interacted with human LDHA. Structural variation of the screening hit resulted in improvements in LDHA biochemical inhibition and pharmacokinetic properties. A crystal structure of an improved compound bound to human LDHA was also obtained and it explained many of the observed structure-activity relationships., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH) was identified by high-throughput screening (IC50=8.1 μM). Biochemical, surface plasmon resonance, and saturation transfer difference NMR experiments indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.48 μM). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure-activity relationships., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Activation of a cellular senescence program is a common response to prolonged oncogene activation or tumor suppressor loss, providing a physiological mechanism for tumor suppression in premalignant cells. The link between senescence and tumor suppression supports the hypothesis that a loss-of-function screen measuring bona fide senescence marker activation should identify candidate tumor suppressors. Using a high-content siRNA screening assay for cell morphology and proliferation measures, we identify 12 senescence-regulating kinases and determine their senescence marker signatures, including elevation of senescence-associated β-galactosidase, DNA damage and p53 or p16 (INK4a) expression. Consistent with our hypothesis, SNP array CGH data supports loss of gene copy number of five senescence-suppressing genes across multiple tumor samples. One such candidate is the EPHA3 receptor tyrosine kinase, a gene commonly mutated in human cancer. We demonstrate that selected intracellular EPHA3 tumor-associated point mutations decrease receptor expression level and/or receptor tyrosine kinase (RTK) activity. Our study therefore describes a new strategy to mine for novel candidate tumor suppressors and provides compelling evidence that EPHA3 mutations may promote tumorigenesis only when key senescence-inducing pathways have been inactivated.

Receptor tyrosine kinase (RTK) signals regulate the specification of a varied array of tissue types by utilizing distinct modules of proteins to elicit diverse effects. The RSK proteins are part of the RTK signal transduction pathway and are thought to relay these signals by acting downstream of extracellular signal-regulated kinase (ERK). In this study we report the identification of ribosomal S6 kinase 4 (Rsk4) as an inhibitor of RTK signals. Among the RSK proteins, RTK inhibition is specific to RSK4 and, in accordance, is dependent upon a region of the RSK4 protein that is divergent from other RSK family members. We demonstrate that Rsk4 inhibits the transcriptional activation of specific targets of RTK signaling as well as the activation of ERK. Developmentally, Rsk4 is expressed in extraembryonic tissue, where RTK signals are known to have critical roles. Further examination of Rsk4 expression in the extraembryonic tissues demonstrates that its expression is inversely correlated with the presence of activated ERK 1/2. These studies demonstrate a new and divergent function for RSK4 and support a role for RSK proteins in the specification of RTK signals during early mouse development.

Signaling between tissues is essential to form the complex, three-dimensional organization of an embryo. Because many receptor tyrosine kinases signal through the RAS-MAPK pathway, phosphorylated ERK can be used as an indicator of when and where signaling is active during development. Using whole-mount immunohistochemistry with antibodies specific to phosphorylated ERK1 and ERK2, we analyzed the location, timing, distribution, duration and intensity of ERK signaling during mouse embryogenesis (5-10.5 days postcoitum). Spatial and temporal domains of ERK activation were discrete with well-defined boundaries, indicating specific regulation of signaling in vivo. Prominent, sustained domains of ERK activation were seen in the ectoplacental cone, extra-embryonic ectoderm, limb buds, branchial arches, frontonasal process, forebrain, midbrain-hindbrain boundary, tailbud, foregut and liver. Transient activation was seen in neural crest, peripheral nervous system, nascent blood vessels, and anlagen of the eye, ear and heart. In the contiguous domains of ERK signaling, phospho-ERK staining was cytoplasmic with no sign of nuclear translocation. With few exceptions, the strongest domains of ERK activation correlated with regions of known or suspected fibroblast growth factor (FGF) signaling, and brief incubation with an inhibitor of the fibroblast growth factor receptor (FGFR) specifically diminished the phospho-ERK staining in these regions. Although many domains of ERK activation were FGFR-dependent, not all domains of FGF signaling were phospho-ERK positive. These studies identify key domains of sustained ERK signaling in the intact mouse embryo, give significant insight into the regulation of this signaling in vivo and pinpoint regions where downstream target genes can be sought.

The absence of the antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) is shown here to cause vacuolar fragmentation in Saccharomyces cerevisiae. Wild-type yeast have 1-3 large vacuoles whereas the sod1Delta yeast have as many as 50 smaller vacuoles. Evidence that this fragmentation is oxygen-mediated includes the findings that aerobically (but not anaerobically) grown sod1Delta yeast exhibit aberrant vacuoles and genetic suppressors of other oxygen-dependent sod1 null phenotypes rescue the vacuole defect. Surprisingly, iron also is implicated in the fragmentation process as iron addition exacerbates the sod1Delta vacuole defect while iron starvation ameliorates it. Because the vacuole is reported to be a site of iron storage and iron reacts avidly with reactive oxygen species to generate toxic side products, we propose that vacuole damage in sod1Delta cells arises from an elevation of iron-mediated oxidation within the vacuole or from elevated pools of "free" iron that may bind nonproductively to vacuolar ligands. Furthermore, additional pleiotropic phenotypes of sod1Delta cells (including increased sensitivity to pH, nutrient deprivation, and metals) may be secondary to vacuolar compromise. Our findings support the hypothesis that oxidative stress alters cellular iron homeostasis which in turn increases oxidative damage. Thus, our findings may have medical relevance as both oxidative stress and alterations in iron homeostasis have been implicated in diverse human disease processes. Our findings suggest that strategies to decrease intracellular iron may significantly reduce oxidatively induced cellular damage.

Mutations in superoxide dismutase 1 (SOD1) polypeptides cause a form of familial amyotrophic lateral sclerosis (FALS). In different kindreds, harboring different mutations, the duration of illness tends to be similar for a given mutation. For example, patients inheriting a substitution of valine for alanine at position four (A4V) average a 1.5 year life expectancy after the onset of symptoms, whereas patients harboring a substitution of arginine for histidine at position 46 (H46R) average an 18 year life expectancy after disease onset. Here, we examine a number of biochemical and biophysical properties of nine different FALS variants of SOD1 polypeptides, including enzymatic activity (which relates indirectly to the affinity of the enzyme for copper), polypeptide half-life, resistance to proteolytic degradation and solubility, in an effort to determine whether a specific property of these enzymes correlates with clinical progression. We find that although all the mutants tested appear to be soluble, the different mutants show a remarkable degree of variation with respect to activity, polypeptide half-life and resistance to proteolysis. However, these variables do not stratify in a manner that correlates with clinical progression. We conclude that the basis for the different life expectancies of patients in different kindreds of sod1-linked FALS may result from an as yet unidentified property of these mutant enzymes.

CTF7 (chromosome transmission fidelity) gene in budding yeast encodes an essential protein that is required for high-fidelity chromosome transmission and contains regions of identity conserved from yeast to man. ctf7 mutant cells arrested prior to anaphase onset contain separated sister chromatids. Thus, Ctf7p is essential for cohesion. Cohesion is established during S phase and then maintained until mitosis. However, Ctf7p activity is required only during S phase, suggesting that Ctf7p functions in the establishment of cohesion. In addition, ctf7 genetically interacts with DNA metabolism mutations pol30 (PCNA) and ctf18 (an RF-C like protein) and ctf7 temperature sensitivity and chromosome loss are rescued by high levels of POL30. These findings provide the first evidence that links the establishment of sister chromatid cohesion to the DNA replication machinery and suggest that the assembly of cohesion (and possibly condensation) complexes are coupled to PCNA-dependent DNA replication. The analysis of Ctf7p also reveals an important connection between sister chromatid cohesion, spindle integrity and the spindle assembly checkpoint.

Copper trafficking in mammalian cells is highly regulated. CCS is a copper chaperone that donates copper to the antioxidant enzyme copper/zinc superoxide dismutase 1 (SOD 1). Mutations of SOD1 are responsible for approximately 20% of familial amyotrophic lateral sclerosis (FALS). Monospecific antibodies were generated to evaluate the localization and cellular distribution of this copper chaperone in human and mouse brain as well as other organs. CCS is found to be ubiquitously expressed by multiple tissues and is present in particularly high concentrations in kidney and liver. In brain and spinal cord, CCS was found throughout the neuropil, with expression largely confined to neurons and some astrocytes. Like SOD1, CCS immunoreactivity was intense in Purkinje cells, deep cerebellar neurons, and pyramidal cortical neurons, whereas in spinal cord, CCS was highly expressed in motor neurons. In cortical neurons, CCS was present in the soma and proximal dendrites, as well as some axons. Although the distribution of CCS paralleled that of SOD1, there was a 12-30-fold molar excess of SOD1 over CCS. That both SOD1 and CCS are present, together, in cells that degenerate in ALS also emphasizes the potential role of CCS in mutant SOD1-mediated toxicity.

Mutations in Cu, Zn superoxide dismutase (SOD1) cause the neurodegenerative disease familial amyotrophic lateral sclerosis from an as-yet-unidentified toxic property(ies). Analysis in Saccharomyces cerevisiae of a broad range of human familial amyotrophic lateral sclerosis-linked SOD1 mutants (A4V, G37R, G41D, H46R, H48Q, G85R, G93C, and I113T) reveals one property common to these mutants (including two at residues that coordinate the catalytic copper): Each does indeed bind copper and scavenge oxygen-free radicals in vivo. Neither decreased copper binding nor decreased superoxide scavenging activity is a property shared by all mutants. The demonstration that shows that all mutants tested do bind copper under physiologic conditions supports a mechanism of SOD1 mutant-mediated disease arising from aberrant copper-mediated chemistry catalyzed by less tightly folded (and hence less constrained) mutant enzymes. The mutant enzymes also are shown to acquire the catalytic copper in vivo through the action of CCS, a specific copper chaperone for SOD1, which in turn suggests that a search for inhibitors of this SOD1 copper chaperone may represent a therapeutic avenue.