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Boopathy, Sivakumar, Tania V. Silvas, Maeve Tischbein, Silvia Jansen, Shivender M. Shandilya, Jill A. Zitzewitz, John E. Landers, Bruce L. Goode, Celia A. Schiffer, and Daryl A. Bosco. "Structural basis for mutation-induced destabilization of profilin 1 in ALS." Proceedings of the National Academy of Sciences 112, no. 26 (June 8, 2015): 7984–89. http://dx.doi.org/10.1073/pnas.1424108112.
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Mutations in profilin 1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS); however, the pathological mechanism of PFN1 in this fatal disease is unknown. We demonstrate that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization is illuminated by the X-ray crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis.
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Schmidt, Eric J., Salome Funes, Jeanne E. McKeon, Brittany R. Morgan, Sivakumar Boopathy, Lauren C. O’Connor, Osman Bilsel, Francesca Massi, Antoine Jégou, and Daryl A. Bosco. "ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization." Proceedings of the National Academy of Sciences 118, no. 23 (June 1, 2021): e2024605118. http://dx.doi.org/10.1073/pnas.2024605118.
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Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin–PFN1–polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.
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Yang, Chunxing, Eric W. Danielson, Tao Qiao, Jake Metterville, Robert H. Brown, John E. Landers, and Zuoshang Xu. "Mutant PFN1 causes ALS phenotypes and progressive motor neuron degeneration in mice by a gain of toxicity." Proceedings of the National Academy of Sciences 113, no. 41 (September 28, 2016): E6209—E6218. http://dx.doi.org/10.1073/pnas.1605964113.
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Mutations in the profilin 1 (PFN1) gene cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease caused by the loss of motor neurons leading to paralysis and eventually death. PFN1 is a small actin-binding protein that promotes formin-based actin polymerization and regulates numerous cellular functions, but how the mutations in PFN1 cause ALS is unclear. To investigate this problem, we have generated transgenic mice expressing either the ALS-associated mutant (C71G) or wild-type protein. Here, we report that mice expressing the mutant, but not the wild-type, protein had relentless progression of motor neuron loss with concomitant progressive muscle weakness ending in paralysis and death. Furthermore, mutant, but not wild-type, PFN1 forms insoluble aggregates, disrupts cytoskeletal structure, and elevates ubiquitin and p62/SQSTM levels in motor neurons. Unexpectedly, the acceleration of motor neuron degeneration precedes the accumulation of mutant PFN1 aggregates. These results suggest that although mutant PFN1 aggregation may contribute to neurodegeneration, it does not trigger its onset. Importantly, these experiments establish a progressive disease model that can contribute toward identifying the mechanisms of ALS pathogenesis and the development of therapeutic treatments.
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Stritt, Simon, Markus Bender, Paquita Nurden, Judith van Eeuwijk, Barbara Zieger, Karim Kentouche, Harald Schulze, et al. "Aberrant Microtubule Organization and Wiskott-Aldrich Syndrome-like Defects in Platelets and Megakaryocytes of Profilin1-Deficient Mice." Blood 124, no. 21 (December 6, 2014): 4200. http://dx.doi.org/10.1182/blood.v124.21.4200.4200.
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Abstract Wiskott-Aldrich syndrome (WAS) is a rare, X-chromosomal recessive disorder which is caused by mutations in the WAS gene and characterized by eczema, immunodeficiency and microthrombocytopenia (Thrasher and Burns, Nat Rev Immunol 2010). Interestingly, WAS protein (WASp)-deficient mice have normal-sized platelets and thus the molecular link between WAS mutations and its central hallmark microthrombocytopenia remains elusive. Profilin1 (Pfn1) is a key actin-regulating protein that, besides actin, interacts with phosphoinositides and multiple proline-rich proteins including the WAS protein (WASp)/WASp-interacting protein (WIP) complex (Witke, Trends Cell Biol 2004; Ramesh et al., Proc Natl Acad Sci USA, 1997). Interestingly, similar to WAS patients, mice with Pfn1-null megakaryocytes/platelets suffered from microthrombocytopenia. We identified accelerated platelet clearance by macrophages and pre-mature platelet release into the bone marrow compartment as the major cause of the reduced platelet count in Pfn1-deficient mice. Both, platelets from Pfn1-null mice and WAS patients contained abnormally organized and hyperstable microtubules. We next tested, if increased microtubule stability could account for the reduced size of Pfn1-deficient platelets. Treatment of control platelets with microtubule stabilizing toxins, such as the histone-deacetylase inhibitor trichostatin A (TSA) or taxol resulted in a decreased platelet size. This finding indicates that increased microtubule stability could account for the reduced platelet size in Pfn1-deficient mice but also in WAS patients. Based on these results we speculate that WASp might modulate Pfn1 function and dysregulation of this interaction leads to increased stability and altered organization of microtubules. In support of this, the subcellular localization of Pfn1 was altered in platelets of three WAS patients. Together, these results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients. Disclosures No relevant conflicts of interest to declare.
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Chen, YongPing, Zhen-Zhen zheng, Rui Huang, Ke Chen, Wei Song, Bi Zhao, XuePing Chen, Yuan Yang, LiXing Yuan, and Hui-Fang Shang. "PFN1 mutations are rare in Han Chinese populations with amyotrophic lateral sclerosis." Neurobiology of Aging 34, no. 7 (July 2013): 1922.e1–1922.e5. http://dx.doi.org/10.1016/j.neurobiolaging.2013.01.013.
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Syriani, Enrique, Candi Salvans, Maria Salvadó, Miguel Morales, Laura Lorenzo, Sonia Cazorla, and Josep Gamez. "PFN1 mutations are also rare in the Catalan population with amyotrophic lateral sclerosis." Journal of Neurology 261, no. 12 (September 24, 2014): 2387–92. http://dx.doi.org/10.1007/s00415-014-7501-x.
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Chi, Jieshan, Junling Chen, Yan Li, Zhiheng Huang, Lijuan Wang, and Yuhu Zhang. "A Familial Phenotypic and Genetic Study of Mutations in PFN1 Associated with Amyotrophic Lateral Sclerosis." Neuroscience Bulletin 36, no. 2 (December 4, 2019): 174–78. http://dx.doi.org/10.1007/s12264-019-00448-8.
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Garitano-Trojaola, Andoni, Ana Sancho, Ralph Goetz, Susanne Walz, Hardikkumar Jetani, Eva Teufel, Nadine Rodhes, et al. "RAC1 Inhibitor EHT1864 and Venetoclax Overcome Midostaurin Resistance in Acute Myeloid Leukemia." Blood 134, Supplement_1 (November 13, 2019): 1277. http://dx.doi.org/10.1182/blood-2019-129762.
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Introduction Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by clonal expansion of immature myeloid progenitor cells in the bone marrow (BM). Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of AML cases, with Internal Tandem Duplications (ITD) being the most common type of mutation. Several FLT3 specific inhibitors (TKI) have been developed such as quizartinib, crenolanib and midostaurin (recently approved for clinical use). Nevertheless FLT3-ITD is associated with unfavorable prognosis and patients develop drug resistance with the underlying mechanisms remaining largely unexplained. Recently, changes within the actin cytoskeleton were associated with drug resistance development in various cancers. FLT3-ITD mutations are associated with RAC1 activation. RAC1 belongs to the family of RHO GTPases and enhances the actin polymerization by inducing the expression of N-WASP or WAVE2 and ARP2/3 complex. Therefore, we investigated actin cytoskeleton rearrangements through RAC1 activation as a potential mechanism contributing to Midostaurin resistance in AML. Material and methods First, we developed two Midostaurin resistant AML cell lines (MID-RES, MV4-11 and MOLM-13). Single cell measurements of Cell Stiffnes, cell adhesion forces between tumor and HS5 stroma cells and Actin filaments were performed by Atomic Force Microscopy (FluidFM®) and SIM microscopy, respectively. RAC1 activation was measured by RAC1 activation kit provided by Cytoskeleton. FLT3 surface and intracellular expression was measured by Flow cytometry and western blot, respectively. Cell death was analyzed by Annexin/PI staining in flow cytometry. Results The MID-RES cell lines MV4-11/MOLM-13 showed higher FLT3 surface and intracellular expression compared to their MID sensitive parental cells. In line with our expectations, we observed RAC1 activation, as well as an up-regulation of actin polymerization positive regulators such as N-WASP, WAVE2, PFN1 and ARP2/3 complex and the inhibition of actin polymerization negative regulator P-ser3 CFL1 in MID-RES cells. FLT3 receptor knock down by siRNAs reversed the MID resistance and reduced RAC1 activation and actin polymerization inducers expression. Likewise, bioinformatic analysis from publicly available microarray expression data (E-MTAB-3444), confirmed positive correlation between actin polymerization inducers and FLT3 signaling expression in 178 FLT3-ITD (r=0,67) and 461 FLT3 WT(r= 0,57) de novoAML patients. RAC1 induced Actin polymerization positively correlates with actin filaments growth and cell stiffness, which was observed in our MID-RES cells, higher load of actin filaments and increased cell stiffness. The combination between RAC1 specific inhibitor, EHT1864 and Midostaurin synergistically induces cell death in MID-RES cells by arresting cell cycle in G0/G1 phase and activating apoptosis. Beside, this combination reduced the adhesion forces to stroma cells, decreased the expression of PFN1/N-WASP/ARP2 and consequently reduced drastically the number of actin filaments and cell stiffness in MID-RES cells. EHT1864 and Midostaurin (alone and in combination) were not toxic in PBMCs obtained from healthy donors. Interestingly, this combination increase >45 % cell death in cells obtained from refractory FLT3-mutated AML patient (this patient was relapsed (≥ 50% residual blasts in the bone marrow)under Chemotherapy+Midostaurin combination).The specific knock down of PFN1/N-WASP/ARP2 with siRNAs equally reversed the resistance to Midostaurin. Of note, RAC1 regulates the anti-apoptotic BCL2. Indeed, EHT1864 in combination with Midostaurin reduced anti-apoptotic family BCL2/MCL1 expression and increases the pro-apoptotic proteins BAX/PUMA. As expected, our MID-RES cells showed higher sensitivity to BCL2 inhibitor Venetoclax, than their parental cells. The combinations EHT1864+venetoclax, venetoclax+midostaurin and venetoclax+Midostaurin+EHT1864 synergistically induced cell death in MID-RES cells. Conclusion Actin polymerization inducers N-WASP, ARP2/3 complex and PFN1 may provide a valuable approach to overcome Midostaurin resistance in AML. Our data further suggest that the addition of BCL2 inhibition through EHT1864 and venetoclax could represent an interesting strategy to potentiate the activity of Midostaurin in FLT3 mutated AML. Disclosures Duell: Regeneron Pharmaceuticals, Inc.: Research Funding. Rosenwald:MorphoSys: Consultancy.
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Voskoboinik, Ilia, Marie-Claude Thia, Annette De Bono, Kylie Browne, Erika Cretney, Jacob T. Jackson, Phillip K. Darcy, Stephen M. Jane, Mark J. Smyth, and Joseph A. Trapani. "The Functional Basis for Hemophagocytic Lymphohistiocytosis in a Patient with Co-inherited Missense Mutations in the Perforin (PFN1) Gene." Journal of Experimental Medicine 200, no. 6 (September 13, 2004): 811–16. http://dx.doi.org/10.1084/jem.20040776.
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About 30% of cases of the autosomal recessive immunodeficiency disorder hemophagocytic lymphohistiocytosis are believed to be caused by inactivating mutations of the perforin gene. We expressed perforin in rat basophil leukemia cells to define the basis of perforin dysfunction associated with two mutations, R225W and G429E, inherited by a compound heterozygote patient. Whereas RBL cells expressing wild-type perforin (67 kD) efficiently killed Jurkat target cells to which they were conjugated, the substitution to tryptophan at position 225 resulted in expression of a truncated (∼45 kD) form of the protein, complete loss of cytotoxicity, and failure to traffic to rat basophil leukemia secretory granules. By contrast, G429E perforin was correctly processed, stored, and released, but the rat basophil leukemia cells possessed reduced cytotoxicity. The defective function of G429E perforin mapped downstream of exocytosis and was due to its reduced ability to bind lipid membranes in a calcium-dependent manner. This study elucidates the cellular basis for perforin dysfunctions in hemophagocytic lymphohistiocytosis and provides the means for studying structure–function relationships for lymphocyte perforin.
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Lattante, Serena, Isabelle Le Ber, Agnès Camuzat, Alexis Brice, and Edor Kabashi. "Mutations in the PFN1 gene are not a common cause in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration in France." Neurobiology of Aging 34, no. 6 (June 2013): 1709.e1–1709.e2. http://dx.doi.org/10.1016/j.neurobiolaging.2012.10.026.
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Merlotti, Daniela, Maria Materozzi, Simone Bianciardi, Vito Guarnieri, Domenico Rendina, Luca Volterrani, Cristiana Bellan, et al. "Mutation of PFN1 Gene in an Early Onset, Polyostotic Paget-like Disease." Journal of Clinical Endocrinology & Metabolism 105, no. 8 (May 11, 2020): 2553–65. http://dx.doi.org/10.1210/clinem/dgaa252.
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Abstract Context Paget disease of bone (PDB) is a metabolic bone disease whose genetic cause remains unknown in up to 50% of familial patients. Objective Our aim was to investigate the underlying genetic defect in a large pedigree with a severe, early onset, autosomal dominant form of PDB across 3 generations. Methods Whole exome sequencing was performed in affected and unaffected family members, and then mutation screening was replicated in a sample of PDB patients with early-onset, polyostotic PDB. Results We identified a frameshift D107Rfs*3 mutation in PFN1 (encoding for profilin 1, a highly conserved regulator of actin-polymerization and cell motility) causing the truncation of the C-terminal part of the protein. The mutation was also detected in a 17-year-old asymptomatic family member who upon biochemical and radiological analyses was indeed found to be affected. Sequencing of the entire PFN1 coding region in unrelated PDB patients identified the same mutation in 1 patient. All mutation carriers had a reduced response to bisphosphonates, requiring multiple zoledronate infusions to control bone pain and achieve biochemical remission over a long term. In vitro osteoclastogenesis in peripheral blood mononuclear cells (PBMCs) from mutation carriers showed a higher number of osteoclasts with PDB-like features. A similar phenotype was observed upon PFN1 silencing in murine bone marrow-derived monocytes, suggesting that the frameshift PFN1 mutation confers a loss of function in profilin 1 activity that induces PDB-like features in the osteoclasts, likely due to enhanced cell motility and actin ring formation. Conclusions Our findings indicate that PFN1 mutation causes an early onset, polyostotic PDB-like disorder.
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Nekouei, Mina, Atousa Aliahmadi, Mahmoud Kiaei, and Ali Reza Ghassempour. "Mutant Profilin1 Aggregation in Amyotrophic Lateral Sclerosis: An in Vivo Biochemical Analysis." Basic and Clinical Neuroscience Journal 12, no. 2 (March 1, 2021): 213–22. http://dx.doi.org/10.32598/bcn.12.2.1631.1.
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Introduction: Profilin1 (PFN1) is a ubiquitously expressed protein known for its function as a regulator of actin polymerization and dynamics. A recent discovery linked mutant PFN1 to Amyotrophic Lateral Sclerosis (ALS), which is a fatal and progressive motor neuron disease. We have also demonstrated that Gly118Val mutation in PFN1 is a cause of ALS, and the formation of aggregates containing mutant PFN1 may be a mechanism for motor neuron death. Hence, we were interested in investigating the aggregation of PFN1 further and searching for co-aggregated proteins in our mouse model overexpressing mutant PFN1. Methods: We investigated protein aggregation in several tissues of transgenic and no-transgenic mice using western blotting. To further understand the neurotoxicity of mutant PFN1, we conducted a pull-down assay using an insoluble fraction of spinal cord lysates from hPFN1G118V transgenic mice. For this assay, we expressed His6-tagged PFN1WT and PFN1G118V in E. coli and purified these proteins using the Ni-NTA column. Results: In this study, we demonstrated that mutant PFN1 forms aggregate in the brain and spinal cord of hPFN1G118V mice, while WT-PFN1 remains soluble. Among these tissues, spinal cord lysates were found to have PFN1 bands at higher molecular weights recognized with anti-PFN1. Moreover, the pull-down assay using His6-PFN1G118V showed that Myelin Binding Protein (MBP) was present in the insoluble fraction. Conclusion: Our analysis of PFN1 aggregation in vivo revealed further details of mutant PFN1 aggregation and its possible complex formation with other proteins, providing new insights into the ALS mechanism.
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Zou, Zhang-Yu, Shi-Dong Chen, Shu-Yan Feng, Chang-Yun Liu, Mei Cui, Shu-fen Chen, Shu-Man Feng, Qiang Dong, Huapin Huang, and Jin-Tai Yu. "Familial flail leg ALS caused by PFN1 mutation." Journal of Neurology, Neurosurgery & Psychiatry 91, no. 2 (August 10, 2019): 223–24. http://dx.doi.org/10.1136/jnnp-2019-321366.
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Haarer, B. K., A. Corbett, Y. Kweon, A. S. Petzold, P. Silver, and S. S. Brown. "SEC3 Mutations Are Synthetically Lethal With Profilin Mutations and Cause Defects in Diploid-Specific Bud-Site Selection." Genetics 144, no. 2 (October 1, 1996): 495–510. http://dx.doi.org/10.1093/genetics/144.2.495.
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Abstract Replacement of the wild-type yeast profilin gene (PFY1) with a mutated form (pfy1-111) that has codon 72 changed to encode glutamate rather than arginine results in defects similar to, but less severe than, those that result from complete deletion of the profilin gene. We have used a colony color-sectoring assay to identify mutations that cause pfy1-111, but not wild-type, cells to be inviable. These profilin synthetic lethal (psl) mutations result in various degrees of abnormal growth, morphology, and temperature sensitivity in PFY1 cells. We have examined psl1 strains in the most detail. Interestingly, these strains display a diploid-specific defect in bud-site selection; haploid strains bud normally, while homozygous diploid strains show a dramatic increase in random budding. We discovered that PSL1 is the late secretory gene, SEC3, and have found that mutations in several other late secretory genes are also synthetically lethal with pfy1-111. Our results are likely to reflect an interdependence between the actin cytoskeleton and secretory processes in directing cell polarity and growth. Moreover, they indicate that the secretory pathway is especially crucial for maintaining budding polarity in diploids.
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Velmurugan, Soundarapandian, Zita Lobo, and Pabitra K. Maitra. "Suppression of pdc2 Regulating Pyruvate Decarboxylase Synthesis in Yeast." Genetics 145, no. 3 (March 1, 1997): 587–94. http://dx.doi.org/10.1093/genetics/145.3.587.
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Mutants lacking pyruvate decarboxylase cannot grow on glucose. We have isolated three different complementation groups of extragenic suppressors that suppress mutations in pdc2, a regulatory locus required for the synthesis of the glycolytic enzyme pyruvate decarboxylase. The most frequent of these is a recessive mutation in the structural gene PFK1 of the soluble phosphofructokinase. The other class XSP18 (extragenic suppressor of pdc2) is a dominant temperature-sensitive suppressor that allows the cells to grow on glucose only at 30° but not at 36°. It also affects the normal induction of the glucose-inducible enolase 2, which can be rescued by providing a copy of wild-type xsp18 in trans-heterozygotes. The pyruvate decarboxylase activity in the triple mutant pdc2 pfk1 XSP18 is nearly equal to the sum of the activities in the two double mutants pdc2 pfk1 and pdc2 XSP18, respectively. This implies that the two suppressors act through independent pathways or that there is no cooperativity between them. In the pdc2 pfk1 XSP18 strain, pfk1 suppresses the loss of induction of glucose-inducible enolase 2 brought about by XSP18, but fails to rescue temperature sensitivity. The third class (xsp37) supports the growth of the pdc2 mutant on glucose but fails to support growth on gluconeogenic carbon sources. All the three suppressors suppress pdc2Δ as well and act on PDC1 at the level of transcription.
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Daoud, Hussein, Sylvia Dobrzeniecka, William Camu, Vincent Meininger, Nicolas Dupré, Patrick A. Dion, and Guy A. Rouleau. "Mutation analysis of PFN1 in familial amyotrophic lateral sclerosis patients." Neurobiology of Aging 34, no. 4 (April 2013): 1311.e1–1311.e2. http://dx.doi.org/10.1016/j.neurobiolaging.2012.09.001.
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Soriano, Ignacio, Enrique Vazquez, Nagore De Leon, Sibyl Bertrand, Ellen Heitzer, Sophia Toumazou, Zhihan Bo, et al. "Expression of the cancer-associated DNA polymerase ε P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication." PLOS Genetics 17, no. 7 (July 6, 2021): e1009526. http://dx.doi.org/10.1371/journal.pgen.1009526.
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Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few indel mutations. The mutations are equally distributed across different genomic regions, but in the immediate vicinity there is an asymmetry in AT frequency. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.
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Wang, Victoria, Chenming Cui, Lei Yang, Gerald Li, Alexa Betzig Schrock, Meijuan Li, Jeffrey Michael Venstrom, and Khaled A. Tolba. "Off-label targeted therapy (TT) use in recurrent/metastatic NSCLC." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e18688-e18688. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e18688.
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e18688 Background: Comprehensive genomic profiling (CGP) is an established diagnostic approach to select patients for TT. As CGP gains wide adoption, an increasing number of patients are found to harbor driver mutations for which no approved TT is available. This is often addressed through use of matched TKI and mAb approved for other mutations or anatomic sites. In this work, we examined the clinical efficacy of off-label TT in R/M NSCLC. Methods: Using a de-identified NSCLC clinico-genomic database (CGDB), we identified 6590 NSCLC patients who underwent Foundation Medicine CGP, of whom 17.8% harbored an actionable genomic alteration (GA) for which an FDA-approved TT was available and 2% (133) whose GA (MET ex-14, uncommon EGFRm, EGFR ex20ins, HER2 amp/mut, RET fusion, BRAF class 2/3) lacked an FDA-approved TT (62 in 1L and 71 in ≥2L ) who received matched off-label TT. ESMO Scale for Clinical Actionability (ESCAT) was used to grade levels of evidence. For patients who progressed on initial chemotherapy (range 2 – 9 lines, median 3), we calculated clinical efficacy using the ratio of real world PFS on targeted therapy (rw-PFS2) to rw-PFS on the last prior line of therapy (rw-PFS1) and used a cut-off of PFS2/PFS1 > 1.3 to determine off-label drug efficacy. Results: Of the 133 patients reviewed, 72 were classified as ESCAT level IB (uncommon EGFRm, MET-ex14), 45 IIB (HER2m/amp, EGFR ex-20ins), 7 IC (RET fusions). PFS varied significantly by mutation and line of therapy (table 1) with uncommon EGFRm+ and MET-ex14 exhibiting best response while EGFR ex20 ins, BRAF class 2/3 and HER2 amp fared significantly worse. 55.8% of the patients (39 of 71) reached a PFS2/PFS1 ratio > 1.3 (two-sided 95% CI, 45.3 % – 68.7 %), ranging from 93% in uncommon EGFRm+ down to 20% in HER2 amp and 44% in ex20ins. Conclusions: We provide real-world evidence to assess off-label TT in NSCLC. Clinical benefit derived from off-label TT is unevenly distributed across various mutations with most survival advantage accruing to specific mutations (MET-ex14 and uncommon EGFRm) at the expense of others (HER2 amp). Survival advantage was highly influenced by two factors: A) the timing of CGP with the earlier recipients of genomic profiling achieving better outcome, B) the identity of the driver mutation, highlighting the role of clinical actionability tier system to define level of evidence supporting such intervention.[Table: see text]
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Nekouei, Mina, Parviz Ghezellou, Atousa Aliahmadi, Sareh Arjmand, Mahmoud Kiaei, and Alireza Ghassempour. "Changes in biophysical characteristics of PFN1 due to mutation causing amyotrophic lateral sclerosis." Metabolic Brain Disease 33, no. 6 (September 10, 2018): 1975–84. http://dx.doi.org/10.1007/s11011-018-0305-4.
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Lin, Jianzhen, Xu Yang, Yinghao Cao, Guangyu Li, Songhui Zhao, Junping Shi, Jie Pan, et al. "Genomics and translational precision oncology for 803 patients with biliary tract cancer." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 4589. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.4589.
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4589 Background: Both incidence and mortality of biliary tract cancer (BTC) are increasing, and BTCs are characterized by poor prognosis and limited antitumoral treatments. There is no well-received regimen as the non-first-line treatment in patients with advanced BTCs, leading to the urgency of umbrella-setting personalized therapies according to genomic alterations. Methods: We performed genomic sequencing in a total of 803 BTCs, including 160 patients with whole-exome sequencing and 643 patients with hybrid capture–based comprehensive genomic profiling. Our molecular tumor board developed precise targeted therapies for patients with actionable targets. Results: Overall, the median tumor mutation burden was 3.0 (IQR: 0.8-6.1) Mut/Mb, with 10.5% patients of hypermutated BTCs. The most frequently mutated genes included TP53 (51%), KRAS (23%), ARID1A (16%) and SMAD4 (11%). The most common genes with significantly amplified oncogenes were CCND1 (6.97%), MET (6.72%) and MDM2 (6.6%), while the frequently deleted tumor-suppressor genes are CDKN2A (5.73%) and CDKN2B (5.35%). The mutational map of BTCs highlighted pathways of receptor-tyrosine kinase (RTK)/RAS and p53 signaling were frequently altered. Somatic truncating mutations of mismatch repair genes were identified in 6.1% (49/803) of patients, and germline pathogenic mutations in DNA damage response genes occurred in 8% (64/803) of BTCs. In addition, we demonstrate the amplified chromosomal focal at 7q31.2 was an oncogenic factor and it independently predicts both disease-free survival and overall survival of BTC patients. When molecular screening was linked to targeted therapies, 25.4% (204/803) of patients could match biomarker-assigned drug treatment (BADT). The frequent actionable biomarkers included amplifications of ERBB2 and MET, FGFR2/3 fusions and IDH1 mutations. For 46 patients with refractory BTCs received BADT, the objective response rate was 26.1%, with a median progression-free survival (mPFS) of 5.0 (95%CI: 3.5-6.5) months, and 56.8% patients achieved a ≥1.3 ratio of PFS2/PFS1. 4 of 6 (67%) patients with high microsatellite instability (MSI-H) BTCs had a responsive status after immunotherapy of PD1 inhibitor, confirming that MSI-H status was a robust biomarker of anti-PD1 treatments. Conclusions: Our study established the largest cohort in Chinese BTC patients to investigate the tumor mutational profiling and its translational clinical applications. Clinical trial information: NCT02715089 .
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Yang, Shu, Jennifer A. Fifita, Kelly L. Williams, Sadaf T. Warraich, Roger Pamphlett, Garth A. Nicholson, and Ian P. Blair. "Mutation analysis and immunopathological studies of PFN1 in familial and sporadic amyotrophic lateral sclerosis." Neurobiology of Aging 34, no. 9 (September 2013): 2235.e7–2235.e10. http://dx.doi.org/10.1016/j.neurobiolaging.2013.04.003.
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Yuan, W., D. L. Tuttle, Y. J. Shi, G. S. Ralph, and W. A. Dunn. "Glucose-induced microautophagy in Pichia pastoris requires the alpha-subunit of phosphofructokinase." Journal of Cell Science 110, no. 16 (August 15, 1997): 1935–45. http://dx.doi.org/10.1242/jcs.110.16.1935.
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We have characterized biochemically, morphologically, and genetically two distinct pathways for the selective degradation of peroxisomes in Pichia pastoris. These pathways are independently regulated and analogous to microautophagy and macroautophagy that have been defined in mammalian cells. When P. pastoris is grown in methanol, cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized. During adaptation from methanol to glucose, these enzymes are rapidly and selectively degraded within the yeast vacuole by microautophagy. We have isolated gsa mutants that are defective in glucose-induced selective autophagy of peroxisomes. In this study, we have shown that gsa1 is unable to sequester peroxisomes into the yeast vacuole. In addition, we provide evidence that the glucose-induced selective autophagy 1 (GSA1) protein is the alpha subunit of the phosphofructokinase enzyme complex encoded by PFK1. First, we can rescue the gsa1 mutant by transformation with a vector containing PFK1. Second, cellular levels of both PFK1 mRNA and phosphofructokinase activity are dramatically reduced in gsa1 when compared to the parental GS115. Third, a PFK1 knockout (delta pfk1) is unable to degrade alcohol oxidase during glucose adaptation. As observed in gsa1, the peroxisomes in delta pfk1 remain outside the vacuole during adaptation. Our data are consistent with the concept that PFK1 protein is required for an event upstream of vacuole degradation (i.e. signaling, selection, or sequestration). However, the degradation of peroxisomes does not require a catalytically active phosphofructokinase. The inability of delta pfk1 cells to degrade alcohol oxidase can be rescued by transformation with either normal PFK1 or mutant pfk1 whose catalytic site had been inactivated by a single amino acid mutation. We propose that PFK1 protein directly modulates glucose-induced microautophagy independent of its ability to metabolize glucose intermediates.
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Yochum, Gregory S., and Donald E. Ayer. "Pf1, a Novel PHD Zinc Finger Protein That Links the TLE Corepressor to the mSin3A-Histone Deacetylase Complex." Molecular and Cellular Biology 21, no. 13 (July 1, 2001): 4110–18. http://dx.doi.org/10.1128/mcb.21.13.4110-4118.2001.
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ABSTRACT The mSin3A-histone deacetylase corepressor is a multiprotein complex that is recruited by DNA binding transcriptional repressors. Sin3 has four paired amphipathic alpha helices (PAH1 to -4) that are protein-protein interaction motifs and is the scaffold upon which the complex assembles. We identified a novel mSin3A-interacting protein that has two plant homeodomain (PHD) zinc fingers we term Pf1, for PHD factor one. Pf1 associates with mSin3A in vivo and recruits the mSin3A complex to repress transcription when fused to the DNA binding domain of Gal4. Pf1 interacts with Sin3 through two independent Sin3 interaction domains (SIDs), Pf1SID1 and Pf1SID2. Pf1SID1 binds PAH2, while Pf1SID2 binds PAH1. Pf1SID1 has sequence and structural similarity to the well-characterized 13-amino-acid SID of the Mad bHLHZip repressor. Pf1SID2 does not have sequence similarity with either Mad SID or Pf1SID1 and therefore represents a novel Sin3 binding domain. Mutations in a minimal fragment of Pf1 that encompasses Pf1SID1 inhibited mSin3A binding yet only slightly impaired repression when targeted to DNA, implying that Pf1 might interact with other corepressors. We show that Pf1 interacts with a mammalian homolog of the Drosophila Groucho corepressor, transducin-like enhancer (TLE). Pf1 binds TLE in an mSin3A-independent manner and recruits functional TLE complexes to repress transcription. These findings suggest that Pf1 may serve to bridge two global transcription networks, mSin3A and TLE.
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Juneja, Manisha, Abdelkrim Azmi, Jonathan Baets, Andreas Roos, Matthew J. Jennings, Paola Saveri, Chiara Pisciotta, et al. "PFN2 and GAMT as common molecular determinants of axonal Charcot-Marie-Tooth disease." Journal of Neurology, Neurosurgery & Psychiatry 89, no. 8 (February 15, 2018): 870–78. http://dx.doi.org/10.1136/jnnp-2017-317562.
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BackgroundCharcot-Marie-Tooth type 2 (CMT2) neuropathy is characterised by a vast clinical and genetic heterogeneity complicating its diagnosis and therapeutic intervention. Identification of molecular signatures that are common to multiple CMT2 subtypes can aid in developing therapeutic strategies and measuring disease outcomes.MethodsA proteomics-based approach was performed on lymphoblasts from CMT2 patients genetically diagnosed with different gene mutations to identify differentially regulated proteins. The candidate proteins were validated through real-time quantitative PCR and western blotting on lymphoblast samples of patients and controls, motor neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) and sciatic nerves of CMT2 mouse models.ResultsProteomic profiling of patient lymphoblasts resulted in the identification of profilin 2 (PFN2) and guanidinoacetate methyltransferase (GAMT) as commonly downregulated proteins in different genotypes compared with healthy controls. This decrease was also observed at the transcriptional level on screening 43 CMT2 patients and 22 controls, respectively. A progressive decrease in PFN2 expression with age was observed in patients, while in healthy controls its expression increased with age. Reduced PFN2 expression was also observed in motor neurons differentiated from CMT2 patient-derived iPSCs and sciatic nerves of CMT2 mice when compared with controls. However, no change in GAMT levels was observed in motor neurons and CMT2 mouse-derived sciatic nerves.ConclusionsWe unveil PFN2 and GAMT as molecular determinants of CMT2 with possible indications of the role of PFN2 in the pathogenesis and disease progression. This is the first study describing biomarkers that can boost the development of therapeutic strategies targeting a wider spectrum of CMT2 patients.
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Ding, Zhen-Yu, and Yue-Yun Chen. "The first- or second-line use of pemetrexed sequentially with EGFR-TKI did not differ in PFS for advanced NSCLC patients harboring EGFR-mutant tumors." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): e20531-e20531. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e20531.
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e20531 Background: Both EGFR-TKI and pemetrexed doublet chemotherapy (P) are two effective therapies in advanced NSCLC patients with EGFR mutant tumors. But which one should be used in first-line remains controversial. Methods: This was a retrospective study where patients prescribed with EGFR-TKI (gefitinib or icotinib) from Jan 2013 to Jan 2016 were screened. Patients must have metastastic diseases harboring TKI-sensitizing EGFR mutation. They must be older than 18 years, and have evaluable target lesions. Whether TKI or P was used in the first line, it must be switched to the other in the second line. PFS in both first line (PFS1) and second line (PFS2) was collected. Results: We screened totally 550 patients (gefitinib n = 455, icotinib n = 95) to enroll a cohort of 36 patients. They were all adenocarcinoma except 1 adenosquamous carcinoma. Gender (M or F), PS (0 or 1), mutation type (Exon19Ddel or other) were equally distributed. The median age was 50.5 years. For the whole cohort, the total PFS (PFS1+PFS2) was 18.1 m (95%CI: 15.2-21.1 m). For those (n = 24) receiving first-line TKI, PFS1, PFS2, and total PFS were 10.3 m, 6.6 m, and 19.2 m. And for those receiving first-line P (n = 12), they were were 3.4 m, 11.5 m, and 16.5m. The total PFS in both groups did not differ significantly (p = 0.548). Conclusions: Our results argued the sequential usage of TKI and P achieve a comparable PFS irrespective of the sequence.
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Hu, Yong-hua, Chun-sheng Liu, Jin-hui Hou, and Li Sun. "Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain." Applied and Environmental Microbiology 75, no. 13 (May 15, 2009): 4333–40. http://dx.doi.org/10.1128/aem.00159-09.
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ABSTRACT A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50°C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.
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Wang, Yuxiang, Wenjian Yu, Jian Shi, Nan Jiang, Zhoufan Wang, Jie Yang, Zhongfei Jia, and Meng Song. "Factors on survival for patients of EGFR mutation advanced lung adenocarcinoma with EGFR-TKIs combined with radiotherapy: Real-world data from single center." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e21126-e21126. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e21126.
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e21126 Background: The aim was to retrospectively evaluate survival and prognostic factors for patients of advanced lung adenocarcinoma with EGFR mutation by a real world data. Methods: From Jan, 2015 to Dec, 2020, lung adenocarcinoma with EGFR positive mutation, advanced (clinical stage III-IV), without surgery, received EGFR-TKIs and radiotherapy (RT) were enrolled. OS and PFS were calculated. Results: 238 patients were included, 101 in males and 137 in females; the median ages was 61. The RT was performed at the time of no-PD in 71 and PD in 167 cases. The median dose of RT was 50 Gy. The rate of acute AE during RT was 36.1% in grade 1-2 and 4.8% in grade 3-4. The follow-up ended at 30, Dec, 2020. The 1-, 2-, 3-years OS,PFS1and PFS2 were 84.4%,59.7%, 38.7%, 52.0%,29.4%, 16.3%, 86.6%, 64.6%, 45.6%. mOS, mPFS1and mPFS2 were 30.3, 14.1, 33.6 months, respectively. Multivariate analysis showed the independent factors for OS were ages (median were 34.0 and 24.9 months for ≤65 and > 65) and clinical stage (median were 22.8, 34.8 and 27.4 months for stage III (33), IVA (84) and IVB (121 cases), respectively), chemotherapy (CT) (median were 27.4, 28.1, 32.5, 44.3 months for TKIs alone(120), TKIs concurrent CT (43), TKIs sequential CT (32), CT sequential TKIs (43 cases), respectively) and total response (the medians were 32.5, 27.2, 33.3 months for CR(7)+PR(107), SD(70) and PD(54 cases), respectively); Independent factors: PFS1 was the time of RT at no-PD and PD (the median were 17.8 and13.2 months), chemotherapy, (the median were 16.1, 12.1, 11.0, 13.5 for TKIs alone, TKIs concurrent CT, TKIs sequential CT, CT sequential TKIs), and total response (the medians were 18.8, 11.7, 10.9 months for CR+PR, SD and PD); PFS2 was the total response (the medians were 58.3, 21.6, 20.9 months for CR+PR, SD and PD, respectively). Univariate analysis also showed doses of RT (the median was 12.3 months for < 50 Gy and 15.5 months for ≥ 50 Gy, p = 0.018) was associated with PFS1, the first-line drugs of TKIs (the median were 39.1, 35.0, 25.6 and 38.6 months for Gefitinib (89), Icotinib (109), Erlotinib (29) and others (11 cases), respectively, p = 0.044) and chemotherapy (the medians were 45.6, 30.6, 18.7, 41.4 months for TKIs alone, TKIs concurrent CT, TKIs sequential CT, CT sequential TKIs, respectively, p = 0.007) were related with PFS2; but the type of EGFR mutation (94 in 19 exon, 118 in 21 exon and 26 cases in others) was not related with survival. Conclusions: EGFR-TKIs combined RT was tolerable and efficient for patients of advanced lung adenocarcinoma with EGFR mutation. TKIs add RT at the time of no-PD could improve PFS1. CT sequential TKIs was probably in favour of OS, even PFS. The better total response (CR+PR) was associated with longer OS, PFS1 and PFS2. But the result need to be proved furtherly.
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Cheng, Jia-Tao, Jin-Ji Yang, and Yi-Long Wu. "Impact of next-generation sequencing on clinical outcomes in advanced EGFR-mutant lung cancer patients after resistance to osimertinib." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e20726-e20726. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e20726.
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e20726 Background: Osimertinib is used to treat EGFR-mutant non–small-cell lung cancer (NSCLC) with acquired T790M mutation. Next-generation sequencing (NGS) is helpful to understand mechanisms of resistance to osimertinib. However, whether NGS after resistance to osimertinib has an impact on clinical outcomes of patients treated with subsequent treatments has been elusive. Methods: We retrospectively identified advanced, EGFR-mutant T790M positive NSCLC patients treated with the 2nd or further-line osimertinib from January 27th, 2015 to January 31th, 2019 at our institute. Genetic profiles and clinical outcomes were analyzed. These patients were divided into 2 groups based on NGS data after resistance to osimertinib. Progression-free survival1 (PFS1) was calculated from the start of osimertinib to progression or death. PFS2 was calculated from the start of subsequent-line treatment to progression or death. Objective response rate (ORR) of subsequent-line treatments was evaluated by RECIST1.1. Results: Among 187 patients treated with osimertinib, 66 had NGS data and 27 had no NGS data after progression. Maintained EGFR T790M was detected in 23 patients (34.8%), and loss of T790M was seen in 43 patients (65.2%). Mutations of EGFR C797S were detected in 12 patients (18.1% overall; 52.2% of those with retained T790M), 11 in cis with a maintained T790M, 1 in trans with a maintained T790M. There was no significant difference in median PFS1 between the maintained T790M group and the loss of T790M group (10.8 vs. 7.0 months, P = 0.085).The NGS group was treated with TKIs according to the results of NGS strictly (n = 36), the non-NGS group received chemotherapy or best supportive care (n = 11).There was a significant difference in median PFS2 between the NGS and non-NGS groups (5.4 vs. 2.9 months, P = 0.043). The ORR of the NGS group was significantly superior to that of the non-NGS group (16.2% vs 11.1%, P < 0.001). Conclusions: NGS after resistance to osimertinib might favor clinical outcomes of advanced EGFR-mutant NSCLC patients. Further more investigations are warranted.
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Lee, Belinda, Angelyn Anton, Margaret Lee, Rachel Wong, Phillip Parente, Jeremy David Shapiro, Desmond Yip, et al. "Examining progression-free survival in first- and second-line treatment for BRAF-mutant metastatic colorectal cancer (CRC)." Journal of Clinical Oncology 35, no. 4_suppl (February 1, 2017): 728. http://dx.doi.org/10.1200/jco.2017.35.4_suppl.728.
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728 Background: BRAF mutated (BRAFm) CRC represents ~10% of all CRC and is associated with significantly poorer prognosis. However, responses to chemotherapy do still occur. Some data suggest that the poor prognosis associated with BRAFm CRC is dominated by substantially poorer second line PFS (PFS2), whereas first line PFS (PFS1) was similar for both BRAFm and BRAF wildtype (BRAFwt) CRC. Using a large multicenter dataset, our study aimed to examine PFS1 and PFS2 in BRAFm versus BRAFwt CRC. Methods: Prospectively collected data from the Treatment of Recurrent and Advanced Colorectal Cancer (TRACC) database was interrogated. PFS was calculated and compared in patients with BRAFm versus BRAFwt CRC. Median survival was determined by the Kaplan-Meier method and compared using the log rank test. Results: TRACC identified 523 CRC patients with known BRAF mutation status, who received first-line chemotherapy: 53 (10%) were BRAFm, while 470 (90%) were BRAFwt. At the time of data analysis, only 231 (44%) CRC patients had received second-line chemotherapy, of which 21 (9%) were BRAFm and 210 (91%) were BRAFwt. PFS1 analyses demonstrated significantly poorer survival in the BRAFm population (Median 7.8mo versus 11.5mo, HR 1.72, p = 0.0026). PFS2 analyses revealed similar findings for the BRAFm population, albeit non-significant due to smaller numbers (Median 5.5mo versus 7.7mo, HR1.26, p = 0.44). Conclusions: Our study demonstrated that BRAFm CRC was associated with poorer PFS in both first- and second-line settings. Additional analyses will be performed to examine the impact of different treatment strategies and other clinicopathological features.
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Wolven, Amy K., Lisa D. Belmont, Nicole M. Mahoney, Steven C. Almo, and David G. Drubin. "In Vivo Importance of Actin Nucleotide Exchange Catalyzed by Profilin." Journal of Cell Biology 150, no. 4 (August 21, 2000): 895–904. http://dx.doi.org/10.1083/jcb.150.4.895.
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The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP2 and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin–cofilin generated during filament disassembly.
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Cheng, Weixiao, Jon A. Doering, Carlie LaLone, and Carla Ng. "Integrative Computational Approaches to Inform Relative Bioaccumulation Potential of Per- and Polyfluoroalkyl Substances Across Species." Toxicological Sciences 180, no. 2 (January 23, 2021): 212–23. http://dx.doi.org/10.1093/toxsci/kfab004.
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Abstract Predictive toxicology is increasingly reliant on innovative computational methods to address pressing questions in chemicals assessment. Of importance is the evaluation of contaminant impact differences across species to inform ecosystem protection and identify appropriate model species for human toxicity studies. Here we evaluated 2 complementary tools to predict cross-species differences in binding affinity between per- and polyfluoroalkyl substances (PFAS) and the liver fatty acid-binding protein (LFABP): the Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool and molecular dynamics (MD). SeqAPASS determined that the structure of human LFABP, a key determinant of PFAS bioaccumulation, was conserved in the majority of vertebrate species, indicating these species would have similar PFAS bioaccumulation potentials. Level 3 SeqAPASS evaluation identified several potentially destabilizing amino acid differences across species, which were generally supported by DUET stability change predictions. Nine single-residue mutations and 7 whole species sequences were selected for MD evaluation. One mutation (F50V for PFNA) showed a statistically significant difference with stronger affinity than wild-type human LFABP. Predicted binding affinities for 9 different PFAS across 7 species showed human, rat, chicken, and rainbow trout had similar binding affinities to one another for each PFAS, whereas Japanese medaka and fathead minnow had significantly weaker LFABP-binding affinity for some PFAS. Based on these analyses, the combined use of SeqAPASS and MD provides rapid screening for potential species differences with deeper structural insight. This approach can be easily extended to other important biological receptors and potential ligands.
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Finger, F. P., and P. Novick. "Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae." Molecular Biology of the Cell 8, no. 4 (April 1997): 647–62. http://dx.doi.org/10.1091/mbc.8.4.647.
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Two new temperature-sensitive alleles of SEC3, 1 of 10 late-acting SEC genes required for targeting or fusion of post-Golgi secretory vesicles to the plasma membrane in Saccharomyces cerevisiae, were isolated in a screen for temperature-sensitive secretory mutants that are synthetically lethal with sec4-8. The new sec3 alleles affect early as well as late stages of secretion. Cloning and sequencing of the SEC3 gene revealed that it is identical to profilin synthetic lethal 1 (PSL1). The SEC3 gene is not essential because cells depleted of Sec3p are viable although slow growing and temperature sensitive. All of the sec3 alleles genetically interact with a profilin mutation, pfy1-111. The SEC3 gene in high copy suppresses pfy1-111 and sec5-24 and causes synthetic growth defects with ypt1, sec8-9, sec10-2, and sec15-1. Actin structure is only perturbed in conditions of chronic loss of Sec3p function, implying that Sec3p does not directly regulate actin. All alleles of sec3 cause bud site selection defects in homozygous diploids, as do sec4-8 and sec9-4. This suggests that SEC gene products are involved in determining the bud site and is consistent with a role for Sec3p in determining the correct site of exocytosis.
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Gadelha, Catarina, Bill Wickstead, Wanderley de Souza, Keith Gull, and Narcisa Cunha-e-Silva. "Cryptic Paraflagellar Rod in Endosymbiont-Containing Kinetoplastid Protozoa." Eukaryotic Cell 4, no. 3 (March 2005): 516–25. http://dx.doi.org/10.1128/ec.4.3.516-525.2005.
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ABSTRACT Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.
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Wadman, Renske I., Marc D. Jansen, Chantall A. D. Curial, Ewout J. N. Groen, Marloes Stam, Camiel A. Wijngaarde, Jelena Medic, et al. "Analysis of FUS, PFN2, TDP-43, and PLS3 as potential disease severity modifiers in spinal muscular atrophy." Neurology Genetics 6, no. 1 (January 3, 2020): e386. http://dx.doi.org/10.1212/nxg.0000000000000386.
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ObjectiveTo investigate mutations in genes that are potential modifiers of spinal muscular atrophy (SMA) severity.MethodsWe performed a hypothesis-based search into the presence of variants in fused in sarcoma (FUS), transactive response DNA-binding protein 43 (TDP-43), plastin 3 (PLS3), and profilin 2 (PFN2) in a cohort of 153 patients with SMA types 1–4, including 19 families. Variants were detected with targeted next-generation sequencing and confirmed with Sanger sequencing. Functional effects of the identified variants were analyzed in silico and for PLS3, by analyzing expression levels in peripheral blood.ResultsWe identified 2 exonic variants in FUS exons 5 and 6 (p.R216C and p.S135N) in 2 unrelated patients, but clinical effects were not evident. We identified 8 intronic variants in PLS3 in 33 patients. Five PLS3 variants (c.1511+82T>C; c.748+130 G>A; c.367+182C>T; c.891-25T>C (rs145269469); c.1355+17A>G (rs150802596)) potentially alter exonic splice silencer or exonic splice enhancer sites. The variant c.367+182C>T, but not RNA expression levels, corresponded with a more severe phenotype in 1 family. However, this variant or level of PLS3 expression did not consistently correspond with a milder or more severe phenotype in other families or the overall cohort. We found 3 heterozygous, intronic variants in PFN2 and TDP-43 with no correlation with clinical phenotype or effects on splicing.ConclusionsPLS3 and FUS sequence variants do not modify SMA severity at the population level. Specific variants in individual patients or families do not consistently correlate with disease severity.
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Newman, James, Chung-Shien Lee, Kerri McGovern, and Nagashree Seetharamu. "Back to the well: Can patients with advanced non-small cell lung cancer benefit from changing PD-1/PD-L1 inhibitors after progression?" Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e21194-e21194. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e21194.
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e21194 Background: Immune checkpoint inhibitors (ICIs) have transformed the standard of care of non-small cell lung cancer (NSCLC) and are capable of inducing a sustained response in a cohort of patients. For those who progress while on ICIs and do not have a targetable mutation, options are typically reduced to chemotherapeutic regimens which have a higher probability of toxicity. In patients with a suboptimal performance status or those who are opposed to receiving chemotherapy and are not eligible or interested in clinical trials, treatments are unfortunately limited. Patients who progress on one ICI do not receive a different ICI as part of standard care in subsequent lines of therapy. Data behind switching ICIs, particularly to those with a different mechanism of action (ie anti-PD-1 followed by anti-PD-L1 or vice versa), are lacking. We evaluated the efficacy of receiving a 2nd ICI in patients with NSCLC. Methods: A single-center, IRB-approved retrospective analysis was conducted of NSCLC patients treated with two different ICIs from March 2015 to July 2020. ICIs were given either in combination with chemotherapy or as monotherapy. Patient and tumoral characteristics, including PD-L1 status (if available) and sequence of ICIs, were captured. A positive PD-L1 was defined as PD-L1 expression > 0%. Progression-free survival (PFS) of each ICI (defined as PFS1 and PFS2) were calculated in months (m) for each patient. Median PFS2 was compared between groups stratified by a cutoff median PFS1 of 3m, sequence of PD-1 and PD-L1 inhibitors, and PD-L1 positive and negative subsets. Mood’s median test was used to compare medians. Results: 26 patients were included in the final analysis. 19/26 patients had a PFS1 > 3m. For this cohort, the median PFS2 was 2.5m (Range: 0.1-36.2) compared to median PFS2 of 1.6m (Range: 0.6-15.9) in patients with PFS1 < 3m (p=0.1847). 15 patients received a PD-1 inhibitor followed by a PD-L1 inhibitor. In this subgroup, the median PFS2 was 1.2m (Range: 0.2-36.2). Alternatively, 7 patients received a PD-L1 inhibitor followed by a PD-1 inhibitor and the median PFS2 in this cohort was 2.3m (Range: 0.1-15.9) (p=0.6471). PD-L1 data was available for 19 patients. 8/19 patients with positive PD-L1 had a median PFS2 of 3.6m (Range: 0.2-13.3) compared to median PFS2 of 1.3m (Range: 0.1-36.2) in patients with negative PD-L1 (p=0.2599). Conclusions: Treatment with a 2nd ICI can potentially provide a modest benefit in patients with advanced NSCLC, and some may even experience a very prolonged response. PFS1, sequence of PD-1/PD-L1 inhibitors, and PD-L1 expression did not show any significant correlation with PFS2. Using a 2nd ICI should be considered for advanced NSCLC patients with diminished performance status or limited treatment options.
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Newcomb, Laura L., Jasper A. Diderich, Matthew G. Slattery, and Warren Heideman. "Glucose Regulation of Saccharomyces cerevisiae Cell Cycle Genes." Eukaryotic Cell 2, no. 1 (February 2003): 143–49. http://dx.doi.org/10.1128/ec.2.1.143-149.2003.
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ABSTRACT Nutrient-limited Saccharomyces cerevisiae cells rapidly resume proliferative growth when transferred into glucose medium. This is preceded by a rapid increase in CLN3, BCK2, and CDC28 mRNAs encoding cell cycle regulatory proteins that promote progress through Start. We have tested the ability of mutations in known glucose signaling pathways to block glucose induction of CLN3, BCK2, and CDC28. We find that loss of the Snf3 and Rgt2 glucose sensors does not block glucose induction, nor does deletion of HXK2, encoding the hexokinase isoenzyme involved in glucose repression signaling. Rapamycin blockade of the Tor nutrient sensing pathway does not block the glucose response. Addition of 2-deoxy glucose to the medium will not substitute for glucose. These results indicate that glucose metabolism generates the signal required for induction of CLN3, BCK2, and CDC28. In support of this conclusion, we find that addition of iodoacetate, an inhibitor of the glyceraldehyde-3-phosphate dehydrogenase step in yeast glycolysis, strongly downregulates the levels CLN3, BCK2, and CDC28 mRNAs. Furthermore, mutations in PFK1 and PFK2, which encode phosphofructokinase isoforms, inhibit glucose induction of CLN3, BCK2, and CDC28. These results indicate a link between the rate of glycolysis and the expression of genes that are critical for passage through G1.
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Liu, Yongmei, Hao Wei, Xiaojuan Zhou, Hui Yang, Youling Gong, Jin Wang, Yong Xu, et al. "Stereotactic body radiotherapy to the lung primary lesion improves the survival of patients with non-oligometastatic NSCLC harboring EGFR activating mutation with first-line EGFR-TKIs: A real-world study." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e21131-e21131. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e21131.
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e21131 Background: This study aimed to explore the clinical value of SBRT for lung primary lesions of EGFR-mutant NSCLC patients with non-oligometastatic disease during first-line EGFR-TKI treatment. Methods: Patients with stage IV EGFR-mutant NSCLC and more than five metastases at diagnosis were identified. All patients were treated with first-line EGFR-TKIs and SBRT for their primary lesions. The primary end points were the progression-free survival-1 (PFS1, time of first TKI dose relative to disease progression based on RECIST), and PFS2 (time of first TKI dose relative to disease progression after SBRT). The secondary endpoints were overall survival (OS) and safety. Results: 79 patients were enrolled, including 45 patients who received SBRT for their primary tumor at the maximal response of EGFR-TKI (the preemptive RT group) and 34 patients who received SBRT for their primary tumor after the occurrence of oligo-progression (the delayed RT group). The preemptive RT group had a significantly better median PFS1 than the delayed RT group (22.3 months vs. 12.9 months, P = 0.0031). The median PFS2 in the preemptive RT and delayed RT groups were 22.3 and 28.9 months, respectively (P = 0.17). The median OS did not differ significantly between the preemptive RT group and the delayed RT group (46.6 versus 51.3 months, P = 0.54). No severe toxicities (≥ grade 3) were recorded. Conclusions: This real-world study showed that preemptive RT to lung primary tumors is a feasible option for patients with EGFR-mutant non-oligometastatic NSCLC who had stable disease during first-line EGFR-TKI treatment, with significantly improved PFS and OS.
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Zhou, Jin-Qiu, Haiyan Qi, Vincent P. Schulz, Maria K. Mateyak, Ellen K. Monson, and Virginia A. Zakian. "Schizosaccharomyces pombe pfh1+Encodes an Essential 5′ to 3′ DNA Helicase That Is a Member of thePIF1 Subfamily of DNA Helicases." Molecular Biology of the Cell 13, no. 6 (June 2002): 2180–91. http://dx.doi.org/10.1091/mbc.02-02-0021.
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The Saccharomyces cerevisiae Pif1p DNA helicase is the prototype member of a helicase subfamily conserved from yeast to humans. S. cerevisiae has two PIF1-like genes, PIF1 itself and RRM3, that have roles in maintenance of telomeric, ribosomal, and mitochondrial DNA. Here we describe the isolation and characterization ofpfh1 + , aSchizosaccharomyces pombe gene that encodes a Pif1-like protein. Pfh1p was the only S. pombe protein with high identity to Saccharomyces Pif1p. Unlike the twoS. cerevisiae Pif1 subfamily proteins, the S. pombe Pfh1p was essential. Like SaccharomycesPif1p, a truncated form of the S. pombe protein had 5′ to 3′ DNA helicase activity. Point mutations in an invariant lysine residue in the ATP binding pocket of Pfh1p had the same phenotype as deleting pfh1 + , demonstrating that the ATPase/helicase activity of Pfh1p was essential. Although mutant spores depleted for Pfh1p proceeded through S phase, they arrested with a terminal cellular phenotype consistent with a postinitiation defect in DNA replication. Telomeric DNA was modestly shortened in the absence of Pfh1p. However, genetic analysis demonstrated that maintenance of telomeric DNA was not the sole essential function of S. pombe Pfh1p.
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KIRCHBERGER, Jürgen, Anke EDELMANN, Gerhard KOPPERSCHLÄGER, and Jürgen J. HEINISCH. "A single point mutation leads to an instability of the hetero-octameric structure of yeast phosphofructokinase." Biochemical Journal 341, no. 1 (June 24, 1999): 15–23. http://dx.doi.org/10.1042/bj3410015.
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Yeast phosphofructokinase is an oligomeric enzyme whose detectable activity in vitro depends on its hetero-octameric structure. Here we provide data demonstrating that an alanine residue at positions 874 (for the PFK1-encoded α-subunit) or 868 (for the PFK2-encoded β-subunit) is crucial to achieve this structure. Thus subunits carrying substitutions by either aspartate or lysine of this residue cause a lack of phosphofructokinase activity in vitro and signals of the subunits are poorly detectable in Western blots. Size-exclusion HPLC in conjunction with ELISA detection of the enzyme protein confirmed that no functional octamer is produced in such mutants. Our data suggest that the mutant subunits, not being assembled, tend to aggregate and subsequently become degraded. Substitution of the alanine by valine in either subunit leads to a reduction in specific activities, as expected from a conservative exchange. The kinetic data of the latter mutant revealed a higher affinity to the substrate fructose 6-phosphate, a lower extent of ATP inhibition and a lower degree of activation by fructose 2,6-bisphosphate. In addition, the affinity of mutants carrying a valine instead of an alanine in either the α- or the β-subunit to fructose 2,6-bisphosphate was increased. As no X-ray data on eukaryotic phosphofructokinases are available yet, our data provide the first evidence that a non-charge amino acid at position 874 or 868 is essential for the formation of the functional oligomer. This conclusion is substantiated by comparison with the structure of the well-known prokaryotic enzyme.
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Ray-Coquard, Isabelle Laure, Philipp Harter, Antonio Gonzalez Martin, Claire Cropet, Sandro Pignata, Keiichi Fujiwara, Christian Marth, et al. "PAOLA-1: An ENGOT/GCIG phase III trial of olaparib versus placebo combined with bevacizumab as maintenance treatment in patients with advanced ovarian cancer following first-line platinum-based chemotherapy plus bevacizumab." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): TPS5605. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.tps5605.
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TPS5605 Background: Olaparib (Lynparza) is an oral PARP inhibitor indicated in the EU for the maintenance treatment of patients (pts) with platinum-sensitive relapsed BRCA-mutated high grade serous ovarian cancer (HGSOC). Bevacizumab is an anti-VEGF monoclonal antibody indicated in the EU in first line or relapse for the treatment of OC in combination with specific chemotherapeutic agents. Bevacizumab treatment is associated with increasing hypoxia-induced homologous recombination repair deficiencies in tumor cells, and is hypothesized to increase ovarian tumor sensitivity to olaparib. Methods: PAOLA-1 (ENGOT-ov25) is a randomized, placebo-controlled trial evaluating the efficacy and safety of olaparib (tablet formulation) in pts with advanced HGSOC receiving bevacizumab maintenance therapy. Eligible pts are those in complete or partial response following first-line platinum chemotherapy plus bevacizumab, and for whom bevacizumab maintenance therapy is planned. Approximately 762 European and 24 Japanese pts will be randomized 2:1 to olaparib 300 mg twice daily or placebo for up to 24 months. All pts will receive standard maintenance care of bevacizumab (15 mg/kg every three weeks) for up to 15 months. Primary objective: PFS1 according to RECIST 1.1 Secondary objectives: PFS2, OS, Safety, PRO/QoL, TFST, TSST All pts will undergo tumor BRCA testing prior to randomization. Central BRCA testing (tumor) will be performed in five screening platforms in France. Tumor BRCA test results have to be available within two months of sample provision. PFS will be evaluated using a log-rank test stratified by response to first-line treatment and BRCA mutation status. Treatment effect hazard ratio of 0.7 is expected and final PFS1 analysis will be performed after 372 events. The first pt from eight ENGOT groups plus Japan (10 participating countries) was randomized in July 2015. As of 31 January 2017, 549 pts have been randomized. The median period between the provision of a tumor sample and returned BRCA test result is 40 days. Accrual is expected to be complete before July 2017. Clinical trial information: NCT02477644.
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Rodríguez-Rojas, Alexandro, and Jesús Blázquez. "The Pseudomonas aeruginosa pfpI Gene Plays an Antimutator Role and Provides General Stress Protection." Journal of Bacteriology 191, no. 3 (November 21, 2000): 844–50. http://dx.doi.org/10.1128/jb.01081-08.
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ABSTRACT Hypermutator Pseudomonas aeruginosa strains, characterized by an increased spontaneous-mutation rate, are found at high frequencies in chronic lung infections. Hypermutability is associated with the loss of antimutator genes related to DNA repair or damage avoidance systems. Only a few antimutator genes have been described in P. aeruginosa, although there is some evidence that additional genes may be involved in naturally occurring hypermutability. In order to find new P. aeruginosa antimutator genes, we constructed and screened a library of random insertions in the PA14 strain. Some previously described P. aeruginosa and/or Escherichia coli antimutator genes, such as mutS, mutL, uvrD, mutT, ung, and mutY, were detected, indicating a good coverage of our insertional library. One additional mutant contained an insertion in the P. aeruginosa PA14-04650 (pfpI) gene, putatively encoding a member of the DJ-1/ThiJ/PfpI superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1a. The pfpI-defective mutants in both PAO1 and PA14 showed higher spontaneous mutation rates than the wild-type strains, suggesting that PfpI plays a key role in DNA protection under nonstress conditions. Moreover, the inactivation of pfpI resulted in a dramatic increase in the H2O2-induced mutant frequency. Global transcription studies showed the induction of bacteriophage Pf1 genes and the repression of genes related to iron metabolism, suggesting that the increased spontaneous-mutant frequency may be due to reduced protection against the basal level of reactive oxygen species. Finally, pfpI mutants are more sensitive to different types of stress and are affected in biofilm formation.
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Leparc-Goffart, Isabelle, Susan T. Hingley, Ming Ming Chua, Joanna Phillips, Ehud Lavi, and Susan R. Weiss. "Targeted Recombination within the Spike Gene of Murine Coronavirus Mouse Hepatitis Virus-A59: Q159 Is a Determinant of Hepatotropism." Journal of Virology 72, no. 12 (December 1, 1998): 9628–36. http://dx.doi.org/10.1128/jvi.72.12.9628-9636.1998.
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ABSTRACT Previous studies of a group of mutants of the murine coronavirus mouse hepatitis virus (MHV)-A59, isolated from persistently infected glial cells, have shown a strong correlation between a Q159L amino acid substitution in the S1 subunit of the spike gene and a loss in the ability to induce hepatitis and demyelination. To determine if Q159L alone is sufficient to cause these altered pathogenic properties, targeted RNA recombination was used to introduce a Q159L amino acid substitution into the spike gene of MHV-A59. Recombination was carried out between the genome of a temperature-sensitive mutant of MHV-A59 (Alb4) and RNA transcribed from a plasmid (pFV1) containing the spike gene as well as downstream regions, through the 3′ end, of the MHV-A59 genome. We have selected and characterized two recombinant viruses containing Q159L. These recombinant viruses (159R36 and 159R40) replicate in the brains of C57BL/6 mice and induce encephalitis to a similar extent as wild-type MHV-A59. However, they exhibit a markedly reduced ability to replicate in the liver or produce hepatitis compared to wild-type MHV-A59. These viruses also exhibit reduced virulence and reduced demyelination. A recombinant virus containing the wild-type MHV-A59 spike gene, wtR10, behaved essentially like wild-type MHV-A59. This is the first report of the isolation of recombinant viruses containing a site-directed mutation, encoding an amino acid substitution, within the spike gene of any coronavirus. This technology will allow us to begin to map the molecular determinants of pathogenesis within the spike glycoprotein.
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Reinacher-Schick, Anke C., Stefanie Noepel-Duennebacke, Jan Hertel, Andrea Tannapfel, Dirk Arnold, Axel Hinke, and Susanna Hegewisch-Becker. "Localization of the primary tumor (LPT) and maintenance strategies after first line oxaliplatin (Ox), fluoropyrimidine (FP), and bevacizumab (Bev) in metastatic colorectal cancer (mCRC): Results from the AIO 0207 trial." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 3543. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.3543.
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3543 Background: Numerous trials have examined the prognostic and predictive value of the LPT in mCRC, but little is known about the predictive value of LPT on different maintenance strategies. We analyzed progression-free survival (PFS) and overall survival (OS) from start of maintenance according to LPT in patients (pts) from the AIO KRK 0207 trial. Methods: Following a 24-week standard induction 471 pts were randomized to FP/Bev, Bev mono or no treatment with 454 pts being evaluable for PFS. Right sided primary tumors were defined as located in the caecum, ascending colon, transverse colon up to the splenic flexure; left colon was defined as splenic flexure, descending and sigmoid colon and rectum. Results: Data on LPT was available in 414 pts. for PFS (91%). LPT was left sided (LPTl) in 291 (70%) and right-sided (LPTr) in 123 (30%) of pts, respectively (remaining pts: status was either unknown, n = 37 or LPT was located in both regions, n = 3). Median PFS1 was 3.9 months (mos.) for LPTr and 5.3 mos. for LPTl (p = 0.11; HR 1.19, 95%CI 0.96 - 1.48). Analyses on PFS did not demonstrate a major predictive impact of LPT on the efficacy of the three maintenance strategies. The pairwise comparison of treatment arms showed a better PFS for FP/Bev vs no treatment independent from LPT (left: p < 0.0001; HR = 2.39, 95%CI 1.73-3.31; right: p = 0.011; HR 1.78, 95%CI 1.14-2.80). In addition, Bev mono vs no treatment was superior in LPTl (p = 0.0032; HR 1.54, 95%CI 1.15-2.06) with less difference in LPTr (p = 0.17; HR 1.36, 95%CI 0.87-2.14). Analysis for OS (429 evaluable pts) confirmed the strong prognostic impact of LPT (left vs right: 24.0 vs 16.7 months; p < 0.0001; HR = 1.65, 95%CI 1.32 - 2.06), but without major interaction between LPT and maintenance arms. The impact related to RAS mutational status will be reported. Conclusions: The strong prognostic factor of the LPT is confirmed in pts with mCRC undergoing Ox/FP/Bev induction therapy while there seems to be no major predictive impact of LPT on different maintenance strategies. Clinical trial information: EudraCT-Nr: 2008-007974-39.
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Yan, Xiao-Jie, Florencia Palacios, Wentian Li, Sophia Yancopoulos, Carlo Calissano, Jacqueline C. Barrientos, Steven L. Allen, Jonathan E. Kolitz, Kanti Rai, and Nicholas Chiorazzi. "B Cells of the Proliferative Fraction of CLL Clones Exhibit Activated B-Cell and Myeloid-Cell Signatures Suggesting Enhanced Antigen-Presentation, Integrin Responsiveness, and IL-4 Receptiveness: Additional Targets for CLL Therapy." Blood 128, no. 22 (December 2, 2016): 302. http://dx.doi.org/10.1182/blood.v128.22.302.302.
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Abstract CLL results from the accumulation of monoclonal B lymphocytes that derive from a small fraction of cells with proliferative activity. Because expression of the DNA mutator, activation-induced cytidine deaminase (AID) is restricted to these dividing cells, they can develop new DNA abnormalities leading to more lethal disease. Hence, such cells are important targets for therapy. Our previous study indicated that the B-cell subset with low levels of CXCR4 and high levels of CD5 (CXCR4DimCD5Bright) is enriched in these cells ("proliferative fraction", PF), whereas the less vital, resting cells exhibit a CXCR4BrightCD5Dim phenotype ("resting fraction", RF). In this study, we focused on analyzing the significantly differentially expressed genes (DEGs) between PF and RF. PF and RF were isolated from 26 CLL (13 U-CLL and 13 M-CLL) and microarrays (llumina HT12) were performed. Selected DEGs between PF and RF were confirmed by rtQ-PCR and/or by flow cytometry. Array data were interpreted using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). First, focusing on the Immunologic Signature of B lymphocytes with GSEA, we found the PF was enriched in gene sets shared with IgM memory cells and pre-germinal center B cells, whereas RF was enriched in gene sets in common with naïve, IgD+ B cells. Notably, the PF also shared gene sets with myeloid dendritic cells and monocytes. Protein expression of 10 myeloid markers (CD68, CD1c, CD11a, CD11b, CD11c, CD13, CD31, CD205, CXCR3 and CLECL1) was documented in PF B cells from 11 U-CLL and 11 M-CLL patients, and each was more highly expressed in the PF than RF. No difference in myeloid markers was observed between U-CLL and M-CLL, suggesting that this expression is independent of IGHV mutation status. DEGs were also determined based on expression ratios for PF and RF for each patient; t tests were performed using R. With a fold change cutoff of > 1.5 or < -1.5 and a level of significance of P < 0.01, we identified 198 genes significantly upregulated in PF and 88 in RF. The top biological-function categories identified using IPA indicated that these genes related to cellular development (n=37), cellular growth and proliferation (n=49), cellular movement (n=42) and cell survival and death (n=50). In addition, these DEGs mapped to 8 canonical pathways with Z-scores ≥2, the highest being the integrin signaling pathway (Z-score = 2.887). Twelve of 13 genes were upregulated in the PF and correlated significantly with the integrin pathway: ACTG, ARPC5, ARPC1B, BCAR3, CAPNS1, ITGAX, ITGB1, ITGB2, ITGB7, PFN1, RAC2 and RHOC. Upregulation of integrin subunits was confirmed by Q-PCR and cell surface staining by flow cytometry. Preliminary cellular adhesion experiments suggest PF bind to fibronectin coated plates, and those cells that bind survive better. Next, comparing PF and RF expression ratios for U-CLL vs. M-CLL revealed 502 DEGs in U-CLL and 179 in M-CLL; 144 genes were shared by both U-CLL and M-CLL. IPA analysis of the latter genes correlated best with integrin signaling, and the potential upstream regulators of these were IFNg, IL1, F2 and IFNa. The Rho Family GTPases signaling pathway was significant (10 genes) for DEGs unique to U-CLL. No significant pathway or bio-function relationship was observed in DEGs only in M-CLL. Finally, IPA analysis showed IL4 as an upstream regulator of DEGs unique to the PF of U-CLL based on upregulation of 12 IL4 target genes (APRT, CCL5, CDKN1A, DECR1, IL17RB, ITGAL, ITGB1, LTA, MAPKAPK3, PIGR, SAMSN1, TIMP1). Three other IL-4 targeted genes were paradoxically under-represented in the U-CLL PF (BCL2, CCR7 and VIPR1). The upregulated findings are consistent with PF B cells inducing T cells to produce IL-4 via co-receptors on B/myeloid cells that foster a Th2 response (e.g., CLECL1). In conclusion, gene expression profiling indicates that cells of the PF display a dual activated B cell/myeloid cell phenotype suggesting enhanced antigen-presentation capacities. Integrin signaling appears to be a key pathway for these cells which could foster cell proliferation, survival, and migration, especially in U-CLL clones where integrin activation can lead to Rho GTPases activation. Finally, genes regulated by IL-4 in the PF could be induced by interactions of autologous T cells with CLL B cells. These findings suggest antigen-presentation and integrin and IL-4 signaling pathways as therapeutic targets in CLL, particularly for U-CLL. Disclosures Barrientos: AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Janssen: Consultancy.
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Sadr, Ahmad Shahir, Changiz Eslahchi, Alireza Ghassempour, and Mahmoud Kiaei. "In silico studies reveal structural deviations of mutant profilin-1 and interaction with riluzole and edaravone in amyotrophic lateral sclerosis." Scientific Reports 11, no. 1 (March 25, 2021). http://dx.doi.org/10.1038/s41598-021-86211-4.
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AbstractThis study aimed to investigate four of the eight PFN-1 mutations that are located near the actin-binding domain and determine the structural changes due to each mutant and unravel how these mutations alter protein structural behavior. Swapaa’s command in UCSF chimera for generating mutations, FTMAP were employed and the data was analyzed by RMSD, RMSF graphs, Rg, hydrogen bonding analysis, and RRdisMaps utilizing Autodock4 and GROMACS. The functional changes and virtual screening, structural dynamics, and chemical bonding behavior changes, molecular docking simulation with two current FDA-approved drugs for ALS were investigated. The highest reduction and increase in Rg were found to exist in the G117V and M113T mutants, respectively. The RMSF data consistently shows changes nearby to this site. The in silico data described indicate that each of the mutations is capable of altering the structure of PFN-1 in vivo. The potential effect of riluzole and edaravone two FDA approved drugs for ALS, impacting the structural deviations and stabilization of the mutant PFN-1 is evaluated using in silico tools. Overall, the analysis of data collected reveals structural changes of mutant PFN-1 protein that may explain the neurotoxicity and the reason(s) for possible loss and gain of function of PFN-1 in the neurotoxic model of ALS.
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Bertucci, François, Anthony Gonçalves, Arnaud Guille, José Adelaïde, Séverine Garnier, Nadine Carbuccia, Emilien Billon, et al. "Prospective high-throughput genome profiling of advanced cancers: results of the PERMED-01 clinical trial." Genome Medicine 13, no. 1 (May 18, 2021). http://dx.doi.org/10.1186/s13073-021-00897-9.
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Abstract Background The benefit of precision medicine based on relatively limited gene sets and often-archived samples remains unproven. PERMED-01 (NCT02342158) was a prospective monocentric clinical trial assessing, in adults with advanced solid cancer, the feasibility and impact of extensive molecular profiling applied to newly biopsied tumor sample and based on targeted NGS (t-NGS) of the largest gene panel to date and whole-genome array-comparative genomic hybridization (aCGH) with assessment of single-gene alterations and clinically relevant genomic scores. Methods Eligible patients with refractory cancer had one tumor lesion accessible to biopsy. Extracted tumor DNA was profiled by t-NGS and aCGH. We assessed alterations of 802 “candidate cancer” genes and global genomic scores, such as homologous recombination deficiency (HRD) score and tumor mutational burden. The primary endpoint was the number of patients with actionable genetic alterations (AGAs). Secondary endpoints herein reported included a description of patients with AGA who received a “matched therapy” and their clinical outcome, and a comparison of AGA identification with t-NGS and aCGH versus whole-exome sequencing (WES). Results Between November 2014 and September 2019, we enrolled 550 patients heavily pretreated. An exploitable complete molecular profile was obtained in 441/550 patients (80%). At least one AGA, defined in real time by our molecular tumor board, was found in 393/550 patients (71%, two-sided 90%CI 68–75%). Only 94/550 patients (17%, 95%CI 14–21) received an “AGA-matched therapy” on progression. The most frequent AGAs leading to “matched therapy” included PIK3CA mutations, KRAS mutations/amplifications, PTEN deletions/mutations, ERBB2 amplifications/mutations, and BRCA1/2 mutations. Such “matched therapy” improved by at least 1.3-fold the progression-free survival on matched therapy (PFS2) compared to PFS on prior therapy (PFS1) in 36% of cases, representing 6% of the enrolled patients. Within patients with AGA treated on progression, the use of “matched therapy” was the sole variable associated with an improved PFS2/PFS1 ratio. Objective responses were observed in 19% of patients treated with “matched therapy,” and 6-month overall survival (OS) was 62% (95%CI 52–73). In a subset of 112 metastatic breast cancers, WES did not provide benefit in term of AGA identification when compared with t-NGS/aCGH. Conclusions Extensive molecular profiling of a newly biopsied tumor sample identified AGA in most of cases, leading to delivery of a “matched therapy” in 17% of screened patients, of which 36% derived clinical benefit. WES did not seem to improve these results. Trial registration ID-RCB identifier: 2014-A00966-41; ClinicalTrials.gov identifier: NCT02342158.
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Huang, Yen-Hsiang, Jeng-Sen Tseng, Kuo-Hsuan Hsu, Kun-Chieh Chen, Kang-Yi Su, Sung-Liang Yu, Jeremy J. W. Chen, Tsung-Ying Yang, and Gee-Chen Chang. "The impact of different first-line EGFR-TKIs on the clinical outcome of sequential osimertinib treatment in advanced NSCLC with secondary T790M." Scientific Reports 11, no. 1 (June 8, 2021). http://dx.doi.org/10.1038/s41598-021-91657-7.
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AbstractThe impact of different first-line epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI)s to the clinical efficacy of osimertinib in EGFR-mutant non-small-cell lung cancer (NSCLC) patients with acquired T790M was still unclear. We enrolled 733 advanced EGFR-mutant NSCLC patients with gefitinib, erlotinib or afatinib as first-line EGFR-TKIs treatment for analysis. 373 patients received re-biopsies after progressive disease to first-line EGFR-TKIs treatment, and the total positive rate of T790M was 51.7%. 151 patients who harbored T790M received osimertinib as subsequent treatment. Among them, the median progression-free survival (PFS) of first-line EGFR-TKI (PFS1) was 14.0 months, and the median PFS of osimertinib (PFS2) was 10.1 months. The median PFS1 + PFS2 was 27.5 months, and the median overall survival from first-line EGFR-TKI was 61.3 months. Concerning different first-line EGFR-TKIs, the median PFS2 was 10.9 months in the gefitinib group, 10.0 months in the erlotinib group, and 6.7 months in the afatinib group (p = 0.534). The median PFS1 + PFS2 was 27.7 months, 26.8 months and 24.0 months in the gefitinib, erlotinib, and afatinib group, respectively (p = 0.575). In conclusion, both first-generation and second-generation EGFR-TKIs sequential osimertinib treatment provided good clinical efficacy in advanced EGFR-mutant NSCLC patients with acquired T790M mutation.
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Su, Buli, Anzhang Li, Ming-Rong Deng, and Honghui Zhu. "Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces cerevisiae via ethanol-induced adaptive laboratory evolution." Bioresources and Bioprocessing 8, no. 1 (June 11, 2021). http://dx.doi.org/10.1186/s40643-021-00402-5.
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AbstractCarotenoids are a large family of health-beneficial compounds that have been widely used in the food and nutraceutical industries. There have been extensive studies to engineer Saccharomyces cerevisiae for the production of carotenoids, which already gained high level. However, it was difficult to discover new targets that were relevant to the accumulation of carotenoids. Herein, a new, ethanol-induced adaptive laboratory evolution was applied to boost carotenoid accumulation in a carotenoid producer BL03-D-4, subsequently, an evolved strain M3 was obtained with a 5.1-fold increase in carotenoid yield. Through whole-genome resequencing and reverse engineering, loss-of-function mutation of phosphofructokinase 1 (PFK1) was revealed as the major cause of increased carotenoid yield. Transcriptome analysis was conducted to reveal the potential mechanisms for improved yield, and strengthening of gluconeogenesis and downregulation of cell wall-related genes were observed in M3. This study provided a classic case where the appropriate selective pressure could be employed to improve carotenoid yield using adaptive evolution and elucidated the causal mutation of evolved strain.