5 results on '"Yingqi Lin"'
Search Results
2. Identification and Characterization of a B-Raf Kinase α-Helix Critical for the Activity of MEK Kinase in MAPK Signaling
- Author
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Christopher Chung, Ronen Marmorstein, Naomi Bronkema, Twan W Sia, YongJoon Shin, Daniel J Boehlmer, Diep Nguyen, Emily Kibby, Jeffrey O. Zhou, Ming-Ray Xu, Rajiv Potluri, Linda Yingqi Lin, Narine Vapuryan, Michael Grasso, Daniela Fera, Tiara Tillis, Caroline Burkhard, Shirley H Zeng, Elizabeth Erler, and Lucila A. Acevedo
- Subjects
MAPK/ERK pathway ,Models, Molecular ,Protein Conformation, alpha-Helical ,Proto-Oncogene Proteins B-raf ,MAP Kinase Signaling System ,Allosteric regulation ,Static Electricity ,MAP Kinase Kinase 1 ,In Vitro Techniques ,Biochemistry ,Article ,03 medical and health sciences ,Drug Discovery ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Binding site ,Phosphorylation ,Melanoma ,0303 health sciences ,Kinase ,Drug discovery ,Effector ,Chemistry ,030302 biochemistry & molecular biology ,HEK 293 cells ,Recombinant Proteins ,Cell biology ,Kinetics ,HEK293 Cells ,Drug Resistance, Neoplasm ,Mutation ,Mutagenesis, Site-Directed ,Allosteric Site - Abstract
In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ~3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-Raf(V600E) expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-Raf(V600E). We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-Raf(V600E)-induced melanomas.
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- 2020
3. Identification and Characterization of a B-Raf Kinase Alpha Helix Critical for the Activity of MEK Kinase in MAPK Signaling
- Author
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Ming-Ray Xu, YongJoon Shin, Caroline Burkhard, Daniela Fera, Lucila A. Acevedo, Rajiv Potluri, Narine Vapuryan, Twan Sia, Naomi Bronkema, Shirley H Zeng, Linda Yingqi Lin, Jeffrey O. Zhou, Michael Grasso, Ronen Marmorstein, Diep Nguyen, Daniel J Boehlmer, Christopher Chung, Emily Kibby, Tiara Tillis, and Elizabeth Erler
- Subjects
MAPK/ERK pathway ,Mutation ,Chemistry ,Kinase ,Effector ,Mutant ,Allosteric regulation ,Raf kinase ,medicine.disease_cause ,Biochemistry ,Cell biology ,Mapk signaling ,Genetics ,medicine ,Phosphorylation ,Identification (biology) ,Binding site ,Molecular Biology ,Alpha helix ,Biotechnology - Abstract
In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ∼3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.
- Published
- 2020
4. Combined +58 and +55 BCL11A enhancer Editing Yields Exceptional Efficiency, Specificity and HbF Induction in Human and NHP Preclinical Models
- Author
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Myriam Armant, Selami Demirci, David R. Williams, Amornrat Tangprasittipap, Stacy Maitland, Pengpeng Liu, Daniel E. Bauer, J. Keith Joung, Karl Petri, Danilo Pellin, Jing Zeng, John F. Tisdale, Linda Yingqi Lin, John P. Manis, Kevin Luk, Daniela Abriss, Scot A. Wolfe, Yuxuan Wu, Christian Brendel, Shengdar Q. Tsai, Luca Pinello, Varun Katta, Jonathan Y. Hsu, Chokdee Vong, Robert E. Donahue, Shondra M. Pruett-Miller, My Anh Nguyen, Khaled Essawi, Naoya Uchida, Duantida Songdej, Shaina N. Porter, Vikram Pattanayak, Suradej Hongeng, Marioara-Felicia Ciuculescu, and Esther Mintzer
- Subjects
Chemistry ,Immunology ,Cell Biology ,Hematology ,Computational biology ,Enhancer ,Biochemistry - Abstract
Targeting the BCL11A erythroid enhancer by gene editing is a promising approach to fetal hemoglobin induction for beta-hemoglobinopathies. HbF levels vary widely among individuals, suggesting potential heterogeneity in HbF responses after therapeutic intervention. We hypothesize that maximizing both gene edit frequency and HbF induction potential could promote consistently favorable clinical outcomes. Here we compared CRISPR-Cas9 endonuclease editing of the BCL11A +58 enhancer with alternative gene modification approaches, including +55 erythroid enhancer editing alone or in combination with the +58 enhancer, as well as editing targeting the HBG1/2 promoter -115 BCL11A binding site and transduction by an shRNA knocking down the BCL11A transcript in erythroid precursors. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in the most potent HbF induction (52.4%±6.3%) of tested approaches (BCL11A +58 editing alone, 29.1%±3.9%; BCL11A +55 editing alone, 34.8±5.1%; HBG1/2 promoter editing, 34.1% ±5.4%; shmiR-BCL11A, 32.2%±4.4%; mock, 7.6%±3.4%). Based on assays in bulk and single cell derived erythroid cultures and xenografted immunodeficient mice, we found that disruption of core half E-box/GATA motifs at both the +58 and +55 enhancers was associated with greatest HbF induction, whether by small indels, interstitial 3.1 kb deletion, or 3.1 kb inversion. Rare gene edited clones with alleles that only partially disrupted these motifs were associated with intermediate HbF induction phenotypes. Combined editing of BCL11A +58 and +55 enhancers was compatible with HSC self-renewal in primary and secondary xenotransplant, with intact lymphoid, myeloid and erythroid repopulation. We conducted gene-edited cell product manufacturing process development and developed conditions using a MaxCyte electroporation instrument achieving mean 97.3±1.8% gene edits and 88.9%±6.4% viability 24 hours after electroporation in 3 engineering runs at clinical scale. We obtained similar results at small-scale with plerixafor-mobilized HSPCs from sickle cell disease (SCD) donors or G-CSF mobilized PBMCs from transfusion-dependent beta-thalassemia (TDT) donors, including 94.2%±4.4%, 99.5%±0.3% and 91.8%±6.3% of gene edits in engrafting cells from NBSGW 16 week mouse bone marrow of healthy, SCD and TDT donors respectively. Off-target analyses by pooled amplicon sequencing of 601 candidate off-target sites for the +58 and +55 targeting sgRNAs, nominated by a range of computational (CRISPRme) and experimental (GUIDE-seq and ONE-seq) methods, did not identify reference genome off-target edits at a sensitivity of 0.1% allele frequency. We evaluated +58/+55 enhancer combined targeting in nonhuman primates by performing ribonucleoprotein (RNP) electroporation in rhesus macaque mobilized peripheral blood CD34+ HSPCs with autologous re-infusion following busulfan myeloablation. We observed highly efficient gene edit frequency (85.2%, 88.8% and 84.9%) and durable HbF induction (26.4%, 57.5%, and 45.9% F-cells and 12.7%, 41.9%, and 28% gamma-globin) in the peripheral blood in 3 animals at most recent recorded time point post infusion (127, 78, and 54 weeks respectively). Single colony analyses and bulk ddPCR and unidirectional sequencing demonstrated that the long-term engrafting cells displayed a similar distribution of indels, 3.1 kb deletions, and 3.1 kb inversions as the input cell products. Erythroid stress due to hydroxyurea treatment, with or without phlebotomy, was associated with substantially augmented HbF responses (to 75.9%, 88.2%, and 57.8% F-cells and 47.9%, 68%, and 35.7% gamma-globin). No hematologic or other toxicities attributable to gene editing were observed. Together these results suggest that combined BCL11A +58 and +55 erythroid enhancer editing produces highly efficient on-target allelic disruption, erythroid-specific BCL11A downregulation, heightened HbF induction capacity compared to alternative approaches, preserved long-term multilineage engraftment potential by both human xenotransplant and rhesus autotransplant assays, and absence of evident genotoxicity, under clinically relevant SpCas9 RNP electroporation conditions. Disclosures No relevant conflicts of interest to declare.
- Published
- 2021
5. Human Genetic Diversity Alters Therapeutic Gene Editing Off-Target Outcomes
- Author
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Samuele Cancellieri, Rosalba Giugno, Francesco Masillo, Nicola Bombieri, Stacy Maitland, Daniel E. Bauer, Myriam Armant, Linda Yingqi Lin, Luca Pinello, Shengdar Q. Tsai, Marioara-Felicia Ciuculescu, Scot A. Wolfe, My Anh Nguyen, Jing Zeng, and Varun Katta
- Subjects
Genome editing ,Immunology ,Cell Biology ,Hematology ,Human genetic variation ,Computational biology ,Biology ,Biochemistry - Abstract
CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia (Frangoul et al. NEJM 2021). We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF) = 4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (~9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We expect that variant-aware off-target assessment will be required for therapeutic genome editing efforts going forward, including both ongoing and future clinical trials, and we provide a powerful approach for comprehensive off-target prediction. CRISPRme is available at crisprme.di.univr.it. Disclosures No relevant conflicts of interest to declare.
- Published
- 2021
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