10 results on '"Amanda Heard"'
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2. Costimulatory domains direct distinct fates of CAR-driven T cell dysfunction
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Mehmet Emrah Selli, Jack Landmann, Marina Terekhova, John Lattin, Amanda Heard, Yu-Sung Hsu, Tien-Ching Chang, Ju-fang Chang, John M Warrington, Helen Ha, Natalie L Kingston, Graham Hogg, Michael Slade, Melissa M Berrien-Elliott, Mark Foster, Samantha Kersting-Schadek, Agata Gruszczynska, David DeNardo, Todd A Fehniger, Maxim Artyomov, and Nathan Singh
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Immunology ,Cell Biology ,Hematology ,Biochemistry ,Article - Abstract
T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B cell cancers yet fail to induce durable remissions for nearly half of patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven anti-tumor T cell function. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation of CAR T cells from a patient with progressive lymphoma confirmed activation of this novel program in a failing clinical product. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.
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- 2023
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3. Costimulatory Domains Direct Distinct Fates of CAR T Cell Dysfunction
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Mehmet Emrah Selli, Jack Landmann, John Lattin, Amanda Heard, John Warrington, Helen Ha, Jufang Chang, Natalie Kingston, Graham Hogg, Mark Foster, Samantha Kersting-Schadek, Marina Terekhova, David DeNardo, Todd A. Fehniger, Maxim Artyomov, and Nathan Singh
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
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4. LIGAND-ASSISTED LOW-TEMPERATURE GROWTH OF SnO2 NANOAGGREGATES
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Amanda Heard, Faisal Rafi, Harshini Vasudevanallur Subra, Saeid Vafaei, Stephan Lucas, Daichi Inoue, Takashi Sugiura, and Kazuhiro Manseki
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- 2022
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5. U2af1 is a haplo-essential gene required for hematopoietic cancer cell survival in mice
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Ajay Khanna, Timothy A. Graubert, Brian A. Wadugu, Matthew Ndonwi, Michael O. Alberti, Tanzir Ahmed, Matthew J. Walter, Jie Liu, Jin Shao, Dennis L. Fei, Amanda Heard, Christopher A. Miller, Cara Lunn Shirai, Joseph Bradley, Sarah Grieb, and Sridhar Nonavinkere Srivatsan
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Mice, Knockout ,Heterozygote ,Leukemia ,Somatic cell ,Mutant ,Neoplasms, Experimental ,General Medicine ,Biology ,Splicing Factor U2AF ,medicine.disease ,Molecular biology ,Neoplasm Proteins ,Mice ,Haematopoiesis ,Essential gene ,Hematologic Neoplasms ,Cancer cell ,medicine ,Animals ,Allele ,Gene ,Alleles ,Research Article - Abstract
Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained wild-type allele is expressed, suggesting that mutant hematopoietic cells may require the residual wild-type allele to be viable. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knock-out mice was similar to control mice, but that deletion of the wild-type allele in U2AF1(S34F) heterozygous mutant expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of wild-type U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1 (S34F) expressing cells were also more sensitive to reduced expression of wild-type U2AF1 than non-mutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the wild-type U2af1 allele was deleted compared to when it was not deleted. These results suggest that selectively targeting the wild-type U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.
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- 2021
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6. U2AF1is a haplo-essential gene required for cancer cell survival
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Michael O. Alberti, Ajay Khanna, Cara Lunn Shirai, Christopher A. Miller, Sarah Grieb, Dennis L. Fei, Jin Shao, Matthew J. Walter, Tanzir Ahmed, Matthew Ndonwi, Timothy A. Graubert, Brian A. Wadugu, Sridhar Nonavinkere Srivatsan, Amanda Heard, and Joseph Bradley
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Haematopoiesis ,Leukemia ,Essential gene ,Somatic cell ,Cancer cell ,Mutant ,medicine ,Allele ,Biology ,medicine.disease ,Gene ,Molecular biology - Abstract
Somatic mutations in the spliceosome geneU2AF1are common in patients with myelodysplastic syndromes.U2AF1mutations that code for the most common amino acid substitutions are always heterozygous, and the retained wild-type allele is expressed, suggesting that mutant hematopoietic cells may require the residual wild-type allele to be viable and cause disease. We show that hematopoiesis and RNA splicing inU2af1heterozygous knock-out mice was similar to control mice, but that deletion of the wild-type allele in U2AF1(S34F) heterozygous mutant expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of wild-type U2AF1 for survivalin vivoand thatU2AF1is a haplo-essential cancer gene. Mutant U2AF1 (S34F) expressing cells were also more sensitive to reduced, but not absent, expression of wild-type U2AF1 than non-mutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the wild-typeU2af1allele was deleted compared to when it was not deleted. These results suggest that selectively targeting the wild-typeU2AF1allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboringU2AF1mutations.
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- 2020
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7. Antigen Glycosylation Is a Central Regulator of CAR T Cell Efficacy
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Katharina E. Hayer, Balraj Doray, Marco Ruella, Amanda Heard, Jufang Chang, Matthew D. Weitzman, Saar Gill, John Lattin, Nathan Singh, Regina Fluhrer, Jack Landmann, Mehmet Emrah Selli, Helen Ha, and Abby M. Green
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chemistry.chemical_compound ,Glycosylation ,Antigen ,Chemistry ,Immunology ,Regulator ,Cell Biology ,Hematology ,Car t cells ,Biochemistry ,Cell biology - Abstract
Chimeric antigen receptor-engineered T cells targeting CD19 (CART19) have revolutionized the management of relapsed and refractory B cell malignancies. Despite high initial response rates, many patients with acute lymphoblastic leukemia (ALL) ultimately relapse after CART19. In contrast, most patients with non-Hodgkin lymphoma experience only partial or no responses. Collectively, To identify pathways responsible for enabling tumor-intrinsic resistance to CART19 we performed a genome-wide loss-of-function screen in the Nalm6 ALL cell line. The second-most enriched gene in this screen was SPPL3 (Figure 1a), encoding a Golgi-resident aspartyl protease. Previous studies have determined that SPPL3 functions to broadly limit protein glycosylation by cleaving glycosyltransferases from the Golgi membrane, impairing their ability to add complex glycans to proteins as they pass through the Golgi (Voss M. et al. EMBO, 2014). Using targeted genomic disruption, we confirmed that loss of SPPL3 results in resistance to CART19 in human ALL and non-Hodgkin lymphoma models (Figures 1b-c). CART19 cells exposed to SPPL3KO ALL demonstrated significantly lower expression of CD69, PD1, Tim3 and CD107a, as well as less activation of the central T cell transcription factors NFAT and NFκB, indicating a global suppression of T cell stimulation. Consistent with its known function, loss of SPPL3 resulted in increased addition of complex glycans to CD19. Surface staining of SPPL3KO cells revealed that CD19 antibodies were less capable of binding this hyperglycosylated CD19. This included decreased binding of the antibody used to construct the anti-CD19 CAR (clone FMC63). Protein modeling revealed that an asparagine residue known to be normally glycosylated on CD19 (N125) is in close physical proximity to the FMC63 binding site (Figure 1d), suggesting that the addition of complex glycans at this site may be responsible for disruption of CAR binding that led to impaired T cell activation. We next turned our attention to CD22, another B cell antigen that is normally glycosylated and the target of CAR therapy. In contrast to CD19, loss of SPPL3 had no impact on CD22 glycosylation or antibody binding. Similarly, loss of SPPL3 did not enable resistance to CD22-targeted CAR T cells. These findings substantiated our hypothesis loss of SPPL3 lead to CART19 failure directly via modifying CD19 glycosylation, and not through another CD19-independent mechanism. To further validate the impact of CD19 glycosylation in regulating CART19 efficacy, we over-expressed SPPL3 in ALL cells, previously shown to promote global hypoglycosylation. We confirmed decreased glycosylation of CD19 (Figure 1e), and found that this resulted in loss of FMC63 binding to CD19 and complete resistance to CART19 activity (Figure 1f). In summary, our findings identify that changes to CD19 glycosylation, either enhanced or decreased, impair the ability of CARs to bind and initiate T cell effector function against malignant B cells. Further, these data identify post-translational protein modification as a novel mechanism of antigen escape from CAR-based T cell immunotherapy. Figure 1 Figure 1. Disclosures Ruella: AbClon: Consultancy, Research Funding; viTToria biotherapeutics: Research Funding; BMS, BAYER, GSK: Consultancy; Novartis: Patents & Royalties; Tmunity: Patents & Royalties. Gill: Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Novartis: Other: licensed intellectual property, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.
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- 2021
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8. Advances in CAR design
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John M. Warrington, Amanda Heard, Nathan Singh, and Jufang Chang
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B-Lymphocytes ,Computer science ,T-Lymphocytes ,T cell ,Clinical Biochemistry ,Cell ,Receptors, Antigen, T-Cell ,Computational biology ,Protein engineering ,Immunotherapy, Adoptive ,Chimeric antigen receptor ,Synthetic biology ,medicine.anatomical_structure ,Immune system ,Oncology ,Structural biology ,Neoplasms ,medicine ,Humans ,B cell - Abstract
Chimeric antigen receptor (CAR) T cells have revolutionized the management of B cell malignancies. These synthetic molecules are composed of peptide fragments from several distinct immune cell proteins and link highly-specific antigen recognition with potent T cell activation. Despite impressive results in many, less than half of patients treated will achieve durable remission after CAR therapy. Recent studies have identified the central role that each structural component of the CAR molecule plays in regulating T cell function. Significant effort has been dedicated to exploring strategies to improve the design of CARs themselves or integrate their activity with other regulatory circuits to enable more precise function. In this review, we will summarize recent pre-clinical and clinical studies that have evaluated novel CAR design formats.
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- 2021
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9. U2AF1(S34F) Mutant Hematopoietic Cells Require Expression of Wild-Type U2af1 for Survival
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Matthew J. Walter, Jin J Shao, Joseph Bradley, Matthew Ndonwi, Amanda Heard, and Brian A. Wadugu
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Mutation ,Immunology ,Mutant ,Wild type ,Heterozygote advantage ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,Biochemistry ,Molecular biology ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Bone marrow ,Stem cell ,Gene - Abstract
Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.
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- 2018
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10. Abstract 5112: U2af1, a spliceosome gene commonly mutated in MDS, is required for hematopoiesis
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Matthew J. Walter, Brian A. Wadugu, Joseph Bradley, Matthew Ndonwi, and Amanda Heard
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Cancer Research ,Haematopoiesis ,Spliceosome ,Oncology ,Biology ,Gene ,Cell biology - Abstract
Somatic mutations in genes encoding factors involved in pre-mRNA splicing (i.e., spliceosome genes) have been identified in more than half of myelodysplastic syndrome (MDS) patients. We are interested in the role of U2AF1, a spliceosome gene mutated in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1 have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type (WT) U2AF1 in normal hematopoiesis has not been studied. A complete understanding of the role of WT U2AF1 on hematopoiesis and RNA splicing is critical to our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of WT U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse. Homozygous embryonic deletion of U2af1 using Vav1-Cre was lethal, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wt or Mx1-Cre/U2af1wt/wt into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1 deletion, homozygous U2af1 KO mice, but not other genotypes, became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KO mice compared to all other genotypes (p ≤ 0.001, n=5). Deletion of U2af1 also led to rapid bone marrow failure and a reduction in bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (p ≤ 0.001, n=5). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and WT congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cells (HSCs). As early as 10 days, and up to 4 months following Mx1-Cre-induction, we observed a significant decrease in white blood cell count chimerism and complete loss of homozygous U2af1 KO HSCs, neutrophils and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). Collectively, the data indicate that normal hematopoiesis is dependent on U2af1 expression. In ongoing studies, we crossed the U2af1 KO mouse to existing transgenic mutant U2AF1-expressing mice to determine if mutant U2AF1 expression is sufficient to rescue cell survival and normal hematopoiesis when endogenous WT U2af1 is deleted. If U2AF1 mutant cells are vulnerable to loss of the WT U2AF1 allele, then selectively targeting the WT U2AF1 allele in heterozygous mutant cells could be a novel therapeutic strategy. Citation Format: Brian A. Wadugu, Amanda Heard, Joseph Bradley, Matthew Ndonwi, Matthew J. Walter. U2af1, a spliceosome gene commonly mutated in MDS, is required for hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5112.
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- 2018
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