p53, Aging, and Cancer

p53，衰老与癌症

• 批准号: 10262348
• 负责人: Curtis Harris
• 金额: $ 216.9万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262348
• 关键词: Acute; Advanced Malignant Neoplasm; Adverse effects; Aging; Alzheimer' s Disease; Alzheimer' s disease patient; Amyloid beta-Protein; Aorta; Apoptosis; Astrocytes; Autophagocytosis; B-Lymphocytes; Binding; Blood - brain barrier anatomy; Brain; Breeding; CAR T cell therapy; CD19 gene; CD28 gene; CD8-Positive T-Lymphocytes; CD8B1 gene; Cancer Patient; Cancer Survivor; Carcinogens; Cell Aging; Cell Death; Cell Survival; Cells; Chemical Structure; Chemotherapy-Oncologic Procedure; Chromosomal Instability; Chronic; Chronic stress; Coculture Techniques; Collaborations; DNA Damage; DNA Repair; DNA cassette; Data; Development; Disease; Dominant-Negative Mutation; Down-Regulation; Drug Targeting; Drug usage; ERBB2 gene; Elderly; Event; Fibroblasts; Future; Gamma-H2AX; Generations; Genes; Genome Stability; Genotype; Glial Fibrillary Acidic Protein; Glioblastoma; Heat-Shock Proteins 70; Human; Immunodeficient Mouse; In Vitro; Incidence; Insulin-Like Growth Factor I; Interleukin-6; Interruption; Investigation; Knock-in; Knock-out; Laboratories; Longevity; Lymphoblastic lymphoma; Malignant - descriptor; Malignant Neoplasms; Mediating; Memory; Modeling; Molecular; Molecular Chaperones; Monitor; Mus; Mutation; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Normal Cell; Oncogenes; Oncogenic; Organ; Pathologic; Pathway interactions; Pennsylvania; Persons; Pharmaceutical Preparations; Phenotype; Physiological; Pluripotent Stem Cells; Population; Production; Progeria; Property; Protein Isoforms; Proteins; RNA Splicing; Radiation; Radiation therapy; Reporting; Research; Role; SELL gene; Sampling; Series; Solid Neoplasm; Syndrome; System; T-Lymphocyte; TP53 gene; Tamoxifen; Tauopathies; Testing; Therapeutic; Tissues; Trans-Activators; Transgenes; Transgenic Mice; Tumorigenicity; Universities; Up-Regulation; age related; base; brain tissue; cancer cell; cancer immunotherapy; cancer radiation therapy; carcinogenesis; cell injury; cell type; checkpoint receptors; chimeric antigen receptor; chimeric antigen receptor T cells; clinical application; cytotoxic; cytotoxicity; drug candidate; drug discovery; exhaust; extracellular; genome-wide; high throughput screening; histopathological examination; humanized mouse; immune checkpoint; improved; in vivo; in vivo Model; inducible gene expression; knock-down; malignant breast neoplasm; malignant phenotype; mouse model; mutant; neoplastic cell; neurotoxic; neurotrophic factor; non-oncogenic; overexpression; oxidative damage; p53-binding protein 1; premature; promoter; protein expression; senescence; small molecule; small molecule libraries; stem cell biology; stem cells; stem-like cell; tau Proteins; temozolomide; therapeutic target; transcriptomics; tumor; tumor xenograft; ubiquitin ligase; vector

Project 1A, Delta133p53alpha in cancer immunotherapy: To examine whether delta133p53alpha-expressing, CD28-restored CD8+ T cells gain a cytotoxic activity on tumor cells, quantitative analyses of tumor cell survival and death have been performed in established co-culture systems in collaboration with Dr. Carl June, University of Pennsylvania, a world-leading expert of chimeric antigen receptor (CAR)-T cell therapy. We have integrated the expression cassette of delta133p53alpha in Dr. June's CAR-T vectors and successfully generated CAR-T cells that express delta133p53alpha. First, in a widely used CD19-expressing B-ALL (B-cell acute lymphoblastic lymphoma) model co-cultured with CAR-T cells in vitro, delta133p53alpha-expressing CAR-T cells showed longer-lasting and higher-efficient anti-tumor activity compared with currently standard CAR-T cells. The beneficial effect of delta133p53alpha was evident especially at high tumor:CAR-T ratio (such as 10-fold more tumor cells), suggesting a promise for applications to difficult-to-treat cases. Mechanistic studies such as transcriptomic and genome-wide binding analyses are ongoing to show the delta133p53alpha-induced CAR-T reprogramming, to dissect the delta133p53alpha-regulated pathways for improved effects, and to verify the non-oncogenic nature of delta133p53alpha in contrast to the oncogenicity by total knockout/knockdown of all p53 activities. We are also moving forward to in vivo models, in which human tumor xenografts (i.e., the above B-ALL and solid tumor cells such as HER2-positive breast cancer) are established in immunodeficient mice, followed by administration of delta133p53alpha-expressing or control CAR-T cells. Of particular importance is to examine the effect of delta133p53alpha in cases where standard CAR-T cells failed and in elderly person-derived samples likely enriched for senescent/exhausted T cells. This study will be a significant step towards clinical application of delta133p53alpha in advanced cancer immunotherapy. Project 1B, Delta133p53alpha in neurodegeneration: We have shown that treatment of normal human astrocytes with Abeta in vitro induces cellular senescence with decreased expression of endogenous delta133p53alpha, increased production of SASP factors (e.g., IL-6 and IL-1beta) and decreased neurotrophic factors (e.g., NGF and IGF-1), similar to replicatively and radiation-induced senescent astrocytes as we previously reported (Turnquist et al., Neuro Oncol 21: 474, 2019; Turnquist et al., Cell Death Differ 23: 1515, 2016). This Abeta-induced astrocyte senescence, like radiation-induced senescence, has been associated with accumulated DNA damage (i.e., gamma-H2AX and 53BP1 foci). Importantly, delta133p53alpha overexpression in human astrocytes counteracts Abeta-induced senescence and SASP, resulting in increased survival of co-cultured neurons. We have recently shown that extracellular tau also induces astrocyte senescence and SASP. These data suggest that DNA damage by endogenous (e.g., Abeta and tau) or exogenous (e.g., radiation) insults, cellular senescence and SASP represent a series of disease-causing events amplified in astrocytes leading to neurodegeneration, which may be therapeutically interrupted by enhancement of astrocytic delta133p53alpha expression. Drugs that cross the BBB and enhance delta133p53alpha expression are to be identified via high-throughput screening (see below Project 1E). Project 1C, Generation of mouse models: Prompted by in vitro effects of delta133p53alpha on HGPS-derived cells (i.e., rescue from premature senescence and accelerated DNA damage) (von Muhlinen et al., Oncogene 37: 2379, 2018), HGPS model mice (LmnaG609G) have been crossed with Hupki mice, confirmed to be expressing delta133p53alpha, and examined for aging phenotypes and organismal lifespan. We have not observed a significant difference in lifespan among wt(wild-type)/wt, wt/Hupki and Hupki/Hupki genotypes in the heterozygous and homozygous LmnaG609G background, suggesting that constitutive expression of endogenous levels of delta133p53alpha may not rescue HGPS phenotypes in vivo and prompting us to generate delta133p53alpha transgenic mice. We are currently generating mice carrying the "CAG-lox-STOP-lox- delta133p53alpha" transgene at the safe harbor ROSA26 locus. Breeding these transgenic mice with mice carrying Cre-ERT2 under the control of various promoters (either ubiquitous or tissue-specific one such as GFAP for astrocytes; available from Jackson Laboratory), followed by treatment with tamoxifen or 4-OHT, will result in ubiquitous or tissue-specific inducible expression of delta133p53alpha. These inducible expression models and Hupki mice, with different levels of delta133p53alpha expression, will be essential for further in vivo functional studies of delta133p53alpha's effects on AD, radiation-induced neurodegeneration (recapitulating a chronic adverse effect in cancer survivors) and HGPS. We also plan to enhance endogenous delta133p53alpha expression in Hupki mice by small molecule activators to be identified (see below Project 1E). Project 1D, Non-oncogenic delta133p53alpha: We have shown that overexpression of delta133p53alpha does not cause chromosome instability, increased mutation rates, or malignant transformation in human fibroblasts and pluripotent stem cells (Horikawa et al., Cell Death Differ 24: 1017, 2017). To obtain further evidence for the non-oncogenic nature of delta133p53alpha, we are currently accumulating data from the cell type actually used in therapeutic applications (i.e., T cells). The transcriptomic analysis of delta133p53alpha-expressing, p53-knocked-out and control CAR-T cells (see above Project 1A) should provide further mechanistic rationale for the therapeutic application of delta133p53alpha. We will also examine these CAR-T and CD8+ T cells for malignant phenotypes in vitro and tumorigenicity in immunodeficient mice. Examinations of spontaneous and carcinogen-induced tumor incidence and spectrum in Hupki mice (ongoing) and inducible delta133p53alpha-transgenic mice (in the near future) provide in vivo data. Project 1E, Drug discovery: Towards development of p53 isoform-based therapies in cancer- and aging-associated diseases, we aim at identifying small molecule compounds that upregulate the expression of delta133p53alpha protein, in particular those that cross the BBB. Using a cell-based, robust high-throughput screening strategy at NCATS, we have been identifying candidate compounds. Computational approaches revealed enrichment in specific chemical structures and drug targets, some of which are consistent with our previous data showing that the protein level of delta133p53alpha is mainly regulated via chaperone-assisted selective autophagy involving Hsp70 family and STUB1/CHIP ubiquitin ligase (Horikawa et al., Nat Commun 5: 4706, 2014). The hit compounds are being tested for modulation of delta133p53alpha protein expression and senescence rescue in several normal human cell types in vitro and Hupki mice in vivo. Project 1F, p53 isoforms in GBM: We have shown that temozolomide (TMZ), a widely used drug for GBM treatment, induces DNA damage, cellular senescence and SASP in p53-wild-type GBM cells via downregulation of delta133p53alpha. We have also found that GBM stem-like cell populations express abundant levels of delta133p53alpha. These data suggest delta133p53alpha as a therapeutic target to be downregulated in GBM. Knockdown of SRSF3, a splicing factor we previously identified as the regulator of p53beta (Tang et al., Oncogene 32: 2792, 2013), has been shown to induce GBM senescence via p53beta upregulation. Synergistic or additional effects of TMZ and SRSF3 knockdown on GBM senescence are under investigation. We will also examine the oncogenic or tumor-suppressive roles of mutant p53 isoforms using p53-mutant and wild-type GBM cells.

项目1A，Delta133p53Alpha在癌症免疫疗法中：要检查Delta133p53表达Alpha，CD28填充的CD8+ T细胞是否在肿瘤细胞上获得细胞毒性活性，，在肿瘤细胞存活和死亡的定量分析中，在培训中的pen pen pen pen pen pen pen carl offer car carl carl carl carl carl carl car carl car car car car car car car car car car n penteriia中是否进行了定量分析。嵌合抗原受体（CAR）-T细胞治疗。我们已经整合了Delta133p53alpha的表达式盒，并在June的Car-t载体中，成功地生成了表达Delta133p53alpha的CAR-T细胞。 First, in a widely used CD19-expressing B-ALL (B-cell acute lymphoblastic lymphoma) model co-cultured with CAR-T cells in vitro, delta133p53alpha-expressing CAR-T cells showed longer-lasting and higher-efficient anti-tumor activity compared with currently standard CAR-T cells. Delta133p53alpha的有益作用是显而易见的，尤其是在高肿瘤：CAR-T比（例如肿瘤细胞多10倍），这表明对难以治疗病例的应用有望。正在进行诸如转录组和全基因组结合分析之类的机理研究，以显示Delta133p53Alpha诱导的CAR-T重编程，以剖定Delta133p53Alpha调节的途径，以改善效果，并验证所有Delta133p53alpha inc inc inc inc inc inc inc inc inc inc inc inc inc inc的效果P53活动。我们还向前迈进了体内模型，其中人类肿瘤异种移植物（即上述B-All和实体瘤细胞（例如HER2阳性乳腺癌））在免疫缺陷的小鼠中建立，然后在delta133p53p53plpha表达或对照CAR-T细胞中施用。特别重要的是，在标准的CAR-T细胞失败并且在老年人衍生的样品中可能富含衰老/耗尽的T细胞的情况下，Delta133p53alpha的影响。这项研究将是Delta133p53alpha在晚期癌症免疫疗法中临床应用迈出的重要一步。项目1B，Delta133p53Alpha在神经变性中：我们已经表明，正常人类星形胶质细胞在体外的正常人星形胶质细胞会诱导细胞衰老，而内源性Delta133p53Alpha表达降低，而SASP因子的产生增加，IL-6和IL-1BETA和IL-1BETA和IL-1BETA和IL-NERFICER FINFER FINTIS（E. IGF-1），类似于复制和辐射诱导的衰老星形胶质细胞（Turnquist等人，Neuro oncol 21：474，2019; Turnquist等人，Turnquist等人，细胞死亡不同，23：1515，2016）。这种ABETA诱导的星形胶质细胞衰老（如辐射引起的衰老）与累积的DNA损伤有关（即γ-H2AX和53BP1灶）。重要的是，人类星形胶质细胞中的Delta133p53Alpha过表达抵消Abeta诱导的衰老和SASP，从而增加了共培养神经元的存活率。我们最近表明，细胞外TAU还诱导星形胶质细胞衰老和SASP。这些数据表明，内源性（例如Abeta和Tau）或外源性（例如辐射）损伤DNA损害，细胞衰老和SASP代表了一系列导致神经变性的引起疾病的事件，导致神经变性的事件可以通过治疗性的人星形胶质细胞的增强而受到治疗中的deltacyty delta13333333 pp533 pp533alparpha。穿越BBB并增强delta133p53Alpha表达的药物将通过高通量筛选来鉴定（请参阅下面的项目1E）。 Project 1C, Generation of mouse models: Prompted by in vitro effects of delta133p53alpha on HGPS-derived cells (i.e., rescue from premature senescence and accelerated DNA damage) (von Muhlinen et al., Oncogene 37: 2379, 2018), HGPS model mice (LmnaG609G) have been crossed with Hupki mice, confirmed to be表达delta133p53alpha，并检查了衰老表型和生物寿命。我们尚未观察到WT（野生型）/WT，WT/Hupki和Hupki/Hupki/Hupki基因型在杂合和纯合LMNAG609G背景中存在显着差异Delta133p53Alpha转基因小鼠。我们目前正在生产携带“ Cag-lox-Stop-Stop-lox-delta133p53p53alpha” transgene的小鼠在Safe Harbour Rosa26基因座上。 Breeding these transgenic mice with mice carrying Cre-ERT2 under the control of various promoters (either ubiquitous or tissue-specific one such as GFAP for astrocytes; available from Jackson Laboratory), followed by treatment with tamoxifen or 4-OHT, will result in ubiquitous or tissue-specific inducible expression of delta133p53alpha.这些具有不同水平的Delta133p53Alpha表达的诱导型表达模型和Hupki小鼠对于进一步的Delta133p53p53Alpha对AD的影响，对辐射诱导的神经变性的影响（重新对癌症幸存者的慢性不良效应）和HGPS和HGPS和HGPS进行进一步的体内功能研究至关重要。我们还计划通过要鉴定的小分子激活剂来增强Hupki小鼠中的内源性Delta133p53Alpha表达（请参见下面的项目1E）。项目1d，非康复的Delta133p53alpha：我们已经表明，Delta133p53alpha的过表达不会引起人类成纤维细胞中的染色体不稳定性，突变率的增加或恶性转化，并使细胞死亡差异（Horikawa et al。为了获得Delta133p53Alpha的非癌性性质的进一步证据，我们目前正在积累来自治疗应用中实际使用的细胞类型的数据（即T细胞）。 delta133p53表达阿尔法，p53敲除和对照CAR-T细胞的转录组分析（请参见上文项目1A），应为Delta133p53alpha的治疗应用提供进一步的机械基础。我们还将检查这些CAR-T和CD8+ T细胞的体外恶性表型和免疫缺陷小鼠的肿瘤性。 Hupki小鼠（正在进行的）和诱导的Delta133p53Alpha-Transgenic小鼠（在不久的将来）对自发和致癌诱导的肿瘤发生率和光谱的检查（在不久的将来）提供了体内数据。项目1E，药物发现：朝着癌症和衰老相关疾病的p53基于p53同工型的疗法开发，我们的目的是鉴定出上调delta133p53alpha蛋白表达的小分子化合物，特别是那些穿越BBB的蛋白。使用NCAT的基于细胞的强大高通量筛选策略，我们一直在识别候选化合物。计算方法揭示了特定化学结构和药物靶标的富集，其中一些与我们先前的数据一致，表明Delta133p53p53alpha的蛋白质水平主要通过伴侣辅助辅助的选择性自噬调节，涉及HSP70家族和HSP70家族和chip1/chip1/chip ubiquitin-ligigitin-ligigitin-ligigitin-ligigitin-ligigitin-ligigitin-ligigitin ligigitin ligigitin ligigitin ligigitin（Horikawa et alikawa et al.horikawa et al ant ant nat nat nat nat nat nat nat smocclion。正在测试HIT化合物，以调节体外几种正常的人类细胞类型中的Delta133p53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53P53p GBM中的项目1F，P53同工型：我们已经表明，替莫唑胺（TMZ）是一种用于GBM治疗的广泛使用的药物，可通过delta133p53alpha下调p53-wild-type GBM细胞中DNA损伤，细胞衰老和SASP。我们还发现，GBM干细胞群体表达大量的Delta133p53alpha。这些数据表明Delta133p53alpha是在GBM中下调的治疗靶标。 SRSF3的敲低是我们先前确定为p53beta的调节剂的一种剪接因子（Tang等，Oncogene 32：2792，2013），已显示通过P53Beta上调诱导GBM衰老。 TMZ和SRSF3敲低对GBM衰老的协同或其他影响正在研究中。我们还将使用p53突变剂和野生型GBM细胞检查突变p53同工型的致癌或肿瘤抑制作用。