p53, Aging, and Cancer

1A项目，Delta133p53alpha在癌症免疫治疗中的应用：为了检验表达Delta133p53alpha、cd28修复的CD8+ T细胞是否对肿瘤细胞具有细胞毒性活性，我们与世界领先的嵌合抗原受体(CAR)-T细胞治疗专家、宾夕法尼亚大学Carl June博士合作，在已建立的共培养系统中对肿瘤细胞存活和死亡进行了定量分析。我们将delta133p53alpha的表达盒整合到June博士的CAR-T载体中，并成功生成了表达delta133p53alpha的CAR-T细胞。首先，在与CAR-T细胞体外共培养的广泛使用的表达cd19的B-ALL （b细胞急性淋巴母细胞淋巴瘤）模型中，与目前标准的CAR-T细胞相比，表达delta133p53alpha的CAR-T细胞表现出更持久、更高效的抗肿瘤活性。delta133p53alpha的有益效果是明显的，特别是在高肿瘤：CAR-T比率（如10倍以上的肿瘤细胞），这表明它有望应用于难以治疗的病例。机制研究，如转录组学和全基因组结合分析正在进行中，以显示delta133p53α诱导的CAR-T重编程，剖析delta133p53α调节的途径以改善效果，并验证delta133p53α的非致癌性质，而不是通过完全敲除/敲除所有p53活性来致癌。我们也正在推进体内模型，在免疫缺陷小鼠中建立人类肿瘤异种移植物（即上述B-ALL和实体肿瘤细胞，如her2阳性乳腺癌），然后给药表达delta133p53α或对照CAR-T细胞。特别重要的是，在标准CAR-T细胞失败的情况下，以及在可能富含衰老/耗尽T细胞的老年人来源的样本中，检测delta133p53alpha的作用。该研究将是delta133p53alpha在晚期癌症免疫治疗中临床应用的重要一步。项目1B， Delta133p53alpha在神经变性中的作用：我们已经证明，体外用β处理正常人类星形胶质细胞诱导细胞衰老，内源性Delta133p53alpha的表达降低，SASP因子（如IL-6和il -1 β）的产生增加，神经营养因子（如NGF和IGF-1）的减少，类似于我们之前报道的复制和辐射诱导的星形胶质细胞衰老(Turnquist et al., neurooncol 21: 474, 2019；Turnquist et al., Cell Death Differ 23: 1515, 2016)。这种β诱导的星形胶质细胞衰老，与辐射诱导的衰老一样，与累积的DNA损伤（即γ - h2ax和53BP1病灶）有关。重要的是，人类星形胶质细胞中delta133p53alpha的过表达抵消了β诱导的衰老和SASP，导致共培养神经元的存活率增加。我们最近发现细胞外tau蛋白也能诱导星形胶质细胞衰老和SASP。这些数据表明，内源性（如Abeta和tau）或外源性（如辐射）损伤、细胞衰老和SASP引起的DNA损伤代表了星形胶质细胞中放大的一系列致病事件，导致神经变性，这可能通过增强星形胶质细胞delta133p53alpha表达而在治疗上中断。通过高通量筛选确定穿过血脑屏障并增强delta133p53alpha表达的药物（见下文项目1E）。项目1C，小鼠模型的生成：由于delta133p53alpha对HGPS来源细胞的体外作用（即从过早衰老和加速DNA损伤中恢复）（von Muhlinen等人，Oncogene 37: 2379, 2018）， HGPS模型小鼠（LmnaG609G）与Hupki小鼠杂交，证实表达delta133p53alpha，并检查衰老表型和机体寿命。在杂合子和纯合子LmnaG609G背景下，我们没有观察到野生型/野生型、wt/Hupki和Hupki/Hupki基因型在寿命上的显著差异，这表明内源性delta133p53alpha的组成性表达可能不会在体内挽救HGPS表型，并促使我们产生delta133p53alpha转基因小鼠。我们目前正在培养在安全港ROSA26位点携带“CAG-lox-STOP-lox- delta133p53alpha”转基因的小鼠。在各种启动子（如星形胶质细胞的GFAP，可从Jackson实验室获得）的控制下，将携带ccreert2的小鼠与这些转基因小鼠一起繁殖，然后用他莫昔芬或4-OHT治疗，将导致δ ta133p53alpha的普遍或组织特异性诱导表达。这些诱导表达模型和具有不同水平delta133p53alpha表达的Hupki小鼠，对于进一步研究delta133p53alpha对AD、辐射诱导的神经变性（癌症幸存者的慢性不良反应）和HGPS的作用至关重要。我们还计划通过待鉴定的小分子激活剂增强Hupki小鼠内源性delta133p53alpha的表达（见下文项目1E）。1D项目，非致癌的delta133p53alpha：我们已经证明，delta133p53alpha的过表达不会导致人类成纤维细胞和多能干细胞的染色体不稳定、突变率增加或恶性转化（Horikawa等人，Cell Death Differ 24: 1017, 2017）。为了获得delta133p53alpha非致癌性质的进一步证据，我们目前正在收集实际用于治疗应用的细胞类型（即T细胞）的数据。对delta133p53alpha表达、p53敲除和对照CAR-T细胞的转录组学分析（见上文1A项目）将为delta133p53alpha的治疗应用提供进一步的机制基础。我们还将在体外检测这些CAR-T和CD8+ T细胞的恶性表型和免疫缺陷小鼠的致瘤性。在Hupki小鼠（正在进行）和可诱导的delta133p53α转基因小鼠（在不久的将来）中对自发和致癌物诱导的肿瘤发病率和频谱的检查提供了体内数据。项目1E，药物发现：在癌症和衰老相关疾病中开发基于p53亚型的治疗方法，我们的目标是鉴定上调delta133p53α蛋白表达的小分子化合物，特别是那些穿过血脑屏障的小分子化合物。利用NCATS基于细胞的高通量筛选策略，我们已经确定了候选化合物。计算方法揭示了特定化学结构和药物靶点的富集，其中一些与我们之前的数据一致，表明delta133p53alpha的蛋白水平主要通过伴侣辅助的选择性自噬调节，涉及Hsp70家族和STUB1/CHIP泛素连接酶（Horikawa et al., Nat comm5: 4706, 2014）。hit化合物正在体外和Hupki小鼠体内测试几种正常人类细胞类型的delta133p53α蛋白表达调节和衰老修复。项目1F， p53在GBM中的异构体：我们已经证明，替莫唑胺（TMZ）是一种广泛用于GBM治疗的药物，通过下调delta133p53alpha来诱导p53野生型GBM细胞的DNA损伤、细胞衰老和SASP。我们还发现GBM干细胞样细胞群表达大量的delta133p53alpha。这些数据表明，在GBM中，delta133p53alpha是下调的治疗靶点。SRSF3是我们之前确定的p53beta的调节因子（Tang et al., Oncogene 32: 2792, 2013），其敲低已被证明通过p53beta上调诱导GBM衰老。TMZ和SRSF3基因下调对GBM衰老的协同或附加作用正在研究中。我们还将使用p53突变型和野生型GBM细胞来研究突变型p53亚型的致癌或抑瘤作用。

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.