p53, Aging, and Cancer

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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.