Transient P53 Inhibition Research Articles

Molecular dynamics of genome editing with CRISPR-Cas9 and rAAV6 virus in human HSPCs to treat sickle cell disease

Exvivo gene correction with CRISPR-Cas9 and a recombinant adeno-associated virus serotype 6 (rAAV6) in autologous hematopoietic stem/progenitor cells (HSPCs) to treat sickle cell disease (SCD) has now entered early-phase clinical investigation. To facilitate the progress of CRISPR-Cas9/rAAV6 genome editing technology, we analyzed the molecular changes in key reagents and cellular responses during and after the genome editing procedure in human HSPCs. We demonstrated the high stability of rAAV6 to serve as the donor DNA template. We assessed the benefit of longer HSPC pre-stimulation in terms of increased numbers of edited cells. We observed that the p53 pathway was transiently activated, peaking at 6 h, and resolved over time. Notably, we revealed a strong correlation between p21 mRNA level and rAAV6 genome number in cells and beneficial effects of transient inhibition of p53 with siRNA on genome editing, cell proliferation, and cell survival. In terms of potential immunogenicity, we found that rAAV6 capsid protein was not detectable, while a trace amount of residual Cas9 protein was still detected at 48h post-genome editing. We believe this information will provide important insights for future improvements of gene correction protocols in HSPCs.

Transient inhibition of p53 enhances prime editing and cytosine base-editing efficiencies in human pluripotent stem cells

Precise gene editing in human pluripotent stem cells (hPSCs) holds great promise for studying and potentially treating human diseases. Both prime editing and base editing avoid introducing double strand breaks, but low editing efficiencies make those techniques still an arduous process in hPSCs. Here we report that co-delivering of p53DD, a dominant negative fragment of p53, can greatly enhance prime editing and cytosine base editing efficiencies in generating precise mutations in hPSCs. We further apply PE3 in combination with p53DD to efficiently create multiple isogenic hPSC lines, including lines carrying GBA or LRRK2 mutations associated with Parkinson disease and a LMNA mutation linked to Hutchinson-Gilford progeria syndrome. We also correct GBA and LMNA mutations in the patient-specific iPSCs. Our data show that p53DD improves PE3 efficiency without compromising the genome-wide safety, making it feasible for safe and routine generation of isogenic hPSC lines for disease modeling.

A CRISPR-p53 interactome with potential implications for clinical CRISPR/Cas9 use

CRISPR/Cas9-based tools are anticipated to transform the gene therapy field by facilitating the correction of disease-causing mutations. However, CRISPR/Cas9 generates DNA damage, which triggers a DNA damage response centered around the tumor-suppressor p53. In this research perspective, we discuss implications of this and describe a CRISPR-p53 interactome with cancer-related genes that, if mutated, can give cells a selective advantage following exposure to CRISPR/Cas9. We propose that the genes in the CRISPR-p53 interactome should be monitored in the clinical setting and describe that transient p53 inhibition could be used to limit the enrichment of cells with such mutations.

CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based Therapies.

Inactivating p53 mutations are the most abundant genetic alterations found in cancer. Here we show that CRISPR/Cas9-induced double-stranded DNA breaks enrich for cells deficient in p53 and in genes of a core CRISPR-p53 tumor suppressor interactome. Such enrichment could predispose to cancer development and thus pose a challenge for clinical CRISPR use. Transient p53 inhibition could suppress the enrichment of cells with these mutations. The level of DNA damage response induced by an sgRNA influenced the enrichment of p53-deficient cells and could be a relevant parameter in sgRNA design to limit cellular enrichment. Furthermore, a dataset of >800 human cancer cell lines identified additional factors influencing the enrichment of p53-mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, these data provide details about p53 biology in the context of CRISPR-induced DNA damage and identify strategies to enable safer CRISPR use. SIGNIFICANCE: CRISPR-mediated DNA damage enriches for cells with escape mutations in a core CRISPR-p53 interactome, which can be suppressed by transient inhibition of p53.

Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking.

Targeted gene editing in hematopoietic stem cells (HSCs) is a promising treatment for several diseases. However, the limited efficiency of homology-directed repair (HDR) in HSCs and the unknown impact of the procedure on clonal composition and dynamics upon transplantation have hampered clinical translation. Here, we apply a barcoding strategy to clonal tracking of edited cells (BAR-Seq) and show that editing activates p53, which significantly shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserved multilineage and self-renewing capacity. Transient p53 inhibition restored polyclonal graft composition. We increased HDR efficiency by forcing cell cycle progression and upregulating components of the HDR machinery through transient expression of the Adenovirus 5 E4orf6/7 protein, which recruits the cell cycle controller E2F on its target genes. Combined E4orf6/7 expression and p53 inhibition resulted in HDR editing efficiencies of up to 50% in the long-term human graft, without perturbing repopulation and self-renewal of edited HSCs. This enhanced protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.

Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response.

SummaryPrecise gene editing in hematopoietic stem and progenitor cells (HSPCs) holds promise for treating genetic diseases. However, responses triggered by programmable nucleases in HSPCs are poorly characterized and may negatively impact HSPC engraftment and long-term repopulation capacity. Here, we induced either one or several DNA double-stranded breaks (DSBs) with optimized zinc-finger and CRISPR/Cas9 nucleases and monitored DNA damage response (DDR) foci induction, cell-cycle progression, and transcriptional responses in HSPC subpopulations, with up to single-cell resolution. p53-mediated DDR pathway activation was the predominant response to even single-nuclease-induced DSBs across all HSPC subtypes analyzed. Excess DSB load and/or adeno-associated virus (AAV)-mediated delivery of DNA repair templates induced cumulative p53 pathway activation, constraining proliferation, yield, and engraftment of edited HSPCs. However, functional impairment was reversible when DDR burden was low and could be overcome by transient p53 inhibition. These findings provide molecular and functional evidence for feasible and seamless gene editing in HSPCs.

Transient p53 inhibition sensitizes aged white adipose tissue for beige adipocyte recruitment by blocking mitophagy.

Aging of white adipose tissue (WAT) is associated with reduced insulin sensitivity, which contributes to whole-body glucose intolerance. WAT aging in mice impairs cold-induced beige adipocyte recruitment (beiging), which has been attributed to the senescence of adipose progenitor cells. Tumor suppressor p53 has also been implicated in WAT aging. However, whether p53-related cellular aging in mature white adipocytes is causative of age-impaired WAT beiging remains unknown. It is also unclear whether transient p53 inhibition can rescue WAT beiging. Herein, we report that p53 increased in adipose tissues of 28-wk-old (aged) mice with impaired beiging capability. Cold exposure decreased p53 in beiging WAT of young mice but not in aged mice. In aged mice, inducible p53 ablation in differentiated adipocytes restored cold-induced WAT beiging and augmented whole-body energy expenditure and insulin sensitivity. Transient pharmacological inhibition of p53 led to the same beneficial effects. Mechanistically, cold exposure repressed autophagy in beiging WAT of young mice yet increased autophagy in aged WAT. p53-ablation reduced microtubule-associated protein light chain 3-mediated mitochondria clearance (mitophagy) and hence facilitated the increase of mitochondria during beiging. These findings suggest that p53-induced mitophagy in aged white adipocytes impedes WAT beiging and may be therapeutically targeted to improve insulin sensitivity in aged WAT.-Fu, W., Liu, Y., Sun, C., Yin, H. Transient p53 inhibition sensitizes aged white adipose tissue for beige adipocyte recruitment by blocking mitophagy.

P53-Based strategy to reduce hematological toxicity of chemotherapy: A proof of principle study

p53 activation is a primary mechanism underlying pathological responses to DNA damaging agents such as chemotherapy and radiotherapy. Our recent animal studies showed that low dose arsenic (LDA)-induced transient p53 inhibition selectively protected normal tissues from chemotherapy-induced toxicity.Study objectives were to: 1) define the lowest safe dose of arsenic trioxide that transiently blocks p53 activation in patients and 2) assess the potential of LDA to decrease hematological toxicity from chemotherapy.Patients scheduled to receive minimum 4 cycles of myelosuppressive chemotherapy were eligible. For objective 1, dose escalation of LDA started at 0.005 mg/kg/day for 3 days. This dose satisfied objective 1 and was administered before chemotherapy cycles 2, 4, and 6 for objective 2. p53 level in peripheral lymphocytes was measured on day 1 of each cycle by ELISA assay. Chemotherapy cycles 1, 3, and 5 served as the baseline for the subsequent cycles of 2, 4, and 6 respectively. If p53 level for the subsequent cycle was lower (or higher) than the baseline cycle, p53 was defined as “suppressed” (or “activated”) for the pair of cycles. Repeated measures linear models of CBC in terms of day, cycle, p53 activity and interaction terms were used.Twenty-six patients treated with 3 week cycle regimens form the base of analyses. The mean white blood cell, hemoglobin and absolute neutrophil counts were significantly higher in the “suppressed” relative to the “activated” group.These data support the proof of principle that suppression of p53 could lead to protection of bone marrow in patients receiving chemotherapy.This trial is registered in ClinicalTrials.gov. Identifier: NCT01428128.

Abstract 4317: Low dose arsenic-induced transient p53 inhibition: A novel strategy for chemotherapy protection

Abstract Because of its effectiveness in killing cancer cells, chemotherapy remains to be one of the common modalities for cancer treatment, but the resulting severe side effects often pose a significant threat to cancer patients. Recognizing p53 activation in the sensitive tissues as the primary mechanism for chemotherapy-induced acute toxicity and the universal p53 inactivation in human cancers, we exploited the use of low dose arsenic to inhibit p53 for a selective protection of normal tissues from chemotherapeutics-induced damages. Colon carcinoma cell line SW-480 was used to generate a mouse xenograft model. Treatments were initiated three weeks after implantation when the tumor size reached approximately 100 mm3. Arsenic pretreatment was carried out by feeding sex-matched mice (4-6 weeks of age) with water (1.0 mg/L) for three days. As expected, treatment of tumor-bearing mice with 5FU for 7 days resulted in 77% decrease in tumor volume when compared with the untreated group. The brief treatment with low doses of arsenic did not affect the ability of 5FU to induce tumor regression as evidenced by an 80% reduction of tumor size. However, arsenic treatment was associated with a marked alleviation of 5FU-induced systemic toxicity as demonstrated by the finding that arsenic pretreatment reduced 5FU-induced body weight loss from 25% to 4%. The protecting effect of arsenic was also clearly evident in reducing 5FU-induced damage in gastrointestinal (GI) track and hematopoietic (HP) system, two of the most sensitive tissues. Using mutant p53 expressing mice, we demonstrate that arsenic-mediated protection primarily depends on functional p53. Our preclinical study implicates that the brief use of low doses arsenic to inhibit p53 is an effective approach of chemotherapy protection. If successfully validated in clinical studies, this strategy may not only minimize side effects, but would also allow escalated therapeutic doses to improve treatment efficacy in a safe and effective approach for cancer therapy protection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4317. doi:10.1158/1538-7445.AM2011-4317

A Novel p53-Based Strategy for Protection of Bone Marrow and Gastrointestinal (GI) Track From Total Body Irradiation (TBI)-Induced Damage.

AbstractAbstract 1454 Purpose:Recognizing p53 activation as the primary mechanism that underlies radiation-induced damage to bone marrow and GI track, we aim to explore the use of low dose arsenic-induced transient p53 inhibition as a novel strategy for radiotherapy protection. Methods:Expanding from our recent finding that low-dose of arsenic can suppress radiation-induced p53 activation and subsequent apoptosis, we used a mouse model to test the potential of arsenic in protection of bone marrow and GI track against TBI induced damage. Arsenic pretreatment was carried out by feeding sex-matched mice (4-6 weeks of age) with water with or without 1.0 mg/L sodium arsenic for three days. Mice were randomized into following groups; control; arsenic only; irradiation only; arsenic and irradiation. Mice were then irradiated with 2Gy TBI daily for 7 days. Radiation-induced bone marrow and GI track damages were analyzed histologically with H&E staining 4 weeks after last treatment. Animal body weight was monitored as a measure of toxicity. To test the prediction that arsenic would not provide protection for malignant cells because of their defect in p53, a lung carcinoma cell line NCI-H358 was used to generate a mouse xenograft model. Treatments were initiated three weeks after implantation when the lung carcinoma cells developed into readily visible tumor with an average volume of approximately 100 mm3. Tumor volumes were measured periodically. Tumor volume was calculated using the equation: (volume = length × width × depth × 0.5236 mm3). Two independent experiments were done and the tumor volumes are means ± SE from total of 10 mice per group. [Display omitted] Results:Consistent with published results, mice treated with irradiation significantly lost their body weight. TBI-induced body weight loss was effectively prevented by arsenic pretreatment (Fig. 1A). In line with the data of body weight change, radiation treatments were associated with severe damages to small intestine and bone marrow cells, and remarkably, such damages were significantly reduced by low-dose arsenic pretreatment (Fig. 1B). The results together demonstrate the potential of low-dose arsenic to effectively protect bone marrow and GI track to radiation-induced damage. [Display omitted] In tumor xenograft models, the tumor volume of the control group continued to increase with time. Arsenic treatment did not have any detectable effect on growth of the implanted tumors. As expected, irradiation with 2Gy of TBI daily for 7 days resulted in marked tumor regression. Arsenic pretreatment showed little effect on irradiation-induced tumor suppression (Fig. 2), indicating that low-dose arsenic pretreatment does not detectably affect the efficacy of radiation, at least in the human lung carcinoma xenograft mouse model. Collectively, the results demonstrate that a brief treatment with low-dose arsenic is associated with a marked protection of bone marrow and GI track without compromising the ability of irradiation to kill carcinoma cells. Conclusion:The animal studies have provided proof-of-principle for the use of low-dose arsenic in radiotherapy protection of bone marrow and GI track, serving as strong rationales to initiate clinical trials. Disclosures:No relevant conflicts of interest to declare.

Short-term inhibition of p53 combined with keratinocyte growth factor improves thymic epithelial cell recovery and enhances T-cell reconstitution after murine bone marrow transplantation

AbstractMyeloablative conditioning before bone marrow transplantation (BMT) results in thymic epithelial cell (TEC) injury, T-cell immune deficiency, and susceptibility to opportunistic infections. Conditioning regimen–induced TEC damage directly contributes to slow thymopoietic recovery after BMT. Keratinocyte growth factor (KGF) is a TEC mitogen that stimulates proliferation and, when given before conditioning, reduces TEC injury. Some TEC subsets are refractory to KGF and functional T-cell responses are not fully restored in KGF-treated BM transplant recipients. Therefore, we investigated whether the addition of a pharmacologic inhibitor, PFT-β, to transiently inhibit p53 during radiotherapy could spare TECs from radiation-induced damage in congenic and allogeneic BMTs. Combined before BMT KGF + PFT-β administration additively restored numbers of cortical and medullary TECs and improved thymic function after BMT, resulting in higher numbers of donor-derived, naive peripheral CD4+ and CD8+ T cells. Radiation conditioning caused a loss of T-cell zone fibroblastic reticular cells (FRCs) and CCL21 expression in lymphoid stroma. KGF + PFT-β treatment restored both FRC and CCL21 expression, findings that correlated with improved T-cell reconstitution and an enhanced immune response against Listeria monocytogenes infection. Thus, transient p53 inhibition combined with KGF represents a novel and potentially translatable approach to promote rapid and durable thymic and peripheral T-cell recovery after BMT.

Short-Term Inhibition of p53 Combined with Keratinocyte Growth Factor Improves Thymic Epithelial Cell Recovery and Enhances T-Cell Reconstitution After Murine Bone Marrow Transplantation.

Abstract 4485Myeloablative conditioning prior to bone marrow transplantation (BMT) results in thymic epithelial cell (TEC) injury, T cell immune deficiency, and susceptibility to opportunistic infections. Conditioning regimen-induced TEC damage directly contributes to slow thymopoietic recovery following BMT. Keratinocyte growth factor (KGF) is a TEC mitogen that stimulates proliferation and, when given prior to conditioning, reduces TEC injury. Some TEC subsets are refractory to KGF and functional T-cell responses are not fully restored in KGF-treated BMT recipients. Therefore, we investigated whether the addition of a pharmacological inhibitor, PFT-β to transiently inhibit p53 during radiotherapy could spare TEC from radiation-induced damage in congenic and allogeneic BMT. The combined pre-BMT administration of KGF plus PFT-β additively restored numbers of cortical and medullary TEC and improved thymic function post-BMT, resulting in higher numbers of donor-derived, naïve peripheral CD4+ and CD8+ T-cells. Improved T-cell reconstitution correlated with enhanced recovery of fibroblastic reticular cells in the lymph node stromal compartment, and a superior immune response against L. monocytogenes infection. Thus, transient p53-inhibition combined with KGF represents a novel and potentially translatable approach to promote rapid and durable thymic and peripheral T-cell recovery following BMT. Disclosures:No relevant conflicts of interest to declare.

Improvement of rat liver graft quality by pifithrin-alpha-mediated inhibition of hepatocyte necrapoptosis.

Early graft dysfunction due to ischemia reperfusion injury remains a major clinical challenge in liver transplantation. Because apoptosis may contribute to graft dysfunction, we studied whether transient inhibition of p53 is capable of improving graft quality by reducing apoptotic cell death. Rat livers were harvested and stored for 24 hours or 48 hours in a 4 degrees C solution containing either pifithrin-alpha (PFT-alpha), a specific p53-inhibitor, or the vehicle dimethyl-sulfoxide. Storage was followed by 2-hour reperfusion with 37 degrees C Krebs-Henseleit buffer in an isolated liver perfusion system. Besides caspase-3 activation, apoptosis was quantified using fluorescence microscopy and hematoxylin-eosin histology. Trypan blue allowed for assessment of cell membrane damage, indicating both secondary apoptosis and primary necrosis. Bile flow, oxygen consumption, K(+)-excretion and enzyme release served as indicators of overall graft quality. Upon 2-hour reperfusion, livers developed procaspase activation as well as a mixture of apoptotic and necrotic cell death, representing necrapoptosis. In livers that had been stored for 48 hours, necrapoptotic injury was more pronounced compared with that after 24-hour storage. PFT-alpha effectively attenuated caspase activation as well as hepatocellular apoptosis and necrosis. Attenuation of both modes of cell death by PFT-alpha was associated with improved liver function, metabolism, and integrity. Experiments with the caspase inhibitor z-VAD-fmk confirmed that apoptosis is one mode of cell death in cold ischemia reperfusion. In conclusion, inhibition of p53-dependent apoptosis by PFT-alpha reduces hepatic preservation-reperfusion injury and improves primary organ function and metabolism. Fortification of the preservation solution with PFT-alpha may represent a promising and easily applicable approach to mitigate reperfusion injury in liver transplants.

Acceleration of Cutaneous Wound Healing by Transient p53 Inhibition

The increase of cell proliferation during early wound healing is thought to be regulated by a decrease of apoptosis. In contrast, the reduction of cellularity during final wound maturation may be controlled by an increase of apoptotic cell death. Herein we studied whether p53 is involved in wound healing–associated apoptosis and whether transient inhibition of p53 is effective to improve the early healing process of cutaneous wounds. Using intravital microscopic and immunohistochemical techniques in hairless mice, we demonstrated that in vivo inhibition of p53 by pifithrin-α (PFT-α; 2.2 mg/kg ip) accelerates early epithelialization and neovascularization of cutaneous wounds by (i) promoting leukocyte recruitment, (ii) increasing cell proliferation, and (iii) reducing apoptotic cell death. We further show that final wound closure with down-regulation of cell proliferation is not inhibited by PFT-α treatment, indicating that transient blockade of p53 function does not affect the process of wound maturation. Western blot analysis revealed that PFT-α lowered nuclear but not cytoplasmic p53, implying that cytoplasmic retention of p53 mediates the antiapoptotic effects of PFT-α. Furthermore, PFT-α significantly increased expression of proliferating cell nuclear antigen protein in whole extracts of cutaneous tissue and caused a rise in proliferation of wild-type, but not mutant, p53-expressing keratinocytes. From our study we conclude that transient inhibition of p53 supports the early cell proliferation required for rapid tissue repair and that this may represent an attractive approach in the treatment of delayed wound healing.

Inhibition of Transcription Results in Accumulation of Wt p53 Followed by Delayed Outburst of p53-Inducible Proteins: p53 as a Sensor of Transcriptional Integrity

Here we investigate activation of the p53 pathway by inhibition of transcription. Comparison of cells with either mutant p53 or wt p53 indicated that inhibition of p53-dependent transcription is necessary and sufficient for wt p53 accumulation. In addition to Mdm-2, p21 is required for effective p53 degradation. Transient inhibition of transcription resulted in initial downregulation of p21 and Mdm-2 leading to accumulation of wt p53. This was followed by induction of p21 and Mdm-2, normalization of p53 levels, and p21-dependent growth arrest. Although simultaneous induction of p53 and p21 could be detected by immunoblot, levels of p53 and p21 were discordant in individual cells. By inducing p21 and Mdm-2, p53 discriminates between transient and sustained inhibition of transcription. Transient inhibition results in p21-dependent growth, while sustained inhibition of transcription leads to p53-facilitated cell death. One can envision p53 as a physiological sensor of transcriptional integrity. Transient inhibition of p53- stimulated transcription by numerous stimuli including nucleotide depletion, hypoxia, UV light may be an prevalent mechanism of activation of wt p53 and its downstream pathways. Key Words Apoptosis, Growth Arrest, Mdm-2, p21, p53