P53 Hotspot Mutants Research Articles

Functional Investigations of p53 Acetylation Enabled by Heterobifunctional Molecules.

Post-translational modifications (PTMs) dynamically regulate the critical stress response and tumor suppressive functions of p53. Among these, acetylation events mediated by multiple acetyltransferases lead to differential target gene activation and subsequent cell fate. However, our understanding of these events is incomplete due to, in part, the inability to selectively and dynamically control p53 acetylation. We recently developed a heterobifunctional small molecule system, AceTAG, to direct the acetyltransferase p300/CBP for targeted protein acetylation in cells. Here, we expand AceTAG to leverage the acetyltransferase PCAF/GCN5 and apply these tools to investigate the functional consequences of targeted p53 acetylation in human cancer cells. We demonstrate that the recruitment of p300/CBP or PCAF/GCN5 to p53 results in distinct acetylation events and differentiated transcriptional activities. Further, we show that chemically induced acetylation of multiple hotspot p53 mutants results in increased stabilization and enhancement of transcriptional activity. Collectively, these studies demonstrate the utility of AceTAG for functional investigations of protein acetylation.

Design, Synthesis, and Evaluation of p53Y220C Acetylation Targeting Chimeras (AceTACs).

The well-known tumor suppressor p53 is mutated in approximately half of all cancers. The Y220C mutation is one of the major p53 hotspot mutations. Several small-molecule stabilizers of p53Y220C have been developed. We recently developed a new technology for inducing targeted protein acetylation, termed acetylation targeting chimera (AceTAC), and the first p53Y220C AceTAC that effectively acetylated p53Y220C at lysine 382. Here, we report structure-activity relationship (SAR) studies of p53Y220C AceTACs, which led to the discovery of a novel p53Y220C AceTAC, compound 11 (MS182). 11 effectively acetylated p53Y220C at lysine 382 in a time- and concentration-dependent manner via inducing the ternary complex formation between p300/CBP acetyltransferase and p53Y220C. 11 was more effective than the parent p53Y220C stabilizer in suppressing the proliferation and clonogenicity in cancer cells harboring the p53Y200C mutation and was bioavailable in mice. Overall, 11 is a potentially valuable chemical tool to investigate the role of p53Y220C acetylation in cancer.

Remodeling of anti-tumor immunity with antibodies targeting a p53 mutant

Backgroundp53, the most frequently mutated gene in cancer, lacks effective targeted drugs.MethodsWe developed monoclonal antibodies (mAbs) that target a p53 hotspot mutation E285K without cross-reactivity with wild-type p53. They were delivered using lipid nanoparticles (LNPs) that encapsulate DNA plasmids. Western blot, BLI, flow cytometry, single-cell sequencing (scRNA-seq), and other methods were employed to assess the function of mAbs in vitro and in vivo.ResultsThese LNP-pE285K-mAbs in the IgG1 format exhibited a robust anti-tumor effect, facilitating the infiltration of immune cells, including CD8+ T, B, and NK cells. scRNA-seq revealed that IgG1 reduces immune inhibitory signaling, increases MHC signaling from B cells to CD8+ T cells, and enriches anti-tumor T cell and B cell receptor profiles. The E285K-mAbs were also produced in the dimeric IgA (dIgA) format, whose anti-tumor activity depended on the polymeric immunoglobulin receptor (PIGR), a membrane Ig receptor, whereas that of IgG1 relied on TRIM21, an intracellular IgG receptor.ConclusionsTargeting specific mutant epitopes using DNA-encoded and LNP-delivered mAbs represents a potential precision medicine strategy against p53 mutants in TRIM21- or PIGR-positive cancers.

Abstract B009: Exploring the putative Kras-p53 mutational interface for vulnerability

Abstract Introduction: Kras Gain of Function mutations are frequently detected in lung, colorectal and pancreatic cancers, in addition to others. Activating Kras mutations results in a constantly active protein, which we reasoned is quite unphysiological, yet it is tolerated by cells since this mutation is seen in hyperplasia. Activating this oncogene is insufficient alone for malignant transformation. Mutations in the tumor suppressor gene (TSG) p53 (the most frequently mutated gene in human cancer), cooperate with mutant Kras and is sufficient to permit full display of the actions of oncogenic Ras (as confirmed in Genetically Engineered Mouse Models and in clinical cases). The phenomenon of oncogene addiction may in fact be the result of obligatory requirements (cellular metabolism or otherwise) brought on by the constant oncogene signaling. This required adjustment in cellular circuitry can be afforded by specific cooperating TSG and since it too is a DNA constant change, becomes an new rigid reality for a cell with a given set of oncogene-TSG pair. p53 mutational spectrum was described as ‘enigmatic’ presumably because they almost never completely abrogate the function of this major regulator suggesting to us a possible essential mechanistic role for the retained transcriptional targets within the (well annotated) p53 transcriptional network. This could represent a putative synthetic lethality opportunity against activated Kras, an oncogene that has proven difficult to drug. We hypothesized that specific mutations in p53 with their respective transcriptional lesions cooperate with unique mutant Kras in tissue specific manner identifying a short list of gene targets for synthetic lethality experiments. Methods: To examine this hypothesis, we analyzed the TCGA database to determine a conserved pair cooperation between common Kras 12C; D or V and the top 6 reported p53 (hot spot) mutations [on residues 175; 245;248;249;273 and 282] in a stage-agnostic manner. To examine whether those putative interactions are cell-type specific, we performed this analysis in 3 different histologies (lung, colon, and pancreas). Results: The results suggested a non-random distribution of p53 mutants among the Kras driven cancers. KRAS (12 C, 12 V, 12 D were reported in 80% of Colon, Pancreas, and Lung Cancer. p53 (175 - 30%) (245 - 30%) (248 - 15%) (249 - 12%) (273 - 6.5%) (282 - 6.5%). More detailed analysis is planned for the poster session. Conclusion: Despite having a single activated oncogene, the distribution of the cooperating p53 mutations is nonrandom so examining the transcriptionally retained gene list represents a novel approach to explore for gene editing experiments. This provides and approach to drug the addiction of cancers to their oncogenes in a cancer specific, and occasionally to target essential proto-oncogene such as Myc where direct inhibition is highly undesirable due to its physiologic roles. Citation Format: Nishanth Thalambedu, Shallya Anand, Haya Safar, Farah Mazahreh, Ahmad Mazin M. Safar. Exploring the putative Kras-p53 mutational interface for vulnerability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B009.

Abstract 4099: AMP-peptide vaccination against multiple p53 mutant epitopes promotes lymph node delivery to generate potent, functional T cell immunity

Abstract Background p53 is the most commonly mutated gene in human cancer, prevalent in nearly all tumor types. Despite its significant medical relevance and the knowledge that human T cells recognize these mutations[1,2], immunotherapies designed to promote responses against mutated p53 (mp53) have not had the hoped-for clinical impact[3]. The Amphiphile (AMP) platform improves the potency of vaccine immunotherapy by programming the delivery of vaccine components to the lymph nodes where efficient uptake by immune cells initiates tumor-targeted immune responses. AMP-modification of vaccine components (peptide antigens, molecular adjuvants) results in covalent conjugation to albumin-binding lipids. Upon injection, AMP-vaccines associate with tissue-resident albumin which efficiently distributes to draining lymph nodes. This approach was shown to promote activation of polyfunctional, cytotoxic T cells with promising safety and improved clinical outcomes in a Phase 1 trial of ELI-002, an mKRAS AMP therapeutic vaccine (AMPLIFY-201 NCT05726864). Application of this strategy to p53 mutations with ELI-008 offers the potential for improved immunotherapeutic activity in a setting of significant unmet need. Methods Following immunization of C57BL/6J mice with AMP-modified or soluble comparator vaccines consisting of mp53 peptides and CpG adjuvant, analyses were performed 7 days after the third bi-weekly dose. To assess antigen-specific T cell responses, ELISpot (IFNγ, GzmB), multiplexed proteomic, and flowcytometric analysis of effector cytokines (IFNγ, TNFα, IL-2) were performed following antigenic stimulation. Responses were determined in secondary lymphoid tissues and lung. Cytolytic capabilities of antigen-specific T cells were evaluated by monitoring specific killing of IV-transferred antigen-pulsed target cells. Results AMP-immunization generated robust immune responses yielding strong T cell activation against common p53 hot spot mutations (R248W, R248Q, R175H, G245S, R273H, Y220C, C135Y, R158H, H214R). Responses to AMP-vaccination were characterized by the generation of increased frequency of polyfunctional T cells (IFNγ, TNFα, IL2) specific to mp53 epitopes. Induced T cells demonstrated significant levels of cytolytic activity including GzmB production and elimination of target cells in vivo. Non-lymph targeted vaccines using soluble comparators were inactive. Conclusions AMP-conjugation permits efficient delivery directly to the lymph nodes and thus improves the immunogenicity of peptide vaccination. These substantially improved immune responses induced by ELI-008 represent a promising therapeutic opportunity for targeting cancer in a large fraction of human tumors. The AMP-platform technology is simple, rapid and scalable, promising broad clinical, off-the-shelf application for treating p53 mutated tumors. Citation Format: Martin P. Steinbuck, Xavier Cabana-Puig, Erica Palmer, Mimi M. Jung, Thomas Williams, Kristen Osaer, Jeff Zhang, Christopher M. Haqq, Peter C. DeMuth. AMP-peptide vaccination against multiple p53 mutant epitopes promotes lymph node delivery to generate potent, functional T cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4099.

Jun Dimerization Protein 2 (JDP2) Increases p53 Transactivation by Decreasing MDM2.

The AP-1 protein complex primarily consists of several proteins from the c-Fos, c-Jun, activating transcription factor (ATF), and Jun dimerization protein (JDP) families. JDP2 has been shown to interact with the cAMP response element (CRE) site present in many cis-elements of downstream target genes. JDP2 has also demonstrates important roles in cell-cycle regulation, cancer development and progression, inhibition of adipocyte differentiation, and the regulation of antibacterial immunity and bone homeostasis. JDP2 and ATF3 exhibit significant similarity in their C-terminal domains, sharing 60-65% identities. Previous studies have demonstrated that ATF3 is able to influence both the transcriptional activity and p53 stability via a p53-ATF3 interaction. While some studies have shown that JDP2 suppresses p53 transcriptional activity and in turn, p53 represses JDP2 promoter activity, the direct interaction between JDP2 and p53 and the regulatory role of JDP2 in p53 transactivation have not been explored. In the current study, we provide evidence, for the first time, that JDP2 interacts with p53 and regulates p53 transactivation. First, we demonstrated that JDP2 binds to p53 and the C-terminal domain of JDP2 is crucial for the interaction. Second, in p53-null H1299 cells, JDP2 shows a robust increase of p53 transactivation in the presence of p53 using p53 (14X)RE-Luc. Furthermore, JDP2 and ATF3 together additively enhance p53 transactivation in the presence of p53. While JDP2 can increase p53 transactivation in the presence of WT p53, JDP2 fails to enhance transactivation of hotspot mutant p53. Moreover, in CHX chase experiments, we showed that JDP2 slightly enhances p53 stability. Finally, our findings indicate that JDP2 has the ability to reverse MDM2-induced p53 repression, likely due to decreased levels of MDM2 by JDP2. In summary, our results provide evidence that JDP2 directly interacts with p53 and decreases MDM2 levels to enhance p53 transactivation, suggesting that JDP2 is a novel regulator of p53 and MDM2.

Arsenic trioxide extends survival of Li–Fraumeni syndrome mimicking mouse

Li-Fraumeni syndrome (LFS) is characterized by germline mutations occurring on one allele of genome guardian TP53. It is a severe cancer predisposition syndrome with a poor prognosis, partly due to the frequent development of subsequent primary tumors following DNA-damaging therapies. Here we explored, for the first time, the effectiveness of mutant p53 rescue compound in treating LFS-mimicking mice harboring a deleterious p53 mutation. Among the ten p53 hotspot mutations in IARC LFS cohorts, R282W is one of the mutations predicting the poorest survival prognosis and the earliest tumor onset. Among the six clinical-stage mutant p53 rescue compounds, arsenic trioxide (ATO) effectively restored transactivation activity to p53-R282W. We thus constructed a heterozygous Trp53 R279W (corresponding to human R282W) mouse model for the ATO treatment study. The p53R279W/+ (W/+) mice exhibited tumor onset and overall survival well mimicking the ones of human LFS. Further, 35 mg/L ATO addition in drink water significantly extended the median survival of W/+ mice (from 460 to 596 days, hazard ratio = 0.4003, P = 0.0008). In the isolated tumors from ATO-treated W/+ mice, the representative p53 targets including Cdkn1a, Mdm2, and Tigar were significantly upregulated, accompanying with a decreased level of the proliferation marker Ki67 and increased level of apoptosis marker TUNEL. Together, the non-genotoxic treatment of p53 rescue compound ATO holds promise as an alternative for LFS therapeutic.

CSIG-08. GAIN-OF-FUNCTION MUTANT P53 REGULATES LONG-NONCODING RNAS TO CONTROL HIF1Α TRANSCRIPTION IN GLIOBLASTOMA

Abstract P53 is mutated in a large subset of glioblastoma (GBM). Most hot-spot p53 mutants are gain-of-function (with only partially understood mechanisms). To investigate the role of gain-of-function mutant p53 (GOF-Mut-p53), we performed ChIP-seq analysis of GOF-Mut-p53 R273H, the most common p53 mutation in GBM. We identified and verified numerous GOF-Mut-p53 targets that differed from those of wildtype p53. Among the 2,830 targets of GOF-Mut-p53 were 535 long non-coding RNAs (lncRNAs), the majority of which we also found enriched in Mut-p53 human tumors and deregulated in the TCGA database. LncRNAs play important regulatory roles in cancer. One of the most highly enriched lncRNAs was LINC00643. We first performed ChIP confirmation of GOF-Mut-p53 binding to the promoter of LINC00643. We then showed that GOF-Mut-p53 inhibits LINC00643 expression. Furthermore, we found that LINC00643 is deregulated in human GBM and LGG and correlated with patient survival. Overexpression of LINC00643 led to a significant inhibition of GBM cell proliferation, migration, invasion and in vivo xenograft growth. Co-expression of human LINC00643 or its mouse homologue with PDGF and Cre in RCAS transgenic mice (N/tv-a; Ink4a-arf-/-; Pten-fl/fl) reduced tumor growth and improved animal survival. These data show that GOF-Mut-p53 exerts oncogenic effects in GBM by downregulating LINC00643. To elucidate the mechanisms of action of LINC00643, we performed Chromatin Isolation by RNA purification high-throughput sequencing (CHIRP-seq) to identify its binding targets. We found that LINC00643 binds to the HIF1α enhancer region. Overexpression of LINC00643 and knockdown of GOF-Mut-p53 in GBM cells under hypoxia growth conditions reduced HIF1α mRNA and protein expression. LINC00643 also regulated the expression of numerous hypoxia-regulating genes downstream of HIF1α. Our study shows for the first time that GOF-Mut-p53 transcriptionally regulates a subset of lncRNAs and that GOF-Mut-p53-regulated LINC00643 regulates hypoxia in GBM.

Adoptive Transfer of T Cells Genetically Engineered to Express T-Cell Receptors Targeting Neoantigens Arising from p53 and Ras Hotspot Mutations in Hematologic Malignancies

The tumor suppressor gene p53 and the oncogenes NRAS and KRAS (shortened to Ras) are frequently mutated in many difficult-to-treat hematologic malignancies including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and relapsed multiple myeloma. Driver mutations in p53 and Ras are associated with treatment-refractory malignancies. There are no Food and Drug Administration (FDA)-approved therapies specifically targeting hematologic malignancies with p53 or Ras mutations. Some missense mutations in p53 and Ras lead to immunogenic peptides (neoepitopes) that are presented by human leukocyte antigens (HLA). Members of our group have identified T-cell receptors (TCRs) targeting some of these neoepitopes and used T cells expressing these TCRs as treatments for epithelial tumors (Levin et al. Clin Cancer Res, 2021 and Kim et al. Cancer Immunol Res, 2022). Adoptively transferred T cells expressing some of these TCRs have caused objective responses of advanced epithelial cancers in patients. Here, we report preclinical experiments with T cells genetically engineered to target neoantigens arising from p53 and Ras mutations in hematologic malignancies. p53 mutations occur in approximately 5-10% of de novo AML, 25-50% of treatment-related or complex-karyotype MDS/AML, 5-10% of multiple myeloma, and 25% of plasma cell leukemia. Mutations in Ras occur in 15-20% of AML and 20% of multiple myeloma. Our TCR panel targets neoepitopes arising from 2 of the 5 most common p53 mutation sites and 2 of the 3 most common Ras mutation sites . The human leukocyte antigen (HLA) restriction of these TCRs is shown along with the HLA frequencies ( Table 1). In all experiments, human donor peripheral blood mononuclear cells were activated in culture with an anti-CD3 antibody. Two days later, T cells were transduced with a gamma-retroviral vector encoding a neoepitope-specific TCR. Presence of mutations in target cell lines was confirmed by DNA sequencing. Cell lines which did not naturally express the appropriate HLA were transduced with a gamma-retroviral construct encoding the HLA. We assessed the general specificity of TCRs for each targeted neoepitope. For each neoepitope-specific TCR, T cells expressing the TCR were cultured overnight with target cell lines expressing the neoepitope plus the HLA targeted by that TCR. We cultured T cells from the same cultures with 12 to 16 negative-control target cell lines that lacked the appropriate mutation and/or HLA. Negative control cell lines were from a variety of human tissues. Enzyme-linked immunosorbent assays (ELISA) performed on the culture supernatants showed that IFN-gamma release was much higher when T cells expressing a neoepitope-specific TCR were cultured with target cells having both the targeted mutation and the appropriate HLA (Table 1). We conducted another set of experiments to evaluate the neoepitope-specificity of the TCRs. For each neoepitope, COS7 cells with the appropriate HLA were transfected with plasmids encoding either wild-type or mutant versions of p53 or Ras differing from wild-type by 1 amino acid. The COS7 cells were cultured with T cells expressing the appropriate TCR. IFN-gamma was released in a neoepitope-specific manner. Higher levels of T-cell degranulation and interleukin-2 release occurred when T cells expressing neoepitope-specific TCRs were cultured with target cells having the targeted mutation and HLA versus target cells lacking the appropriate mutation and/or HLA. We evaluated efficacy of the T53-R175H-A2 TCR in vivo. Immunocompromised NOD-scid common gamma-chain-deficient mice received intravenous injections of the KMS26-luciferase multiple myeloma cell line, which naturally expresses HLA-A*02:01 and the p53 R175H mutation. One week later when all mice had detectable tumor burdens, mice received either 10 million R175H-targeted T cells, T cells expressing an irrelevant TCR, or untransduced T cells. All groups received 3 doses of interleukin-2. Mice treated with R175H-targeted T cells cleared the tumor while mice in all other groups had progressive tumor growth (Figure 1). In vivo studies of additional TCRs are in progress. In conclusion, we have demonstrated activity of neoantigen-targeted T cells against hematologic malignancies in preclinical models. We plan to evaluate adoptively transferred autologous T cells targeting p53 and Ras mutations in an upcoming clinical trial.

Abstract 2608: Defining the function of tumor suppressor p53 arginine 181 residue in tumorigenesis

Abstract The gene Tp53 encodes for the protein p53 which has many tumor suppressive functions. Li-Fraumeni syndrome is characterized by germline Tp53 mutations that significantly increase the risk of cancer. The p53 amino acid residue arginine 181 is one such mutation site that promotes tumorigenesis by impacting a phenomenon known as p53 cooperativity. This is the ability of one p53 molecule to interact with another in order to efficiently bind specific sites on DNA to enact specific p53 functional pathways. Two p53 molecules interact with each other through the formation of a salt bridge between H1 helices found in respective DNA binding domains. The two key amino acid residues in this salt bridge are positively charged arginine (R) 181 and negatively charged glutamic acid (E) 180 which form electrostatic interactions with the residues on the opposite p53 H1 helix. Cooperativity mutants such as R181H and R181C show a hypomorphic behavior wherein much of p53’s canonical functions remain intact. Colony suppression data show that H1299 cells with p53 R181H and R181C yield a similar number of colonies as p53 wild type, which is significantly fewer than the yield of p53 hotspot mutation R175H and the absence of p53. Despite retaining some wild-type behavior, R181 mutants are found in LFS patients with cancer. Discovering the importance of the R181 residue for p53’s role in defending against tumorigenesis can lead to unique treatment opportunities for LFS patients with cooperativity mutants. Citation Format: Renyta Moses, Gregory Kelly, Alexandra Indeglia, Sven Miller, John Karanicolas, Maureen Murphy, Kara Maxwell. Defining the function of tumor suppressor p53 arginine 181 residue in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2608.

Specific regulation of BACH1 by the hotspot mutant p53R175H reveals a distinct gain-of-function mechanism.

Although the gain of function (GOF) of p53 mutants is well recognized, it remains unclear whether different p53 mutants share the same cofactors to induce GOFs. In a proteomic screen, we identified BACH1 as a cellular factor that recognizes the p53 DNA-binding domain depending on its mutation status. BACH1 strongly interacts with p53R175H but fails to effectively bind wild-type p53 or other hotspot mutants in vivo for functional regulation. Notably, p53R175H acts as a repressor for ferroptosis by abrogating BACH1-mediated downregulation of SLC7A11 to enhance tumor growth; conversely, p53R175H promotes BACH1-dependent tumor metastasis by upregulating expression of pro-metastatic targets. Mechanistically, p53R175H-mediated bidirectional regulation of BACH1 function is dependent on its ability to recruit the histone demethylase LSD2 to target promoters and differentially modulate transcription. These data demonstrate that BACH1 acts as a unique partner for p53R175H in executing its specific GOFs and suggest that different p53 mutants induce their GOFs through distinct mechanisms.

Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile.

TP53 is the most commonly mutated gene in cancer, and gain-of-function mutations have wide-ranging effects. Efforts to reactivate wild-type p53 function and inhibit mutant functions have been complicated by the variety of TP53 mutations. Identified from a screen, the NSC59984 compound has been shown to restore activity to mutant p53 in colorectal cancer cells. Here, we investigated its effects on esophageal adenocarcinoma cells with specific p53 hot-spot mutations. NSC59984 treatment of cells reactivated p53 transcriptional regulation, inducing mitochondrial intrinsic apoptosis. Analysis of its effects on cellular metabolism demonstrated increased utilization of the pentose phosphate pathway and inhibition of glycolysis at the fructose-1,6-bisphosphate to fructose 6-phosphate junction. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; these effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. We found that the effects of NSC59984 were substantially greater in cells harboring the p53 R248W mutation. Overall, these findings demonstrate p53-dependent effects of NSC59984 on cellular metabolism, with increased activity in cells harboring the p53 R248W mutation. This research highlights the importance of defining the mutational status of a particular cancer to create a patient-centric strategy for the treatment of p53-driven cancers.

Unraveling the Structural Changes in the DNA-Binding Region of Tumor Protein p53 (TP53) upon Hotspot Mutation p53 Arg248 by Comparative Computational Approach

The vital tissue homeostasis regulator p53 forms a tetramer when it binds to DNA and regulates the genes that mediate essential biological processes such as cell-cycle arrest, senescence, DNA repair, and apoptosis. Missense mutations in the core DNA-binding domain (109-292) simultaneously cause the loss of p53 tumor suppressor function and accumulation of the mutant p53 proteins that are carcinogenic. The most common p53 hotspot mutation at codon 248 in the DNA-binding region, where arginine (R) is substituted by tryptophan (W), glycine (G), leucine (L), proline (P), and glutamine (Q), is reported in various cancers. However, it is unclear how the p53 Arg248 mutation with distinct amino acid substitution affects the structure, function, and DNA binding affinity. Here, we characterized the pathogenicity and protein stability of p53 hotspot mutations at codon 248 using computational tools PredictSNP, Align GVGD, HOPE, ConSurf, and iStable. We found R248W, R248G, and R248P mutations highly deleterious and destabilizing. Further, we subjected all five R248 mutant-p53-DNA and wt-p53-DNA complexes to molecular dynamics simulation to investigate the structural stability and DNA binding affinity. From the MD simulation analysis, we observed increased RMSD, RMSF, and Rg values and decreased protein-DNA intermolecular hydrogen bonds in the R248-p53-DNA than the wt-p53-DNA complexes. Likewise, due to high SASA values, we observed the shrinkage of proteins in R248W, R248G, and R248P mutant-p53-DNA complexes. Compared to other mutant p53-DNA complexes, the R248W, R248G, and R248P mutant-p53-DNA complexes showed more structural alteration. MM-PBSA analysis showed decreased binding energies with DNA in all five R248-p53-DNA mutants than the wt-p53-DNA complexes. Henceforth, we conclude that the amino acid substitution of Arginine with the other five amino acids at codon 248 reduces the p53 protein's affinity for DNA and may disrupt cell division, resulting in a gain of p53 function. The proposed study influences the development of rationally designed molecular-targeted treatments that improve p53-based therapeutic outcomes in cancer.

Chk1 inhibition-induced BRCAness synergizes with olaparib in p53-deficient cancer cells

ABSTRACT Although targeting DNA-damage repair by inhibition of PARP exhibits weak or modest single-agent activity due to the existence of functional BRCA1/2 alleles, PARP inhibitors have been gradually applicable in BRCA-proficient cancers. Checkpoint kinase 1 (Chk1) inhibition selectively disrupts homologous recombination (HR)-mediated DNA repair and confers synthetic lethality in p53-deficient tumors, we therefore aim at expounding the chemopotentiating effects of Chk1 inhibition on PARPi in BRCA-proficient and p53-deficient cancer cells. Initially, BRCA wild-type, p53-null cells including AsPC-1 and H1299 demonstrated innate resistance to PARP inhibitor olaparib compared to BRCA1-mutant, p53-null MDA-MB-436 cells. We quantified the interaction between olaparib and a selective Chk1 inhibitor MK-8776, which produced synergistic effects under sub-IC50 concentrations in p53-depleted AsPC-1 and H1299 cells. Olaparib in combination with MK-8776 showed enhanced antitumor effects through prohibiting proliferation and secondarily inducing apoptosis in two cell lines. Of note, we observed that MK-8776 significantly sensitized cells to olaparib by broad DNA and chromosomal breaks. Mechanistically, MK-8776 abrogated olaparib-induced BRCA1 intranuclear foci formation, MCM7-mediated replication machineries, and ultimately triggered an accumulation of γH2AX, a well-recognized marker of DNA double-strand breaks. Additionally, we established ectopic expression of hotspot mutant p53 in H1299 cells. Introduction of p53R175 H promoted olaparib resistance as single-agent treatment, but the synergy between olaparib and MK-8776 was still achievable and the region of synergy was produced by lower combination concentrations. These data provide insight into how Chk1 inhibition could be effectively targeted and confer sensitivity to olaparib toward p53-deficient and HR-proficient cancers.

Discovery of compounds that reactivate p53 mutants in vitro and in vivo

The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers. Here we report a virtual screening approach that identified several small molecules with p53 reactivation activities. The UCI-LC0023 compound series was studied in detail and was shown to bind p53, induce a conformational change in mutant p53, restore the ability of p53 hotspot mutants to associate with chromatin, reestablish sequence-specific DNA binding of a p53 mutant in a reconstituted invitro system, induce p53-dependent transcription programs, and prevent progression of tumors carrying mutant p53, but not p53null or p53WT alleles. Our study demonstrates feasibility of a computation-guided approach to identify small molecule corrector drugs for p53 hotspot mutations.

Abstract 1035: VCP/p97 promotes pancreatic cancer growth by enhancing mutant p53 activity

Abstract Pancreatic cancer is the most lethal malignant neoplasms across the world with the lowest overall five-year survival rate (9%). TP53 is mutated in ~70% of pancreatic ductal adenocarcinomas (PDACs). Hotspot p53 mutants promote cancer cell proliferation, metastasis and metabolism through their gain-of-function (GOF). Among the p53 “hotspot” mutations, R273 is the most frequent mutation position (~12%) with R273H as the most commonly mutated codon (~8%) in PDACs. However, the mechanisms underlying regulation of mutant p53s’ GOF in PDACs remain incompletely understood. In our attempt to address this question, we recently identified the ATPase valosin-containing protein (VCP)/p97 as a novel mutant p53-binding protein by performing immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) analysis. VCP bound to p53-R273H via the DNA-binding domain. VCP inhibition either by genetic depletion or pharmacological inhibition by CB-5083 resulted in the increase of MDM2-mediated ubiquitination and degradation of p53-R273H. VCP did so by binding to MDM2 and disturbing its interaction with mutant p53. As a result, VCP protected mutant p53 from degradation and enhanced its GOF in cancer development. Ablation of VCP retarded PDAC cell growth in vitro and in vivo. Our further studies also unveiled the negative regulation of wt p53 by VCP in cancer cells. Together, these results demonstrate that VCP can strengthen mutant p53’s oncogenic function by stabilizing it and negating wt p53 function, suggesting VCP as a resistant factor for chemotherapy against PDACs and thus a potential therapeutic target of the mutant p53-harboring pancreatic cancers as well as of the wt p53-containing cancer cells. Citation Format: Jieqiong Wang. VCP/p97 promotes pancreatic cancer growth by enhancing mutant p53 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1035.

Correlation of gemcitabine sensitization by Chk1 inhibition with p53 status

s Chk1 inhibition can selectively improve gemcitabine sensitivity in p53-deficient cells by checkpoints abrogation throughout the cell cycle. However, the dependency of p53 status is still controversial for predicting the priority of such synergy. This study aimed at expounding the differential therapeutic properties of gemcitabine sensitization by Chk1 inhibition potentially affected by p53 status. We introduced wild-type and hotspot mutant p53 in p53-null H1299 cells, and quantified combination of gemcitabine with two Chk1 inhibitors using Chou–Talalay method. As a result, depletion of p53 preferentially produced synergistic effects. Wild-type and mutant p53 also conferred drug synergy but gradually showed compromised potency of growth inhibition. These data provide increased evidence that p53 status is a weak predictor for identifying an effective synergy, but genetic loss of p53 is relatively favorable for combination treatment. Further efforts on validation in more cell lines and clinical models could improve the predictive validity in this study.

Different hotspot p53 mutants exert distinct phenotypes and predict outcome of colorectal cancer patients

The TP53 gene is mutated in approximately 60% of all colorectal cancer (CRC) cases. Over 20% of all TP53-mutated CRC tumors carry missense mutations at position R175 or R273. Here we report that CRC tumors harboring R273 mutations are more prone to progress to metastatic disease, with decreased survival, than those with R175 mutations. We identify a distinct transcriptional signature orchestrated by p53R273H, implicating activation of oncogenic signaling pathways and predicting worse outcome. These features are shared also with the hotspot mutants p53R248Q and p53R248W. p53R273H selectively promotes rapid CRC cell spreading, migration, invasion and metastasis. The transcriptional output of p53R273H is associated with preferential binding to regulatory elements of R273 signature genes. Thus, different TP53 missense mutations contribute differently to cancer progression. Elucidation of the differential impact of distinct TP53 mutations on disease features may make TP53 mutational information more actionable, holding potential for better precision-based medicine.

Differential Gain-of-Function Activity of Three p53 Hotspot Mutants In Vivo.

p53 hotspot mutants inhibit wild-type p53 similarly but differ in their GOF activities, with stronger tumor-promoting activity in contact mutants and distinct protein partners of each mutant driving tumorigenesis and metastasis.

Mutant p53 Attenuates Oxidative Phosphorylation and Facilitates Cancer Stemness through Downregulating miR-200c-PCK2 Axis in Basal-Like Breast Cancer.

miR-200c is a tumor suppressor miRNA that plays a critical role in regulating epithelial phenotype and cancer stemness. p53 deficiency downregulates the expression of miR-200c and leads to epithelial-mesenchymal transition (EMT) and stemness phenotype, which contributes to the progression of breast cancers. In this study, we demonstrated that CRISPR-mediated knockout (KO) of miR-200c induces metabolic features similar to the metabolic rewiring caused by p53 hot-spot mutations, and that impairing this metabolic reprogramming interferes with miR-200c deficiency-induced stemness and transformation. Moreover, restoring miR-200c expression compromised EMT, stem-cell properties, and the Warburg effect caused by p53 mutations, suggesting that mutant p53 (MTp53) induces EMT-associated phenotypes and metabolic reprogramming by downregulating miR-200c. Mechanistically, decreased expression of PCK2 was observed in miR-200c- and p53-deficient mammary epithelial cells, and forced expression of miR-200c restored PCK2 in p53 mutant-expressing cells. Reduced PCK2 expression not only led to attenuated oxidative phosphorylation (OXPHOS) and increased stemness in normal mammary epithelial cells but also compromised the enhanced OXPHOS and suppression of cancer stemness exerted by miR-200c in p53 mutation-bearing basal-like breast cancer (BLBC) cells. Clinically, PCK2 expression is negatively associated with EMT markers and is downregulated in basal-like subtype and cases with low miR-200c expression or p53 mutation. Notably, low expression of PCK2 is associated with poor overall survival (OS) in patients with breast cancer. IMPLICATIONS: Together, our results suggest that p53 and miR-200c regulate OXPHOS and stem/cancer stemness through PCK2, and loss of the p53-miR-200c-PCK2 axis might provide metabolic advantages that facilitate cancer stemness, leading to the progression of BLBCs.