Mutant P53 Protein Research Articles

Polo-like kinase 2 targeting as novel strategy to sensitize mutant p53-expressing tumor cells to anticancer treatments.

Polo-like kinase 2 (Plk2) belongs to a family of serine/threonine kinases, and it is involved in tumorigenesis of diverse kind of tissues. We previously reported that Plk2 gene was a transcriptional target of the mutant p53/NF-Y oncogenic complex. Plk2 protein can bind to and phosphorylate mutant p53 triggering an oncogenic autoregulatory feedback loop involved in cancer cell proliferation and chemoresistance. In this study, we aimed to assess whether the specific inhibition of Plk2 kinase activity by the selective TC-S 7005 inhibitor could decrease cell proliferation and migration inhibiting mutant p53 phosphorylation, thus disarming its oncogenic potential. We found that the Plk2 inhibitor treatment sensitized the cells to the irradiation and chemotherapy drugs, thereby overcoming the mutant p53-dependent chemoresistance. Taken together, we provided results that Plk2 could be considered a tractable pharmacological target for cancers expressing mutant p53 proteins. The combined treatment with conventional chemotherapeutic drugs and Plk2 inhibitors may represent a new candidate intervention approach, which may be considered for improving tumor cell sensitivity to DNA damaging drugs. KEY MESSAGES : Missense mutations are present in the TP53 gene in about half of all human cancers and correlate with poor patient outcome. Mutant p53 proteins exert gain of function (GOF) activities in tumor cells such as increased proliferation, genomic instability and resistance to therapies. Polo-like kinase 2 (PLK2) binds and phosphorylates mutant p53 protein strengthening its GOF activities. Pharmacologically targeting PLK2 weakens mutant p53 proteins and sensitizes tumor cells to therapeutic treatments.

Mutant p53 regulates cancer cell invasion in complex three-dimensional environments through mevalonate pathway-dependent Rho/ROCK signaling.

This study addresses the contribution of mutant p53 to the process of cancer cell dissemination in physiologically relevant three-dimensional environments - a key characteristic of metastatic disease. Several mutant p53 proteins display pro-oncogenic activity with respect to cancer cell invasion in 3D environments via mevalonate pathway-dependent Rho/ROCK signaling axis.

Abstract A026: Studying the synergistic potential of a p53 reactivator with ROS-mediated therapies for the treatment of pancreatic cancer

Abstract Introduction: Pancreatic cancer, with a dismal five-year survival rate of just 12%, demands innovative approaches due to its resistance to current therapies. TP53 mutations, present in 75% of patients, make mutant p53 an appealing target. Thus, p53 reactivation, restoring its tumour suppressor functions, represents a promising strategy to address this challenge. While the proposed reactivator has shown efficacy in other malignancies, its potential in pancreatic cancer therapy requires further exploration. Methods: In this project, we have assessed the potential of repurposing a reactivator of mutant p53 protein, to inactivate pancreatic cancer cells. We have performed RNA-sequencing analysis in patient-derived models available in the lab, to study the signalling pathways regulated by the p53 reactivator anticancer activity. We have also studied the possibility to synergistically combine this repurposing strategy with reactive oxygen species (ROS)-mediated therapeutic approaches, as we previously observed p53 reactivation inhibits the glutathione antioxidant pathway. Results: The combination treatment induced synergistic cell death (3-fold increase) compared to monotherapy as evaluated by different molecular assays (e.g. MTT and Live/Death Assay). It also increased ROS levels (Flow cytometry) and induced paraptosis and apoptosis (Western Blot, Tomography Electron Microscopy). The RNA-seq data revealed differential expression of more than 200 genes, confirming the activation of oxidative stress and a cellular response to unfolded proteins. Moreover, Sanger sequencing results demonstrated that the sensibility of each pancreatic model to treatment was dependent on the mutational status of TP53. Conclusion/discussion: The combination therapy of a TP53 reactivator and a ROS mediated therapeutic strategy works synergistically in patient derived models, inducing cell death and changes in the expression of certain RNAs directly involved in unfolded protein response. This pilot study shows that this combination is a feasible strategy to treat pancreatic cancer, setting the path for its testing on in vivo models. Citation Format: Daniel Parra Sánchez, Andrés García-Sampedro, Isabela Laughland, Sylvain Peuget, Galina Selivanova, Stephen P Pereira, Pilar Acedo. Studying the synergistic potential of a p53 reactivator with ROS-mediated therapies for the treatment of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A026.

Pyrazolone-Based Zn(II) Complexes Display Antitumor Effects in Mutant p53-Carrying Cancer Cells.

The synthesis and characterization of nine Schiff bases of pyrazolone ligands HLn (n = 1-9) and the corresponding zinc(II) complexes 1-9 of composition [Zn(Ln)2] (n = 1-9) are reported. The molecular structures of complexes 2, 3, 4, 8, and 9 were determined by single-crystal X-ray diffraction analysis, highlighting in all cases a distorted tetrahedral geometry around the Zn(II) ion. Density functional theory studies are performed on both the HLn ligands and the derived complexes. A mechanism of dissociation and hydrolyzation of the coordinated Schiff base ligands is suggested, confirmed experimentally by powder X-ray diffraction study and photophysical studies. Complexes 1-9 were investigated in vitro as anticancer agents, along with mutant p53 (mutp53) protein levels in human cancer cell lines carrying R175H and R273H mutp53 proteins. Only those complexes with the highest Zn(II) ion release via dissociation have shown a significant cytotoxic activity with reduction of mutp53 protein levels.

The pathological and immunohistochemical profile of tumor angiogenesis in perforated sigmoid carcinoma–Case report and short literature review

<b>Background:</b> Little is known about the physiopathological factors or mechanisms that underlie tumor invasion of the serosa and lead to perforation in the peritoneal cavity. The aim of the work was to analyze the pathological and immunohistochemical factors of tumor neoangiogenesis which could influence tumor perforation in colorectal cancer.<br /> <b>Results and discussions:</b> 451 cases of complicated colorectal carcinomas were statistically analyzed, of which 19 cases were perforated sigmoid tumors. The immunohistochemical detection of mutant p53 proteins was the first molecular parameter examined in the context of the search for markers predicting the natural evolution mode in colorectal carcinomas.<br /> <b>Conclusions</b>: Both loss of p53 and overexpression of bcl-2 proteins confer immortalization on cancer cells by inhibiting the processes leading to apoptosis. The paper proposes a review of the specialized literature, but also the presentation of a clinical case.

Targeting of mutant-p53 and MYC as a novel strategy to inhibit oncogenic SPAG5 activity in triple negative breast cancer

Triple negative breast cancer (TNBC) is an aggressive disease which currently has no effective therapeutic targets and prominent biomarkers. The Sperm Associated antigen 5 (SPAG5) is a mitotic spindle associated protein with oncogenic function in several human cancers. In TNBC, increased SPAG5 expression has been associated with tumor progression, chemoresistance, relapse, and poor clinical outcome. Here we show that high SPAG5 expression in TNBC is regulated by coordinated activity of YAP, mutant p53 and MYC. Depletion of YAP or mutant p53 proteins reduced SPAG5 expression and the recruitment of MYC onto SPAG5 promoter. Targeting of MYC also reduced SPAG5 expression and concomitantly tumorigenicity of TNBC cells. These effects of MYC targeting were synergized with cytotoxic chemotherapy and markedly reduced TNBC oncogenicity in SPAG5-expression dependent manner. These results suggest that mutant p53-MYC-SPAG5 expression can be considered as bona fide predictors of patient’s outcome, and reliable biomarkers for effective anticancer therapies.

GRAIL1 Stabilizes Misfolded Mutant p53 through a Ubiquitin Ligase-Independent, Chaperone Regulatory Function.

Frequent (>70%) TP53 mutations often promote its protein stabilization, driving esophageal adenocarcinoma (EAC) development linked to poor survival and therapy resistance. We previously reported that during Barrett's esophagus progression to EAC, an isoform switch occurs in the E3 ubiquitin ligase RNF128 (aka GRAIL-gene related to anergy in lymphocytes), enriching isoform 1 (hereby GRAIL1) and stabilizing the mutant p53 protein. Consequently, GRAIL1 knockdown degrades mutant p53. But, how GRAIL1 stabilizes the mutant p53 protein remains unclear. In search for a mechanism, here, we performed biochemical and cell biology studies to identify that GRAIL has a binding domain (315-PMCKCDILKA-325) for heat shock protein 40/DNAJ. This interaction can influence DNAJ chaperone activity to modulate misfolded mutant p53 stability. As predicted, either the overexpression of a GRAIL fragment (Frag-J) encompassing the DNAJ binding domain or a cell-permeable peptide (Pep-J) encoding the above 10 amino acids can bind and inhibit DNAJ-Hsp70 co-chaperone activity, thus degrading misfolded mutant p53. Consequently, either Frag-J or Pep-J can reduce the survival of mutant p53 containing dysplastic Barrett's esophagus and EAC cells and inhibit the growth of patient-derived organoids of dysplastic Barrett's esophagus in 3D cultures. The misfolded mutant p53 targeting and growth inhibitory effects of Pep-J are comparable with simvastatin, a cholesterol-lowering drug that can degrade misfolded mutant p53 also via inhibiting DNAJA1, although by a distinct mechanism. Implications: We identified a novel ubiquitin ligase-independent, chaperone-regulating domain in GRAIL and further synthesized a first-in-class novel misfolded mutant p53 degrading peptide having future translational potential.

NFKBIE is a predictive factor of survival and is correlated with immune infiltration and antigen processing and presentation in hepatocellular carcinoma.

The important role of the nuclear factor κB (NFκB) pathway in tumour development has long been recognized; however, the role of the NFκB inhibitor family in liver cancer has not been elucidated. Hepatocellular carcinoma (HCC) is a serious public health burden with a high incidence, poor prognosis, and early detection, especially in Asia, where hepatitis is prevalent. In the present study, the mRNA expression level of the NFκB inhibitor family was assessed in HCC and normal tissues using the Metabolic Gene Rapid Visualizer, University of Alabama at Birmingham Cancer Data Analysis Portal, and the Tumor Immune Estimation Resource database (TIMER). Survival curves of nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor (NFKBI)E were obtained using the Kaplan-Meier method. Genes co-expressed with NFKBIE in HCC samples were studied using data from the LinkedOmics and the Hepatocellular Carcinoma Databases. Protein-protein interaction networks, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment pathway analyses were used to assess the NFKBIE mechanism in HCC. Using the TIMER database, the association between immune infiltration and NFKBIE was determined. RNA-sequencing (RNA-seq) was used to evaluate the function of NFKBIE in HCC and its impact on proliferation and migration. Western blotting was used to confirm the expression of NFKBIE in HCC cell lines. In addition, NFKBIE overexpression in HCC was demonstrated using tissue microarrays encompassing 80 pairs of HCC and normal liver tissues. NFKBIE was the only NFκB inhibitor with high expression and an improved prognosis in HCC compared with other NFκB inhibitors. NFKBIE was correlated with clinical characteristics, such as tumour grade, tumour protein P53 mutation status and tumour stage. Data obtained from Gene Set Cancer Analysis suggested that NFKBIE may inhibit the PI3K/AKT, RAS/MAPK, RTK and TSC/mTOR pathways. In addition, NFKBIE was significantly associated with B-cell immune infiltration and the RNA-seq data demonstrated that knockdown of NFKBIE significantly affected 'Antigen processing and presentation' and 'hepatocellular carcinoma' pathways. Immunohistochemistry of microarrays of tissue samples revealed that NFKBIE was overexpressed in several stages of HCC. Finally, inhibition of NFKBIE decreased the proliferation and migration of HCC cells. In conclusion, due to its prognostic value and overexpression in HCC, NFKBIE distinguished itself from other NFκB inhibitors. As such, it may provide a novel prognostic indicator and immunotherapeutic target for HCC.

Impacts of cytoplasmic p53 aggregates on the prognosis and the transcriptome in lung squamous cell carcinoma.

The tumor suppressor TP53 gene, the most frequently mutated gene in human cancers, produces the product tumor protein p53, which plays an essential role in DNA damage. p53 protein mutations may contribute to tumorigenesis by loss of tumor suppressive functions and malignancy of cancer cells via gain-of-oncogenic functions. We previously reported that mutant p53 proteins form aggregates and that cytoplasmic p53 aggregates were associated with poor prognosis in human ovarian cancer. However, the prognostic impact of p53 aggregation in other tumors including lung squamous cell carcinoma (SCC) is poorly understood. Here, we demonstrated that lung SCC cases with cytoplasmic p53 aggregates had a significantly poor clinical prognosis. Analysis via patient-derived tumor organoids (PDOs) established from lung SCC patients and possessing cytoplasmic p53 aggregates showed that eliminating cytoplasmic p53 aggregates suppressed cell proliferation. RNA sequencing and transcriptome analysis of p53 aggregate-harboring PDOs indicated multiple candidate pathways involved in p53 aggregate oncogenic functions. With lung SCC-derived cell lines, we found that cytoplasmic p53 aggregates contributed to cisplatin resistance. This study thus shows that p53 aggregates are a predictor of poor prognosis in lung SCC and suggests that detecting p53 aggregates via p53 conventional immunohistochemical analysis may aid patient selection for platinum-based therapy.

Recent Advances on Mutant p53: Unveiling Novel Oncogenic Roles, Degradation Pathways, and Therapeutic Interventions.

The p53 protein is the master regulator of cellular integrity, primarily due to its tumor-suppressing functions. Approximately half of all human cancers carry mutations in the TP53 gene, which not only abrogate the tumor-suppressive functions but also confer p53 mutant proteins with oncogenic potential. The latter is achieved through so-called gain-of-function (GOF) mutations that promote cancer progression, metastasis, and therapy resistance by deregulating transcriptional networks, signaling pathways, metabolism, immune surveillance, and cellular compositions of the microenvironment. Despite recent progress in understanding the complexity of mutp53 in neoplastic development, the exact mechanisms of how mutp53 contributes to cancer development and how they escape proteasomal and lysosomal degradation remain only partially understood. In this review, we address recent findings in the field of oncogenic functions of mutp53 specifically regarding, but not limited to, its implications in metabolic pathways, the secretome of cancer cells, the cancer microenvironment, and the regulating scenarios of the aberrant proteasomal degradation. By analyzing proteasomal and lysosomal protein degradation, as well as its connection with autophagy, we propose new therapeutical approaches that aim to destabilize mutp53 proteins and deactivate its oncogenic functions, thereby providing a fundamental basis for further investigation and rational treatment approaches for TP53-mutated cancers.

Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts

The p53 tumor suppressor, encoded by the TP53 gene, serves as a major barrier against malignant transformation. Patients with Li-Fraumeni syndrome (LFS) inherit a mutated TP53 allele from one parent and a wild-type TP53 allele from the other. Subsequently, the wild-type allele is lost and only the mutant TP53 allele remains. This process, which is termed loss of heterozygosity (LOH), results in only mutant p53 protein expression. We used primary dermal fibroblasts from LFS patients carrying the hotspot p53 gain-of-function pathogenic variant, R248Q to study the LOH process and characterize alterations in various pathways before and after LOH. We previously described the derivation of mutant p53 reactivating peptides, designated pCAPs (p53 Conformation Activating Peptides). In this study, we tested the effect of lead peptide pCAP-250 on LOH and on its associated cellular changes. We report that treatment of LFS fibroblasts with pCAP-250 prevents the accumulation of mutant p53 protein, inhibits LOH, and alleviates its cellular consequences. Furthermore, prolonged treatment with pCAP-250 significantly reduces DNA damage and restores long-term genomic stability. pCAPs may thus be contemplated as a potential preventive treatment to prevent or delay early onset cancer in carriers of mutant p53.

Abstract PO2-24-01: Kif11 Inhibition Preferentially Kills TP53-mutant Breast Cancer Cells

Abstract Background Triple-Negative Breast Cancer (TNBC) makes up 15-20% of breast cancer diagnoses and is more common in younger women, those with BRCA mutations, and Black patients. Furthermore, TNBCs are defined by their lack of expression of the targetable receptors: estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR), resulting in few targeted therapy options for TNBC patients. One potential therapeutic target in TNBC is mutant p53, since 85-90% of TNBCs harbor TP53 mutations. However, the mutant p53 protein is challenging to target directly. Therefore, we have sought to identify survival pathways critical to TP53 mutant cells. Through a combined in vitro and in silico drug screen, we identified that the Kif11 inhibitor SB-743921 differentially kills TP53 mutant breast cancer cells compared to TP53 wild-type cells. Hypothesis Based on this screening data, we hypothesized that Kif11 inhibition causes TP53 mutant cells to undergo mitotic catastrophe resulting in cell death due to failure of activation of p53-mediated cell cycle checkpoints. Methods Clinical data was obtained from cBioPortal from the TCGA and METABRIC cohorts. Expression data analysis and survival analyses were performed using GraphPad Prism. Cell growth assays were conducted by seeding cells in 96 well plates, treating under designated conditions, then staining with Hoechst 33342 for cell counting on the ImageXpress Pico. Cell death was determined by co-staining with Hoechst 33342 and DRAQ7 followed by imaging and analysis on the ImageXpress Pico and by Annexin V and Propidium Iodide Staining followed by flow cytometry analysis. Cell cycle analysis was conducted following synchronization with Lovastatin and Mevalonate release followed by collection of samples, fixation, PI staining, and flow cytometry analysis. Immunofluorescence imaging was conducted by staining with anti-alpha-tubulin-AlexaFluor488 and DAPI and imaging at 20x and 63x on the ImageXPress PICO. In vitro expression experiments were carried out through qPCR and western blotting. < Results In human breast cancers, KIF11 is more highly expressed in TP53 mutant, as compared to wild-type breast cancers, and in TNBCs, as compared to other breast cancer subtypes. Patients with tumors that had higher expression of Kif11 had poorer overall survival outcomes. Inhibition of Kif11 with the small molecule inhibitor SB-743921 induces greater death of TP53 mutant as compared to wild-type breast cancer cells. Kif11 inhibition leads to a cell cycle block in both TP53 mutant and wild-type cells, but TP53 mutant cells then die following this cell cycle block. Kif11 inhibition causes mitotic dysfunction, including monopolar spindle formation, in mutant and wild-type cells, but greater incidence of spindle abnormalities and multinucleated cells in TP53 mutants. Introduction of a TP53 mutation into TP53 wild-type cells also induces cell death following Kif11 inhibition. Conclusions The Kif11 mitotic kinesin is more highly expressed in TP53 mutant and triple-negative breast cancers than in TP53 wild-type breast cancers, and high expression is associated with poorer survival outcomes. In TP53 mutant cells, Kif11 inhibition results in mitotic dysfunction and cell death due to mitotic catastrophe. Acknowledgments Research reported in this publication was supported by the John Charles Cain Endowment and the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Numbers TL1TR003169 and UL1TR003167. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We would also like to thank the MDACC NORTH Campus Flow Cytometry and Cellular Imaging Core Facility for their assistance. Citation Format: Amanda Lanier, William Tahaney, Jing Qian, Cassandra Moyer, Yanxia Ma, Banu Arun, Abhijit Mazumdar, Powel Brown. Kif11 Inhibition Preferentially Kills TP53-mutant Breast Cancer Cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-01.

Abstract LB303: Suppression of stress granule formation is a novel vulnerability imposed by mutant p53

Abstract Mutation of the tumor suppressor p53 is the most common cancer specific event. Most of these mutations are missense mutations in the DNA-binding domain of p53, causing loss of wild-type p53 (wtp53) transcriptional activity. Missense mutant p53 (mutp53) also enhances cancer malignancy in a manner independent of wtp53. Hence, mutp53 is an ideal therapeutic target due to its uniqueness and high expression levels in cancers; however, directly targeting mutp53 protein itself has been challenging. To better understand the mechanism of oncogenic function of mutp53, we performed in silico TCGA database analysis and found that patients with missense mutp53-carrying hepatocellular carcinoma (HCC), but not other types of cancer, had unexpectedly better overall survival than patients with p53-null HCC. Since sorafenib (SOR) has historically been used as a single agent chemotherapy for HCC, unlike other cancer types, we hypothesized that SOR treatment may have yet unappreciated advantages for treating mutp53-expressing tumors. Indeed, knockdown of mutp53 rendered HCC cells and xenografts more resistant to SOR. Consistent with a report showing that SOR induces ER stress-mediated pro-survival stress granule (SG) formation, mutp53-expressing HCC cells, treated with SOR or other ER stress inducers, formed significantly fewer SGs with increased apoptosis, as compared with mutp53-knockdown cells. This result was partly due to mutp53’s ability to interact with an eIF2α kinase PERK and a key SG core protein, G3BP1. Moreover, the oligomerization domain of mutp53 was required for the interaction of mutp53 with G3BP1 and subsequent suppression of ER-stress-mediated SG formation and survival. These results suggest that inhibition of SG formation is a novel vulnerability imposed by mutp53, leading to sensitization of cancer cells to ER stress. Our results also emphasize the use of ER stress inducers to treat mutp53-expressing tumors and mutp53 as a biomarker of SOR sensitivity. Citation Format: Elizabeth Thoenen, Atul Ranjan, Alejandro Parrales, Shigeto Nishikawa, Dan Dixon, Sugako Oka, Tomoo Iwakuma. Suppression of stress granule formation is a novel vulnerability imposed by mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB303.

Abstract 4641: Preclinical evaluation of the novel curcumin Analogue AKT-100 demonstrates therapeutic effectiveness in p53-mutated cancer cells

Abstract Among the myriad of molecular alterations associated with cancer, loss of the normal tumor suppressive functions of p53, the “guardian of the genome,” is the most consistent. Thus, the possibility of targeting oncogenic, mutant p53 proteins has long been a therapeutic goal. However, that aspiration has not been fully realized, leaving patients vulnerable to aggressive disease that is resistant to chemotherapy and to targeted agents such as PARP inhibitors and immunotherapy owing to the loss of wild type p53 functionality and the gain of new oncogenic functions when mutant p53 is expressed. An effective therapeutic strategy is urgently needed for p53 mutant cancers, and our team is developing novel analogues of curcumin with the potential to convert mutant p53 to a wild type conformation and restore its tumor suppressive functions. In this study, we investigated the effects of AKT-100, which was developed from the parent molecule and curcumin analogue HO-3867. Effects were compared to another drug believed to reactivate p53 functionality, APR-246, using CyQUANT IC50 cell proliferation assays and bulk and single cell RNA sequencing. Structural analyses are underway investigating the specific sites of interaction of AKT-100 with various mutant forms of p53. Our findings indicate that, first, AKT-100 exhibits impressive cell killing in multiple ovarian and serous endometrial cancer cells at concentrations ranging from 100-300 nM and is synergistic with the PARP inhibitor olaparib. Second, RNA sequencing demonstrates the reactivation of wild type p53 genes associated with normal cell cycle regulation (induction of CDKN1A encoding p21 and GADD45A), apoptosis (induction of PMAIP1 encoding Noxa and DR5 encoding Death Receptor 5), and the inhibition of DNA replication and multiple alternative mechanisms of DNA repair employed by malignant cells. Third, RNA sequencing indicates that AKT-100 acts through both p53-dependent and independent pathways to inhibit cancer cell proliferation. Gene expression is impacted in ways that portend a therapeutic effect as well as highlight potential resistance pathways (e.g. significant induction of PLK5 encoding polo like kinase 5) that can be further targeted to create synthetic lethal drug combinations with AKT-100 chosen specifically to target the various mutant forms of p53. Taken together, these results identify novel curcumin analogues in early development with the potential to improve therapy for p53 mutant cancers. Through this strategy it is also possible to identify and block resistance pathways that are activated by different forms of mutant p53 in response to AKT-100 and to create synthetic lethal precision drug combinations. Citation Format: Kimberly K. Leslie, Jamie L. Padilla, Geneva L. Williams, Hua-Ying Fan, Cristian G. Bologa, Eric R. Prossnitz, Jun-Yong Choe, Lane E. Smith, Alexander Goss, Robert J. Lake, Avinash D. Sahu. Preclinical evaluation of the novel curcumin Analogue AKT-100 demonstrates therapeutic effectiveness in p53-mutated cancer cells [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 4641.

Abstract 631: Functional genetics of APR-246 rescuable TP53 mutants

Abstract TP53, often referred to as the guardian of the genome, encodes for the p53 tumor suppressor protein, and is one of the most frequently mutated genes in human cancers. Some cancer sub-types, such as non-small cell lung carcinoma (NSCLC) and high grade serous carcinoma (HGSC), have high TP53 mutation rates reaching up to 56% and 96%, respectively. Given its multi-faceted role in human cancers, there have been several emerging therapeutic approaches targeting mutant p53. APR-246 is a small molecule drug currently in clinical trials for the functional rescue of p53 missense mutants, the most common type of p53 mutant protein, across various cancer types. APR-246 is proposed to bind mutant p53 and promote thermodynamic stabilization of the protein allowing it to regain wild-type-like conformation and consequentially regain WT-like functionality. However, there has yet to be a functional genomic screening of p53 mutants to systematically catalog which mutants are rescuable by APR-246. To address this critical gap, we have performed functional genetic screenings using a TP53 mutagenesis library. This pooled lentiviral library contains ~8,000 amino acid variants, spanning the entirety of the TP53 gene. Following stable transduction of this library into p53-null NSCLC and HGSOC cell lines, we performed functional screenings against sublethal doses of APR-246. Next-generation sequencing of the untreated and treated cell population allows for comparison of variant representation and determination of APR-246-sensitivity of p53 mutants. Preliminary statistical analysis of our screens has identified a pool of 117 “hit” mutants which are significantly depleted following treatment, indicative of APR-246-sensitivity. Two of these mutants, C242F and G266V, are non-functional, damaging mutations which occur in patients. Identifying p53 mutants that can be rescued by APR-246 will help identify which patients may benefit most from treatment with the drug. Citation Format: Anais Saunders, Caili Tong, Anthony N. Karnezis, Gary S. Leiserowitz, Jeremy R. Chein. Functional genetics of APR-246 rescuable TP53 mutants [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 631.

Final results on effectiveness and safety of Ibrutinib in patients with chronic lymphocytic leukemia from the non-interventional FIRE study.

We conducted an observational study (FIRE) to understand the effectiveness and safety outcomes of ibrutinib in patients with chronic lymphocytic leukemia (CLL) in France, after a maximum follow-up of five years. Patients were included according to the French marketing authorization in 2016 (i.e. patients with relapsed or refractory CLL or to previously untreated CLL patients with deletion 17p and/or tumor protein p53 mutations unsuitable for chemoimmunotherapy) and could have initiated ibrutinib more than 30days prior their enrolment in the study (i.e. retrospective patients) or between 30days before and 14days after their enrolment (i.e. prospective patients). The results showed that in the effectiveness population (N = 388), the median progression-free survival (PFS) was 53.1 (95% CI: 44.5-60.5) months for retrospective patients and 52.9 (95% CI: 40.3-60.6) months for prospective patients and no difference was shown between the PFS of patients who had at least one dose reduction versus the PFS of patients without dose reduction (p = 0.7971 for retrospective and p = 0.3163 for prospective patients). For both retrospective and prospective patients, the median overall survival was not reached. The most frequent treatment-emergent adverse event of interest was infections (57.6% retrospective; 71.4% prospective). A total of 14.6% of the retrospective patients and 22.4% of the prospective patients had an adverse event leading to death. Our findings on effectiveness were consistent with other studies and the fact that patients with dose reductions had similar PFS than patients without dose reduction is reassuring. No additional safety concerns than those already mentioned in previous studies could be noticed.Trial registrationClinicalTrials.gov, NCT03425591. Registered 1 February 2018 - Retrospectively registered.

TP53 gain-of-function mutations promote osimertinib resistance via TNF-α–NF-κB signaling in EGFR-mutated lung cancer

EGFR tyrosine kinase inhibitors (TKIs) are effective against EGFR-mutated lung cancer, but tumors eventually develop resistance to these drugs. Although TP53 gain-of-function (GOF) mutations promote carcinogenesis, their effect on EGFR-TKI efficacy has remained unclear. We here established EGFR-mutated lung cancer cell lines that express wild-type (WT) or various mutant p53 proteins with CRISPR-Cas9 technology and found that TP53-GOF mutations promote early development of resistance to the EGFR-TKI osimertinib associated with sustained activation of ERK and expression of c-Myc. Gene expression analysis revealed that osimertinib activates TNF-α–NF-κB signaling specifically in TP53-GOF mutant cells. In such cells, osimertinib promoted interaction of p53 with the NF-κB subunit p65, translocation of the resulting complex to the nucleus and its binding to the TNF promoter, and TNF-α production. Concurrent treatment of TP53-GOF mutant cells with the TNF-α inhibitor infliximab suppressed acquisition of osimertinib resistance as well as restored osimertinib sensitivity in resistant cells in association with attenuation of ERK activation and c-Myc expression. Our findings indicate that induction of TNF-α expression by osimertinib in TP53-GOF mutant cells contributes to the early development of osimertinib resistance, and that TNF-α inhibition may therefore be an effective strategy to overcome such resistance in EGFR-mutant lung cancer with TP53-GOF mutations.

Novel oncogenic transcriptional targets of mutant p53 in esophageal squamous cell carcinoma.

Missense mutations in the DNA binding domain of p53 are observed frequently in esophageal squamous cell carcinoma (ESCC). Recent studies have revealed the potentially oncogenic transcriptional networks regulated by mutant p53 proteins. However, majority of these studies have focused on common "hotspot" p53 mutations while rarer mutations are poorly characterized. In this study, we report the characterization of rare, "non-hotspot" p53 mutations from ESCC. In vitro tumorigenic assays performed following ectopic-expression of certain "non-hotspot" mutant p53 proteins caused enhancement of oncogenic properties in squamous carcinoma cell lines. Genome-wide transcript profiling of ESCC tumor samples stratified for p53 status, revealed several genes exhibiting elevated transcript levels in tumors harboring mutant p53. Of these, ARF6, C1QBP, and TRIM23 were studied further. Reverse transcription-quantitative PCR (RT-qPCR) performed on RNA isolated from ESCC tumors revealed significant correlation of TP53 transcript levels with those of the three target genes. Ectopic expression of wild-type and several mutant p53 forms followed by RT-qPCR, chromatin affinity-purification (ChAP), and promoter-luciferase assays indicated the exclusive recruitment of p53 mutants-P190T and P278L, to the target genes leading to the activation of expression. Several functional assays following knockdown of the target genes revealed a significant suppression of tumorigenicity in squamous carcinoma cell lines. Rescue experiments confirmed the specificity of the knockdown. The tumorigenic effects of the genes were confirmed in nude mice xenograft assays. This study has therefore identified novel oncogenic targets of "non-hotspot" mutant p53 proteins relevant for ESCC besides validating the functional heterogeneity of the spectrum of tumor-specific p53 mutations.

Mutant p53 Gain-of-Function in the Spotlight: Are We Suffering a GOF Delusion?

Mutant p53 proteins are often highly expressed in human cancers and have been thought to have oncogenic driver gain-of-function (GOF) properties. Wang and colleagues show, surprisingly, that this is not the case because removing the TP53-mutant gene from human and mouse cancer cells using CRISPR technology has no effect on cancer cell growth in vitro or in vivo. See related article by Wang et al., p. 362 (10) .

Abstract B061: Discovering co-driver genes and pathways of mutant TP53 in breast cancer by CRISPR screening and multi-omics approaches

Abstract Triple-Negative Breast Cancer (TNBC) is the most aggressive and highly heterogeneous molecular subtype of breast cancer lacking the progesterone, HER2 and estrogen receptors. The heterogeneity of these receptors makes it difficult to target and treat. The TP53 gene which encodes the p53 protein is mutated in 80% of TNBC cases. We hypothesized that, in addition to the neo-morphic activities of different p53 mutants themselves, the heterogeneity in clinical phenotypes observed in TNBC is due to functional interactions of the p53 driver mutants with other functionally important co-existing mutations called ‘co-drivers’. To identify co-driver mutations, our lab performed a genome-wide screening by using lentiviral CRISPR libraries on a cell line over-expressing p53-R273C (a prevalent mutant form of p53), and MYC (an oncogene). Upon injection of the library-transduced cells into mice, large tumor was formed, which was extracted and subjected to targeted PCR and whole exome sequencing. This identified truncation mutations in the EVC2 (EvC Ciliary Complex Subunit 2) and the NR1D1 (Nuclear Receptor 1D1) genes. This suggested that they are tumor suppressors in their wild type functions, and loss of this native function would co-drive the formation of tumors implicated with mutant p53-R273C to promote tumor formation in vivo. EVC2 positively modulates the Hedgehog signaling pathway. Interestingly, a majority of mutations in NR1D1 were an in-frame deletion of a tyrosine residue, which may be a critical regulatory residue of NR1D1. It is known to modulate the JAK/STAT3 pathway and, just like p53, NR1D1 can inhibit the circadian clock genes. We are testing whether deleting EVC2 and NR1D1 genes by CRISPR or expressing the mutant forms in the presence of p53-R273C increase invasion and/or other cancer hallmark phenotypes and also whether the amino acid, tyrosine is critical to the regulation of NR1D1, with the goal of understanding their contributions to the development of various hallmarks of cancer and the underlying mechanisms which will identify how these mutations contribute to the progression of TNBC and could potentially provide molecularly targeted therapies for TNBC. In parallel, our lab previously carried out RNA-Seq on cell lines expressing 10 different mutant p53 proteins, where pathway analysis identified the Hippo pathway, a tumor suppressor pathway, highly associated with aggressive cellular phenotypes in the mutant p53 contexts. In cancer, it is known that, upon suppression of the upstream Hippo pathway, unphosphorylated TAZ (or WWTR1) localizes in the nucleus binds to TEAD, leading to the activation of a series of genes to promote cancer hallmark phenotypes. We are investigating the molecular mechanisms of actions through which Hippo pathway drives the progression of TNBC and assess the pathway as the target for potential therapy for TNBC with specific TP53 mutations. This study will reveal how different mutant p53 proteins regulate Hippo pathway, TAZ activation/localization, and TEAD-mediated transcription to promote invasiveness. Citation Format: Lilian Nwekwo, Anasuya Pal, Yining Zheng, Lydia Sakala, Laura Gonzalez-Malerva, Dustin Grief, Dustin Lord, Jin Park, Joshua LaBaer. Discovering co-driver genes and pathways of mutant TP53 in breast cancer by CRISPR screening and multi-omics approaches [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B061.