Inactivation Of P53 Research Articles

Translational readthrough of nonsense mutant TP53 by mRNA incorporation of 5-Fluorouridine

TP53 nonsense mutations in cancer produce truncated inactive p53 protein. We show that 5-FU metabolite 5-Fluorouridine (FUr) induces full-length p53 in human tumor cells carrying R213X nonsense mutant TP53. Ribosome profiling visualized translational readthrough at the R213X premature stop codon and demonstrated that FUr-induced readthrough is less permissive for canonical stop codon readthrough compared to aminoglycoside G418. FUr is incorporated into mRNA and can potentially base-pair with guanine, allowing insertion of Arg tRNA at the TP53 R213X UGA premature stop codon and translation of full-length wild-type p53. We confirmed that full-length p53 rescued by FUr triggers tumor cell death by apoptosis. FUr also restored full-length p53 in TP53 R213X mutant human tumor xenografts in vivo. Thus, we demonstrate a novel strategy for therapeutic rescue of nonsense mutant TP53 and suggest that FUr should be explored for treatment of patients with TP53 nonsense mutant tumors.

Abstract A051: Understanding the Arf-p53 axis in PDAC suppression

Abstract Understanding the gene regulatory networks that contribute to PDAC initiation and progression is critical for improving early detection and more effectively treating this disease. Activating mutations in KRAS are often accompanied by mutations in the TP53, CDKN2A, and SMAD4 tumor suppressor genes in ~72%, 30%, and 32% of PDAC cases (Raphael et al. 2017). Understanding the consequences of deletion of individual tumor suppressor genes can delineate specific genotype-phenotype correlations that can provide new clinical insights. Using a genetically engineered mouse PDAC model based on KrasG12D expression and tumor suppressor gene inactivation through Ptf1aCreER expression in adult acinar cells, we showed previously that p53 deletion or point mutation dramatically accelerates PDAC relative to mice with intact p53. To understand tumor evolution in mice with different p53 statuses, we analyzed the transcriptomic profiles of PDACs developing with intact or mutant p53 by spatial transcriptomics and both bulk and single cell RNA-sequencing. These experiments have provided insight into the pathways that become dysregulated as PDAC develops in the context of different p53 alleles. In addition, as oncogenic signals trigger p53 stabilization via the p19ARF (ARF) protein in some contexts, we sought to determine whether ARF acts upstream of p53 in PDAC suppression. Indeed, the frequent deletion of the CDKN2A locus, which encodes both the ARF and p16INK4A (p16) tumor suppressor genes, suggests the importance of both these genes in suppressing PDAC, but the specific role of ARF in PDAC suppression has not been tested. Using mouse models to definitively interrogate the importance of ARF in suppressing PDAC, we found that Arf inactivation promotes KrasG12D-driven PDAC, and with a latency similar to p53 inactivation. Interestingly, concurrent inactivation of both p53 and Arf accelerated oncogenic KRAS-driven PDAC development relative to p53 inactivation alone, suggesting a p53-independent role for ARF in PDAC suppression. Transcriptomic profiling of tumors from these mice revealed both p53-dependent and p53-independent programs of ARF-mediated tumor suppression, which provides insight into how tumors develop in the absence of ARF. Characterization of the immune and fibroblast components of the tumor microenvironment in tumors with intact tumor suppressors, p53 deficiency, Arf deficiency and combined p53;Arf deficiency reveals genotype-dependent differences, including different CAF profiles observed in tumors of different genotypes. Ongoing analysis of human PDAC will help to further parse the roles of ARF and p16 in human pancreatic cancer development. Together, these studies will help to illuminate how p53 and Arf inactivation contribute to cancer cell evolution and crosstalk with the tumor microenvironment during PDAC development, understanding with potential for improving clinical interventions in PDAC. Citation Format: Laura D. Attardi, Brittany M. Flowers, Kathryn Hanson, Abigail S. Mulligan, Sofia Ferreira, Sohinee Bhattacharyya, Hannes Vogel, Laura D. Wood, Mara Sherman. Understanding the Arf-p53 axis in PDAC suppression [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A051.

Abstract A060: Characterizing acinar cell and ductal cell derived PDACs in mouse models

Abstract Understanding the origins of PDAC is critical for developing better clinical interventions. Modeling in mice has revealed that either pancreatic acinar or ductal cells can serve as the cell of origin for PDAC. Using tamoxifen-inducible Ptf1aCreER and Sox9CreER alleles to induce genetic alterations in adult pancreatic acinar and ductal cells, respectively, we showed that oncogenic Kras expression, coupled with p53 inactivation or mutation, can drive PDAC from either pancreatic cell type. PDACs derived from acinar cells resemble the classical subtype of human PDAC, while those derived from ductal cells resemble the basal-like subtype of human PDAC, suggesting that cell of origin is one factor that can influence PDAC subtype. Moreover, the histological lesions preceding cancer are notably different, suggesting that oncogenic events in each cell type result in distinct paths of tumor evolution. Such findings highlight the importance of the earliest events of tumorigenesis in influencing phenotypes and outcomes, and better understanding these different paths of PDAC evolution can therefore be critical for improving early diagnosis and treatment of PDAC. To deepen our understanding of these paths of PDAC development, we have used spatial transcriptomics to characterize the acinar and ductal cell-derived mouse PDACs, to contextualize cell-specific expression programs in cancer cell and stromal cell compartments. The segmentation of transcriptional programs by cell type is of great importance in PDAC due to a characteristically pervasive desmoplastic stroma and low neoplastic cellularity. These studies have validated differences in transcriptomic profiles of the cancer cells in these two types of PDAC, with HIF1 signaling and glycolysis pathway being upregulated in ductal cell-derived PDAC and programs that mirror tissue development and morphogenesis being more prominent in acinar cell-derived PDAC. Our analysis also revealed differences in the fibroblast and immune compartments of acinar and ductal cell-derived tumors, which we are further characterizing, and which will reveal novel strategies for tumor targeting based on the tumor microenvironment. Together, these findings suggest that these two types of PDAC are driven and sustained by distinct pathways and tumor-stromal crosstalk. To help characterize pathways central for cancer cell growth, we also generated cell lines from acinar cell and ductal cell-derived PDACs, and we are characterizing their sensitivity to different agents that modulate pathways specific to each PDAC subtype. Ultimately, this work will help to elucidate how the two major types of human PDAC develop, paving the way in finding subtype-specific vulnerabilities that may be exploited therapeutically. Citation Format: Sofia Ferreira, Brittany M. Flowers, Kathryn J. Hanson, Alberto Gatto, Sohinee Bhattacharyya, Mara H. Sherman, Laura D. Attardi. Characterizing acinar cell and ductal cell derived PDACs in mouse models [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A060.

Characterize Biomarkers and Mechanisms of Resistance for MDM2 Inhibitors in AML

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease with an aggressive phenotype and poor prognosis. Inactivation of the wild-type p53, through upregulation of the TP53 negative regulator MDM2, is observed in around 50% of AML patients, providing a rationale for targeting MDM2 for the treatment of AML. Small molecule inhibitors of MDM2, including Idasanutlin, have shown efficacy as monotherapy and in combinations. However, patients exhibit heterogeneous responses with frequent drug resistance. We integrated ex vivo drug sensitivity assay, clinical parameters, whole exome sequence (WES), RNAseq, cytokine stimulation, and CRISPR/Cas9 dropout screening data to identify biomarkers that predict idasanutlin response and to elucidate the underlying drug-resistant mechanisms. Surprisingly, besides TP53 itself, we did not find an essential role in losing any of the major TP53 downstream targets alone leading to Idasanutlin resistance from Idasanutlin treated resistant cell lines, CRISPR screening, or individually targeted knockout experiments. Besides TP53 mutations, other major AML mutations, including RAS pathway mutations do not drive Idasanutlin resistance indicated by the patient sample functional screening data and in vitro mutation overexpression models. We observed instead that AML with a high differentiation or mature myeloid signature: including low blast, a high percentage of monocytes/granulocytes/T cells, AML FAB M4/M5 morphology, and a high expression of a myeloid immune signature gene are relatively resistant to Idasanutlin. We further characterized that both healthy/leukemia-associated T cells, granulocytes, and monocytes are intrinsically resistant to idasnutlin due to low expression levels of MDM2 and/or TP53. To examine whether the presence of differentiated cells protects leukemia blast cells from Idasanutlin, we performed a co-culture drug treatment experiment. Strikingly, only leukemia-associated monocytes, but not healthy monocytes, healthy/leukemia T cells or granulocytes, or leukemia blast cells protected leukemia blast cells from apoptosis in the presence or absence of Idasanutlin. Similar results were obtained for the other two MDM2 inhibitors, AMG232 and DS-3032b, and a BCL2 inhibitor, venetoclax. To further uncover which cytokines secreted from the leukemia-associated monocytes confer MDM2 inhibitor resistance, we tested 11 common cytokines, including IL-1a, IL-1b, IL-3, IL-6, IL-8, M-CSF GM-CSF, FLT3, MCP-1, TNFa, and SCF. We observed that IL-1α, IL-1β, IL-3, and GM-CSF markedly induced idasanutlin treatment resistance for AML, but not ALL or CLL patient cells. Interestingly, IL-1α and IL-1β only protect leukemia blasts, but not healthy CD34 hematopoietic stem and progenitor cells from Idasanutlin-induced apoptosis. We further performed RNAseq and immunoblots on IL-1α and IL-1β treated AML, CLL, and ALL cells. We observed the major TP53 downstream pathways remain activated by idasanutlin in the presence of IL-1α and IL-1β. Interestingly, GATA2 is significantly upregulated in IL-1α and IL-1β treated AML, but not CLL or ALL cells in the presence or absence of Idasanutlin. Furthermore, overexpressing GATA2 conferred Idasanutlin resistance at high concentrations. Finally, we observed that combining a FLT3/IRAK dual inhibitor, pacritinib, or an IRAK1 /IL-1 receptor antagonist, anakinra, partially rescued IL-1α and IL-1β mediated drug resistance of MDM2 inhibitors. As such, we uncovered the role of leukemia-associated monocyte in driving intrinsic and extrinsic MDM2 inhibitor resistance and the potential underlying mechanisms.

A Descriptive Analysis of TP53 Y220C Mutations in Patients with Hematologic Malignancies

Background: Mutations in the key tumor suppressor gene TP53 lead to inactivation of the protein p53, which leads to genome instability, oncogenesis, treatment resistance and an overall unfavorable prognosis in various malignancies. The specific TP53 Y220C variant has been described as one of the most frequent "hot spot" mutations associated with solid cancers. Recently, a Phase 1 clinical trial studying the novel Y220C-targeted small molecule PC14586, has shown preliminary safety and efficacy in patients with heavily treated solid tumors. No data has been reported about TP53 Y220C in hematologic malignancies to date. The aim of the present analysis is to evaluate the clinicopathologic characteristics of TP53 Y220C in hematologic malignancies. Methods: We searched our molecular laboratory database for hematologic neoplasms with TP53 Y220C mutations and summarized the patient characteristics, disease features and outcomes for patients treated within the Department of Leukemia at MD Anderson. Overall survival (OS) was assessed by the Kaplan-Meier method calculated from the date of TP53 Y220C detection to the date of death from any cause or date of last follow-up. Results: We identified 126 patients with hematologic malignancies harboring a TP53 Y220C mutation. Nine patients with lymphoma or myeloma were treated outside our Department and were excluded from subsequent analysis. 117 patients had a diagnosis of acute or chronic leukemia or myelodysplastic syndrome (MDS) [Table 1]. Median age was 69 years (31-91) and 59% of patients were male. Acute myeloid leukemia (AML) and MDS accounted for 33% and 38%, respectively. Consistent with the increased incidence in TP53-mutated myeloid disorders, 28% of AML and 43% of MDS cases were therapy-related. Cytogenetics was abnormal in 85 patients (73%), with a complex karyotype occurring in 70 patients (60%). Among 73 patients with detailed VAF data, the TP53 Y220C mutation had a median VAF of 35% (1-96%). Seventy-eight patients (67%) had additional co-mutations; 12 patients (10%) had only additional TP53 mutations and the remaining 66 patients (57%) had additional variants involving genes other than TP53 (56 patients with known somatic mutations and 10 patients with variants of uncertain significance -VUS). With a median follow-up of 49 months, the median OS of patients with TP53 Y220C mutations was 10 months, recognizing that the treatments received were variable based on diagnosis, age, and comorbidities among other variables. Median OS was 6.7, 9.3 and 60 months for AML, MDS and CLL patients, respectively. Additional outcomes data will be forthcoming. Conclusions: TP53 Y220C mutation is present in hematologic diseases, primarily in myeloid neoplasms (MDS and AML) and with higher frequency in therapy-related disease. Targeted therapies for this specific TP53 mutation would be of interest in this difficult to treat patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Vκ*MYC Mouse Model Recapitulates Key Genomic Defining Events in Multiple Myeloma

Background: The Vk*MYC transgenic model of multiple myeloma (MM) is based on the sporadic AID-induced activation of MYC in a single germinal center (GC) B-cell, in a mouse strain (C57Bl6) that spontaneously develops monoclonal gammopathy. With age, Vk*MYC mice universally develop a progressive expansion of class-switched, somatically hypermutated, monoclonal plasma cells. Myeloma defining events occur only after long latency, suggesting that additional mutations are required to induce malignancy. Here, we interrogated the genomic landscape of Vk*MYC MM to capture somatic mutations selected during progression. Methods: We interrogated whole genome (WGS) and exome sequencing (WES) from 55 purified plasma cell (PC) from Vκ*MYC transgenic mice tumors. Additional 27 and 12 Vκ*MYC mice were investigated with array comparative genomic hybridization (aCGH) and mate-pair low coverage WGS, respectively. Our analysis included 34 individual de novo tumors from aged Vk*MYC mice, 37 individual tumors from recipient mice transplanted with tumor cell lines established from de novo donors, and 13 tumors selected to propagate in vitro. Results: Tumor mutational burden was lowest in de novo samples (median 2.3 mutations/Mb, range 0.3-4.3 mutations/Mb) and highest in in vitro samples (median 5.8 mutations/Mb, range 1.2-12.7 mutations/Mb), similar to that of human MGUS and MM, respectively. To identify genes affected by nonsynonymous mutations more frequently than expected by chance (i.e., driver genes under positive selection), we ran dndscv. A total of 11 significant driver genes were detected, 7 of which are known driver genes in human MM: Dusp2, Trp53, Nfkbia, Pim1, Tent5c (Fam46c), Hist1h1c, Hist1h1e. Driver genes not previously described in MM were: Apobec3, Pten, Cd74, Cd79b. Additional nonsynonymous mutations were detected in driver genes frequently altered in MM including Kras, Nras, Dis3, as well as histone modifiers and NF-kB regulators. Differential mutation analysis revealed bi-allelic inactivation of Trp53 and Pten were more frequent in in vitro samples compared to de novo and transplanted tumor, in line with the almost universal p53 inactivation reported in human MM cell lines. As in human hyperdiploid MM, GISTIC and molecular time analyses revealed evidence of simultaneous multi-gain events of chromosomes 1, 9, 11, and 15. As reported previously, we observed frequent deletion of chromosome 5. There were numerous recurrent focal CNAs involving oncodrivers. These included focal deletion of tumor suppressors genes Cdkn2a, Cdkn2b, Cdh11, Ncor1, Rb1, Trp53, Kdm6a and Pten, as well as amplification of oncogenes Mdm4, Mcl1, and Kras. We detected several structural variants and complex events throughout the Vκ*MYC genome, similar to human MM. Importantly, we found chromothripsis in five (14%) cases. Additionally, in three Vκ*MYC mice we detected chromosomal translocations involving the immunoglobulin heavy or light chain loci that partnered and induced overexpression of MM associated genes such as Irf4, Map3k14 and Pou2af1. Overall, mutations activating the NF-kB and STAT3 pathways were the most common events, each present in more than half MM. Overall, the genomic landscape of the Vκ*MYC model resembles hyperdiploid MM in humans, with MYC translocations and enrichment of events affecting the NF-κB pathway. Finally, analysis of mutational signatures uncovered similar signatures present in human MM, including aging, APOBEC, and poly-eta in the GC, among others. In particular, APOBEC mutational activity was detected in more than half of Vκ*MYC MM, further supporting the similarities with human MM where APOBEC mutational activity is detectable in 80-90% of human MM and progressive precursor, but it is undetectable in the stable precursor conditions. Conclusions: Overall, these data reveal a progressive accumulation of genomic events from de novo, to transplant, to in vitro tumors that mimic the natural progression from newly diagnosed, to relapsed disease, to cell lines noted in human MM. Thus, our results highlight how the transformation pathways to malignant PC are conserved between mice and men. Given the long lead time between precursor diagnosis and malignant transformation to MM that limit the ability to investigate the earliest phases of tumorigenesis in human MM, the Vκ*MYC transgenic mouse model of human MM represents a unique opportunity to overcome this temporal limitation.

LGR6-dependent conditional inactivation of E-cadherin and p53 leads to invasive skin and mammary carcinomas in mice

Tissue-specific inactivation of E-cadherin combined with tumor suppressor loss leads to invasive and metastatic cancers in mice. While epidermal E-cadherin loss in mice induces squamous cell carcinomas, inactivation of E-cadherin in the mammary gland leads to invasive lobular carcinoma. To further explore the carcinogenic consequences of cell-cell adhesion loss in these compartments, we developed a new conditional mouse model inactivating E-cadherin (Cdh1) and p53 (Trp53) simultaneously in cells expressing the leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6), a putative epithelial stem cell marker in the skin and alveolar progenitor marker in the mammary gland.Compound Lgr6-CreERT2;Cdh1F;Trp53F female mice containing either heterozygous or homozygous Cdh1F alleles were bred, and Lgr6-driven Cre expression was activated in pre-puberal mice using tamoxifen. We observed that 41% of the mice (16/39) developed mostly invasive squamous-type skin carcinomas, but also a non-lobular mammary tumor was formed. In contrast to previous K14cre or WAPcre E-cadherin and p53 compound models, no significant differences were detected in the tumor-free survival of Lgr6-CreERT2 heterozygous Cdh1F/WT;Trp53F/F versus homozygous Cdh1F/F;Trp53F/F mice (778 versus 754 days, p=0.5). One Cdh1F homozygous mouse presented with lung metastasis that originated from a non-lobular and ERα negative invasive mammary gland carcinoma with squamous metaplasia. In total, 2/8 (25%) Cdh1F heterozygous and 3/12 (25%) Cdh1F homozygous mice developed metastases to lungs, liver, lymph nodes, or the gastro-intestinal tract.In conclusion, we show that inducible and conditional Lgr6-driven inactivation of E-cadherin and p53 in mice causes squamous cell carcinomas of the skin in approximately 40% of the mice and an occasional ductal-type mammary carcinoma after long latency periods.

HPV-18 E7 Interacts with Elk-1 Leading to Elevation of the Transcriptional Activity of Elk-1 in Cervical Cancer.

The human papillomavirus (HPV)-18 E7 (E7) oncoprotein is a major transforming protein that is thought to be involved in the development of cervical cancer. It is well-known that E7 stimulates tumour development by inactivating pRb. However, this alone cannot explain the various characteristics acquired by HPV infection. Therefore, we examined other molecules that could help explain the acquired cancer properties during E7-induced cancer development. Using the yeast two-hybrid (Y2H) method, we found that the Elk-1 factor, which is crucial for cell proliferation, invasion, cell survival, anti-apoptotic activity, and cancer development, binds to the E7. By determining which part of E7 binds to which domain of Elk-1 using the Y2H method, it was found that CR2 and CR3 of the E7 and parts 1-206, including the ETS-DNA domain of Elk-1, interact with each other. As a result of their interaction, the transcriptional activity of Elk-1 was increased, thereby increasing the expression of target genes EGR-1, c-fos, and E2F. Additionally, the colony forming assay revealed that overexpression of Elk-1 and E7 promotes C33A cell proliferation. We expect that the discovery of a novel E7 function as an Elk-1 activator could help explain whether the E7 has novel oncogenic activities in addition to p53 inactivation. We also expect that it will offer new methods for developing improved strategies for cervical cancer treatment.

Molecular Targeting of the Most Functionally Complex Gene in Precision Oncology: p53.

While chemotherapy is a key treatment strategy for many solid tumors, it is rarely curative, and most tumor cells eventually become resistant. Because of this, there is an unmet need to develop systemic treatments that capitalize on the unique mutational landscape of each patient's tumor. The most frequently mutated protein in cancer, p53, has a role in nearly all cancer subtypes and tumorigenesis stages and therefore is one of the most promising molecular targets for cancer treatment. Unfortunately, drugs targeting p53 have seen little clinical success despite promising preclinical data. Most of these drug compounds target specific aspects of p53 inactivation, such as through inhibiting negative regulation by the mouse double minute (MDM) family of proteins. These treatment strategies fail to address cancer cells' adaptation mechanisms and ignore the impact that p53 loss has on the entire p53 network. However, recent gene therapy successes show that targeting the p53 network and cellular dysfunction caused by p53 inactivation is now possible and may soon translate into successful clinical responses. In this review, we discuss p53 signaling complexities in cancer that have hindered the development and use of p53-targeted drugs. We also describe several current therapeutics reporting promising preclinical and clinical results.

Identifying Potential p53‐MDM2 Interaction Antagonists: An Integrated Approach of Pharmacophore‐Based Virtual Screening, Interaction Fingerprinting, MD Simulation and DFT Studies

AbstractThe inactivation of p53 in tumor cells is as a result of the deletion or mutation of the TP3 gene, while the inhibition of its activity is by interaction with overexpressed mouse double minute 2 (MDM2). Small‐molecule inhibitors that target the interaction between p53 and MDM2 are now being pursued as potential therapeutic agents for cancer therapy. To date, many small‐molecule inhibitors of the p53‐MDM2 interaction have been identified which, however, possess toxicity, bioavailability and/or drug resistance limitations. In this study, we directed a ligand‐based pharmacophore modeling protocol towards the search of novel inhibitors of the p53‐MDM2 interaction. Generated pharmacophore features from known and novel inhibitors of the p53‐MDM2 interaction were used in searching the ZINC database for drug‐like molecules. With stringent search parameters set, we generated a ligand library of 7000 ZINC compounds and a sub‐library of 907 compounds from the Selleckchem's “Apoptosis Compound Library”. Upon the docking of both compound libraries to the target receptor, the resulting top 100 binders were subjected to in silico ADMET screening, out of which the top 3 compounds with the most favorable properties (ZINC71932671, ZINC02375540 and ZINC08870467) were selected for further assessment. These 3 hits also displayed favorable interactions with important residues involved in the p53‐MDM2 protein‐protein interaction, while the molecular dynamics simulation study suggest a stable conformation for the protein‐ligand complexes. These three compounds can be used in the development of potential cancer therapeutics that are targeted at restoring the function of p53 by blocking its interaction with MDM2.

Defect in Ser312 phosphorylation of Tp53 dysregulates lipid metabolism for fatty accumulation and fatty liver susceptibility: Revealed by lipidomics

The Tp53 gene is a well-known tumour suppressor, mutation of which (e.g. prevention of Ser312 phosphorylation) induces deletion or expression of an inactive p53 protein to increase the susceptibility of tumour occurance. However, the role of Tp53 gene in maintaining metabolic homeostasis for regulating physio-pathological activities is still not well-understood. This study aimed to use the lipidomics study as a systematic approach to understand the relationship between the phenotypic effects of Tp53 mutation on lipid-related endogenous metabolites. Plasma and liver samples from mice carrying a Tp53 Ser312 to Ala mutation and wild type mice were collected, lipids were extracted by liquid–liquid extraction method and analyzed by the RPLC-LTQ-FTMS for the lipidomics study. Our results indicated that defect in Ser312 phosphorylation of Tp53 leads the lipid disturbance (e.g. triacylglycerols) for fatty accumulation and fatty liver susceptibility, which is with preference of females. Histological observation by staining with haematoxylin and eosin further validated our lipidomics findings. To our conclusion, fatty liver occurrence may have different phenotypes, one of which is strongly linked with the Tp53 mutation and is susceptible in females. Lipidomics as a technique to detect a great number of endogenous compounds provides precise metabolic information that may further help improve personalized diagnosis of Chronic hepatic diseases.

77P Targeted compounds for modulation of mutant p53 activity

The study of the role of oncosuppressors in tumor transformations and in the death of tumor cells is an important aspect in many areas of biochemistry. One of the most well-known tumor suppressors is the p53 protein. The function of the p53 protein is to regulate the expression of genes whose products lead to cell cycle arrest and apoptosis. Many types of cancer are associated with inactivation of the tumor suppressor p53 as a result of mutation. The p53Y220C oncogenic mutation it creates an extended surface pocket in the DNA-binding domain.

78P Small molecule compounds targeting the p53 mutants

The tumor suppressor p53 is inactivated by mutation in about half of all tumors, making mutant p53 a prime target for cancer therapy. Missense mutations R248W, R273H and R248Q are included in the five most frequent p53 mutations and in total lead to more than 630 000 new diagnosed cases of tumor diseases in the world every year. These mutations, located at or near the protein-DNA interface, lead to p53 inactivation by loss of direct p53-DNA interactions, and by causing conformational changes in the protein, resulting in lowering its stability.

FBXW7 inactivation induces cellular senescence via accumulation of p53

F-box and WD repeat domain containing 7 (FBXW7) acts as a substrate receptor of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase and plays crucial roles in the regulation of several cellular processes, including cell growth, division, and differentiation, by targeting diverse key regulators for degradation. However, its role in regulating cellular senescence remains elusive. Here, we found that FBXW7 inactivation by siRNA-based knockdown or CRISPR/Cas9-based knockout induced significant cellular senescence in p53 wild-type cells, but not in p53 mutant or null cells, along with activation of both the p53/p21 and p16INK4a/Rb pathways. Simultaneous p53 inactivation abrogated senescence and cell growth arrest induced by FBXW7 deficiency as well as the alteration of both the p53/p21 and p16INK4a/Rb pathways. Moreover, Fbxw7 deletion accelerated replicative senescence of primary mouse embryonic fibroblasts in a p53-dependent manner. In addition, FBXW7 deletion induced the senescence-associated secretory phenotype to trigger secondary senescence. Importantly, in a radiation-induced senescence mouse model, simultaneous deletion of p53 rescued accelerated senescence and aging caused by Fbxw7 loss. Thus, our study uncovered a novel role for FBXW7 in the regulation of senescence by eliminating p53.

Small-molecule MX-C2/3 suppresses non-small cell lung cancer progression via p53 activation

p53 inactivation is a common feature in non-small cell lung cancer (NSCLC) resulting in NSCLC malignant transformation. Targeting serine 392 phosphorylation to restore p53 anticancer activity has proven to be an effective therapeutic strategy against NSCLC. A synthetic p53 activator, NA-17, has been developed that shows promise in preclinical models of NSCLC. However, NA-17 exhibits limited therapeutic efficacy in oncogene-driven tumors as well as relatively high toxicity to normal cells. It is possible that high efficiency and low toxicity p53 activators can be obtained by optimizing the leading molecule. Here, we performed high-throughput screening of compounds optimized based on NA-17 to identify new p53 activators. Two promising candidates named MX-C2 and MX-C3 were identified, both exhibited considerable therapeutic efficacy in oncogene-driven tumor models. Similar to NA-17, MX-C2/3 induced p53 activation via phosphorylating serine-392 without DNA damage. Both compounds showed broad antitumor activity in NSCLC cells and limited toxicity in normal cell lines. Moreover, MX-C2/3 suppressed tumor progression by arresting the cell cycle at G2/M phase, exhibiting a different mechanism of cell cycle arrest than NA-17. In addition, MX-C2/3 promoted the enrichment of p-p53 (s392) in mitochondria, leading to the conformational activation of Bak for cell apoptosis, which is consistent with NA-17. Finally, we demonstrated that MX-C2 significantly inhibited tumor growth without obvious systemic toxicity in oncogene-driven HCC-827 xenograft models. Collectively, we report two p53 activators with high-efficiency and low-toxicity that target p53 serine 392 phosphorylation for anticancer translational investigation.

P53 positively regulates the proliferation of hepatic progenitor cells promoted by laminin-521

Hepatic progenitor cells (HPCs) hold tremendous potential for liver regeneration, but their well-known limitation of proliferation hampers their broader use. There is evidence that laminin is required for the proliferation of HPCs, but the laminin isoform that plays the dominant role and the key intracellular downstream targets that mediate the regulation of HPC proliferation have yet to be determined. Here we showed that p53 expression increased gradually and reached maximal levels around 8 days when laminin α4, α5, β2, β1, and γ1 subunit levels also reached a maximum during HPC activation and expansion. Laminin-521 (LN-521) promoted greater proliferation of HPCs than do laminin, matrigel or other laminin isoforms. Inactivation of p53 by PFT-α or Ad-p53V143A inhibited the promotion of proliferation by LN-521. Further complementary MRI and bioluminescence imaging analysis showed that p53 inactivation decreased the proliferation of transplanted HPCs in vivo. p53 was activated by LN-521 through the Integrin α6β1/FAK-Src-Paxillin/Akt axis. Activated p53 was involved in the nuclear translocation of CDK4 and inactivation of Rb by inducing p27Kip1. Taken together, this study identifies LN-521 as an ideal candidate substrate for HPC culture and uncovers an unexpected positive role for p53 in regulating proliferation of HPCs, which makes it a potential target for HPC-based regenerative medicine.

New Advances in Supportive Care: Chemoprotective Agents as Novel Opportunities in Geriatric Oncology.

To explore the effectiveness of trilaciclib and ALRN-6924 in the prevention of cancer chemotherapy-induced toxicity in older patients. New chemoprotective agents are necessary because age is the main risk factor for chemotherapy complications that account largely for the poorer outcome of cancer in the elderly. Trilaciclib and ALRN-6924 cause a reversible block of the proliferation of normal cells through cell cycle arrest (CCA). With this mechanism, they may prevent the toxicity of cycle-active cancer treatment including neutropenia, anemia, thrombocytopenia, lymphopenia, mucositis, and alopecia. Myelopoietic growth factors may prevent neutropenia in the aged, but they may cause severe bone pain, may aggravate thrombocytopenia and anemia, and may cause myelodysplasia and acute leukemia as a late complication. The prevention of thrombocytopenia, anemia, mucositis, and alopecia is unsatisfactory at present. These complications may jeopardize the treatment outcome as they require a reduction of treatment dose/intensity and because many patients find the resulting symptoms intolerable. In three studies of patients with extensive disease small cell lung cancer (ES-SCLC), trilaciclib reduced the severity and duration of neutropenia and thrombocytopenia as well as the need for blood transfusions. In addition, it produced a significant expansion of T-cell clones. Trilaciclib received FDA approval for the prevention of chemotherapy-induced myelosuppression in patients with ES-SCLC. ALRN-6924 is currently studied in phase II study of ES-SCLC. In a phase IB of 38 patients, ALRN-6924 prevented myelosuppression to an extent comparable with trilaciclib. Both drugs proved as effective in patients 65 and older as they were in the younger ones. In an "ex vivo" study, ALRN-6924 protected the epithelial stem cells of hair follicles from taxanes and promised to prevent alopecia. The possibility that CCA of tumor cells may reduce the effectiveness of cycle-active chemotherapy is a major concern. For this reason, the use of trilaciclib, an inhibitor of CDK 4/6, should be limited to tumors with inactivated RB1, and the use of ALRN-6924, an inhibitor of P53, should be limited to tumors with inactivated P53. Chemotherapy-related toxicities limit dose intensity and contribute to significant morbidity and mortality in elderly cancer patients. Trilaciclib and ALRN-6924 are of particular interest to geriatric oncologists because of their novel mechanism of action. Ameliorating chemotherapy-induced toxicities holds the promise of transforming the practice of geriatric oncology by enabling chemotherapeutic regimens that are currently not feasible for this patient population. Specifically, these agents may prevent chemotherapy-induced neutropenia and thrombocytopenia, perhaps the most life-threatening complications of cytotoxic chemotherapy, thereby obviating the need for the use of rescue strategies such as hematopoietic growth factors. In addition, these agents offer the potential for broad tissue protection from other chemotherapy-related toxicities, including mucositis, diarrhea, and alopecia, which historically have been poorly managed. Importantly, by preventing a spectrum of chemotherapy-related toxicities, these agents may permit the administration of chemotherapy at full-dose intensity, prevent functional decline, and grant maintenance of resilience to older cancer patients. As a result, the successful prevention of chemotherapy-induced side effects may not only mitigate the costs of care but also improve patient outcomes and quality of life. Finally, chemoprotective strategies offer the opportunity to apply geriatric principles to clinical trials of cancer treatment. In particular, they may allow the testing of prolongation of "active life expectancy" as a major goal of clinical trials in elderly patients. They may also enable novel and more practical forms of clinical trials. By assessing the risk of chemotherapy-related toxicity with the Chemotherapy Risk Assessment Scale for High Age Patients (CRASH) or the Cancer and Aging Research Group (CARG) instruments, these agents may permit researchers to utilize patients as their own controls and endorse the approval of supportive care drugs based upon the risk profile of individual patients.

Ordered and deterministic cancer genome evolution after p53 loss

Although p53 inactivation promotes genomic instability1 and presents a route to malignancy for more than half of all human cancers2,3, the patterns through which heterogenous TP53 (encoding human p53) mutant genomes emerge and influence tumorigenesis remain poorly understood. Here, in a mouse model of pancreatic ductal adenocarcinoma that reports sporadic p53 loss of heterozygosity before cancer onset, we find that malignant properties enabled by p53 inactivation are acquired through a predictable pattern of genome evolution. Single-cell sequencing and in situ genotyping of cells from the point of p53 inactivation through progression to frank cancer reveal that this deterministic behaviour involves four sequential phases—Trp53 (encoding mouse p53) loss of heterozygosity, accumulation of deletions, genome doubling, and the emergence of gains and amplifications—each associated with specific histological stages across the premalignant and malignant spectrum. Despite rampant heterogeneity, the deletion events that follow p53 inactivation target functionally relevant pathways that can shape genomic evolution and remain fixed as homogenous events in diverse malignant populations. Thus, loss of p53—the ‘guardian of the genome’—is not merely a gateway to genetic chaos but, rather, can enable deterministic patterns of genome evolution that may point to new strategies for the treatment of TP53-mutant tumours.

A case series of androgen receptor (AR) inhibition by bicalutamide in combination with leuprolide acetate and exemestane in recurrent AR positive adult-type ovarian granulosa cell tumor (AGCT) (158)

Objectives: There remains a need to develop targeted therapies for recurrent AGCT that produce measurable tumor responses. The FOXL2C134W mutation is identified in nearly all AGCT. In a genetically engineered mouse model utilized by Cluzet et al., tumorigenesis was associated with the inactivation of p53 and Rb pathways, increased circulating androgens, estradiol, and antimullerian hormone along with decreased FOXL2 abundance [1]. By examining the pathways involved in granulosa cell development, including limitations on GnRH-induced apoptosis, aromatase induction, and increased androgen expression, a novel treatment regimen is proposed for recurrent AGCT: bicalutamide (androgen receptor antagonist), exemestane (aromatase inhibitor), and leuprolide acetate (gonadotropin-releasing hormone receptor agonist) to block excess steroidogenesis seen in AGCT. Methods: Six patients identified at our institution with recurrent AGCT and tumor AR positive (by Leica IHC-AR27 antibody) were treated with bicalutamide 50mg PO once daily, Exemestane 25mg PO once daily, and Leuprolide acetate 3.75mg IM every four weeks. All patients were heavily pre-treated, with two to seven prior lines of therapy and had undergone two to seven surgeries. Prior treatment lines consisted of prior chemotherapy, hormonal therapy, and targeted therapies, including bevacizumab and everolimus. Patients were monitored for toxicities and for response to treatment via surveillance exams, inhibin B levels and CT scans. Results: Based on RECIST v1.1 criteria, of the six patients receiving bicalutamide, exemestane, and leuprolide acetate, two patients had a partial response, four patients had stable disease and one patient had progressive disease. Strong AR expression in 95% of cells was identified in the tumors of patients who had a partial response. This drug combination has been well-tolerated. There has been one hospitalization for disease-related complications; no other grade 3 or higher toxicities reported thus far. Conclusions: Based on the response of our institutional cohort and the rationale behind the antiandrogen/aromatase inhibitor/GnRH agonist combination in recurrent AGCT, this regimen of bicalutamide/exemestane/leuprolide acetate is a promising treatment. An NRG concept has been submitted to proceed with a single-arm, multicenter phase II study of this regimen in patients who meet the criteria of recurrent AGCT, AR expression, measurable disease, and one prior line of therapy. Objectives: There remains a need to develop targeted therapies for recurrent AGCT that produce measurable tumor responses. The FOXL2C134W mutation is identified in nearly all AGCT. In a genetically engineered mouse model utilized by Cluzet et al., tumorigenesis was associated with the inactivation of p53 and Rb pathways, increased circulating androgens, estradiol, and antimullerian hormone along with decreased FOXL2 abundance [1]. By examining the pathways involved in granulosa cell development, including limitations on GnRH-induced apoptosis, aromatase induction, and increased androgen expression, a novel treatment regimen is proposed for recurrent AGCT: bicalutamide (androgen receptor antagonist), exemestane (aromatase inhibitor), and leuprolide acetate (gonadotropin-releasing hormone receptor agonist) to block excess steroidogenesis seen in AGCT. Methods: Six patients identified at our institution with recurrent AGCT and tumor AR positive (by Leica IHC-AR27 antibody) were treated with bicalutamide 50mg PO once daily, Exemestane 25mg PO once daily, and Leuprolide acetate 3.75mg IM every four weeks. All patients were heavily pre-treated, with two to seven prior lines of therapy and had undergone two to seven surgeries. Prior treatment lines consisted of prior chemotherapy, hormonal therapy, and targeted therapies, including bevacizumab and everolimus. Patients were monitored for toxicities and for response to treatment via surveillance exams, inhibin B levels and CT scans. Results: Based on RECIST v1.1 criteria, of the six patients receiving bicalutamide, exemestane, and leuprolide acetate, two patients had a partial response, four patients had stable disease and one patient had progressive disease. Strong AR expression in 95% of cells was identified in the tumors of patients who had a partial response. This drug combination has been well-tolerated. There has been one hospitalization for disease-related complications; no other grade 3 or higher toxicities reported thus far. Conclusions: Based on the response of our institutional cohort and the rationale behind the antiandrogen/aromatase inhibitor/GnRH agonist combination in recurrent AGCT, this regimen of bicalutamide/exemestane/leuprolide acetate is a promising treatment. An NRG concept has been submitted to proceed with a single-arm, multicenter phase II study of this regimen in patients who meet the criteria of recurrent AGCT, AR expression, measurable disease, and one prior line of therapy.

Validation of a novel model of ovarian carcinogenesis: Digital spatial profiling of premalignant fallopian tube lesions in the MADM mouse and the potential impact on primary prevention of epithelial ovarian cancer (124)

Validation of a novel model of ovarian carcinogenesis: Digital spatial profiling of premalignant fallopian tube lesions in the MADM mouse and the potential impact on primary prevention of epithelial ovarian cancer (124)