Oncogenic P53 Research Articles

Anticancer therapeutic strategies for targeting mutant p53-Y220C.

The tumor suppressor p53 is a transcription factor with a powerful antitumor activity that is controlled by its negative regulator murine double minute 2 (MDM2, also termed HDM2 in humans) through a feedback mechanism. At the same time, TP53 is the most frequently mutated gene in human cancers. Mutant p53 proteins lose wild-type p53 tumor suppression functions but acquire new oncogenic properties, among which are deregulating cell proliferation, increasing chemoresistance, disrupting tissue architecture, and promoting migration, invasion and metastasis as well as several other pro-oncogenic activities. The oncogenic p53 mutation Y220C creates an extended surface crevice in the DNA-binding domain destabilizing p53 and causing its denaturation and aggregation. This cavity accommodates stabilizing small molecules that have therapeutic values. The development of suitable small-molecule stabilizers is one of the therapeutic strategies for reactivating the Y220C mutant protein. In this review, we summarize approaches that target p53-Y220C, including reactivating this mutation with small molecules that bind Y220C to the hydrophobic pocket and developing immunotherapies as the goal for the near future, which target tumor cells that express the p53-Y220C neoantigen.

DNAJA1- and conformational mutant p53-dependent inhibition of cancer cell migration by a novel compound identified through a virtual screen

Cancers are frequently addicted to oncogenic missense mutant p53 (mutp53). DNAJA1, a member of heat shock protein 40 (HSP40), also known as J-domain proteins (JDPs), plays a crucial role in the stabilization and oncogenic activity of misfolded or conformational mutp53 by binding to and preventing mutp53 from proteasomal degradation. However, strategies to deplete mutp53 are not well-established, and no HSP40/JDPs inhibitors are clinically available. To identify compounds that bind to DNAJA1 and induce mutp53 degradation, we performed an in silico docking study of ~10 million of compounds from the ZINC database for the J-domain of DNAJA1. A compound 7-3 was identified, and its analogue A11 effectively reduced the levels of DNAJA1 and conformational mutp53 with minimal effects on the levels of wild-type p53 and DNA-contact mutp53. A11 suppressed migration and filopodia formation in a manner dependent on DNAJA1 and conformational mutp53. A mutant DNAJA1 with alanine mutations at predicted amino acids (tyrosine 7, lysine 44, and glutamine 47) failed to bind to A11. Cells expressing the mutant DNAJA1 became insensitive to A11-mediated depletion of DNAJA1 and mutp53 as well as A11-mediated inhibition of cell migration. Thus, A11 is the first HSP40/JDP inhibitor that has not been previously characterized for depleting DNAJA1 and subsequently conformational mutp53, leading to inhibition of cancer cell migration. A11 can be exploited for a novel treatment against cancers expressing conformational mutp53.

Discovery of Nanomolar-Affinity Pharmacological Chaperones Stabilizing the Oncogenic p53 Mutant Y220C.

The tumor suppressor protein p53 is inactivated in the majority of human cancers and remains a prime target for developing new drugs to reactivate its tumor suppressing activity for anticancer therapies. The oncogenic p53 mutant Y220C accounts for approximately 125,000 new cancer cases per annum and is one of the most prevalent p53 mutants overall. It harbors a narrow, mutationally induced pocket at the surface of the DNA-binding domain that destabilizes p53, leading to its rapid denaturation and aggregation. Here, we present the structure-guided development of high-affinity small molecules stabilizing p53-Y220C in vitro, along with the synthetic routes developed in the process, in vitro structure-activity relationship data, and confirmation of their binding mode by protein X-ray crystallography. We disclose two new chemical probes displaying sub-micromolar binding affinity in vitro, marking an important milestone since the discovery of the first small-molecule ligand of Y220C in 2008. New chemical probe JC744 displayed a K d = 320 nM, along with potent in vitro protein stabilization. This study, therefore, represents a significant advance toward high-affinity Y220C ligands for clinical evaluation.

76P Modulation of mutant p53 activity using indazole derivatives

Oncogenic mutation p53 Y220C is the ninth most common mutation of p53 protein. The Y220C mutation creates an expanded surface pocket in the DNA-binding domain, rapidly unfolds and denatures under physiological conditions, which effectively cancels p53 signaling and leads to the development of a tumor. In this work the novel indazole derived compounds were tested. It was found that these molecules bind to the mutated p53 protein and stabilize its structure in vitro.

The C-terminus of Gain-of-Function Mutant p53 R273H Is Required for Association with PARP1 and Poly-ADP-Ribose.

The C-terminal region of mtp53 confers GOF activity that mediates mtp53-PARP1 and PAR interactions assisting DNA replication, thus implicating new biomarkers for PARP inhibitor therapy.

HPV16-E6/E7 Oncogene Mutation and p53 Expression among Indonesian Women with Cervical Cancer.

To characterize HPV16 E6/E7 mutation and its association with p53 expression among Indonesian women with cervical cancer. This is a cross-sectional study involving 31 Indonesian women with pathologically proven cervical cancer and HPV16 infection. Data about the clinical characteristics of the study population were obtained from the medical records. Biopsy specimen of the cervical cancer mass from each study participant was obtained for DNA isolation. The ORFs of E6 and E7 genes were amplified using specific primer designed according to K02718/HPV16R gene sequence obtained from GenBank. Sequencing was performed using software program MEGA10. HPV16 E6 and E7 prototype sequences for nucleotide alignment (HPv16. P, GenBank Access code: NC_001526) was selected from European variant. The sequence of nucleotide and amino acid was aligned using software program BioEdit. p53 expression was evaluated through immunohistochemistry and quantified using immunoreactivity score (IRS). Twelve subjects (38.7%) present with E6 and E7 mutation. Median age, parity, stage and histologic type of the tumour did not associate with E6/E7 mutation. E6 and E7 mutation rate was 25.8% (8/31) and 12.9% (4/31), respectively. Seven single nucleotide changes were identified within the E6 and E7 oncogenes, including four non-synonymous and three synonymous mutations. E6 T27C was the most prevalent mutation (16.1%). Nonsynonymous mutations were more prevalent within E7 gene (9.6%) (N29T, N29S, and R77C). Median IRS did not differ between HPV16-E6/E7 variants and wildtype (p value = 0.990). There was no association between E6/E7 mutations and p53 expression in Indonesian women with cervical cancer (PR 1.4, 95% CI: 0.29-6.77, p value = 0.704). HPV16 E6 mutation was more prevalent than E7 mutation among Indonesian women. There was no association between E6/E7 mutation and p53 expression level.

Abstract 422: Novel approach to destabilization of oncogenic mutant p53 and therapeutic implications

Abstract Introduction: Mutant p53 drives several hallmarks of cancer through a gain-of-function oncogenic program. The oncogenicity of mutant p53 includes sustained proliferation, increased survival and resistance to therapy, increased invasion and metastasis, and genomic instability. The oncogenicity of mutant p53 is related to its stability and accumulation in cells by evading degradation by the proteasome. Therefore, destabilization of mutant p53 has been sought as a therapeutic strategy, but so far without clinical success. We and others observe that proteasome inhibitors cause paradoxical depletion of mutant p53 in lung cancer cell lines. Objectives: In this project, we sought to determine the mechanism(s) of mutant p53 depletion by proteasome inhibition and its therapeutic implications in lung cancer. Methods: Human lung cancer cell lines bearing mutant p53 and wildtype p53 (WTp53) were used in this study. An isogenic panel of cell lines with mutant p53, WTp53 and a vector control was also used to determine mutant p53 dependency of the observed effects. Western blotting, immunoprecipitation, siRNA technology, DNA transfection were used for protein detection, protein-protein interactions, gene knock-down and overexpression respectively. Cycloheximide chase assay was used to determine protein degradation. The cancer genome atlas (TCGA) analysis was performed using the cBioPortal platform. Results: Proteasome inhibitors resulted in depletion of mutant p53 in the lung cancer cell line H1975. This effect was also confirmed using the isogenic model described above. Depletion of mutant p53 occurred through a degradation mechanism and was time dependent. While WTp53 accumulated at initial timepoints of proteasome inhibition, mutant p53 did not accumulate but instead depleted with time. This suggested that the proteasome is not the primary mechanism for mutant p53, in contrast to WTp53. Since autophagy is an alternative mechanism of protein degradation in eukaryotes, we evaluated for, and found evidence of autophagy upon proteasome inhibition. We further showed that autophagic degradation of mutant p53 could involve the heat shock protein 70. Our TCGA data analysis showed proteasome genes amplification in mutant p53 lung cancers, suggesting a possible functional dependency. Furthermore, RNA sequencing analysis showed upregulation of most proteasome genes upon stable expression of the R273H mutant in p53-null lung cancer cell line. Finally, in vitro cell viability assays using two different proteasome inhibitors showed increased sensitivity of the lung cancer cell line with mutant p53 compared to WTp53. Conclusion: The cumulative evidence of proteasome hyperactivity, degradation of mutant p53 and in vitro evidence of increased susceptibility of mutant p53 lung cancer cell lines suggests that proteasome inhibitors warrant a closer attention for therapeutic targeting of mutant p53 in lung cancer. Citation Format: Eziafa I. Oduah, Larisa Litovchick, Steven R. Grossman. Novel approach to destabilization of oncogenic mutant p53 and therapeutic implications [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 422.

Abstract 143: Novel HSP40 or J domain protein inhibitors that deplete misfolded mutant p53

Abstract Background: Accumulation of oncogenic mutant p53 (mutp53) greatly contributes to cancer progression. Heat shock protein 40 (HSP40), also known as J-domain proteins (JDPs), has been implicated in stabilization of misfolded mutp53. Specifically, knockdown of DNAJA1, a member of HSP40/JDPs, results in degradation of mainly misfolded mutp53, leading to inhibition of tumor progression. Since no DNAJA1 inhibitors are currently available in clinics, these findings prompted us to identify potential DNAJA1 inhibitors which can induce mutp53 degradation. Methods/Design: An in-silico docking study was performed for binding of the DNAJA1 J-domain to compounds in zinc data base, which identified compound Number7-3. Further analysis suggests three critical amino acids in DNAJA1 for the binding, including Y7, K44, and Q47. Results: Eighteen commercially available analogs of Number7-3 were further examined. Of these, a compound, namely A11, had the strongest activity to deplete both DNAJA1 and misfolded mutp53. A11 reduced protein levels of misfolded mutp53 in concentration- and time-dependent manners without affecting mRNA levels of DNAJA1 and mutp53. Importantly, A11 showed minimal effects on the levels of wild-type p53 and DNA contact mutp53. Treatment of cells expressing misfolded mutp53 with A11 resulted in reduced filopodia formation and cell migration, which was not observed in cells lacking DNAJA1. Moreover, protein levels of a triple mutant DNAJA1 (Y7A/K44A/Q47A) were not altered by A11. Intriguingly, A11 reduced protein levels of several other HSP40/JDPs. Conclusions: Our study, for the first time, has identified a small compound that depletes DNAJA1, leading to reduced levels of misfolded mutp53 and inhibited malignant properties of cancer cells. Citation Format: Tomoo Iwakuma, Shigeto Nishikawa, Atsushi Kaida, Atul Ranjan, Alejandro Parrales, Mohamed A. Alalem, David K. Johnson. Novel HSP40 or J domain protein inhibitors that deplete misfolded mutant p53 [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 143.

Abstract 1826: Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human cancers

Abstract Background: Disulfiram (DSF) in combination with copper pills has emerged as a safe and exciting anticancer strategy and is undergoing clinical trials in multiple human cancers. However, DSF breaks down in the gut or blood immediately and chances of cupric diethyldithiocarbamate (Cu-DDC), the active cytotoxic principle being formed at tumor sites is very low. Therefore, we have found that synthetic Cu-DDC is directly and potently cytotoxic against p53 mutant cancers. We report here for the first time the differential effects of Cu-DDC in robustly eliminating the mutant p53 proteins from tumor cells in contrast to its augmentation of the wild-type p53 protein and activation. Destabilization of Gain Of Function (GOF) p53 mutant proteins for overcoming oncogenic addiction and improving the therapeutic efficacy has been a long-sought, but unmet anticancer avenue. Methods: A panel of 7 P53 mutant cell lines (R248W, R273H,R173H), and p53 wt tumor and normal cells (HPBLs, dental pulp stem cells, IMR90 and HUVEC) were used. Immunoprecipitation, Western blotting, Annexin-V apoptotic assays, flow cytometry, immunocytochemistry were used to quantitate p53 protein. Mia-Paca2 xenografts developed in nude mice were administered Cu-DDC/BSA. Results: Cu-DDC generated peroxides but not superoxides in tumor cells. The GOF mutant p53 proteins, R248W and R273H in three different tumor cell lines were eliminated after Cu-DDC treatment in a time- and concentration-dependent manner. The loss of mutant p53 proteins was confirmed by three different procedures. The degradation occurred rapidly in 8-10 h at 100 nM Cu-DDC and was inhibited by proteasome blockers. The expression of the GRO-α oncogene, positively regulated by GOF R248W was decreased in Cu-DDC treated Mia-Paca2 cells. Cellular proteasome activity was not inhibited by Cu-DDC. Ubiquitination of mutant p53 proteins was not altered by Cu-DDC. In contrast, the wild-type p53 present in HCT116 and MCF-7 cells, and normal cell counterparts (HPBL, IMR-90 lung cells) was significantly upregulated and activated with enhanced p21cip1 expression. Cu-DDC triggered strong apoptosis irrespective of the p53 status, although the mutant p53 containing cells were 2 to 3-fold more sensitive in cytotoxicity assays (IC50 range 0.25-0.66 µM). A significant tumor growth delay accompanied by loss of mutant p53 protein was observed in MiaPaca2 xenografts. Conclusions: In moving the DSF anticancer repurposing forward, we describe the unique ability of Cu-DDC to rapidly eliminate oncogenic p53 mutant proteins and confer potent antitumor efficacy. The degradation was unrelated to the proteostasis involving the inactivation of NPL4 by Cu-DDC described previously (supported by a Cancer Prevention and Research Institute of Texas [CPRIT] grant RP170207 to KSS). Citation Format: Viswanath Arutla, Kalkunte S. Srivenugopal. Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human cancers [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 1826.

Caspase 9b Drives Cellular Transformation, Lung Inflammation, and Lung Tumorigenesis.

Our findings show that C9b can directly activate NF-κB pathway in vivo to modulate lung inflammation, immune cell influx, and peripheral immune responses, which demonstrates that C9b is key factor in driving cell transformation and lung tumorigenesis.

Impedimetric sensing platform for human papillomavirus and p53 tumor suppressor gene in cervical samples

The oncogenic human papillomavirus (HPV) is an excellent biomarker for cervical cancer screening. The present research reports the construction of an integrated sensor platform (biosensor MY11 and biosensor p53 ) for the impedimetric detection of the HPV L1 sequence and p53 tumor suppressor gene. The nanostructured biosensor is composed of electrosynthesized polypyrrole (PPy) and gold nanoparticles (AuNPs). Methodological strategies based on cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), and atomic force microscopy (AFM) were used. The exposure of the genetic biosensors to molecular targets triggered changes in the analytical signals. Calibration plots for the biosensor MY11 showed a linear range between 0.001 to 100 pg μL −1 for all analyzed genotypes (HPV6, 11, 16, 18, 45, 53, 54, and 58). The detection limits were measured between 0.11 to 0.61 pg μL −1 and limits of quantification between 0.33 to 1.86 pg μL −1 . The sensors exhibited reproducibility with a relative standard deviation of 4.21% and high repeatability (less than 2.5%). Two electrochemical profiles were obtained for high and low-risk HPV groups, suggesting the differential diagnosis using a degenerate oligonucleotide probe. Our results allow correlating the p53 gene expression in clinical samples infected with papillomavirus. The proposed biosensor represents an innovative device for the integrated monitoring of biomarkers related to cervical cancer with ultra-sensitive analytical performance. • A nanointegrated system was designed for the differential diagnosis of papillomavirus. • Polypyrrole conjugated to gold particles acted as an ultrasensitive signal transducer. • High and low-risk HPV groups were identified using a degenerate oligonucleotide probe. • Molecular screening for oncogenic HPV and p53 gene was performed in clinical samples. • The biosensor showed high specificity with a linear range from 0.001 to 100 pg μL −1 .

Identifying pathways regulating the oncogenic p53 family member \u0394Np63 provides therapeutic avenues for squamous cell carcinoma

BackgroundΔNp63 overexpression is a common event in squamous cell carcinoma (SCC) that contributes to tumorigenesis, making ΔNp63 a potential target for therapy.MethodsWe created inducible TP63-shRNA cells to study the effects of p63-depletion in SCC cell lines and non-malignant HaCaT keratinocytes. DNA damaging agents, growth factors, signaling pathway inhibitors, histone deacetylase inhibitors, and metabolism-modifying drugs were also investigated for their ability to influence ΔNp63 protein and mRNA levels.ResultsHaCaT keratinocytes, FaDu and SCC-25 cells express high levels of ΔNp63. HaCaT and FaDu inducible TP63-shRNA cells showed reduced proliferation after p63 depletion, with greater effects on FaDu than HaCaT cells, compatible with oncogene addiction in SCC. Genotoxic insults and histone deacetylase inhibitors variably reduced ΔNp63 levels in keratinocytes and SCC cells. Growth factors that regulate proliferation/survival of squamous cells (IGF-1, EGF, amphiregulin, KGF, and HGF) and PI3K, mTOR, MAPK/ERK or EGFR inhibitors showed lesser and inconsistent effects, with dual inhibition of PI3K and mTOR or EGFR inhibition selectively reducing ΔNp63 levels in HaCaT cells. In contrast, the antihyperlipidemic drug lovastatin selectively increased ΔNp63 in HaCaT cells.ConclusionsThese data confirm that ΔNp63-positive SCC cells require p63 for continued growth and provide proof of concept that p63 reduction is a therapeutic option for these tumors. Investigations of ΔNp63 regulation identified agent-specific and cell-specific pathways. In particular, dual inhibition of the PI3K and mTOR pathways reduced ΔNp63 more effectively than single pathway inhibition, and broad-spectrum histone deacetylase inhibitors showed a time-dependent biphasic response, with high level downregulation at the transcriptional level within 24 h. In addition to furthering our understanding of ΔNp63 regulation in squamous cells, these data identify novel drug combinations that may be useful for p63-based therapy of SCC.

New Benzimidazole-, 1,2,4-Triazole-, and 1,3,5-Triazine-Based Derivatives as Potential EGFRWT and EGFRT790M Inhibitors: Microwave-Assisted Synthesis, Anticancer Evaluation, and Molecular Docking Study.

A new series of benzimidazole, 1,2,4-triazole, and 1,3,5-triazine derivatives were designed and synthesized using a microwave irradiation synthetic approach utilizing 2-phenylacetyl isothiocyanate (1) as a key starting material. All the new analogues were evaluated as anticancer agents against a panel of cancer cell lines utilizing doxorubicin as a standard drug. Most of the tested derivatives exhibited selective cytotoxic activity against MCF-7 and A-549 cancer cell lines. Furthermore, the new target compounds 5, 6, and 7 as the most potent antiproliferative agents have been assessed as in vitro EGFRWT and EGFRT790M inhibitors compared to the reference drugs erlotinib and AZD9291. They represented more potent suppression activity against the mutated EGFRT790M than the wild-type EGFRWT. Moreover, the compounds 5, 6, and 7 down-regulated the oncogenic parameter p53 ubiquitination. A docking simulation of compound 6b was carried out to correlate its molecular structure with its significant EGFR inhibition potency and its possible binding interactions within the active site of EGFRWT and the mutant EGFRT790M.

IASPP suppresses Gp78-mediated TMCO1 degradation to maintain Ca2+ homeostasis and control tumor growth and drug resistance

Ca2+ release from the endoplasmic reticulum (ER) is an essential event in the modulation of Ca2+ homeostasis, which is coordinated by multiple biological processes, ranging from cell proliferation to apoptosis. Deregulated Ca2+ homeostasis is linked with various cancer hallmarks; thus, uncovering the mechanisms underlying Ca2+ homeostasis dynamics may lead to new anticancer treatment strategies. Here, we demonstrate that a reported Ca2+-channel protein TMCO1 (transmembrane and coiled-coil domains 1) is overexpressed in colon cancer tissues at protein levels but not at messenger RNA levels in colon cancer. Further study revealed that TMCO1 is a substrate of ER-associated degradation E3 ligase Gp78. Intriguingly, Gp78-mediated TMCO1 degradation at K186 is under the control of the iASPP (inhibitor of apoptosis-stimulating protein of p53) oncogene. Mechanistically, iASPP robustly reduces ER Ca2+ stores, mainly by competitively binding with Gp78 and interfering with Gp78-mediated TMCO1 degradation. A positive correlation between iASPP and TMCO1 proteins is further validated in human colon tissues. Inhibition of iASPP-TMCO1 axis promotes cytosolic Ca2+ overload-induced apoptotic cell death, reducing tumor growth both invitro and invivo. Thus, iASPP-TMCO1 represents a promising anticancer treatment target by modulating Ca2+ homeostasis.

T cell receptors employ diverse strategies to target a p53 cancer neoantigen

Adoptive cell therapy with tumor-specific T cells can mediate durable cancer regression. The prime target of tumor-specific T cells are neoantigens arising from mutations in self-proteins during malignant transformation. To understand T cell recognition of cancer neoantigens at the atomic level, we studied oligoclonal T cell receptors (TCRs) that recognize a neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by the major histocompatibility complex class I molecule HLA-A2. We previously reported the structures of three p53R175H-specific TCRs (38-10, 12-6, and 1a2) bound to p53R175H and HLA-A2. The structures showed that these TCRs discriminate between WT and mutant p53 by forming extensive interactions with the R175H mutation. Here, we report the structure of a fourth p53R175H-specific TCR (6-11) in complex with p53R175H and HLA-A2. In contrast to 38-10, 12-6, and 1a2, TCR 6-11 makes no direct contacts with the R175H mutation, yet is still able to distinguish mutant from WT p53. Structure-based in silico mutagenesis revealed that the 60-fold loss in 6-11 binding affinity for WT p53 compared to p53R175H is mainly due to the higher energetic cost of desolvating R175 in the WT p53 peptide during complex formation than H175 in the mutant. This indirect strategy for preferential neoantigen recognition by 6-11 is fundamentally different from the direct strategies employed by other TCRs and highlights the multiplicity of solutions to recognizing p53R175H with sufficient selectivity to mediate T cell killing of tumor but not normal cells.

Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

Tumor suppressor p53, a critical regulator of cell fate, is frequently mutated in cancer. Mutation of p53 abolishes its tumor-suppressing functions or endows oncogenic functions. We recently found that p53 binds via its proline-rich domain to peptidase D (PEPD) and is activated when the binding is disrupted. The proline-rich domain in p53 is rarely mutated. Here, we show that oncogenic p53 mutants closely resemble p53 in PEPD binding but are transformed into tumor suppressors, rather than activated as oncoproteins, when their binding to PEPD is disrupted by PEPD knockdown. Once freed from PEPD, p53 mutants undergo multiple posttranslational modifications, especially lysine 373 acetylation, which cause them to refold and regain tumor suppressor activities that are typically displayed by p53. The reactivated p53 mutants strongly inhibit cancer cell growth in vitro and in vivo. Our study identifies a cellular mechanism for reactivation of the tumor suppressor functions of oncogenic p53 mutants.

Structural basis of reactivation of oncogenic p53 mutants by a small molecule: methylene quinuclidinone (MQ)

In response to genotoxic stress, the tumor suppressor p53 acts as a transcription factor by regulating the expression of genes critical for cancer prevention. Mutations in the gene encoding p53 are associated with cancer development. PRIMA-1 and eprenetapopt (APR-246/PRIMA-1MET) are small molecules that are converted into the biologically active compound, methylene quinuclidinone (MQ), shown to reactivate mutant p53 by binding covalently to cysteine residues. Here, we investigate the structural basis of mutant p53 reactivation by MQ based on a series of high-resolution crystal structures of cancer-related and wild-type p53 core domains bound to MQ in their free state and in complexes with their DNA response elements. Our data demonstrate that MQ binds to several cysteine residues located at the surface of the core domain. The structures reveal a large diversity in MQ interaction modes that stabilize p53 and its complexes with DNA, leading to a common global effect that is pertinent to the restoration of non-functional p53 proteins.

Abstract 2417: Mutant p53 and oncogenic KRAS converge on CREB1 to drive pancreatic cancer metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most p53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis. However, the extent of direct cooperation or synergism between KRAS effectors and mutant p53 remain undefined. To study how KRAS effectors and mutant p53 cooperate to drive PDAC development, we developed a novel somatic model that expresses mutant KRAS and p53 only in pancreas tumor cells, leaving the tumor microenvironment and immune system intact. PDAC genetically engineered mouse models (GEMMs) were generated by crossing a conditional oncogenic KRasG12D (K) allele with a conditional null p53 allele (Pfl) or a novel, conditional knock-in mutant p53R172H allele (Pwm) that selectively expresses mutant p53 in tumor cells while preserving wildtype p53 expression in all other animal cells. The incidence of metastatic lesions in the liver and lungs of KPwmC mice was over 2-fold greater (p=.03) relative to KPflC mice. Through orthogonal analyses of this model and human PDAC samples, we show that oncogenic KRAS effectors phosphorylate and activate cyclic AMP responsive element binding protein 1 (CREB1), allowing physical interactions with mutant p53 to transcriptionally upregulate the pro-metastatic transcription factor, FOXA1, which enhances β-catenin stabilization and activity. Targeting KRAS and mutant p53 cooperativity through pharmacologic inhibition of CREB1 dramatically dampens FOXA1 expression and PDAC metastasis. Our findings demonstrate a direct, mechanistic link between key oncogenic KRAS signaling elements and mutant p53 that result in a broad, multiplexed activation of cancer-associated transcriptional networks. Moreover, we identify CREB1 as a viable therapeutic strategy to undermine oncogenic KRAS and mutant p53 cooperation to mitigate PDAC metastasis. Citation Format: Michael Paul Kim, Xinqun Li, Jenying Deng, Yun Zhang, Bingbing Dai, Kendra Allton, Tara Hughes, Christian Siangco, Jithesh Augustine, Yaan Kang, Joy M. McDaniel, Shunbin Xiong, Eugene Koay, Florencia McAllister, Christopher Bristow, Timothy Heffernan, Anirban Maitra, Bin Liu, Michelle Barton, Amanda Wasylischen, Jason Fleming, Guillermina lozano. Mutant p53 and oncogenic KRAS converge on CREB1 to drive pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2417.

Abstract 2487: disrupting binding of p53 mutants to PEPD unleashes their tumor suppressor activities

Abstract Background & Goals: The p53 tumor suppressor is one of the most important determinants of cell fate and is critical for tumor suppression. It functions by regulating the expression of a plethora of genes via sequence-specific DNA binding and also by transcription-independent, mitochondria-mediated mechanisms. Not surprisingly, the TP53 gene, which encodes p53, is frequently mutated in human cancer. For example, TP53 is mutated in about 75% of HER2-positive breast cancer and about 85% of triple negative breast cancer. The majority of TP53 mutations are missense mutations, changing one amino acid per mutant, most of which occur in its DNA-binding domain. p53 mutants show loss of tumor suppressor functions or gain of oncogenic functions. We recently found that peptidase D (PEPD), also known as prolidase, binds via its C-terminal sequence to the proline-rich domain (PRD) of wild type p53 and that disrupting the binding activates p53. The enzymatic activity of PEPD is not required for this function. Our objective in the present study is to determine whether PEPD also regulates p53 mutants. We focused on some of the most common oncogenic p53 mutants, including R175H, R248Q, R273H and R280K. Results: Our study shows that 1) each p53 mutant binds directly to PEPD and binds to PEPD in cells in a significant quantity, 2) disrupting their binding in cells by PEPD knockdown reactivates each p53 mutant, unleashing both transcription-dependent and -independent tumor suppressing activities, 3) after leaving PEPD, p53 mutants undergo acetylation, mono-ubiquitination and phosphorylation, which drive their refolding and reactivation, 4) Lysine 373 acetylation is essential for their reactivation, and 5) p53 mutants and p53WT show almost no difference in PEPD-binding and response to PEPD KD. Conclusions & Therapeutic Relevance: In conclusion, we have identified a cellular mechanism by which the tumor suppressor functions of p53 mutants are strongly reactivated, which is neither cell-specific nor mutant-specific. Mainly, p53 mutants bind to PEPD in cancer cells, and disrupting the complex causes posttranslational modifications of mutants freed from PEPD, which drive their refolding and unleash their tumor suppressor activities. This discovery advances our understanding of the function and regulation of p53 mutants and breaks new ground for developing novel cancer treatment strategies. Citation Format: Lu Yang. disrupting binding of p53 mutants to PEPD unleashes their tumor suppressor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2487.

Breast cancer after IVF: Can ovary stimulation and follicular response affect prognostic factors?

e12538 Background: The ovary stimulation and the follicular response is related with estradiol level. Study in breast cancer patients after IVF if ovarian response or number of IVF cycles affects the prognostic factors. Methods: Patients with breast cancer who underwent IVF are studied the prognostic factors (Ki67, HER2, estrogen receptor (ER), progesterone receptor (PR), oncogene p53, histologic grade) in relation to the ovary response and number of IVF cycles. Results: 73 patients with breast cancer after IVF are studied. They performed 135 cycles of IVF, 36 (49’3%) with 1 IVF and 37 (50’7%) with more than one IVF. Hyper response was present in at least one IVF in 24 (32.9%) patients and there was no hyper response in any IVF in 49 (67.1%) patients. The prognostic factors were: Ki 67> 20 in 31'91% (15/47) Ki 67 <20 in 68'08% (32/47), HER2 + 31'94% (23/72) HER2- 68'05% (49/72), p53 + 45'09% (23/51), p53-54'90% (28/51), HG II-III 56'36% (31/55), HG I 43'63% ( 24/55), RE + 87'5% (63/72), RE- 12'5% (9/72), RP + 76'38% (55/72), RP- 23'61% (17/72). None of prognostic factors varied with the ovary response (hyper response in at least one IVF cycle, normal response, normal or low response) (p=ns). The only prognostic factor that varied with the IVF number was p53 +. Patients with p53 + (23/51), 7 (30’43%) has one IVF, and 16 (69’53%) have more one IVF (p<0’05). Conclusions: In breast cancer after IVF, the ovary stimulation and the follicular response not affect Ki67, HER2, estrogen receptor, progesterone receptor, p53, and histologic grade. p53 positive is more frequent in patients with more than one IVF.