P53 Tumor Suppressor Function Research Articles

Abstract NG02: Distinct genomic and immunologic tumor evolution in germline TP53-driven breast cancers

Abstract NG02: Distinct genomic and immunologic tumor evolution in germline TP53-driven breast cancers

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 5682: Micro RNA level changes in prostate cancer cells treated with MDMX and MDM2 inhibitors

Abstract INTRODUCTION: Prostate Cancer (PCa) is the most common non-cutaneous cancer and the second leading cause of cancer death among men in the United States. Most of the PCa cases are diagnosed in men between the ages of 65-74. Due to the recent push for frequent prostate screenings, these cancers are being detected early and treated much faster, leading to a 5-year survival of a localized or regional PCa to >99%. However, the 5-year survival of PCa with distant metastasis is significantly reduced to 32%, especially with involvement of osteoblastic bone lesions, lung lesions, and brain lesions. Therefore, there is a clinical need to identify prognostic markers and new targets for developing therapies and effectively treating advanced PCa. Previous studies have shown that levels of MDMX and MDM2, oncogenic inhibitors of p53 tumor suppressor protein, are significantly elevated in PCa cells. Inhibition of MDM2 and MDMX using specific inhibitors has been shown to elevate the levels of p21, p27, and p53 in LNCaP (Lymph Node Carcinoma of the Prostate) cells leading to cell death. Recent studies have demonstrated that micro RNAs (miRNAs) play an important role in supporting the tumor suppressor function of p53 through regulating the balance between p53 and MDM2. In this regard, several miRNAs were found to be significantly altered to modulate the level and intracellular mechanisms of p53 and MDM2 in various cancers. OBJECTIVES: To determine the relative levels of miRNA31, miRNA-34a and miRNA-128 in LNCaP cells that were treated with NSC-207895 (MDMX inhibitor), SJ-172550 (MDMX inhibitor), and RG-7388 (MDM2 inhibitor). METHODS: LNCaP cells were treated with NSC-207895 (20 uM), SJ-172550 (20 uM) or RG-7388 (2 uM) for 24 hrs. After completing the treatments, the miRNAs were extracted from the LNCaP cells using the miRNeasy Tissue/Cells Advanced Kit from Qiagen. We analyzed the levels of miRNA-31, miRNA-34a, and miRNA-128 in the treated samples using qRT-PCR analysis method and compared the levels to the control. During the qRT-PCR analysis the cDNA was amplified using Meridian Biosciences qPCR Master Mix and specific primers for miRNAs from Origene were used according to the manufacturer's protocol. RESULTS: Our experimental results indicate that inhibition of MDMX using NSC-207895 and SJ-172550 can elevate the levels of p21, p27, and p53 leading to cell death. Determination of cell death markers in the past revealed induction of necroptosis following drug treatments. Our current results indicate measurable changes in the levels of miRNA-31 and miRNA-34a following inhibition of MDMX using SJ-172550. We are in the process of determining the possible link between the changes in the levels of miRNAs and cell death. ACKNOWLEDGEMENT: This research was supported by the Royal Dames of Cancer Research, Ft. Lauderdale, Florida. Citation Format: Rojin Fatirkhorani, Ahmed Saleh Alsarrani, Shyam Sundar Jaganathan, Umamaheswari Natarajan, Appu Rathinavelu. Micro RNA level changes in prostate cancer cells treated with MDMX and MDM2 inhibitors [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 5682.

Abstract 640: In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations

Abstract p53 tumor suppressor mutations result in loss of function of p53 in approximately 50% of all human cancers.TP53 missense, nonsense mutations and p53 deletion are present in about 50%, 12% and 5% of all p53 driven cancers, respectively. To date there is no effective treatment for rescuing p53 function in cancers using a pan-p53 approach. The use of ADGN-531 is a new potent strategy combining mRNA with a tumor selective peptide nanocarrier to rescue of p53 tumor suppressor function. ADGN-531 contains proprietary p53-mRNA complexed with proprietary short amphipathic peptides in the form of nanoparticles. ADGN-531 was evaluated on 20 different cancer cell lines harboring various p53 mutations. In-vivo efficacy of IV-administered ADGN-531 nanoparticles was evaluated on colorectal SW403 (p53 nonsense/KRASG12V), osteosarcoma SaOs2 (p53 null) and NSCLC NCI-H358 (p53 null/KRASG12C) mouse xenografts at a dose of 0.25-1.0 mg/kg weekly. ADGN-531/sotorasib synergy was evaluated in vitro on NCI-H358 (p53 null/KRASG12C) and Mia-PACA (p53R48W/KRASG12C) cells and in vivo on NCI-H358 mouse xenograft. ADGN-531 markedly inhibits the growth of a large panel of cancer cells harboring p53-missense or nonsense mutations or p53-deletion, by rescuing p53 transcriptional activity and subsequent expression of its target proteins. ADGN-531 induces cell cycle arrest in G1 due to p21 upregulation and apoptosis following BAX and PUMA activation. IV-administration of ADGN-531 resulted in dose responsive tumor growth inhibition in SaOs2, SW403 and H358 xenografts independent of KRAS status. We demonstrated a strong synergy in a NSCLC H358 xenograft (tumor regression 80%) when restoring P53 function with ADGN-531 (0.5mg/kg, weekly) along with inhibition of KRASG12C mutant using sotorasib (10mg/kg, daily). ADGN-531 potentiated the effect of sotorasib on sotorasib resistant cells harboring high level of KRASG12C in permanent active state. ADGN-531 treatments are well tolerated, no sign of clinical toxicity was detected after single or repeated administrations. ADGN-531 is effective in rescuing p53 function both in vitro and in vivo across a wide range of p53 alterations. Our study provides a proof-of-concept that restoration of tumor suppressor function by ADGN-531 could be used as a single agent therapy in P53 altered tumors independent of other driver mutations e.g., KRAS, or with other therapies for potent combinatorial cancer treatment. Citation Format: Gilles Divita, Audrey Grunenberger, Elodie Czuba, Veronique Josserand, Melanie Guidetti, Neil Desai. In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations [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 640.

Abstract 3027: p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas

Abstract High-Grade Serous Ovarian Cancer (HGSOC) is the most common and aggressive gynecological malignancy. It is typically diagnosed in late stages due to lack of diagnostic tests and current therapies available are not efficacious when the tumor is advanced. HGSOC is characterized by ubiquitous loss of functional p53 tumor suppressor protein function, largely due to point mutations that arise very early in carcinogenesis. P53 mutations are detected in pre-neoplastic cells of the fallopian tube mucosa, called p53 signature, and in early precursor lesions called serous tubal intraepithelial carcinoma (STIC). Some mutations promote the misfolding and aggregation of p53, transforming the protein into a cancer-promoting oncogene. Here, we hypothesize that the transition from folded, soluble to aggregated mutant p53 underlies the transformation from a pre-neoplastic lesion (p53 signature, STILs), to STICs and eventually HGSOC. We first set to determine the proportion of cells carrying soluble vs misfolded mutant p53 in fallopian tube tissues collected from n=10 patients undergoing salpingo-oophorectomy. We performed conformation-sensitive staining and quantification of folded and unfolded p53 cases and demonstrated that p53 misfolding, a mandatory precursor to aggregation, is present in STICs and HGSOCs, but notably absent from p53 signatures and surrounding healthy tissue. To better characterize the role of aggregated p53 in the progression of HGSOC, we took advantage of primary human fallopian tube secretory epithelial cells (FTSEC) that mimic early and more advanced stages of carcinogenesis. We utilize the FTSEC cells line FT282, immortalized by introducing hTERT and the aggregation-prone mutant TP53R175H that is found in p53 signature lesions and STILs, as well as the FT282-CCNE1, derived from FT282 cell line with overexpression of CCNE1, representing a more malignant phenotype.We used these models to investigate p53 aggregation, characterize the mutant p53 interactome and test the efficacy of ReACp53, a p53 aggregation-targeting peptide we developed. Our results demonstrated that p53 aggregation is more severe in the FT282-CCNE1 line, with a higher susceptibility to ReACp53 compared to the FT282 cells. Overall, our results demonstrated that aggregation of mutant p53 is a structural defect that can distinguish between pre-malignant and malignant HGSOC lesions and can potentially be targeted, offering a potential window for early intervention in halting ovarian cancer progression. Citation Format: Sara Sartini, Lexi Omholt, Neda A. Moatamed, Alice Soragni. p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas [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 3027.

P53 exerts anticancer effects by regulating enhancer formation and activity.

The abnormality of p53 tumor suppressor is crucial in lung cancer development, and p53 may regulate target gene promoters to combat cancer. Recent studies have shown extensive p53 binding to enhancer elements. However, whether p53 exerts a tumor suppressor role by shaping the enhancer landscape remains poorly understood. In the current study, we employed several functional genomics approaches to assess the enhancer activity at p53 binding sites throughout the genome based on our established TP53 knockout human bronchial epithelial cells (BEAS-2B). A total of 943 active regular enhancers and 370 super-enhancers (SEs) disappeared upon the deletion of p53, indicating that p53 modulates the activity of hundreds of enhancer elements. We found that one p53-dependent SE, located on chromosome 9 and designated as KLF4-SE, regulated the expression of the Krüppel-like factor 4 ( KLF4) gene. Furthermore, deletion of p53 significantly decreased the KLF4-SE enhancer activity and the KLF4 expression, but increased colony formation ability in the nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced cell transformation model. Subsequently, in TP53 knockout cells, the overexpression of KLF4 partially reversed the increased clonogenic capacity caused by p53 deficiency. Consistently, KLF4 expression also decreased in lung cancer tissues and cell lines. Overexpression of KLF4 significantly suppressed lung cancer cell proliferation and migration. Collectively, our results suggest that the regulation of enhancer formation and activity by p53 is an integral component of the p53 tumor suppressor function. Therefore, our findings offer novel insights into the regulation mechanism of p53 in lung oncogenesis and introduce a new strategy for screening therapeutic targets.

The influence of a modified p53 C-terminal peptide by using a tumor-targeting sequence on cellular apoptosis and tumor treatment.

The restoration of the function of p53 in tumors is a therapeutic strategy for the highly frequent mutation of the TP53 tumor suppressor gene. P460 is a wild-type peptide derived from the p53 C-terminus and has been proven to be capable of restoring the tumor suppressor function of p53. The poor accumulation of drugs in tumors is a serious hindrance to tumor treatment. For enhancing the activity of P460, the tumor-targeting sequence Arg-Gly-Asp-Arg (RGDR, C-end rule peptide) was introduced into the C-terminus of P460 to generate the new peptide P462. P462 presented better activity than P460 in inhibiting the proliferation of cancer cells and increasing the number of tumor cells undergoing apoptosis. Cell adhesion analysis and tumor imaging results revealed that P462 showed more specific and extensive binding with tumor cells and greater accumulation in tumors than the wild-type peptide. Importantly, treatment with P462 was more efficacious than that with P460 in vivo and was associated with considerably improved tumor-homing activity. This study highlights the importance of the roles of the tumor-homing sequence RGDR in the enhancement in cell attachment and tumor accumulation. The results of this work indicate that P462 could be a novel drug candidate for tumor treatment.

Abstract PR04: Deciphering radiotherapy resistance mechanisms in HPV negative and positive head and neck cancers

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. The prognosis of HNSCC is poor with a 5-year survival rate for all stages around 55%. HNSCC can be classified into two subtypes, human papilloma virus (HPV)-negative, caused mainly by tobacco use, and HPV-positive subtype. Radiotherapy is one of the cornerstones of the treatment of HNSCC. However, radioresistance is common and is associated with a high risk of recurrence. Radioresistance may be pre-existing, or acquired, and may be direct (tumor-cell autonomous) or indirect, mediated by RT-mediated changes to the TME such as cellular and/or extracellular matrix (ECM) changes to the stroma. These pathways are not fully understood. Better understanding of the intrinsic as well as RT-induced resistance is needed to improve survival of hard-to-treat HNSCC patients. HPV-positive HNSCC have significantly better RT response and prognosis, however currently they are treated with similarly high dose RT which causes significant adverse side-effects. As the incidence of HPV-induced HNSCC is increasing very fast, the current focus is on treatment de-escalation to reduce toxicity without compromising outcome. EGFR upregulation is an established biomarker of treatment resistance and aggressiveness in head HNSCC. EGFR-targeted therapies have shown benefits for HPV-negative HNSCC; surprisingly, inhibiting EGFR in HPV-associated HNSCC led to inferior therapeutic outcomes suggesting opposing roles for EGFR in the two HNSCC subtypes. We have recently shown that EGFR activation regulates the DNA damage response pathway differently in the two HNSCC subtype. Here, we further investigated the link between EGFR with HPV-infected HNSCC particularly the regulation of HPV oncoproteins E6 and E7. We demonstrate that EGFR overexpression suppresses cellular proliferation and increases radiosensitivity of HPV-positive HNSCC cell lines. EGFR overexpression was shown to inhibit protein expression of BRD4, a known cellular transcriptional regulator of HPV E6/E7 expression and DNA damage repair facilitator. Using in vitro 2D and 3D spheroid models of HNSCC we showed that EGFR inhibition by cetuximab restored the expression of BRD4 leading to increased HPV E6 and E7 transcription. Concordantly, pharmacological inhibition of BRD4 led to suppression of HPV E6 and E7 transcription, delayed cellular proliferation and sensitised HPV-positive HNSCC cells to ionising radiation. This effect was shown to be mediated through EGFR-induced upregulation of microRNA-9-5p and consequent silencing of its target BRD4 at protein translational level, repressing HPV E6 and E7 transcription and restoring p53 tumor suppressor functions. These results suggest a novel mechanism for EGFR inhibition of HPV E6/E7 oncoprotein expression through an epigenetic pathway, mediated through microRNA-9-5p/BRD4 regulation. The results may help better future design of radiotherapy combination treatment of HPV-positive and negative HNSCC, such as combination with BRD4 inhibitors to improve HNSCC therapeutic outcome. Citation Format: Mahvash Tavassoli. Deciphering radiotherapy resistance mechanisms in HPV negative and positive head and neck cancers [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR04.

Targeting Oncogenic Mutant p53 and BCL-2 for Small Cell Lung Cancer Treatment.

Through a unique genomics and drug screening platform with ~800 solid tumor cell lines, we have found a subset of SCLC cell lines are hypersensitive to venetoclax, an FDA-approved inhibitor of BCL-2. SCLC-A (ASCL1 positive) and SCLC-P (POU2F3 positive), which make up almost 80% of SCLC, frequently express high levels of BCL-2. We found that a subset of SCLC-A and SCLC-P showed high BCL-2 expression but were venetoclax-resistant. In addition, most of these SCLC cell lines have TP53 missense mutations, which make a single amino acid change. These mutants not only lose wild-type (WT) p53 tumor suppressor functions, but also acquire novel cancer-promoting activities (oncogenic, gain-of-function). A recent study with oncogenic mutant (Onc)-p53 knock-in mouse models of SCLC suggests gain-of-function activity can attenuate chemotherapeutic efficacy. Based on these observations, we hypothesize that Onc-p53 confers venetoclax resistance and that simultaneous inhibition of BCL-2 and Onc-p53 induces synergistic anticancer activity in a subset of SCLC-A and SCLC-P. We show here that (1) down-regulation of Onc-p53 increases the expression of a BH3-only pro-apoptotic BIM and sensitizes to venetoclax in SCLC-P cells; (2) targeting Onc-p53 by the HSP90 inhibitor, ganetespib, increases BIM expression and sensitizes to venetoclax in SCLC-P and SCLC-A cells. Although there are currently many combination studies for venetoclax proposed, the concept of simultaneous targeting of BCL-2 and Onc-p53 by the combination of venetoclax and HSP90 inhibitors would be a promising approach for SCLC treatment.

Mutant p53 in head and neck squamous cell carcinoma: Molecular mechanism of gain‑of‑function and targeting therapy (Review).

Head and neck squamous cell carcinoma (HNSCC) is one of the most widespread malignancies worldwide. p53, as a transcription factor, can play its role in tumor suppression by activating the expression of numerous target genes. However, p53 is one of the most commonly mutated genes, which frequently harbors missense mutations. These missense mutations are nucleotide substitutions that result in the substitution of an amino acid in the DNA binding domain. Most p53 mutations in HNSCC are missense mutations and the mutation rate of p53 reaches 65‑85%. p53 mutation not only inhibits the tumor suppressive function of p53 but also provides novel functions to facilitate tumor recurrence, called gain‑of‑function (GOF). The present study focused on the prevalence and clinical relevance of p53 mutations in HNSCC, and further described how mutant p53 accumulates. Moreover, mutant p53 in HNSCC can interact with proteins, RNA, and exosomes to exert effects on proliferation, migration, invasion, immunosuppression, and metabolism. Finally, several treatment strategies have been proposed to abolish the tumor‑promoting function of mutant p53; these strategies include reactivation of mutant p53 into wild‑type p53, induction of mutant p53 degradation, enhancement of the synthetic lethality of mutant p53, and treatment with immunotherapy. Due to the high frequency of p53 mutations in HNSCC, a further understanding of the mechanism of mutant p53 may provide potential applications for targeted therapy in patients with HNSCC.

An iASPP‐derived short peptide restores p53‐mediated cell death in cancers with wild‐type p53

AbstractBackgroundInhibitor of apoptosis‐stimulating protein of p53 (iASPP) is an evolutionarily conserved p53 inhibitor. Mechanistically, iASPP can accelerate tumorigenesis by inhibiting the transactivation function of p53. Targeting the interaction between iASPP and p53 may be a potential therapy for restoring the activity of p53 in tumors.MethodsWe constructed an iASPP‐derived peptide, called A8, that was derived from the C‐terminus of iASPP. Here, we transfected A8 into two wild‐type (WT) p53 cell lines, U2OS and A549, and then determined the number of apoptotic cells. The mechanism by which A8 affected apoptosis was further examined by immunoprecipitation (IP), Dual‐Luciferase reporter assays, and chromatin IP assays. Real‐time polymerase chain reaction and western blots were also used to examine the expression levels of apoptosis‐related factors.ResultsOur data demonstrate that A8 can increase apoptosis rates in WT p53 cell lines. Functional analysis suggested that A8 restored the transcriptional function and DNA binding activities of p53 toward the Bax and PUMA gene promoters. Moreover, A8 reduced cell proliferation and inhibited tumor growth in xenograft nude mice.ConclusionsThese data provide a new approach for restoring the tumor suppressor function of p53 in cancer cells that express WT p53 and therefore may serve as a novel cancer treatment strategy.

NOTOS: A pivotal study of navtemadlin, a first-in-class mouse double minute 2 inhibitor (MDM2i), in patients (pts) with TP53 wild-type (TP53WT) Merkel cell carcinoma (MCC) for whom anti PD-1/L1 therapy has failed.

TPS9600 Background: There are no approved therapies for pts with MCC for whom an anti PD-1/L1 agent has failed. The high rate of non-responsive disease after anti PD-1/L1 therapy together with high mortality rate and the absence of a standard of care in this setting highlight the urgent unmet need for effective novel therapies. Approximately 80% of MCC tumors carry the oncogenic Merkel cell polyoma virus (MCPyV) and most cases are TP53WT. Oncoproteins from MCPyV inhibit p53 tumor suppressor functions by activating MDM2. Navtemadlin, a potent, selective, orally available MDM2i, overcomes MDM2 dysregulation by restoring p53 activity and inducing apoptosis of TP53WT MCC tumors. Navtemadlin is the first targeted therapy to show single-agent activity in pts with metastatic MCC for whom anti-PD-1/L1 has failed (Wong ASCO 2022). In the dose finding study, evaluable pts (n=8) receiving the recommended phase 2 dose of navtemadlin (180 mg QD on Day 1-5/28-day cycle) demonstrated a 25% confirmed objective response rate (ORR), 38% unconfirmed + confirmed ORR, and 63% disease control rate. The median duration of response was not reached (range, 6-16.2+ months [mos]) and median time to treatment response was 4.1 mos (range, 1.2-7). Responses to navtemadlin were highest in those pts with no prior chemotherapy (chemo; Wong ASCO oral 2022). In pts receiving navtemadlin at doses of ≥180 mg, an ORR of 40% was reported with no prior chemo (n=15) versus 14% in those who received prior chemo (n=14, Wong ASCO oral 2022). Navtemadlin demonstrated an acceptable safety profile with the most common treatment emergent adverse events (TEAEs) cytopenias, nausea, vomiting, diarrhea, and fatigue. Grade 3/4 TEAEs were 32% anemia, 32% lymphopenia, and 19% thrombocytopenia (Wong 2022). Based on this encouraging data, the NOTOS study (navtemadlin induces p53-driven apoptosis in Merkel cell carcinoma) will evaluate the safety and efficacy of navtemadlin in two post PD-1/L1 treated cohorts: chemo-naive pts and chemo treated pts. Methods: Adults with MCC for whom anti PD-1/L1 therapy has failed, who have ≥1 measurable lesion per RECIST v1.1, ECOG PS 0-1 and TP53WT MCC by central testing will receive 180 mg QD on days 1-5 of a 28-day cycle until disease progression, unacceptable toxicity, death or withdrawal of consent. The primary endpoint is ORR per RECIST v1.1 by blinded independent review. Secondary endpoints include ORR per investigator, duration of response, progression-free survival, overall survival, clinical benefit rate (including stable disease for ≥10 wk, partial response or complete response), and safety. The NOTOS trial is currently enrolling (NCT03787602). Clinical trial information: NCT03787602 .

Abstract LB003: BI 907828: A highly potent MDM2-p53 antagonist suitable for intermittent dose schedules

Abstract The E3 ligase MDM2 controls the tumor suppressor function of p53, which is encoded by the TP53gene. Compounds designed to bind to MDM2 preventing its interaction with p53 restore the ability of p53 to combat the pathogenic processes that underly cancer development. It has become clear from early clinical experience that thrombocytopenia represents an on-target, dose-limiting toxicity that may restrict the therapeutic utility of first-generation MDM2 inhibitors. Dosing less frequently, while maintaining efficacious exposure levels, has been proposed as an approach to mitigate side effects and improve the therapeutic window. In this -Late Breaking- session we present the discovery and preclinical evaluation of BI 907828, a highly potent, orally bioavailable and selective molecule binding to MDM2 and preventing its interaction with p53. The compound was designed to have a long half-life to enable intermittent dosing schedules in the clinic where it showed a manageable safety profile in a Phase Ia/Ib, dose-escalation/expansion study and encouraging signs of antitumor activity in patients with advanced DDLPS and WDLPS. BI 907828 is actively investigated in several clinical trials including the Phase II/III Brightline-1 study that aims to evaluate whether it is superior to doxorubicin in the first-line treatment of advanced/metastatic DDLPS. BI 907828 belongs to the class of spiro-oxindole MDM2-p53 antagonists and was discovered starting from an optimized core structure with improved chemical stability. Structure-based medicinal chemistry optimization including rigidification of the scaffold were key to accomplish the desired profile which was tested preclinically in several in vivo MDM2-amplified xenograft models where treatment with BI 907828 showed efficient tumor growth inhibition and regression. Citation Format: Andreas Gollner, Patrizia Sini, Dorothea Rudolph, Harald Weinstabl, Juergen Ramharter, Ulrike Weyer-Czernilofsky, Bojana Golubovic, Rolf Grempler, Alejandro Pérez Pitarch, Reinhard Sailer, Darryl B. McConnell, Norbert Kraut. BI 907828: A highly potent MDM2-p53 antagonist suitable for intermittent dose schedules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB003.

Abstract 2606: Requirement of MDM2 E3 ligase activity for regulating p53 during normal development, cell cycle regulation and genome integrity

Abstract p53 tumor suppressor, the most mutated gene in human cancer, is negatively regulated by MDM2 and MDM4 proteins. Two distinct mechanisms of MDM2/MDM4 regulation of p53 include, one, suppression of p53 transactivation activity by direct interaction of MDM2/MDM4 with p53 protein, thus blocking its transactivation interface and two, targeting p53 to proteasomal degradation by MDM2 E3 ligase activity. However, the importance of these mechanisms in normal p53 regulation in vivo has remained controversial. To genetically separate these two mechanisms of p53 regulation we generated a novel Mdm2 mutant mouse (Mdm2L466A) that lacks E3 ligase activity but retains the ability of Mdm2 to heterodimerize with Mdm4 and inhibit p53 transactivation. Homozygous Mdm2L466A mice are embryonic lethal due to dysregulated p53 activity, thus demonstrating Mdm2 E3 ligase mediated regulation of p53 is essential during embryonic development. Additionally, we uncovered novel p53-independent functions of MDM2 E3 ligase in cell cycle regulation and genome integrity in cells. Cells lacking MDM2 E3 ligase activity have defective G2-M transition during cell cycle and developed elevated levels of aneuploidy regardless of p53 status. This study unequivocally demonstrates the requirement of Mdm2 E3 ligase activity for normal regulation of p53 and uncovers previously unknown p53-independent role of Mdm2 in cell cycle regulation and maintaining genome integrity. A current therapeutic approach involves blocking MDM2-p53 interaction to promote p53 tumor suppression function in cancer cells. Our findings suggest that this approach alone is insufficient because MDM2 has other potential oncogenic mechanisms independent of its role as negative regulator of p53. Hence, targeting MDM2 E3 ligase activity is likely to be more effective cancer therapy as it addresses both p53-dependent and p53-independent oncogenic mechanisms of MDM2. Citation Format: Meenalakshmi Chinnam, Rati Lama, Chao Xu, Xiaojing Zhang, Carlos Cedeno, Yanqing Wang, Aimee B. Stablewski, David W. Goodrich, Xinjiang Wang. Requirement of MDM2 E3 ligase activity for regulating p53 during normal development, cell cycle regulation and genome integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2606.

Therapeutic Strategies to Activate p53.

The p53 protein has appropriately been named the "guardian of the genome". In almost all human cancers, the powerful tumor suppressor function of p53 is compromised by a variety of mechanisms, including mutations with either loss of function or gain of function and inhibition by its negative regulators MDM2 and/or MDMX. We review herein the progress made on different therapeutic strategies for targeting p53.

BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network.

Inhibition of the tumour suppressive function of p53 (encoded by TP53) is paramount for cancer development in humans. However, p53 remains unmutated in the majority of cases of glioblastoma (GBM)-the most common and deadly adult brain malignancy1,2. Thus, how p53-mediated tumour suppression is countered in TP53 wild-type (TP53WT) GBM is unknown. Here we describe a GBM-specific epigenetic mechanism in which the chromatin regulator bromodomain-containing protein 8 (BRD8) maintains H2AZ occupancy at p53 target loci through the EP400 histone acetyltransferase complex. This mechanism causes a repressive chromatin state that prevents transactivation by p53 and sustains proliferation. Notably, targeting the bromodomain of BRD8 displaces H2AZ, enhances chromatin accessibility and engages p53 transactivation. This in turn enforces cell cycle arrest and tumour suppression in TP53WT GBM. In line with these findings, BRD8 is highly expressed with H2AZ in proliferating single cells of patient-derived GBM, and is inversely correlated with CDKN1A, a canonical p53 target that encodes p21 (refs. 3,4). This work identifies BRD8 as a selective epigenetic vulnerability for a malignancy for which treatment has not improved for decades. Moreover, targeting the bromodomain of BRD8 may be a promising therapeutic strategy for patients with TP53WT GBM.

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

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

Evaluation of a Novel Oncolytic Adenovirus Silencing SYVN1

Oncolytic adenoviruses are promising new anticancer agents. To realize their full anticancer potential, they are being engineered to express therapeutic payloads. Tumor suppressor p53 function contributes to oncolytic adenovirus activity. Many cancer cells carry an intact TP53 gene but express p53 inhibitors that compromise p53 function. Therefore, we hypothesized that oncolytic adenoviruses could be made more effective by suppressing p53 inhibitors in selected cancer cells. To investigate this concept, we attenuated the expression of the established p53 inhibitor synoviolin (SYVN1) in A549 lung cancer cells by RNA interference. Silencing SYVN1 inhibited p53 degradation, thereby increasing p53 activity, and promoted adenovirus-induced A549 cell death. Based on these observations, we constructed a new oncolytic adenovirus that expresses a short hairpin RNA against SYVN1. This virus killed A549 cells more effectively in vitro and inhibited A549 xenograft tumor growth in vivo. Surprisingly, increased susceptibility to adenovirus-mediated cell killing by SYVN1 silencing was also observed in A549 TP53 knockout cells. Hence, while the mechanism of SYVN1-mediated inhibition of adenovirus replication is not fully understood, our results clearly show that RNA interference technology can be exploited to design more potent oncolytic adenoviruses.

MEX3A Mediates p53 Degradation to Suppress Ferroptosis and Facilitate Ovarian Cancer Tumorigenesis.

Degradation of p53 mediated by MEX3A drives ovarian cancer growth by circumventing p53 tumor suppressive functions, suggesting targeting MEX3A as a potential strategy for treating of ovarian cancer expressing WT p53.

EGFR-induced suppression of HPV E6/E7 is mediated by microRNA-9-5p silencing of BRD4 protein in HPV-positive head and neck squamous cell carcinoma

EGFR upregulation is an established biomarker of treatment resistance and aggressiveness in head and neck cancers (HNSCC). EGFR-targeted therapies have shown benefits for HPV-negative HNSCC; surprisingly, inhibiting EGFR in HPV-associated HNSCC led to inferior therapeutic outcomes suggesting opposing roles for EGFR in the two HNSCC subtypes. The current study aimed to understand the link between EGFR and HPV-infected HNSCC particularly the regulation of HPV oncoproteins E6 and E7. We demonstrate that EGFR overexpression suppresses cellular proliferation and increases radiosensitivity of HPV-positive HNSCC cell lines. EGFR overexpression inhibited protein expression of BRD4, a known cellular transcriptional regulator of HPV E6/E7 expression and DNA damage repair facilitator. Inhibition of EGFR by cetuximab restored the expression of BRD4 leading to increased HPV E6 and E7 transcription. Concordantly, pharmacological inhibition of BRD4 led to suppression of HPV E6 and E7 transcription, delayed cellular proliferation and sensitised HPV-positive HNSCC cells to ionising radiation. This effect was shown to be mediated through EGFR-induced upregulation of microRNA-9-5p and consequent silencing of its target BRD4 at protein translational level, repressing HPV E6 and E7 transcription and restoring p53 tumour suppressor functions. These results suggest a novel mechanism for EGFR inhibition of HPV E6/E7 oncoprotein expression through an epigenetic pathway, independent of MAPK, but mediated through microRNA-9-5p/BRD4 regulation. Therefore, targeting EGFR may not be the best course of therapy for certain cancer types including HPV-positive HNSCC, while targeting specific signalling pathways such as BRD4 could provide a better and potentially new treatment to improve HNSCC therapeutic outcome.