P53 Tumor Suppressor Function Research Articles

Targeting Apoptosis Pathways With BCL2 and MDM2 Inhibitors in Adult B-cell Acute Lymphoblastic Leukemia.

In adult patients, the treatment outcome of acute lymphoblastic leukemia (ALL) remains suboptimal. Here, we used an ex vivo drug testing platform and comprehensive molecular profiling to discover new drug candidates for B-ALL. We analyzed sensitivity of 18 primary B-ALL adult patient samples to 64 drugs in a physiological concentration range. Whole-transcriptome sequencing and publicly available expression data were used to examine gene expression biomarkers for observed drug responses. Apoptotic modulators targeting BCL2 and MDM2 were highly effective. Philadelphia chromosome–negative (Ph–) samples were sensitive to both BCL2/BCL-W/BCL-XL-targeting agent navitoclax and BCL2-selective venetoclax, whereas Ph-positive (Ph+) samples were more sensitive to navitoclax. Expression of BCL2 was downregulated and BCL-W and BCL-XL upregulated in Ph+ ALL compared with Ph– samples, providing elucidation for the observed difference in drug responses. A majority of the samples were sensitive to MDM2 inhibitor idasanutlin. The regulatory protein MDM2 suppresses the function of tumor suppressor p53, leading to impaired apoptosis. In B-ALL, the expression of MDM2 was increased compared with other hematological malignancies. In B-ALL cell lines, a combination of BCL2 and MDM2 inhibitor was synergistic. In summary, antiapoptotic proteins including BCL2 and MDM2 comprise promising targets for future drug studies in B-ALL.

Abstract P5-09-01: Using isogenic model systems to determine mechanisms regulating mutant p53 protein stability in breast cancer cells

Abstract Two of the most common events in triple-negative breast cancers (TNBC) are mutation of the tumor suppressor gene TP53 and development of aneuploidy. This is often the case in aggressive and molecularly heterogeneous cancer subtypes such as TNBC, where ~80% of cases harbor a mutation in TP53. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. Mutant p53 proteins can accumulate to high levels in tumors, and this stabilization is thought to be a pre-requisite for mutant p53 to exert its GOF effects. Mechanisms underlying the accumulation of mutant p53 are not well understood. To study mutant p53 GOF mechanisms and phenotypes, we used CRISPR/Cas9-mediated genome editing and developed two isogenic TNBC cell line models (one non-transformed and one tumor-derived) that express the most frequently occurring p53 missense mutations (R175H and R273H), are deficient for functional p53 protein (null), or retain the wild-type (WT) protein. In these engineered models, endogenous p53 expression is regulated by the native p53 promoter, thus providing a controlled system for rigorous functional experimentation across different p53 states. Through functional genomics analyses comparing isogenic TNBC cell lines, which initially differed only by the TP53 genotype, we have evaluated the relationship between mutant p53 and development of aneuploidy and determined that development of aneuploidy and not TP53 genotype contributed to several previously reported mutant p53 GOF phenotypes in vitro and in vivo1. Immunoblotting, immunofluorescence, and immunohistochemistry experiments revealed that mutant p53-expressing cell lines display a range of p53 protein levels, with several maintaining low levels of p53, similar to the cell lines which contain WT p53. In addition, mutant p53 levels correlate with aneuploidy, but not mean nuclear area or whole-genome doubling status, and protein levels increase over time in cell lines that become aneuploid. Using our various clonal cell lines that exhibit differing p53 protein stability, we are currently determining if p53 stability is dysregulated in aneuploid cell lines after copy number gain or loss of genes encoding critical p53 regulators, leading to the accumulation of the mutant protein. Analysis of cancer cell line genetic dependencies and human tumor data from The Cancer Genome Atlas (TCGA) has identified genetic and clinical correlates with increased mutant p53 protein levels. Utilizing biochemical and bioinformatic approaches, we aim to uncover mechanisms for increased mutant p53 stability and identify novel regulators of both WT and mutant p53 proteins. The dissection of mechanisms that contribute to stabilization of p53 protein will not only give insight into the regulation and tumor suppressive function of WT p53, but it has the potential for clinical translation in human cancers that have high-frequency p53 mutation and high levels of mutant p53 protein, as targeting mutant p53 through pharmacologic inhibition or antibody-based therapies is an active area of investigation. 1Article in press in Nature Communications Citation Format: Lindsay Redman-Rivera, Hailing Jin, Brian D Lehmann, Jennifer A Pietenpol. Using isogenic model systems to determine mechanisms regulating mutant p53 protein stability in breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-01.

Inhibition of triple‑negative breast cancer proliferation and motility by reactivating p53 and inhibiting overactivated Akt.

Mutations of p53 tumor suppressors occur more frequently in cancers at advanced stages or in more malignant cancer subtypes such as triple‑negative breast cancer. Thus, restoration of p53 tumor suppressor function constitutes a valuable cancer therapeutic strategy. In the present study, it was revealed that a specific inhibitor of histone deacetylase6, ACY‑1215, caused increased acetylation of p53 in breast cancer cells with mutated p53, which was accompanied by increased expression of p21. These results suggested that ACY‑1215 may lead to enhanced transcriptional activity of p53. It was also determined that ACY‑1215 treatment resulted in G1 cell cycle arrest and apoptosis in these cancer cells. Furthermore, ACY‑1215 displayed a synergistic effect with specific inhibitors of ATM, an activator of Akt, in inducing cancer cell apoptosis and inhibiting their motility. More importantly, it was observed that combination of ACY‑1215 and ATM inhibitors exhibited markedly more potent antitumor activity than the individual compound in xenograft mouse models of breast cancer with mutant p53. Collectively, our results demonstrated that ACY‑1215 is a novel chemotherapeutic agent that could restore mutant p53 function in cancer cells with strong antitumor activity, either alone or in combination with inhibitors of the ATM protein kinase.

Molecular Insights into the Antistress Potentials of Brazilian Green Propolis Extract and Its Constituent Artepillin C.

Propolis, also known as bee-glue, is a resinous substance produced by honeybees from materials collected from plants they visit. It contains mixtures of wax and bee enzymes and is used by bees as a building material in their hives and by humans for different purposes in traditional healthcare practices. Although the composition of propolis has been shown to depend on its geographic location, climatic zone, and local flora; two largely studied types of propolis: (i) New Zealand and (ii) Brazilian green propolis have been shown to possess Caffeic Acid Phenethyl Ester (CAPE) and Artepillin C (ARC) as the main bioactive constituents, respectively. We have earlier reported that CAPE and ARC possess anticancer activities, mediated by abrogation of mortalin-p53 complex and reactivation of p53 tumor suppressor function. Like CAPE, Artepillin C (ARC) and the supercritical extract of green propolis (GPSE) showed potent anticancer activity. In this study, we recruited low doses of GPSE and ARC (that did not affect either cancer cell proliferation or migration) to investigate their antistress potential using in vitro cell based assays. We report that both GPSE and ARC have the capability to disaggregate metal- and heat-induced aggregated proteins. Metal-induced aggregation of GFP was reduced by fourfold in GPSE- as well as ARC-treated cells. Similarly, whereas heat-induced misfolding of luciferase protein showed 80% loss of activity, the cells treated with either GPSE or ARC showed 60–80% recovery. Furthermore, we demonstrate their pro-hypoxia (marked by the upregulation of HIF-1α) and neuro-differentiation (marked by differentiation morphology and upregulation of expression of GFAP, β-tubulin III, and MAP2). Both GPSE and ARC also offered significant protection against oxidative stress and, hence, may be useful in the treatment of old age-related brain pathologies.

Abstract P203: Milademetan is a potent, murine double minute 2 (MDM2) inhibitor, highly active in TP53 wild-type (p53WT) Merkel cell carcinoma (MCC) cell lines

Abstract Background: MCC is a highly aggressive neuroendocrine carcinoma of the skin with a poor overall prognosis. Current treatment options include surgery and radiation therapy for local MCC tumor and checkpoint blockade therapy for advanced disease. However, primary and acquired resistance can reduce response to therapy. Around 80% of all MCCs have integrated copies of Merkel cell polyomavirus (MCV). Virus-Positive MCC (MCCP) tumors typically contain few somatic mutations and express wild type (WT) p53 (TP53). In contrast, Virus-Negative MCC (MCCN) tumors have a high mutational burden, with a predominantly UV mutational signature. MCCP express two viral proteins: MCV small T antigen (ST) and a truncated form of large T antigen (LT). The MCV ST recruits MYCL and EP400 to form the SLaP complex that specifically activates several genes contributing to oncogenesis. A direct target of the SLaP complex is MDM2, an E3 ligase for p53. In p53WT MCCP, SLaP-dependent activation of MDM2 inhibits the tumor suppressive functions of p53. Here, we analyzed the efficacy of milademetan (RAIN-32), a potent, selective, and orally available MDM2 inhibitor, in MCC. Experimental procedures: Established MCCP cell lines with WT (MKL-1, WaGa, PeTa) or mutant p53 (MS-1), were treated with vehicle or several concentrations of milademetan or another MDM2 inhibitor AMG232 and cell viability was analyzed. Similar viability assays were also performed using MKL-1 p53 knockout (KO) cells and two newly established p53WT primary MCC cell lines. The p53 response in MCC cells treated with vehicle, milademetan or AMG232 was assessed by western blot (WB) analysis of p53 and its downstream effectors p21, PUMA and PARP-1. For in vivo testing, an initial tolerability study was conducted with once daily administration of milademetan by oral gavage in NSG mice. Milademetan activity was evaluated in MKL-1 xenograft and patient-derived xenograft (PDX) models in NSG mice. Pharmacodynamic markers of response in tumor samples from mice treated with vehicle or milademetan is analyzed by q-PCR and WB analysis. Summary of Results: Nanomolar concentrations of milademetan reduced cell viability of p53WT but not p53mutantMCCP MCC cell lines. Milademetan treatment increased levels of p21, PUMA and cleaved PARP-1 in MCCP cell lines MKL-1 and WaGa. Using p53 KO MKL-1 cells, we show that the effect of milademetan on MCC cell viability is p53 dependent. In vitro data show that milademetan is more potent than AMG232 in the context of MCC. Tolerability studies show that mice safely tolerate 100 mg/kg of milademetan. Milademetan treatment in the MKL-1 xenograft tumor model shows a dose-dependent response in tumor growth inhibition. In the DFMC-33043 PDX model, milademetan significantly inhibited tumor growth. Conclusion: Milademetan is a promising drug effective against p53WT MCC cell lines, xenograft, and PDX models. Ongoing in vivo testing of the anti-cancer cell activity of milademetan will provide evidence for clinical exploration of milademetan in MCC refractory to current therapies. Citation Format: Varsha Ananthapadmanabhan, Aine Knott, Kara M. Soroko, Prafulla C. Gokhale, Vijaya Tirunagaru, Robert Doebele, James A. DeCaprio. Milademetan is a potent, murine double minute 2 (MDM2) inhibitor, highly active in TP53 wild-type (p53WT) Merkel cell carcinoma (MCC) cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P203.

TMOD-28. MYC GENERATES AGGRESSIVE MEDULLOBLASTOMA BY HSP90 PATHWAY ACTIVATION AND ARF SILENCING

Abstract Medulloblastoma, the most common malignant pediatric brain tumor, often shows amplification or overexpression of the MYC transcription factor and arises in the presence of a functional p53 tumor suppressor protein. To elucidate the mechanism behind this inexplicable tumor development we generated an inducible, immunocompetent transgenic mouse model of MYC-expressing medulloblastoma. Aggressive tumors developed clonally in the presence of an unaltered p53 gene that molecularly resembled Group 3 medulloblastoma. Compared to MYCN-expressing medulloblastoma driven from the same promoter, we instead discovered pronounced and MIZ1-independent silencing of the ARF suppressor, which was also suppressed in MYC-amplified as compared to MYCN-amplified human medulloblastoma. While MYCN-driven tumor malignancy was more sensitive to ARF depletion, it dramatically increased metastatic spread of MYC-driven tumors. DNMT inhibition could restore ARF levels in MYC-expressing tumors but did not show any therapeutic advantage in tumors in vivo. Bioinformatics analysis further showed a strong correlation of the HSP90 pathway with MYC in human Group 3 MB and in the MYC-driven mouse model. The HSP90 inhibitor Onalespib showed significant selectivity for targeting MYC-driven as compared to MYCN-driven tumors. The drug promoted ARF restoration and increased the survival in our animal model which suggests that it could be potentially used in the treatment of MYC-driven ARF-silenced brain cancer patients.

Recent Advances of WEE1 Inhibitors and Statins in Cancers With p53 Mutations.

p53 is among the most frequently mutated tumor suppressor genes given its prevalence in >50% of all human cancers. One critical tumor suppression function of p53 is to regulate transcription of downstream genes and maintain genomic stability by inducing the G1/S checkpoint in response to DNA damage. Tumor cells lacking functional p53 are defective in the G1/S checkpoint and become highly dependent on the G2/M checkpoint to maintain genomic stability and are consequently vulnerable to Wee1 inhibitors, which override the cell cycle G2/M checkpoint and induce cell death through mitotic catastrophe. In addition to the lost tumor suppression function, many mutated p53 (Mutp53) proteins acquire gain-of-function (GOF) activities as oncogenes to promote cancer progression, which manifest through aberrant expression of p53. In cancer cells with GOF Mutp53, statins can induce CHIP-mediated degradation of Mutp53 within the mevalonate pathway by blocking the interaction between mutp53 and DNAJA1. Therefore, targeting critical downstream pathways of Mutp53 provides an alternative strategy for treating cancers expressing Mutp53. In this review, we summarize recent advances with Wee1 inhibitors, statins, and mevalonate pathway inhibitors in cancers with p53 mutations.

Matrix Metalloproteinases (MMPs) in skin and oral pathology - Premalignant Diseases, Spinocellular Carcinoma and Basocellular Carcinoma (markers).

Matrix Metalloproteinases (MMPs) in skin and oral pathology - Premalignant Diseases, Spinocellular Carcinoma and Basocellular Carcinoma (markers).

Mutated p53 in HGSC-From a Common Mutation to a Target for Therapy.

Simple SummaryOvarian high-grade serous cancer (HGSC), the most common and the deadliest subtype of epithelial ovarian cancer, is characterized by frequent mutations in the TP53 tumor suppressor gene, encoding for the p53 protein in nearly 100% of cases. This makes p53 the focus of many studies trying to understand its role in HGSC. The aim of our review paper is to provide updates on the latest findings related to the role of mutant p53 in HGSC. This includes the clinical outcomes of TP53 mutations in HGSC, upstream regulators and downstream effectors of p53, its function in the earliest stages of HGSC development and in the interplay between the tumor cells and their microenvironment. We summarize with the likelihood of p53 mutants to serve as biomarkers for early diagnosis and as targets for therapy in HGSC.Mutations in tumor suppressor gene TP53, encoding for the p53 protein, are the most ubiquitous genetic variation in human ovarian HGSC, the most prevalent and lethal histologic subtype of epithelial ovarian cancer (EOC). The majority of TP53 mutations are missense mutations, leading to loss of tumor suppressive function of p53 and gain of new oncogenic functions. This review presents the clinical relevance of TP53 mutations in HGSC, elaborating on several recently identified upstream regulators of mutant p53 that control its expression and downstream target genes that mediate its roles in the disease. TP53 mutations are the earliest genetic alterations during HGSC pathogenesis, and we summarize current information related to p53 function in the pathogenesis of HGSC. The role of p53 is cell autonomous, and in the interaction between cancer cells and its microenvironment. We discuss the reduction in p53 expression levels in tumor associated fibroblasts that promotes cancer progression, and the role of mutated p53 in the interaction between the tumor and its microenvironment. Lastly, we discuss the potential of TP53 mutations to serve as diagnostic biomarkers and detail some more advanced efforts to use mutated p53 as a therapeutic target in HGSC.

TP53/NPM1-mutated acute myeloid leukemia as a molecularly distinct disease entity.

7030 Background: TP53-mutated acute myeloid leukemia (AML) is a distinct disease entity associated with a dismal prognosis. This disease group is distinguishable by its low frequency of SNVs, unremarkable transcriptional signatures, and lower leukocyte and myeloblast counts compared to TP53 wildtype disease. Response to gold-standard hypomethylating agents is typically transient. NPM1 mutations in this disease subset are rare despite the fact that NPM1c has been shown to negatively regulate the tumor suppressive functions of p53 .Methods: Bone marrow, peripheral blood, or FFPE tissue samples from 10,118 patients with suspected myeloid disease were sequenced using a dual DNA/RNA 297 gene myeloid panel. Results were validated in a separate independent dataset using a 54 gene TruSight myeloid panel (N = 2463). FISH/cytogenetic data was analyzed across myeloid disease. Patients with confirmed AML (n = 460) were included in the NGS portion of this study. Statistics were performed using Fisher’s exact test for categorical variables and two-tailed T-test for continuous variables. Results: All TP53-mutated myeloid disease (n = 1282 / 10,118) was associated with fewer co-mutations except DNMT3A (13.4%; n = 172), and complex cytogenetics (36.4%; n = 134/381). TP53+/NPM1+ status across all myeloid disease was not associated with a complex karyotype (7.6% vs 38.5%; 1/13 vs. 133/368, p = 0.02). Among AML patients, NPM1+/TP53+ patients (n = 18) were more co-mutated with DNMT3A (33.3% vs. 10.3%, P = 0.01), FLT3 (33.33% vs. 2.5%, P < 0.0001), IDH1 (27.8% vs. 4.4%, P = 0.002), IDH2 (22.2% vs. 6.4%, P = 0.03); and PTPN11 (22.2% vs. 2.5%, P = 0.003) when compared to TP53+/NPM1- patients. NPM1+/TP53+ AML had more mutations in recurrently mutated genes (4.5 vs 2.1; P < 0.0001) than TP53+/NPM1- AML. Conclusions: TP53+/NPM1+ AML harbors molecular signatures which clearly distinguish it from ordinary TP53-mutated AML, and is more reflective of de novo and NPM1+ AML. Further clinical outcome studies are needed to determine the therapeutic and prognostic implications of this subset, and whether other TP53-mutated patients can be better risk stratified.[Table: see text]

Trial in progress: A phase I/II trial of novel MDM2 inhibitor alrizomadlin (APG-115), with or without platinum chemotherapy, in patients with p53 wild-type salivary gland carcinoma.

TPS6094 Background: Salivary gland carcinoma is a rare tumor that accounts for 6% of all head and neck cancers. This histologically and anatomically heterogeneous malignant tumor type is largely resistant to platinum and other chemotherapies but commonly has wild-type TP53 based on next-generation sequencing analysis. Alrizomadlin is a novel, orally active, small molecular agent that binds to MDM2, restoring p53 tumor suppressor function and inducing apoptosis in tumor cells retaining wild-type p53. Preliminary clinical evidence suggests promising antitumor activity and a favorable safety profile for alrizomadlin in the treatment of solid tumors (Rasco 2019). Methods: This US multicenter open-label trial is evaluating alrizomadlin with or without platinum chemotherapy in adults with histologically documented wild-type TP53 salivary gland carcinoma, including primary or metastatic lesions, an ECOG performance status 0-1, and a life expectancy of at least 12 weeks. In addition, subjects need to have measurable disease by computed tomography according to RECIST v1.1, with radiographic disease progression within the prior 12 months, high-grade status with or without metastases, and/or not amenable to curative treatment. An initial randomized phase (Part 1) will be followed by a single-arm phase (Part 2). Treatment arms include a cycle length of 21 days, and the study is using a time-to-event continual reassessment method. In Part 1 (42 patient target), patients are randomly allocated (in a 1:2 ratio) to one of two arms: single-agent alrizomadlin at a starting dose of 150 mg (Arm A) or at a starting dose of 150 mg with concomitant IV carboplatin administered at starting AUC = 4.5 (Arm B). Based on overall response rate (ORR; complete or partial response after Cycle 2) and safety profile, the most promising treatment arm will be advanced to Part 2, which has a target enrollment of 20 patients. Study endpoints are (1) dose-limiting toxicity (DLT), which is defined by the rate of drug-related grade ≥ 3 adverse events (by NCI CTCAE v5.0) over the first 2 cycles (6 weeks) of study treatment; (2) maximum tolerated dose based on these DLTs; and (3) ORR by RECIST v1.1 observed at up to 12 months. As of January 27, 2021, 11 of 42 patients had been enrolled in Part 1. Internal study identifier APG-115SG101. Clinical trial registration: NCT03781986. Clinical trial information: NCT03781986.

Selinexor versus doxorubicin in dedifferentiated liposarcoma PDXs: evidence of greater activity and apoptotic response dependent on p53 nuclear accumulation and survivin down\u2010regulation

BackgroundDedifferentiated liposarcoma (DDLPS), a tumor that lacks effective treatment strategies and is associated with poor outcomes, expresses amplified MDM2 in the presence of wild-type p53. MDM2 ubiquitination of p53 facilitates its XPO1-mediated nuclear export, thus limiting p53 tumor suppressor functions. Consequently, nuclear export is a rational target in DDLPS. We directly compared the antitumor activity of the first-in class XPO1 inhibitor selinexor and doxorubicin, the standard front-line therapy in sarcomas, in DDLPS patient-derived xenografts (PDXs) and primary cell lines.MethodsDrug activity was assessed in three PDXs (and two corresponding cell lines) established from the dedifferentiated component of primary untreated retroperitoneal DDLPS with myogenic (N = 2) and rhabdomyoblastic (N = 1) differentiation from patients who underwent surgery. These models were marked by amplification of MDM2, CDK4 and HMGA2 genes.ResultsSelinexor was moderately active in the three PDXs but achieved greater tumor response compared to doxorubicin (maximum tumor volume inhibition: 46–80 % vs. 37–60 %). The PDX harboring rhabdomyoblastic dedifferentiation showed the highest sensitivity to both agents. PDX response to selinexor and doxorubicin was not associated with the extent of MDM2 and CDK4 gene amplification. Interestingly, the most chemosensitive PDX model showed the lowest extent of HMGA2 amplification. Selinexor was also more efficient than doxorubicinin in inducing an apoptotic response in PDXs and cell lines. Consistently, an increased nuclear accumulation of p53 was seen in all selinexor-treated models. In addition, a time-dependent decrease of survivin expression, with an almost complete abrogation of the cytoplasmic anti-apoptotic pool of this protein, was observed as a consequence of the decreased acetylation/activation of STAT3 and the increased ubiquitination of nuclear survivin.ConclusionsSelinexor showed a moderate antitumor activity in three DDLPS PDXs, which was, however, consistently higher than doxorubicin across all different models regardless the extent of MDM2 amplification and the histological differentiation. The depletion of survivin protein seems to significantly contribute to the induction of apoptosis through which selinexor exerts its antitumor activity.

Identification of a druggable protein–protein interaction site between mutant p53 and its stabilizing chaperone DNAJA1

The TP53 gene is the most frequently mutated gene in human cancers, and the majority of TP53 mutations are missense mutations. As a result, these mutant p53 (mutp53) either directly lose wildtype p53 (wtp53) tumor suppressor function or exhibit a dominant negative effect over wtp53. In addition, some mutp53 have acquired new oncogenic function (gain of function). Therefore, targeting mutp53 for its degradation may serve as a promising strategy for cancer prevention and therapy. Based on our previous finding that farnesylated DNAJA1 is a crucial chaperone in maintaining mutp53 stabilization, and by using an in silico approach, we built 3D homology models of human DNAJA1 and mutp53R175H proteins, identified the interacting pocket in the DNAJA1–mutp53R175H complex, and found one critical druggable small molecule binding site in the DNAJA1 glycine/phenylalanine-rich region. We confirmed that the interacting pocket in the DNAJA1–mutp53R175H complex was crucial for stabilizing mutp53R175H using a site-directed mutagenesis approach. We further screened a drug-like library to identify a promising small molecule hit (GY1-22) against the interacting pocket in the DNAJA1–mutp53R175H complex. The GY1-22 compound displayed an effective activity against the DNAJA1–mutp53R175H complex. Treatment with GY1-22 significantly reduced mutp53 protein levels, enhanced Waf1p21 expression, suppressed cyclin D1 expression, and inhibited mutp53-driven pancreatic cancer growth both in vitro and in vivo. Together, our results indicate that the interacting pocket in the DNAJA1–mutp53R175H complex is critical for mutp53’s stability and oncogenic function, and DNAJA1 is a robust therapeutic target for developing the efficient small molecule inhibitors against oncogenic mutp53.

CDK9 activity is critical for maintaining MDM4 overexpression in tumor cells

The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs – enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

Abstract 3427: Finding critical cancer driving and cancer suppressing genes using functional genomics screening in vivo

Abstract P53 is one of the most potent tumour suppressors and its activity is also required for cancer cells to respond to diverse chemotherapeutic drugs. Focused functional genetic screens in haematopoietic cells in vivo revealed DNA repair to be one of the most critical downstream pathways for p53's tumour suppressive function. Recently, we extended our search for novel tumour suppressor genes acting potentially outside the p53 tumour suppressor pathway. To this end, we performed whole genome CRISPR/Cas9 knockout screening in vivo, using the Eµ-Myc transgenic mouse model. This identified previously unknown genes and hence pathways, which upon deletion/mutation lead to the acceleration of tumour onset. Amongst these novel tumour suppressor genes, we identified the GATOR1 members DEPDC5 and NPRL3, which are known inhibitors of the mTOR pathway. Interestingly, drug mediated inhibition of mTOR activity with rapamycin or Torin1 in GATOR1 deficient Eµ-Myc cells led to efficient killing of these malignant cells, which was in striking contrast to control Eµ-Myc lymphoma cells that were resistant to this treatment. In parallel we conducted CRISPR/Cas9 screens for identifying p53 bound promoter and enhancer elements in haematopoietic cells in vivo. Excitingly, we observed that targeting the NPRL3 promoter led to accelerated tumour onset in the Eµ-Myc transgenic model, suggesting that NPRL3 is under direct transcriptional control of p53. These results indicate that p53 mediated shutdown of the mTOR pathway through upregulation of the GATOR1 member NPRL3 is a critical tumour suppressive function of p53. Citation Format: Marco Josef Herold, Shinsuke Mizutani, Yexuan Deng, Ana Janic, Andrew Kueh, Martin Pal, Stephen Wilcox, Lin Tai, Gemma L. Kelly, Andreas Strasser. Finding critical cancer driving and cancer suppressing genes using functional genomics screening in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3427.

KRT-232, a first-in-class, murine double minute 2 inhibitor (MDM2i), for TP53 wild-type (p53WT) Merkel cell carcinoma (MCC) after anti–PD-1/L1 immunotherapy.

10072 Background: MCC is an aggressive neuroendocrine skin cancer with very poor prognosis. Immune checkpoint inhibition was recently shown to benefit some patients (pts). There are few effective treatments (tx) and no standard of care for those who relapse on or are refractory to anti-PD-1/L1 agents (R/R). In p53WT MCC, oncoproteins from the Merkel cell polyomavirus can inhibit p53 tumor suppressor functions via L-MYC/EP400-dependent activation of MDM2. KRT-232, a potent, selective, orally available MDM2i, is being evaluated in pts with p53WTMCC who are R/R to anti-PD-1/L1 tx. Methods: In stage 1 of this open-label, multicenter, phase 2 study (NCT03787602) pts initially received KRT-232 240mg QD days 1-7 of a 21 day (d) cycle (cy). This cohort was closed due to Grade (Gr) 3/4 cytopenias and pts were moved to 240mg QD days 1-5 of a 28d cy to allow for hematologic recovery. Two new arms were opened: 240mg and 180mg QD days 1-5 of a 28d cy. The primary endpoint is objective response rate (ORR) by RECIST 1.1. Secondary endpoints include duration of response, progression-free survival, overall survival, and safety and tolerability of KRT-232. Results: At the time of this analysis, 11 pts were treated with KRT-232: 6 on the 240mg 7/21d, 3 on the 240mg 5/28d and 2 on the 180mg 5/28d schedules. Median age was 66; 46% of pts had ECOG 1 (range 0-1), the median prior lines of systemic therapy was 3 (range 1-4) and 82% had prior radiation tx. Treatment-emergent adverse events (AEs), regardless of grade or causality, were reported in all pts: 55% had Gr 3-4 AEs and 36% had serious AEs (SAEs). The most common AEs included neutropenia (55%), anemia, leukopenia and thrombocytopenia (each 45%), diarrhea, nausea and fatigue (each 36%), and lymphopenia, hypomagnesaemia, lipase increase and sinus tachycardia (each 27%). SAEs were mainly due to cytopenias. One Gr 5 AE of respiratory failure/ascites was attributed to progression. Median time on study was 11.3 wks (range 1.3-20.9). Two of 11 pts on active tx have not yet reached the first response assessment (wk 6). Of the 9 pts who have reached wk 6, 2 PRs and 1 SD (converted to PR at wk 12) were reported. At the second response assessment (wk 12), 2 PRs were reported; one of the PRs at wk 6 has not yet reached wk 12. The ORR was 33% (3/9 pts) . Conclusions: KRT-232 demonstrates promising monotherapy activity in MCC p53WT pts who failed anti-PD-1/L1 tx. This is the first clinical proof-of-concept for inhibiting the MDM2 pathway in MCC. Safety and efficacy continue to inform KRT-232 dose and schedule optimization. Clinical trial information: NCT03787602.

Recent Synthetic Approaches towards Small Molecule Reactivators of p53.

The tumor suppressor protein p53 is often called “the genome guardian” and controls the cell cycle and the integrity of DNA, as well as other important cellular functions. Its main function is to trigger the process of apoptosis in tumor cells, and approximately 50% of all cancers are related to the inactivation of the p53 protein through mutations in the TP53 gene. Due to the association of mutant p53 with cancer therapy resistance, different forms of restoration of p53 have been subject of intense research in recent years. In this sense, this review focus on the main currently adopted approaches for activation and reactivation of p53 tumor suppressor function, focusing on the synthetic approaches that are involved in the development and preparation of such small molecules.

10-Hydroxydecanoic acid inhibits LPS-induced inflammation by targeting p53 in microglial cells

Neuroinflammation, characterized by the activation of microglia and astrocytes, is important in the pathogenesis of many neurological disorders, such as Alzheimer’s disease. Nonsteroidal anti-inflammatory drugs (NSAIDs), a group of chemically heterogenous medications, are used widely in the treatment of inflammation. However, the safety of these drugs is a growing concern due to their side effects on the gastrointestinal tract and liver. Royal jelly (RJ) is a potential functional food produced by the hypopharynx and mandibular salivary glands of nurse bees. In this study, we explored the anti-neuroinflammatory effect of 10-hydroxydecanoic acid (10-HDAA), which is the second most abundant but less studied fatty acid in RJ. We showed that 10-HDAA decreased the lipopolysaccharide (LPS)-induced elevation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels in both microglial BV-2 and N9 cell lines. Compared to the LPS group, the 10-HDAA/LPS treated BV-2 cells had a higher level of the phagocytic receptor TREM2. RNAseq transcriptomic results showed a different transcriptional profile between the LPS group and the 10-HDAA/LPS group in BV-2 cells and the 10-HDAA pre-treatment significantly decreased levels of pro-inflammatory mediators, which were further confirmed by qRT-PCR analysis. Moreover, we found that p53 was a target of 10-HDAA. p53 may mediate the anti-inflammation effect of 10-HDAA in two ways: first by directly deactivating the NLRP3 inflammatory pathway, second by indirectly promoting autophagy. Taken together, our results reveal a novel function of tumor suppressor p53 in the inhibition of neuroinflammation and provide a theoretical basis for broadening the application range of 10-HDAA and RJ.

Transcriptional Coactivator TAZ Negatively Regulates Tumor Suppressor p53 Activity and Cellular Senescence.

Transcriptional coactivator with a PDZ-binding motif (TAZ) is one of the mammalian orthologs of Drosophila Yorkie, a transcriptional coactivator of the Hippo pathway. TAZ has been suggested to function as a regulator that modulates the expression of cell proliferation and anti-apoptotic genes in order to stimulate cell proliferation. TAZ has also been associated with a poor prognosis in several cancers, including breast cancer. However, the physiological role of TAZ in tumorigenesis remains unclear. We herein demonstrated that TAZ negatively regulated the activity of the tumor suppressor p53. The overexpression of TAZ down-regulated p53 transcriptional activity and its downstream gene expression. In contrast, TAZ knockdown up-regulated p21 expression induced by p53 activation. Regarding the underlying mechanism, TAZ inhibited the interaction between p53 and p300 and suppressed the p300-mediated acetylation of p53. Furthermore, TAZ knockdown induced cellular senescence in a p53-dependent manner. These results suggest that TAZ negatively regulates the tumor suppressor functions of p53 and attenuates p53-mediated cellular senescence.

MicroRNA-29a plays a prominent role in PRIMA-1Met-induced apoptosis in ovarian cancer cells

The structural analog of the small 2,2-bis(hydroxymethyl)-1-azabicyclo[2,2,2]octan-3-one molecule named PRIMA-1Metfor ?p53 reactivation and induction of massive apoptosis? has been shown to inhibit cell growth and induce apoptosis in human tumor cells by restoring the tumor suppressor function of tumor protein p53. In several microRNA (miRNA) profiling studies related to ovarian cancer, different miRNAs associated with PRIMA-1Met have been reported, but miRNAs related to PRIMA-1Met-induced apoptosis remain unclear. This study was designed to explain the potential mechanism of PRIMA-1-induced apoptosis. According to the MTSassay and fluorescence-activated cell sorting (FACS) analysis results, PRIMA-1Met induced a significant decrease in cell viability and an increase in apoptosis in both A2780 and Caov-3 cells, regardless of p53 status. PRIMA-1Met upregulated miRNA-29a in both cell lines. To determine the effect of miRNA-29a on PRIMA-1Met-induced apoptosis, A2780 and Caov-3 cells were transfected with miRNA-29a inhibitor. After treatment with PRIMA-1Met, cell viability increased and apoptosis decreased in the transfected cells. The results of this study suggest that miRNA-29a potentially regulates PRIMA-1Met-induced apoptosis in ovarian cancer cells.