P53 Mutation Status Research Articles

Estrogen-related receptor alpha directly binds to p53 and cooperatively controls colon cancer growth through the regulation of mitochondrial biogenesis and function

BackgroundOf the genes that control mitochondrial biogenesis and function, ERRα emerges as a druggable metabolic target to be exploited for cancer therapy. Of the genes mutated in cancer, TP53 remains the most elusive to target. A clear understanding of how mitochondrial druggable targets can be accessed to exploit the underlying mechanism(s) explaining how p53-deficient tumors promote cell survival remains elusive.MethodsWe performed protein-protein interaction studies to demonstrate that ERRα binds to p53. Moreover, we used gene silencing and pharmacological approaches in tandem with quantitative proteomics analysis by SWATH-MS to investigate the role of the ERRα/p53 complex in mitochondrial biogenesis and function in colon cancer. Finally, we designed in vitro and in vivo studies to investigate the possibility of targeting colon cancers that exhibit defects in p53.ResultsHere, we are the first to identify a direct protein-protein interaction between the ligand-binding domain (LBD) of ERRα and the C-terminal domain (CTD) of p53. ERRα binds to p53 regardless of p53 mutational status. Furthermore, we show that the ERRα and p53 complex cooperatively control mitochondrial biogenesis and function. Targeting ERRα creates mitochondrial metabolic stresses, such as production of reactive oxygen species (ROS) and mitochondrial membrane permeabilization (MMP), leading to a greater cytotoxic effect that is dependent on the presence of p53. Pharmacological inhibition of ERRα impairs the growth of p53-deficient cells and of p53 mutant patient-derived colon xenografts (PDX).ConclusionsTherefore, our data suggest that by using the status of the p53 protein as a selection criterion, the ERRα/p53 transcriptional axis can be exploited as a metabolic vulnerability.

Radiologic findings and the molecular expression profile of diffuse midline glioma H3 K27M mutant.

Diffuse midline glioma H3 K27M-mutant (DMG) are reported to show heterogeneous radiologic imaging features in children. We hypothesized that other genetic mutations may contribute to this heterogeneity. To describe the magnetic resonance imaging (MRI) findings of DMG in adult patients and to correlate the imaging findings with the molecular expression profile. Eighteen patients with pathologically proven DMG were enrolled. On preoperative MRI, the following were evaluated: location; size of the lesion; ratio of non-enhancing (NE) and contrast-enhancing (CE) area; presence of cortical invasion and necrotic component; maximum relative cerebral blood volume ratio (rCBV ratio) of NE and CE portions; and minimum apparent diffusion coefficient (ADC) of NE and CE portions, among others. Molecular profiles including ATRX expression and p53 mutation were reviewed to find correlation with imaging features. Thalamus was the most commonly involved location, followed by pons and tectum. Five patients showed loss of normal ATRX expression. p53 mutation was positive in 12 patients. 40% of normal ATRX expression patients had cortical involvement and 20% had leptomeningeal seeding; none of the patients with ATRX loss had cortical involvement or leptomeningeal seeding. Patients with normal ATRX expression showed significantly higher rCBV ratio and lower ADC value in the NE area than patients with ATRX loss (P=0.04, 0.016). p53 mutation status did not correlate with any imaging finding. Cortical invasion, leptomeningeal tumor spread, lower ADC value and higher rCBV ratio in NE areas of DMG may be related to normal expression of ATRX.

Derivation of induced pluripotent stem cell lines from New Zealand donors

ABSTRACT We aimed to generate human induced pluripotent stem cell (iPSC) lines from New Zealand donors. These lines are the first to be generated in New Zealand. Human dermal fibroblasts were collected from two individual donors and reprogrammed with the human OSKM transcription factors using the Sendai virus system. Emerging iPSC colonies were picked, expanded and karyotyped. Clones with normal karyotype were characterised for pluripotency marker expression, p53 mutational status and trilineage differentiation potential. The MANZ-2-2 and MANZ-4-37 iPSC lines showed normal karyotype and expressed pluripotency markers at RNA and protein levels without detectable transgene expression. Both lines differentiated into the three germ layers in vitro and passed the hPSC Scorecard assay for pluripotency and trilineage differentiation. Furthermore, both lines were susceptible to cell apoptosis mediated by nutlin-3a indicative of their wildtype p53 status. This study presents the successful derivation and characterisation of iPSC lines derived from New Zealand donors. These lines will facilitate iPSC-based research in New Zealand and beyond.

BCL11B suppresses tumor progression and stem cell traits in hepatocellular carcinoma by restoring p53 signaling activity

Accumulating evidence indicates that hepatocellular carcinoma (HCC) tumorigenesis, recurrence, metastasis, and therapeutic resistance are strongly associated with liver cancer stem cells (CSCs), a rare subpopulation of highly tumorigenic cells with self-renewal capacity and differentiation potential. Previous studies identified B cell leukemia/lymphoma-11b (BCL11B) as a novel tumor suppressor with impressive capacity to restrain CSC traits. However, the implications of BCL11B in HCC remain unclear. In this study, we found that low BCL11B expression was an independent indicator for shorter overall survival (OS) and time to recurrence (TTR) for HCC patients with surgical resection. In vitro and in vivo experiments confirmed BCL11B as a tumor suppressor in HCC with inhibitory effects on proliferation, cell cycle progression, apoptosis, and mobility. Furthermore, BCL11B could suppress CSC traits, as evidenced by dramatically decreased tumor spheroid formation, self-renewal potential and drug resistance. A Cignal Finder Array and dual-luciferase activity reporter assays revealed that BCL11B could activate the transcription of P73 via an E2F1-dependent manner. Thus, we concluded that BCL11B is a strong suppressor of retaining CSC traits in HCC. Ectopic expression of BCL11B might be a promising strategy for anti-HCC treatment with the potential to cure HBV-related HCC regardless of P53 mutation status.

Dual targeting of Polo-like kinase 1 and baculoviral inhibitor of apoptosis repeat-containing 5 in TP53-mutated hepatocellular carcinoma.

BACKGROUNDHepatocellular carcinoma (HCC), often diagnosed at advanced stages without curative therapies, is the fifth most common malignant cancer and the second leading cause of cancer-related mortality. Polo-like kinase 1 (PLK1) is activated in the late G2 phase of the cell cycle and is required for entry to mitosis. Interestingly, PLK1 is overexpressed in many HCC patients and is highly associated with poor clinical outcome. Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) is also highly overexpressed in HCC and plays key roles in this malignancy.AIMTo determine the expression patterns of PLK1 and BIRC5, as well as their correlation with p53 mutation status and patient clinical outcome.METHODSThe expression patterns of PLK1 and BIRC5, and their correlation with p53 mutation status or patient clinical outcome were analyzed using a TCGA HCC dataset. Cell viability, cell apoptosis, and cell cycle arrest assays were conducted to investigate the efficacy of the PLK1 inhibitors volasertib and GSK461364 and the BIRC5 inhibitor YM155, alone or in combination. The in vivo efficacy of volasertib and YM155, alone or in combination, was assessed in p53-mutated Huh7-derived xenograft models in immune-deficient NSIG mice.RESULTSOur bioinformatics analysis using a TCGA HCC dataset revealed that PLK1 and BIRC5 were overexpressed in the same patient subset and their expression was highly correlated. The overexpression of both PLK1 and BIRC5 was more frequently detected in HCC with p53 mutations. High PLK1 or BIRC5 expression significantly correlated with poor clinical outcome. PLK1 inhibitors (volasertib and GSK461364) or a BIRC5 inhibitor (YM155) selectively targeted Huh7 cells with mutated p53, but not HepG2 cells with wild-type p53. The combination treatment of volasertib and YM155 synergistically inhibited the viability of Huh7 cells via apoptotic pathway. The efficacy of volasertib and YM155, alone or in combination, was validated in vivo in a Huh7-derived xenograft model.CONCLUSIONPLK1 and BIRC5 are highly co-expressed in p53-mutated HCC and inhibition of both PLK1 and BIRC5 synergistically compromises the viability of p53-mutated HCC cells in vitro and in vivo.

Abstract 1451: Hyperthermic conditions improve curcumin efficacy for the treatment of peritoneal carcinomatosis of colon cancer origin

Abstract Patients with peritoneal metastases from colorectal cancer (CRC) have poor prognosis. Systemic chemotherapy by itself is often ineffective in the treatment of peritoneal carcinomatosis. Cytoreductive surgery (CRS) with heated intraperitoneal chemotherapy (HIPEC) is offered to appropriately selected patients. HIPEC consists of direct application of high-dose chemotherapy to the peritoneal cavity under hyperthermic conditions for 90-120 minutes. Although there is strong evidence to demonstrate improved survival with cytoreductive surgery, the role of HIPEC needs further evaluation. Furthermore, despite optimal cytoreduction, a subset of patients will suffer early peritoneal recurrence which indicates that the intraperitoneal chemotherapy was ineffective in controlling the microscopic disease. In vitro studies focused on optimizing the drug, duration of exposure, and temperature based on the biology of the cancer are critically needed to inform the design of future clinical trials. The combination of the HIPEC treatment modality with curcumin as the therapeutic agent is attractive since the naturally occurring curcumin has fewer side effects than drugs currently used for HIPEC. Additionally, although curcumin possesses antitumor effects on various cancers including colon, its efficacy is hindered by a low systemic bioavailability. For HIPEC, curcumin could be delivered directly into the peritoneal cavity, thereby increasing bioavailability. We hypothesize that curcumin used alone or in combination with standard regimen mitomycin C treatment in HIPEC will exert antitumor effects superior to the standard regimen HIPEC. To assess this hypothesis, we developed an in vitro model in which colon cancer cell lines (SK-CO-1, COLO205 and SNU-C1) were treated with curcumin (25 µM) or mitomycin C (1 µM) alone or in combination at different exposure times (1, 2 or 3 hours), and then incubated at either 37°C (normothermia) or 42 °C (hyperthermia), simulating conditions used in clinical practice. Cell lines were chosen in order to determine the effects of p53 (SNU-C1 & COLO205), KRAS (SK-CO-1) and BRAF (COLO205) mutation status on this proposed intraperitoneal chemotherapy strategy. After treatment, cells were maintained at 37°C for 24, 48, and 72 hours after which cell viability was assessed using Annexin V/PI, trypan blue and Hoffman microscopy. Our results indicate that hyperthermia enhances the efficacy of curcumin treatment as evidenced by a significant two-fold decrease in cell viability compared to normothermia in vitro in all three cell lines. No mutation-associated effect was quantified. Though additional comparisons are still forthcoming, the addition of curcumin to HIPEC may be a promising novel therapeutic option. Citation Format: Janviere Kabagwira, Paul A. Vallejos, Audrey H. Choi, Chase Sugiono, Vola-Masoandro Andrianarijaona, Jazmine Chism, William HR Langridge, Maheswari Senthil, Nathan R. Wall. Hyperthermic conditions improve curcumin efficacy for the treatment of peritoneal carcinomatosis of colon cancer origin [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 1451.

P-27 Retrospective observational analysis of p53 mutational status as a prognostic factor in TAS-102 treated metastatic colorectal cancer patients

Tipiracil-trifluridine combination (TAS-102) has recently been introduced as a treatment option in pretreated metastatic colorectal cancer (MCRC) patients. Owing to the favourable safety profile of the drug, the number of patients that are currently been treated is increasing. Based on the results of the registration trials, about 40% patients achieve disease control (either stable disease or partial response) at their first radiological evaluation. In previous preclinical models it has been shown that p53 mutational status might affect tumor cells' sensitivity to the drug: p53 wild-type cells seem to have increased sensitivity to trifluridine compared with p53 mutated cells.

BNIP3 contributes to cisplatin-induced apoptosis in ovarian cancer cells.

BNIP3 is a proapoptotic protein that mediates apoptosis, necrosis and autophagy. However, the involvement of BNIP3 in cisplatin‐induced apoptosis in ovarian cancer is not clear. In this study, we examined the role of BNIP3 in ovarian cancer during cisplatin treatment and its correlation with clinical outcomes. We first measured cisplatin cytotoxicity and BNIP3 levels before and after cisplatin exposure for ovarian cancer cell lines A2780, SKOV3, OVCAR4, OV2008, ES2 and HO8910. BNIP3 was observed to be differentially expressed in these cell lines, and cisplatin induced a significant increase in BNIP3 levels in A2780 and OVCAR4. BNIP3 knockdown with siRNA in A2780 and OVCAR4 significantly reduced cisplatin cytotoxicity in these two cell lines and alleviated cisplatin‐induced apoptosis. We searched the online databases Gene Expression Omnibus and The Cancer Genome Atlas to analyze the correlation between BNIP3 level and overall survival and progression‐free survival in patients with ovarian cancer. Pooled analyses showed that higher BNIP3 level was correlated with poorer overall survival (95% confidence intervals; hazard ratio = 1.18, 1.04–1.34; P = 0.013) and progression‐free survival (95% confidence intervals; hazard ratio = 1.26, 1.10–1.43; P = 0.00049). However, the results of individual datasets and stratification analyses of histology, FIGO (Federation Internationale de Gynecolgie et d’Obstetrique) stage, chemotherapy regimen and P53 mutation status varied. These findings indicate that cisplatin‐induced apoptosis is dependent on BNIP3 level in ovarian cancer cell lines. Targeting BNIP3 may therefore be a potential way of restoring cisplatin sensitivity.

Abstract 15: Imputing a more targeted therapy for triple-negative breast cancers

Abstract Triple-negative breast cancer (TNBC) is a clinically defined subset of breast cancers that are inherently difficult to treat since, unlike the other breast cancer subtypes, they are defined by their lack of a distinct molecular target. Previously our lab developed a computational framework to impute drug response in patients that accurately mirrored the observed patient response. In short, this methodology involves building unsupervised linear regression models between cell line baseline gene expression values and the respective drug sensitivity metric (EC50 or AUC), which allows us to generate imputed sensitivity scores for cell line or patient tumor samples for which we have gene expression data. For this study, we applied this methodology to prospectively identify agents that are predicted to be more effective specifically in triple-negative breast cancer. We began by expanding the drug imputation training dataset to the Broad’s Cell Therapeutics Response Portal, which contains 545 drugs and over 1,000 cancer cell lines (CCLs) and imputed sensitivity in The Cancer Genome Atlas’s (TCGA’s) breast cancer cohort. Our imputation data indicated that TNBC tumors were expected to be comparatively sensitive to cell cycle pathway inhibitors. Among these compounds, the Wee1 inhibitor MK1775 (aka adavosertib and AZD1775) was identified for having the most significant predicted effect in TNBC patients compared to the receptor-positive patients. We were able to validate the accuracy of the MK1775 imputation model using traditional cross-validation approaches as well as in independent cell line dataset and showed that the measured and our predicted response to Wee1 inhibition strongly correlated. Additionally, we were able to uses our predicted MK1775 response scores to recapitulate the significant association between MK1775 response and p53 mutation status. We then looked to validate the efficacy of MK1775’s efficacy in TNBC cell line and xenograft experiments. We observed strong inhibition of cancer growth by MK1775 alone but also identified a combination effect between MK1775 and the TNBC standard-of-care agent paclitaxel as assayed by both in vitro viability experiments as well as in vivo tumor growth assays. We believe these results indicate that this methodology could generally be applied as a hypothesis-generating tool for identifying targeted agents for other disease subtypes as well as supporting the use of MK1775, especially in combination with paclitaxel, for TNBC. Citation Format: Robert F. Gruener, Geoffrey L. Greene, R. Stephanie Huang. Imputing a more targeted therapy for triple-negative breast cancers [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 15.

Abstract B20: p53 regulation of repetitive elements and the interferon response in cancer

Abstract Here we characterize the effects of p53 status on the immune microenvironment of tumors. Specifically, we focus on the immune response induced by treatment with epigenetic modulators like 5-azacytidine (Aza), a DNA methyltransferase inhibitor (DNMTi), and whether this differs in tumors with and without functional p53. We and others have previously shown that cancer cells elicit an immune response when treated with epigenetic modulators such as DNMTis. These drugs inhibit methylation-induced gene silencing and cause re-expression of repetitive elements. Furthermore, they trigger interferon signaling in tumors by upregulation of double-stranded RNA (dsRNA) including DNA methylated endogenous retroviruses (ERVs), remains of exogenous retroviruses that integrated into the germline millions of years ago. The tumor suppressor protein p53, which is mutated in over half of all cancers, acts as a transcriptional repressor of ERVs. Little is known about the interaction between ERVs and p53 in a pathophysiologic model such as cancer. We hypothesize that p53 mutations influence the epigenetic regulation and ERV-induced immune signaling in cancer cells treated with Aza. We injected mouse ovarian cancer cells with p53 wild-type or null status into a syngeneic animal model for ovarian cancer and treated these mice with vehicle or Aza, an FDA-approved epigenetic modulator for myelodysplastic syndrome. We report that after 15 weeks, mice injected with p53 null cells present with a significantly higher tumor burden reflected in occurrence and volume of abdominal ascites fluid, weight gain over time, and shorter survival compared to mice injected with p53 wild-type cells. Further, Aza treatment compared to mock has a significant positive effect on tumor burden and survival but exclusively in mice with p53 null cells. No significant differences between treatments in the groups containing p53 wild-type cells were observed, indicating that the presence of p53 in these tumor cells likely abrogates the immunologic antitumor effects of Aza. This makes sense as p53 is a major transcriptional regulator of repetitive elements such as ERVs, which are upregulated after Aza treatment in this tumor model. We are currently characterizing differences in immune regulation of these treatment groups, in particular, infiltrating lymphocytes and Type I interferon signaling, which is crucial for the antitumor effect of Aza. We have studied how functional or lack of p53 contributes to the epigenetic regulation of ERVs and their potential to sensitize patients to immune therapy. We are now creating isogenic ovarian cancer cells containing wild-type or null as well as p53 point mutations, which occur in the majority of cancers. In doing so, we will gain novel information about the effects of mutant p53 on the ERV-induced immune response in tumor cells. Considering the vast amount of cancers harboring p53 mutations, assessing the p53 mutational status in cancers could become a useful biomarker for epigenetic therapies activating ERVs. Citation Format: Elisa Arthofer, Noor Diab, Katherine Bakshian Chiappinelli. p53 regulation of repetitive elements and the interferon response in cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B20.

Small‐molecule MDM2 inhibitor LQFM030‐induced apoptosis in p53‐null K562 chronic myeloid leukemia cells

Our group designed and synthesized the N-phenyl-piperazine LQFM030 [1-(4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl) piperazin-1-yl) ethanone], a small molecule derived from molecular simplification of the Nutlin-1, an inhibitor of the human homologue of murine double minute 2 (MDM2) protein that is expressed in several types of cancer. To better investigate the effects of LQFM030 regarding the p53 mutation status, this study investigated the antiproliferative activity of LQFM030 against the p53-null K562 leukemia cells as well as the cell death pathways involved. In addition, the effects of LQFM030 on the levels of the p53/MDM2 complex were also carried out using 3T3 cells as a p53 wild-type model. Our data suggest that LQFM030 triggered apoptosis in K562 cells via different mechanisms including cell cycle arrest, caspase activation, reduction of mitochondrial activity, decrease in MDM2 expression, and transcriptional modulation of MDMX, p73, MYC, and NF-ĸB. Additionally, it promoted effects in p53/MDM2 binding in p53 wild-type 3T3 cells. Therefore, LQFM030 has antiproliferative effects in cancer cells by a p53 mutation status-independent manner with different signaling pathways. These findings open new perspectives to the treatment of leukemic cells considering the resistance development associated with cancer treatment with conventional cytotoxic drugs.

Immunohistochemical staining patterns of Ki-67 and p53 in florid reactive urothelial atypia and urothelial carcinoma in situ demonstrate significant overlap

Flat urothelial lesions with atypia may pose significant diagnostic challenges. Given frequent increased proliferation rates in florid reactive urothelial atypia and limited studies on the interpretation of p53 stains in the urothelium (following current standard guidelines for correlation with P53 mutation status), we sought to further study the discriminatory value of Ki-67 and p53 for florid reactive urothelial atypia versus urothelial carcinoma in situ (CIS). Bladder specimens diagnosed as reactive urothelial atypia (n=40) and CIS (n=40) were assessed by immunohistochemical staining with antibodies for Ki-67, p53, CD44, and CK20. Immunoreactivity was scored based on percent cells positive for Ki-67 and pattern of reactivity with p53 (aberrant: diffuse strong positive or negative; normal: patchy/wild type). CD44 and CK20 reactivity patterns served as adjunctive internal validation controls for reactive urothelial atypia and CIS, as previously described. In reactive urothelial atypia, Ki-67 ranged from 0% to 90% (mean, 34%±26) with 30 cases (75%) having >10%. In CIS, Ki-67 ranged from 5% to 95% (mean, 50%±25) with 17 cases (43%) having >50%. In all 40 cases (100%) of reactive urothelial atypia, p53 expression had a wild-type pattern. In CIS, aberrant p53 expression was identified in 15 cases (37%): 3 cases (7%) were p53 negative (i.e. null phenotype) and 12 cases (30%) showed strong and diffuse nuclear reactivity (in >85% of cells). The remaining 25 cases (63%) of CIS had a p53 wild-type pattern of expression. Cytoplasmic CK20 immunoreactivity in umbrella cells was seen in 34 cases (85%) of reactive urothelial atypia, and 6 cases (15%) were negative. In addition, 35 cases (88%) of reactive urothelial atypia demonstrated full-thickness CD44 expression, while 5 cases (12%) had expression confined to the basal/parabasal layers of the urothelium. Strong and diffuse CK20 positivity was present in 39 cases (98%) of CIS, and patchy positivity was detected in 1 case (2%). None of the CIS cases overexpressed CD44: 16 cases (40%) showed focal expression in the nonneoplastic basal cell layer; 24 cases (60%) demonstrated no staining. In summary, Ki-67 has poor discriminatory value for reactive urothelial atypia versus CIS and adds little to the classic CK20/CD44 immunophenotype. While p53 sensitivity for CIS is relatively low (30%) and interpretation as either wild type or negative may be challenging in a small subset of cases, strong and diffuse nuclear reactivity was 100% specific in the distinction from florid reactive urothelial atypia in this cohort.

Associations of sirtuins with clinicopathological variables and prognosis in human ovarian cancer.

Ovarian cancer (OC) is the fifth most frequent cause of cancer-associated mortality worldwide, and is accompanied by asymptomatic progression. Sirtuins (SIRTs) are a family of nicotinamide adenine dinucleotide-dependent protein deacetylases, comprising seven members (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 and SIRT7). Accumulating evidence has demonstrated that SIRTs act as prognostic estimators in certain types of cancer such as lung cancer, prostate cancer, gastric cancer, breast cancer and colorectal cancer. However, it remains unknown whether individual SIRTs can serve as independent prognostic factors in OC. In the present study, the Kaplan-Meier plotter online database was utilized to examine the prognostic values of SIRT mRNA expression in patients with OC. The results demonstrated that the overexpression of SIRT3, SIRT5, SIRT6 and SIRT7 mRNAs was associated with a good prognosis in patients, whereas elevated mRNA levels of SIRT1 and SIRT4 indicated poor survival in patients with OC. In addition, among the favorable predictors, SIRT3, SIRT5, SIRT6 and SIRT7 overexpression were associated with overall survival (OS), according to clinical characteristics, such as histological classification, clinical stage, pathology grade, drug therapy and tumor protein p53 mutation status in patients with OC. Similarly, SIRT4 mRNA overexpression was associated with poor OS in pathological grade III cancer. High SIRT1 and SIRT4 expression were associated with unfavorable OS at all clinical stages. Furthermore, SIRT1 and SIRT4 were negatively associated with OS in drug-treated patients. In summary, the present study demonstrated that the SIRT family is associated with the prognosis of human OC, suggesting that individual SIRTs may also act as prognostic predictors in patients.

Combined Targeting of Mutant p53 and Jumonji Family Histone Demethylase Augments Therapeutic Efficacy of Radiation in H3K27M DIPG.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor with a 5-year survival of <1%. Up to 80% of the DIPG tumors contain a specific K27M mutation in one of the two genes encoding histone H3 (H3K27M). Furthermore, p53 mutations found in >70–80% of H3K27M DIPG, and mutant p53 status is associated with a decreased response to radiation treatment and worse overall prognosis. Recent evidence indicates that H3K27M mutation disrupts tri-methylation at H3K27 leading to aberrant gene expression. Jumonji family histone demethylases collaborates with H3K27 mutation in DIPG by erasing H3K27 trimethylation and thus contributing to derepression of genes involved in tumorigenesis. Since the first line of treatment for pediatric DIPG is fractionated radiation, we investigated the effects of Jumonji demethylase inhibition with GSK-J4, and mutant p53 targeting/oxidative stress induction with APR-246, on radio-sensitization of human H3K27M DIPG cells. Both APR-246 and GSK-J4 displayed growth inhibitory effects as single agents in H3K27M DIPG cells. Furthermore, both of these agents elicited mild radiosensitizing effects in human DIPG cells (sensitizer enhancement ratios (SERs) of 1.12 and 1.35, respectively; p < 0.05). Strikingly, a combination of APR-246 and GSK-J4 displayed a significant enhancement of radiosensitization, with SER of 1.50 (p < 0.05) at sub-micro-molar concentrations of the drugs (0.5 μM). The molecular mechanism of the observed radiosensitization appears to involve DNA damage repair deficiency triggered by APR-246/GSK-J4, leading to the induction of apoptotic cell death. Thus, a therapeutic approach of combined targeting of mutant p53, oxidative stress induction, and Jumonji demethylase inhibition with radiation in DIPG warrants further investigation.

Metabolic Pathways Enhancement Confers Poor Prognosis in p53 Exon Mutant Hepatocellular Carcinoma.

RNA-Sequencing (RNA-Seq), the most commonly used sequencing application tool, is not only a method for measuring gene expression but also an excellent media to detect important structural variants such as single nucleotide variants (SNVs), insertion/deletion (Indels), or fusion transcripts. The Cancer Genome Atlas (TCGA) contains genomic data from a variety of cancer types and also provides the raw data generated by TCGA consortium. p53 is among the top 10 somatic mutations associated with hepatocellular carcinoma (HCC). The aim of the present study was to analyze concordant different gene profiles and the priori defined set of genes based on p53 mutation status in HCC using RNA-Seq data. In the study, expression profile of 11 799 genes on 42 paired tumor and adjacent normal tissues was collected, processed, and further stratified by the mutated versus normal p53 expression. Furthermore, we used a knowledge-based approach Gene Set Enrichment Analysis (GSEA) to compare between normal and p53 mutation gene expression profiles. The statistical significance (nominal P value) of the enrichment score (ES) genes was calculated. The ranked gene list that reflects differential expression between p53 wild-type and mutant genotypes was then mapped to metabolic process by KEGG, an encyclopedia of genes and genomes to assign functional meanings. These approaches enable us to identify pathways and potential target gene/pathways that are highly expressed in p53 mutated HCC. Our analysis revealed 2 genes, the hexokinase 2 (HK2) and Enolase 1 (ENO1), were conspicuous of red pixel in the heatmap. To further explore the role of these genes in HCC, the overall survival plots by Kaplan-Meier method were performed for HK2 and ENO1 that revealed high HK2 and ENO1 expression in patients with HCC have poor prognosis. These results suggested that these glycolysis genes are associated with mutated-p53 in HCC that may contribute to poor prognosis. In this proof-of-concept study, we proposed an approach for identifying novel potential therapeutic targets in human HCC with mutated p53. These approaches can take advantage of the massive next-generation sequencing (NGS) data generated worldwide and make more out of it by exploring new potential therapeutic targets.

Human papillomavirus infection and p53 mutation in esophageal squamous cell carcinoma and its impact on treatment outcome.

Human papillomavirus (HPV) is emerging as a risk factor for esophageal squamous carcinoma. The prognostic value of the HPV status has been investigated. However, the results are much controversial. This study aims to document the association of HPV infection and mutation of p53 gene in esophageal squamous cell carcinoma (ESCC) and its impact on treatment outcome. The study was conducted over a period of 12 months. A total of 30 cases of ESCC who were primarily to be treated with radiotherapy/chemoradiotherapy were included in the study. All the tissue samples for biopsy were subjected to immunohistochemistry to study p53 and p16 expression, which is a surrogate marker for HPV. The patients were treated by radiotherapy alone or concurrent chemoradiotherapy depending on performance status and stage of disease. The impact of p16 and p53 on overall survival (OS) and disease-free survival (DFS) was determined. The median OS of HPV-positive patients was 22 months (95% confidence interval [CI] 12-31) as compared to 13 months (95% CI 7-18) for HPV-negative patients (P = 0.298). The median DFS for HPV-positive patients was 16 months (95% CI 7-24) as compared to 5 months (95% CI 4-6) for HPV-negative patients (P = 0.048). The median OS of p53-positive patients was 13 months (95% CI 6.7-19) as compared to 22 months (95% CI 12.7-31.2) for p53-negative patients (P = 0.080). The median DFS for p53-positive patients was 5 months (95% CI 3.7-6.2) as compared to 22 months (95% CI 15.7-29.4) for p53-negative patients (P = 0.014). Clinical findings of our result can be used to sum up that both HPV infection and p53 mutation status are reliable biomarkers and can help clinicians to predict treatment outcome and prognosticate patients better.

The single nucleotide polymorphism arg399gln rs25487 in XRCC1 gene is a breast cancer risk factor, but is not related to tp53 mutation status

Genetic changes in DNA repair genes, such as X-ray cross-complementing group1 (XRCC1), can cause modifications in the capacity of damaged DNA repair and affect the risk of cancer. Several mutations in TP53, which is a tumor suppressor gene, have been associated with breast cancer. In this study, it is aimed to evaluate the association of genetic variation in XRCC1rs25487 single nucleotide polymorphism (SNP) with TP53 mutation and breast cancer risk. In this research, 200 breast cancer women and 200 controls from the Iranian-Azeri population, Iran, were enrolled. Genomic DNA was extracted from the whole blood of controls patients. PCR-RFLP was carried out to genotype all participants for XRCC1rs25487SNP. Determination of possible mutations of TP53 in exons 5,6,7, and 8 were performed on 30 cancerous breast tissues through sequencing the amplified fragments. Our results of the case-control study indicated that the GA genotype of XRCC1 gene in rs25487polymorphism has a significantly risk effect on the breast cancer in the dominant genetic model (OR: 1.580, 95% CI: (1.025-2.436); p-value =0.049) and also GA genotype of XRCC1 gene in rs25487polymorphism has a protective effect on breast cancer in overdominant genetic model (OR: 0.591, 95% CI (0.395-0.886), p-value = 0.014). Furthermore, genotypes of this SNP did not associate with the clinical specifications of the patients and P53 mutation status. Sequencing results showed the mutational spectrum of P53 in the studied cases. According to the results of this study, in some of the genetic models, XRCC1SNP appears to be a modulator of breast cancer risk in Iranian East-Azerbaijan women. However, there was no correlation between XRCC1SNP and TP53 mutation status of the tumor.

Targeting the Ribosome Biogenesis Key Molecule Fibrillarin to Avoid Chemoresistance.

Inherent or acquired chemo resistance in cancer patients has been a perpetual limitation in cancer treatment. Expanding knowledge on essential cellular processes opens a new window for therapeutic targeting. Ribosome biogenesis is a process that shows potential due to its fundamental role in cell development and contribution to tumorigenesis as a result of its upregulation. Inhibiting components of ribosome biogenesis has been explored and has shown interesting results. Yet, an important key component, methyltransferase Fibrillarin (FBL), which influences both the abundance and composition of ribosomes, has not been exploited thus far. In this literature review, we describe relevant aspects of ribosome biogenesis in cancer to emphasize the potential of FBL as a therapeutic target, in order to lower the genotoxic effects of anti-cancer treatment. Remarkably, the amplification of the 19q13 cytogenetic band, including the gene coding for FBL, correlated to cell viability and resistance in pancreatic cells as well as to a trend toward a shorter survival in pancreatic cancer patients. Targeting ribosome biogenesis, more specifically compared to the secondary effects of chemotherapeutics such as 5-fluorouracil or oxaliplatin, has been achieved by compound CX-5461. The cell dependent activity of this Pol I inhibitor has been reported in ovarian cancer, melanoma and leukemia models with active or mutated p53 status, presenting a promising mechanism to evade p53 resistance. Targeting critical ribosome biogenesis components in order to decrease the genotoxic activity in cancer cell looks promising. Hence, we believe that targeting key protein rRNA methyltransferase FBL shows great potential, due to its pivotal role in ribosome biogenesis, its correlation to an improved survival rate at low expression in breast cancer patients and its association with p53.

Therapeutic Implications of p53 Status on Cancer Cell Fate Following Exposure to Ionizing Radiation and the DNA-PK Inhibitor M3814.

Inhibition of DNA double-strand break (DSB) repair in cancer cells has been proposed as a new therapeutic strategy for potentiating the anticancer effects of radiotherapy. M3814 is a novel, selective pharmacologic inhibitor of the serine/threonine kinase DNA-dependent protein kinase (DNA-PK), a key driver of nonhomologous end-joining, one of the main DSB-repair pathways, currently under clinical investigation. Here, we show that M3814 effectively blocks the repair of radiation-induced DSBs and potently enhances p53 phosphorylation and activation. In p53 wild-type cells, ataxia telangiectasia-mutated (ATM) and its targets, p53 and checkpoint kinase 2 (CHK2), were more strongly activated by combination treatment with M3814 and radiation than by radiation alone, leading to a complete p53-dependent cell-cycle block and premature cell senescence. Cancer cells with dysfunctional p53 were unable to fully arrest their cell cycle and entered S and M phases with unrepaired DNA, leading to mitotic catastrophe and apoptotic cell death. Isogenic p53-null/wild-type A549 and HT-1080 cell lines were generated and used to demonstrate that p53 plays a critical role in determining the response to ionizing radiation and M3814. Time-lapse imaging of cell death and measuring apoptosis in panels of p53 wild-type and p53-null/mutant cancer lines confirmed the clear differences in cell fate, dependent on p53 status. IMPLICATIONS: Our results identify p53 as a possible biomarker for response of cancer cells to combination treatment with radiation and a DNA-PK inhibitor and suggest that p53 mutation status should be considered in the design of future clinical trials. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/17/12/2457/F1.large.jpg.

Efficacy of OR21, a Novel Oral Demethylating Agent in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease induced by t(9;22)(q34;q11) translocation. The prognosis of patients with CML has dramatically improved since tyrosine kinase inhibitors (TKIs) were introduced, and recent studies show that approximately 40 to 50 % of CML patients achieved deep molecular response (DASISION and ENESTnd trial) within several years. However, therapeutic options for patients with CML who are resistant for TKIs are limited. Besides BCR-ABL kinase domain mutation, somatic mutations associated with epigenetic gene alteration (e.g. TET, DNMT3A) are reportedly involved in TKI resistance and disease progression. Thus, we investigated the efficacy of DNA demethylating agents in CML. Azacitidine (AZA) and decitabine (DAC), currently available as DNA demethylating agents, have low bioavailability for oral administration because they are easily degraded by cytidine deaminase. Then, we are developing a novel demethylating agent, OR21, with possible oral absorbability as a prodrug of DAC. In vivo analysis using cynomolgus monkeys, the area under the curve of DAC after intraduodenally administration of DAC (1.5mg/kg, 6.6µmol/kg) or OR21 (2.25mg/kg, 6.6µmol/kg) was 0.01 µM·h, 0.298 µM·h respectively, indicating OR21 have high oral absorbability. To assess the demethylating activity of OR21 for CML, we performed western blot analysis and bisulfite pyrosequencing assay to measure LINE1 methylation, using CML cell-lines (K562, BV173). OR21 decreased DNMT1 protein level as a result of demethylating and LINE1 methylation in K562 and BV173 comparable to DAC. Next, we performed cell growth inhibition and cell apoptosis assay after 72 hours exposure of OR21 to assess anti-tumor effect in vitro. OR21 inhibited cell growth comparable to DAC in CML cell-lines (K562, BV173, KCL22, MYL) in a dose-dependent manner. Notably, OR21 inhibited the cell growth and apoptosis against BV173 with an extremely low concentration (IC50; 5nM) than that of AZA (IC50; 122nM) via significant accumulation of reactive oxygen species. Whereas, OR21 weakly induced cell apoptosis against K562. OR21 induced G2/M phase cell-cycle arrest in CML cell-lines except for BV173 via pRb downregulation. These results indicated mechanisms of anti-tumor effect in OR21 were induction of cell apoptosis (BV173) or cell cycle arrest (K562, KCL22, MYL). Because of the different mechanism of action, we assessed whether OR21 and TKIs combination can enhance the anti-tumor effect of CML. We investigated the combination effects of OR21 with imatinib (IM) or dasatinib (DAS) in K562. Combination index values at IC80 calculated by Calucusyn software showed 0.642±0.129 (with IM), 1.182±0.2 (with DAS), respectively. Accordingly, OR21 combined with TKIs showed at least additive or synergistic effect for K562. TKI resistance, which can be associated with somatic mutations leading to epigenetic gene alteration or loss of function of p53, is an obstacle for molecular remission in patients with CML, thus we examined the effects of OR21 in TKI resistant cell lines or efficacy on p53 mutational status. OR21 inhibited the cell growth in IM-resistant cell-line MYL-R, a derivative of MYL, which had overexpression of Lyn, and Ba/F3 BCR-ABLT315I, which exogenously expressed Bcr-Abl (T315I), indicating OR21 could overcome TKI resistance in CML. OR21 or cytarabine did not enhance cell apoptosis against K562, MYL and KCL22 (p53 deficient or mutant cell lines) combination with nutlin-3a (MDM-2 inhibitor), while increased cell apoptosis was observed in BV173 (p53 wild type) treated with cytarabine and nutlin-3a, but not with OR21 and nutlin-3a. These results suggested the effects of OR21 did not depend on p53 mutational status. Finally, we used a mouse xenograft model to evaluate anti-tumor effect of OR21 in vivo. BALB/c Rag-2/JAK3 double-deficient (BRJ) mice were injected intravenously via tail vein with 5 ×106 BV173 cells. OR21 were administered at a dose of 2.7mg/kg (equivalent to DAC 1.0mg/kg in AUC) and PBS (vehicle) twice weekly. OR21 significantly prolonged survival in a xenograft mice model (median 35 days vs not reached, P&lt;0.01). In conclusion, a novel orally available demethylating agent OR21 is effective for CML cells including TKI resistant clones. The efficacy and safety of OR21 for CML is expected to be verified by early-phase clinical trials. Disclosures Kamachi: Ohara Pharmaceutical Co.: Research Funding. Ureshino:OHARA Pharmaceutical Co.: Research Funding. Yoshida:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:Ohara Pharmaceutical Co.: Employment. Watanabe:Ohara Pharmaceutical Co.: Research Funding. Okada:Japan Agency for Medical Research and Development: Research Funding; Bristol-Myers Squibb: Research Funding. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.