P53 Wt Cells Research Articles

COTI-2 reactivates mutant p53 and inhibits growth of triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) currently lacks an approved targeted therapy. The tumour suppressor TP53 gene is mutated in approximately 80% of TNBC cases. COTI-2 is a third-generation thiosemicarbazone engineered for high efficacy and low toxicity which acts by reactivating mutant p53 to a WT form. The aim of this study was to investigate COTI-2 as a targeted therapy for TNBC patients. Using a panel of 18 breast cell lines, we carried out MTT assay. p53 protein folding was determined by immunofluorescent staining with the p53 mutant-specific antibody PAb240 and the p53 WT-specific PAb1620. Surface plasmon resonance was used to determine binding affinity of COTI-2 to full length (FL) p53, and the DNA-binding domain (DBD). Flow cytometry was used to measure apoptosis. TNBC cell lines were significantly more responsive to COTI-2 than non-TNBC cell lines (p = 0.04). Furthermore, lower IC50 values were found in p53 mutant compared to p53 WT cells (p = 0.001). COTI-2 was shown to bind to FL and DBD of mutant p53. Treatment resulted in an increase in staining with PAb1620 which coincided with a decrease in staining with PAb240, suggesting refolding of the mutant protein. In addition, COTI-2 was found to induce apoptosis in TNBC cell lines. We conclude that targeting mutant p53 with COTI-2 is a potential approach for treating p53-mutated TNBC.

Helping the Released Guardian: Drug Combinations for Supporting the Anticancer Activity of HDM2 (MDM2) Antagonists.

The protein p53, known as the “Guardian of the Genome”, plays an important role in maintaining DNA integrity, providing protection against cancer-promoting mutations. Dysfunction of p53 is observed in almost every cancer, with 50% of cases bearing loss-of-function mutations/deletions in the TP53 gene. In the remaining 50% of cases the overexpression of HDM2 (mouse double minute 2, human homolog) protein, which is a natural inhibitor of p53, is the most common way of keeping p53 inactive. Disruption of HDM2-p53 interaction with the use of HDM2 antagonists leads to the release of p53 and expression of its target genes, engaged in the induction of cell cycle arrest, DNA repair, senescence, and apoptosis. The induction of apoptosis, however, is restricted to only a handful of p53wt cells, and, generally, cancer cells treated with HDM2 antagonists are not efficiently eliminated. For this reason, HDM2 antagonists were tested in combinations with multiple other therapeutics in a search for synergy that would enhance the cancer eradication. This manuscript aims at reviewing the recent progress in developing strategies of combined cancer treatment with the use of HDM2 antagonists.

Abstract 386: Targeting MDM4 to reactivate p53 signaling in neuroblastoma

Abstract Introduction: Despite aggressive therapeutic regimens, long-term survival of patients with high risk neuroblastoma (NB) is less than 50%. The p53 tumor suppressor gene is mutated in more than 50% of human cancers, however, unlike in most adult tumors, p53 is wild-type and functional in 99% of NB cases at diagnosis. Reactivation of p53 tumor suppressor function by pharmacological inhibition of MDM4, a negative regulator of p53, is a novel strategy in NB treatment. We hypothesized that targeting MDM4 to reactivate p53 signaling would have effective anti-tumor effects on NB. Methods: Levels of gene expression of MDM4 and its association with event-free and overall survival in NB patients were analyzed using the publically available patient datasets in the R2 Genomics Analysis and Visualization Platform. MDM4 gene and protein expression were also examined in six NB cell lines with qPCR and immunoblotting assays. The effects of MDM4 inhibitor NSC207895 on p53 wild-type (p53wt) and mutated cell lines were analyzed for cytotoxicity, growth in soft agar, and expression of apoptosis markers and p53 downstream targets. Moreover, an orthotopic xenograft NB mouse model was used to test the in vivo efficacy of NSC207895. Results: MDM4 expression is significantly correlated with both event-free and overall survival of NB patients in the Kocak (n=476) and Oberthuer (n=251) datasets in the R2 Genomics Analysis and Visualization Platform, with higher expressions correlated with decreased survival (*** p <0.001). All six NB cell lines showed gene and protein expression of MDM4. NSC207895 had strong cytotoxic effects at relatively low concentrations (IC50 from 0.45-2.46 μM) after 48 hours of treatment with p53wt IMR-32, NGP, NB-19 and SH-SY5Y cells, but not with p53 mutant SK-N-AS (IC50:10.55 μM). NSC207895 significantly inhibited the colony formation ability of several NB cells. NSC207895 exposure also led to upregulation of p53 targets p21, Puma, and Bax in p53wt cells but not in p53 mutant cells. In addition, NSC207895 is more potent in p53wt SH-SY5Y (IC50=0.46 uM) than in the p53 knock out SH-SY5Y cells (IC50=0.98 uM). Moreover, NSC207895 significantly enhanced the cytotoxicity of doxorubicin on p53wt cells but not in p53 mutant cells. Above all, NSC207895 inhibited the tumor growth in an orthotopic xenograft NB mouse model (Mann-Whitney test, ** p <0.01) with no significant side-effects. In addition, compared with the placebo group, NSC207895 treated NB tumors demonstrated elevated expression levels of cleaved PARP, together with p53 downstream targets p21, Puma, and Bax, suggesting that NSC207895 induced p53-mediated apoptosis in vivo. Conclusion: Novel MDM4 inhibitor NSC207895 shows anti-tumor efficacy in p53 wild-type NB by reactivating p53 tumor suppressor signaling and inducing p53-mediated apoptosis, both in vitro and in vivo. Inhibition of MDM4 by small molecules is a promising treatment strategy for high risk patients with wild-type p53. Citation Format: Zhenghu Chen, Sarah E. Woodfield, Roma H. Patel, Aryana M. Ibarra, Sanjeev A. Vasudevan. Targeting MDM4 to reactivate p53 signaling in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 386.

Abstract 4440: Genetic knockdown of the E3 ubiquitin ligase Itch increases pancreatic cell line sensitivity to cancer therapeutics: A comparison between different gene editing techniques

Abstract Introduction: E3 ligases are responsible for tagging ubiquitin onto substrates to either activate, relocate or target the substrate for degradation via the proteasome. Previous studies have shown that genetic knockdown of the E3 ubiquitin ligase Itch caused an increase in sensitivity to gemcitabine in pancreatic cancer models both in-vitro (cell lines) and in-vivo (mouse xenografts). Here we test the hypothesis that Itch knockdown sensitises pancreatic cell lines to existing cancer therapeutics, using both transient and stable knockdown methods. Experimental Procedures: Knockdown of Itch in MiaPaCa2 (p53 mutant) and Capan 2 (p53 WT) cells was performed using both siRNA and CRISPR-Cas9 techniques; validated by western blot and RT-PCR. Alongside parental and scrambled controls, cells were treated with either doxorubicin, gemcitabine or γ-radiation. For doxorubicin and gemcitabine treatment, cells were exposed to the drug for 24 hours before it was removed. In the case of irradiation; cells were subject to 0-4Gy of radiation before being passaged onto plates. Cell viability was assessed in 96-well plates using a combination of a Sulforhodamine B assay (SRB) to quantify live cells 4 days post-treatment, and automated cytometry analysis (Celigo) to assess well confluence over each of the 4 days. Clonogenicity was also assessed by aliquoting cells at low initial density and treating the cells with distinct doses dependent on mode of treatment and cell line. After 7 days number of colonies using a colony formation algorithm on Celigo. Data Summary: Transient knockdown of Itch in p53-/- cells was sufficient to decrease cell viability. This decrease in cell viability was further potentiated by the application of gemcitabine, doxorubicin or radiation. Stable knockdown using CRISPR-Cas9 increased MiaPaCa2 cell sensitivity to low-dose doxorubicin and radiation (5-10nM and 0.5-1Gy respectively). Whilst statistically significant, the change in response was not as large as that observed with the transient knockdown. In p53 WT cells there was no significant difference in cell sensitivity upon Itch knockdown. Conclusions: The data from this investigation support the hypothesis of Itch being a possible target for sensitising pancreatic cell lines to cancer therapeutics. However the discrepancies in response to treatment between the transient and stable Itch knockdown scenarios raises the possibility that, in the case of the stable knockdown, the cells have adapted to the absence of Itch. This presents a potential weakness in using CRISPR-Cas9 in long-term experiments where the downstream consequences of gene silencing are not immediate. Citation Format: Oliver J. Read, David J. Harrison, Paul A. Reynolds. Genetic knockdown of the E3 ubiquitin ligase Itch increases pancreatic cell line sensitivity to cancer therapeutics: A comparison between different gene editing techniques [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4440.

Abstract 4278: A comparison of drug sensitivity in isogenic tumor cell lines confirms that cytoprotective autophagy confers intrinsic resistance to cisplatin

Abstract Although autophagy inhibition often increases tumor cell sensitivity to chemotherapy and radiation, these observations do not provide definitive proof that autophagy is actually an intrinsic mechanism of resistance since virtually all tumor cells undergo autophagy in response to therapy. To directly address whether autophagy induction can confer chemoresistance, the current studies utilized a paired set of isogenic cell lines in which (cisplatin-induced) autophagy was either cytoprotective (crp53 cells where p53 was knocked out) or nonprotective (parental p53 wt cells) in function, but where the overall extent of autophagy was essentially identical. Cisplatin sensitivity was significantly lower in the crp53 cells where cisplatin-induced autophagy was protective compared to the parental p53 wt cells where cisplatin-induced autophagy was nonprotective, in support of the premise that cytoprotective autophagy could confer a relative degree of intrinsic resistance to chemotherapy. Accordingly, we can draw the following conclusions: (i) radiation and chemotherapy can promote both protective and nonprotective autophagy in tumor cells; (ii) although p53 can act as an autophagic switch, it cannot be reliably predicted whether autophagy will be protective or nonprotective based solely on p53 function; and (iii) even in the case(s) where autophagy is cytoprotective, it cannot be assumed that autophagy will confer drug or radiation resistance, but only that autophagy inhibition may, in select cases, increase drug or radiation sensitivity. These findings highlight the complexity of efforts to exploit autophagy inhibition as a therapeutic strategy to enhance tumor response to chemotherapy and radiation. Citation Format: Jingwen Xu, Nipa Patel, Tareq Saleh, Yingliang Wu, Santiago Lima, David A. Gewirtz. A comparison of drug sensitivity in isogenic tumor cell lines confirms that cytoprotective autophagy confers intrinsic resistance to cisplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4278.

A therapeutic patent overview of MDM2/X-targeted therapies (2014–2018)

ABSTRACTIntroduction: MDM2 and MDMX proteins provide the inhibition of p53 tumor suppressor, thus allowing for accelerated mutation-driven cancer microevolution. A pharmacological blockade of MDM2/X-p53 interaction results in p53 reactivation in p53wt cells, leading to cancer growth inhibition. Throughout the past 20 years, multiple chemical entities have been proposed to reactivate p53 by antagonizing MDM2/X proteins.Areas covered: This manuscript reviews 2014–2018 therapeutic patents in the field of MDM2/X antagonists and is a continuation of previous reviews on similar matter. The patents covering the use of MDM2/X antagonists in drug combinations are also presented in this review, as they constitute an important trend in the field of cancer treatment with MDM2/X antagonists.Expert opinion: In the years 2014–2018, several previously-known chemical scaffolds have been further developed and disclosed. Importantly, in the same time period, many lead compounds have entered clinical trials for the treatment of cancer patients. Meanwhile, several important reports have pointed to serious limitations of anticancer properties of MDM2 antagonists. As a result, many efforts have been made to seek for positive, synergistic therapeutic effects of combined anti-cancer treatment strategies. One recent example is a dual targeting of MDM2 and additional protein targets by utilizing the PROTAC technology.Trial registration: ClinicalTrials.gov identifier: NCT02264613.Trial registration: ClinicalTrials.gov identifier: NCT02624986.Trial registration: ClinicalTrials.gov identifier: NCT02670044.Trial registration: ClinicalTrials.gov identifier: NCT02545283.

A fluorinated indole-based MDM2 antagonist selectively inhibits the growth of p53wt osteosarcoma cells.

The p53 protein is engaged in the repair of DNA mutations and elimination of heavily damaged cells, providing anticancer protection. Dysregulation of p53 activity is a crucial step in carcinogenesis. This dysregulation is often caused by the overexpression of negative regulators of p53, among which MDM2 is the most prominent one. Antagonizing MDM2 with small molecules restores the activity of p53 in p53 wild-type (p53wt ) cells and thus provides positive outcomes in the treatment of p53wt cancers. Previously, we have reported the discovery of a panel of fluoro-substituted indole-based antagonists of MDM2. Here, we demonstrate the biological activity and stereoselectivity of the most active compound from this series. Both enantiomers of the esterified form of the compound, as well as its corresponding carboxylic acids, were found active in fluorescence polarization (FP) assay, nuclear magnetic resonance (NMR) and microscale thermophoresis (MST) assay, with Ki and KD values around 1μm. From these four compounds, the esterified enantiomer (R)-5a was active in cells, which was evidenced by the increase of p53 levels, the induced expression of p53-target genes (CDKN1A and MDM2), the selective induction of cell cycle arrest, and selective growth inhibition of p53wt U-2 OS and SJSA-1 compared to p53del SAOS-2 cells. The analysis of the crystal structure of human MDM2 in complex with the compound (R)-6a (carboxylic acid of the active (R)-5a compound) revealed the classical three-finger binding mode. Altogether, our data demonstrate the activity of the compound and provide the structural basis for further structure optimization.

In Vitro Characterization of a Potent p53-MDM2 Inhibitor, RG7112 in Neuroblastoma Cancer Cell Lines.

Background: Neuroblastoma (NB) is one of the most aggressive and common solid tumors in pediatrics. Development of effective new therapeutics for NB is in progress to help reduce mortality and morbidity of the disease, particularly in relapsed patients. The tumor suppressor protein p53 plays a critical role in multiple signaling pathways to maintain cellular hemostasis. Dysregulation of p53 protein and/or molecular aberrations have been associated with multiple human malignancies. p53 stability and protein activity is negatively regulated by the E3 ubiquitin ligase (MDM2). Thus, targeting p53-MDM2 protein-protein interaction is a feasible and promising therapeutic strategy to restore the physiological function of p53 in cancer cells. RG7112 is a highly potent and selective small molecule inhibitor, which target a unique structure located within p53 binding motif of MDM2. Methods: The efficacy of RG7112 in vitro using NB cell lines was examined. Two wild-type (WT)-p53 NB cell lines IMR5 and LAN-5, a mutant p53 cell line SK-N-BE(2), and a WT-p53/p14 deleted cell line SH-EP were employed. Results: Data showed that RG7112 significantly reduced cellular viability of IMR5 (IC50, 562 nM) and LAN-5 (IC50, 430 nM), but not SK-N-BE(2) and SH-EP cells. Further, RG7112 restores p53 and p21 protein levels in IMR5 and LAN-5 in a dose-dependent manner. RG7112 induces cell cycle arresting (60% G1 arresting) in WT-p53 cells (IMR5), but no pronounced effect observed in SK-N-BE(2). In this study, 15 different drugs in combination with RG7112 in IMR5 cell line and identified venetoclax (Bcl-2/Bcl-xL inhibitor) as a promising candidate were evaluated. Conclusions: Taken together, these findings provide initial proof-of-concept data for further investigations of RG7112 in selected subgroups of NB patients.

Inhibition of WIP1 Phosphatase in Multiple Myeloma Overcomes Bortezomib Resistance and Promotes Cell Death Via ER Stress-Induced Apoptotic JNK/c-Jun Signaling and Downregulation of Inhibitors of Apoptosis Proteins (IAPs)

Acquired or de novo resistance to the traditional and novel anti-multiple myeloma (MM) agents remains a major treatment obstacle, therefore novel therapies are in need. Wild-type p53-induced phosphatase 1 (WIP1) is an oncogenic serine/threonine phosphatase implicated in silencing of cellular responses to genotoxic stress. WIP1 overexpression was documented in various solid cancers in correlation with aggressive features and poor prognosis. Thus, we studied WIP1 in MM addressing its potential role in mediating resistance and aggressive phenotype.Increased expression of WIP1 was detected in MM cell lines (n=8) and primary samples (n=18) at both mRNA and protein level as compared with normal PBMCs (n=5). Furthermore, a positive correlation between WIP1 and CXCR4 levels (p<0.02, R2=0.5) was revealed. The latter is a well-known oncogenic receptor in MM. WIP1 expression levels were significantly up-regulated following bortezomib (Bort) treatment. Using MM cell lines with acquired resistance to Bort (RPMI8226BortRes and CAGBortRes), a higher induction of WIP1 upon Bort exposure could be demonstrated, suggesting a possible role for WIP1 in the acquisition of MM drug resistance to proteasome inhibitors. WIP1 was also upregulated in MM cells cultured on human BM stroma (BMSC) known to protect the tumor cells from Bort-induced apoptosis, further supporting its function in mediating resistance.GSK2830371 (GSK), a novel allosteric inhibitor of WIP1, significantly suppressed MM cells proliferation (p<0.01) and induced apoptosis, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization (ψm), caspase 3 and PARP cleavage, and DNA fragmentation. Moreover, combined treatment with GSK and Bort synergistically potentiated cell death in both Bort-sensitive and resistant MM cells and overcame BMSC protection (CI<0.5). The robust apoptosis induced by Bort/GSK treatment was accompanied by increased mitochondrial ROS accumulation, subsequent mitochondrial destabilization and extensive DNA damage. GSK treatment resulted in a reduction of WIP1 basal expression and abrogated WIP1 induction upon Bort treatment. Thus, we defined that GSK can regulate WIP1 expression in MM cells.To determine the molecular mechanism of Bort/GSK synergism we performed gene and protein expression analysis. Combination of both agents significantly reduced expression of anti-apoptotic proteins such as cIAP1, cIAP2, XIAP and Survivin. Previous studies indicate that maintaining IAPs expression is part of an adaptive unfolded protein response that promotes MM survival upon Bort-induced endoplasmic reticulum (ER) stress. Therefore, it is conceivable that targeting IAPs upon WIP1 inhibition may overcome protective responses, inducing unresolved ER stress and MM cell death.Indeed, we found that combination of Bort and GSK significantly enhanced ER stress, as indicated by increase in the pro-apoptotic factors ATF4, CHOP and GADD34. Concomitantly, mitosis-inducing factors Cyclin B1, CDK1 and PLK1 were prominently reduced upon Bort/GSK treatment.To assess the potential role of p53 activation in GSK-mediated effects, p53-stabilizing agents nutlin3a and PRIMA1 were applied in combination with WIP1 inhibition. We observed a significant (p<0.01) increase in the responsiveness of both p53WT and p53mut MM cells to GSK-mediated apoptosis. Consistently, combined GSK/Bort treatment upregulated p53 targets, including PUMA, NOXA, GADD45A and p21 genes. These data suggest that p53 may potentiate the WIP1 inhibition mediated stress induction.Finally, we assessed the signaling pathways that may be involved in WIP1 mediated cessation of stress response. GSK profoundly augmented Bort-induced phosphorylation of JNK and c-Jun, without affecting p38 phosphorylation. Accordingly, JNK inhibitor SP600125 successfully reverted both the apoptosis and the downregulation of IAPs induced by Bort/GSK treatment. Altogether, these results identify pro-apoptotic JNK/c-Jun signaling being preferential target of WIP1 in the process of dampening Bort-induced stress response.To conclude, we disclose the role of WIP1 in blunting stress response and promoting resistance to bortezomib. Collectively, WIP1 suppression prevents MM cell adaptation and recovery upon ER stress. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. [Display omitted] DisclosuresPeled:Cellect Biotherapeutics Ltd: Consultancy.

Prolonged Idasanutlin (RG7388) Treatment Leads to the Generation of p53-Mutated Cells.

The protein p53 protects the organism against carcinogenic events by the induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually all cancers inactivate p53 either by mutations/deletions of the TP53 gene or by boosting negative regulation of p53 activity. The overexpression of MDM2 protein is one of the most common mechanisms utilized by p53wt cancers to keep p53 inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer potential in vitro and is now tested in clinical trials. However, the prolonged treatment of p53wt cells with MDM2 antagonists leads to the development of secondary resistance, as shown first for Nutlin-3a, and later for three other small molecules. In the present study, we show that secondary resistance occurs also after treatment of p53wt cells with idasanutlin (RG7388, RO5503781), which is the only MDM2 antagonist that has passed phase II and entered phase III clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the induction of p21 expression and potent cell cycle arrest in all the three cell lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 OS cells could be cultured for long time periods in the presence of the drug. This prolonged treatment led to the generation of p53-mutated resistant cell populations. This resistance was generated de novo, as evidenced by the utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin presents much improved activities compared to its precursor, it displays the similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug-resistant subpopulations.

Inhibition of kinesin family member 20B sensitizes hepatocellular carcinoma cell to microtubule-targeting agents by blocking cytokinesis.

Kinesin family member 20B (KIF20B, also known as MPHOSPH1) is a kinesin protein that plays a critical role in cytokinesis. Previously, we and others have demonstrated the oncogenic role of KIF20B in several cancers; however, the exact mechanisms underlying its tumorigenic effects remain unclear. Herein, we showed overexpression of KIF20B in human hepatocellular carcinoma (HCC) and reported a negative correlation between KIF20B level and prognosis of patients. Mechanistically, reducing KIF20B blockades mitotic exit of HCC cells at telophase in a spindle assembly checkpoint independent way. Importantly, reducing KIF20B acts synergistically with three microtubule‐associated agents (MTA) to p53‐ or p14ARF‐dependently suppress p53‐wt or p53‐null HCC cells. In addition to taxol, reducing KIF20B also enhanced the toxicity of two chemotherapeutic drugs, hydroxycamptothecin and mitomycin C. In conclusion, we found a novel mechanism in that blocking cytokinesis by KIF20B inhibition increases the efficacy of MTA; our results thus suggested a dual‐mitotic suppression approach against HCC by combining MTA with KIF20B inhibition, which simultaneously blocks mitosis at both metaphase and telophase.

Growth factor independence 1 expression in myeloma cells enhances their growth, survival, and osteoclastogenesis

BackgroundIn spite of major advances in treatment, multiple myeloma (MM) is currently an incurable malignancy due to the emergence of drug-resistant clones. We previously showed that MM cells upregulate the transcriptional repressor, growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSCs) that induces prolonged inhibition of osteoblast differentiation. However, the role of Gfi1 in MM cells is unknown.MethodsHuman primary CD138+ and BMSC were purified from normal donors and MM patients’ bone marrow aspirates. Gfi1 knockdown and overexpressing cells were generated by lentiviral-mediated shRNA. Proliferation/apoptosis studies were done by flow cytometry, and protein levels were determined by Western blot and/or immunohistochemistry. An experimental MM mouse model was generated to investigate the effects of MM cells overexpressing Gfi1 on tumor burden and osteolysis in vivo.ResultsWe found that Gfi1 expression is increased in patient’s MM cells and MM cell lines and was further increased by co-culture with BMSC, IL-6, and sphingosine-1-phosphate. Modulation of Gfi1 in MM cells had major effects on their survival and growth. Knockdown of Gfi1 induced apoptosis in p53-wt, p53-mutant, and p53-deficient MM cells, while Gfi1 overexpression enhanced MM cell growth and protected MM cells from bortezomib-induced cell death. Gfi1 enhanced cell survival of p53-wt MM cells by binding to p53, thereby blocking binding to the promoters of the pro-apoptotic BAX and NOXA genes. Further, Gfi1-p53 binding could be blocked by HDAC inhibitors. Importantly, inoculation of MM cells overexpressing Gfi1 in mice induced increased bone destruction, increased osteoclast number and size, and enhanced tumor growth.ConclusionsThese results support that Gfi1 plays a key role in MM tumor growth, survival, and bone destruction and contributes to bortezomib resistance, suggesting that Gfi1 may be a novel therapeutic target for MM.

Chemosensitivity of human colon cancer cells is influenced by a p53-dependent enhancement of ceramide synthase 5 and induction of autophagy

Resistance against chemotherapy is a life-threatening complication in colon cancer therapy. To increase response rate, new additional targets that contribute to chemoresistance are still needed to be explored. Ceramides, which belong to the group of sphingolipids, are well-known regulators of cell death and survival, respectively. Here, we show that in human wild-type (wt) p53 HCT-116 colon cancer cells treatment with oxaliplatin or 5-fluorouracil (5-FU) leads to a strong increase in ceramide synthase 5 (CerS5) expression and C16:0-ceramide levels, which was not shown in HCT-116 lacking p53 expression (HCT-116 p53−/−). The increase in CerS5 expression occurs by stabilizing CerS5 mRNA at the 3′-UTR. By contrast, in the p53-deficient cells CerS2 expression and CerS2-related C24:0- and C24:1-ceramide levels were elevated which is possibly related to enhanced polyadenylation of the CerS2 transcript in these cells. Stable knockdown of CerS5 expression using CerS5-targeting shRNA led to an increased sensitivity of HCT-116 p53wt cells, but not of p53−/− cells, to oxaliplatin and 5-FU. Enhanced sensitivity was accompanied by an inhibition of autophagy and inhibition of mitochondrial respiration in these cells. However, knockdown of CerS2 had no significant effects on chemosensitivity of both cell lines.In conclusion, in p53wt colon cancer cells chemosensitivity against oxaliplatin or 5-FU could be enhanced by downregulation of CerS5 expression leading to reduced autophagy and mitochondrial respiration.

PO-037 Targeting mutant p53 with COTI-2: a new approach for the treatment of patients with triple-negative breast cancer?

IntroductionThe identification of a targeted therapy for triple-negative breast cancer (TNBC) is one of the most urgent needs in breast cancer therapeutics. Since the p53 gene is mutated in approx. 80% of TNBC tumours, it is an attractive target. COTI-2 is a clinical stage, small molecule which claims to target p53. The aim of this study was to investigate COTI-2 as a new treatment for TNBC.Material and methodsCell viability was determined by MTT assay. p53 protein levels were quantified by ELISA and immunofluorescent staining. P53 binding kinetics were measured by Surface Plasmon Resonance. Apoptosis was measured using Annexin V-FITC Apoptosis Detection Kit. Caspase 3/7 was measured by CellEventCaspase-3/7Green Flow Cytometry Assay Kit. CI values were calculated using Calcusyn software.Results and discussionsUsing a panel of 18 breast cell lines, TNBC cell lines were more responsive to COTI-2 than non-TNBC cells (p=0.04). Lower IC50 values for COTI-2 were found in p53 mutant vs p53 WT cells (p=0.001). Additionally, the higher the endogenous p53 protein, the more sensitive the cell line was to COTI-2 (p=0.035, r=−0.51, n=18). By staining with antibodies specific for folded WT p53 (PAb1620) or unfolded mutant p53 (PAb240), we showed that COTI-2 can induce refolding of mutant p53. Moreover, by SPR, we showed that COTI-2 binds to fl-mut-p53 protein, in a concentration dependent manner.In addition to inhibiting proliferation, COTI-2 induced apoptosis in a concentration dependent manner. Furthermore, inhibition of caspase activity with Z-VAD-FMK reduced apoptosis, suggesting that COTI-2 induces caspase-dependent apoptosis. Accordingly, COTI-2 induced a significant increase in caspase 3/7.In an effort to enhance response, COTI-2 was combined with a number of cytotoxic agents. Highly synergistic growth inhibition, i.e. CI <1, was found when COTI-2 was combined with doxorubicin in 6 different cell lines. In addition, COTI-2 plus docetaxel or eribulin was synergistic in 4/6 cell lines, plus carboplatin was synergistic in 3/6, while plus cisplatin was synergistic in 2/6. Finally, we compared response to COTI-2 with that of APR-246, the best studied p53 reactivating compound. Overall, the mean COTI-2 IC50 value was 64 fold lower than that for APR-246 (p=0.0006). Furthermore, no correlation was seen between response to COTI-2 or APR-246, suggesting that the compounds act differently in inhibiting cell growth.ConclusionWe conclude that targeting mutant p53 with COTI-2 is a potential new approach for treating p53-mutated TNBC.

Abstract 2413: TP53 gene mutations differently regulate ovarian cancer metabolism: Ex vivo and in vitro studies

Abstract Background: Even in the presence of oxygen, cancer cells tend to metabolize glucose into lactate instead of undergoing oxidative phosphorylation. Knowing that glucose is crucial for tumor's proliferation, new patient's treatment strategies tend to combine chemotherapy to hypoglycemic drugs. p53 is a tumor suppressor and key regulator of glycolysis in cancer cells, however highly mutated in tumors. In ovarian cancer, the majority of studies concerning p53 mutations focus on the DNA binding domain since the majority of hotspot mutations affects this region of the gene. However, mutations affecting other regions such as the proline rich domain linked directly to apoptosis may also affect the protein's expression and activity. The aim of this study is to investigate the effect of various positions of mutations in TP53 gene on glycolysis, apoptosis and transcription of p53 target genes. Material and Methods: this study was divided into two parts. The in vitro study was done using 3 ovarian cancer cell lines SKOV-3 (p53 null), Igrov-1 (WT p53) and Ovcar-3 (DNA domain mutant p53), along with the transfection of SKOV-3 cells by three different vectors: WTp53, P72R p53 and R249S p53. The ex vivo part was performed on 30 ovarian cancer biopsies. The biopsies were subjected to Tp53 immunohistochemistry and sequencing to study the mutations and their effect on p53 expression. The effect of Tp53 mutations on glucose metabolism was assayed by glucose consumption measurements, lactate production and mRNA expression of various glycolytic enzymes. To study the effect of mutations on the transcriptional activity of p53, p21 and MDM2 expression was quantified in these cells. Annexin V assay was performed on transfected SKOV-3 to study the apoptotic activity of WT and mutant p53. Results: DNA binding domain mutant cells (Ovcar-3 and R249S SKOV-3) presented a 30% increase in glucose consumption and lactate production when compared to IGROV-1 and WT SKOV-3. P72R SKOV-3 exhibited the same pattern as WTp53 cells. The increase in the mRNA expression of glucose transporters GLUT-1 and 3 and of the glycolytic enzymes PK, PFK, G6PDH and aldolase in cells with DNA binding domain mutations varied from 20 to 30%, whereas the levels of PDHa and TIGAR decreased significantly (40%). The transcriptional activity of DNA binding domain mutant cells showed a 50% decrease in comparison to WT and P72R mutant cells. The apoptotic activity of p53 was unaltered in P72R transfected SKOV-3 cells. The ex vivo results revealed a high frequency of the P72R variant in the ovarian biopsies (69%), associated with an overexpression of the p53 protein. The glycolytic and transcriptional patterns of cells extracted from these biopsies were similar to those obtained in transfected cells. Conclusion: Though both DNA-binding and proline-rich domains mutations lead to p53 overexpression, only the first type promotes Warburg effect and could have the worst patient's prognosis. Citation Format: Stephanie Antoun, David Atallah, Roula Tahtouh, Malak Moubarak, Nada Alaeddine, George Hilal. TP53 gene mutations differently regulate ovarian cancer metabolism: Ex vivo and in vitro studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2413.

Abstract 5523: Allele-specific restoration of biological function of the mutant p53 by Baylis-Hillman adducts

Abstract p53 is the most frequently mutated gene in human cancer, and the p53R175 mutant is the third most frequently found missense mutant that confers resistance to anticancer drugs and apoptosis. Therefore, small-molecule reactivation of mutant p53 function(s) represents an important and novel anticancer strategy. We have previously reported that induction of oxidative protein modifications primarily underlies the mechanistic conversion of mutant to wt or wt-like p53 forms (Eur J Medchem 107:233, 2016; Int J Oncol 48:1426, 2016). Here, we describe the identification of Baylis-Hillman (BH) adducts as potent and novel mutant p53 reactivators through cell-based, high-throughput small-molecule screening procedures. An extensive structure-activity relationship studies indicated that carbon-carbon double bond and the presence of an electron-withdrawing substituent at the aromatic ring were essential for the activity. Cell lines used in this study includes SKBR3, AU565 (p53R175H), MCF7 (p53 wt), MDA-MB-231 (p53del), MDA-MB-468 (p53R273H) and MCF10a normal breast epithelial cells. BH adducts displayed preferential cytotoxicity against cancer cells expressing the allele-specific R175H mutation (SKBR3 and AU565) in comparison with the wt p53 or p53-null cancer cell lines. Additionally, the BH adducts did not show significant cytotoxicity against normal cells (MCF10a). Further, we developed isogenic cell lines expressing the p53R175H in the p53-null H1299 lung cancer cells and evaluated their sensitivities towards BH adducts; these models again verified the greater drug efficacy. To prove the hypothesis the higher efficacy of BH adduct stems from the reactivation of mutant p53, the conformation-specific antibodies (Pab1620 for wt and Pab420 for mutant) were employed. Immunoprecipitation and immunostaining using confocal microscopy showed that BH adduct treatment of p53R175H cells resulted in a marked reciprocal loss of mutant protein and gradual increase of wt-like protein. EMSA revealed a time-dependent restoration of DNA-binding of the mutant p53, which was accompanied by a distinct upregulation of pro-apoptotic signaling pathways mediated by PUMA, Bax, p21cip1 and MDM2 proteins. A strong G2/M arrest and significant apoptosis were observed with BH adduct in a time and concentration-dependent manner. Other data showed that BH adduct restores the wt-structure and function by covalently binding with the mutant p53R175H. In conclusion, BH adduct is an attractive lead compound for further development because of its potent p53 reactivation of nonfunctional mutant protein (supported by CPRIT grants RP130266 and RP170207 to KSS). Citation Format: Surendra R. Punganuru, Viswanath Arutla, Hanumantha Rao Madala, Kalkunte S. Srivenugopal. Allele-specific restoration of biological function of the mutant p53 by Baylis-Hillman adducts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5523.

PO-172 Role of lithocholic acid-induced cell signalling in oesophageal cancer progression model

IntroductionA p53-checkpoint operates in the development of oesophageal adenocarcinoma (OAC) defined by prevalent TP53 gene mutations in patients with Barrett’s and high-grade dysplasia (HGD). The molecular genetic relationship between OAC and its precursor lesion, Barrett’s oesophagus, is poorly understood. The effective therapeutic targets should focus on early mutations of the disease, which become clonal in the later stage. Mutational context of some specific single nucleotide variants are common throughout the progression of the disease, suggesting exposure to common mutagens throughout the progression course.Material and methodsTP53 null or truncated cell lines were generated by targeting exon 4 of TP53 gene using CRISPR/Cas9 technology. Cell cycle analysis and Annexin V5 stained cells were analysed by FACS after treatment with lithocholic acid (LCA) to recapitulate the reflux of bile acids in OAC. Changes in proteins levels and signalling pathways upon LCA exposure were identified using mass spectrometry and validated with western blotting. Immunohistochemistry (IHC) analysis of OAC and normal tissues was performed to check the expression of identified bile acid-induced genes in tumours.Results and discussionsOesophageal Barrett’s wt-p53 cell line CPA was used to define the effects of TP53 gene ablation on stress responses, cell survival, and mutation rates. FACS analysis revealed TP53 null cells sensitivity towards LCA via apoptotic pathway. Mass spectrometry analysis identified disrupted NDRG1 and TGFβ pathways. SMAD4 driver mutations have previously been reported exclusively to OAC, which provide a clear genetic boundary between OAC and HGD. Western blot analysis after LCA exposure showed increased levels of SMAD4-independent of p53 pathway. SMAD4 depletion in TP53 null cells stimulates cell-migration in the presence of inhibitory levels of LCA, corroborates the role of SMAD4 loss in metastasis. Loss of p53 also upregulates NDRG1 which increases migration in presence of LCA and was found to be a pro-invasive factor. IHC staining of OAC, lymph node and normal tissues showed heterogeneous expression of NDRG1 in tumour, mostly in membrane, suggesting its dynamic expansion on LCA exposure in vivo.ConclusionThe recurrent reflux of bile acids in Barrett’s cells, especially LCA, could sensitise cells to harbour further mutations. LCA induces p53-independent upregulation of SMAD4. Depletion of NDRG1 and SMAD4 in p53 null cells stimulates cell-migration in the presence of LCA, corroborating their role in metastasis.

Abstract 885: Anthracycline-induced apoptosis through caspase3/7 activity is p53-dependent in bladder cancer cells

Abstract Doxorubicin (Dox) is a chemotherapy drug widely used for treatment of several cancers including bladder cancer. However, cardiovascular side effects caused by doxorubicin-induced cardiotoxicity has necessitated the synthesis and validation of next generation chemotherapy drugs that are as effective but not cardiotoxic. AD 312 (N-Nitrosureidodaunorubicin; Daunomustine®, Paradox Pharmaceuticals, Inc.) and AD 198 (N- benzyladriamycin-14-valerate; Benzarubicin®, Paradox Pharmaceuticals, Inc.) are novel anthracyclines with confirmed non-cardiotoxicity in mice. Compared to Dox, AD 312 and AD 198 induce apoptosis in cells through distinct mechanisms. The tumor suppressor gene p53 is a key regulator of cell-cycle arrest and apoptosis. Dysregulation of p53 expression and function was identified in over 70% of the bladder cancers and reported as a potential target for therapy. As a result, small molecule compounds that can restore mutated p53 function to wild-type, including PRIMA-1, are under intensive investigation. In this study, we have compared the efficacy of AD 312 and AD 198 to Dox to induce apoptosis in human bladder transitional cell carcinoma (TCC) cells by MTT assay and assessed the possible mechanisms of anthracyclines-induced apoptosis in TCC cells expressing wild-type (wt) p53 (RT4, SW780) and mutated (mt) p53 (UM-UC-3, 5637, J82, and TCCSUP) protein. We have also investigated the effects of co-treatment of anthracyclines with PRIMA-1 in J82 cells, as a representative mt-p53 TCC cell line, and compared the co-treatment effects in RT4, as a representative wt-p53 cell line. Our results demonstrated that anthracyclines inhibited cell viability of all tested TCC cells in vitro, with AD 198 demonstrating highest efficacy. Furthermore, anthracyclines upregulated p53 protein expression in wt-p53 RT4 and SW780 cells, but not in tested mt-p53 UM-UC-3, 5637, J82, and TCCSUP cells. A strong correlation was observed between p53 mutation status and activation of caspase 3/7 and cleavage of PARP. The upregulated caspase 3/7 activity and PARP cleavage was observed in wt-p53, but not in mt-p53 TCC cells. AD198 blocked c-myc expression in mt-p53 TCC cells and no expression of c-myc was detected in wt-p53 TCC cells. Furthermore, sensitivity to AD 198 treatment in mt-p53 J82 cells was significantly improved by a co-treatment with PRIMA-1. However, PRIMA-1 had no effect on cell viability in RT4, the wt-p53 TCC cells. Overall, our results demonstrated that AD 312 and AD 198 are potent inhibitors of bladder cancer cells in vitro and are promising candidates for new therapeutic strategies to replace Dox for bladder cancer treatment. PRIMA-1 even further sensitized mt-p53 cells to AD 198 treatment and thereby this co-treatment improved the inhibition of bladder cancer in vitro. Citation Format: Sony Pandey, Leonard Lothstein, Maria Cekanova. Anthracycline-induced apoptosis through caspase3/7 activity is p53-dependent in bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 885.

Synthesizing a Genetic Sensor Based on CRISPR-Cas9 for Specifically Killing p53-Deficient Cancer Cells.

Cancer is still one of the greatest medical challenges in the world. The p53 protein plays an important role in the process of cancer formation. In addition, p53 is found as the most common mutant gene in cancers. Because of the central role of p53 in oncology, it is necessary to construct effective sensors to detect this protein. However, there are few methods to detect wild type p53 protein (WTP53) or to distinguish the wild type and mutant p53 proteins. In our study, we designed and constructed a p53 genetic sensor that detected the expression of WTP53 in cells. Moreover, we combined the p53 sensor with diphtheria toxin using the CRISPR-Cas9 system to construct a p53 genetic sensor that specifically killed p53-deficient cells such as tumor cells. Our study therefore developed a new way to treat cancers by using an available genetic sensor based on p53 protein.

A Recombinant Fragment of Human Surfactant Protein D induces Apoptosis in Pancreatic Cancer Cell Lines via Fas-Mediated Pathway.

Human surfactant protein D (SP-D) is a potent innate immune molecule, which is emerging as a key molecule in the recognition and clearance of altered and non-self targets. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis via p53-mediated apoptosis pathway in an eosinophilic leukemic cell line, AML14.3D10. Here, we report the ability of rfhSP-D to induce apoptosis via TNF-α/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53mt), MiaPaCa-2 (p53mt), and Capan-2 (p53wt) cell lines. Treatment of these cell lines with rfhSP-D for 24 h caused growth arrest in G1 cell cycle phase and triggered transcriptional upregulation of pro-apoptotic factors such as TNF-α and NF-κB. Translocation of NF-κB from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed via immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed via qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed via western blot at 48 h. The cell number following the rfhSP-D treatment was reduced in the order of Panc-1 (~67%) > MiaPaCa-2 (~60%) > Capan-2 (~35%). This study appears to suggest that rfhSP-D can potentially be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype.