Negative Regulator Of P53 Research Articles

Fibrinogen activates focal adhesion kinase (FAK) promoting colorectal adenocarcinoma growth

BackgroundWe previously showed that fibrinogen is a major determinant of the growth of a murine model of colorectal cancer (CRC). ObjectiveOur aim was to define the mechanisms coupling fibrin(ogen) to CRC growth. ResultsCRC tumors transplanted into the dorsal subcutis of Fib− mice were less proliferative and demonstrated increased senescence relative to those grown in Fib+ mice. RNA‐seq analyses of Fib+ and Fib− tumors revealed 213 differentially regulated genes. One gene highly upregulated in tumors from Fib− mice was stratifin, encoding 14‐3‐3σ, a master regulator of proliferation/senescence. In a separate cohort, we observed significantly increased protein levels of 14‐3‐3σ and its upstream and downstream targets (i.e., p53 and p21) in tumors from Fib− mice. In vitro analyses demonstrated increased tumor cell proliferation in a fibrin printed three‐dimensional environment compared with controls, suggesting that fibrin(ogen) in the tumor microenvironment promotes tumor growth in this context via a tumor cell intrinsic mechanism. In vivo analyses showed diminished activation of focal adhesion kinase (FAK), a key negative regulator of p53, in Fib− tumors. Furthermore, nuclear magnetic resonance–based metabolomics demonstrated significantly reduced metabolic activity in tumors from Fib− relative to Fib+ mice. Together, these findings suggest that fibrin(ogen)‐mediated engagement of colon cancer cells activates FAK, which inhibits p53 and its downstream targets including 14‐3‐3σ and p21, thereby promoting cellular proliferation and preventing senescence. ConclusionsThese studies suggest that fibrin(ogen) is an important component of the colon cancer microenvironment and may be exploited as a potential therapeutic target.

UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11.

Simple SummaryHerein, the oncogenic role of UBE2M as an E2 NEDD8-conjugating enzyme was explored in hepatocellular carcinoma (HCC) cells, since neddylation plays a critical role in tumorigenesis. To address this issue, human tissue array and TCGA analysis were conducted in HCCs to find overexpression of UBE2M in HCCs. In addition, a differential profile was confirmed in UBE2M-depleted HepG2 cells. Furthermore, UBE2M depletion activated p53 expression and stability, while the ectopic expression of UBE2M disturbed p53 activation and enhanced degradation of exogenous p53 mediated by MDM2 in HepG2 cells via binding to MDM2 and ribosomal protein L11 by immunoprecipitation and immunofluorescence. These findings provide evidence that UBE2M is critically involved in liver cancer progression as a p53 negative regulator by binding to MDM2 and ribosomal protein L11.Though UBE2M, an E2 NEDD8-conjugating enzyme, is overexpressed in HepG2, Hep3B, Huh7 and PLC/PRF5 HCCs with poor prognosis by human tissue array and TCGA analysis, its underlying oncogenic mechanism remains unclear. Herein, UBE2M depletion suppressed viability and proliferation and induced cell cycle arrest and apoptosis via cleavages of PARP and caspase 3 and upregulation of p53, Bax and PUMA in HepG2, Huh7 and Hep3B cells. Furthermore, UBE2M depletion activated p53 expression and stability, while the ectopic expression of UBE2M disturbed p53 activation and enhanced degradation of exogenous p53 mediated by MDM2 in HepG2 cells. Interestingly, UBE2M binds to MDM2 or ribosomal protein L11, but not p53 in HepG2 cells, despite crosstalk between p53 and UBE2M. Consistently, the colocalization between UBE2M and MDM2 was observed by immunofluorescence. Notably, L11 was required in p53 activation by UBE2M depletion. Furthermore, UBE2M depletion retarded the growth of HepG2 cells in athymic nude mice along with elevated p53. Overall, these findings suggest that UBE2M promotes cancer progression as a p53 negative regulator by binding to MDM2 and ribosomal protein L11 in HCCs.

Investigation of the molecular causes underlying physical abnormalities in Diamond‐Blackfan anemia patients with RPL5 haploinsufficiency

Diamond-Blackfan anemia (DBA) is a genetic disorder caused by mutations in genes encoding ribosomal proteins and characterized by erythroid aplasia and various physical abnormalities. Although accumulating evidence suggests that defective ribosome biogenesis leads to p53-mediated apoptosis in erythroid progenitor cells, little is known regarding the underlying causes of the physical abnormalities. In this study, we established induced pluripotent stem cells from a DBA patient with RPL5 haploinsufficiency. These cells retained the ability to differentiate into osteoblasts and chondrocytes. However, RPL5 haploinsufficiency impaired the production of mucins and increased apoptosis in differentiated chondrocytes. Increased expression of the pro-apoptotic genes BAX and CASP9 further indicated that RPL5 haploinsufficiency triggered p53-mediated apoptosis in chondrocytes. Murine double minute 2 (MDM2), the primary negative regulator of p53, plays a crucial role in erythroid aplasia in DBA patient. We found the phosphorylation level of MDM2 was significantly decreased in RPL5 haploinsufficient chondrocytes. In stark contrast, we found no evidence that RPL5 haploinsufficiency impaired osteogenesis. Collectively, our data support a model in which RPL5 haploinsufficiency specifically induces p53-mediated apoptosis in chondrocytes through MDM2 inhibition, which leads to physical abnormalities in DBA patients.

Inhibition of nonsense-mediated decay rescues p53β/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.

Andrographolide Induces Apoptosis in Gastric Cancer Cells through Reactivation of p53 and Inhibition of Mdm-2.

Andrographolide is a labdane diterpenoid isolated from Andrographis paniculata. The plant extract and andrographolide has long been used in traditional medicine practices mainly for gastrointestinal diseases and improving liver function. Andrographolide has shown various pharmacological properties including anti-inflammatory, antioxidant and anticancer activity. This study evaluated the effect of andrographolide on proliferation of human gastric carcinoma cells in relevance to p53 and Mdm-2 pathways. Andrographolide inhibited the proliferation of SGC7901 and AGS cells in a dose-dependent manner with estimated IC50 values 38 and 44 μM respectively. Effect of andrographolide on p53 activity was ascertained by using a p53 activator (RITA) which showed synergistic inhibition of cell proliferation. While andrographolide when used in combination with a p53 inhibitor (pifithrin-α) showed potent restriction over its response. Andrographolide caused decrease in mitochondrial membrane potential as an indicator of apoptotic activity. Andrographolide activated the expression of p53 protein and gene and downregulated the levels of Mdm-2 (negative regulator of p53). Andrographolide inhibited the colony formation abilities in SGC7901 in a p53-dependent manner followed by induction of mitochondrial intrinsic apoptosis through activation of caspases-9 and -3, cleavage of PARP, and inhibition of pro-apoptotic Bcl-2. Andrographolide induced p53 mediated apoptosis in gastric carcinoma cells which adds to a novel approach in anticancer therapies.

Ribosomal L1 domain-containing protein 1 coordinates with HDM2 to negatively regulate p53 in human colorectal Cancer cells

BackgroundRibosomal L1 domain-containing protein 1 (RSL1D1) is a nucleolar protein that is essential in cell proliferation. In the current opinion, RSL1D1 translocates to the nucleoplasm under nucleolar stress and inhibits the E3 ligase activity of HDM2 via direct interaction, thereby leading to stabilization of p53.MethodsGene knockdown was achieved in HCT116p53+/+, HCT116p53−/−, and HCT-8 human colorectal cancer (CRC) cells by siRNA transfection. A lentiviral expression system was used to establish cell strains overexpressing genes of interest. The mRNA and protein levels in cells were evaluated by qRT-PCR and western blot analyses. Cell proliferation, cell cycle, and cell apoptosis were determined by MTT, PI staining, and Annexin V-FITC/PI double staining assays, respectively. The level of ubiquitinated p53 protein was assessed by IP. The protein-RNA interaction was investigated by RIP. The subcellular localization of proteins of interest was determined by IFA. Protein-protein interaction was investigated by GST-pulldown, BiFC, and co-IP assays. The therapeutic efficacy of RSL1D1 silencing on tumor growth was evaluated in HCT116 tumor-bearing nude mice.ResultsRSL1D1 distributed throughout the nucleus in human CRC cells. Silencing of RSL1D1 gene induced cell cycle arrest at G1/S and cell apoptosis in a p53-dependent manner. RSL1D1 directly interacted with and recruited p53 to HDM2 to form a ternary RSL1D1/HDM2/p53 protein complex and thereby enhanced p53 ubiquitination and degradation, leading to a decrease in the protein level of p53. Destruction of the ternary complex increased the level of p53 protein. RSL1D1 also indirectly decreased the protein level of p53 by stabilizing HDM2 mRNA. Consequently, the negative regulation of p53 by RSL1D1 facilitated cell proliferation and survival and downregulation of RSL1D1 remarkably inhibited the growth of HCT116p53+/+ tumors in a nude mouse model.ConclusionWe report, for the first time, that RSL1D1 is a novel negative regulator of p53 in human CRC cells and more importantly, a potential molecular target for anticancer drug development.

OMRT-6. Optimizing MDM2 inhibition for the treatment of high-grade glioma

Over 80% of high-grade gliomas have alterations in members of the p53 pathway, a central regulator of cell cycle progression and apoptosis that becomes activated in response to cellular stress and DNA damage. For tumors that retain wild-type p53, pathway deregulation frequently occurs through the amplification of negative regulators of p53, including the E3 ubiquitin ligase MDM2. The p53/MDM2 interaction axis has served as basis for the development of several classes of MDM2 inhibitors, with AMG232 being the most potent molecule currently undergoing clinical evaluation. As the effects of MDM2 inhibition (MDM2i) remain poorly understood in high-grade glioma, we performed genomic and transcriptomic analyses in patient-derived models to better characterize sensitive tumors and identify putative biomarkers of drug response. Treatment with AMG232 impaired the growth of cell lines with wild-type p53 status, particularly in tumors with additional amplification of MDM4 or PPM1D activating mutations. Treatment with AMG232 upregulated both cell cycle arrest and apoptotic cellular responses, as measured by annexin V/PI staining and immunoblotting. Interestingly, the dynamics of these two downstream p53 signaling axis were dependent on treatment duration across models. In addition to p53 pathway activation and apoptotic induction, RNA-sequencing revealed MDM2i to be associated with the activation of oncogenic MAPK and KRAS signaling as well as epithelial to mesenchymal transition markers. In most solid tumors, resistance to MDM2i is mainly mediated by acquisition of p53 inactivating mutations. We hypothesized that resistance mechanisms in glioma may be partially driven by transcriptional changes, as these tumors consist of subpopulations with diverse cell differentiation states. By chronic AMG232 treatment, we have developed in vitro and in vivo models of acquired MDM2i resistance that are not mediated by p53 inactivation. Ongoing work is focused on characterizing the transcriptional profile of these cells to identify transcriptional changes leading to decreased drug response.

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and invivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how invivo tissue-specific phenotypes emerge in ribosomopathies.

Puma, noxa, p53, and p63 differentially mediate stress pathway induced apoptosis

Cellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.

Open Label Phase I Study of Single Agent Oral RG7388 (idasanutlin) in Patients with Polycythemia Vera and Essential Thrombocythemia

Background: Polycythemia vera (PV) and essential thrombocythemia (ET) are associated with an increased risk of thrombosis, progression to myelofibrosis (MF) and significant symptom burden. While hydroxyurea, interferon, and JAK2 inhibitors are used to treat these myeloproliferative neoplasms (MPNs), hematopoietic stem cell-depleting therapies may result in an alternative mechanism to alter disease course and improve outcome. MDM2, a negative regulator of P53, is overexpressed in CD34+ MPN cells harboring wild type P53. It is the target of idasanutlin (IDA), an oral MDM2 antagonist that results in up-regulation of P53 and down-stream activators of apoptosis. Preclinical data from our laboratory group supports the clinical evaluation of IDA in patients (pts) with ET/PV (Lu et al Blood. 2014;124(5):771-9). We evaluated the safety and tolerability of IDA in a phase I dose escalation trial of pts with JAK2V617F-positive ET or PV [NCT02407080].Methods: 13 pts (1 withdrew prior to treatment) resistant/intolerant to hydroxyurea and/or interferon therapy and without prior JAK2 inhibitor therapy were enrolled in a single institution phase I trial of oral IDA at two dose levels of 100 mg and 150 mg daily for five consecutive days and repeated every 28 day cycles. The first cycle consisted of 56 days for the evaluation of dose limiting toxicities (DLT). A DLT was defined as any non-hematologic AE of grade 3+ or a hematologic AE of grade 2+ thrombocytopenia, or grade 3+ neutropenia or anemia. The absence of DLT in the initial cohort of 100 mg daily in 3 evaluable pts allowed for dose escalation in a new cohort to 150 mg and after a total of 6 pts were treated at this level an additional 3 pts were treated at 100mg to complete the intended study. Pts that did not attain at least a partial response (PR) by modified European LeukemiaNet (ELN) criteria after cycle 6 and met eligibility were able to continue receiving IDA in combination with pegylated interferon-α (PEG) at 45ug weekly. Correlative studies included baseline mutational profiling, MDM2 protein levels, changes in serum MIC-1 levels and JAK2V617F variant allele frequency (VAF) with therapy.Results: 12 pts (6 at 100 mg; 6 at 150 mg) were treated and their baseline characteristics are shown in Table 1. Pts received a median of 7 cycles (range, 1-12) over a median of 33 weeks (range, 8-107) on study. Three pts discontinued treatment due to pt decision (n=2) and physician decision (n=1). A DLT was not identified for either dose level during cycle 1, and no hematologic treatment emergent adverse events (TEAE) were seen at any time point. Table 2 shows the non-hematologic TEAEs that occurred in at least 2 pts regardless of attribution. Grade 3 fatigue (n=1) and grade 3 headache (n=1) were the only significant TEAE noted at any time on study (both in 100mg cohort). The overall response rate by modified ELN criteria by cycle 7 for the 9 evaluable pts was 7/9 (78%). 7 of 10 (70%) evaluable pts achieved a ≥50% improvement in total symptom score (TSS) from baseline. There were no thrombotic or major hemorrhagic episodes, or progression to MF. Bone marrow pathologic response was assessed in 2 pts after 5 cycles. One case showed histological improvement, with normalization of overall marrow cellularity and megakaryocyte number, and less pronounced megakaryocytic atypia. The median JAK2V617F VAF at baseline, 7 cycles, and 9 cycles of therapy was 45% (n=12), 12% (n=6) and 13% (n=6), respectively. One pt was eligible to receive combination therapy and achieved phlebotomy freedom, normalization of palpable spleen (baseline 18cm), leukocyte count (baseline 44 x109/L), and PV-related symptoms by cycle 8. This pt also attained a 20% reduction in JAK2V617F VAF.The percentage of mononuclear cells expressing MDM2 was dramatically increased as compared to normal controls (p=0.01) (Figure 1). Serum levels of MIC-1 was used to assess pharmacodynamic effects of IDA therapy. Serum MIC-1 levels were elevated (~ 5 fold) after 5 days of administration of IDA in 85% of the pts (Figure 2), indicating activation of P53 by the low doses of IDA administered. Persistent elevation in MIC-1 levels was observed on day 15 in 6 of the 12 pts treated.Conclusion: IDA is well tolerated and demonstrates a clear signal of clinical activity in pts with therapy refractory PV. Correlatives support on-target activity. Mature follow up on the entire cohort will be presented. A multicenter phase II trial of IDA at a dose of 150 mg is underway [Display omitted] DisclosuresMascarenhas:CTI Biopharma: Research Funding; Janssen: Research Funding; Promedior: Research Funding; Novartis: Other: DSMB member , Research Funding; Merck: Research Funding; Incyte: Other: Clinical Trial Steering Committee , Research Funding.

Combination Treatment with an MDM2 Antagonist and a BET Inhibitor Targets Both Myelofibrosis Hematopoietic Stem/ Progenitor Cells and Their Tumor Promoting Microenvironment

Introduction: Myelofibrosis (MF) arises at the level of hematopoietic stem and progenitor cells (HSC/HPC) due to a sequence of mutational events that results in increased JAK/STAT signaling. JAK2 has served as a target for drug development and has led to the development of several novel small molecule inhibitors. Clinical trials of these agents have revealed their ability to decrease the degree of splenomegaly and to improve systemic symptoms of PMF patients, but not to substantially affect the progeny of the malignant clone or the rate of disease progression. MF HSC/HPCs and their progeny are known to elaborate a number pro-inflammatory mediators including lipocalin (LCN2) (Lu et al, Blood, 2015), and IL-8 which are capable of creating a tumor promoting microenvironment. LCN2 promotes osteoblast formation and marrow fibrosis (Lu et al, Blood, 2013) while IL8 promotes endothelial cell proliferation. We have previously reported that plasma LCN2 levels are increased in MF patients (Lu et al, ASH, 2016) and show now that plasma IL-8 levels are also dramatically increased (Figure 1). Furthermore the levels of IL-8 as shown by IHC were increased in splenic myeloid cells especially megakaryocytes and the IL-8 receptor CXCR2 was expressed at greater levels by MF splenic littoral cells that line the sinusoids as well as endothelial cells as compared to normal spleens. We hypothesize that effective treatment of MF would require a combination of drugs that directly deplete the malignant HSC/HPCs as well as dampen the tumor promoting microenvironment. In a previous study, we have found the MDM2 protein, a negative regulator of p53, are highly expressed in PV CD34+ cells from PV patients and can be targeted by nutlin therapy (Lu et al, Blood, 2013). In order to assess if nutlin therapy would deplete MF CD34+ cells, using flow cytometry method, we documented that MDM2 protein expression was significantly increased in MF CD34+ cells as compared to normal CD34+ cells and was higher than that in PV CD34+ cells (Figure 2). This finding suggests that MF malignant HSC/HPC might also be sensitive to the treatment with a nutlin. Furthermore, MF HSC/HPCs reside in the BM and extra medullary sites containing dysfunctional microenvironments which promote the predominance of the malignant stem cells. We assume that BET inhibitors might dampen the MF inflammatory milieu since BET proteins play a central role in regulation of inflammatory responses. BET proteins interact with transcription factors (TF) including NF-KB, P53 and c-Myc, and downregulate the transcriptional activation of numerous cytokines including IL-6 and IL-8. We then hypothesize that treatment with low doses of nutlin combined with JQ1, a BET inhibitor, will target MF malignant cells as well as correct MF HSC tissue specific supportive microenvironments by decreasing pro-inflammatory cytokine generation.Results: 1. Treatment with a combination of low dose RG7112 and JQ1 selectively decreased hematopoietic colony formation by MF CD34+ cells. Exposure to either low dose RG7112 (200nM) or JQ1 (50nM) alone for 2 days increased the degree of MF CD34+ cell apoptosis modestly, while a combination of the 2 drugs significantly induced MF CD34+ cell apoptosis (p<0.05). By contrast, treatment with each drug alone or in combination however did not significant induce apoptosis of normal CD34+ cells. Combination treatment of RG7112 and JQ1 inhibited total number of colonies generated by MF CD34+ cells by 60%, as compared to the treatment with each drug alone (p=0.004 and p=0.03, respectively). Combination treatment with RG7112 and JQ1 did not, however, decrease colony formation by normal HPC. In a small number of cases, combination therapy decreased the numbers of homozygous JAK2V617F+ colonies assayed from MF CD34+ cells (at range from 8% to 20%).2. Both RG7112 and JQ1 treatment decrease the elaboration of MF pro-inflammatory cytokines. Treatment of MF mononuclear cells (MNC) with RG7112 but not JQ1 decreased LCN2 levels in conditioned medium (CM) (at range from 5% to 39%, p=0.012); while treatment with JQ1 at 50nM strongly decreased the levels of IL-8 in MF MNC CM by 50% (p=0.0003).Summary: These data indicate that JQ1 combined with RG7112 might be a potential effective strategy for the treatment of MF. This drug combination will not only selectively target MF HSC/HPCs but also alter the MF HSC promoting microenvironment by inhibiting the elaboration of pro-inflammatory mediators. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Deferasirox Induces Apoptosis in Mantle Cell Lymphoma in a Mechanism Involving Cyclin-D1, p53 and the Ribonucleotide Reductase

Mantle cell lymphoma (MCL) is a difficult to treat B-cell malignancy characterized by a rapid progressive course and poor prognosis. Despite advances in conventional and novel agents such as bortezomib, lenalidomide and ibrutinib, many MCL patients are still subject to rapid relapses of the disease, responsible for the relatively short overall survival. MCL is characterized by the t(11,14) translocation, resulting in cyclin-D1 (CD1) overexpression and dysregulation of the cell cycle. Recently, it has been demonstrated that depletion of CD1 results in cell cycle arrest, apoptosis and cell death of MCL cells thus labeling CD1 as a therapeutic target of this aggressive malignancy. Accumulating findings indicate that not only overexpressed CD1 but also deregulated signaling pathways such as PI3K/AKT/mTOR and Wnt are accountable for the tumorigenesis of MCL and for the maintenance of its proliferative and survival capacity. We thus believe that MCL treatment should target multiple deregulated signaling pathways and key molecules. Iron chelators have emerged as a novel strategy to treat tumors due to their capacity to target a multitude of key molecules, signaling pathways and cellular processes. Yet, the application of common iron chelators is limited due to their low bioavailability and high toxicities. Deferasirox (DFX), a clinically approved iron chelator applied for the treatment of iron overload, in contrast to the majority of iron chelators, is highly bioavailable and exhibits little to no side effects. To date, DFX is reported as being an orally effective antitumor agent against solid tumors by various mechanisms. In our project, we examined the in vitro impact of DFX on three MCL cell lines; Jeko-1, REC-1 and Z138; aiming thereby to resolve its molecular mode of action. DFX inhibited the growth of the MCL cell lines with 50% inhibitory concentrations (IC50) of 8.07±1.08µM, 1.8±1.03µM and 1.52±1.54µM for Jeko-1, REC-1 and Z138 cell lines, respectively. Moreover, we demonstrated that DFX treatment trigged a rapid and intense (~2-fold) production of reactive oxygen species (ROS), induced severe DNA damage and arrested cells at the G1 phase within 6 hrs. Ultimately, a 72h treatment of 10µM DFX led to apoptosis in 60% to 86% of the MCL cells. Most importantly, we found DFX to substantially reduce CD1 protein levels in a dose- and time-dependent manner. The depletion of CD1 levels was accompanied with its phosphorylation at Thr-286 (pCD1), a phosphorylation known to sentence CD1 to proteasomal degradation. In agreement with previous studies that have shown a correlation between ROS induction, CD1-phosphorylation, CD1 depletion and DNA damage, we found CD1 levels to negatively correlate with increasing DNA damage levels following DFX treatment.Interestingly, the efficacy of DFX treatment was dependent on the p53-status of the respective cell line. The wild-type p53 expressing lines, REC-1 and Z138, were found to be ~5-fold more sensitive to DFX than the p53 negative Jeko-1 cell line. In an attempt to understand this intriguing finding, we found DFX treatment to post-translationally elevate and stabilize p53 levels, most likely by depleting the intracellular heme pool which serves as a negative p53-regulator. Furthermore, we showed that DFX plays an important role in the attenuation of the iron dependent ribonucleotide reductase enzyme (RR); the rate limiting enzyme in dNTP biosynthesis and thus the key enzyme in DNA synthesis, cell cycle regulation and proliferation. DFX inhibited the enzymatic activity of RR with an IC50 of ~25 µM; which is ~20-fold more effective than other established inhibitors such as deferoxamine.In conclusion and pending further analysis, we demonstrated a potent impact of DFX on MCL cells. The vigorous anti-tumoral activity of DFX is most likely mediated via its ability to induce oxidative stress, to inhibit the RR enzyme and to eliminate the main oncogene in MCL pathogenesis; CD1. The depleting of intracellular iron/heme pools and the modulation of p53 only intensify the effect of DFX. The ability to inhibit the RR activity qualifies DFX as a sensitizer to existing DNA damaging radio- and chemotherapy options of MCL. DFX possesses the unique capacity to affect a multitude of cellular mechanisms in an auto-potentiating manner that propose this clinically established molecule as a potential therapeutic option for mantle cell lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Tifab Deficiency Protects Against Stress-Induced Bone Marrow Failure at the Expense of Pre-Leukemic Selection

Interstitial deletion of a single copy of chromosome 5q is the most frequent cytogenetic alteration in Myelodysplastic Syndromes (MDS), which results in reduced dosage of numerous genes. TRAF-interacting protein with forkhead-associated domain B (TIFAB) resides within the proximal commonly-deleted region on band 5q31.1, and belongs to a family of forkhead-associated domain proteins. TIFAB is deleted in nearly all reported cases of del(5q) MDS and AML. As expected, TIFAB expression is significantly lower in CD34+ and BM mononuclear cells isolated from MDS patients with del(5q) as compared with cells from MDS patients diploid at chr 5q. Recently we have shown that hematopoietic-specific deletion of Tifab in mice results in progressive BM and blood defects, including aberrant HSPC proportions, altered myeloid differentiation, and progressive cytopenia (Varney and Niederkorn et al., JEM 2015). Gene expression analysis of Tifab KO lineage-Sca1+cKit+ (LSK) cells identified dysregulation of immune-related signatures, hypersensitivity to Toll-like receptor stimulation, and enrichment of a p53 regulatory gene-set.Importantly, p53 is implicated in the pathogenesis of del(5q) MDS: 1) BM cells from murine models and BM from del(5q) patients exhibit increased p53 activity, which is thought to contribute to ineffective hematopoiesis and anemia; and 2) del(5q) MDS patients often acquire concurrent TP53 mutations that result in rapid transformation to AML and poor treatment response. To examine the effects of TIFAB on p53 function, we examined p53 target genes in TIFAB-overexpressing and -deficient cells. Gene expression profiling and qRT-PCR analysis of Tifab KOHSPCs revealed significant upregulation of p53 regulatory genes. In contrast, overexpression of TIFAB in a p53-competent cell line reduced the expression of p53 target gene, p21 . We also observed increased p53 protein in Tifab KO total BM, lineage- BM, and spleen. At steady-state and after in vitro treatment with 2 Gy irradiation, Tifab KO lineage- BM cells exhibit increased p53 protein compared to WT cells.To test whether this increased p53 stability renders Tifab -deficient HSPCs more sensitive to stress, we treated Tifab KO HSPCs both in vivo and ex vivo using irradiation. When treated with low dose irradiation ex vivo, Tifab KO HSPCs initially exhibit significantly reduced colony forming ability; however, upon secondary re-plating, Tifab KO HSPCs strikingly form more colonies than WT HSPC, resembling a clonal selection event. To model this effect in vivo, we transplanted WT or Tifab KO BM into lethally-irradiated recipient mice. After 4 weeks of hematopoietic reconstitution, recipient mice were treated with 6.5 Gy sublethal irradiation. We find that Tifab KO recipient mice are less likely to succumb to IR-induced bone marrow failure as compared to mice transplanted with WT BM cells. Instead, after 3+ months, the remaining Tifab KO recipient mice have an increased tendency to develop hematologic malignancies, evident by myeloid blasts. Collectively, these data suggest that TIFAB-deficiency protects against cellular stress by eliminating “less fit” HSPCs, at the expense of selecting for pre-leukemic populations, paralleling observations in patients with transformed MDS.To investigate the mechanism of this phenomenon, we performed a tandem-affinity tag purification and mass-spectrometry analysis of TIFAB complexes in a del(5q) AML cell line (HL60), and identified unique TIFAB-interacting proteins. The top interacting candidate was an ubiquitin-specific peptidase (USP), USP15. USPs play a major role in ubiquitin-dependent processes including DNA damage response signaling, protein degradation, and kinase activation. Specifically, USP15 has been shown to promote p53 degradation via deubiquitination and stabilization of its major negative regulator, MDM2. Through biochemical assays and a series of deletion mutants, we confirmed that TIFAB interacts with USP15 and enhances the deubiquitination of biological USP15 substrates, including MDM2. Thus, our data implicate TIFAB as an important negative regulator of p53 and modulator of USP15 DUB activity.Altogether, our findings identify a novel role for TIFAB as a modifier of p53 function, potentially via a USP15-MDM2 axis. These findings have important implications in the potential role of TIFAB and p53 signaling in the pathogenesis of del(5q) MDS and transformation to AML. DisclosuresNo relevant conflicts of interest to declare.

MDMX Is Widely Expressed in Adult Pre-Cursor B Cell B Acute Lymphoblastic Leukemia and Is a Potential Therapeutic Target.

MDMX is a human homolog of murine double minute 2 (HDM2), which like HDM2, is a negative regulator of p53. In pediatric precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL), p53 expression is associated with poor prognosis. In this study, we assessed MDMX, HDM2, p53 and p21 levels in bone marrow samples from 55 adult patients with pre-B ALL diagnosis. A 110-core tissue microrray was subject to immunohistochemical analysis; in which 10% or more tumor cells stained were considered positive. Results were compared with Philadelphia chromosome (Ph) status (37 Ph-, 18 Ph+) and clinical data. MDMX was found to be expressed in 39 of 49 cases (80%), whereas HDM2 was expressed in 14 of 54 cases (26%). All 14 cases positive for HDM2 were also positive for MDMX (P < 0.05). Median survival duration was 138 weeks in the Ph- group and 48 weeks in the Ph+ group (P < 0.001). p53 was expressed more often in the Ph+ group than in Ph- group (10 of 17 versus 10 of 36, P < 0.05), as was p21 (11 of 17 versus 6 of 34, P < 0.003). In the Ph+ group, patients with tumor cells positive for p53, HDM2, and MDMX had a worse prognosis than other patients (35 weeks versus 61 weeks, P < 0.002). Western blot analysis of protein extracts of blasts isolated from patients with pre-B ALL confirmed the expression of MDMX or its variants. Furthermore, only 1 of 5 cases demonstrated missense mutation of p53 genes. Thus MDMX was expressed in most of these cases of pre-B ALL, suggesting that MDMX may be a potential therapeutic target. The higher incidence of MDMX expression seemed to be independent of p53. P21 expression by the blasts of a subset of ALL cases indicated that the p53 pathway was functional.

Association of Murine Double Minute 2 polymorphisms with gastric cancer: A systematic review with meta-analysis.

Gastric cancer (GC) is the 5th most common type of cancer, with the 3rd highest mortality rate worldwide in both sexes. Murine double minute 2 (MDM2) protein is the major negative regulator of p53, and genetic polymorphisms in this gene have shown to be associated with several types of cancer. In the present study, a literature search was performed using PubMed and Scopus with the following key word combinations 'gastric cancer AND polymorphism AND MDM2'. Studies were carefully revised according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines to identify eligible studies that matched the inclusion criteria. Statistical analysis was performed to assess the association between the different genetic polymorphisms and GC risk, by calculating the odds ratios (OR) and the confidence intervals (CI), with a 5% level of significance. A total of 11 manuscripts studied MDM2 polymorphisms in GC: rs937283 (n=1), rs3730485 (n=1) and rs2279744 (n=9). Both the rs937283 and rs3730485 reports showed an association with GC; however, there was only one study on each of these polymorphisms in the literature. A meta-analysis was performed for the rs2279744 polymorphism, of which studies showed a positive association between the G allele and risk of GC, either in the dominant model (OR=1.46; 95% CI 1.21-1.75; P<0.001) or recessive model (OR 1.65; 95% CI 1.45-1.87; P<0.001). In conclusion, genetic polymorphisms in MDM2 seemed to be associated with an increased risk of GC development, nevertheless, the number of studies were relatively low and the studied populations were primarily Chinese. The present meta-analysis emphasizes the need for additional studies in other populations to corroborate the association of these polymorphisms with GC.

PRPF19 regulates p53-dependent cellular senescence by modulating alternative splicing of MDM4 mRNA

Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53–p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.

Cullin-4B promotes cell proliferation and invasion through inactivation of p53 signaling pathway in colorectal cancer

Cullin 4B (CUL4B) is a member of the Cullin RING E3 ligase family, which is found to be overexpressed in multiple cancers, thus facilitating tumorigenesis and progression. However, the correlation between CUL4B and p53 in colorectal cancer cells (CRC) remains to be further elucidated. In this study, we newly identified that CUL4B functions as a negative regulator of p53, thereby facilitating CRC tumorigenesis and progression. Our data has demonstrated that CUL4B was frequently overexpressed in CRC tissues, and its upregulation was closely correlated with disease progression and poor prognosis. Moreover, CUL4B knockdown suppressed cell proliferation, invasion and epithelial-mesenchymal transition (EMT) of CRC cells. Mechanistically, CUL4B depletion increased the expression of p53 protein and its downstream targets p21, PUMA and MDM2. Furthermore, CUL4B depletion prolonged the half-life of p53 protein, and CUL4B is a binding partner of MDM2. In conclusion, our study shed new lights on the complex regulatory network between CUL4B and p53, and clarifies this CUL4B-p53 axis contributes greatly to CRC tumorigenesis and progression.

ATRT-08. TARGETING THE TP53-MDM2 ENHANCES RADIATION SENSITIVITY IN ATRT

Atypical Teratoid Rhabdoid Tumor is a highly aggressive pediatric brain tumor. Despite radiation, aggressive chemotherapy and autologous stem cell rescue, the children usually have poor survival. A functional genomic screen identified the TP53-MDM2 axis as a therapeutic vulnerability in ATRT. Gene expression demonstrates that all ATRT sub-groups have high level of MDM2 a negative regulator of p53. We demonstrate that MDM2 inhibition by shRNA or with small-molecule drugs, Nutlin3 and Idasanutlin resulted in decreased ATRT cell growth, inhibition of clonogenic potential and induction of apoptosis in vitro and in vivo. MRI imaging of intracranial tumors shows that Apparent Diffusion Coefficient (ADC), a good marker for successful treatment, significantly increased with Idasanutlin treatment showing tumor necrosis. Moreover, Idasanutlin significantly decreased growth of intracranial orthotopic ATRT brain tumors as evaluated by T2 MRI imaging and prolonged survival compared to control animals. Further idasanutlin potentiated radiation induced DNA damage and increased sensitivity to radiaton of ATRT cells. These findings highlight the TP53-MDM2 axis as a rational therapeutic target in ATRT.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Functional display of bioactive peptides on the vGFP scaffold

Grafting bioactive peptides into recipient protein scaffolds can often increase their activities by conferring enhanced stability and cellular longevity. Here, we describe use of vGFP as a novel scaffold to display peptides. vGFP comprises GFP fused to a bound high affinity Enhancer nanobody that potentiates its fluorescence. We show that peptides inserted into the linker region between GFP and the Enhancer are correctly displayed for on-target interaction, both in vitro and in live cells by pull-down, measurement of target inhibition and imaging analyses. This is further confirmed by structural studies highlighting the optimal display of a vGFP-displayed peptide bound to Mdm2, the key negative regulator of p53 that is often overexpressed in cancer. We also demonstrate a potential biosensing application of the vGFP scaffold by showing target-dependent modulation of intrinsic fluorescence. vGFP is relatively thermostable, well-expressed and inherently fluorescent. These properties make it a useful scaffold to add to the existing tool box for displaying peptides that can disrupt clinically relevant protein–protein interactions.