Search Results (10)

The p53 tumor suppressor protein activates various sets of genes depending on its covalent modifications, which are controlled by the nature and intensity of cellular stress. We observed that actinomycin D and nutlin-3a (A + N) collaborate in inducing activating phosphorylation of p53. Our recent transcriptomic data demonstrated that these substances strongly synergize in the upregulation of DUSP13, a gene with an unusual pattern of expression, coding for obscure phosphatase having two isoforms, one expressed in the testes and the other in skeletal muscles. In cancer cells exposed to A + N, DUSP13 is expressed from an alternative promoter in the intron, resulting in the expression of an isoform named TMDP-L1. Luciferase reporter tests demonstrated that this promoter is activated by both endogenous and ectopically expressed p53. We demonstrated for the first time that mRNA expressed from this promoter actually produces the protein, which can be detected with Western blotting, in all examined cancer cell lines with wild-type p53 exposed to A + N. In some cell lines, it is also induced by clinically relevant camptothecin, by nutlin-3a acting alone, or by a combination of actinomycin D and other antagonists of p53-MDM2 interaction—idasanutlin or RG7112. This isoform, fused with green fluorescent protein, localizes in the perinuclear region of cells. Full article

Chronic lymphocytic leukemia (CLL) is a genetically and clinically diverse hematological cancer affecting middle-aged and elderly individuals. Novel targeted therapy options are needed for patients who relapse following initial responses or who are intrinsically resistant to current treatments. There is a growing body of investigation currently underway on MDM2 inhibitors in clinical trials, reflecting the increasing interest in including these drugs in cancer treatment regimens. One of the developed compounds, idasanutlin (RG7388), has shown promise in early-stage clinical trials. It is a second-generation MDM2–p53-binding antagonist with enhanced potency, selectivity, and bioavailability. In addition to the TP53 status, which is an important determinant of the response, we have shown in our previous studies that the SF3B1 mutational status is also an independent predictive biomarker of the ex vivo CLL patient sample treatment response to RG7388. The objective of this study was to identify novel biomarkers associated with resistance to RG7388. Gene set enrichment analysis of differentially expressed genes (DEGs) between RG7388-sensitive and -resistant CLL samples showed that the increased p53 activity led to upregulation of pro-apoptosis pathway genes while DNA damage response pathway genes were additionally upregulated in resistant samples. Furthermore, differential expression of certain genes was detected, which could serve as the backbone for novel combination treatment approaches. This research provides preclinical data to guide the exploration of drug combination strategies with MDM2 inhibitors, leading to future clinical trials and associated biomarkers that may improve outcomes for CLL patients. Full article

Chronic lymphocytic leukemia (CLL) is a genetically and clinically heterogeneous malignancy affecting older individuals. There are a number of current treatment options for CLL, including monoclonal antibodies, targeted drugs, chemotherapy, and different combinations of these. However, for those patients who are intrinsically treatment resistant, or relapse following initial responses, novel targeted therapies are still needed. Targeting the mouse double-minute-2 human homolog (MDM2), a primary negative regulator of p53, is an appealing therapeutic strategy for non-genotoxic reactivation of p53, since the TP53 gene is in its wild-type state at diagnosis in approximately 90% of patients. Mutated SF3B1 and TP53 are both associated with more aggressive disease, resistance to therapies and poorer overall survival for CLL. In this study, we performed a screen for SF3B1 and TP53 mutations and tested RG7388 (idasanutlin), a second-generation MDM2 inhibitor, in a cohort of CLL primary patient samples. SF3B1 mutations were detected in 24 of 195 cases (12.3%) and found associated with poor overall survival (hazard ratio [HR] 2.12, p = 0.032) and high CD38 expression (median CD38 (%) 32 vs. 5; p = 0.0087). The novel striking finding of this study was an independent link between SF3B1 mutational status and poor response to RG7388. Overall, SF3B1 mutations in CLL patient samples were associated with resistance to treatment with RG7388 ex vivo, and patients with the wild type for both SF3B1 and TP53 are more likely to benefit from treatment with MDM2 inhibitors. Full article

Several molecular subtypes of cancer are highly dependent on splicing for cell survival. There is a general interest in the therapeutic targeting of splicing by small molecules. E7107, a first-in-class spliceosome inhibitor, showed strong growth inhibitory activities against a large variety of human cancer xenografts. Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous hematologic malignancy, with approximately 90% of cases being TP53 wild-type at diagnosis. An increasing number of studies are evaluating alternative targeted agents in CLL, including MDM2–p53 binding antagonists. In this study, we report the effect of splicing modulation on key proteins in the p53 signalling pathway, an important cell death pathway in B cells. Splicing modulation by E7107 treatment reduced full-length MDM2 production due to exon skipping, generating a consequent reciprocal p53 increase in TP53^WT cells. It was especially noteworthy that a novel p21^WAF1 isoform with compromised cyclin-dependent kinase inhibitory activity was produced due to intron retention. E7107 synergized with the MDM2 inhibitor RG7388, via dual MDM2 inhibition; by E7107 at the transcript level and by RG7388 at the protein level, producing greater p53 stabilisation and apoptosis. This study provides evidence for a synergistic MDM2 and spliceosome inhibitor combination as a novel approach to treat CLL and potentially other haematological malignancies. Full article

The transcription factor p53 has well-recognized roles in regulating cell cycle, DNA damage repair, cell death, and metabolism. It is an important tumor suppressor and pharmacological activation of p53 by interrupting its interaction with the ubiquitin E3 ligase mouse double minute 2 homolog (MDM2) is actively explored for anti-tumor therapies. In immune cells, p53 modulates inflammatory responses, but the impact of p53 on macrophages remains incompletely understood. In this study, we used the MDM2 antagonist idasanutlin (RG7388) to investigate the responses of primary human macrophages to pharmacological p53 activation. Idasanutlin induced a robust p53-dependent transcriptional signature in macrophages, including several pro-apoptotic genes. However, idasanutlin did not generally sensitize macrophages to apoptosis, except for an enhanced response to a Fas-stimulating antibody. In fully differentiated macrophages, idasanutlin did not affect pro-inflammatory gene expression induced by toll-like receptor 4 (TLR4), TLR3, and TLR7/8 agonists, but inhibited interleukin-4-induced macrophage polarization. However, when present during monocyte to macrophage differentiation, idasanutlin attenuated inflammatory responses towards activation of TLR4 and TLR7/8 by low doses of lipopolysaccharide or resiquimod (R848). This was accompanied by a reduced expression of CD14, TLR7, and TLR8 in macrophages differentiated in the presence of idasanutlin. Our data suggest anti-inflammatory effects of pharmacological p53 activation in differentiating human macrophages. Full article

3,3′-Diindolylmethane (DIM) is a naturally derived chemopreventive compound. It comes from glucobrassicin, an indole glucosinolate enriched in cruciferous vegetables, and is formed in the acidic environment of the stomach after ingestion. Mouse double minute 2 homolog (MDM2) is an important, multi-functional oncogenic protein and it has been well recognized for its negative regulation of the tumor suppressor protein p53. We discovered a novel mechanism of action of DIM, that it directly inhibits MDM2 in multiple colorectal cancer (CRC) cell lines. Treatment with DIM decreased MDM2 at messenger RNA (mRNA) and protein levels, inhibited cancer cell proliferation, and induced cell cycle arrest and apoptosis. DIM-induced decrease of MDM2 is p53-independent and is partly mediated by proteasome degradation of MDM2, as blocking of the proteasome activity reversed MDM2 protein inhibition. Overexpression of MDM2 blocked DIM’s effects in growth suppression and apoptosis induction. When combined with imidazoline MDM2 inhibitors (Nutlin-3a and Idasanutlin/RG-7388), synergism was observed in cancer cell growth inhibition. In summary, our data support a new mechanism of action for DIM in direct inhibition of MDM2. The identification of MDM2 as a novel DIM target may help develop a new strategy in CRC prevention. Full article

Dysfunction of p53 is observed in the many malignant tumors. In cervical cancer, p53 is inactivated by degradation through the complex with human papilloma virus (HPV) oncoprotein E6 and E6-associated protein (E6AP), an E3 ubiquitin protein ligase. In endometrial cancer, overexpression of p53 in immunohistochemistry is a significant prognostic factor. A discrepancy between p53 overexpression and TP53 mutations is observed in endometrioid endometrial cancer, indicating that the accumulation of p53 protein can be explained by not only gene mutations but also dysregulation of the factors such as ERβ and MDM2. Furthermore, the double-positive expression of immunoreactive estrogen receptor (ER) β and p53 proteins is closely associated with the incidence of metastasis and/or recurrence. High-grade serous ovarian carcinoma (HGSC) arises from secretary cells in the fallopian tube. The secretary cell outgrowth (SCOUT) with TP53 mutations progresses to HGSC via the p53 signature, serous intraepithelial lesion (STIL), and serous intraepithelial carcinoma (STIC), indicating that TP53 mutation is associated with carcinogenesis of HGSC. Clinical application targeting p53 has been approved for some malignant tumors. Gene therapy by the adenovirus-mediated p53 gene transfer system is performed for head and neck cancer. A clinical phase III trial using MDM2/X inhibitors, idasanutlin (RG7388) combined with cytarabine, is being performed involving relapse/refractory acute myeloid leukemia patients. The use of adenoviruses as live vectors which encode wild-type p53 has given promising results in cervical cancer patients. Full article

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 p53^wt 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. Full article

Background and Objective: Alterations in gene expressions are often due to epigenetic modifications that can have a significant influence on cancer development, growth, and progression. Lately, histone deacetylase inhibitors (HDACi) such as suberoylanilide hydroxamic acid (SAHA, or vorinostat, MK0683) have been emerging as a new class of drugs with promising therapeutic benefits in controlling cancer growth and metastasis. The small molecule RG7388 (idasanutlin, R05503781) is a newly developed inhibitor that is specific for an oncogene-derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. These two drugs have shown the ability to induce p21 expression through distinct mechanisms in MCF-7 and LNCaP cells, which are reported to have wild-type TP53. Our understanding of the molecular mechanism whereby SAHA and RG7388 can induce cell cycle arrest and trigger cell death is still evolving. In this study, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 using MCF-7 and LNCaP cells. Materials and Methods: The cytotoxicity, cell cycle arrest, and apoptosis/necroptosis effects of the SAHA and RG7388 treatments were assessed using the Trypan Blue dye exclusion (TBDE) method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence assay with DEVD-amc substrate, and immunoblotting methods. Results: The RG7388 treatment was able to induce cell death by elevating p21^WAF1/CIP1 through inhibition of MDM2 in LNCaP, but not in MCF-7 cells, even though there was evidence of p53 elevation. Hence, we suspect that there is some level of uncoupling of p53-mediated transcriptional induction of p21^WAF1/CIP1 in MCF-7 cells. Conclusion: Our results from MCF-7 and LNCaP cells confirmed that SAHA and RG7388 treatments were able to induce cell death via a combination of cell cycle arrest and cytotoxic mechanisms. We speculate that our findings could lead to the development of newer treatments for breast and prostate cancers with drug combinations including HDACi. Full article

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 p53^wt 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 p53^wt 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 p53^wt 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. Full article