Functions of MDMX in the Modulation of the p53-Response

The MDM family proteins MDM2 and MDMX are two critical regulators of the p53 tumor suppressor protein. Expression of both proteins is necessary for allowing the embryonal development by keeping the activity of p53 in check. Upon stresses that need to activate p53 to perform its function as guardian of the genome, p53 has to be liberated from these two inhibitors. In this review, we will discuss the various mechanisms by which MDMX protein levels are downregulated upon various types of stress, including posttranslational modifications of the MDMX protein and the regulation of mdmx mRNA expression, including alternative splicing. In addition, the putative function(s) of the described MDMX splice variants, particularly in tumor development, will be discussed. Lastly, in contrast to common belief, we have recently shown the existence of a p53-MDMX feedback loop, which is important for dampening the p53-response at later phases after genotoxic stress

Oncogenic functions of hMDMX in in vitro transformation of primary human fibroblasts and embryonic retinoblasts

<p>Abstract</p> <p>Background</p> <p>In around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels.</p> <p>Methods</p> <p>We made use of an <it>in vitro </it>transformation model using a retroviral system of RNA interference and gene overexpression in primary human fibroblasts and embryonic retinoblasts. Consecutive knockdown of RB and p53, overexpression of SV40-small t, oncogenic HRasV12 and HA-hMDMX resulted in a number of stable cell lines representing different stages of the transformation process, enabling a comparison between loss of p53 and hMDMX overexpression. The cell lines were tested in various assays to assess their oncogenic potential.</p> <p>Results</p> <p>Both p53-knockdown and hMDMX overexpression accelerated proliferation and prevented growth suppression induced by introduction of oncogenic Ras, which was required for anchorage-independent growth and the ability to form tumors <it>in vivo</it>. Furthermore, we found that hMDMX overexpression represses basal p53 activity to some extent. Transformed fibroblasts with very high levels of hMDMX became largely resistant to the p53 reactivating drug Nutlin-3. The Nutlin-3 response of hMDMX transformed retinoblasts was intact and resembled that of retinoblastoma cell lines.</p> <p>Conclusions</p> <p>Our studies show that hMDMX has the essential properties of an oncogene. Its constitutive expression contributes to the oncogenic phenotype of transformed human cells. Its main function appears to be p53 inactivation. Therefore, developing new drugs targeting hMDMX is a valid approach to obtain new treatments for a subset of human tumors expressing wild-type p53.</p

Targeting of the MAPK and AKT pathways in conjunctival melanoma shows potential synergy

Purpose: Conjunctival melanoma (CM) is a rare but lethal form of cancer. Similar to cutaneous melanoma, CM frequently carries activating mutations in BRAF and NRAS. We studied whether CM as well as conjunctival benign and premalignant melanocytic lesions express targets in the mitogen-activated protein kinase (MAPK) and AKT pathways, and whether specific inhibitors can suppress CM growth in vitro. Methods: 131 conjunctival lesions obtained from 129 patients were collected. The presence of BRAF V600E mutation and expression of phosphorylated (p)-ERK and p-AKT were assessed by immunohistochemistry. We studied cell proliferation, phosphorylation, cell cycling and apoptosis in three CM cell lines using two BRAF inhibitors (Vemurafenib and Dabrafenib), a MEK inhibitor (MEK162) and an AKT inhibitor (MK2206). Results: The BRAF V600E mutation was present in 19% of nevi and 26% of melanomas, but not in primary acquired melanosis (PAM). Nuclear and cytoplasmic p-ERK and p-AKT were expressed in all conjunctival lesions. Both BRAF inhibitors suppressed growth of both BRAF mutant CM cell lines, but only one induced cell death. MEK162 and MK2206 inhibited proliferation of CM cells in a dose-dependent manner, and the combination of these two drugs led to synergistic growth inhibition and cell death in all CM cell lines. Conclusion: ERK and AKT are constitutively activated in conjunctival nevi, PAM and melanoma. While BRAF inhibitors prohibited cell growth, they were not always cytotoxic. Combining MEK and AKT inhibitors led to more growth inhibition and cell death in CM cells. The combination may benefit patients suffering from metastatic conjunctival melanoma

HDMX-L is expressed from a functional P53-responsive promoter in the first intron of the HDMX gene, and participates in an auto-regulatory feedback loop to control P53 activity.

The p53 regulatory network is critically involved in preventing the initiation of cancer. In unstressed cells p53 is maintained at low levels and is largely inactive, mainly through the action of its two essential negative regulators, HDM2 and HDMX. p53 abundance and activity are upregulated in response to various stresses including DNA damage and oncogene activation. Active p53 initiates transcriptional and transcription-independent programs that result in cell cycle arrest, cellular senescence or apoptosis. p53 also activates transcription of HDM2, which initially leads to the degradation of HDMX, creating a positive feedback loop to obtain maximal activation of p53. Subsequently, when stress-induced post-translational modifications start to decline, HDM2 becomes effective in targeting p53 for degradation, thus attenuating the p53 response. To date, no clear function for HDMX in this critical attenuation phase has been demonstrated experimentally. Like HDM2, the HDMX gene contains a promoter (P2) in its first intron that is potentially inducible by p53. We show that p53 activation in response to a plethora of p53-activating agents induces the transcription of a novel HDMX mRNA transcript from the HDMX-P2 promoter. This mRNA is more efficiently translated than that expressed from the constitutive HDMX-P1 promoter, and it encodes a long form of HDMX protein, HDMX-L. Importantly, we demonstrate that HDMX-L cooperates with HDM2 to promote the ubiquitination of p53, and that p53-induced HDMX transcription from the P2 promoter can play a key role in the attenuation phase of the p53-response, to effectively diminish p53 abundance as cells recover from stress

mTor inhibitor GDC-0349 improves ASO induced SAMMSON knock down resulting in enhanced anti-tumor efficacy in uveal melanoma

Uveal melanoma (UM) is the most common intraocular malignancy in adults. The lack of an effective treatment results in a median survival time of less than one year for patients with metastatic disease and shows the high unmet need for the development of effective treatments. Recently, the melanoma-specific lncRNA SAMMSON was shown to be essential for skin melanoma survival. Analysis of a PAN cancer RNA-sequencing dataset revealed consistent expression of SAMMSON in uveal melanoma tumors. Targeting SAMMSON by means of antisense oligonucleotides (ASOs) results in a strong reduction in cell viability with induction of apoptosis of UM cells and slows down tumor growth in multiple UM PDX models. These effects were driven by impaired mitochondrial function and protein translation, resulting in cell death. To identify potential synergistic combinations, we combined SAMMSON knockdown with a library of 2911 FDA-approved drugs and quantified cell viability in a uveal melanoma cell line. The strongest synergy was obtained with the mTOR inhibitor GDC-0349. Combining SAMMSON knockdown with mTOR inhibition resulted in enhanced impairment of mitochondrial function and protein synthesis. Interestingly, we observed a more pronounced knockdown of SAMMSON when combining SAMMSON targeting ASOs with GDC-0349, suggesting mTOR inhibition facilitates ASO uptake in uveal melanoma cells. Further experiments are ongoing to confirm this mechanism. Taken together, these results demonstrate that SAMMSON inhibition in combination with mTOR inhibition could be a novel treatment option for uveal melanoma patients

So close, yet so far : discrepancies between uveal and other melanomas. A Position Paper from UM Cure 2020

Despite much progress in our understanding of uveal melanoma (UM) over the past decades, this rare tumour is still often misclassified. Although UM, like other melanomas, is very probably derived from melanocytes, it is drastically different from cutaneous melanoma and most other melanoma subtypes in terms of epidemiology, aetiology, biology and clinical features, including an intriguing metastatic hepatotropism. UM carries distinctive prognostic chromosome alterations, somatic mutations and gene expression profiles, allowing an active tailored surveillance strategy and dedicated adjuvant clinical trials. There is no standard systemic treatment for disseminated UM at present. In contrast to cutaneous melanoma, UMs are not BRAF-mutated, thus curtailing the use of B-Raf inhibitors. Although these tumours are characterised by some immune infiltrates, immune checkpoint inhibitors are rarely effective, possibly due to a low mutation burden. UM patients across the world not only face rare cancer-related issues (e.g., specific management strategies, access to information and to expert centres), but also specific UM problems, which can be exacerbated by the common misconception that it is a subtype of cutaneous melanoma. As a European Consortium dedicated to research on UM and awareness on the disease, “UM Cure 2020” participants urge medical oncologists, pharmaceutical companies, and regulatory agencies to acknowledge UM as a melanoma with specific issues, in order to accelerate the development of new therapies for patients

HDAC inhibition increases HLA class I expression in uveal melanoma

The treatment of uveal melanoma (UM) metastases or adjuvant treatment may imply immunological approaches or chemotherapy. It is to date unknown how epigenetic modifiers affect the expression of immunologically relevant targets, such as the HLA Class I antigens, in UM. We investigated the expression of HDACs and the histone methyl transferase EZH2 in a set of 64 UMs, using an Illumina HT12V4 array, and determined whether a histone deacetylase (HDAC) inhibitor and EZH2 inhibitor modified the expression of HLA Class I on three UM cell lines. Several HDACs (HDAC1, HDAC3, HDAC4, and HDAC8) showed an increased expression in high-risk UM, and were correlated with an increased HLA expression. HDAC11 had the opposite expression pattern. While in vitro tests showed that Tazemetostat did not influence cell growth, Quisinostat decreased cell sur