Background: In all giant-cell-rich lesions (GCRL) occurring in bone, a common underlying excessive RANKL expression is held responsible for the osteolytic activity. Apart from giant cell tumour of... Show moreBackground: In all giant-cell-rich lesions (GCRL) occurring in bone, a common underlying excessive RANKL expression is held responsible for the osteolytic activity. Apart from giant cell tumour of bone (GCTB), systematic outcome analysis of RANKL inhibition in other GCRL is unavailable. The aim of this study is to assess the efficacy and safety of a 1-year denosumab protocol in giant cell lesions of the jaw (GCLJ).Methods: A retrospective cohort study was conducted compromising patients treated with a 1year protocol of monthly subcutaneously administered 120 mg denosumab. Objective tumour response based on histology and imaging was used to calculate objective tumour response rate, progression-free survival (PFS) and time to progression. Type, severity and frequency of adverse events were recorded in a standardised way to assess safety.Results: Twenty patients, predominantly female (90%), were included. Fifty-five per cent of lesions were located in the mandible; most classified as aggressive lesions (90%). Thirty-five per cent (7/20) of cases were either recurrent after prior treatment or progressive, while on other drug treatment. Objective tumour response rate was 100% after 12 months of treatment. Median PFS was 50.4 months (95% CI 38.0-62.8) with a cumulative PFS rate of 22.6% (95% CI 1.8-43.4) at 5 years follow-up. Median time to progression was 38.4 months (95% CI 26.0 -50.8). Treatment was well tolerated, and none of the patients had to interrupt therapy for toxicity.Conclusion: High-dose denosumab is effective and safe in achieving a complete response in GCLJ within 12 months. The high long-term relapse rate after treatment cessation is the main obstacle for denosumab to become standard treatment for GCLJ. 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
Non-ossifying fibroma (NOF) and central giant cell granuloma (CGCG) are both benign tumours of bone with overlapping morphology and similar mutations in the RAS/MAPK pathway. However, NOF is... Show moreNon-ossifying fibroma (NOF) and central giant cell granuloma (CGCG) are both benign tumours of bone with overlapping morphology and similar mutations in the RAS/MAPK pathway. However, NOF is located in the long bones with regression after puberty in contrast to CGCG which is located in the jaw bones and does not regress spontaneously. We hypothesised that endocrine regulation by oestrogen plays a role in the spontaneous regression in NOF. Therefore, we examined the expression of ER alpha in a series of NOF and CGCG. ER alpha expression (EP1) was determined using immunohistochemistry on 16 NOFs (whole slides), and 47 CGCGs (tissue microarrays (TMA's n = 41 and whole slide n = 6)). As comparison, we included TMAs of other giant cell containing bone lesions: giant cell tumour of bone (n = 75), chondroblastoma (n = 12), chondromyxoid fibroma (n = 12), aneurysmal bone cyst (n = 6) and telangiectatic osteosarcoma (n = 6). All 16 NOF samples demonstrated ER alpha protein expression, while all 47 CGCG and all other giant cell containing bone tumours were negative. Most NOF samples had moderate staining intensity and between 24 and 49% of the spindle cells were ER alpha-positive. Our findings further support the role of endocrine regulation via oestrogen in the spontaneous regression in NOF. Whether oestrogen signalling at puberty is involved in the induction of senescence in the neoplastic cells of NOF harbouring RAS/MAPK pathway mutations needs further research. Since ER alpha expression was not observed in other giant cell containing bone lesions with overlapping morphological features, positive ER alpha expression may favour the diagnosis of NOF in challenging diagnostic cases. Show less
Weegen, Y. van der; Lint, K. de; Heuvel, D. van den; Nakazawa, Y.; Mevissen, T.E.T.; Schie, J.J.M. van; ... ; Luijsterburg, M.S. 2021
Two side-by-side papers report that the transcription elongation factor ELOF1 drives transcription-coupled repair and prevents replication stress.Cells employ transcription-coupled repair (TCR) to... Show moreTwo side-by-side papers report that the transcription elongation factor ELOF1 drives transcription-coupled repair and prevents replication stress.Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4(CSA) ubiquitin ligase. How CRL4(CSA) is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4(CSA) for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication. Show less
Chen, H.; Li, Y.G.; Reiber, J.H.C.; Lange, J. de; Tu, S.X.; Stelt, P. van der; ... ; Aarab, G. 2018
The p53 tumor suppressor pathway is inactivated in cancer either via direct mutation or via deregulation of upstream regulators or downstream effectors. P53 mutations are rare in uveal melanoma.... Show moreThe p53 tumor suppressor pathway is inactivated in cancer either via direct mutation or via deregulation of upstream regulators or downstream effectors. P53 mutations are rare in uveal melanoma. Here we investigated the role of the p53 inhibitor Hdmx in uveal melanoma. We found Hdmx over-expression in a subset of uveal melanoma cell lines and fresh-frozen tumor samples. Hdmx depletion resulted in cell-line dependent growth inhibition, apparently correlating with differential Hdm2 levels. Surprisingly, p53 knockdown hardly rescued cell cycle arrest and apoptosis induction upon Hdmx knockdown, whereas it effectively prevented growth suppression induced by the potent p53 activator Nutlin-3. In addition, two compounds inhibiting Hdmx function or expression, SAH-p53-8 and XI-011, also elicited a growth inhibitory effect in a partly p53-independent manner. These findings suggest a novel, growth-promoting function of Hdmx that does not rely on its ability to inhibit p53. We provide evidence for a contribution of p27 protein induction to the observed p53-independent G1 arrest in response to Hdmx knockdown. In conclusion, our study establishes the importance of Hdmx as an oncogene in a subset of uveal melanomas and widens the spectrum of its function beyond p53 inhibition. Show less
The p53 tumor suppressor protein plays a key role in cancer and its direct or indirect inactivation is an almost universal feature of human tumors. P53 has a central position in the prevention of... Show moreThe p53 tumor suppressor protein plays a key role in cancer and its direct or indirect inactivation is an almost universal feature of human tumors. P53 has a central position in the prevention of genomic instability and protection of tumorigenesis. This thesis presents novel studies regarding the role of Hdmx in p53 inactivation during tumorigenesis, as well as the use of specific drugs for p53 reactivation as cancer treatment. Chapter 2 shows that constitutive Hdmx overexpression contributes to the neoplastic transformation of human fibroblasts and embryonic retinoblasts, thereby functionally resembling loss of p53. Chapter 3 establishes the importance of Hdmx as an oncogene in a subset of uveal melanomas. Importantly, the results described in this chapter extend the function of Hdmx beyond p53 inhibition. Chapter 4 evaluates the use of the specific p53 activating drugs Nutlin-3 and RITA in synergy studies as potential therapy for uveal melanoma. Chapter 5 is a more detailed analysis of the cellular responses to RITA. In particular, Chk2 is shown to be an essential mediator of the RITA-induced effects. Chapter 6 is a general discussion of the results presented in this thesis, and their implications for clinical exploitation and future research. Show less
Lange, J. de; Ly, L.V.; Lodder, K.; Verlaan-de Vries, M.; Teunisse, A.F.A.S.; Jager, M.J.; Jochemsen, A.G. 2012
UNLABELLED ABSTRACT: BACKGROUND 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... Show moreUNLABELLED ABSTRACT: BACKGROUND 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. METHODS We made use of an in vitro 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. RESULTS 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 in vivo. 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. CONCLUSIONS 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. Show less
Spinnler, C.; Hedstrom, E.; Li, H.; Lange, J. de; Nikulenkov, F.; Teunisse, A.F.A.S.; ... ; Selivanova, G. 2011
The prognosis of patients with uveal melanoma is poor. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. Because p53 mutations are uncommon in... Show moreThe prognosis of patients with uveal melanoma is poor. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. Because p53 mutations are uncommon in uveal melanoma, reactivation of p53 may be used to achieve tumor regression. We investigated the use of combination therapies for intraocular melanoma, based on the p53 activators Nutlin-3 and reactivation of p53 and induction of tumor cell apoptosis (RITA) and the topoisomerase I inhibitor Topotecan. Nutlin-3 treatment induced p53-dependent growth inhibition in human uveal melanoma cell lines. The sensitivity to Nutlin-3 of the investigated cell lines did not correlate with basal Hdm2 or Hdmx levels. Nutlin-3 synergized with RITA and Topotecan to induce apoptosis in uveal melanoma cell lines and short-term cultures. Drug synergy correlated with enhanced induction of p53-Ser46 phosphorylation, which was attenuated by ATM inhibition. Nutlin-3 and Topotecan also significantly delayed tumor growth in vivo in a murine B16F10 model for ocular melanoma. Combination treatment appeared to inhibit tumor growth slightly more efficient than either drug alone. Nutlin-3, RITA and Topotecan lead to comparable p53 activation and growth inhibition under normoxia and hypoxia. Treatment with Nutlin-3 or RITA had no effect on HIF-1α induction by hypoxia, whereas the combination of these two drugs did inhibit hypoxia-induced HIF-1α. Also Topotecan, alone or in combination with Nutlin-3, reduced HIF-1α protein levels, suggesting that a certain level of DNA damage response is required for p53-mediated downregulation of HIF-1α. In conclusion, combination treatments based on small-molecule-induced p53 activation may have clinical potential for uveal melanoma.Oncogene advance online publication, 18 July 2011;doi:10.1038/onc.2011.309. Show less
Phillips, A.; Teunisse, A.; Lam, S.; Lodder, K.; Darley, M.; Emaduddin, M.; ... ; Jochemsen, A.G. 2010
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... Show moreThe 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 up-regulated 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. Show less
