Inge H. Briaire-De Bruijn

Background: Chondrosarcomas are well known for their resistance to conventional chemotherapy and radiotherapy treatment regimens, which is particularly detrimental in patients who have unresectable tumors. Recently, inhibition of poly(ADP-ribose) polymerase (PARP) by talazoparib was shown to sensitize chondrosarcoma cell lines to chemotherapy (temozolomide) or radiotherapy, irrespective of isocitrate dehydrogenase (IDH) mutation status. Because two-dimensionally grown cell lines have limitations and may not accurately represent the clinical response to drug treatment, we aimed to use a more representative three-dimensional alginate spheroid chondrosarcoma model. It is important to test therapeutic agents in vitro before testing them in animals or humans; therefore, we aimed to determine the effectiveness of a PARP inhibitor in reducing the viability of chondrosarcoma spheroids. Using a more stringent, complex in vitro model refines future therapeutic options for further investigation in animal models, increasing efficiency, reducing unnecessary animal use, and saving time and cost. Questions/purposes: (1) Does talazoparib treatment slow or inhibit the growth of chondrosarcoma spheroids, and does an increased treatment duration change the drug’s effect? (2) Does talazoparib work in synergy with temozolomide treatment to reduce the viability of chondrosarcoma spheroids? (3) Does talazoparib work in synergy with radiotherapy treatment to reduce the viability of chondrosarcoma spheroids? Methods: Three representative conventional chondrosarcoma cell lines (CH2879 [IDH wildtype], JJ012 [IDH1 mutant], and SW1353 [IDH2 mutant]) were cultured as alginate spheroids and treated with talazoparib (0.001 to 10 µM), temozolomide (0.01 to 100 µM), or combinations of these drugs for 3, 7, and 14 days, representing different stages of spheroid growth. The cell lines were selected to represent a variety of IDH mutation statuses and were previously validated in spheroid culturing. Temozolomide was chosen because of its previous success when combined with PARP inhibitors, dissimilar to other commonly used chemotherapies. The effect on spheroid viability was assessed using three cell viability assays. Additionally, spheroid count, morphology, proliferation, and apoptosis were assessed. The effect of talazoparib (5 to 10 nM) combined with ƴ-radiation applied using a ¹³⁷C source (0 to 6 Gy) was assessed as surviving fractions by counting the number of spheroids (three). The therapeutic synergy of low-concentration talazoparib (5 to 10 nM) with temozolomide or radiotherapy was determined by calculating Excess over Bliss scores. Results: Talazoparib treatment reduced the spheroid viability of all three cell lines after 14 days (IC₅₀ ± SD of CH2879: 0.1 ± 0.03 µM, fold change: 220; JJ012: 12 ± 1.4 µM, fold change: 4.8; and SW1353: 1.0 ± 0.2 µM, fold change: 154), compared with 3-day treatments of mature spheroids. After 14 days of treatment, the Excess over Bliss scores for 100 µM temozolomide and talazoparib indicated synergistic efficacy (Excess over Bliss scores: CH2879 59% [lower 95% CI 52%], JJ012 18% [lower 95% CI 8%], and SW1353 55% [lower 95% CI 25%]) of this combination treatment. A stable synergistic effect of talazoparib and radiotherapy was present only in JJ012 spheroids at a 4Gƴ radiation dose (Excess over Bliss score: 22% [lower 95% CI 6%]). Conclusion: In our study, long-term PARP inhibition was more effective than short-term treatment, and only one of the three chondrosarcoma spheroid lines was sensitive to combined PARP inhibition and radiotherapy. These findings suggest subsequent animal studies should focus on long-term PARP inhibition, and temozolomide combined with talazoparib has a higher chance of success than combination with radiotherapy. Clinical Relevance: Combination treatment of talazoparib and temozolomide was effective in reducing the viability of chondrosarcoma spheroids and spheroid growth, regardless of IDH mutation status, providing rationale to replicate this treatment combination in an animal chondrosarcoma model.

The neoplastic “stromal” cells in giant cell tumor of bone (GCTB) harbor a mutation in the H3F3A gene, which causes alterations in the epigenome. Current systemic targeted therapies, such as denosumab, do not affect the neoplastic cells, resulting in relapse upon treatment discontinuation. Therefore, this study examined whether targeting the epigenome could eliminate the neoplastic cells from GCTB. We established four novel cell lines of neoplastic “stromal” cells that expressed the H3F3A p.G34W mutation. These cell lines were used to perform an epigenetics compound screen (n = 128), which identified histone deacetylase (HDAC) inhibitors as key epigenetic regulators in the neoplastic cells. Transcriptome analysis revealed that the neoplastic cells expressed all HDAC isoforms, except for HDAC4. Therefore, five HDAC inhibitors targeting different HDAC subtypes were selected for further studies. All GCTB cell lines were very sensitive to HDAC inhibition in both 2D and 3D in vitro models, and inductions in histone acetylation, as well as apoptosis, were observed. Thus, HDAC inhibition may represent a promising therapeutic strategy to eliminate the neoplastic cells from GCTB lesions, which remains the paramount objective for GCTB patients who require life-long treatment with denosumab.

Multiple components of the SUMOylation machinery are deregulated in various cancers and could represent potential therapeutic targets. Understanding the role of SUMOylation in tumor progression and aggressiveness would increase our insight in the role of SUMO in cancer and clarify its potential as a therapeutic target. Here we investigate SUMO in relation to conventional chondrosarcomas, which are malignant cartilage forming tumors of the bone. Aggressiveness of chondrosarcoma increases with increasing histological grade, and a multistep progression model is assumed. High-grade chondrosarcomas have acquired an increased number of genetic alterations. Using immunohistochemistry on tissue microarrays (TMA) containing 137 chondrosarcomas, we showed that higher expression of SUMO1 and SUMO2/3 correlates with increased histological grade. In addition, high SUMO2/3 expression was associated with decreased overall survival chances (p = 0. 0312) in chondrosarcoma patients as determined by log-rank analysis and Cox regression. Various chondrosarcoma cell lines (n = 7), especially those derived from dedifferentiated chondrosarcoma, were sensitive to SUMO inhibition in vitro. Mechanistically, we found that SUMO E1 inhibition interferes with cell division and as a consequence DNA bridges are frequently formed between daughter cells. In conclusion, SUMO expression could potentially serve as a prognostic biomarker.

For osteosarcoma (OS), the most common primary malignant bone tumor, overall survival has hardly improved over the last four decades. Especially for metastatic OS, novel therapeutic targets are urgently needed. A hallmark of cancer is aberrant metabolism, which justifies targeting metabolic pathways as a promising therapeutic strategy. One of these metabolic pathways, the NAD+ synthesis pathway, can be considered as a potential target for OS treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the classical salvage pathway for NAD+ synthesis, and NAMPT is overexpressed in OS. In this study, five OS cell lines were treated with the NAMPT inhibitor FK866, which was shown to decrease nuclei count in a 2D in vitro model without inducing caspase-driven apoptosis. The reduction in cell viability by FK866 was confirmed in a 3D model of OS cell lines (n = 3). Interestingly, only OS cells with low nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1) RNA expression were sensitive to NAMPT inhibition. Using a publicly available (Therapeutically Applicable Research to Generate Effective Treatments (TARGET)) and a previously published dataset, it was shown that in OS cell lines and primary tumors, low NAPRT1 RNA expression correlated with NAPRT1 methylation around the transcription start site. These results suggest that targeting NAMPT in osteosarcoma could be considered as a novel therapeutic strategy, where low NAPRT expression can serve as a biomarker for the selection of eligible patients.

A simple bone cyst (SBC) is a cystic bone lesion predominantly affecting young males. The cyst is lined by a fibrous membrane and filled with serosanguinous fluid. EWSR1/FUS-NFATC2 rearrangements were recently identified in SBC. We here report exactly the same rearrangement in 3 lesions diagnosed as vascular malformations of 2 elderly patients. In total, through Archer FusionPlex, fluorescence in situ hybridization and/or reverse transcriptase-polymerase chain reaction the EWSR1-NFATC2 rearrangement was identified in 6 of 9 SBC, 3 of 12 benign vascular tumors, and none of 5 aneurysmal bone cyst lacking USP6 fusion. Using fluorescence in situ hybridization, it was apparent that amplification of the fusion, as seen in EWSR1-NFATC2 round cell sarcomas, was absent, and that in the vascular tumors the fusion was present both in the lining cells as well as in the surrounding spindle cells. Of note, not all of the spaces in the vascular malformations were lined by endothelial cells. Aggrecan was positive in all cases but was not specific. NKX2-2 and NKX3-1 staining were negative in all cases. Thus, even though the overlap between the 2 entities is limited to the presence of few thick-walled cysts lacking endothelial lining in the benign vascular malformations, the spectrum of benign tumors containing NFATC2 fusions should be expanded and contains not only SBC in the young, but also vascular malformation/hemangioma in elderly patients.

Mutations in the isocitrate dehydrogenase (IDH1 or IDH2) genes are common in enchondromas and chondrosarcomas, and lead to elevated levels of the oncometabolite D-2-hydroxyglutarate causing widespread changes in the epigenetic landscape of these tumors. With the use of a DNA methylation array, we explored whether the methylome is altered upon progression from IDH mutant enchondroma towards high-grade chondrosarcoma. High-grade tumors show an overall increase in the number of highly methylated genes, indicating that remodeling of the methylome is associated with tumor progression. Therefore, an epigenetics compound screen was performed in five chondrosarcoma cell lines to therapeutically explore these underlying epigenetic vulnerabilities. Chondrosarcomas demonstrated high sensitivity to histone deacetylase (HDAC) inhibition in both 2D and 3D in vitro models, independent of the IDH mutation status or the chondrosarcoma subtype. siRNA knockdown and RNA expression data showed that chondrosarcomas rely on the expression of multiple HDACs, especially class I subtypes. Furthermore, class I HDAC inhibition sensitized chondrosarcoma to glutaminolysis and Bcl-2 family member inhibitors, suggesting that HDACs define the metabolic state and apoptotic threshold in chondrosarcoma. Taken together, HDAC inhibition may represent a promising targeted therapeutic strategy for chondrosarcoma patients, either as monotherapy or as part of combination treatment regimens.

Chondrosarcomas are chemo- and radiotherapy resistant and frequently harbor mutations in isocitrate dehydrogenase (IDH1 or IDH2), causing increased levels of D-2-hydroxyglutarate (D-2-HG). DNA repair defects and synthetic lethality with poly(ADP-ribose) polymerase (PARP) inhibition occur in IDH mutant glioma and leukemia models. Here we evaluated DNA repair and PARP inhibition, alone or combined with chemo- or radiotherapy, in chondrosarcoma cell lines with or without endogenous IDH mutations. Chondrosarcoma cell lines treated with the PARP inhibitor talazoparib were examined for dose–response relationships, as well as underlying cell death mechanisms and DNA repair functionality. Talazoparib was combined with chemo- or radiotherapy to evaluate potential synergy. Cell lines treated long term with an inhibitor normalizing D-2-HG levels were investigated for synthetic lethality with talazoparib. We report that talazoparib sensitivity was variable and irrespective of IDH mutation status. All cell lines expressed Ataxia Telangiectasia Mutated (ATM), but a subset was impaired in poly(ADP-ribosyl)ation (PARylation) capacity, homologous recombination, and O-6-methylguanine-DNA methyltransferase (MGMT) expression. Talazoparib synergized with temozolomide or radiation, independent of IDH1 mutant inhibition. This study suggests that talazoparib combined with temozolomide or radiation are promising therapeutic strategies for chondrosarcoma, irrespective of IDH mutation status. A subset of chondrosarcomas may be deficient in nonclassical DNA repair pathways, suggesting that PARP inhibitor sensitivity is multifactorial in chondrosarcoma.

Conventional chondrosarcomas are malignant cartilage tumors considered radioresistant. Nevertheless, retrospective series show a small but significant survival benefit for patients with locally advanced disease treated with radiotherapy. And, in daily practice when considered inoperable their irradiation is an accepted indication for proton beam radiotherapy. Therefore, we investigated the sensitivity of chondrosarcoma cell lines and -tissue samples towards radiotherapy and screened for biomarkers to identify predictors of radiosensitivity. Proliferation and clonogenic assays were performed in chondrosarcoma cell lines after γ-radiation in combination with mutant IDH1 inhibitor AGI-5198. In addition, glutathione levels were measured using mass spectrometry. Chondrosarcoma tumor explants were irradiated after which γ-H2AX foci were counted. Mutation analysis was performed using the Ion AmpliSeq™ Cancer Hotspot Panel and immunohistochemical staining’s were performed for P-S6, LC-3B, P53, Bcl-2, Bcl-xl and Survivin. Results were correlated with the number of γ-H2AX foci. Chondrosarcoma cell lines were variably γ-radiation resistant. No difference in radiosensitivity, nor glutathione levels was observed after treatment with AGI-5198. Irradiated chondrosarcoma patient tissue presented a variable increase in γ-H2AX foci compared to non-radiated tissue. Samples were divided into two groups, high and low radioresistant, based on the amount of γ-H2AX foci. All four highly resistant tumors exhibited mutations in the pRb pathway, while none of the less radioresistant tumors showed mutations in these genes. Chondrosarcoma cell lines as well as primary tumors are variably radioresistant, particularly in case of a defective Rb pathway. Whether selection for radiotherapy can be based upon an intact Rb pathway should be further investigated.

Mutations in isocitrate dehydrogenase (IDH)1 or -2 are found in ~50% of conventional central chondrosarcomas and in up to 87% of their assumed benign precursors enchondromas. The mutant enzyme acquires the activity to convert α-ketoglutarate into the oncometabolite d-2-hydroxyglutarate (d-2-HG), which competitively inhibits α-ketoglutarate dependent enzymes such as histone- and DNA demethylases. We therefore evaluated the effect of IDH1 or -2 mutations on histone modifications (H3K4me3, H3K9me3 and H3K27me3), chromatin remodeler ATRX expression, DNA modifications (5-hmC and 5-mC), and TET1 subcellular localization in a genotyped cohort (IDH, succinate dehydrogenase (SDH) and fumarate hydratase (FH)) of enchondromas and central chondrosarcomas (n = 101) using immunohistochemistry. IDH1 or -2 mutations were found in 60.8% of the central cartilaginous tumours, while mutations in FH and SDH were absent. The mutation status did not correlate with outcome. Chondrosarcomas are strongly positive for the histone modifications H3K4me3, H3K9me3 and H3K27me3, which was independent of the IDH1 or -2 mutation status. Two out of 36 chondrosarcomas (5.6%) show complete loss of ATRX. Levels of 5-hmC and 5-mC are highly variable in central cartilaginous tumours and are not associated with mutation status. In tumours with loss of 5-hmC, expression of TET1 was more prominent in the cytoplasm than the nucleus (p = 0.0001). In summary, in central chondrosarcoma IDH1 or -2 mutations do not affect immunohistochemical levels of 5-hmC, 5mC, trimethylation of H3K4, -K9 and K27 and outcome, as compared to wildtype.

Previous studies on high grade sarcomas using mass spectrometry imaging showed proteasome activator complex subunit 1 (PSME1) to be associated with poor survival in soft tissue sarcoma patients. PSME1 is involved in immunoproteasome assembly for generating tumor antigens presented by MHC class I molecules. In this study, we aimed to validate PSME1 as a prognostic biomarker in an independent and larger series of soft tissue sarcomas by immunohistochemistry. Tissue microarrays containing leiomyosarcomas (n = 34), myxofibrosarcomas (n = 14), undifferentiated pleomorphic sarcomas (n = 15), undifferentiated spindle cell sarcomas (n = 4), pleomorphic liposarcomas (n = 4), pleomorphic rhabdomyosarcomas (n = 2), and uterine leiomyomas (n = 7) were analyzed for protein expression of PSME1 using immunohistochemistry. Survival times were compared between high and low expression groups using Kaplan–Meier analysis. Cox regression models as multivariate analysis were performed to evaluate whether the associations were independent of other important clinical covariates. PSME1 expression was variable among soft tissue sarcomas. In leiomyosarcomas, high expression was associated with overall poor survival (p = 0.034), decreased metastasis-free survival (p = 0.002) and lower event-free survival (p = 0.007). Using multivariate analysis, the association between PSME1 expression and metastasis-free survival was still significant (p = 0.025) and independent of the histological grade. High expression of PSME1 is associated with poor metastasis-free survival in soft tissue leiomyosarcoma patients, and might be used as an independent prognostic biomarker.