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 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.

Chondrosarcomas are malignant cartilage-producing tumours that frequently harbour isocitrate dehydrogenase 1 and -2 (IDH) gene mutations. Several studies have confirmed that these mutations are key players in the early stages of cartilage tumour development, but their role in later stages remains ambiguous. The prognostic value of IDH mutations remains unclear and preclinical studies have not identified effective treatment modalities (in)directly targeting these mutations. In contrast, the IDH mutation status is a prognostic factor in other cancers, and IDH mutant inhibitors as well as therapeutic strategies targeting the underlying vulnerabilities induced by IDH mutations seem effective in these tumour types. This discrepancy in findings might be ascribed to a difference in tumour type, elevated D-2-hydroxyglutarate levels, and the type of in vitro model (endogenous vs. genetically modified) used in preclinical studies. Moreover, recent studies suggest that the (epi)genetic landscape in which the IDH mutation functions is an important factor to consider when investigating potential therapeutic strategies or patient outcomes. These findings imply that the dichotomy between IDH wildtype and mutant is too simplistic and additional subgroups indeed exist within chondrosarcoma. Future studies should focus on the identification, characterisation, and tailoring of treatments towards these biological subgroups within IDH wildtype and mutant chondrosarcoma.

Recently, specific driver mutations were identified in chondroblastoma, giant cell tumour of bone and central cartilaginous tumours (specifically enchondroma and central chondrosarcoma), sharing the ability to induce genome-wide epigenetic alterations. In chondroblastoma and giant cell tumour of bone, the neoplastic mononuclear stromal-like cells frequently harbour specific point mutations in the genes encoding for histone H3.3 (H3F3A and H3F3B). The identification of these driver mutations has led to development of novel diagnostic tools to distinguish between chondroblastoma, giant cell tumour of bone and other giant cell containing tumours. From a biological perspective, these mutations induce several global and local alterations of the histone modification marks. Similar observations are made for central cartilaginous tumours, which frequently harbour specific point mutations in the metabolic enzymes IDH1 or IDH2. Besides an altered methylation pattern on histones, IDH mutations also induce a global DNA hypermethylation phenotype. In all of these tumour types, the mutation-driven epigenetic alterations lead to a highly altered transcriptome, resulting for instance in alterations in differentiation. These genomic alterations have diagnostic impact. Further research is needed to identify the genes and signalling pathways that are affected by the epigenetic alterations, which will hopefully lead to a better understanding of the biological mechanism underlying tumourigenesis.

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.