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.

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.

Osteosarcoma is a high-grade bone-forming neoplasm, with a complex genome. Tumours frequently show chromothripsis, many deletions, translocations and copy number alterations. Alterations in the p53 or Rb pathway are the most common genetic alterations identified in osteosarcoma. Using spontaneously transformed murine mesenchymal stem cells (MSCs) which formed sarcoma after subcutaneous injection into mice, it was previously demonstrated that p53 is most often involved in the transformation towards sarcomas with complex genomics, including osteosarcoma. In the current study, not only loss of p53 but also loss of p16^Ink4a is shown to be a driver of osteosarcomagenesis: murine MSCs with deficient p15^Ink4b, p16^Ink4a, or p19^Arf transform earlier compared to wild-type murine MSCs. Furthermore, in a panel of nine spontaneously transformed murine MSCs, alterations in p15^Ink4b, p16^Ink4a, or p19^Arf were observed in eight out of nine cases. Alterations in the Rb/p16 pathway could indicate that osteosarcoma cells are vulnerable to CDK4/CDK6 inhibitor treatment. Indeed, using two-dimensional (n = 7) and three-dimensional (n = 3) cultures of human osteosarcoma cell lines, it was shown that osteosarcoma cells with defective p16^INK4A are sensitive to the CDK4/CDK6 inhibitor palbociclib after 72-hour treatment. A tissue microarray analysis of 109 primary tumour biopsies revealed a subset of patients (20–23%) with intact Rb, but defective p16 or overexpression of CDK4 and/or CDK6. These patients might benefit from CDK4/CDK6 inhibition, therefore our results are promising and might be translated to the clinic.

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.