Exploiting vulnerabilities induced by recurrent mutations in chondrosarcoma and giant cell tumour of bone: therapeutic targeting of the altered epigenome and beyond

Chondrosarcoma and giant cell tumour of bone (GCTB) are bone tumours characterized by recurrent mutations (IDH1/IDH2 and H3F3A, respectively) that induce remodelling of the epigenetic landscape. The standard of care for both of these sarcoma subtypes is surgery and alternative treatment options for patients with inoperable disease are currently lacking (chondrosarcoma) or suboptimal (GCTB). Therefore, the aim of this thesis was to identify novel therapeutic targets for high-grade chondrosarcoma as well as GCTB, with a focus on potential therapies that could counteract the remodelling of the epigenome. PARP and HDAC inhibition, alone or in combination treatment strategies, were identified as promising therapeutic strategies for chondrosarcoma or both of these bone tumours, respectively. Additionally, this thesis describes the development and use of novel 3D cell culture models which can be used to improve the translation of preclinical findings to the clinic.LUMC / Geneeskund

Inhibition of PARP Sensitizes Chondrosarcoma Cell Lines to Chemo- and Radiotherapy Irrespective of the IDH1 or IDH2 Mutation Status

Chondrosarcomas are chemo- and radiotherapy resistant and frequently harbor mutationsin 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 repairand PARP inhibition, alone or combined with chemo- or radiotherapy, in chondrosarcoma celllines with or without endogenous IDH mutations. Chondrosarcoma cell lines treated with thePARP inhibitor talazoparib were examined for dose–response relationships, as well as underlyingcell death mechanisms and DNA repair functionality. Talazoparib was combined with chemo- orradiotherapy to evaluate potential synergy. Cell lines treated long termwith an inhibitor normalizingD-2-HG levels were investigated for synthetic lethality with talazoparib. We report that talazoparibsensitivity was variable and irrespective of IDH mutation status. All cell lines expressed AtaxiaTelangiectasia Mutated (ATM), but a subset was impaired in poly(ADP-ribosyl)ation (PARylation)capacity, homologous recombination, andO-6-methylguanine-DNAmethyltransferase (MGMT) expression.Talazoparib synergized with temozolomide or radiation, independent of IDH1 mutant inhibition.This study suggests that talazoparib combined with temozolomide or radiation are promisingtherapeutic strategies for chondrosarcoma, irrespective of IDH mutation status. A subset ofchondrosarcomas may be deficient in nonclassical DNA repair pathways, suggesting that PARPinhibitor sensitivity is multifactorial in chondrosarcoma.Toxicolog

A screening-based approach identifies cell cycle regulators AURKA, CHK1 and PLK1 as targetable regulators of chondrosarcoma cell survival

Chondrosarcomas are malignant cartilage tumors that are relatively resistant towards conventional therapeuticapproaches. Kinase inhibitors have been investigated and shown successful for several different cancer types. Inthis study we aimed at identifying kinase inhibitors that inhibit the survival of chondrosarcoma cells and therebyserve as new potential therapeutic strategies to treat chondrosarcoma patients.An siRNA screen targeting 779 different kinases was conducted in JJ012 chondrosarcoma cells in parallelwith a compound screen consisting of 273 kinase inhibitors in JJ012, SW1353 and CH2879 chondrosarcoma celllines. AURKA, CHK1 and PLK1 were identified as most promising targets and validated further in a morecomprehensive panel of chondrosarcoma cell lines. Dose response curves were performed using tyrosine kinaseinhibitors: MK-5108 (AURKA), LY2603618 (CHK1) and Volasertib (PLK1) using viability assays and cell cycleanalysis. Apoptosis was measured at 24 h after treatment using a caspase 3/7 assay. Finally, chondrosarcomapatient samples (N = =34) were used to examine the correlation between AURKA, CHK1 and PLK1 RNAexpression and documented patient survival.Dose dependent decreases in viability were observed in chondrosarcoma cell lines after treatment with MK-5108, LY2603618 and volasertib, with cell lines showing highest sensitivity to PLK1 inhibition. In additionincreased sensitivity to conventional chemotherapy was observed after CHK1 inhibition in a subset of the celllines. Interestingly, whereas AURKA and CHK1 were both expressed in chondrosarcoma patient samples, PLK1expression was found to be low compared to normal cartilage. Analysis of patient samples revealed that highCHK1 RNA expression correlated with a worse overall survival.AURKA, CHK1 and PLK1 are identified as important survival genes in chondrosarcoma cell lines. Althoughfurther research is needed to validate these findings, inhibiting CHK1 seems to be the most promising potentialtherapeutic target for patients with chondrosarcoma.Toxicolog

Comparative Proteomic Analysis of Serum from Patients with Systemic Sclerosis and Sclerodermatous GVHD. Evidence of Defective Function of Factor H

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by immunological and vascular abnormalities. Until now, the cause of SSc remains unclear. Sclerodermatous graft-versus-host disease (ScGVHD) is one of the most severe complications following bone marrow transplantation (BMT) for haematological disorders. Since the first cases, the similarity of ScGVHD to SSc has been reported. However, both diseases could have different etiopathogeneses. The objective of this study was to identify new serum biomarkers involved in SSc and ScGVHD. METHODOLOGY: Serum was obtained from patients with SSc and ScGVHD, patients without ScGVHD who received BMT for haematological disorders and healthy controls. Bi-dimensional electrophoresis (2D) was carried out to generate maps of serum proteins from patients and controls. The 2D maps underwent image analysis and differently expressed proteins were identified. Immuno-blot analysis and ELISA assay were used to validate the proteomic data. Hemolytic assay with sheep erythrocytes was performed to evaluate the capacity of Factor H (FH) to control complement activation on the cellular surface. FH binding to endothelial cells (ECs) was also analysed in order to assess possible dysfunctions of this protein. PRINCIPAL FINDINGS: Fourteen differentially expressed proteins were identified. We detected pneumococcal antibody cross-reacting with double stranded DNA in serum of all bone marrow transplanted patients with ScGVHD. We documented higher levels of FH in serum of SSc and ScGVHD patients compared healthy controls and increased sheep erythrocytes lysis after incubation with serum of diffuse SSc patients. In addition, we observed that FH binding to ECs was reduced when we used serum from these patients. CONCLUSIONS: The comparative proteomic analysis of serum from SSc and ScGVHD patients highlighted proteins involved in either promoting or maintaining an inflammatory state. We also found a defective function of Factor H, possibly associated with ECs damage

Non-IDH1-R132H IDH1/2 mutations are associated with increased DNA methylation and improved survival in astrocytomas, compared to IDH1-R132H mutations

Somatic mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 occur at high frequency in several tumour types. Even though these mutations are confined to distinct hotspots, we show that gliomas are the only tumour type with an exceptionally high percentage of IDH1(R132H) mutations. Patients harbouring IDH1(R132H) mutated tumours have lower levels of genome-wide DNA-methylation, and an associated increased gene expression, compared to tumours with other IDH1/2 mutations ("non-R132H IDH1/2 mutations"). This reduced methylation is seen in multiple tumour types and thus appears independent of the site of origin. For 1p/19q non-codeleted glioma (astrocytoma) patients, we show that this difference is clinically relevant: in samples of the randomised phase III CATNON trial, patients harbouring tumours with IDH mutations other than IDH1(R132H) have a better outcome (hazard ratio 0.41, 95% CI [0.24, 0.71], p = 0.0013). Such non-R132H IDH1/2-mutated tumours also had a significantly lower proportion of tumours assigned to prognostically poor DNA-methylation classes (p < 0.001). IDH mutation-type was independent in a multivariable model containing known clinical and molecular prognostic factors. To confirm these observations, we validated the prognostic effect of IDH mutation type on a large independent dataset. The observation that non-R132H IDH1/2-mutated astrocytomas have a more favourable prognosis than their IDH1(R132H) mutated counterpart indicates that not all IDH-mutations are identical. This difference is clinically relevant and should be taken into account for patient prognostication.MTG6Molecular tumour pathology - and tumour genetic

Mutation-driven epigenetic alterations as a defining hallmark of central cartilaginous tumours, giant cell tumour of bone and chondroblastoma

Molecular tumour pathology - and tumour geneticsMTG

Rainfall characteristics and regionalization in peninsular malaysia based on a high resolution gridded data set

Daily gridded rainfall data over Peninsular Malaysia are delineated using an objective clustering algorithm, with the objective of classifying rainfall grids into groups of homogeneous regions based on the similarity of the rainfall annual cycles. It has been demonstrated that Peninsular Malaysia can be statistically delineated into eight distinct rainfall regions. This delineation is closely associated with the topographic and geographic characteristics. The variation of rainfall over the Peninsula is generally characterized by bimodal variations with two peaks, i.e., a primary peak occurring during the autumn transitional period and a secondary peak during the spring transitional period. The east coast zones, however, showed a single peak during the northeast monsoon (NEM). The influence of NEM is stronger compared to the southwest monsoon (SWM). Significantly increasing rainfall trends at 95% confidence level are not observed in all regions during the NEM, with exception of northwest zone (R1) and coastal band of west coast interior region (R3). During SWM, most areas have become drier over the last three decades. The study identifies higher variation of mean monthly rainfall over the east coast regions, but spatially, the rainfall is uniformly distributed. For the southwestern coast and west coast regions, a larger range of coefficients of variation is mostly obtained during the NEM, and to a smaller extent during the SWM. The inland region received least rainfall in February, but showed the largest spatial variation. The relationship between rainfall and the El Niño Southern Oscillation (ENSO) was examined based on the Multivariate ENSO Index (MEI). Although the concurrent relationships between rainfall in the different regions and ENSO are generally weak with negative correlations, the rainfall shows stronger positive correlation with preceding ENSO signals with a time lag of four to eight months

Effects of renal failure on complement C3d levels

Elevated plasma concentrations of complement split product C3d have been reported to represent activation of the complement system. In the present study the effect of renal function on C3d concentrations was investigated in patients with various degrees of renal impairment, in patients with chronic renal failure and in CAPD patients. It appeared that elevated plasma C3d concentrations were present in patients with plasma creatinine concentrations in excess of 200 mumol/l regardless of the type of kidney disease. It is very likely that this can be attributed to renal handling (i.e. glomerular filtration, tubular reabsorption and renal catabolism) of C3d in a similar way as has been demonstrated for other low molecular weight proteins. The peritoneal permeability to C3d was slightly less than could be expected on the basis of its molecular weight without evidence of local production of C3d. Renal function should be taken into account in the interpretation of elevated plasma concentrations of C3

Rainfall characteristics and regionalization in peninsular malaysia based on a high resolution gridded data set

Daily gridded rainfall data over Peninsular Malaysia are delineated using an objective clustering algorithm, with the objective of classifying rainfall grids into groups of homogeneous regions based on the similarity of the rainfall annual cycles. It has been demonstrated that Peninsular Malaysia can be statistically delineated into eight distinct rainfall regions. This delineation is closely associated with the topographic and geographic characteristics. The variation of rainfall over the Peninsula is generally characterized by bimodal variations with two peaks, i.e., a primary peak occurring during the autumn transitional period and a secondary peak during the spring transitional period. The east coast zones, however, showed a single peak during the northeast monsoon (NEM). The influence of NEM is stronger compared to the southwest monsoon (SWM). Significantly increasing rainfall trends at 95% confidence level are not observed in all regions during the NEM, with exception of northwest zone (R1) and coastal band of west coast interior region (R3). During SWM, most areas have become drier over the last three decades. The study identifies higher variation of mean monthly rainfall over the east coast regions, but spatially, the rainfall is uniformly distributed. For the southwestern coast and west coast regions, a larger range of coefficients of variation is mostly obtained during the NEM, and to a smaller extent during the SWM. The inland region received least rainfall in February, but showed the largest spatial variation. The relationship between rainfall and the El Niño Southern Oscillation (ENSO) was examined based on the Multivariate ENSO Index (MEI). Although the concurrent relationships between rainfall in the different regions and ENSO are generally weak with negative correlations, the rainfall shows stronger positive correlation with preceding ENSO signals with a time lag of four to eight months

Selection of Effective Therapies Using Three-Dimensional in vitro Modeling of Chondrosarcoma

Purpose: Chondrosarcomas are a group of cartilaginous malignant neoplasms characterized by the deposition of chondrogenic extracellular matrix. Surgical resection is currently the only curative treatment option, due to their high resistance to conventional chemotherapy and radiotherapy. Novel therapeutic treatment options may improve outcome. Predominantly used cell line monolayer in vitro models lack in vivo complexity, such as the presence of extracellular matrix, and differing oxygen access. Hence, we aimed to improve pre-clinical chondrosarcoma research by developing an alginate-based 3D cell culture model.Method: An alginate scaffold was applied to generate spheroids of three chondrosarcoma cell lines (CH2879, JJ012, SW1353). Morphological, histological and immunohistochemical assessment of the spheroids were used to characterize the chondrosarcoma model. Presto blue assay, morphological and immunohistochemical assessment were applied to assess spheroid response to a panel of chemotherapeutics and targeted therapies, which was compared to conventional 2D monolayer models. Synergistic effect of doxorubicin and ABT-737 (Bcl-2 inhibitor) was compared between monolayer and spheroid models using excess over Bliss. A 3D colony formation assay was developed for assessment of radiotherapy response.Results: Chondrosarcoma spheroids produced chondrogenic matrix and remained proliferative after 2 weeks of culture. When treated with chemotherapeutics, the spheroids were more resistant than their monolayer counterparts, in line with animal models and clinical data. Moreover, for sapanisertib (mTOR inhibitor) treatment, a recovery in chondrosarcoma growth, previously observed in mice models, was also observed using long-term treatment. Morphological assessment was useful in the case of YM-155 (survivin inhibitor) treatment where a fraction of the spheroids underwent cell death, however a large fraction remained proliferative and unaffected. Synergy was less pronounced in 3D compared to 2D. A 3D clonogenic assay confirmed increased resistance to radiotherapy in 3D chondrosarcoma spheroids.Conclusion: We demonstrate that the chondrosarcoma alginate spheroid model is more representative of chondrosarcoma in vivo and should be used instead of the monolayer model for therapy testing. Improved selection at in vitro stage of therapeutic testing will increase the amount of information available for experimental design of in vivo animal testing and later, clinical stages. This can potentially lead to increased likelihood of approval and success at clinical trials.Molecular tumour pathology - and tumour geneticsMTG