Anti-tick and pathogen transmission blocking vaccines.

Ticks and tick-borne diseases are a challenge for medical and veterinary public health and often controlled through the use of repellents and acaricides. Research on vaccination strategies to protect humans, companion animals, and livestock from ticks and tick-transmitted pathogens has accelerated through the use of proteomic and transcriptomic analyses. Comparative analyses of unfed versus engorged and uninfected versus infected ticks have provided valuable insights into candidates for anti-tick and pathogen transmission blocking vaccines. An intricate interplay between tick saliva and the host's immune system has revealed potential antigens to be used in vaccination strategies. Immunization of hosts with targeted anti-tick vaccines would ideally lead to a reduction in tick numbers and prevent transmission of tick-borne pathogens. Comprehensive control of tick-borne diseases would come from successful anti-tick vaccination, vaccination preventing transmission of tick-borne diseases or a combination. Due to the close interaction with wildlife and ticks, with wildlife reservoirs enabling propagation of pathogens between ticks, the vaccination of these reservoirs is an attractive target to reduce human contact with ticks and tick-borne diseases through a one-health approach. Wildlife vaccination presents formulation and regulatory challenges which should be considered early in the development of reservoir-targeted vaccines.

Small mitochondrial Arf (smArf) protein corrects p53-independent developmental defects of Arf tumor suppressor-deficient mice.

The mouse p19Arf (human p14ARF) tumor suppressor protein, encoded in part from an alternative reading frame of the Ink4a (Cdkn2a) gene, inhibits the Mdm2 E3 ubiquitin ligase to activate p53. Arf is not expressed in most normal tissues of young mice but is induced by high thresholds of aberrant hyperproliferative signals, thereby activating p53 in incipient tumor cells that have experienced oncogene activation. The single Arf mRNA encodes two distinct polypeptides, including full-length p19Arf and N-terminally truncated and unstable p15smArf ("small mitochondrial Arf") initiated from an internal in-frame AUG codon specifying methionine-45. Interactions of p19Arf with Mdm2, or separately with nucleophosmin (NPM, B23) that localizes and stabilizes p19Arf within the nucleolus, require p19Arf N-terminal amino acids that are not present within p15smArf We have generated mice that produce either smARF alone or M45A-mutated (smArf-deficient) full-length p19Arf proteins. BCR-ABL-expressing pro/pre-B cells producing smArf alone are as oncogenic as their Arf-null counterparts in generating acute lymphoblastic leukemia when infused into unconditioned syngeneic mice. In contrast, smArf-deficient cells from mice of the ArfM45A strain are as resistant as wild-type Arf+/+ cells to comparable oncogenic challenge and do not produce tumors. Apart from being prone to tumor development, Arf-null mice are blind, and their male germ cells exhibit defects in meiotic maturation and sperm production. Although ArfM45A mice manifest the latter defects, smArf alone remarkably rescues both of these p53-independent developmental phenotypes.

Field evaluation of a novel oral reservoir-targeted vaccine against Borrelia burgdorferi utilizing an inactivated whole-cell bacterial antigen expression vehicle.

Blacklegged ticks (Ixodes scapularis) are the principal vector for Borrelia burgdorferi, among other infectious agents, in the northeastern, mid-Atlantic, and upper midwestern USA. White-footed mice (Peromyscus leucopus) are the primary and most competent reservoir host of B. burgdorferi in the Northeast. Live reservoir-targeted vaccines (RTVs) to limit enzootic transmission of B. burgdorferi were previously developed and successfully evaluated in laboratory and controlled field trials. A novel, inactivated RTV was developed to minimize regulatory and market challenges facing previous RTVs based on live bacterial or viral vehicles. Thirty-two residential properties in Redding, Connecticut, participated in a field trial of an orally delivered, inactivated RTV efficacy study (2015-2016). During the two-year vaccination period, a significant decrease in the percentage of B. burgdorferi-infected I. scapularis larvae parasitizing P. leucopus was observed, as was a significant reduction in the percentage of infected P. leucopus on RTV-treated properties when compared to control properties. This novel inactivated RTV was effective in reducing numbers of B. burgdorferi-infected I. scapularis and B. burgdorferi-infected P. leucopus on properties where it was distributed.

Screening for potential targets for therapy in mesenchymal, clear cell, and dedifferentiated chondrosarcoma reveals Bcl-2 family members and TGFß as potential targets.

The mesenchymal, clear cell, and dedifferentiated chondrosarcoma subtypes are extremely rare, together constituting 10% to 15% of all chondrosarcomas. Their poor prognosis and lack of efficacious treatment emphasizes the need to elucidate the pathways playing a pivotal role in these tumors. We constructed tissue microarrays containing 42 dedifferentiated, 23 clear cell, and 23 mesenchymal chondrosarcomas and performed immunohistochemistry to study the expression of growth plate-signaling molecules and molecules shown to be involved in conventional chondrosarcoma. We observed high expression of SOX-9 and FGFR-3, as well as aberrant cellular localization of heparan sulfate proteoglycans, in all subtypes. TGFß signaling through p-SMAD2 and PAI-1 was highly active in all chondrosarcoma subtypes, which suggests that TGFß inhibitors as a possible therapeutic strategy in rare chondrosarcoma subtypes. As in conventional chondrosarcoma, antiapoptotic proteins (Bcl-2, and/or Bcl-xl) were highly expressed in all subtypes. Inhibition with the BH-3 mimetic ABT-737 rendered dedifferentiated chondrosarcoma cell lines sensitive to doxorubicin or cisplatin. Our data indicate that antiapoptotic proteins may play an important role in chemoresistance, suggesting a promising role for targeting Bcl-2 family members in chondrosarcoma treatment, irrespective of the subtype.

Orally delivered Bacillus subtilis expressing chicken NK-2 peptide stabilizes gut microbiota and enhances intestinal health and local immunity in coccidiosis-infected broiler chickens.

We recently reported a stable Bacillus subtilis-carrying chicken NK-lysin peptide (B. subtilis-cNK-2) as an effective oral delivery system of an antimicrobial peptide to the gut with therapeutic effect against Eimeria parasites in broiler chickens. To further investigate the effects of a higher dose of an oral B. subtilis-cNK-2 treatment on coccidiosis, intestinal health, and gut microbiota composition, 100 (14-day-old) broiler chickens were allocated into 4 treatment groups in a randomized design: 1) uninfected control (CON), 2) infected control without B. subtilis (NC), 3) B. subtilis with empty vector (EV), and 4) B. subtilis with cNK-2 (NK). All chickens, except the CON group, were infected with 5,000 sporulated Eimeria acervulina (E. acervulina) oocysts on d 15. Chickens given B. subtilis (EV and NK) were orally gavaged (1 × 1012 cfu/mL) daily from d 14 to 18. Growth performances were measured on d 6, 9, and 13 postinfection (dpi). Spleen and duodenal samples were collected on 6 dpi to assess the gut microbiota, and gene expressions of gut integrity and local inflammation makers. Fecal samples were collected from 6 to 9 dpi to enumerate oocyst shedding. Blood samples were collected on 13 dpi to measure the serum 3-1E antibody levels. Chickens in the NK group showed significantly improved (P < 0.05) growth performance, gut integrity, reduced fecal oocyst shedding and mucosal immunity compared to NC. Interestingly, there was a distinct shift in the gut microbiota profile in the NK group compared to that of NC and EV chickens. Upon challenge with E. acervulina, the percentage of Firmicutes was reduced and that of Cyanobacteria increased. In NK chickens, however, the ratio between Firmicutes and Cyanobacteria was not affected and was similar to that of CON chickens. Taken together, NK treatment restored dysbiosis incurred by E. acervulina infection and showed the general protective effects of orally delivered B. subtilis-cNK-2 on coccidiosis infection. This includes reduction of fecal oocyst shedding, enhancement of local protective immunity, and maintenance of gut microbiota homeostasis in broiler chickens.

Three new chondrosarcoma cell lines: one grade III conventional central chondrosarcoma and two dedifferentiated chondrosarcomas of bone.

BACKGROUND: Chondrosarcoma is the second most common primary sarcoma of bone. High-grade conventional chondrosarcoma and dedifferentiated chondrosarcoma have a poor outcome. In pre-clinical research aiming at the identification of novel treatment targets, the need for representative cell lines and model systems is high, but availability is scarce. METHODS: We developed and characterized three cell lines, derived from conventional grade III chondrosarcoma (L835), and dedifferentiated chondrosarcoma (L2975 and L3252) of bone. Proliferation and migration were studied and we used COBRA-FISH and array-CGH for karyotyping and genotyping. Immunohistochemistry for p16 and p53 was performed as well as TP53 and IDH mutation analysis. Cells were injected into nude mice to establish their tumorigenic potential. RESULTS: We show that the three cell lines have distinct migrative properties, L2975 had the highest migration rate and showed tumorigenic potential in mice. All cell lines showed chromosomal rearrangements with complex karyotypes and genotypic aberrations were conserved throughout late passaging of the cell lines. All cell lines showed loss of CDKN2A, while TP53 was wild type for exons 5-8. L835 has an IDH1 R132C mutation, L2975 an IDH2 R172W mutation and L3252 is IDH wild type. CONCLUSIONS: Based on the stable culturing properties of these cell lines and their genotypic profile resembling the original tumors, these cell lines should provide useful functional models to further characterize chondrosarcoma and to evaluate new treatment strategies.

Preclinical and Pilot Study of Type I FLT3 Tyrosine Kinase Inhibitor, Crenolanib, with Sorafenib in Acute Myeloid Leukemia and FLT3-Internal Tandem Duplication.

PURPOSE: To evaluate the safety, activity, and emergence of FLT3-kinase domain (KD) mutations with combination therapy of crenolanib and sorafenib in acute myeloid leukemia (AML) with FLT3-internal tandem duplication (ITD). PATIENTS AND METHODS: After in vitro and xenograft efficacy studies using AML cell lines that have FLT3-ITD with or without FLT3-KD mutation, a pilot study was performed with crenolanib (67 mg/m2/dose, three times per day on days 1-28) and two dose levels of sorafenib (150 and 200 mg/m2/day on days 8-28) in 9 pediatric patients with refractory/relapsed FLT3-ITD-positive AML. Pharmacokinetic, pharmacodynamic, and FLT3-KD mutation analysis were done in both preclinical and clinical studies. RESULTS: The combination of crenolanib and sorafenib in preclinical models showed synergy without affecting pharmacokinetics of each agent, inhibited p-STAT5 and p-ERK for up to 8 hours, and led to significantly better leukemia response (P < 0.005) and survival (P < 0.05) compared with single agents. Fewer FLT3-KD mutations emerged with dose-intensive crenolanib (twice daily) and low-intensity sorafenib (three times/week) compared with daily crenolanib or sorafenib (P < 0.05). The crenolanib and sorafenib combination was tolerable without dose-limiting toxicities, and three complete remissions (one with incomplete count recovery) and one partial remission were observed in 8 evaluable patients. Median crenolanib apparent clearance showed a nonsignificant decrease during treatment (45.0, 40.5, and 20.3 L/hour/m2 on days 1, 7, and 14, respectively) without drug-drug interaction. Only 1 patient developed a FLT3-KD mutation (FLT3 F691L). CONCLUSIONS: The combination of crenolanib and sorafenib was tolerable with antileukemic activities and rare emergence of FLT3-TKD mutations, which warrants further investigation.

Resistance to Ticks and the Path to Anti-Tick and Transmission Blocking Vaccines.

The medical and veterinary public health importance of ticks and tick-borne pathogens is increasing due to the expansion of the geographic ranges of both ticks and pathogens, increasing tick populations, growing incidence of tick-borne diseases, emerging tick transmitted pathogens, and continued challenges of achieving effective and sustained tick control. The past decades show an increasing interest in the immune-mediated control of tick infestations and pathogen transmission through the use of vaccines. Bovine tick resistance induced by repeated infestations was reported over a century ago. This review addresses the phenomena and immunological underpinning of resistance to tick infestation by livestock and laboratory animals; the scope of tick countermeasures to host immune defenses; and the impact of genomics, functional genomics, and proteomics on dissecting complex tick-host-pathogen interactions. From early studies utilizing tick tissue extracts to salivary gland derived molecules and components of physiologically important pathways in tick gut and other tissues, an increased understanding of these relationships, over time, impacted the evolution of anti-tick vaccine antigen selection. Novel antigens continue to emerge, including increased interest in the tick microbiome. Anti-tick and transmission blocking vaccines targeting pathogen reservoirs have the potential to disrupt enzootic cycles and reduce human, companion, domestic animal, and wildlife exposure to infected ticks.

Oral Delivery of Bacillus subtilis Expressing Chicken NK-2 Peptide Protects Against Eimeria acervulina Infection in Broiler Chickens.

Chicken NK-lysin peptide 2 (cNK-2) is a natural lytic peptide with direct cytotoxicity against many apicomplexan parasites including Eimeria. Developing an effective oral delivery strategy to express cNK-2 in the intestine, where Eimeria parasites interact with the host's gut epithelial cells, may effectively reduce the fecundity of parasites and minimize intestinal damage. Furthermore, cNK-2 modulates gut immune responses to decrease local inflammation elicited by Eimeria infection in the intestine. Therefore, we developed a stable strain of Bacillus subtilis (B. subtilis) that carries cNK-2 to the gut to determine its effectiveness in ameliorating the negative impacts of coccidiosis and to replace the use of antibiotics in controlling coccidiosis in commercial broiler chicken production. Chickens were randomly allocated into eight treatment groups: two control groups (NC: E. acervulina infected non-B. subtilis control; CON: non-infected control); three B. subtilis-empty vector (EV) groups (EV6: 106 cfu/day/bird; EV8: 108 cfu/day/bird; EV10: 1010 cfu/day/bird), and three B. subtilis-cNK-2 groups (NK6: 106 cfu/day/bird; NK8: 108 cfu/day/bird; NK10: 1010 cfu/day/bird). All chickens, except those in the CON group, were challenged with 5,000 freshly sporulated E. acervulina oocysts through oral gavage on day 15. Chickens were given an oral dose of B. subtilis on days 14, 15, and 16. Body weight, weight gains, and fecal oocyst shedding were measured. To investigate the efficacy of oral B. subtilis-cNK-2 against coccidiosis, gene expression of gut health-related biomarkers was measured using RT-PCR. Markers included SOD1, CAT, and HMOX1 for oxidative stress in the spleen and intestinal mucosa, OCLN, ZO-1, and JAM2 for tight junction proteins, and MUC2 for mucin gene expression in the gut. The results showed that oral treatment of young chickens with B. subtilis-cNK-2 improved growth performance, enhanced gut integrity, and reduced fecal oocyst shedding. Altogether, these results confirm B. subtilis-cNK-2 treatment as a promising and effective alternative strategy to replace antibiotics against coccidiosis based on its ability to reduce parasite survival, to reduce coccidiosis-induced body weight loss, and to decrease gut damage based on the enhanced expression of proteins associated with gut integrity and intestinal health.

Administration of an Orally Delivered Substrate Targeting a Mammalian Zoonotic Pathogen Reservoir Population: Novel Application and Biomarker Analysis.

Reservoir-targeted vaccines (RTVs) have the potential to be effective at breaking the transmission cycle of many tick-borne pathogens including, but not limited to, Borrelia burgdorferi, B. miyamotoi, B. mayonii, Babesia microti, and Anaplasma phagocytophilum. To determine what proportion of a wild reservoir species we could effectively target, we distributed an experimental non-RTV Rhodamine B (RhB)-coated pellet formulation devoid of nutrient supplementation using bait boxes with ad libitum access, in battery-operated time-release bait stations, and by hand broadcast. Regardless of distribution method, a total of 208 of 242 (86%) white-footed mouse (Peromyscus leucopus) captures were positive for RhB by either pelage staining or by detecting fluorescent expression in vibrissae under a microscope. In bait box locations, 91% of captured mice were RhB-positive, 89% in hand broadcast locations, and 80% in time-release station locations. Based on results, we are confident that the bait formulation was readily accepted regardless of distribution technique, reached a substantial proportion of the reservoir population, and provides an effective vehicle to deliver a range of RTVs to targeted, wild, pathogen reservoir populations.

Sexually dimorphic tumor suppression by small mitochondrial Arf.

Internal translational initiation of the mRNA encoding the Arf tumor suppressor yields an N-terminally truncated small Arf protein (smArf) that lacks amino acid residues required for Mdm2 binding and p53 activation. Here, we report that female, but not male, mice engineered to produce only smArf in lieu of the full-length Arf protein retain residual, sexually dimorphic tumor suppressive activity.

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 therapeutic approaches. Kinase inhibitors have been investigated and shown successful for several different cancer types. In this study we aimed at identifying kinase inhibitors that inhibit the survival of chondrosarcoma cells and thereby serve as new potential therapeutic strategies to treat chondrosarcoma patients. An siRNA screen targeting 779 different kinases was conducted in JJ012 chondrosarcoma cells in parallel with a compound screen consisting of 273 kinase inhibitors in JJ012, SW1353 and CH2879 chondrosarcoma cell lines. AURKA, CHK1 and PLK1 were identified as most promising targets and validated further in a more comprehensive panel of chondrosarcoma cell lines. Dose response curves were performed using tyrosine kinase inhibitors: MK-5108 (AURKA), LY2603618 (CHK1) and Volasertib (PLK1) using viability assays and cell cycle analysis. Apoptosis was measured at 24 h after treatment using a caspase 3/7 assay. Finally, chondrosarcoma patient samples (N = =34) were used to examine the correlation between AURKA, CHK1 and PLK1 RNA expression 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 addition increased sensitivity to conventional chemotherapy was observed after CHK1 inhibition in a subset of the cell lines. Interestingly, whereas AURKA and CHK1 were both expressed in chondrosarcoma patient samples, PLK1 expression was found to be low compared to normal cartilage. Analysis of patient samples revealed that high CHK1 RNA expression correlated with a worse overall survival. AURKA, CHK1 and PLK1 are identified as important survival genes in chondrosarcoma cell lines. Although further research is needed to validate these findings, inhibiting CHK1 seems to be the most promising potential therapeutic target for patients with chondrosarcoma.

Bcl-xl as the most promising Bcl-2 family member in targeted treatment of chondrosarcoma.

Chondrosarcomas are malignant cartilage tumors showing relative resistance to conventional chemo- and radiotherapy. Previous studies showed that chondrosarcoma cells could be sensitized to chemotherapy by inhibiting the Bcl-2 family members Bcl-2, Bcl-xl and Bcl-w using ABT-737. In this study we explored the specific role of Bcl-2 family members to identify the most important player in chondrosarcoma cell survival and chemo resistance. Immunohistochemistry was performed on tissue microarrays containing 137 conventional chondrosarcomas of different grades. Selective inhibition of Bcl-2 (S55746) or Bcl-xl (WEHI-539 or A-1155463) and the combination with doxorubicin or cisplatin was investigated in a panel of 8 chondrosarcoma cell lines using presto blue viability assays and caspase 3/7 glo apoptosis assays. In addition Bcl-2 and Bcl-xl inhibition was investigated in an orthotopic Swarm Rat Chondrosarcoma (SRC) model. Bcl-2 and Bcl-xl were most abundantly expressed in the primary tumors, and expression increased with increasing histological grade. A subset of chondrosarcoma cell lines was sensitive to selective inhibition of Bcl-xl, and synergy was observed with doxorubicin or cisplatin in 3 out of 8 chondrosarcoma cell lines resulting in apoptosis. Conversely, selective inhibition of Bcl-2 was not effective in chondrosarcoma cell lines and could not sensitize to chemotherapy. In vivo, selective inhibition of Bcl-xl, but not Bcl-2 resulted in a decrease in tumor growth rate, even though no sensitization to doxorubicin was observed. These results suggest that among the Bcl-2 family members, Bcl-xl is most important for chondrosarcoma survival. Further research is needed to validate whether single or combination treatment with chemotherapy will be beneficial for chondrosarcoma patients.

Hypoxia-induced upregulation of BMX kinase mediates therapeutic resistance in acute myeloid leukemia.

Oncogenic addiction to the Fms-like tyrosine kinase 3 (FLT3) is a hallmark of acute myeloid leukemia (AML) that harbors the FLT3-internal tandem duplication (FLT3-ITD) mutation. While FLT3 inhibitors like sorafenib show initial therapeutic efficacy, resistance rapidly develops through mechanisms that are incompletely understood. Here, we used RNA-Seq-based analysis of patient leukemic cells and found that upregulation of the Tec family kinase BMX occurs during sorafenib resistance. This upregulation was recapitulated in an in vivo murine FLT3-ITD-positive (FLT3-ITD+) model of sorafenib resistance. Mechanistically, the antiangiogenic effects of sorafenib led to increased bone marrow hypoxia, which contributed to HIF-dependent BMX upregulation. In in vitro experiments, hypoxia-dependent BMX upregulation was observed in both AML and non-AML cell lines. Functional studies in human FLT3-ITD+ cell lines showed that BMX is part of a compensatory signaling mechanism that promotes AML cell survival during FLT3 inhibition. Taken together, our results demonstrate that hypoxia-dependent upregulation of BMX contributes to therapeutic resistance through a compensatory prosurvival signaling mechanism. These results also reveal the role of off-target drug effects on tumor microenvironment and development of acquired drug resistance. We propose that the bone marrow niche can be altered by anticancer therapeutics, resulting in drug resistance through cell-nonautonomous microenvironment-dependent effects.

An orthotopic mouse model for chondrosarcoma of bone provides an in vivo tool for drug testing.

Chondrosarcoma is a malignant cartilaginous tumor of the bone. Recently, mutations in isocitrate dehydrogenase-1 (IDH1) and isocitrate dehydrogenase-2 (IDH2) were identified in central chondrosarcomas. As chondrosarcomas are notoriously resistant to conventional treatment modalities, the need for model systems to screen new treatment options is high. We used two chondrosarcoma cell lines (CH2879 and SW1353) to generate a bioluminescent orthotopic chondrosarcoma mouse model. Cell lines were stably transduced with a lentiviral luciferase expression vector, and after clonal selection, luciferase-expressing clones were subcutaneously and orthotopically implanted in nude mice. Mice injected with CH2879 cells were treated with doxorubicin over a period of 6 weeks. Both cell lines resulted in tumor growth. CH2879 tumors were consistently larger than SW1353 tumors. No difference in size could be observed between subcutaneous and orthotopic tumors. Tumor growth could be monitored over time through assessment of luciferase activity, without harming the mice. Using this model, we show that doxorubicin does not have a significant effect on in vivo tumor growth. We describe an orthotopic chondrosarcoma mouse model that can be used to test new treatment strategies evolving from in vitro research.

Update on targets and novel treatment options for high-grade osteosarcoma and chondrosarcoma.

Osteosarcoma and chondrosarcoma are the 2 most common malignant bone tumors. This review discusses the clinicopathologic features, recent preclinical developments, and targets currently being or to be validated in the clinic.

Functional profiling of receptor tyrosine kinases and downstream signaling in human chondrosarcomas identifies pathways for rational targeted therapy.

PURPOSE: Chondrosarcomas are notoriously resistant to cytotoxic chemotherapeutic agents. We sought to identify critical signaling pathways that contribute to their survival and proliferation, and which may provide potential targets for rational therapeutic interventions. EXPERIMENTAL DESIGN: Activation of receptor tyrosine kinases (RTK) was surveyed using phospho-RTK arrays. S6 phosphorylation and NRAS mutational status were examined in chondrosarcoma primary tumor tissues. siRNA or small-molecule inhibitors against RTKs or downstream signaling proteins were applied to chondrosarcoma cells and changes in biochemical signaling, cell cycle, and cell viability were determined. In vivo antitumor activity of BEZ235, a phosphoinositide 3-kinase (PI3K)/mTOR inhibitor, was evaluated in a chondrosarcoma xenograft model. RESULTS: Several RTKs were identified as critical mediators of cell growth, but the RTK dependencies varied among cell lines. In exploration of downstream signaling pathways, strong S6 phosphorylation was found in 69% of conventional chondrosarcomas and 44% of dedifferentiated chondrosarcomas. Treatment with BEZ235 resulted in dramatic reduction in the growth of all chondrosarcoma cell lines. Tumor growth was similarly inhibited in a xenograft model of chondrosarcoma. In addition, chondrosarcoma cells with an NRAS mutation were sensitive to treatment with a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor. Functional NRAS mutations were found in 12% of conventional central chondrosarcomas. CONCLUSIONS: RTKs are commonly activated in chondrosarcoma, but because of their considerable heterogeneity, targeted inhibition of the PI3K/mTOR pathway represents a rational therapeutic strategy. Chondrosarcomas with NRAS mutations may benefit from treatment with MEK inhibitors.

Cartilage tumour progression is characterized by an increased expression of heparan sulphate 6O-sulphation-modifying enzymes.

Chondrosarcomas are malignant cartilage-forming tumours that can arise centrally (in the medulla) or peripherally (at the surface) of the bone. They are classified into three histological grades which correspond to the clinical severity. Previous studies by our group have shown altered signal transduction of the fibroblast growth factor and Wnt signalling pathways during peripheral chondrosarcoma progression. Heparan sulphate (HS) is a glycosaminoglycan that facilitates receptor binding of multiple growth factors, in which the sulphation of 6O position plays a pivotal role. 6O-Sulphation occurs through three HS 6O-sulphotransferases (HS6ST1-3) and is fine-tuned by two endosulphatases (SULF1-2) that remove 6O-sulphate groups. We have investigated whether the expression of HS6STs and SULFs changes during chondrosarcoma progression and have determined 6O-sulphation levels in two chondrosarcoma cell lines. Immunohistochemistry on tissue microarrays of chondrosarcomas showed that HS6ST3 and SULF1 were highly expressed in most chondrosarcomas, whereas SULF2 expression was absent in most cases. HS6ST1 and HS6ST2 expression are significantly increased during chondrosarcoma progression, which suggest that 6O-sulphation is increased during progression. This was confirmed in one grade III chondrosarcoma cell line, which showed a dramatically increased 6O-sulphation compared to an articular chondrocyte cell line by HPLC; another cell line showed an increased expression of one 6O-sulphated HS disaccharide. In conclusion, our results show increased HS6ST1 and HS6ST2 expression during chondrosarcoma progression and increased HS 6O-sulphation in vitro. As 6O-sulphation plays an important role in signal transduction, altered HS6ST expression might be associated with changes in signal transduction pathways in chondrosarcoma progression.

Somatic mosaic IDH1 and IDH2 mutations are associated with enchondroma and spindle cell hemangioma in Ollier disease and Maffucci syndrome.

Ollier disease and Maffucci syndrome are non-hereditary skeletal disorders characterized by multiple enchondromas (Ollier disease) combined with spindle cell hemangiomas (Maffucci syndrome). We report somatic heterozygous mutations in IDH1 (c.394C>T encoding an R132C substitution and c.395G>A encoding an R132H substitution) or IDH2 (c.516G>C encoding R172S) in 87% of enchondromas (benign cartilage tumors) and in 70% of spindle cell hemangiomas (benign vascular lesions). In total, 35 of 43 (81%) subjects with Ollier disease and 10 of 13 (77%) with Maffucci syndrome carried IDH1 (98%) or IDH2 (2%) mutations in their tumors. Fourteen of 16 subjects had identical mutations in separate lesions. Immunohistochemistry to detect mutant IDH1 R132H protein suggested intraneoplastic and somatic mosaicism. IDH1 mutations in cartilage tumors were associated with hypermethylation and downregulated expression of several genes. Mutations were also found in 40% of solitary central cartilaginous tumors and in four chondrosarcoma cell lines, which will enable functional studies to assess the role of IDH1 and IDH2 mutations in tumor formation.