Novel models of myxoid liposarcoma xenografts mimicking the biological and pharmacologic features of human tumors.

PURPOSE: Myxoid liposarcoma is a common subtype of liposarcoma. It is associated in more than 90% of cases with the chromosomal translocation t(12;16)(q13;p11) leading to the fusion FUS-CHOP gene that is responsible for the oncogenic transformation of preadipocytes. Recently the marine natural product trabectedin has shown highly selective activity for myxoid liposarcoma, even in the most aggressive round-cell subtype. EXPERIMENTAL DESIGN: Fragments of 17 sarcomas were transplanted s.c. in female athymic NCr-nu/nu mice. Xenografts were established and characterized by morphology, fluorescence in situ hybridization analysis for the translocation and reverse transcriptase-PCR analysis for fusion transcripts. Trabectedin was injected i.v. RESULTS: Seven of 17 tumors grew as continuous xenografts, five of them being myxoid liposarcoma of the round-cell subtype. The chromosomal rearrangement and fusion transcripts in different passages were the same as in the human tumors from which they were derived. The responsiveness to trabectedin in type II myxoid liposarcoma xenografts was as high as in patients. The pathologic response was associated with the presence of the FUS-CHOP fusion gene, indicating that the drug does not totally eradicate the disease. Type III myxoid liposarcoma xenografts seemed much less sensitive to trabectedin, confirming previous clinical observations. CONCLUSIONS: This study reports for the first time the characterization of human myxoid liposarcoma xenografts that adequately mimic the biological and pharmacologic features of the human tumor. These models offer a useful tool for investigating the mechanism of selectivity of trabectedin, testing new combinations with this drug and evaluating novel therapies for myxoid liposarcoma.

Functional mapping of receptor tyrosine kinases in myxoid liposarcoma.

PURPOSE: The aim of this study was to analyze receptor tyrosine kinases (RTK) and their downstream signaling activation profile in myxoid liposarcomas (MLS) by investigating 14 molecularly profiled tumors: 7 naive and 7 treated with conventional chemotherapy/radiotherapy or the new drug trabectedin. EXPERIMENTAL DESIGN: Frozen and matched formalin-fixed, paraffin-embedded material from surgical specimens were analyzed using biochemical, molecular, and molecular/cytogenetic approaches, complemented by immunohistochemistry and confocal microscopy. RESULTS: In the absence of any RTK and downstream effector deregulation, the naive cases revealed epidermal growth factor receptor, platelet-derived growth factor receptor B, RET, and MET activation sustained by autocrine/paracrine loops, and RTK cross-talk as a result of heterodimerization. Interestingly, RET and MET activation seems to play a major role in the pathogenesis of MLS by involving different targets through different mechanisms. RET activation (which may activate MET) involves the tumoral vascular component by means of RET/MET cross-talk and VEGFA (vascular endothelial growth factor A)/GFRalpha3 (glial cell-derived neurotrophic factor family receptor alpha3)/artemin-mediated signaling as revealed by VEGF receptor 2/RET coimmunoprecipitation. MET activation involves the cellular tumor component by means of a direct ligand-dependent loop and indirect GFRalpha3 (RET coreceptor)/artemin-mediated signaling. About downstream signaling, the association of AKT activation with the round cell variant is interesting. No relevant changes in the original RTK activation profiles were observed in the posttreatment cases, a finding that is in keeping with the nontargeted treatments used. CONCLUSIONS: These findings highlight the particular cell-specific activation profile of RET/GFRalpha3 and MET in MLS, and the close correlation between AKT activation and the round cell variant, thus opening up new therapeutic perspectives for MET/AKT inhibitors and antagonistic small molecules binding GFRalpha3.

Antitumor and anti-inflammatory effects of trabectedin on human myxoid liposarcoma cells.

Inflammatory mediators present in the tumor milieu may promote cancer progression and are considered promising targets of novel biological therapies. We previously reported that the marine antitumor agent trabectedin, approved in Europe in 2007 for soft tissue sarcomas and in 2009 for ovarian cancer, was able to downmodulate the production of selected cytokines/chemokines in immune cells. Patients with myxoid liposarcoma (MLS), a subtype characterized by the expression of the oncogenic transcript FUS-CHOP, are highly responsive to trabectedin. The drug had marked antiproliferative effects on MLS cell lines at low nanomolar concentrations. We tested the hypothesis that trabectedin could also affect the inflammatory mediators produced by cancer cells. Here, we show that MLS express several cytokines, chemokines, and growth factors (CCL2, CCL3, CCL5, CXCL8, CXCL12, MIF, VEGF, SPARC) and the inflammatory and matrix-binder protein pentraxin 3 (PTX3), which build up a prominent inflammatory environment. In vitro treatment with noncytotoxic concentrations of trabectedin selectively inhibited the production of CCL2, CXCL8, IL-6, VEGF, and PTX3 by MLS primary tumor cultures and/or cell lines. A xenograft mouse model of human MLS showed marked reduction of CCL2, CXCL8, CD68+ infiltrating macrophages, CD31+ tumor vessels, and partial decrease of PTX3 after trabectedin treatment. Similar findings were observed in a patient tumor sample excised after several cycles of therapy, indicating that the results observed in vitro might have in vivo relevance. In conclusion, trabectedin has dual effects in liposarcoma: in addition to direct growth inhibition, it affects the tumor microenvironment by reducing the production of key inflammatory mediators.

Analysis of receptor tyrosine kinases (RTKs) and downstream pathways in chordomas.

We have previously demonstrated that chordomas express activated platelet-derived growth factor receptor (PDGFRB) and that treatment with imatinib, which is capable of switching off the activation of various receptor tyrosine kinases (RTKs) including PDGFRB, benefits a number of patients. The aim of this study was to identify the possible presence of other activated RTKs and their downstream signaling effectors. Cryopreserved material from 22 naïve sporadic chordomas was investigated for the presence of activated RTKs and their cognate ligands and downstream signaling effectors by means of human phospho-RTK antibody arrays, Western blotting, and molecular analysis; immunohistochemistry and fluorescence in situ hybridization were used to analyze the corresponding formalin-fixed and paraffin-embedded samples. We detected activated PDGFRB, FLT3, and colony stimulating factor 1 receptor (CSF1R) of the PDGFR family and highly phosphorylated EGFR, HER2/neu, and (to a lesser extent) HER4 of the EGFR family. The detection of PDGFRB/PDGFB confirmed our previous data. The presence of activated EGFR was paralleled by the finding of high levels of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) and PDGFB co-expression and PDGFRB co-immunoprecipitation. Of the downstream effectors, the PI3K/AKT and RAS/MAPK pathways were both activated, thus leading to the phosphorylation of mammalian target of rapamycin (mTOR) and 4E-BP1 among the regulators involved in translational control. Taken together, our results (i) provide a rationale for tailored treatments targeting upstream activated receptors, including the PDGFR and EGFR families; (ii) support the idea that a combination of upstream antagonists and mTOR inhibitors enhances the control of tumor growth; and (iii) indicate that the 4E-BP1/eIF4E pathway is a major regulator of protein synthesis in chordoma.

Trabectedin (ET-743) promotes differentiation in myxoid liposarcoma tumors.

Differentiation is a complex set of events that can be blocked by rearrangements of regulatory genes producing fusion proteins with altered properties. In the case of myxoid liposarcoma (MLS) tumors, the causative abnormality is a fusion between the CHOP transcription factor and the FUS or EWS genes. CHOP belongs to and is a negative regulator of the large CAAT/enhancer binding protein family whose alpha, beta, and delta members are master genes of adipogenesis. Recent clinical data indicate a peculiar sensitivity of these tumors to the natural marine compound trabectedin. One hypothesis is that the activity of trabectedin is related to the inactivation of the FUS-CHOP oncogene. We find that trabectedin causes detachment of the FUS-CHOP chimera from targeted promoters. Reverse transcription-PCR and chromatin immunoprecipitation analysis in a MLS line and surgical specimens of MLS patients in vivo show activation of the CAAT/enhancer binding protein-mediated transcriptional program that leads to morphologic changes of terminal adipogenesis. The activity is observed in cells with type 1 but not type 8 fusions. Hence, the drug induces maturation of MLS lipoblasts in vivo by targeting the FUS-CHOP-mediated transcriptional block. These data provide a rationale for the specific activity of trabectedin and open the perspective of combinatorial treatments with drugs acting on lipogenic pathways.

T670X KIT mutations in gastrointestinal stromal tumors: making sense of missense.

BACKGROUND: Chronic myeloid leukemia, gastrointestinal stromal tumors (GISTs), and idiopathic hypereosinophilic syndrome are associated with pathological deregulation of the tyrosine kinases BCR-ABL, KIT, and PDGFRA, respectively. Patients who become resistant to imatinib treatment often develop secondary mutations, the most common of which results in a substitution of isoleucine for threonine at the same location in the ATP-binding domain in all three kinases (in KIT this occurs at amino acid 670). We sought to determine why Thr is always replaced by Ile. METHODS: All possible point mutations in the DNA triplet codon that could result in amino acid substitutions at Thr670 (Thr670Arg, Thr670Ile, Thr670Lys, Thr670Ala, Thr670Ser, Thr670Pro) were introduced by site-specific mutagenesis of the complementary DNA for a constitutively active, imatinib-sensitive form of the KIT receptor, Delta559/KIT. The resulting mutant KIT proteins were transiently expressed in COS1 African green monkey kidney cells grown with and without imatinib, and cell extracts were analyzed for KIT activation by immunoprecipitation and immunoblotting to determine autophosphorylation levels. We also performed molecular modeling to estimate the relative affinities of wild-type (Thr670) KIT and the KIT mutants for ATP and imatinib. RESULTS: Like the parental strain, Thr670Ala, Thr670Ser, and Thr670Lys mutants were inhibited by 5 microM imatinib, but in comparison, they were only weakly active and Thr670Pro and Thr670Arg were not active at all. Only the Thr670Ile mutant was fully active (autophosphorylated) and resistant to imatinib. These findings were consistent with computer modeling predictions that ranked these mutants Thr - Ile > Ala, Ser > Lys >> Pro according to their affinity for ATP but Thr > Ala, Ser > Lys >Pro - Arg - Ile according to their affinity for imatinib. CONCLUSIONS: This combination of in vitro and molecular modeling analyses shows why, among all possible amino acid substitutions at position 670 of KIT, only Ile is naturally selected as a resistance mutant in imatinib-treated GIST patients.

A genomewide search using an original pairwise sampling approach for large genealogies identifies a new locus for total and low-density lipoprotein cholesterol in two genetically differentiated isolates of Sardinia.

A powerful approach to mapping the genes for complex traits is to study isolated founder populations, in which genetic heterogeneity and environmental noise are likely to be reduced and in which extended genealogical data are often available. Using graph theory, we applied an approach that involved sampling from the large number of pairwise relationships present in an extended genealogy to reconstruct sets of subpedigrees that maximize the useful information for linkage mapping while minimizing calculation burden. We investigated, through simulation, the properties of the different sets in terms of bias in identity-by-descent (IBD) estimation and power decrease under various genetic models. We applied this approach to a small isolated population from Sardinia, the village of Talana, consisting of a unique large and complex pedigree, and performed a genomewide search through variance-components linkage analysis for serum lipid levels. We identified a region of significant linkage on chromosome 2 for total serum cholesterol and low-density lipoprotein (LDL) cholesterol. Through higher-density mapping, we obtained an increased linkage for both traits on 2q21.2-q24.1, with a LOD score of 4.3 for total serum cholesterol and of 3.9 for LDL cholesterol. A replication study was performed in an independent and larger set from a genetically differentiated isolated population of the same region of Sardinia, the village of Perdasdefogu. We obtained consistent linkage to the region for total serum cholesterol (LOD score 1.4) and LDL cholesterol (LOD score 2.2), with a level of concordance uncommon for complex traits, and refined the location of the quantitative-trait locus. Interestingly, the 2q21.1-22 region has also been linked to premature coronary heart disease in Finns, and, in the adjacent 2q14 region, significant linkage with triglycerides has been reported in Hutterites.