Search

Purpose The relevance of the subfamily A members of ATP-binding cassette (ABCA) transporters as biomarkers of risk and response is emerging in different tumors, but their mechanisms of action have only been partially defined. In this work, we investigated their role in Ewing sarcoma (EWS), a pediatric cancer with unmet clinical issues. Methods The expression of ABC members was evaluated by RT-qPCR in patients with localized EWS. The correlation with clinical outcome was established in different datasets using univariate and multivariate statistical methods. Functional studies were conducted in cell lines from patient-derived xenografts (PDXs) using gain- or loss-of-function approaches. The impact of intracellular cholesterol levels and cholesterol lowering drugs on malignant parameters was considered. Results We found that ABCA6, which is usually poorly expressed in EWS, when upregulated became a prognostic factor of a favorable outcome in patients. Mechanistically, high expression of ABCA6 impaired cell migration and increased cell chemosensitivity by diminishing the intracellular levels of cholesterol and by constitutive IGF1R/AKT/mTOR expression/activation. Accordingly, while exposure of cells to exogenous cholesterol increased AKT/mTOR activation, the cholesterol lowering drug simvastatin inhibited IGF1R/AKT/mTOR signaling and prevented Ser166 phosphorylation of MDM2. This, in turn, favored p53 activation and enhanced pro-apoptotic effects of doxorubicin. Conclusions Our study reveals that ABCA6 acts as tumor suppressor in EWS cells via cholesterol-mediated inhibition of IGF1R/AKT/MDM2 signaling, which promotes the pro-apoptotic effects of doxorubicin and reduces cell migration. Our findings also support a role of ABCA6 as biomarker of EWS progression and sustains its assessment for a more rational use of statins as adjuvant drugs.

Additional materials and methods

Silencing of DNMTs inhibits cell proliferation, induces osteoblastic differentiation and enhances DXR sensitivity

Inhibitory activity of MC3343 against human DNMT1 and human DNMT3a

The identification of new therapeutic strategies against osteosarcoma, the most common primary bone tumor, continues to be a primary goal to improve the outcomes of patients refractory to conventional chemotherapy. Osteosarcoma originates from the transformation of mesenchymal stem cells (MSC) and/or osteoblast progenitors, and the loss of differentiation is a common biological osteosarcoma feature, which has strong significance in predicting tumor aggressiveness. Thus, restoring differentiation through epigenetic reprogramming is potentially exploitable for therapeutic benefits. Here, we demonstrated that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 affected tumor proliferation by blocking osteosarcoma cells in G1 or G2–M phases and induced osteoblastic differentiation through the specific reexpression of genes regulating this physiologic process. Although MC3343 has a similar antiproliferative effect as 5azadC, the conventional FDA-approved nucleoside inhibitor of DNA methylation, its effects on cell differentiation are distinct. Induction of the mature osteoblast phenotype coupled with a sustained cytostatic response was also confirmed in vivo when MC3343 was used against a patient-derived xenograft (PDX). In addition, MC3343 displayed synergistic effects with doxorubicin and cisplatin (CDDP), two major chemotherapeutic agents used to treat osteosarcoma. Specifically, MC3343 increased stable doxorubicin bonds to DNA, and combined treatment resulted in sustained DNA damage and increased cell death. Overall, this nonnucleoside DNMTi is an effective novel agent and is thus a potential therapeutic option for patients with osteosarcoma who respond poorly to preadjuvant chemotherapy. Mol Cancer Ther; 17(9); 1881–92. ©2018 AACR.

Supplementary Table S1: Efficacy of anti-CD99 mAb 0662 in a panel of EWS cell lines Supplementary Table S2: Fold induction/reduction of gene expression in treated samples over control mRNAs, analyzed with F-statistic (error

Purpose: Large-scale sequencing studies have indicated that besides genomic alterations, the posttranscriptional regulation of gene expression or epigenetic mechanisms largely influences the clinical behavior of Ewing sarcoma. We investigated the significance of the RNA-binding protein IGF2BP3 in the regulation of Ewing sarcoma aggressiveness.Experimental Design: Explorative study was performed in 14 patients with localized Ewing sarcoma using RNA sequencing. Next, 128 patients with localized Ewing sarcoma were divided into two cohorts. In the training set, 29 Ewing sarcoma samples were analyzed using Affymetrix GeneChip arrays. In the validation set, 99 Ewing sarcoma samples were examined using qRT-PCR. Patient-derived cell lines and experimental models were used for functional studies.Results:Univariate and multivariate analyses indicated IGF2BP3 as a potent indicator of poor prognosis. Furthermore, ABCF1 mRNA was identified as a novel partner of IGF2BP3. Functional studies indicated IGF2BP3 as an oncogenic driver and ABCF1 mRNA as a sponge that by binding IGF2BP3, partly repressed its functions. The combined evaluation of IGF2BP3 and ABCF1 could identify different patient outcomes—high IGF2BP3 and low ABCF1 levels indicated poor survival (25%), whereas low IGF2BP3 and high ABCF1 levels indicated favorable survival (85.5%). The bromodomain and extraterminal domain inhibitor (BETi) JQ1 decreased IGF2BP3 expression, modified the expression of its validated targets and inhibited the capability of Ewing sarcoma cells to grow under anchorage-independent conditions.Conclusions: The combined assessment of IGF2BP3 and ABCF1 predicts recurrence in Ewing sarcoma patients. Thus, for patients with high expression of IGF2BP3 and poor probability of survival, the use of BETis should be clinically evaluated. Clin Cancer Res; 24(15); 3704–16. ©2018 AACR.

IGF2BP3 expression in ES cells after EWS-FLI1 silencing

Supplementary Tables include Supplementary Table S1, S2, S3, S4, S5, and S6

dAbd C7 activity is related to p53 status, but is ineffective in h-MSC cells.

Effects of 0662 on Gadd45-α mRNA expression and osteoblastic differentiation on mesenchymal stem cells.

Purpose: The paucity of new drugs for the treatment of Ewing sarcoma (EWS) limits the cure of these patients. CD99 has a strong membranous expression in EWS cells and, being also necessary for tumor survival, is a suitable target to aim at. In this article, we described a novel human monospecific bivalent single-chain fragment variable diabody (dAbd C7) directed against CD99 of potential clinical application.Experimental Design:In vitro and in vivo evaluation of cell death and of the molecular mechanisms triggered by anti-CD99 agents were performed alone or in combination with doxorubicin to demonstrate efficacy and selectivity of the new dAbd C7.Results: The dAbd C7 induced rapid and massive EWS cell death through Mdm2 degradation and p53 reactivation. Mdm2 overexpression as well as silencing of p53 in p53wt EWS cells decreased CD99-induced EWS cell death, whereas treatment with nutlin-3 enhanced it. Furthermore, cell death was associated with induction of p21, bax, and mitochondrial depolarization together with substantial inhibition of tumor cell proliferation. Combined treatment of anti-CD99 dAbd C7 with doxorubicin was additive both in vitro and in vivo against EWS xenografts. Normal mesenchymal stem cells showed no p53 activation and were resistant to cell death, unless transformed by EWS-FLI, the oncogenic driver of EWS.Conclusions: These results indicate that dAbd C7 is a suitable candidate tool to target CD99 in patients with EWS able to spare normal stem cells from death as it needs an aberrant genetic context for the efficient delivery of CD99-triggered cell death. Clin Cancer Res; 21(1); 146–56. ©2014 AACR.

Supplementary Data from Integrated Molecular Characterization of Patient-Derived Models Reveals Therapeutic Strategies for Treating CIC-DUX4 Sarcoma

HSP70 levels are unaffected from dAbd C7 treatment.

Effects of combined treatment with doxorubicin and dAbd C7.

Increased avidity and efficacy of dAbd over scFv C7 in 6647 EWS cell line.

Capicua-double homeobox 4 (CIC-DUX4)–rearranged sarcomas (CDS) are extremely rare, highly aggressive primary sarcomas that represent a major therapeutic challenge. Patients are treated according to Ewing sarcoma protocols, but CDS-specific therapies are strongly needed. In this study, RNA sequencing was performed on patient samples to identify a selective signature that differentiates CDS from Ewing sarcoma and other fusion-driven sarcomas. This signature was used to validate the representativeness of newly generated CDS experimental models—patient-derived xenografts (PDX) and PDX-derived cell lines—and to identify specific therapeutic vulnerabilities. Annotation analysis of differentially expressed genes and molecular gene validation highlighted an HMGA2/IGF2BP/IGF2/IGF1R/AKT/mTOR axis that characterizes CDS and renders the tumors particularly sensitive to combined treatments with trabectedin and PI3K/mTOR inhibitors. Trabectedin inhibited IGF2BP/IGF2/IGF1R activity, but dual inhibition of the PI3K and mTOR pathways was required to completely dampen downstream signaling mediators. Proof-of-principle efficacy for the combination of the dual AKT/mTOR inhibitor NVP-BEZ235 (dactolisib) with trabectedin was obtained in vitro and in vivo using CDS PDX-derived cell lines, demonstrating a strong inhibition of local tumor growth and multiorgan metastasis. Overall, the development of representative experimental models (PDXs and PDX-derived cell lines) has helped to identify the unique sensitivity of the CDS to AKT/mTOR inhibitors and trabectedin, revealing a mechanism-based therapeutic strategy to fight this lethal cancer.Significance:This study identifies altered HMGA2/IGF2BP/IGF2 signaling in CIC-DUX4 sarcomas and provides proof of principle for combination therapy with trabectedin and AKT/mTOR dual inhibitors to specifically combat the disease.

Supplementary Methods from CD99 Triggering in Ewing Sarcoma Delivers a Lethal Signal through p53 Pathway Reactivation and Cooperates with Doxorubicin

p53 is necessary for CD99-induced cell-death and is phosphorilated by 0662.

Supplementary Table 1 from Overcoming Glutathione S-Transferase P1–Related Cisplatin Resistance in Osteosarcoma

Supplementary Figure 2 from Overcoming Glutathione S-Transferase P1–Related Cisplatin Resistance in Osteosarcoma

Supplementary Figure and Table Legends from Overcoming Glutathione S-Transferase P1–Related Cisplatin Resistance in Osteosarcoma

Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most used drugs for osteosarcoma chemotherapy. By using a series of CDDP-resistant variants, which were established from the U-2OS and Saos-2 human osteosarcoma cell lines, we found that CDDP resistance was mainly associated with the increase of both the intracellular level and enzymatic activity of glutathione S-transferase P1 (GSTP1). On the basis of these findings, we evaluated the clinical effect of GSTP1 in a series of 34 high-grade osteosarcoma patients and we found that the increased expression of GSTP1 gene was associated with a significantly higher relapse rate and a worse clinical outcome. These indications prompted us to assess the in vitro effectiveness of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), a promising new anticancer agent that is a highly efficient inhibitor of GSTP1. NBDHEX was tested on a panel of 10 human osteosarcoma cell lines and 20 variants of the U-2OS or Saos-2 cell lines that were resistant to CDDP, doxorubicin, or methotrexate. NBDHEX proved to be very active on the vast majority of these cell lines, including those with higher GSTP1 levels and enzymatic activity. Drug combination studies showed that NBDHEX can be used in association with CDDP and provided useful information about the best modality of their combined administration. In conclusion, our findings show that GSTP1 has a relevant effect for both CDDP resistance and clinical outcome of high-grade osteosarcoma and that targeting GSTP1 with NBDHEX may be considered a promising new therapeutic possibility for osteosarcoma patients who fail to respond to conventional chemotherapy. [Cancer Res 2008;68(16):6661–8]

Supplementary Figure 1 from Overcoming Glutathione S-Transferase P1–Related Cisplatin Resistance in Osteosarcoma

Supplementary Table 2 from Overcoming Glutathione S-Transferase P1–Related Cisplatin Resistance in Osteosarcoma

Introduction: Small round cell osteosarcoma (SCOS) is a very rare type of osteosarcoma, composed by small cells producing bone matrix, with limited studies that mainly focus on histological features. In this study we employed RNA sequencing to discover the transcriptional profile of SCOS in order to find a specific gene signatures useful to find new potential therapeutic targets. To date, the tumor is treated according to the osteosarcoma protocols. Material and Methods: We studied 15 primary tumor including 6 SCOS and 9 high grade Osteosarcoma (OS) by RNASeq. Pair-end libraries were synthesized using TruSeq RNA Library Prep Kit for Enrichment and loaded on NextSeq 500 platform (Illumina). After FastqQC quality control, fastq data were aligned to Homo_sapiens GRCh38 with STAR algorithm and counted by featureCounts. In Addition we randomly selected 30 muscle-skeletal normal tissues from 800 available samples from Genotype-Tissue Expression repository (GTEX). Using surrogate variables to remove batch effects and other unwanted variations, we performed differentially expression analysis employing DESeq2 (R package). We considered as differentially expressed genes those BH adj p-value Results: To elucidate the transcriptome landscape of SCOS we first performed differential gene expression analysis between SCOS and normal tissue identifying 5061 deregulated genes (2516 up-regulated and 2545 down-regulated in SCOS). Then, from this signature we filtered out the signature differentially expressed between OS and normal tissue, obtaining a specific SCOS signature of 1371 genes (1013 up-regulated and 358 down-regulated). To study the biological function behind SCOS we performed GSEA and we found that SCOS gene signature resulted enriched in epigenetics mechanisms as well as in embryonic stem cell (ES) gene sets. In particular, we found the overexpression of EZH2, ASXL1, HDAC9, MYCN, MYB genes involved in Polycomb group complex. This on one side confirms the highly undifferentiated nature of this tumor that could explain the more aggressive behaviour of SCOS in comparison to OS; on the other side, offers in epigenetic drugs a new therapeutic perspective. Validation studies are in progress. Conclusions: This work firstly profiled the transcriptome of SCOS and identifies new pathways that can be of interest for diagnostic and/or therapeutic purposes. This work was supported by Ministry of Health 5 × 1000, Anno 2020 Redditi 2019, contributions to the IRCCS, Istituto Ortopedico Rizzoli. Citation Format: Maria Antonella Laginestra, Alessandro Parra, Elisa Simonetti, Laura Formentini, Michela Pasello, Alberto Righi, Davide Maria Donati, Cristina Ferrari, Laura Pazzaglia, Katia Scotlandi. Transcriptome analysis of Small Cell Osteosarcoma reveals an embryonic stem cell-like phenotype. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3958.

Ewing sarcoma (EWS), the second most common malignant bone tumor in children and adolescents, occurs abruptly without clear evidence of tumor history or progression. Previous association studies have identified some inherited variants associated with the risk of developing EWS but a common picture of the germline susceptibility to this tumor remains largely unclear. Here, we examine the association between thirty single nucleotide polymorphisms (SNPs) of the

In this article an incorrect affiliation had inadvertently been added. The original article has been updated.

Overexpression of ATP-binding cassette (ABC) transporters is a cause of drug resistance in a plethora of tumors. More recent evidence indicates additional contribution of these transporters to other processes, such as tumor cell dissemination and metastasis, thereby extending their possible roles in tumor progression. While the role of some ABC transporters, such as ABCB1, ABCC1 and ABCG2, in multidrug resistance is well documented, the mechanisms by which ABC transporters affect the proliferation, differentiation, migration and invasion of cancer cells are still poorly defined and are frequently controversial. This review, summarizes recent advances that highlight the role of subfamily A members in cancer. Emerging evidence highlights the potential value of ABCA members as biomarkers of risk and response in different tumors, but information is disperse and very little is known about their possible mechanisms of action. The only clear evidence is that ABCA members are involved in lipid metabolism and homeostasis. In particular, the relationship between ABCA1 and cholesterol is becoming evident in different fields of biology, including cancer. In parallel, emerging findings indicate that cholesterol, the main component of cell membranes, can influence many physiological and pathological processes, including cell migration, cancer progression and metastasis. This review aims to link the dispersed knowledge regarding the relationship of ABCA members with lipid metabolism and cancer in an effort to stimulate and guide readers to areas that the writers consider to have significant impact and relevant potentialities.

The ATP-binding cassette (ABC) transporter superfamily consists of several proteins with a wide repertoire of functions. Under physiological conditions, ABC transporters are involved in cellular trafficking of hormones, lipids, ions, xenobiotics, and several other molecules, including a broad spectrum of chemical substrates and chemotherapeutic drugs. In cancers, ABC transporters have been intensely studied over the past decades, mostly for their involvement in the multidrug resistance (MDR) phenotype. This review provides an overview of ABC transporters, both related and unrelated to MDR, which have been studied in osteosarcoma and Ewing’s sarcoma. Since different backbone drugs used in first-line or rescue chemotherapy for these two rare bone sarcomas are substrates of ABC transporters, this review particularly focused on studies that have provided findings that have been either translated to clinical practice or have indicated new candidate therapeutic targets; however, findings obtained from ABC transporters that were not directly involved in drug resistance were also discussed, in order to provide a more complete overview of the biological impacts of these molecules in osteosarcoma and Ewing’s sarcoma. Finally, therapeutic strategies and agents aimed to circumvent ABC-mediated chemoresistance were discussed to provide future perspectives about possible treatment improvements of these neoplasms.

Ewing sarcoma, a highly aggressive pediatric tumor, is driven by EWS–FLI1, an oncogenic transcription factor that remodels the tumor genetic landscape. Epigenetic mechanisms play a pivotal role in Ewing sarcoma pathogenesis, and the therapeutic value of compounds targeting epigenetic pathways is being identified in preclinical models. Here, we showed that modulation of CD99, a cell surface molecule highly expressed in Ewing sarcoma cells, may alter transcriptional dysregulation in Ewing sarcoma through control of the zyxin–GLI1 axis. Zyxin is transcriptionally repressed, but GLI1 expression is maintained by EWS–FLI1. We demonstrated that targeting CD99 with antibodies, including the human diabody C7, or genetically inhibiting CD99 is sufficient to increase zyxin expression and induce its dynamic nuclear accumulation. Nuclear zyxin functionally affects GLI1, inhibiting targets such as NKX2–2, cyclin D1, and PTCH1 and upregulating GAS1, a tumor suppressor protein negatively regulated by SHH/GLI1 signaling. We used a battery of functional assays to demonstrate (i) the relationship between CD99/zyxin and tumor cell growth/migration and (ii) how CD99 deprivation from the Ewing sarcoma cell surface is sufficient to specifically affect the expression of some crucial EWS–FLI1 targets, both in vitro and in vivo, even in the presence of EWS–FLI1. This article reveals that the CD99/zyxin/GLI1 axis is promising therapeutic target for reducing Ewing sarcoma malignancy.

Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient’s tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches.

Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated.

Ewing's sarcoma (EWS) is the second most common primary bone tumor in pediatric patients characterized by over expression of CD99. Current management consists in extensive chemotherapy in addition to surgical resection and/or radiation. Recent improvements in treatment are still overshadowed by severe side effects such as toxicity and risk of secondary malignancies; therefore, more effective strategies are urgently needed. The goal of this work was to develop a rapid, inexpensive, and "up-scalable" process of a novel human bivalent single-chain fragment variable diabody (C7 dAbd) directed against CD99, as a new therapeutic approach for EWS. We first investigated different Escherichia coli constructs of C7 dAbd in small-scale studies. Starting from 60 % soluble fraction, we obtained a yield of 25 mg C7 dAbd per liter of bacterial culture with the construct containing pelB signal sequence. In contrast, a low recovery of C7 dAbd was achieved starting from periplasmic inclusion bodies. In order to maximize the yield of C7 dAbd, large-scale fermentation was optimized. We obtained from 75 % soluble fraction 35 mg C7 dAbd per L of cell culture grown in a synthetic media containing 3 g/L of vegetable peptone and 1 g/L of yeast extract. Furthermore, we demonstrated the better efficacy of the cell lysis by homogenization versus periplasmic extraction, in reducing endotoxin level of the C7 dAbd. For gram-scale purification, a direct aligned two-step chromatography cascade based on binding selectivity was developed. Finally, we recovered C7 dAbd with low residual process-related impurities, excellent reactivity, and apoptotic ability against EWS cells.

e23564 Background: Activity of temozolomide (TEM) and irinotecan (IRI) in recurrent Ewing sarcoma (EWS) was demonstrated. Few data are available on TEMIRI use upfront. Biological predictive factors are lacking. Methods: This multi-institutional retrospective study (NCT03542097) included 59 patients with EWS. 8 patients with very high risk (HR) EWS (multivisceral ± bone marrow) received TEMIRI (TEM 100 mg/m2/day oral, and IRI 40 mg/m2/day intravenous, days 1-5, every 21 days) upfront, 51 patients after relapse (28% in 1st line, 72% ≥ 2nd line). Overall response rate (ORR: CR+PR), SD, and PD, progression-free (PFS) and overall survival (OS) were assessed. The relationship between pre-treatment expression of MGMT and ABCG2 (when FFPE tissue available) with ORR, PFS and OS was evaluated. Results: Median age was 27 years (range 4-62 years): 47 patients (80%) were adults (≥18 years), 35 (61%) had ECOG 0 and 42 (71%) presented with multivisceral disease (+ bone marrow in 5). MGMT was positive in 16/30 (53%), ABCG2 in 4/33 (12%). ORR for upfront TEMIRI (n = 8) was 50% (CR + PR = 1 + 3), with SD 50%, while in recurrent EWS (n = 49, 2 patients no measurable by RECIST) ORR was 31% (CR + PR = 4 + 11), SD 38%, and PD 31%. A better ORR was observed in adult (p = 0.008) & ECOG 0 (p = 0.001) patients; MGMT & ABCG2 expression did not influence ORR. 6-mos PFS was 87% after TEMIRI upfront, 43% at recurrence (p = 0.06), and 65% vs 28% (p = 0.02) for ECOG 0 vs ECOG 1-2, respectively. 6-mos PFS was 62% in MGMT+ vs 33% in MGMT- (p = 0.4) and 75% in ABCG2+ vs 50% in ABCG2- (p = 0.7). Median time to progression (TTP) with upfront TEMIRI was 9 months (range 5-28 months), with 1 patient with ongoing CR at 56 months; median TTP at relapse was 3 months (1-29 months). MGMT and ABCG2 expression did not influence 1-yr OS, whereas ECOG (0 vs 1-2) did (88% vs 31% p < 0.001). Grade 3-4 diarrhea, neutropenia, and thrombocytopenia incidence was < 5%, 1 patient with recurrent kidney EWS died of acute liver failure. Conclusions: TEMIRI showed promising activity in a very unfavorable cohort of EWS patients, with manageable toxicity. Performance status correlated with 6-month PFS & OS whereas MGMT & ABCG2 did not. Clinical trial information: NCT03542097 .

Size and variability often represent an obstacle in generating an effective antibody gene library for the detection of an abundant repertoire of antigens. Therefore, optimizing the construction of a large library is essential for the selection of high-affinity reactive fragments. Here, we report a highly efficient method for the construction of a human naïve antibody gene library for the selection of antibodies as single-chain variable fragments. This protocol is based on many different sets of oligonucleotide primers and multistep electroporation and ligation reactions.This advanced method can be adopted by any molecular biology laboratory to generate a naïve library for use in isolating single-chain fragment variables against specific targets.

The identification of new therapeutic strategies against osteosarcoma, the most common primary bone tumor, continues to be a primary goal to improve the outcomes of patients refractory to conventional chemotherapy. Osteosarcoma originates from the transformation of mesenchymal stem cells (MSC) and/or osteoblast progenitors, and the loss of differentiation is a common biological osteosarcoma feature, which has strong significance in predicting tumor aggressiveness. Thus, restoring differentiation through epigenetic reprogramming is potentially exploitable for therapeutic benefits. Here, we demonstrated that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 affected tumor proliferation by blocking osteosarcoma cells in G1 or G2–M phases and induced osteoblastic differentiation through the specific reexpression of genes regulating this physiologic process. Although MC3343 has a similar antiproliferative effect as 5azadC, the conventional FDA-approved nucleoside inhibitor of DNA methylation, its effects on cell differentiation are distinct. Induction of the mature osteoblast phenotype coupled with a sustained cytostatic response was also confirmed in vivo when MC3343 was used against a patient-derived xenograft (PDX). In addition, MC3343 displayed synergistic effects with doxorubicin and cisplatin (CDDP), two major chemotherapeutic agents used to treat osteosarcoma. Specifically, MC3343 increased stable doxorubicin bonds to DNA, and combined treatment resulted in sustained DNA damage and increased cell death. Overall, this nonnucleoside DNMTi is an effective novel agent and is thus a potential therapeutic option for patients with osteosarcoma who respond poorly to preadjuvant chemotherapy. Mol Cancer Ther; 17(9); 1881–92. ©2018 AACR.

Size and variability often represent an obstacle in generating an effective antibody gene library for the detection of an abundant repertoire of antigens. Therefore, optimizing the construction of a large library is essential for the selection of high-affinity reactive fragments. Here, we report a highly efficient method for the construction of a human naive antibody gene library for the selection of antibodies as single-chain variable fragments. This protocol is based on many different sets of oligonucleotide primers and multistep electroporation and ligation reactions.This advanced method can be adopted by any molecular biology laboratory to generate a naive library for use in isolating single-chain fragment variables against specific targets.