Engineering Synthetic Chromosomes by Sequential Loading of Multiple Genomic Payloads over 100 Kilobase Pairs in Size.

Gene delivery vehicles currently in the clinic for treatment of monogenic disorders lack sufficient carrying capacity to efficiently address complex polygenic diseases. Thus, to engineer multifaceted genetic circuits for bioengineering human cells as a therapeutic option for polygenic diseases, we require new tools that are currently in their infancy. Mammalian artificial chromosomes, or synthetic chromosomes, represent a viable approach for delivery of large genetic payloads that are mitotically stable and remain independent of the host genome. Previously, we described a mammalian synthetic chromosome platform, termed the ACE system, that requires a single unidirectional integrase for the introduction of multiple genes onto the ACE platform chromosome. In this report, we provide a proof of concept that the ACE synthetic chromosome bioengineering platform is amenable to sequential delivery of off-the-shelf large genomic fragments. Specifically, large genomic clones spanning the human solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1 or GLUT1, 169 kbp), and human monocarboxylate transporter 1 (SLC16A1 or MCT1, 144 kbp) genetic loci were engineered onto the ACE platform and demonstrated to express and correctly splice both gene transcripts. Thus, the ACE system provides a facile and tractable engineering platform for the development of gene-based therapeutic agents targeting polygenic diseases.

Differential expression of cysteine dioxygenase 1 in complex karyotype liposarcomas.

Altered cysteine dioxygenase 1 (CDO1) gene expression has been observed in several cancers but has not yet been investigated in liposarcomas. The aim of this study was to evaluate CDO1 expression in a cohort of liposarcomas and to determine its association with clinicopathological features. Existing microarray data indicated variable CDO1 expression in liposarcoma subtypes. CDO1 mRNA from a larger cohort of liposarcomas was quantified by real time-PCR, and CDO1 protein expression was determined by immunohistochemistry (IHC) in more than 300 tumor specimens. Well-differentiated liposarcomas (WDLSs) had significantly higher CDO1 gene expression and protein levels than dedifferentiated liposarcomas (DDLSs) (P < 0.001). Location of the tumor was not predictive of the expression level of CDO1 mRNA in any histological subtype of liposarcoma. Recurrent tumors did not show any difference in CDO1 expression when compared to primary tumors. CDO1 expression was upregulated as human mesenchymal stem cells (hMSCs) undergo differentiation into mature adipocytes. Our results suggest that CDO1 is a marker of liposarcoma progression and adipogenic differentiation.

Telomerase suppresses formation of ALT-associated single-stranded telomeric C-circles.

Telomere maintenance is an essential characteristic of cancer cells, most commonly achieved by activation of telomerase. Telomeres can also be maintained by a recombination-based mechanism, alternative lengthening of telomeres (ALT). Cells using ALT are characterized by the presence of ALT-associated promyelocytic leukemia (PML) bodies (APB), long, heterogeneously sized telomeres, extrachromosomal telomeric circular DNA, and elevated telomeric recombination. Consistent with other reports, we found that liposarcomas containing APBs, but lacking telomerase expression, always contained C-rich circles (C-circles), and these C-circles were never present in the absence of APBs, indicating a tight link between these features in ALT cells. However, a rare subgroup of tumors showing evidence of telomere maintenance by both telomerase and ALT did not contain C-circles. To test the hypothesis that telomerase expression disrupts the tight link between APBs and C-circles, we used ALT cell lines that were engineered to express telomerase. Introduction of telomerase activity in these ALT cells resulted in, on average, shorter telomeres with retention of APBs. However, at high passage, the level of C-circles was significantly reduced, which was paralleled by a switch from C-strand overhangs to G-strand overhangs. We propose that by extending critically short telomeres in these cells, telomerase is disrupting a key step in the ALT pathway necessary for production and/or maintenance of C-circles.

Doxorubicin resistance in a novel in vitro model of human pleomorphic liposarcoma associated with alternative lengthening of telomeres.

Soft tissue sarcomas are a diverse set of fatal human tumors where few agents have demonstrable clinical efficacy, with the standard therapeutic combination of doxorubicin and ifosfamide showing only a 25% to 30% response rate in large multi-institutional trials. Although liposarcomas are the most common histologic form of adult soft tissue sarcomas, research in this area is severely hampered by the lack of experimentally tractable in vitro model systems. To this end, here we describe a novel in vitro model for human pleomorphic liposarcoma. The cell line (LS2) is derived from a pleomorphic liposarcoma that uses the alternative lengthening of telomeres (ALT) mechanism of telomere maintenance, which may be important in modulating the response of this tumor type to DNA-damaging agents. We present detailed baseline molecular and genomic data, including genome-wide copy number and transcriptome profiles, for this model compared with its parental tumor and a panel of liposarcomas covering multiple histologies. The model has retained essentially all of the detectable alterations in copy number that are seen in the parental tumor, and shows molecular karyotypic and expression profiles consistent with pleomorphic liposarcomas. We also show the utility of this model, together with two additional human liposarcoma cell lines, to investigate the relationship between topoisomerase 2A expression and the sensitivity of ALT-positive liposarcomas to doxorubicin. This model, together with its associated baseline data, provides a powerful new tool to develop treatments for this clinically poorly tractable tumor and to investigate the contribution that ALT makes to modulating sensitivity to doxorubicin.