Expression Of Chimeric Protein Research Articles

Structural Determination of the Australian Bat Lyssavirus Nucleoprotein and Phosphoprotein Complex.

Australian bat lyssavirus (ABLV) shows similar clinical symptoms as rabies, but there are currently no protein structures available for ABLV proteins. In lyssaviruses, the interaction between nucleoprotein (N) and phosphoprotein (N) in the absence of RNA generates a complex (N0P) that is crucial for viral assembly, and understanding the interface between these two proteins has the potential to provide insight into a key feature: the viral lifecycle. In this study, we used recombinant chimeric protein expression and X-ray crystallography to determine the structure of ABLV nucleoprotein bound to residues 1-40 of its phosphoprotein chaperone. Comparison of our results with the recently generated structure of RABV CVS-11 N0P demonstrated a highly conserved interface in this complex. Because the N0P interface is conserved in the lyssaviruses of phylogroup I, it is an attractive therapeutic target for multiple rabies-causing viral species.

Abstract C099: Establishment and characterization of an NUP98-KDM5A-driven AML XPDX model

Abstract Background: Acute myeloid leukemia (AML) with NUP98 fusions is associated with poor prognosis in adults and especially children. Pediatric AML is a rare and heterogeneous disease characterized by a high prevalence of gene fusions as driver mutations, including the NUP98-KDM5A fusion found in a particular subgroup of patients with complex karyotypes. To better understand this subtype of AML, we established and characterized a XPDX model designated ST1653 representing NUP98-KDM5A-driven disease from a previously treated AML patient. We performed DNA and RNA-based sequencing of the model. Further we evaluated in vivo sensitivity towards cytarabine and inhibitors targeting the JAK and CDK6 pathways, which have been shown to be important in NUP98-KDM5A-driven AML. Methods: ST1653 was established from a bone marrow aspirate collected from a 63-year-old Caucasian male who had AML recurrence following high dose cytarabine. The sample was injected subcutaneously into a triple-deficient mouse and the P0 model passaged after seventy days into CB-17 SCID and athymic nude mice and further developed until stable. WES and RNAseq were then performed. For in vivo studies, ST1653 was evaluated against cytarabine and the JAK inhibitors ruxolitinib, baricitinib and tofacitinib and the CDK4/6 inhibitors palbociclib, abemaciclib and ribociclib. Endpoints in all studies included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C<0%) versus Day 0 tumor volume was also reported. Results: Growth of the ST1653 AML XPDX model was similar in CB-17 SCID or athymic nude mice with a median time to endpoint of forty-five days, although the take rate in the SCID strain was superior. Sequencing identified the presence of the NUP98-KDM5A fusion and chimeric protein expression was confirmed. In vivo, ST1653 was insensitive to cytarabine and the JAK inhibitors but demonstrated limited sensitivity to CDK4/6i treatment. Conclusion: We have established and characterized a XPDX model of NUP98-KDM5A-driven AML including sequencing and in vivo evaluation. This model is an important tool for developing novel therapies to this high-risk leukemia subtype. Citation Format: Armando Diaz III, Alyssa Simonson, Johnnie Flores, Joseph Holahan, Kyriakos P Papadopoulos, Jim Lund, Drew Rasco, Amita Patnaik, Michael J Wick. Establishment and characterization of an NUP98-KDM5A-driven AML XPDX model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C099.

Expression of chimeric proteins based on a backbone of the GII.4 norovirus VP1 and their application in the study of a GII.6 norovirus-specific blockade epitope.

The surface-exposed loop regions of the protruding domain of the norovirus (NoV) major capsid protein VP1 can tolerate the insertion of foreign antigens without affecting its assembly into subviral particles. In this study, we investigated the tolerance of the surface-exposed loop region of the GII.4 NoV VP1 by replacing it with homologous or heterologous sequences. We designed a panel of constructs in which the amino acid sequence from position 298-305 of the GII.4 NoV VP1 was replaced by sequences derived from the same region of GI.3, GII.3, GII.6, and GII.17 NoVs as well as neutralizing epitopes of enterovirus type 71 and varicella-zoster virus. The constructs were synthesized and expressed using a recombinant baculovirus expression system. The expression of target proteins was measured by indirect enzyme-linked immunosorbent assay (ELISA), and the assembly of virus-like particles (VLPs) was confirmed by electron microscopy. Our results showed that all of the constructs expressed high levels of target chimeric proteins, and all of the chimeric proteins successfully assembled into VLPs or subviral particles. An in vitro VLP-histo-blood group antigen (HBGA) binding assay revealed that chimeric-protein-containing VLPs did not bind or showed reduced binding to salivary HBGAs, a ligand for NoV particles. The results of an in vitro VLP-HBGA binding blockade assay indicated that the predicted surface-exposed loop region of the GII.6 NoV VP1 may comprise a blockade epitope. In summary, the surface-exposed loop region of the GII.4 NoV VP1 can be replaced by foreign sequences of a certain length. Using this strategy, we found that the predicted surface-exposed loop region of GII.6 NoV VP1 might contain a blockade epitope.

Belgian expert consensus for tumor-agnostic treatment of NTRK gene fusion-driven solid tumors with larotrectinib.

Fusions of NTRK (neurotrophic tyrosine receptor kinase) genes with 5' partner genes can result in the expression of chimeric proteins that drive oncogenesis through ligand-independent kinase activation. Despite variable frequencies of NTRK fusions in different tumor types, the fact that they are common to a wide range of cancers raises the possibility of developing tumor-agnostic treatments specifically targeting NTRK fusion products, irrespective of tumor type. The first-generation Trk (tropomyosin receptor kinase) inhibitor, larotrectinib, was the first tumor-agnostic treatment of NTRK fusion-positive cancers in adults and children, to be approved in the European Union. This consensus, developed by a Belgian multidisciplinary expert panel, aims to highlight the unmet medical need associated to NTRK fusion-driven cancer treatment and, based on current knowledge of NTRK fusions and larotrectinib treatment outcome and safety, provide comprehensive guidance to oncologists regarding NTRK fusion-driven cancer diagnostics and the best use of larotrectinib in real-world clinical settings.

Designing and determining immunogenicity of a recombinant protein due to producing a new vaccine against Enterotoxigenic Escherichia coli containing CfaE and CotD subunits

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is one of the most common bacterial causes of mortalities in developing countries due to diarrhea. Since mucosal immune responses to CFs can prevent the disease, a chimeric protein containing ETEC’s CFA/I (CfaE) tip subunits and CS2 (CotD) sub-structural units is developed to produce effective vaccine. Using bioinformatics tools, the chimeric construct was analyzed and then the optimized gene was synthesized and expressed in E. coli. The recombinant protein was expressed and purified by the Ni-NTA chromatography column and confirmed by anti-his tag antibody by western blotting. Mice were immunized with recombinant protein, and the IgG and IgA antibodies’ titrations of the sera were analyzed by ELISA. In addition, the immunogenicity and protective efficacy against the live ETEC bacteria in the challenge test were determined. Western blot analysis verified the chimeric protein expression of CotD-CfaE. The outcome of ELISA was a substantial improvement in the IgG antibody titer in immunized mice. In a live ETEC challenge, the survival percentage of 30% was shown for immunized mice. The developed recombinant chimeric protein could be suggested as an effective component in producing an efficient vaccine against Enterotoxigenic E. coli with other crucial subunits, different immunization route, and other factors.

Determination & expression of chimeric protein in colorectal cancer: A bioinformatics Approach

Colorectal cancer (CRC) accounts for about 10% of cancer-related mortality in western countries. Increasing ageing population, undesirable modern dietary and high-risk factors like smoking, obesity and low exercise. Chromosomal instability (CIN), CpG island methylator phenotype (CIMP), and microsatellite instability are the three different mechanism that give rise to CRC. It often grow slowly, and customarily doesn’t produce symptoms until reaching a substantial size of several centimeters, which can block the passage of feces and cause cramping, pain, or bleeding which can present as visible bleeding with bowel movements or, rarely, dark “tarry” stools. Most colon tumors develop via a several different processes involving a series of histological, morphological, and genetical changes that accumulate over time to time. New treatments for primary and metastatic colorectal cancer have emerged, like in variety of therapeutic process by preparing chimeric proteins that triggers the cells and stop it from getting severe.

Abstract 3905: Human liver organoids for disease modeling of fibrolamellar hepatocellular carcinoma

Abstract Fibrolamellar Hepatocellular Carcinoma (FLC) is a usually lethal liver cancer affecting adolescents and young adults without previous liver disease. FLC is characterized by a ~400kb nucleotide deletion resulting in a fusion of a heatshock protein cofactor, DNAJB1, with the catalytic subunit of protein kinase A (PRKACA). One limitation to the study of this disease is the lack of in vitro models. Organoids have shown utility as a model system for studying many diseases, including cancer. This study describes the development and characterization of FLC tumor organoids. With IRB approval, normal liver and FLC tumor tissue was obtained from patients undergoing surgical resection. Liver cells were isolated from the tissue samples and placed into a 3D basement membrane matrix and the media supplemented with growth factors specific to promote liver organoid expansion. Normal and tumor organoids were analyzed by microscopy for morphology, by PCR for presence of the DNAJB1-PRKACA chimeric transcript, by Western blot for chimeric protein expression, and by RNA sequencing for transcriptomic changes. Eight organoid lines have been developed from normal liver and eight from FLC tumor tissue, including multiple independent organoids developed from different metastases from the same patient. A distinct morphological difference between normal liver organoids and FLC tumor organoids can be seen with either brightfield microscopy or with H&E. The DNAJB1-PRKACA chimeric transcript was detected in all of the tumor tissue and tumor organoids by RT-PCR and not detected in any of the normal tissue or organoids. The presence of the DNAJB1-PRKACA fusion protein was verified by Western blot in tumor organoids and tissue but not detectable in the normal tissue. The RNA sequencing analysis shows clustering of tumor organoids with tumor tissue. Tumor organoids injected subcutaneously in mice grew as tumors. Now that the normal and tumor organoids have been developed and validated, they are being used for screening for potential therapeutics. Citation Format: Nicole J. Croteau, David Requena, Gadi Lalazar, Denise Ng, Solomon Levin, Bassem Shebl, Ruisi Wang, William J. Hammond, James A. Saltsman, Helmuth Gehart, Hans Clevers, Michael P. LaQuaglia, Sanford Simon. Human liver organoids for disease modeling of fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3905.

NTRK fusions in osteosarcoma are rare and non-functional events.

Neurotrophic tyrosine receptor kinase (NTRK) fusions are promising molecular targets that have been described in a broad range of malignant tumours. Fusions commonly lead to the expression of chimeric proteins with constitutive tyrosine kinase activation that drives tumorigenesis. Despite a low prevalence among most solid tumours (<1%), the first encouraging results with pan‐NTRK tyrosine kinase inhibitors (TKIs) such as larotrectinib or entrectinib stimulated the search for eligible patients. Here, we report the first three cases of osteosarcoma harbouring NTRK fusions, among 113 patients sequenced. It is also the first report on NTRK fusions within a tumour type characterised by highly rearranged genomes and abundant passenger mutations. Whereas the presence of NTRK gene fusions in many tumours is considered to be one of the main driver events for tumour progression, the three chimeric transcripts described here appear non‐functional and likely represent randomly occurring passenger alterations. Particularly in tumours with complex karyotypes, it may therefore be advisable to specifically investigate the fusion transcripts for functional impact before considering targeted treatment approaches using pan‐NTRK TKIs.

Influence of introduction of interleukin-2 and interleukin-12 into experimental marker DNA-vaccine

Aim. To determine the effects of the combined administration of recombinant expression vectors containing genes of murine interleukin-2 and interleukin-12 on humoral immune response, elicited by the experimental marker DNA-vaccine against classical swine fever. Methods. Expression of chimeric proteins in vitro and in vivo was determined by western-blot analysis. The antibodies specific to target antigens in blood serum have been detected by ELISA. Results. A series of recombinant plasmid vectors containing the genes of murine interleukin-2 and chimeric murine interleukin-12 have been developed. It has been shown that target chimeric proteins were expressed from the developed vectors both in vitro in HEK293 and in vivo in murine muscle tissue. The use of combined administration of murine interleukin-2 and interleukin-12 genes resulted in significant enhancement of titer of the anti-E2 and anti-β-galactosidase IgG, induced by vaccination with experimental marker DNA-vaccine against CSF, and model DNA-vaccine respectively. Conclusions. The data obtained show that the introduction of recombinant expressing vectors containing genes of murine interleukin-2 and interleukin-12 into vaccine preparations enhances humoral immune response elicited by the experimental marker DNA-vaccine against CSF and modelDNA-vaccine. Keywords: DNA-vaccine, humoral immune response, interleukin-2, interleukin-12, classical swine fever.

MS 25.05 Novel Fusions

Chromosomal rearrangements or deletions can generate novel gene fusions that lead to the expression of chimeric proteins with oncogenic activity in lung and other cancers. The paradigm for gene fusions in lung cancer is the EML4-ALK gene fusion which is found in approximately 5% of lung adenocarcinomas.1 Once identified, drug development for this target proceeded rapidly and crizotinib was the first approved tyrosine kinase inhibitor for patients with ALK+ non-small cell lung cancer based on its ability to generate substation objective response rates and prolonged progression free survival (NSCLC).3,4 The development of CNS penetrant and/or next generation ROS1 inhibitors including lorlatinib, entrectinib and TPX-0005 is ongoing. RET gene fusions are also found in 1-2% of NSCLC, but the use of multiple different RET inhibitors have failed to reproduce the success of targeting ALK or ROS1 fusions.5 The development of more selective RET inhibitors such as LOXO-292, BLU-667, and RXDX-105 are currently in clinical trials for RET+ lung and other cancers. NTRK1 fusions were recently identified in NSCLC and homologous NTRK2 and NTRK3 fusions are found in multiple tumor types.6,7, Early clinical trials of larotrectinib showed an impressive objective response rate of 76% for 12 different tumor histologies harboring NTRK1/2/3 fusions and the CNS-penetrant entrectinib is similarly being evaluated in a basket trial. Other rare, novel fusions have recently been identified in NSCLC including EGFR fusions or MET fusions.7 Early evidence suggests that these fusions may also respond to cognate TKIs. EGFR fusions break the paradigm of ALK and ROS1 fusions in which the 5’ end of ALK or ROS1 is replaced with the 5’ portion of another gene. EGFR fusions retain most of the EGFR gene, with the unrelated gene sequencing fusing at the 3’ end of EGFR. A related oncogenic EGFR mutation in which the kinase domain of EGFR is duplicated in tandem has also been described and appears responsive to EGFR TKIs.7 Additional fusions involving the receptor tyrosine kinase (RTK) encoding genes AXL, PDGFRA, and ERBB4 fusions have been described, but little is known about the true incidence in lung cancer.8,9 The fusions described thus far all involve genes that encode RTKs, but additional gene fusions have also been identified in lung cancer including BRAF fusions.7,9 BRAF fusions replace the 5’ region of BRAF, including the Ras-binding domain (RBD), with sequences from another gene. Anecdotal evidence suggests that these alterations may be responsive to MEK inhibition. Analogous splice alteration which remove a region including the RBD have also been described in cancer and are oncogenic. Finally, gene fusions involving NRG1, which encodes the HER3/4 ligand neuregulin-1, have been described, mostly in invasive mucinous adenocarcinomas of the lung.9,10 Several challenges exist to the development of targeted therapies for these novel fusions. Even in the era of next-generation sequencing tests these alterations may go undetected due to limited testing of genes to those with approved therapies (ALK, ROS1, EGFR, and BRAF) and not all assays are designed to detect all of these alterations. Furthermore, the rarity of some of these alterations may make clinical trials for these novel fusions less appealing, although amalgamating some of these alterations with analogous mutations, e.g., MET gene fusions with MET exon 14 splice alterations or MET gene amplification, may allow for a more rapid path to approval.

FTR83, a Member of the Large Fish-Specific finTRIM Family, Triggers IFN Pathway and Counters Viral Infection.

Tripartite motif (TRIM) proteins are involved in various cellular functions and constitute key factors of the antiviral innate immune response. TRIM proteins can bind viral particles directly, sending them to degradation by the proteasome, or ubiquitinate signaling molecules leading to upregulation of innate immunity. TRIM proteins are present in across metazoans but are particularly numerous in vertebrates where genes comprising a B30.2 domain have been often duplicated. In fish, a TRIM subset named finTRIM is highly diversified, with large gene numbers and clear signatures of positive selection in the B30.2 domain suggesting they may be involved in antiviral mechanisms. finTRIM provides a beautiful model to investigate the primordial implication of B30.2 TRIM subsets in the arsenal of vertebrate antiviral defenses. We show here that ftr83, a zebrafish fintrim gene mainly expressed in the gills, skin and pharynx, encodes a protein affording a potent antiviral activity. In vitro, overexpression of FTR83, but not of its close relative FTR82, induced IFN and IFN-stimulated gene expression and afforded protection against different enveloped and non-enveloped RNA viruses. The kinetics of IFN induction paralleled the development of the antiviral activity, which was abolished by a dominant negative IRF3 mutant. In the context of a viral infection, FTR83 potentiated the IFN response. Expression of chimeric proteins in which the B30.2 domain of FTR83 and the non-protective FTR82 had been exchanged, showed that IFN upregulation and antiviral activity requires both the Ring/BBox/Coiled coil domain (supporting E3 ubiquitin ligase) and the B30.2 domain of FTR83. Finally, loss of function experiments in zebrafish embryos confirms that ftr83 mediates antiviral activity in vivo. Our results show that a member of the largest TRIM subset observed in fish upregulates type I IFN response and afford protection against viral infections, supporting that TRIMs are key antiviral factors across vertebrates.

Immunogenicity of Leishmania-derived hepatitis B small surface antigen particles exposing highly conserved E2 epitope of hepatitis C virus.

BackgroundHepatitis C virus (HCV) infection is a major health problem worldwide, affecting an estimated 2–3 % of human population. An HCV vaccine, however, remains unavailable. High viral diversity poses a challenge in developing a vaccine capable of eliciting a broad neutralizing antibody response against all HCV genotypes. The small surface antigen (sHBsAg) of hepatitis B virus (HBV) has the ability to form highly immunogenic subviral particles which are currently used as an efficient anti-HBV vaccine. It also represents an attractive antigen carrier for the delivery of foreign sequences. In the present study, we propose a bivalent vaccine candidate based on novel chimeric particles in which highly conserved epitope of HCV E2 glycoprotein (residues 412–425) was inserted into the hydrophilic loop of sHBsAg.ResultsThe expression of chimeric protein was performed in an unconventional, Leishmania tarentolae expression system resulting in an assembly of particles which retained immunogenicity of both HCV epitope and sHBsAg protein. Direct transmission electron microscopy observation and immunogold staining confirmed the formation of spherical particles approximately 22 nm in diameter, and proper foreign epitope exposition. Furthermore, the sera of mice immunized with chimeric particles proved reactive not only to purified yeast-derived sHBsAg proteins but also HCV E2 412–425 synthetic peptide. Most importantly, they were also able to cross-react with E1E2 complexes from different HCV genotypes.ConclusionsFor the first time, we confirmed successful assembly of chimeric sHBsAg virus-like particles (VLPs) in the L. tarentolae expression system which has the potential to produce high-yields of properly N-glycosylated mammalian proteins. We also proved that chimeric Leishmania-derived VLPs are highly immunogenic and able to elicit cross-reactive antibody response against HCV. This approach may prove useful in the development of a bivalent prophylactic vaccine against HBV and HCV and opens up a new and low-cost opportunity for the production of chimeric sHBsAg VLPs requiring N-glycosylation process for their proper functionality and immunogenicity.

Expression and purification of toxic anti-breast cancer p28-NRC chimeric protein.

Background:Chimeric proteins consisting of a targeting moiety and a cytotoxic moiety are now under intense research focus for targeted therapy of cancer. Here, we report cloning, expression, and purification of such a targeted chimeric protein made up of p28 peptide as both targeting and anticancer moiety fused to NRC peptide as a cytotoxic moiety. However, since the antimicrobial activity of the NRC peptide would intervene expression of the chimeric protein in Escherichia coli, we evaluated the effects of two fusion tags, that is, thioredoxin (Trx) and 6x-His tags, and various expression conditions, on the expression of p28-NRC chimeric protein.Materials and Methods:In order to express the chimeric protein with only 6x-His tag, pET28 expression plasmid was used. Cloning in pET32 expression plasmid was performed to add both Trx and 6x-His tags to the chimeric protein. Expression of the chimeric protein with both plasmids was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis following optimization of expression conditions and host strains.Results:Expression of the chimeric protein in pET28a was performed. However, expression yield of the chimeric protein was low. Optimization of culture conditions and host strains led to reasonable expression yield of the toxic chimeric protein in pET32a vector. In cases of both plasmids, approximately 10 kDa deviation of the apparent molecular weight from the theoretical one was seen in SDS-PAGE of purified chimeric proteins.Conclusions:The study leads to proper expression and purification yield of p28-NRC chimeric protein with Trx tag following optimizing culture conditions and host strains.

Solitary fibrous tumors: loss of chimeric protein expression and genomic instability mark dedifferentiation

Solitary fibrous tumors, which are characterized by their broad morphological spectrum and unpredictable behavior, are rare mesenchymal neoplasias that are currently divided into three main variants that have the NAB2-STAT6 gene fusion as their unifying molecular lesion: usual, malignant and dedifferentiated solitary fibrous tumors. The aims of this study were to validate molecular and immunohistochemical/biochemical approaches to diagnose the range of solitary fibrous tumors by focusing on the dedifferentiated variant, and to reveal the genetic events associated with dedifferentiation by integrating the findings of array comparative genomic hybridization. We studied 29 usual, malignant and dedifferentiated solitary fibrous tumors from 24 patients (including paired samples from five patients whose tumors progressed to the dedifferentiated form) by means of STAT6 immunohistochemistry and (when frozen material was available) reverse-transcriptase polymerase chain reaction and biochemistry. In addition, the array comparative genomic hybridization findings were used to profile 12 tumors from nine patients. The NAB2/STAT6 fusion was detected in all of the tumors, but immunohistochemistry and western blotting indicated that chimeric protein expression was atypical or absent in 9 out of 11 dedifferentiated tumors. The comparative genomic hybridization results revealed that the usual and malignant solitary fibrous tumors had a simple profile, whereas the genome of the dedifferentiated tumors was complex and unstable, and suggested that 13q and 17p deletions and TP53 mutations may be present in malignant lesions before the full expression of a dedifferentiated phenotype. Solitary fibrous tumor dedifferentiation is associated with the loss of chimeric oncoprotein expression, genomic instability, and cell decommitment and reprogramming. The assessment of dedifferentiated solitary fibrous tumors is based on the presence of the fusion transcripts and, in principle, negative STAT6 immunohistochemistry should not rule out a diagnosis of solitary fibrous tumor.

Distinct isoform of FABP7 revealed by screening for retroelement-activated genes in diffuse large B-cell lymphoma.

Remnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences harbor multiple regulatory motifs and hence are capable of influencing expression of host genes. In response to environmental changes, TEs are known to be released from epigenetic repression and to become transcriptionally active. Such activation could also lead to lineage-inappropriate activation of oncogenes, as one study described in Hodgkin lymphoma. However, little further evidence for this mechanism in other cancers has been reported. Here, we reanalyzed whole transcriptome data from a large cohort of patients with diffuse large B-cell lymphoma (DLBCL) compared with normal B-cell centroblasts to detect genes ectopically expressed through activation of TE promoters. We have identified 98 such TE-gene chimeric transcripts that were exclusively expressed in primary DLBCL cases and confirmed several in DLBCL-derived cell lines. We further characterized a TE-gene chimeric transcript involving a fatty acid-binding protein gene (LTR2-FABP7), normally expressed in brain, that was ectopically expressed in a subset of DLBCL patients through the use of an endogenous retroviral LTR promoter of the LTR2 family. The LTR2-FABP7 chimeric transcript encodes a novel chimeric isoform of the protein with characteristics distinct from native FABP7. In vitro studies reveal a dependency for DLBCL cell line proliferation and growth on LTR2-FABP7 chimeric protein expression. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in cancer and suggest that LTR2-FABP7 may contribute to the pathogenesis of DLBCL in a subgroup of patients.

Genomic- and Transcriptomic Profiling Of Acute Lymphoblastic Leukemia With Dicentric Chromosomes

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, comprising multiple subtypes characterized by aneuploidy, recurring submicroscopic gains and losses of DNA, and inter- and intra-chromosomal rearrangements. A common feature in ALL with 44-45 chromosomes (from here on referred to as near-diploid ALL) is the presence of a dicentric chromosome, which may lead to the formation of gene fusions and the expression of chimeric proteins. At present, however, we have a limited understanding of the nature of these chimeric proteins, but some are known or predicted to dysregulate the function of oncoproteins or hematopoietic growth factors.We recently performed genome-wide profiling of over 120 pediatric cases with hypodiploid ALL, including 68 near-haploid cases (harboring 24-31 chromosomes), 34 low-hypodiploid cases (32-39 chromosomes) and 22 near-diploid cases, 20 of which harbored a dicentric chromosome. Near-haploid ALL is characterized by a high frequency of activating mutations of Ras signaling (mainly NF1 alterations) and IKZF3 deletions, and low-hypodiploid ALL by IKZF2, TP53 and RB1alterations. In contrast, these alterations are infrequent in dicentric ALL. To gain insight into the genetic basis of near-diploid ALL with dicentric chromosomes, we examined the 20 cases including 6 with dic(9;20), 3 with dic(7;12) and 2 with dic(9;17), while the remaining 9 cases had dicentric chromosomes involving various chromosomal arms. Affymetrix SNP 6.0 microarrays, gene expression profiling and candidate gene resequencing for 19 genes were performed for all cases, and transcriptome sequencing (mRNA Seq) on tumor and whole exome sequencing on tumor and matched normal genomic DNA have to date been carried out for 10 cases.The most common lesion was focal deletion of CDKN2A/CDKN2B, encoding the tumor suppressors INK4/ARF (77%). Approximately one third of near-diploid cases harbored mutations targeting Ras and receptor tyrosine kinase signaling, including mutations in NRAS (18%), KRAS (9%), PTPN11 (9%) and NF1 (5%). The B-lymphoid transcription factor PAX5 was altered by deletion, sequence mutation or amplification in 59% of near-diploid ALL. PAX5 has previously been reported to be involved in gene fusions created by dicentric chromosomes in ALL, including PAX5-ETV6 in some cases with dic(9;12)(p13;p13). PAX5 has previously also been shown to be targeted by dic(9;20)(p11-13;q11), which creates several non-productive fusion transcripts.The burden of non-synonymous single nucleotide variations (SNVs) and insertion/deletion (indel) mutations in coding- and splice regions in mRNA- and exome sequenced dicentric ALL varied with 8-25 SNVs and 4-15 indels per case. Except for the recurrent lesions stated above, the only recurrently sequence mutated gene identified in these 10 cases was NOTCH1, with mutations found in two cases, both of T-lineage. Singleton mutations were identified in e.g. IL7R, JAK3, STAT3, MYCN, PTEN and DOT1L. Fusion detection in mRNA Seq data identified 1-4 head-to-tail gene fusions per case, including the previously characterized fusions DDX3X-MLLT10, PICALM-MLLT10, MLL-AFF1 and P2RY8-CRLF2, none of which are involved in the dicentric chromosomes. The P2RY8-CRLF2 fusion was by RT-PCR shown to be present in two additional dicentric ALL cases. Further, novel in-frame fusions including ETV6-AMPH and PAX5-FBRSL1 created by dic(7;12)(p11.2;p11.2) and dic(9;12), respectively, were identified, the function of which in tumorigenesis is still unknown.An extended mRNA Seq study is currently ongoing, investigating an additional 30 near-diploid ALL cases with a focus on the dicentric chromosomes dic(7;9), dic(9;12) and dic(9;20). Further, recurrence screening will be performed on a cohort including another 45 cases, and functional evaluation is on an ongoing basis carried out on identified fusions.Altogether, this study is anticipated to provide critical new insights into the genetic basis of near-diploid ALL harboring a dicentric chromosome, and to shed more light on the putative chimeric genes produced by the formation of dicentric chromosomes. Further, we hope to identify new targets for therapy. Disclosures:No relevant conflicts of interest to declare.

The B7-1 Cytoplasmic Tail Enhances Intracellular Transport and Mammalian Cell Surface Display of Chimeric Proteins in the Absence of a Linear ER Export Motif

Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids) and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

A t(1;11) translocation linked to schizophrenia and affective disorders gives rise to aberrant chimeric DISC1 transcripts that encode structurally altered, deleterious mitochondrial proteins.

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a risk factor for psychiatric illness through its disruption by a balanced chromosomal translocation, t(1;11)(q42.1;q14.3), that co-segregates with schizophrenia, bipolar disorder and depression. We previously reported that the translocation reduces DISC1 expression, consistent with a haploinsufficiency disease model. Here we report that, in lymphoblastoid cell lines, the translocation additionally results in the production of abnormal transcripts due to the fusion of DISC1 with a disrupted gene on chromosome 11 (DISC1FP1/Boymaw). These chimeric transcripts encode abnormal proteins, designated CP1, CP60 and CP69, consisting of DISC1 amino acids 1–597 plus 1, 60 or 69 amino acids, respectively. The novel 69 amino acids in CP69 induce increased α-helical content and formation of large stable protein assemblies. The same is predicted for CP60. Both CP60 and CP69 exhibit profoundly altered functional properties within cell lines and neurons. Both are predominantly targeted to mitochondria, where they induce clustering and loss of membrane potential, indicative of severe mitochondrial dysfunction. There is currently no access to neural material from translocation carriers to confirm these findings, but there is no reason to suppose that these chimeric transcripts will not also be expressed in the brain. There is thus potential for the production of abnormal chimeric proteins in the brains of translocation carriers, although at substantially lower levels than for native DISC1. The mechanism by which inheritance of the translocation increases risk of psychiatric illness may therefore involve both DISC1 haploinsufficiency and mitochondrial deficiency due to the effects of abnormal chimeric protein expression.GenBank accession numbers: DISC1FP1 (EU302123), Boymaw (GU134617), der 11 chimeric transcript DISC1FP1 exon 2 to DISC1 exon 9 (JQ650115), der 1 chimeric transcript DISC1 exon 4 to DISC1FP1 exon 4 (JQ650116), der 1 chimeric transcript DISC1 exon 6 to DISC1FP1 exon 3a (JQ650117).

Abstract 1429: Forkhead box M1 (FoxM1) is a direct transcriptional target of EWS/FLI1 and a regulator of Ewing's sarcoma cell morphology, adhesiveness and tumorigenicity

Abstract Ewing's sarcomas (EWS) are tumors typically formed by undifferentiated small round cells that predominantly affect children and young adults. The aggressiveness of EWS is illustrated by both the fact that about 25-30% patients present with metastases at diagnosis and the elevated rate of lethal recurrence among them. In spite of therapeutic combinations including chemotherapy, radiation and surgery, the 5-year disease-free survival rate for EWS patients has remained low for many years. Poor prognosis, lethal recurrence and long-term side effects point to the urgent requirement of novel EWS targeted therapies. EWS is cytogenetically characterized by the presence of chromosomal translocations resulting in the fusion of the EWS gene with several ETS genes, most frequently with FLI1, leading to the expression of chimeric proteins that contain the transcriptional activation domain of EWS and the FLI1 DNA binding domain. The EWS/FLI1 aberrant transcription factors are responsible for the malignant phenotype of EWS and regulate a large repertoire of target genes, many of which are critical determinants for EWS development, and thus have the potential of providing novel therapeutic options for EWS. In order to understand mechanisms of cell cycle regulation by EWS/FLI1, we performed comparative array analyses of EWS cells before and after knocking down EWS/FLI1 expression. Results showed that EWS/FLI1 knockdown consistently down-regulated FoxM1, and chromatin immunoprecipitation (ChIP) assays demonstrated that FoxM1 is a novel direct transcriptional target of EWS/FLI1. Western hybridization and immunohistochemical analyses demonstrated that FoxM1 is indeed abundantly expressed in EWS cell lines, patient tumors and EWS cell derived tumors in nude mouse xenografts. Down-regulation of FoxM1 expression in by shRNA-mediated gene knockdown caused morphological alterations, slowed the growth of EWS cells, and increased their adhesion to the substrate in culture-plates. Accordingly, FoxM1 down-regulation in EWS cells diminished their migration and invasiveness as well as their tumorigenicity in nude mice. Alterations in the pattern of integrin expression observed in FoxM1 knocked-down EWS cells may likely contribute to their altered morphology, increased adhesion and the overall downgrading of their malignant phenotype. These data identify FoxM1 as a direct EWS/FLI1 target and as an important regulator of the proliferation, morphology and tumorigenicity of EWS cells, and imply that targeting FoxM1 may allow the development of new molecular therapeutic strategies for EWS patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1429. doi:1538-7445.AM2012-1429

Current status and perspectives of plant-based candidate vaccines against the human immunodeficiency virus (HIV)

Genetically engineered plants are economical platforms for the large-scale production of recombinant proteins and have been used over the last 21 years as models for oral vaccines against a wide variety of human infectious and autoimmune diseases with promising results. The main inherent advantages of this approach consist in the absence of purification needs and easy production and administration. One relevant infectious agent is the human immunodeficiency virus (HIV), since AIDS evolved as an alarming public health problem implicating very high costs for government agencies in most African and developing countries. The design of an effective and inexpensive vaccine able to limit viral spread and neutralizing the viral entry is urgently needed. Due to the limited efficacy of the vaccines assessed in clinical trials, new HIV vaccines able to generate broad immune profiles are a priority in the field. This review discusses the current advances on the topic of using plants as alternative expression systems to produce functional vaccine components against HIV, including antigens from Env, Gag and early proteins such as Tat and Nef. Ongoing projects of our group based on the expression of chimeric proteins comprising C4 and V3 domains from gp120, as an approach to elicit broadly neutralizing antibodies are mentioned. The perspectives of the revised approaches, such as the great need of assessing the oral immunogenicity and a detailed immunological characterization of the elicited immune responses, are also discussed.