In silico and in vitro screening of small molecule Inhibitors against SYT-SSX1 fusion protein in synovial sarcoma.

Synovial sarcoma (SS) is characterized by a tumour specific chromosomal translocation t(X;18) (p11;q11) which results in the formation of SYT-SSX1 fusion protein. This fusion protein represents a clear therapeutic target and molecules specifically targeting SYT-SSX1 fusion protein are currently not available. In this study, SYT-SSX1 fusion protein sequence was retrieved from Uniprot and 3D structure was generated using I-TASSER modeling program. A structure based computational screening approach has been employed using Glide docking software to identify potential SYT-SSX1 small molecule inhibitors that bind to the junction region of the fusion protein. The obtained inhibitors were further filtered based on the docking score and ADME/T properties. Ten best fit compounds were chosen for in vitro studies. The anti-proliferative activities of these 10 compounds were screened in Yamato, ASKA (carries SYT-SSX1 fusion protein) and other sarcoma cell lines such as A673, 143B to understand the specificity of inhibition of the chosen compounds. The in vitro activity was compared against HEK293 cell lines. The compound 5-fluoro-3-(1-phenyl-1H-tetraazol-5-yl)-1H-indole (FPTI) was found to be selectively cytotoxic in synovial sarcoma cell lines (Yamato and ASKA) and this compound also showed insignificant anti proliferative activity on other cell lines. Further, target gene expression study confirmed that FPTI treatment down-regulated SYT-SSX1 and modulated its downstream target genes. Cell cycle analysis revealed the involvement of an apoptotic mechanism of cell death. Further experimental validations may elucidate the therapeutic potentials of FPTI against SYT-SSX1 fusion protein.

Preclinical Justification of pbi-shRNA EWS/FLI1 Lipoplex (LPX) Treatment for Ewing's Sarcoma.

The EWS/FLI1 fusion gene is well characterized as a driver of Ewing's sarcoma. Bi-shRNA EWS/FLI1 is a functional plasmid DNA construct that transcribes both siRNA and miRNA-like effectors each of which targets the identical type 1 translocation junction region of the EWS/FLI1 transcribed mRNA sequence. Previous preclinical and clinical studies confirm the safety of this RNA interference platform technology and consistently demonstrate designated mRNA and protein target knockdown at greater than 90% efficiency. We initiated development of pbi-shRNA EWS/FLI1 lipoplex (LPX) for the treatment of type 1 Ewing's sarcoma. Clinical-grade plasmid was manufactured and both sequence and activity verified. Target protein and RNA knockdown of 85-92% was demonstrated in vitro in type 1 human Ewing's sarcoma tumor cell lines with the optimal bi-shRNA EWS/FLI1 plasmid. This functional plasmid was placed in a clinically tested, liposomal (LP) delivery vehicle followed by in vivo verification of activity. Type 1 Ewing's sarcoma xenograft modeling confirmed dose related safety and tumor response to pbi-shRNA EWS/FLI1 LPX. Toxicology studies in mini-pigs with doses comparable to the demonstrated in vivo efficacy dose resulted in transient fever, occasional limited hypertension at low- and high-dose assessment and transient liver enzyme elevation at high dose. These results provide the justification to initiate clinical testing.

Sequence-specific electrochemical detection of double-strand PCR amplicons of PML/RARα fusion gene in acute promyelocytic leukemia.

A novel electrochemical method for the sequence-specific detection of double-stranded polymerase chain reaction (PCR) products of PML/RARα fusion gene in acute promyelocytic leukemia (APL) was described in detail. Based on a "sandwich" sensing mode involving a pair of locked nucleic acids probes (capture probe and reporter probe), this DNA sensor exhibited excellent selectivity and specificity. The direct and quantitative analysis of double-stranded complementary was firstly performed by our sensor without the use of alkali, helicase enzymes, or denaturants. Finally, combining PCR technique with electrochemical detection scheme, PCR amplicons (191 bp) of the PML/RARα fusion gene were obtained and rapidly identified with a low detection limit of 79 fmol in the 100-µL hybridization system. The results clearly showed the power of sensor as a promising tool for the sensitive, specific, and portable detection of APL and other diseases.

An enhanced sensing platform for ultrasensitive impedimetric detection of target genes based on ordered FePt nanoparticles decorated carbon nanotubes.

We demonstrate a novel high-performance DNA hybridization biosensor with a carbon nanotubes (CNTs)-based nanocomposite membrane as the enhanced sensing platform. The platform was constructed by homogenously distributing ordered FePt nanoparticles (NPs) onto the CNTs matrix. The surface structure and electrochemical performance of the FePt/CNTs nanocomposite membrane were systematically investigated. Such a nanostructured composite membrane platform could combine with the advantages of FePt NPs and CNTs, greatly facilitate the electron-transfer process and the sensing behavior for DNA detection, leading to excellent sensitivity and selectivity. The complementary target genes from acute promyelocytic leukemia could be quantified in a wide range of 1.0×10⁻¹² mol/L to 1.0×10⁻6 mol/L using electrochemical impedance spectroscopy, and the detection limit was 2.1×10⁻¹³ mol/L under the optimal conditions. In addition, the DNA electrochemical biosensor was highly selective to discriminate single-base or double-base mismatched sequences.

Electrochemical biosensor based on nanoporous gold electrode for detection of PML/RARα fusion gene.

In this study, a kind of nanoporous gold electrode (NPG) prepared with repetitive square-wave oxidation reduction cycle (SWORC) was reported. The active surface area of the proposed NPG electrode was 9.9 times larger than that of a bare flat one characterized by cyclic voltammetry (CV). An electrochemical DNA biosensor based on NPG electrode was fabricated for detection of promyelocytic leukemia/retinoic acid receptor α (PML/RARα) fusion gene in acute promyelocytic leukemia (APL) by using Methylene Blue (MB) as an electroactive indicator. Differential pulse voltammetry (DPV) was employed to monitor the hybridization reaction on the probe modified electrode. The decrease of the peak current of MB was observed upon hybridization of the probe with target DNA. The results indicated that the peak current was linear with the concentration of complementary strand in the range of 60 pM to 220 pM with a detection limit of 6.7 pM. This new biosensor exhibited excellent sensitivity and selectivity and had been used for an assay of PCR real sample with a satisfactory result.

Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML.

Arsenic, an ancient drug used in traditional Chinese medicine, has attracted worldwide interest because it shows substantial anticancer activity in patients with acute promyelocytic leukemia (APL). Arsenic trioxide (As2O3) exerts its therapeutic effect by promoting degradation of an oncogenic protein that drives the growth of APL cells, PML-RARalpha (a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor alpha). PML and PML-RARalpha degradation is triggered by their SUMOylation, but the mechanism by which As2O3 induces this posttranslational modification is unclear. Here we show that arsenic binds directly to cysteine residues in zinc fingers located within the RBCC domain of PML-RARalpha and PML. Arsenic binding induces PML oligomerization, which increases its interaction with the small ubiquitin-like protein modifier (SUMO)-conjugating enzyme UBC9, resulting in enhanced SUMOylation and degradation. The identification of PML as a direct target of As2O3 provides new insights into the drug's mechanism of action and its specificity for APL.

In vitro inhibition of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) expression and leukemogenic activity by DNA/LNA chimeric antisense oligos.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.

A highly conserved NTRK3 C-terminal sequence in the ETV6-NTRK3 oncoprotein binds the phosphotyrosine binding domain of insulin receptor substrate-1: an essential interaction for transformation.

Receptor tyrosine kinases are integral components of cellular signaling pathways and are frequently deregulated in malignancies. The NTRK family of neurotrophin receptors mediate neuronal cell survival and differentiation, but altered NTRK signaling has also been implicated in oncogenesis. The ETV6-NTRK3 (EN) gene fusion occurs in human pediatric spindle cell sarcomas and secretory breast carcinoma, and encodes the oligomerization domain of the ETV6 transcription factor fused to the protein-tyrosine kinase domain of NTRK3. The EN protein functions as a constitutively active protein-tyrosine kinase with potent transforming activity in multiple cell lineages, and EN constitutively activates both the Ras-MAPK and phosphatidylinositol 3-kinase-Akt pathways. EN transformation is associated with constitutive tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Further, IRS-1 functions as the adaptor protein linking EN to downstream signaling pathways. However, the exact nature of the EN-IRS-1 interaction remains unknown. We now demonstrate that EN specifically binds the phosphotyrosine binding domain of IRS-1 via an interaction at the C terminus of EN. An EN mutant lacking the C-terminal 19 amino acids does not bind IRS-1 and lacks transforming ability. Moreover, expression of an IRS-1 polypeptide containing the phosphotyrosine binding domain acts in a dominant negative manner to inhibit EN transformation, and overexpression of IRS-1 potentiates EN transforming activity. These findings indicate that EN.IRS-1 complex formation through the NTRK3 C terminus is essential for EN transformation.

The AF10 leucine zipper is required for leukemic transformation of myeloid progenitors by MLL-AF10.

The t(10;11)(p12;q23) chromosomal translocation in human acute myeloid leukemia results in the fusion of the MLL and AF10 genes. The latter codes for a novel leucine zipper protein, one of many MLL fusion partners of unknown function. In this report, we demonstrate that retroviral-mediated transduction of an MLL-AF10 complementary DNA into primary murine myeloid progenitors enhanced their clonogenic potential in serial replating assays and led to their efficient immortalization at a primitive stage of myeloid differentiation. Furthermore, MLL-AF10-transduced cells rapidly induced acute myeloid leukemia in syngeneic or severe combined immunodeficiency recipient mice. Structure/function analysis showed that a highly conserved 82-amino acid portion of AF10, comprising 2 adjacent alpha-helical domains, was sufficient for immortalizing activity when fused to MLL. Neither helical domain alone mediated immortalization, and deletion of the 29-amino acid leucine zipper within this region completely abrogated transforming activity. Similarly, the minimal oncogenic domain of AF10 exhibited transcriptional activation properties when fused to the MLL or GAL4 DNA-binding domains, while neither helical domain alone did. However, transcriptional activation per se was not sufficient because a second activation domain of AF10 was neither required nor competent for transformation. The requirement for alpha-helical transcriptional effector domains is similar to the oncogenic contributions of unrelated MLL partners ENL and ELL, suggesting a general mechanism of myeloid leukemogenesis by a subset of MLL fusion proteins, possibly through specific recruitment of the transcriptional machinery.

Loss of the exon encoding the juxtamembrane domain is essential for the oncogenic activation of TPR-MET.

TPR-MET, a transforming counterpart of the c-MET proto-oncogene detected in experimental and human cancer, results from fusion of the MET kinase domain with a dimerization motif encoded by TPR. In this rearrangement the exons encoding the Met extracellular, transmembrane and juxtamembrane domains are lost. The juxtamembrane domain has been suggested to be a regulatory region endowed with negative feedback control. To understand whether its absence is critical for the generation of the Tpr-Met transforming potential, we produced a chimeric molecule (Tpr-juxtaMet) with a conserved juxtamembrane domain. The presence of the domain (aa 962-1009) strongly inhibited Tpr-Met dependent cell transformation. Cell proliferation, anchorage-independent growth, motility and invasion were also impaired. The enzymatic behavior of Tpr-Met and Tpr-juxtaMet was the same, while Tpr-juxtaMet ability to associate cytoplasmic signal transducers and to elicit downstream signaling was severely impaired. These data indicate that the presence of the juxtamembrane domain counterbalances the Tpr-Met transforming potential and therefore the loss of the exon encoding the juxtamembrane domain is crucial in the generation of the active TPR-MET oncogene.