Identification of a Novel Oncogenic Fusion Gene SPON1-TRIM29 in Clinical Ovarian Cancer That Promotes Cell and Tumor Growth and Enhances Chemoresistance in A2780 Cells

Gene structure alterations, such as chromosomal rearrangements that develop fusion genes, often contribute to tumorigenesis. It has been shown that the fusion genes identified in public RNA-sequencing datasets are mainly derived from intrachromosomal rearrangements. In this study, we explored fusion transcripts in clinical ovarian cancer specimens based on our RNA-sequencing data. We successfully identified an in-frame fusion transcript SPON1-TRIM29 in chromosome 11 from a recurrent tumor specimen of high-grade serous carcinoma (HGSC), which was not detected in the corresponding primary carcinoma, and validated the expression of the identical fusion transcript in another tumor from a distinct HGSC patient. Ovarian cancer A2780 cells stably expressing SPON1-TRIM29 exhibited an increase in cell growth, whereas a decrease in apoptosis was observed, even in the presence of anticancer drugs. The siRNA-mediated silencing of SPON1-TRIM29 fusion transcript substantially impaired the enhanced growth of A2780 cells expressing the chimeric gene treated with anticancer drugs. Moreover, a subcutaneous xenograft model using athymic mice indicated that SPON1-TRIM29-expressing A2780 cells rapidly generated tumors in vivo compared to control cells, whose growth was significantly repressed by the fusion-specific siRNA administration. Overall, the SPON1-TRIM29 fusion gene could be involved in carcinogenesis and chemotherapy resistance in ovarian cancer, and offers potential use as a diagnostic and therapeutic target for the disease with the fusion transcript.

JAFFAL: detecting fusion genes with long-read transcriptome sequencing

In cancer, fusions are important diagnostic markers and targets for therapy. Long-read transcriptome sequencing allows the discovery of fusions with their full-length isoform structure. However, due to higher sequencing error rates, fusion finding algorithms designed for short reads do not work. Here we present JAFFAL, to identify fusions from long-read transcriptome sequencing. We validate JAFFAL using simulations, cell lines, and patient data from Nanopore and PacBio. We apply JAFFAL to single-cell data and find fusions spanning three genes demonstrating transcripts detected from complex rearrangements. JAFFAL is available at https://github.com/Oshlack/JAFFA/wiki.

RNA-driven JAZF1-SUZ12 gene fusion in human endometrial stromal cells

Oncogenic fusion genes as the result of chromosomal rearrangements are important for understanding genome instability in cancer cells and developing useful cancer therapies. To date, the mechanisms that create such oncogenic fusion genes are poorly understood. Previously we reported an unappreciated RNA-driven mechanism in human prostate cells in which the expression of chimeric RNA induces specified gene fusions in a sequence-dependent manner. One fundamental question yet to be addressed is whether such RNA-driven gene fusion mechanism is generalizable, or rather, a special case restricted to prostate cells. In this report, we demonstrated that the expression of designed chimeric RNAs in human endometrial stromal cells leads to the formation of JAZF1-SUZ12, a cancer fusion gene commonly found in low-grade endometrial stromal sarcomas. The process is specified by the sequence of chimeric RNA involved and inhibited by estrogen or progesterone. Furthermore, it is the antisense rather than sense chimeric RNAs that effectively drive JAZF1-SUZ12 gene fusion. The induced fusion gene is validated both at the RNA and the genomic DNA level. The ability of designed chimeric RNAs to drive and recapitulate the formation of JAZF1-SUZ12 gene fusion in endometrial cells represents another independent case of RNA-driven gene fusion, suggesting that RNA-driven genomic recombination is a permissible mechanism in mammalian cells. The results could have fundamental implications in the role of RNA in genome stability, and provide important insight in early disease mechanisms related to the formation of cancer fusion genes.

SEPT6_TRIM33 Gene Fusion and Mutated TP53 Pathway Associate With Unfavorable Prognosis in Patients With B-Cell Lymphomas

BackgroundMounting studies have sought to identify novel mutation biomarkers having diagnostic and prognostic potentials. Nevertheless, the understanding of the mutated pathways related to development and prognosis of B-cell lymphoma is still lacking. We aimed to comprehensively analyze the mutation alterations in genes of canonical signaling pathways and their impacts on the clinic outcomes of patients with B-cell lymphoma.MethodsCirculating cell-free DNA (cfDNA) samples from 79 patients with B-cell lymphomas were used for targeted sequencing with a 560-gene panel for depicting mutation landscapes and identifying gene fusion events. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses of mutated genes were performed. The associations of mutation status of genes and seven canonical oncogenic pathways with progression-free survival (PFS) were assessed using Kaplan-Meier test and multivariate Cox analysis. The variant allele frequencies (VAFs) of genes in TP53 and Hippo pathways in paired baseline and post-treatment samples from 18 B-cell lymphoma patients were compared. Finally, the associations of identified fusion genes, mutated genes, and pathways with treatment response were evaluated based on objective response rates (ORRs) comparisons of groups.ResultsWe identified 666 mutations from 262 genes in baseline cfDNAs from 79 B-cell lymphoma patients, and found some genes were preferentially mutated in our cohort such as GNAQ, GNAS, H3F3A, DNMT3A, HLA-A, and HLA-B. These frequently mutated genes were significantly associated with negative “regulation of gene expression, epigenetic” and virus infections such as cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus 1 infections. We detected five fusion genes in at least two patients with B-cell lymphoma, and among them, TCF7L2_WT1 gene fusion was most frequently detected in 30.4% of patients (24 of 79 cases). SEPT6_TRIM33 gene fusion, mutated TP53 and Hippo pathways were significantly associated with poor PFS, and SEPT6_TRIM33 fusion gene and mutated TP53 pathway were independent prognostic factors for B-cell lymphoma. A decreased VAF of TP53 p.Y88C and LATS2 p.F972L was detected in patients with complete response to treatments. Moreover, a significant difference in ORR was observed in patients with NPM1_NR4A3 and SEPT6_TRIM33 fusions.ConclusionsSEPT6_TRIM33 gene fusion and mutated TP53 and Hippo pathways may serve as prognostic makers for B-cell lymphoma patients.

Targeting JAK2 Gene Rearrangements with a Novel Kinase Inhibitor in a Preclinical Model of Pediatric Acute Lymphoblastic Leukemia

Background. Although the Event Free Survival for Childhood Acute lymphoblastic leukemia (ALL) reaches 85%, the remaining 15% of patients relapse, and 25-40% of them die. Novel molecular targets may increase the efficacy of therapy and reduce treatment toxicity. Among B-other ALL patients, JAK/STAT pathway alterations represent about 7% of the 'Philadelphia-like' cases. JAK2 gene encodes for a non-receptor tyrosine kinase fundamental for hematopoiesis, cellular proliferation and survival. In last years, JAK2 mutations have been widely studied in leukemia and lymphoma, whereas JAK2 fusion genes are still poorly characterized. Aim. This study aims to identify JAK2 fusion genes among BCP-ALL pediatric patients and develop a target strategy in in vitro and in vivo preclinical models. Methods. A targeted capture RNA Next Generation Sequencing strategy was applied to discover JAK2 fusion genes in a large cohort of PCR-based MRD high risk (HR) BCP-ALL pediatric patients. Fusions were validated by RT-PCR and/or FISH. Primary patients' cells have been in vivo expanded in NSG mice. We performed ex vivo and in vivo drug treatments with JAK2 inhibitors; phosphoflow and apoptosis-viability assays were performed in patients' blasts in co-culture with human bone marrow stroma. Results. We identified 10 pediatric cases carrying a JAK2 fusion gene with different partners in single cases, such as ATF7IP, ZEB2, MPRIP, BCR, TLE4, GIT2 and RAB7, in addition to PAX5, which was the only recurrent in three cases. Cells were available from 3 cases, carrying PAX5-JAK2, ATF7IP-JAK2 and ZEB2-JAK2, respectively. After in vivo expansion, we demonstrated that the JAK2 signaling pathway was active at basal level, through phosphorylation on Y1007-1008 JAK2 residues inside the catalytic activation loop, compared to cases wild type for JAK2 and CRLF2 (+70%, two-tailed P value 0.0355); a positive trend was also shown compared to primary cells with P2RY8-CRLF2 rearrangements and JAK2 mutation, as positive controls (+40% two-tailed P value 0.158). The JAK2 downstream effectors pS727-STAT3 and pY694-STAT5 were also activated. We thus setup a JAK2 targeted drug treatment using CHZ868, a new class-II tyrosine kinase inhibitor (TKI) (Novartis, Basel, CH). After 30 minutes of treatment, we appreciated a mean inhibition of -62% of Y1007-1008 JAK2 residues in PAX5-JAK2, -22% in ATF7IP-JAK2 and -35% in ZEB2- JAK2. Contemporarily, we observed a decrease of pS727-STAT3 (-35-50%) and pY694-STAT5 (-15-50%). After 48h monotherapy treatment by CHZ868, we detected apoptosis induction and cell viability decrease between 20- 75% at IC50. In combination with dexamethasone, we assessed a further decrease of viability between 10 to 95%. A biological variability among the three different patients was appreciated, according to the different partner genes. Exclusively for the PAX5-JAK2 fusion, we also performed treatments with the kinase inhibitor BIBF1120/Nintedanib, targeting LCK, which is activated downstream PAX5 fusions and we observed a 20% reduction of cell viability. Importantly, combination of BIBF1120 and CHZ868 showed a synergistic effect (-45%, at IC50). Moreover, we found that ruxolitinib caused autophagy as observed by higher levels of LC3-II compared to untreated cells (+ 45%, p<0.01), with consequent reduction of apoptosis induction. Indeed, active caspase 3 increased when ruxolitinib was given in combination with chloroquine, an autophagy inhibitor (+20% vs ruxolitinib alone, p<0.01). CHZ868 alone or in combination with chloroquine instead does not induce autophagy as LC3-II and active caspase 3 levels are the same of untreated cells. Finally, we demonstrated the in vivo efficacy of CHZ868 in patient derived xenograft model in presence of PAX5-JAK2 fusion. After two weeks of 30mg/Kg daily treatment of CHZ868, we observed a significant reduction of leukemic CD10+/CD19+ cells both in bone marrow (p<0.01, -43%), spleen (p<0.001, -72%), central nervous system (-40%) and peripheral blood (p<0.05, -46%), compared to vehicle mice. Further in vivo experiments are ongoing in other JAK2 fusion settings. Conclusion. CHZ868 is a promising candidate for treatment of BCP-ALL carrying JAK2 fusions, showing high efficacy and specificity, both ex vivo and in vivo. Further studies will include combination with standard chemotherapy drugs with the aim to maintain its efficacy by reducing the intensity and toxicity of chemotherapy. Disclosures Biondi: Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board; Colmmune: Honoraria; Amgen: Honoraria.

MINTIE: identifying novel structural and splice variants in transcriptomes using RNA-seq data

Calling fusion genes from RNA-seq data is well established, but other transcriptional variants are difficult to detect using existing approaches. To identify all types of variants in transcriptomes we developed MINTIE, an integrated pipeline for RNA-seq data. We take a reference-free approach, combining de novo assembly of transcripts with differential expression analysis to identify up-regulated novel variants in a case sample. We compare MINTIE with eight other approaches, detecting > 85% of variants while no other method is able to achieve this. We posit that MINTIE will be able to identify new disease variants across a range of disease types.

ZNF384 Gene Rearrangement in Acute Lymphocytic Leukemia with Kidney Involvement as the First Manifestation is Associated with Poor Prognosis: A Case Report

Background: Novel fusion genes such as ZNF384 have been identified in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in recent years. Patients harboring ZNF384 rearrangement have a distinctive immunophenotype with weak CD10 and aberrant CD13 and/or CD33 expression, which makes ZNF384-rearranged ALL a unique subtype of BCP-ALL. However, research on the prognostic significance of ZNF384 rearrangement has been limited to date.Case presentation: We described a 17-year-old young woman who was diagnosed with ALL and had kidney involvement as the first manifestation, which was very rare in the existing studies. FISH analysis indicated a rearrangement of ZNF384 according to its probe. The patient had a typical characteristic immunophenotype of ZNF384 rearrangement with CD10 being negative but CD13 and CD33 being positive. She had an unfavorable prognosis since she responded poorly to chemotherapy and developed a relapse shortly after reaching CR.Conclusion: The importance of the ZNF384 gene rearrangement in terms of prognosis remains unclear. We reported a young woman harboring ZNF384 rearrangement in ALL with kidney involvement. She experienced different treatments, but her prognosis remained poor. Since ZNF384 rearrangement may act as a negative prognostic predictor, early detection based on its characteristic immunophenotype is of great necessity.

HUGO Gene Nomenclature Committee (HGNC) recommendations for the designation of gene fusions

Gene fusions have been discussed in the scientific literature since they were first detected in cancer cells in the early 1980s. There is currently no standardized way to denote the genes involved in fusions, but in the majority of publications the gene symbols in question are listed either separated by a hyphen (-) or by a forward slash (/). Both types of designation suffer from important shortcomings. HGNC has worked with the scientific community to determine a new, instantly recognizable and unique separator—a double colon (::)—to be used in the description of fusion genes, and advocates its usage in all databases and articles describing gene fusions.

Fusion Genes Altered in Adult Malignant Gliomas

Malignant gliomas are highly heterogeneous brain tumors in molecular genetic background. Despite the many recent advances in the understanding of this disease, patients with adult high-grade gliomas retain a notoriously poor prognosis. Fusions involving oncogenes have been reported in gliomas and may serve as novel therapeutic targets to date. Understanding the gene fusions and how they regulate oncogenesis and malignant progression will contribute to explore new approaches for personalized treatment. By now, studies on gene fusions in gliomas remain limited. However, some current clinical trials targeting fusion genes have presented exciting preliminary findings. The aim of this review is to summarize all the reported fusion genes in high-grade gliomas so far, discuss the characterization of some of the most popular gene fusions occurring in malignant gliomas, as well as their function in tumorigenesis, and the underlying clinical implication as therapeutic targets.