First report of Serotype-1 Marek’s disease virus (MDV-1) with oncogenic form in backyard turkeys in Turkey: a molecular analysis study

Background Marek’s disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2, MDV-1), which primarily affects chickens. However, the virus is also able to induce tumors and polyneuritis in turkeys, albeit less frequently than in chickens. Results This is the first study in Turkey reporting the molecular characterization of a MDV-1 strain detected in a flock of backyard turkeys exhibiting visceral lymphoma. Here, MEQ, vIL-8, pp38 and 132-bp tandem repeat regions, which are frequently preferred in the pathotyping of MDV-1, were examined. It was determined that the MEQ gene of MDV-1/TR-21/turkey strain obtained in the present study encoded 339 amino acids (1020 nt) and had four proline-rich repeat regions (PPPP). Based on the nucleotide sequence of the MEQ gene of the MDV-1/TR-21/turkey strain, a phylogenetic tree was created using the MEGA-X software with the Maximum Likelihood Method (in 1000 replicates). Our strain was highly identical (> 99.8) to the Italian/Ck/625/16, Polish (Polen5) and some Turkish (Layer-GaHV-2-02-TR-2017, Tr/MDV-1/19) MDV-1 strains. Also, nt and aa sequences of the MEQ gene of our strain were 99.1 and 99.41% identical to another Turkish strain (MDV/Tur/2019) originated from chickens. Sequence analysis of pp38 and vIL-8 genes also supported the above finding. The identity ratios of nucleotide and amino acid sequences of vIL-8 and pp38 genes of MDV-1/TR-21/turkey strain were 99.64–100% and 99.79–100%, respectively, when compared with those of the Polish strain. According to 132-bp tandem repeat PCR results, the MDV-1/TR-21/turkey strain had five copies. Conclusions These results suggested that the MDV-1/TR-21/turkey strain obtained from backyard turkeys can be either very virulent or very virulent plus pathotype, though experimental inoculation is required for precise pathotyping.

Molecular Genetics in Epstein–Barr Virus-Associated Malignancies

Global genomic studies have detected the role of genomic alterations in the pathogenesis of Epstein–Barr virus (EBV)-associated tumors. EBV oncoproteins cause a vital shift of EBV from an infectious virus to an oncogenic form during the latent and lytic phase within the lymphoid B cells and epithelial cells. This epigenetic alteration modulates the virus and host genomes and inactivates and disrupts numerous tumor suppressors and signaling pathways. Genomic profiling has played the main role in identifying EBV cancer pathogenesis and its related targeted therapies. This article reviews the role of genetic changes in EBV-associated lymphomas and carcinomas. This includes the prolific molecular genesis, key diagnostic tools, and target-specific drugs that have been in recent clinical use.

Resolution of a Clinical Dilemma with Whole Genome Sequencing, and Discovery of a New Mechanism for Generating PML-Rara: Insertional Fusion

Abstract 2755 We describe a difficult diagnostic case of t(15;17)-negative acute promyelocytic leukemia (APL). A 39 year-old woman presented with pancytopenia and low grade DIC. Bone marrow biopsy revealed AML with promyelocytic features. She was treated with Cytarabine, Daunorubicin and ATRA. However, ATRA was discontinued after FISH revealed a possible RARA-PML, but no PML-RARA fusion (one fusion, one RARA and two PML signals), and cytogenetics did not demonstrate a translocation involving chromosome 15 or 17. In fact, her cytogenetics revealed a complex pattern that predicted poor prognosis (46 XX del(9)(q12q32),del(12)(q12q21)[6]/46,idem,-6,-16,add(16)(p13.2),+2 mar[13]/46 XX[1]). Following reinduction, she entered complete remission and was empirically consolidated with arsenic. RT-PCR for PML-RARA was not performed at diagnosis, and was negative at the time of consultation in remission. Her complex cytogenetics and uncertain FISH status posed a diagnostic and therapeutic dilemma that could only be resolved by whole genome sequencing in the time frame required for a clinical decision to be made (i.e. allogeneic transplantation vs. ATRA-based consolidation). DNA was therefore generated from bone marrow (cryopreserved at the time of diagnosis) and a skin sample (obtained in remission), and subjected to massively parallel sequencing using paired-end reads (Mardis et al, NEJM 2009). We generated 187 billion bp (tumor) and 200 billion bp (skin) of sequence, corresponding to 43.7x and 46.8x haploid coverage (99.76% and 99.74% diploid coverage) for the respective samples. Within six weeks of sample receipt, validated results were available. We confirmed the del(9)(q12q32) and del(12)(q12q21) somatic events and identified a novel oncogenic form of chromosomal rearrangement, an insertional fusion: 77 Kb of chromosome 15 (chr15:72027045–72104108 containing LOXL1 exon 6 through PML exon 3) were inserted en bloc into RARA intron 2 (chr17:35742678–35742683). This event resulted in the expression of PML-RARA (bcr3 isoform) and two novel fusion transcripts (RARA-LOXL1 and LOXL1-PML), which were all successfully amplified by RT-PCR and sequenced. The RARA-LOXL1 and LOXL1-PML fusions both created stop codons shortly after the fusion events. Re-evaluation of the FISH results revealed that the insertion generated a fusion signal, while loss of 77 Kb from the PML locus did not prevent binding of the 239 Kb commercial PML probe to chromosome 15 (thus generating 1 fusion, 1 RARA and 2 PML signals). These signals represented the PML-RARA insertional fusion event, not the RARA-PML translocation that was originally reported. We further identified and validated deletions on chromosomes 12 (60 Mb), 14 (22 Kb) and 19 (30 Kb) and non-synonymous, somatic single nucleotide variants (SNVs) in the coding regions of ZNF687, DYTN, C3orf54, CH3D19, SLC35A4, GPRC6A, ZFHX4, PTK2, PITPNM1, DEGS2, PCSK2, CDC45L, although the clinical relevance of these deletions and point mutations is not yet known. After validating PML-RARA bcr3 expression, a decision was made to consolidate the patient with an ATRA-containing regimen. Using whole genome sequencing with paired end reads, we have identified a novel oncogenic form of chromosomal rearrangement, an insertional fusion. Similar insertional events may occur in other loci. Small structural events (under a few megabases in size) are often undetectable by conventional cytogenetics and FISH, and are expected to be invisible to standard break-apart probes (commonly used to evaluate the RARA and MYC loci). This case highlights the clinical relevance of whole genome sequencing for informing diagnostic and therapeutic decisions that must be made within weeks after sample acquisition. Disclosures: Off Label Use: Decitabine, Arsenic and Ascorbic acid for the treatment of AML. DiPersio:Genzyme: Honoraria. Westervelt:Novartis: Honoraria; Celgene: Honoraria, Speakers Bureau.

STAT3 induces transcription of the DNA methyltransferase 1 gene (DNMT1) in malignant T lymphocytes

In this study, we demonstrated that STAT3, a well-characterized transcription factor expressed in continuously activated oncogenic form in the large spectrum of cancer types, induces in malignant T lymphocytes the expression of DNMT1, the key effector of epigenetic gene silencing. STAT3 binds in vitro to 2 STAT3 SIE/GAS-binding sites identified in promoter 1 and enhancer 1 of the DNMT1 gene. STAT3 also binds to the promoter 1 region and induces its activity in vivo. Treatment of the malignant T lymphocytes with STAT3 siRNA abrogates expression of DNMT1, inhibits cell growth, and induces programmed cell death. In turn, inhibition of DNMT1 by a small molecule inhibitor, 5-aza-2-deoxy-cytidine, and 2 DNMT1 antisense DNA oligonucleotides inhibits the phosphorylation of STAT3. These data indicate that STAT3 may in part transform cells by fostering epigenetic silencing of tumor-suppressor genes. They also indicate that by inducing DNMT1, STAT3 facilitates its own persistent activation in malignant T cells. Finally, these data provide further rationale for therapeutically targeting STAT3 in T-cell lymphomas and, possibly, other malignancies.

PKCδ plays opposite roles in growth mediated by wild-type Kit and an oncogenic Kit mutant

The Kit receptor tyrosine kinase is critical for normal hematopoiesis. Mutation of the aspartic acid residue encoded by codon 816 of human c-kit or codon 814 of the murine gene results in an oncogenic form of Kit. Here we investigate the role of protein kinase Cδ (PKCδ) in responses mediated by wild-type murine Kit and the D814Y mutant in a murine mast cell-like line. PKCδ is activated after wild-type (WT) Kit binds stem cell factor (SCF), is constitutively active in cells expressing the Kit catalytic domain mutant, and coprecipitates with both forms of Kit. Inhibition of PKCδ had opposite effects on growth mediated by wild-type and mutant Kit. Both rottlerin and a dominant-negative PKCδ construct inhibited the growth of cells expressing mutant Kit, while SCF-induced growth of cells expressing wild-type Kit was not inhibited. Further, overexpression of PKCδ inhibited growth of cells expressing wild-type Kit and enhanced growth of cells expressing the Kit mutant. These data demonstrate that PKCδ contributes to factor-independent growth of cells expressing the D814Y mutant, but negatively regulates SCF-induced growth of cells expressing wild-type Kit. This is the first demonstration that PKCδ has different functions in cells expressing normal versus oncogenic forms of a receptor.

Autoinhibition of the Kit Receptor Tyrosine Kinase by the Cytosolic Juxtamembrane Region

ABSTRACT Genetic studies have implicated the cytosolic juxtamembrane region of the Kit receptor tyrosine kinase as an autoinhibitory regulatory domain. Mutations in the juxtamembrane domain are associated with cancers, such as gastrointestinal stromal tumors and mastocytosis, and result in constitutive activation of Kit. Here we elucidate the biochemical mechanism of this regulation. A synthetic peptide encompassing the juxtamembrane region demonstrates cooperative thermal denaturation, suggesting that it folds as an autonomous domain. The juxtamembrane peptide directly interacted with the N-terminal ATP-binding lobe of the kinase domain. A mutation in the juxtamembrane region corresponding to an oncogenic form of Kit or a tyrosine-phosphorylated form of the juxtamembrane peptide disrupted the stability of this domain and its interaction with the N-terminal kinase lobe. Kinetic analysis of the Kit kinase harboring oncogenic mutations in the juxtamembrane region displayed faster activation times than the wild-type kinase. Addition of exogenous wild-type juxtamembrane peptide to active forms of Kit inhibited its kinase activity in trans, whereas the mutant peptide and a phosphorylated form of the wild-type peptide were less effective inhibitors. Lastly, expression of the Kit juxtamembrane peptide in cells which harbor an oncogenic form of Kit inhibited cell growth in a Kit-specific manner. Together, these results show the Kit kinase is autoinhibited through an intramolecular interaction with the juxtamembrane domain, and tyrosine phosphorylation and oncogenic mutations relieved the regulatory function of the juxtamembrane domain.

GCKR Links the Bcr-Abl Oncogene and Ras to the Stress-Activated Protein Kinase Pathway

The Bcr-Abl oncogene, found in Philadelphia chromosome-positive myelogenous leukemia (CML), activates Ras and triggers the stress-activated protein kinase (SAPK or Jun NH2-terminal kinase [JNK]) pathway. Interruption of Ras or SAPK activation dramatically reduces Bcr-Abl–mediated transformation. Here, we report that Bcr-Abl through a Ras-dependent pathway signals the serine/threonine protein kinase GCKR (Germinal Center Kinase Related) leading to SAPK activation. Either an oncogenic form of Ras or Bcr-Abl enhances GCKR catalytic activity and its activation of SAPK, whereas inhibition of GCKR impairs Bcr-Abl–induced SAPK activation. Bcr-Abl mutants that are impaired for GCKR activation are also unable to activate SAPK. Consistent with GCKR being a functional target in CML, GCKR is constitutively active in CML cell lines and found in association with Bcr-Abl. Our results indicate that GCKR is a downstream target of Bcr-Abl and strongly implicate GCKR as a mediator of Bcr-Abl in its transformation of cells.