Yuan Chang, MD, University of Utah 1987 - US grants

Two DNA sequences, designated KS330Bam and KS627Bam, have been isolated from a KS lesion by representational difference analysis (RDA). Southern blot hybridizations and PCR analyses indicate that these sequences are specifically associated with KS as well as a subset of rare AIDS- associated body cavity-based lymphomas. These sequences are also found in classical KS lesions and KS lesions from HIV-negative gay men suggesting an unified etiology for the clinical subtypes of KS. These sequences are the first reliable molecular markers found for KS. The 627 bp fragment has nucleotide homology to a gamma herpesvirus tegument gene. A 2166 bp region including the 330 bp fragment was sequenced and found to have two open reading frames (ORF) homologous to gamma herpesvirus capsid ORFs. These KS associated sequences appear to be part of the genome of a transmissible DNA virus with an approximately 270 kb genome, homologous to the Gammaherpesvirinae Epstein-Barr virus (EBV) and herpesvirus saimiri (HSVSA). An EBV positive cell line, BCBL1, derived from one of the positive body cavity-based lymphomas has been found to be dually infected with the agent at an average of 50 copies per cell and is an important tool for characterizing the virus. The studies suggest that a newly discovered human herpesvirus is causal for KS and some AIDS lymphomas. Although a formal classification of this agent has not yet been determined, it is designated here as the Kaposi's sarcoma-associated herpes-like virus (KSHV) for purposes of convenience and clarity. This proposal's strategy for characterizing this virus and determining its oncogenic potential will be similar to the approach used by other groups in the study of herpesvirus saimiri and EBV. First, continued bidirectional genomic walking will be used to obtain sequence information on the virus' genomic organization and its phylogenetic relationship to other herpesviruses. Specific genes which may be biologically and clinically important, such as the herpesviral major capsid protein and thymidine kinase ORF homologs, will be targeted. Secondly, in-situ hybridization studies of KS lesions will be performed to identify the infected cell type in KS lesions as well as subcellular localization of various viral antigens. Third, transmission studies will be undertaken to propagate the virus in EBV nonpermissive cell lines. These studies will lead to in vitro transformation experiments to identify specific viral oncogenes. In combination, these sets of studies will begin the characterization of this new and novel emerging human pathogen. These studies will directly lay the groundwork for future therapeutic and vaccine interventions against AIDS-associated KS.

DESCRIPTION: (Adapted from Applicant's Abstract) Tumor viruses, such as KSHV and EBV, are responsible for the majority of AIDS-related malignancies, including Kaposi's sarcoma and CNS lymphoma. Many tumor viruses share the ability to directly or indirectly inhibit p 53 and pRB tumor suppressor pathways. KSHV vIRF and EBV EBNA2 oncoproteins share a third common feature of inhibiting interferon signaling which may contribute to cell transformation. These investigators have found that vIRF and EBNA2, like adenovirus E1A, bind the transscriptional coadaptor p300 involved in interferon and apoptosis-related transcription. VIRF, EBNA2, and EIA induce the cMYC oncogene promotor in a p300 regulated manner through an interferon-responsive element and cMYC induction is required for vIRF-induced cell transformation. This effect is mediated by an undiscovered transcriptional factor, provisionally called PBF-X, may have general importance for the dysregulation of cMYC in both infectious and non-infectious cancers. This is a collaborative research effort designed to bring together two research groups to systematically examine the effects of p300 binding by vIRF and EBNA2 on MYC induction and apoptotic pathway inhibition. This will be achieved by mapping functional domains of vIRF and EBNA2, and by fine mapping the response element sequences in the cMYC promoter. The effects of p300 binding by EBNA3 and vIRF on p53-dependent and independent apoptotic transcriptional responses will be examined using p53-temperature sensitive mutant cells. Mechanistic studies (e.g. using protein synthesis inhibition and dominant negative inhibitors) will broadly define the pathway for cMYC induction and the contributing role of p300. The identity of PBF-X will be sought from among eight known transcription factors (IRFs1-7 , and Blimp-1) and new candidates will be identified from yeast one- and two-hybrid studies. After PBF-X is identified, knockout mice will be generated for physiologic studies of the this transcriptonal factor. PBF-X is p potential tumor suppressor candidate and LOH and chromosomal breakpoint data will be examinded for mutations involving this locus. A pilot study of tumors, particularly non-Burkitt's NHL with germline cMYC will be examined for PBF-X mutations or LOH to test whether this transcription factor plays a role in human tumorigenesis. These studies will lead to novel approaches in the control of EBV and KSHV-related malignancies in AIDS patients.

DESCRIPTION: (Adapted from Applicant's Abstract) KSHV/HHV8, the eighth human herpesvirus, was discovered in applicant's laboratory in 1993. It is associated with three human proliferative diseases ranging from hyperplastic proliferation to neoplasia: Castleman's disease, Kaposi's sarcoma, and primary effusion lymphoma (PEL). These disorders are rarely expressed in the general population, but are common among KSHV seropositive individuals who are immunocompromised, particularly AIDS patients. During the past grant period, the investigators sequenced the viral genome, and discovered and characterized a number of viral genes likely to play a role in KSHV pathogenesis. Their studies have concentrated on latent viral genes expressed in tumor cells that are likely to modify host cell regulatory pathways. By mapping whole genome expression and determining protein expression patterns, they have identified specific viral genes likely to play roles in cell proliferation and prevention of apoptosis in situ. Recently, they have found a latently expressed gene, which is not a component of the previously described major latency transcript locus, LT1 and LT2 encompassing v-cyclin (0RF72) and LANA1 (0RF73). This 1704 bp spliced gene encodes a protein designated latency-associated nuclear antigen (LANA) 2, which is localized to the nucleus and is expressed in all KSHV-infected PEL-derived cells. LANA2 has structural similarities to the IRF4 transcriptional factor present in B cells. This renewal seeks to characterize this latent protein and investigate its potential role in KSHV-related diseases. The four specific aims of this proposal are: 1) to extend the characterization of LANA2 concentrating on the in vivo expression of the protein in KSHV-associated disease tissues, 2) to extend their preliminary studies demonstrating LANA2-mediated upregulation of the CD23 B cell activation antigen, which is also induced by Epstein-Barr virus infection, 3) to identify protein-protein interactions between LANA2 and host cell proteins involved in CD23 transcriptional regulation, and 4) to investigate the potential role of this latent viral gene on cell cycle regulation, apoptosis and in vitro cell transformation. These studies may provide critical insights into the biological role of CD23 signaling in lymphomagenesis and in herpesvirus infection of B cells. Characterization of LANA2 may also contribute to an important future diagnostic and therapeutic target for KSHV infection

DESCRIPTION: (Adapted from Applicant's Abstract) Tumor viruses, such as KSHV and EBV, are responsible for the majority of AIDS-related malignancies, including Kaposi's sarcoma and CNS lymphoma. Many tumor viruses share the ability to directly or indirectly inhibit p 53 and pRB tumor suppressor pathways. KSHV vIRF and EBV EBNA2 oncoproteins share a third common feature of inhibiting interferon signaling which may contribute to cell transformation. These investigators have found that vIRF and EBNA2, like adenovirus E1A, bind the transscriptional coadaptor p300 involved in interferon and apoptosis-related transcription. VIRF, EBNA2, and EIA induce the cMYC oncogene promotor in a p300 regulated manner through an interferon-responsive element and cMYC induction is required for vIRF-induced cell transformation. This effect is mediated by an undiscovered transcriptional factor, provisionally called PBF-X, may have general importance for the dysregulation of cMYC in both infectious and non-infectious cancers. This is a collaborative research effort designed to bring together two research groups to systematically examine the effects of p300 binding by vIRF and EBNA2 on MYC induction and apoptotic pathway inhibition. This will be achieved by mapping functional domains of vIRF and EBNA2, and by fine mapping the response element sequences in the cMYC promoter. The effects of p300 binding by EBNA3 and vIRF on p53-dependent and independent apoptotic transcriptional responses will be examined using p53-temperature sensitive mutant cells. Mechanistic studies (e.g. using protein synthesis inhibition and dominant negative inhibitors) will broadly define the pathway for cMYC induction and the contributing role of p300. The identity of PBF-X will be sought from among eight known transcription factors (IRFs1-7 , and Blimp-1) and new candidates will be identified from yeast one- and two-hybrid studies. After PBF-X is identified, knockout mice will be generated for physiologic studies of the this transcriptonal factor. PBF-X is p potential tumor suppressor candidate and LOH and chromosomal breakpoint data will be examinded for mutations involving this locus. A pilot study of tumors, particularly non-Burkitt's NHL with germline cMYC will be examined for PBF-X mutations or LOH to test whether this transcription factor plays a role in human tumorigenesis. These studies will lead to novel approaches in the control of EBV and KSHV-related malignancies in AIDS patients.

DESCRIPTION (provided by applicant): One in 5 cancer cases worldwide is caused by infection (International Agency for Research on Cancer, 2002) and yet only seven viruses have been established to cause human cancers. Our laboratory discovered and characterized two of these agents, KS herpesvirus (KSHV/HHV8) and Merkel cell polyomavirus (MCV), both of which were discovered through highly-directed genomic searches. These findings initiated new fields in cancer biology and provided new bases for improved diagnosis, treatment and prevention of cancers caused by these viruses. Traditional approaches to cancer genetics have focused almost exclusively on somatic cell mutations and large scale sequencing studies are likely to miss discovery of viruses that might be causing some human tumors. Over the past four years, a group of seven new human polyomaviruses (including MCV, known to cause Merkel cell carcinoma (MCC)) were discovered that encode T antigen oncoproteins and are credible candidate tumor viruses. Although most adults are chronically infected with these viruses, none were found through cancer genome anatomy project sequencing studies. The NCI Director's Provocative Question Initiative #12 arises from our recent MCV-related MCC studies showing that MCV, a common commensal skin infection, initiates tumors after specific mutations to the viral genome. This is a new mechanism for carcinogenesis that will not necessarily be found through cancer cell genome sequencing projects unless a highly directed search for nonhuman viral sequences is performed in a orderly fashion. To identify human cancer viruses, we developed digital transcriptome subtraction (DTS), a deep sequencing approach that depends on generation of high-fidelity sequence databases and provides quantitative data on tumor cell transcription. Although standard deep sequencing has been used by others to search for viruses, successful analysis is highly-dependent on specific technical skills and assumptions. In 2008, we used DTS to discover MCV mRNA sequences in MCC, allowing subsequent full viral genome sequencing and confirmatory studies. In this application, we maximize the likelihood for identifying a novel cancer virus (es) in hematologic malignancies through both directed and unbiased approaches. Our directed approach will survey a large panel of hematologic malignancies for presence of the new human polyomaviruses. Using information gained from the biology of MCV, we will determine whether tumor-specific viral mutation patterns are present that differentiate causal from incidental viral infections. Our unbiased approach will use DTS to examine highly-selected gold-standard cases of EBV-negative post-transplant lymphoproliferative disorder (EN-PTLD) for presence of novel viral transcripts. High PHRED-equivalent sequencing of EN-PTLD tumors to <1 transcript per million (TPM) level will be performed to identify novel viral transcripts. These data will be compared to DTS on EBV-positive PTLD and CD19+ peripheral B cells to 1) confirm virus transcript detection and 2) determine differential cellular gene expression patterns between EBV-negative and EBV- positive disease. We will also initiate an exploratory collaboration with the Pacific Northwest National Laboratory to develop the next generation of tumor virus discovery technology using unsupervised LC- MS/MS proteomics of whole EN-PTLD tissue samples. Comparisons of DTS (transcript) and LC-MS/MS (peptide) data, obtained from the same tissue samples, will allow a more precise subtraction of human sequence data to identify novel viruses present in tumors. With the completion of these aims, we anticipate being able to answer whether one of the six new human polyomaviruses contributes to hematolymphoid malignancies and we will determine whether or not EN- PTLD harbors a novel virus. This systematic approach provides the highest probability to find a new human cancer virus and we will develop new technologic approaches that can be widely used in future searches for infections in human cancer. PUBLIC HEALTH RELEVANCE: Cancer viruses cause a large but underappreciated fraction of human cancer cases. New viruses causing cancer can be discovered with new sequencing technologies but are usually overlooked unless the samples are analyzed in a highly-specialized manner. Once a new tumor virus is found, a major obstacle to overcome is determining whether the virus is the cause for the cancer or is a simply a coincidental infection that plays no role in cancer formation. We plan to approach these problems in two ways. We will first perform a broad search for members of a group of newly-discovered viruses, called polyomaviruses, in hematologic cancers. These viruses all encode oncoproteins (T antigens) that target cellular pathways controlling cell proliferation. For these viruses to cause cancer, they must undergo specific mutations within the cancer cell. These mutations prevent the polyomaviruses from replicating and so finding these mutations distinguishes tumor-causing from coincidental polyomavirus infections in samples that are positive for one or more of the new polyomaviruses. We will next examine in detail one type of hematologic cancer highly likely to be caused by viral infection. Most cases of this malignancy, called post-transplant lymphoproliferative disorder, are caused by a known tumor virus (Epstein-Barr virus, EBV). We will examine cases of post-transplant lymphoproliferative disorder that are negative for EBV and compare them to EBV-positive samples to search for gene products from a new virus. The ability to find EBV fragments in known EBV-positive tumors will serve as a quality- control reference for our studies. If a new virus fragment is found, the whole virus can be rapidly acquired and then tested in a variety of tumors to determine if it is also present in other, more common cancers. We will first use a genomic methodology developed in our laboratory and successfully used to discover one new human cancer virus called Merkel cell polyomavirus (MCV). This method detects messenger RNA from viruses in tumors. We will next couple this technology with a method to detect peptides made in tumor cells through collaboration with scientists at the DOE Pacific Northwest National Laboratory. We anticipate that this combined approach will detect viruses in tumors if they are present and rule out certain classes of viral cause when no virus is found. The data generated by this project can be repurposed to also measure cellular gene expression from tumors so that even if no new viruses are discovered, the critical nonviral genetic changes that contribute to a cancer can be studied.

ABSTRACT 4E-BP1 is the primary gatekeeper for cancer cell, cap-dependent protein translation. It is directly targeted by the mTOR pathway that is frequently dysregulated in cancer. We have found that CDK1/CYCB1 substitutes for mTOR during mitosis to phosphorylate 4E-BP1 generating a novel phosphorylation mark at serine (S) 83 that is not present when mTOR phosphorylates 4E-BP1. A mutant form of 4E-BP1 unable to be phosphorylated at S83 partially reverses cell transformation caused by the Merkel cell polyomavirus (MCV) small T oncoprotein. This project is focused on investigating a novel CDK-1-dependent but mTOR-independent 4E-BP1 regulatory pathway. The central hypothesis for this proposal is that S83 phosphorylation modulates translation of a unique subset of mRNAs to facilitate mitosis-specific protein expression. In Aim I, substitution of a mutant (S83A) and the phosphomimetic (S83D) 4EBP1 proteins into EIF4EBP1 null cells, as well as developing MEFs from the EIF4EBP1 S83A knock-in mice will be used to assay the effects of S83 phosphorylation on basic cell homeostasis, including cell cycle analysis, proliferation and protein synthesis. In Aim II, we will identify differentially translated mRNAs as a result of mitotic 4E-BP1 phosphorylation through two complementary approaches: ribosomal profiling of mitosis-arrested cells, and RNA immunoprecipitation and sequencing (RIPseq). In Aim III we will use live-cell imaging tools to track and quantify dynamics of translation in live cells. Finally in Aim IV, we will explore a unique knock-in mutant mouse model for 4E-BP1 dysregulation. Our studies will advance our fundamental understanding of how a mitosis-specific hyperphosphorylated form of 4E-BP1 functions in normally cycling cells and how its dysregulation in cancer cells may contribute to human malignancies.