Alexander Ishov - US grants

Description (provided by applicant) The Microscopy and Histopathology Laboratory provides a service to these members of the program project, whose work involves the evaluation of tissue or cell alterations that occur in response to HSV infection. Tissue will be embedded, sectioned, and stained. Microscopy services will be provided, including confocal microscopy. Dr Ehud Lavi, neuropathologist, will continue to provide anatomical advice on the sections.

[unreadable] DESCRIPTION (provided by applicant): Taxanes are powerful drugs for breast cancer treatment; however, a large number of patients are resistant to this therapy for unknown reasons. Therefore it will be essential to develop prognostic tools and predictive markers to differentiate the patient population for appropriate chemotherapy selection. This proposal aims to evaluate protein Daxx as a predictive marker for taxane response and is based on our observation that sensitivity to paclitaxel treatment, in breast cancer cell lines and mouse cells, correlates with the level of Daxx. Taxanes block cells in mitosis followed by cell death. Our central hypothesis is that Daxx deficiency can determine resistance to paclitaxel-induced cell death in breast cancer patients upon treatment. The proposed project is to examine the function of Daxx as a paclitaxel sensitivity factor that can be used as a predictive marker in selection of breast cancer patients to receive taxane therapy. We will dissect the mechanism of this sensitivity elucidating the role of Daxx in mitotic progression using mouse Daxx knockout system, human breast cancer cell lines and breast cancer primary specimens. Specifically, we will: 1) examine the role of Daxx in paclitaxel induced cell death, and 2) elucidate the function of Daxx in mitotic progression as a mechanism of Daxx-dependent resistance to paclitaxel treatment. The proposed study is based on our current understanding of Daxx as a trigger of taxanes activity. Identification of Daxx as a novel mitotic checkpoint release protein that determines resistance for taxanes, chemotherapeutic drugs commonly used in breast cancer treatment, will aid in proper selection of breast cancer patients to receive this therapy and add to understanding of mechanisms that connect cell division, genome instability and breast cancer progression. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) causes significant pain and suffering in humans. It is acquired by skin infection (typically mouth/lips for HSV-1 and genitals for HSV-2) where it replicates lytically and then spreads to the nervous system. It establishes a life-long latent infection within sensory nerve ganglia and persists as a double-stranded DNA (episome) within the nuclei of latently infected neurons. Periodically HSV reactivates from this latent state and causes cold sores (HSV-1) or genital lesions (HSV-2). The mechanism of how HSV establishes a latent infection or reactivates from latency is not understood. Over 50 million people in the US experience clinical recurrent HSV disease. HSV-1 is also the leading cause of infectious blindness in the US with over 30,000 new cases a year. While antiviral drugs can reduce the severity of symptoms, they do not block reactivation and they are not curative. Understanding how HSV interacts with the cell to regulate its lytic and latent life cycle could provide new targets for therapeutic intervention. Recent advantages in the field have demonstrated that transcriptional control of viral genes at the chromatin level plays a key role in the delicate balance between latency and reactivation, suggesting that chromatin is the key element in HSV latency. While the details of these regulatory processes are largely unknown, several recent findings have provided clues such as: evidence that HSV lytic genes are silenced by post-translational modifications of histones (epigenetic repression); insulator protein CTCF establishes epigenetic borders on viral DNA during latency, separating repressed lytic genes and active LAT region; nuclear protein DAXX and its interaction partner ATRX play a pivotal role by chaperoning the histone variants that can facilitate formation of mobile, transcriptionally active chromatin; DAXX and ATRX participate in intrinsic antiviral defenses repressing lytic infection. During latency establishing, DAXX complex participates in chromatin formation on HSV-1 DNA. In turn, this may create specific chromatin signature to establish proper loading and maintenance of CTCF at specific CTCF-binding elements of HSV genome thus creating epigenetic boundaries on this genome to regulate latency. These observations allowed us to formulate the main hypothesis of this proposal: the DAXX/ATRX-mediated loading of histone variants regulates dynamic deposition of CTCF on HSV-1 genome thus controlling appropriate chromatin formation that is critical for latency. We expect that the balanced deposition of histone variants represents a key necessary step in latency and predict that DAXX/ATRX complex plays an essential role in this process. In order to test this hypothesis we are combining the expertise of two co-investigators. Dr. Alexander Ishov is an expert in the DAXX/ATRX biology and in the analysis of intra-nuclear aspects of viral infection. Dr. David Bloom is an expert in epigenetic control of HSV gene expression and models to study HSV pathogenesis and latency. The key studies proposed in this project will investigate the involvement of DAXX/ATRX in recruiting histones variants to the regulatory elements of HSV genome and whether this alters the ability of the genome to establish and maintain latency via CTCF deposition in both in vitro and in vivo settings. These studies will determine whether DAXX/ATRX chaperone complex is involved in the regulatory balance between HSV lytic and latent infection and will provide essential data for the mechanistic understanding of this process. In addition to providing insight into the mechanism of HSV latency and reactivation, this highly innovative and focused pilot study has the potential to identify new therapeutic targets for treating HSV infections. If DAXX/ATRX were shown to have a role in regulating the switch between lytic and latent infection, this complex would be highlighted as target for anti-viral therapy.

? DESCRIPTION (provided by applicant): Prostate cancer (PCa) is the second leading cause of cancer-related mortality in American men. Androgen ablation therapies are initially effective in ~90% of patients. Unfortunately, these therapies offer only a temporary relief and the disease eventually recurs with a lethal outcome. PCa that relapsed after hormonal therapies is the major cause of disease lethality and is referred to as castration-resistant PCa (CRPC). A better understanding of mechanisms that control AR activation and allow CRPC to circumvent hormonal ablation therapies is critically important for improving disease outcome. Identification of activators or mediators of AR signaling should help to reveal novel targeted and combinatorial regimens for clinical management of CRPC. Aberrant AR activation in CRPC is achieved via several mechanisms, including gain-of-function mutation. This new-identified molecular mechanism of persistent AR signaling activity is based on expression of AR variants with deletion of ligand-binding domain (AR?LBD); indeed, CRPC that express AR?LBDs are resistant to hormone withdrawal. Due to the constant nuclear localization, AR?LBDs are persistently active for transcription regulation of AR-dependent genes. The goal of this project is to characterize novel targets and to identify new approaches for pharmacological intervention of AR?LBD signaling for CRPC treatment. Transcriptional control of AR-induced genes is regulated by epigenetic modifications of chromatin. The histone variants barcode hypothesis is based on differences between histone variants, and postulates that incorporation of transcription-associated H3.3 and H2A.Z variants creates active territories of chromatin. While the details of these processes are largely unknown, several recent findings and our unpublished data have provided clues: 1) Nuclear protein Daxx and its interactors ATRX and p400 chaperone histone H3 variant, H3.3, and H2A variant, H2A.Z, facilitating formation of transcriptionally active chromatin; 2) both AR and AR?LBD interact with Daxx, recruiting Daxx-containing histone chaperone complexes to ARE in PCa and CRPC; 3) Elevated expressions of Daxx and H2A.Z are associated with bad prognoses of PCa; 4) characterizing expression profiles of genome-edited AR?LBD CRPC cell models, we identified genes that are co-regulated by AR?LBD and Daxx. These findings allowed us to formulate the main hypothesis of this proposal: Constant and uncontrolled recruitment of histone chaperone Daxx by AR?LBDs elevates incorporation of transcription-associated histone variants H3.3 and H2A.Z to ARE, thus modifying expression profile for CRPC progression. Thus, Daxx is potential contributor of CRPC, and Daxx/AR interaction can be investigated as a target for therapeutic intervention, specifically in AR gain-of-function CRPC. In this exploratory grant, we will: I. Evaluate role of histone variants H3.3 and H2A.Z in CRPC; II. Explore mechanisms underlying histone variants H3.3/H2A.Z profiling in CRPC; III. Investigate mechanisms of chaperone recruitment for identification of new therapeutic targets in CRPC. To make progress in PCa treatment, we must develop evidence-based strategies for choosing therapy interventions. If Daxx/AR?LBD were shown to have a role in regulating CRPC progression, this complex would be further highlighted as target for anti-cancer therapy to improve morbidity and mortality of PCa patients.