A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties.

Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3' splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A's terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3' splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing

Stealing the Show: KSHV Hijacks Host RNA Regulatory Pathways to Promote Infection

Kaposi’s sarcoma-associated herpesvirus (KSHV) induces life-long infections and has evolved many ways to exert extensive control over its host’s transcriptional and post-transcriptional machinery to gain better access to resources and dampened immune sensing. The hallmark of this takeover is how KSHV reshapes RNA fate both to control expression of its own gene but also that of its host. From the nucleus to the cytoplasm, control of RNA expression, localization, and decay is a process that is carefully tuned by a multitude of factors and that can adapt or react to rapid changes in the environment. Intriguingly, it appears that KSHV has found ways to co-opt each of these pathways for its own benefit. Here we provide a comprehensive review of recent work in this area and in particular recent advances on the post-transcriptional modifications front. Overall, this review highlights the myriad of ways KSHV uses to control RNA fate and gathers novel insights gained from the past decade of research at the interface of RNA biology and the field of KSHV research

Trapping KSHV in latency - Exploring kinase inhibitors as a class of anti-virals that target the KSHV lytic cycle

KSHV causes Kaposi sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). These malignancies lack effective treatments; therefore, there is need to develop better anti-KSHV therapies. KSHV latent and lytic cycles are essential for tumor progression, and disrupting the balance between these two phases can effectively limit viral pathogenesis, as discussed in Chapter 1 of this dissertation. In Chapter 2, we explore the possibility of blocking the transition to the lytic cycle by inhibiting RTA, the only viral protein necessary and sufficient to induce lytic reactivation. RTA is heavily phosphorylated, therefore, we exploit this phosphorylation by using a kinase inhibitor screen to identify two novel compounds, AZD5438 and BMS-265246 that inhibit RTA transactivation. We show that blocking RTA transactivation with AZD5438 or BMS-265246 is sufficient to prevent viral egress and KSHV transmission in vitro. We also perform a phospho-proteomic analysis of the RTA protein and develop a detailed map of RTA phosphorylation sites. We show that RTA is phosphorylated at numerous amino acids across different protein domains. Treatment with AZD5438 or BMS-265246 inhibits phosphorylation of RTA at some, but not all, phosphorylation sites, suggesting that these compounds target a subset of RTA-phosphorylating kinases. The kinases that (I) phosphorylate RTA and (II) are inhibited by AZD5438 or BMS-265246 are likely important for RTA transactivation; therefore, we compile the list of kinases that may contribute to RTA function. We also test in vivo efficacy and show that BMS-265246, but not AZD5438, inhibits tumor growth in a xenograft mouse model for PEL. BMS-265246 likely inhibits PEL tumor growth in vivo via two mechanisms; (I) disrupting RTA phosphorylation, and (II) silencing expression of RTA-interacting cellular cofactors such as Hey1, c-Jun and FosB. Overall, in this dissertation, we explore RTA as a novel therapeutic target for anti-KSHV treatments due to RTA’s role in inducing the transition from latent to lytic cycles. We identify two kinase inhibitors that prevent RTA transactivation and KSHV egress in vitro, with only BMS-265246 inhibiting PEL tumor growth in vivo, thus laying the groundwork for developing BMS-265246 as a potential anti-KSHV therapeutic.Doctor of Philosoph

CHARACTERIZATION OF THE ROLE OF CELLULAR SIGNALING PATHWAYS ON THE PATHOGENESIS OF KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of multiple malignancies, including Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL). KSHV belongs to the gammaherpesvirus family, and it contains a large double-stranded DNA genome encoding a plethora of viral genes and non-coding RNAs. Understanding the interplay between the virus and the host is crucial for identifying therapeutical targets. KSHV viral interleukin-6 (vIL-6) is a viral homolog of human IL-6. This viral protein is expressed in KSHV-associated malignancies and is known to deregulate signaling pathways. We found that vIL-6 induces the expression of integrin β3 (ITGB3), resulting in the surface expression of integrin αVβ3. We discovered that JAK/STAT signaling pathway is necessary for the induction of ITGB3. Moreover, we found that vIL-6 can induce the expression of ITGB3 in a paracrine manner which is physiologically relevant as vIL-6 can be detected circulating in patients suffering from KSHV-associated malignancies. Importantly, depletion of ITGB3 hindered the ability of vIL-6 to promote angiogenesis. Therefore, we identified ITGB3 as a potential therapeutic target for KSHV-associated malignancies. The PI3K/Akt/mTOR pathway has been shown to contribute to cell survival and proliferation and is constitutively activated by some KSHV proteins. Unsurprisingly, the pathway is commonly deregulated in malignancies including non-Hodgkin lymphomas (NHL). Given the importance of the pathway for cell survival, we reasoned that inhibiting the network with novel inhibitors should be a viable therapeutical approach. Miransertib and MK-4440 are small molecules that effectively inhibit Akt and have entered clinical development. Using in vitro and in vivo models of NHL, we explored targeting the Akt alone or in combination with the mTORC1 inhibitor, sirolimus. We found that the combination of miransertib and sirolimus synergistically reduced cell proliferation and tumor growth in NHL, including the KSHV-associated PEL. These data suggest that targeting the PI3K/Akt/mTOR pathway with Akt inhibitors such as miransertib in combination with mTOR inhibitors serves as a broadly applicable therapeutic in NHL. In sum, with this dissertation, I undertook two different routes to better understand the importance of signaling pathways in KSHV-associated malignancies, with implications beyond viral cancers.Doctor of Philosoph