In this proposal, we will determine how key innate immune response pathways to viral infection are altered in human cancer cells, particularly by comparing primary innate immune sentinel myeloid cells to their transformed counterparts. We will investigate the mechanistic roles of virus sensing pathways and their linkage to downstream innate immune responses via inflammasomes and NF-?B signaling in determining the tropism and sensing of myxoma virus (MYXV), a rabbit specific poxvirus that also exhibits the capacity to infect a wide spectrum of human cancer cells. We will investigate MYXV sensing and infection of human myeloid cells, especially monocyte-derived macrophages and dendritic cells. We will examine the molecular mechanisms used by MYXV-encoded pyrin domain (PYD)-containing host range protein M013 to co-regulate two distinct innate immune pathways of human myeloid cells, particularly as mediated by inflammasomes and by the NF-?B -mediated inflammatory signaling responses. We have reported that M013 binds the cellular adaptor protein ASC-1 (apoptosis-associated speck-like protein containing a CARD) of various inflammasome complexes and NF-?B1/p105 of NF-?B signaling pathway. Investigation of this unique co-regulation will have potential clinical utility for developing drugs having the capacity o target multiple inflammatory signaling pathways. Finally, we will take advantage of our knowledge of regulation of the inflammasome and NF-?B pathways to study the mechanism(s) of oncolysis by MYXV using myeloid leukemia models. Using knock-out mice for key innate immune pathways and MYXV constructed mutated at the M013 locus, we will study the role of these pathways in MYXV virotherapy for myeloid cancer. The three overlapping areas to be investigated in this proposal are:
Aim 1. Study the alteration of sensing pathways due to transformation of human myeloid cells and its impact on MYXV tropism: We will determine the role of RLR and TLR signaling in inducing type I IFN and pro-inflammatory cytokines in human myeloid cells that are permissive for MYXV (eg transformed myeloid cell lines like THP-1) or nonpermissive (eg primary human monocytes/macrophages);determine the role of NLRP3 inflammasome components and NF-?B1 in the synthesis of precursor IL-1? and IL-18 and release of these mature cytokines in response to MYXV infection;study the co-ordinating upstream role of virus-induced MAPK, in particular the role of MEK1/2-ERK1/2 kinases, in co-inducing both the type I IFN and pro-inflammatory cytokine signaling pathways in response to MYXV infection;and identify specifically which cellular sensor/transducer components become dysfunctional in transformed human myeloid cells.
Aim 2. Create engineered variants of the PYD-containing protein M013 that differentially regulate the inflammasome and NF-?B signaling pathways and test these M013 constructs in an in vivo caspase 1- dependent rat transplant vascular disease model: We will generate M013 mutants that selectively inhibit either the inflammasome or NF-?B pathways by interaction with only ASC-1 or NF-?B1 and exploit these mutants to construct recombinant MYXVs to test their effects in the myeloid cells described above;we will also test M013 variants engineered to allow intracellular penetration in a rat allograft transplant vascular disease (TVD) model that has been shown to be regulated by inflammasome-mediated activation of caspase 1.
Aim 3. Elucidate the roles of host inflammasomes and NF-?B signaling in MYXV-induced oncolytic clearance of myeloid cancer in situ: We will compare the roles of key inflammasome and NF-?B components (ie ASC-1, caspase-1, NLRP3 and NF-?B1) in MYXV-induced anti-tumor responses and clearance of human (eg THP-1) or murine (eg RAW426.7) myeloid leukemias in NSG immunocompromised mice and in C57BL/6-based immunocompetent mouse models, respectively;study the role of M013 variants in selective knockout mice using the M013KO vs wild-type MYXV, and the M013 variant-expressing viruses that selectively inhibit only the inflammasome or NF-?B pathways.
This project addresses several important central issues that have broad application in the areas of virus tropism and viral oncolysis of cancer cells. Those include: how transformation of primary innate immune sentinel cells to their transformed counterparts alters the sensing and innate responses to viruses, how and why innate immune signaling pathways are co-regulated by viruses, and why innate immune signaling pathways are important for oncolytic virotherapy. The proposed studies will demonstrate the role of key immune pathways in transformed vs primary human myeloid cells, how co-regulation of innate signaling pathways can benefit the virus and provide new therapeutic strategies to treat various inflammatory diseases. The model virus proposed here is Myxoma virus (MYXV), which infects only rabbits in nature, and has significant potential for the treatment of human cancers like myeloid leukemia and multiple myeloma. The knowledge that will be gained from this project will have broader implications in the treatment of human cancers using MYXV and can be applicable for other oncolytic viruses as well.
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