2019Joint Usage and Research Report
Title of Research Project

The ADP-Ribosyltransferase, TIPARP, andmono-ADP-ribosylation are critical mediators ofAHR-dependent regulation of inflammation and immune
responses

ApplicantInstitution

Institute for Genetic Medicine

Job title

Professor Akinori Takaoka

and name
Visiting

Name

Professor Jason Matthews University of Oslo

researcher
Purpose of the Research Project

The Aryl hydrocarbon receptor (AHR) is a transcription factor(approx. 250 words)

that regulates the expression of many genes at the interfaces
among environmental clues, metabolic alterations, and theimmune system The AHR has historically been studied due to its
ability to mediate the toxic responses of environmental
pollutants,

such

as

2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD/dioxin). It is now recognized as an essential gatekeeper
integrating signals from the diet and endogenous metabolism tomodulate immune cell homeostasis, reactive oxygen species,immunosuppression and tumorigenesis. The AHR regulates T
cell differentiation, immune tolerance and innate immunity.
Through this collaboration, we reported that TIPARP is the key
regulator of AHR-dependent innate immune suppression andfound that it specifically ADP-ribosylates TBK1 reducing its
ability to activate type I interferon responses. (Yamada et al.
Nature Immunology 2016). However, we still know very little
about TIPARP’s role in these processes, how it contributes toadaptive immunity, how the TIPARP-AHR axis is regulated
during the innate/adaptive immune responses, and the role ofTIPARP in AHR-dependent B or T cell function. Moreover, theinhibition of TIPARP may serve as potential therapeuticstrategy to boost cancer immunotherapy by reversing tumour
associated immunosuppression through increases in type I
interferon

signaling.

Our

proposedprojectwill

includecomprehensive studies of Tiparp knockout mice, primary andimmortalize cell line models, as well as genomic and proteomic
methods to characterize the importance of TIPARP in theimmune system and cancer immunotherapy.

Development of the Research

During the past year my lab gained expertise in characterizing

Project and Results

type I interferon response after treatment with synthetic(approx.. 850 words)

pathogen-associated molecular patterns (PAMPs) like 3pRNAand cGAMP), through the sharing of protocols and material fromProf. Takaoka’s group. For these studies we have focused onmouse embyronic fibroblasts (MEFs) from our Tiparp-/- mice andfrom our Tiparp catalytic mutant mice TiparpH532A. We foundthat exposure to PAMPs, as well as LPS dramatically increases
the expression of several proinflammatory cytokines in addition
to type I interferons. Our preliminary data suggest that TIPARP
targets

multiplecomponents

ofthe

nuclearfactorkappa-light-chain-enhancer of activated B (NFkB) signaling
pathwayandthatNFkB

activation

regulatesTIPARP

expression independently of AHR. This suggests that TIPARP ispart of a novel negative feedback loop regulated NFkB signaling,which could have important implications in inflammatory
diseases. We are working to identify the mono-ADP-ribosylated
peptides in NFkB. Another research focus of this work will be toinvestigate the role of TIPARP in T and B cell differentiation offunction. Once isolated, the cells will be differentiation and thepresence of absence of AHR ligands to determine the impact ofTIPARP and AHR and T and B cell differentiation and function.
These studies are ongoing. Together with a collaborator in theUSA, we have identified a selective TIPARP inhibitor that weare currently testing in our various MEF lines. In preliminary
studies, the new inhibitor phenocopied the effect of TIPARP loss
and genetic catalytic inhibition. We are now poised to begin invivo studies to determine if pharmacological inhibition ofTIPARP could be a beneficial anti-viral therapy, but also cancer
immunotherapy because of its increase in type I interferon
signaling. Finally, we are creating a panel of CRISPR/Cas9
generated TIPARP knockout cell lines to determine the potential
role the TIPARP in cancer. We our focusing on breast, liver, lungand pancreatic cancer cells. It has proven to be more difficult tocreate and isolate TIPARP ko cell lines, but we have created twobreast cancer lines, to date. We have altered our strategy toimprove knockout cell generation.
We have also finally generated a mouse monoclonal antibody
that recognizes TIPARP. This antibody has taken us almost 4
years to produce and as far superior to all of the current

commercially available ones. Our unique MEF cell lines andanimal models provide excellent material to test the specificity
and quality of the antibody. This is a major advancement in
TIPARP research, since the majority of studies have been doneusing overexpressed proteins, transient transfection or stable
cell lines. We still know very little about endogenous TIPARP
signaling, its cellular location, cellular targets and how it isregulated at the protein level. Ongoing studies reveal thatTIPARP inhibition either pharmacologically or geneticallyresults in increased stabilization of TIPARP protein. Thissupports our previous view that TIPARP is rapidly degraded andthat it acts to regulate protein levels via ADP-ribosylation. The
antibody cross reacts with human and mouse TIPARP and works
inwesternblots,immunoprecipitations

andimmunohistochemistry, albeit not perfectly. We are starting aseries of experiments to probe the role of endogenous TIPARP inAHR and type I interferon signaling. However, before we can
expand our studies we need to generate more anti-TIPARP
antibody from our hybridoma cells. The antibody was firstisolated in early December of 2019.
Our mouse models will be used to evaluate the role of TIPARP incancer immunotherapy in combination with immune check point
inhibitors to boost therapeutic responses. Inhibiting immune
checkpoint proteins, such as programmed cell death protein 1(PD1), and its ligand, programmed death-ligand 1 (PDL1), haveshown promise in the treatment for multiple cancers by blocking
immunosuppression. PD1 expressed on T cells counters positive
signals through T cell receptors by engaging its ligand PDL1.
During infections and under normal physiological conditions,the PD1/PDL1 axis is an essence negative regulator of T cells.
Blocking the PD1 pathway during cancer, improves T cellfunction and reduces tumour burden. PD1 pathway inhibitors
promoted durable anti-tumour immune responses in several
trials, which led to the approval of the monoclonal antibodies
raised against PD1 (nivolumab and pembrolizumab), or against
PDL1 (atezolizumab, and avelumab) for therapeutic use invarious cancers, including melanoma, non-small-cell lungcancer, breast and hepatocellular carcinoma. However, many
patients do not respond and 1/3 of those that do, experience
relapse.

Thus,efforts

toincrease

theeffectivenessof

immunotherapy are urgently needed. We believe the TIPARP isa promising target that should be considered. In the upcoming
year, if our joint research application is successful, we will
explore the role of TIPARP inhibition to boost the effectiveness ofcancer immunotherapy.
We feel that this novel, research strategy to characterize the roleof TIPARP in type I interferon response offers great potential forboth anti-viral but also anti-cancer therapy. Our complementary
expertise and access to unique animal and cell line models forstudying TIPARP activity, give us an advantage over other labs
pursuing this line of research. Continued support from the joint
research program will ensure the success of this research.
Publication【Conference, symposium, workshop etc.】*Enter the names of conference

Local department seminars at the University of Oslo.

or journal and its vol. No. where
the above work was presented.
【Journals】
Yamada et al Nature Immunol 2016 Jun;17(6):687-94. doi:
10.1038/ni.3422. Epub 2016 Apr 18.