Deciphering antigen receptor-mediated NF-kB – DARN

Engagement of T-cell receptor assembles a large signalosome, which culminates with non degradative ubiquitination of key adaptors. To identify new regulators of this multi-proteins complex, our strategy relies on a proteomic analysis by mass spectrometry. In addition to this proteomic approach, we develop a genetic screen of a siRNA library against the 96 deubiquitinases (DUBs) encoded by the human genome.

Combining proteomic and genetic screens emerges as a usefull tool to identify new regulators of NF-?B activation in lymphocytes, and putative « non-oncogene addiction » genes in lymphoma. Our research also unveiled an unexpected facet of the endoplasmic reticulum, as a milestone in NF-?B signaling: it anchors secondary signalosomes enriched in NUTs to unleash NF-kB.

Our research will help better understanding how NF-?B activation is finely tuned during the course of an immune response, and deregulated in some lymphoma. We predict that our results might inspire the development of new therapeutical strategies targeting some lymphoma addicted to NF-?B.

So far, this project led to 9 scientific publications (7 published, 1 submitted for publication, and 1 book chapter).

The transcription factor NF-kB plays a vital role in cellular development, homeostasis and survival in the immune system as well as in the propagation of certain lymphoma cells. Antigen receptor stimulation assembles a multi-protein complex, which contains the scaffold CARMA1, together with the heterodimer BCL10/MALT1 (CBM complex) linked to the inhibitor of NF-kB kinase complex (IKK). Recently, we identified casein kinase 1a (CK1a) as an essential bimodal regulator of lymphocyte activation. In addition to its positive adaptor function, CK1a phosphorylates CARMA1, in a negative feedback loop to restrain the activating signal to a single pass. Furthermore, silencing of CK1a in the Activated B-Cell like subtype of Diffuse Large B-Cell Lymphoma (ABC DLBCL), whose survival depends on the CBM-NF-kB axis, is toxic. CK1a can be considered as “conditionally essential malignancy (CeMal) ” gene, a new class of genes that are important not for the initiation but for the propagation of the transformed phenotype.

Because NF-kB has pleiotropic effect on a myriad of tissues, strategies aiming at targeting IKK or NF-kB heterodimers might have broad and harmful consequences on an organism. Understanding how T-Cell Receptor (TCR) promotes and subsequently shuts down NF-kB signaling is therefore an intense field of research. Our approach focuses on identifying new molecular components that regulate the IKK-NF-kB nexus. Our current project wants to:

1. Identify new regulators of the CK1a-CBM-IKK nexus through a Mass Spectrometry-based screen. Such strategy was successfully used to identify CK1a, and preliminary experiments provided promising new candidates. For example, we identified the mammalian diaphanous 1 protein (mDIA1) and the tumor suppressor PALS1 as CK1a and CARMA1 partners, respectively. Their siRNA-based knockdown dampened NF-kB activity, and our future work will aim to decipher PALS1 and mDIA1 precise function within the CBM and on IKK activation. In addition, we propose to expand our Mass Spectrometry screen.

2. Analyze the impact of CK1a on development, lymphocytes ontogenesis, and on mounting an immune response. To firmly establish a role for CK1a in vivo, we propose to build and study a CK1a knockout mouse model. Two types of CK1a+/- mice were already generated in collaboration with MJ Lenardo’s group (NIH/NIAID), and animals were shipped in France. This project will allow us to establish animal models in our laboratory, and develop in vivo work in the future.

3. Understand how TCR-induced NF-kB is negatively regulated. Because non-degradative K63-linked ubiquitination play a major role in conveying NF-kB, we propose to set up a siRNA library screen against the Deubiquitinylation proteases (DUBs) family. Our screen will target over 90 DUBs and should identify those functionally involved in TCR-, and TLR-mediated NF-kB activation. Because MALT1 plays an essential role in lymphocyte activation and lymphomagenesis, and is regulated by ubiquitination, we propose to identify the DUBs involved in MALT1 post-translational modifications. This might open new avenues for modulating NF-kB activity.

In summary, our project will help to better understand how IKK is finely tuned during the course of an immune response. Our proposal combine high technology experiments with classical Biochemistry, Cell Biology and Immunology. Of note, we predict that in vivo animal models will unveil innovating and exciting perspectives. We expect our program to inspire the development of specific inhibitors or activators of the CBM-IKK nexus. Such drugs might be helpful to modulate a deregulated NF-kB pathway without broadly affecting the transcription factor.