The adaptor protein Crk in immune responses

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Project Summary: This proposal is a renewal application of a previous R01 (AI130197). During the last grant period, the PI has published more than 30 peer-reviewed papers and applied for 10 patents. One of the most important discoveries directly resulting from the last funding period is that the PI has identified three promising Crk inhibitors and has filed a patent around these inhibitors (Patent #: 63/305,354). In the previous funding cycle, the PI seeks to identify the mechanism(s) by which the small adaptor protein CT10 regulator of the kinase-like (CrkL) and its phosphorylation (pCrkL) control NK cell activation and inhibition by using both human NK cells and novel NK cell-specific conditional knockout mice. The long-term goal is to use this knowledge and the novel imaging techniques to uncover the molecular basis of NK cell activation and inhibition and to develop new treatments for human primary immunodeficiency diseases and chronic diseases such as cancer and viral infection. After discovering the novel pCrkL inhibitors, we hypothesize that the newly discovered pCrkL inhibitor determines Crk’s ability to interact with critical downstream signaling molecules and ultimately shapes the actin cytoskeleton into a functional immunological synapse (IS), which can be potentially used to treat infection and cancer. Guided by strong preliminary data, in this renewal application, we will test these hypotheses via three Specific Aims: 1) Define the precise molecular mechanisms by how phosphorylation of Crk-like (pCrkL) protein inhibitors affects NK cell functions. The proposed work will bring cutting-edge imaging technology to the field of NK cell research. Experiments will determine where and when Crk is phosphorylated at the IS, as well as how it interacts with key receptors, signaling molecules, and the actin cytoskeleton with and without pCrkL inhibitors; 2) Determine the role of CrkL in T cells from patients with partial DiGeorge syndrome (pDGS). By studying one of the most common (1 in 3,000 births) immunodeficiency diseases, pDGS (mainly caused by CrkL haploinsufficiency), leveraging our previous discoveries on pDGS that demonstrate defective integrin activation in NK cells in pDGS, we will determine how the loss of CrkL affects CD8+ T cell integrin signaling and function in pDGS, which can generate novel immunotherapy (e.g., modulating integrin signaling) to treat pDGS patients in the future; 3) Discover the role of pCrkL inhibitors in cancer therapy. We identified several novel pCrkL inhibitors, which can interact with and inhibit the pCrkL protein. It inhibits the proliferation of several solid tumor cell lines in vitro, and tumor cell line-derived (CDX) xenograft and orthotopic patient-derived xenograft (PDX) mouse models; we will determine this inhibitor and its analogs’ roles in immune cell (focusing on NK and T)-mediated immune responses against solid cancers in vivo. The proposed work involves key signaling players and regulatory mechanisms and generates a novel model system to determine the role of CrkL as a master regulatory molecule. It is broadly relevant with direct clinical implications for treating primary immunodeficiency diseases and cancers.