Decoding the Cullin-5 complex to engineer therapeutic T cells with durable effector function - Background: T cell therapies represent one of the most promising recent developments in oncology, with impressive responses in certain leukemias and lymphomas resulting in the FDA approval of six CAR-T cell therapies. However, it has become increasingly clear that there are numerous challenges to the successful application of these therapies to a broader spectrum of malignancies. One significant obstacle is the development of T cell dysfunction that can result from chronic tumor antigen stimulation. Preliminary Data: Our genome-wide CRISPR screens in primary human T cells subjected to the pressure of repeated tumor stimulation identified nearly every core subunit of the Cullin-5 E3 ubiquitin ligase complex as restraining T cell functional persistence. The Cul5 complex is upregulated and activated by TCR stimulation, suggesting that chronic stimulation may trigger excessive activity of this negative regulator. We showed that disabling the Cul5 complex enables therapeutic T cells to resist dysfunction from chronic antigen stimulation and maintain potent long-term effector function. Rationale: The Cul5 complex is a highly modular E3 ubiquitin ligase complex that can host approximately 40 different known substrate receptors, which act as adaptors that each bind a variety of different substrate targets for ubiquitination and proteasomal degradation. We hypothesize that by disrupting the Cul5 complex, loss of multiple key Cul5 substrate receptors results in preservation of key substrates that can collaborate to avert T cell dysfunction. In addition, a number of the Cul5 complex substrate receptors play known roles in negatively regulating cytokine signaling responses. Our preliminary data suggest that by knocking out Cul5, we are lowering the threshold for cytokine responsiveness (signal 3), which may avert T cell dysfunction induced by repeated antigen stimulation (signal 1). Research Strategy: In this proposal, we will build on our preliminary data in three specific aims. In Aim 1, we will identify the key Cul5 complex subunits driving human T cell dysfunction after chronic stimulation through comprehensive genetic and proteomic interrogation. In Aim 2, we will evaluate the therapeutic potential of manipulating this complex in human T cells through a variety of relevant preclinical models. In Aim 3, we will test how the loss of Cul5 complex subunits affects response to key cytokines and determine whether Cul5 complex editing can synergize with antigen- inducible synthetic cytokine circuits. Expected Results: These studies will rigorously test the hypothesis that the Cul5 complex is a central driver of T cell dysfunction due to chronic antigen stimulation and will map the functional network of key Cul5 complex interactors mediating these effects. We will also determine how to combine genetic manipulation of the Cul5 complex with antigen-inducible cytokine circuits to override signal 1 induced dysfunction by boosting signal 3. Altogether, our results will teach us how to reprogram the Cul5 complex for optimal functional persistence in future generations of CRISPR-engineered T cell therapies.
