Alejandro Chavez, Associate Professor at UC San Diego. "Scaled interrogation of protein function via high-throughput epitope tagging". Previous efforts from the CCMI identified a set of 395 protein systems encompassing 548 genes that are highly mutated in cancer. Here we propose to use HITAG, a high-throughput approach for performing targeted insertions into the genome, to tag all 548 tumor associated proteins with the 3xFLAG epitope at their native genomic locus. By tagging proteins at their endogenous locus, we preserve physiologic expression levels while also maintaining any cell type specific variation in splicing which may influence protein activity. Furthermore, as protein behavior can be cell context dependent, we will examine the set of 548 proteins in multiple tumor and control cell lines, to better capture these differences and enable a more holistic understanding of each protein. At the completion of this project, we will have generated a series of renewable cell reagents which in collaboration with the members of the CCMI such as our primary collaborator for this proposal the Ideker lab, will be used to develop more comprehensive knowledge maps.
Alexander Marson, Assistant Professor at UCSF. "The successes of cancer immunotherapy provide strong motivation to discover new therapeutic strategies to enhance the function of T cells as the majority of tumors remain incurable. Thepromise of next-generation immunotherapies depends on the discovery of specific molecular instructions to precisely control T cell functions. In collaboration with the Krogan lab, we envision a pipeline that pairs deep mutagenesis technology with targeted proteomics in primary human T cells to understand the network consequences of mutated variants in the Mediator complex that tune T cell functions. Nucleotide-resolution maps of the Mediator complex that regulate critical T cell functions will nominate variants and inform immunotherapy design. In partnership with the CCMI, we can accelerate our biology-to-clinic investigations by coupling genetics and proteomics to provide detailed molecular instructions to boost T cell functions and program next-generation cancer immunotherapies.
Karin Pelka, Assistant Professor at UCSF, in collaboration with Emma Lundberg. "Comparative multimodal spatial profiling of human patient-derived and mouse organoid-based colorectal cancer liver metastases as foundation for systematically perturbing immune hubs". Current T cell-targeting immunotherapies, such as the checkpoint inhibitor anti-PD-1 and state-of-the-art T cell therapies have limited efficacy in most solid tumors. As T cells cannot execute their functions in isolation, but instead engage in positive and negative feedback loops with both immune and non-immune cells, we urgently need to improve our understanding of the immunologically relevant communication networks in solid tumors in order to design more effective immunotherapies. By performing single cell RNA sequencing and spatial profiling of a large cohort of primary treatment-naïve colorectal cancer (CRC) specimens, we recently discovered several conserved and spatially organized interaction networks between immune and malignant cells and the inter-cellular signaling pathways that likely drive and shape these ‘immune hubs’ in human tumors. Of note, one particular type of hub, the anti-tumor immunity hub, appeared as focal structures of activated T cells together with myeloid and malignant cells that express T cell-attracting chemokines at the interface between malignant cells and the tumor stroma and was associated with immunotherapy response in CRC and non-small-cell lung cancer (NSCLC). Another hub at the luminal surface of primary tumors was characterized by inflammatory fibroblasts and myeloid cells together with neutrophil infiltrates and regulatory T cells, cell types previously implicated in the negative regulation of anti-tumor responses. Thus, inducing or remodeling these spatially coordinated immune cell collectives could be a powerful new therapeutic approach. As our previous work largely focused on treatment-naïve primary CRCs, our understanding of immune hubs in the clinically relevant metastatic setting is limited. Furthermore, commonly used in vivo models are not suitable to systematically dissect the gene programs and functional cell collectives that we have discovered: Transplantable cell lines lack the malignant epithelial gene programs and complex histologic structure seen in human CRC, while autochthonous models are not readily amenable for multiplexed genetic manipulations. CRC organoid-based in vivo models could close this gap. To make these models widely usable to study immune-malignant cell interactions and test the next generation of immunotherapies, we here propose to deeply characterize and compare immune hubs in liver metastases from CRC patients with CRC organoid-based liver metastasis from our in vivo model system by multimodal spatial profiling. To this end, we will combine our expertise in transcriptionally profiling immune hubs using multiplexed error-robust fluorescence in situ hybridization (MERFISH) with the proteomic profiling expertise of CCMI investigator Dr. Emma Lundberg. The proposed approach is expected to define the spectrum of compositionally and functionally distinct immune hub states in human metastatic lesions and identify their similarities and differences to immune hubs in a histologically complex in vivo model amenable to systematic perturbation screens. This will be critical for the identification and test of new drug targets that induce or remodel immune hubs.