Mining the microbiome for immunomodulatory microproteins

Stanford University, Stanford CA

Investigators

Abstract

PROJECT ABSTRACT Cancer immunotherapy, when effective, is lifesaving. Unfortunately, 15-50% of individuals will not respond to therapy; therefore, efforts to ‘flip the switch’ and turn these ‘non-responders’ into ‘responders’ are critical and urgent. Encouragingly, phenomenological studies in humans and preclinical studies in mice have demonstrated that the gut microbiome is associated with cancer immunotherapy response. However, the exact mechanisms that drive microbiome-based changes in immunotherapy response are not known. This project will elucidate how microbial microproteins modulate macrophage function, a critical coordinator of tumor immune response. The central hypothesis of this proposal is that specific gut microbes express microproteins that directly modulate macrophages, thus leading to immunomodulation of cancer. Previous work seeking to deconvolute the signaling interface between microbes and immune cells has done so by: (i) carrying out limited- throughput, arrayed screens, (ii) focusing on microbial small molecule metabolites and (iii) focusing on T-cells. Here, we propose to address many gaps left by these approaches. Namely, we will (i) carry out two orthogonal high-throughput screens (peptide-display polarization assay, Perturb-seq) of 1,000s-10,000s of microbial macromolecules in pooled screens (technical innovation), (ii) focus on proteins, especially an understudied class of microproteins we recently discovered and have now annotated in microbial genomes (conceptual innovation), and (iii) moving one step up in the immunological ‘cascade’ by focusing on macrophages (conceptual innovation). Our strong preliminary data have identified two novel macrophage-modulating microbial proteins that we will mechanistically study in Aim 1, and we have also developed and validated a powerful peptide display system that will be expanded in Aim 2. Building upon these preliminary data, we will: (Aim 1) determine the detailed molecular mechanisms, including target receptors and downstream signaling pathways, that two microproteins (from Klebsiella pneumonia and a Leptotrichia spp.) use to modulate macrophage polarization; (Aim 2) screen a large, data-derived library of candidate microbial microproteins to identify those with immunomodulatory properties; and, (Aim 3) apply scRNAseq to define the dynamic changes in macrophage state induced by these immunomodulating microproteins. Our core team has collaborated for >5 years, and has expertise in microproteins, the human microbiome and translational research (Bhatt – a practicing oncologist) and cancer- focused high-throughput functional genomics and macrophage biology (Bassik). Our preliminary data demonstrates the strength of our environment for this research, and we are supported by experts in multi-omics (Snyder), chemical biology (Bertozzi), immunotherapy/microbiome (Wargo), scRNAseq and tumor immunology (Jerby), and functional assays of microproteins (de la Fuente). Taken together, we are optimally positioned to identify macrophage-modulating microbial factors, which will then be used to develop new, mechanism-based strategies to enhance cancer immunotherapy efficacy.