Metastatic disease accounts for 90% of deaths amongst patients with solid tumors, revealing the dire need for new therapies that block metastatic progression. Although anti-metastatic therapies which specifically kill or limit the dissemination of metastatic tumor cells have been described, these approaches exhibit limited efficacy in the broader population of cancer patients. Modulating the immune system to promote a natural anti-metastatic response represents an alternative anti-metastatic therapeutic paradigm, akin to what has been described for immunotherapies against primary tumors. To discover putative anti-metastatic immunomodulatory compounds, I used MULTI-seq to perform the largest-ever single-cell RNA-seq-coupled immunomodulatory drug screen in human peripheral blood mononuclear cells (PBMCs). Across the 516 drugs assayed in this screen, I made two key discoveries. First, I observed that the histone deacetylase (HDAC) inhibitor entinostat (ENT) enhances expression of genes associated with sustained anti-tumorigenic CD8+ T-cell effector functions. Second, I observed that ENT enhances the expression of genes associated with metastatic progression in macrophages. These are exciting results because ENT has immunomodulatory and anti-metastatic activity in vivo, where it delays, but does not completely halt, metastatic outgrowth in the Lewis Lung Carcinoma (LLC) mouse model. This finding suggests that (i) ENT blocks only a subset of pro-metastatic immune functions, and (ii) more effective immunomodulation could provide lasting anti-metastatic responses, in vivo. Based on these observations, I hypothesize that ENT treatment functions as a `double-edged sword' with respect to metastatic progression by promoting anti-metastatic responses in CD8+ T-cells and pro- metastatic responses in macrophages. In this research proposal, I outline how I will experimentally test this hypothesis using LLC mouse models which enable selective manipulation of CD8+ T-cells and macrophages (Aim 1). Moreover, I propose a series of MULTI-seq experiments which explore (i) whether HDAC inhibitor potencies and molecular targets correlate with anti-metastatic immune cell gene expression signatures, and (ii) whether ENT combination with macrophage-depleting compounds function additively to induce CD8+ T-cell effector function while depleting pro-metastatic macrophages. This research proposal will be carried out under the mentorship of Dr. Zev Gartner and Dr. Zena Werb at UCSF. Dr. Gartner, Dr. Werb, and I have formulated a training plan which includes coursework, experimental training, and professional development which leverage the collaborative and interdisciplinary nature of the UCSF academic environment, as well as Dr. Gartner's expertise in single-cell genomics and Dr. Werb's expertise in cancer immunology.
The immune system is co-opted by primary tumors to facilitate metastatic dissemination to distant organ sites, and treatment with epigenetic modifiers (e.g., entinostat) have shown preliminary potential for reprogramming the immune system to achieve anti-metastatic effects. However, single-cell RNA-sequencing analysis of entinostat-treated immune cells suggests that epigenetic therapy may represent a `double-edged sword' with respect to metastatic progression, simultaneously driving pro- and anti-metastatic gene expression signatures in diverse immune cell types. In this proposal, I describe a series of experiments which test whether entinostat-stimulated immune cells contribute conflictingly to metastatic progression, and then propose targeted single-cell RNA-sequencing experiments which have the potential to reveal novel anti-metastatic therapy pathways.
