Methods to detect endogenous dsRNA induction and recognition.

Nucleic acid sensing is a central mechanism for innate immune defense against foreign molecules that culminates with an activation of interferon signaling pathways. This involves detection of molecular patterns associated with extracellular or intracellular pathogens by specialized receptors within the cell. In addition to foreign molecules, cells also sense endogenous molecules. One specific arm of nucleic acid sensors detects dsRNA structures. In this chapter, we discuss principles of dsRNA recognition and downstream activation of signaling pathways important in the process of antiviral responses. We also discuss various mechanisms by which endogenous dsRNA can form in a cell, in particular, through epigenetic regulation. Finally, we provide approaches for measuring and quantifying dsRNA accumulation and downstream activation in human colorectal cancer cells.

The Mitochondrial Transacylase, Tafazzin, Regulates for AML Stemness by Modulating Intracellular Levels of Phospholipids.

Tafazzin (TAZ) is a mitochondrial transacylase that remodels the mitochondrial cardiolipin into its mature form. Through a CRISPR screen, we identified TAZ as necessary for the growth and viability of acute myeloid leukemia (AML) cells. Genetic inhibition of TAZ reduced stemness and increased differentiation of AML cells both in vitro and in vivo. In contrast, knockdown of TAZ did not impair normal hematopoiesis under basal conditions. Mechanistically, inhibition of TAZ decreased levels of cardiolipin but also altered global levels of intracellular phospholipids, including phosphatidylserine, which controlled AML stemness and differentiation by modulating toll-like receptor (TLR) signaling.

Pharmacological DNA demethylation: Implications for cancer immunotherapy.

Recent studies have demonstrated that DNA demethylation agents can mimic a viral infection by induction of dsRNAs. This viral mimicry leads to an antiviral response mediated by the cytosolic pattern recognition receptor MDA5, followed by MAVS (IPS1) activation, IRF7 nuclear translocation and upregulation of type III Interferon and interferon-stimulated genes.

B7-H4 expression by nonhematopoietic cells in the tumor microenvironment promotes antitumor immunity.

The B7 family plays a critical role in both positive and negative regulation of immune responses by engaging a variety of receptors on lymphocytes. Importantly, blocking coinhibitory molecules using antibodies specific for CTLA-4 and PD-1 enhances tumor immunity in a subset of patients. Therefore, it is critical to understand the role of different B7 family members since they may be suitable therapeutic targets. B7-H4 is another member that inhibits T-cell function, and it is also upregulated on a variety of tumors and has been proposed to promote tumor growth. Here, we investigate the role of B7-H4 in tumor development and show that B7-H4 expression inhibits tumor growth in two mouse models. Furthermore, we show that B7-H4 expression is required for antitumor immune responses in a mouse model of mammary tumorigenesis. We found that the expression levels of B7-H4 correlate with MHC class I expression in both mouse and human samples. We show that IFNγ upregulates B7-H4 expression on mouse embryo fibroblasts and that the upregulation of B7-H4 on tumors is dependent on T cells. Notably, patients with breast cancer with increased B7-H4 expression show a prolonged time to recurrence. These studies demonstrate a positive role for B7-H4 in promoting antitumor immunity.