Project Summary The genome of the bacterium that causes tuberculosis, Mycobacterium tuberculosis (Mtb), harbors an astonishingly large number of specialized growth regulating toxins. We and others have discovered that many of these Mtb toxins are highly selective tRNases with properties that make them attractive candidates for cancer treatment on their own or as protein-conjugated immunoRNases. There is one RNase to go into clinical trials for treatment of cancer, Onconase. However, the Mtb tRNase toxins have much higher intrinsic specificity than the poorly characterized enzymatic activity of Onconase. The unprecedented target selectivity of these Mtb tRNase toxins is important because earlier tRNA microarray expression studies revealed significant increases in a few functional, full length tRNAs in patient-derived breast tumor samples. These tRNA species are thought to be upregulated in order to accommodate higher demand when cells are reprogrammed to produce one or more cancer-promoting proteins rich in this particular amino acid. Therefore, Mtb tRNase toxins constitute a large new untapped reservoir of highly specialized tRNases, each endowed with the ability to recognize a single tRNA as substrate. In this exploratory R21 proposal in response to FOA PAR-19-194 Microbial-based Cancer Therapy - Bugs as Drugs, we hypothesize that these specialized toxins can ultimately be exploited for cancer treatment because they are predicted to cleave and disable tRNAs required for efficient translation of pro-oncogenic proteins. The three specific Aims define the specificity of six of these tRNase toxins in human breast cancer cell lines and tissues.