Project Summary Triple-Negative breast cancer (TNBC), which accounts for 15-20% of all breast cancer, represents an aggressive clinical history, development of distant metastasis, shorter survival and high mortaility rate compared with other subtypes of breast cancer. It is imperative to identity new therapeutic targets that are actionale in TNBC. Our lab has been focusing on studying a family of enzymes that uses oxygen, Fe2+ and 2-oxoglutarate (2-OG) for their enzymatic reactions. This enzyme family has been reported to be involved in the pathogenesis of cancers. We generated the custom siRNA library for all of 2-OG dependent enzymes and developed a stringent screening strategy by combining the functional readouts from both 2-D cell proliferation and 3-D soft agar growth assay with TNBC breast cancer cell lines. Our preliminary data show that gamma-butyrobetaine hydroxylase 1 (BBOX1) involved in carnitine biosynthesis pathway is essential for TNBC cell proliferation on 2-D and 3-D. Mechanistically, we show that BBOX1 binds with the calcium channel inositol-1,4,5-trisphosphate receptor type 3 (IP3R3), therefore promoting calcium release, mitochondrial function and glycolysis in TNBC. We hypothesize that BBOX1-IP3R3 signaling axis promotes TNBC by inducing calcium release and tumor metabolism. This is the first study directed at a pro-oncogenic function for BBOX1 in cancer, with our focus in TNBC. In Specific Aim 1, we will characterize the functional significance of BBOX1-IP3R3 signaling in TNBC. In Specific Aim 2, we will elucidate the molecular mechanism by which BBOX1-IP3R3 signaling promotes oncogenic phenotypes in TNBC. In Specific Aim 3, we will assess the therapeutic implications of targeting BBOX1 in TNBC xenografts and patient derived xenografts (PDXs). Successful completion of this proposal would establish the role of BBOX1 as a new oncogenic driver in TNBC and explore its therapeutic potential in this lethal disease.