Iron-sulfur clusters (ISCs) are essential protein cofactors whose dysregulation is linked to a wide range of debilitating diseases including Friedrch’s ataxia. This pathology is due in part to the use of ISCs in iron-responsive proteins (IRPs), which control the translation of mRNAs in the iron-starvation response. We recently found that by suppressing ISC biosynthesis we can robustly activate this IRP-mediated iron-starvation response, sensitizing cancer cells to cell death by ferroptosis. Our preliminary data, which form the premise of our application, point to an unexpected mode of IRP2 regulation by ISCs and demonstrate that the mechanism by which ISCs modulate the iron-starvation response, and therefore impact human disease, remains unclear. Our long-term goal is to dissect the mechanisms by which ISCs regulates cellular responses to changing iron and oxygen availability and how this response is mediated by IRPs. We anticipate that these discoveries will lead to the identification of pathologies for diseases where prior ISC involvement was unclear, to new treatments for diseases of ISC dysregulation, and will facilitate novel methods to sensitize cancer cells to ferroptosis. The objective of this grant is to dissect how ISCs control IRP2 activation and to comprehensively define the targets of IRP1 and IRP2 and the conditions under which they are activated. Our overarching hypothesis is that ISCs integrate iron and oxygen level inputs to effect specific translational responses via differential regulation of IRPs. Our rationale is that identification of the specific mechanisms by which IRPs are activated and the targets that they activate will enable the discovery of novel strategies to treat cancer and disorders of ISC metabolism. Our specific aims will test the following hypotheses: (Aim 1) ISCs can activate the iron-starvation response through an IRP2-mediated mechanism; (Aim 2) IRPs exhibit differential IRE binding in response to iron and oxygen level modulation. Upon completion of these aims we will (1) gain an understanding of how cells sense iron and oxygen levels and integrate these inputs using ISC sensors and (2) identify IRP regulated target genes and the conditions in which they are regulated. This contribution is significant because dysregulation of IRPs occurs frequently in human pathologies and inducing activation of the iron starvation response sensitizes cancer cells to ferroptotic cell death. This research is innovative because we challenge paradigms in cellular iron-sensing to arrive at a comprehensive mechanism by which cells respond to changes in the level of this important nutrient, and because we utilize heretofore unique approaches to identify RNAs regulated by the iron starvation response and define their activation by upstream stimuli. The outcomes of this study promise disrupt our understanding of iron sensing and have broad implications for the treatment of cancer as well as human diseases related to defects in iron metabolism and storage.