<b>Abstract ID 16706</b> <b>Poster Board 248</b> Triple-negative breast cancer (TNBC) is one of the most aggressive forms of breast cancer that hormonal therapies cannot treat. Current standards of care include surgical removal of the tumor and aggressive chemotherapy. Consequently, there is an urgent need for new, molecular-based therapies to treat these patients effectively<sup>1,2</sup>. On a molecular level, two proteins facilitate finely tuned calcium entry: Orai1, a pore-forming calcium channel, and stromal interaction molecule 1 (STIM1), a calcium sensor embedded in the ER membrane. Upon ER calcium store depletion, STIM1 undergoes a conformational change to bind to Orai1, which triggers extracellular calcium entry to achieve homeostasis<sup>3</sup>. Recently, it has been discovered that a third protein is implicated in this process: adenylyl cyclase type 8 (AC8)<sup>4</sup>. The N-terminus of AC8 can bind to the N-terminus of Orai1 to prolong calcium influx, which ceases upon Orai1 phosphorylation<sup>4</sup>. In normal breast epithelial cells, AC8 is expressed at a basal level and does not interfere with Orai1-STIM1 calcium entry. However, in TNBC cells, AC8 is grossly overexpressed so that all Orai1 channels become bound<sup>4,5</sup>. As AC8 binds to Orai1 on its phosphorylation site, the Orai1 channel cannot be inactivated, leading to excess calcium influx and adverse cellular effects such as proliferation and migration. Thus, <b>we hypothesize</b> that if we inhibit the interaction between AC8 and Orai1 with a therapeutic small molecule, we will observe a decrease in proliferation in TNBC cells. We also expect that inhibiting the interaction between Orai1 and STIM1 will produce similar effects. Ultimately, we aim to screen a library of small molecules against our protein-protein interactions to find therapeutic inhibitors. We will use the NanoBiT system to study each different protein-protein interaction. Briefly, the NanoBiT system tags each protein with a split luciferase, allowing for the generation of a luminescent signal upon protein binding. Using this method, <b>we have found</b> that the interaction between STIM1 and Orai1 is strictly governed by calcium, whereas the interaction between AC8 and Orai1 is more constitutive. Interestingly, we found that forskolin, an AC8 activator, prevents AC8 binding to Orai1, as evident through loss of luminescence. This suggests that AC8 must be inactive to interact with Orai1 and that phosphorylated Orai1 cannot bind to AC8. These findings have allowed us to conduct Z-Score studies to prime for initial screening, which fall within a 0.6-0.8 range for each protein-protein interaction. To date, we have accomplished screening two libraries of small molecule compounds; NCI Diversity Set VI and NCI Approved Oncology Set X, which will be extended further to other diversity libraries. In addition, we plan to test any inhibitors in different TNBC cell lines (MDA-MB-231 and MDA-MB-157) to observe effects on cancer cell migration. <b>In conclusion</b>, understanding the unique interplay between AC8, Orai1, and STIM1 is critical to understanding how to prevent TNBC cell proliferation and migration. <b>References</b> Stovgaard, et. al. Triple-negative breast cancer… <i>Acta Onc</i>. 2018; 57:1, 74-82, Bose. Triple-negative Breast Carcinoma… <i>Adv. in Ana. Path</i>. 2015; 22: 306-313 Lunz, et. al. STIM1 activation of Orai1. <i>Cell Cal</i>. 2019; 77, 29-38 Sanchez-Collado, et al. Adenylyl Cyclase Type 8 Overexpression… <i>Cancers</i>. 2019; 11, 1624 Derler, et. al. Structure, regulation and biophysics of I(CRAC), STIM/Orai1. <i>Adv. Exp. Med. Biol</i>. 2012; 740, 383–410
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