The proteasome inhibitors bortezomib (Btz), carfilzomib (Cfz) and ixazomib (Ixz) are successfully used for the treatment of multiple myeloma (MM), but lack clinical efficacy against solid tumors despite demonstrating significant activity in tissue culture and animal models. In an effort to understand this discrepancy, we decided to focus on triple-negative breast cancers (TNBC), a genetically diverse group that lacks targeted treatments. TNBCs overexpress pro-apoptotic protein NOXA, which plays a key role in proteasome inhibitor-induced apoptosis, and are proteasome-addicted. The proteasome has three pairs of active sites: ß5, ß1, and ß2. The primary target of Btz and Cfz are the ß5 sites. At concentrations of Btz and Cfz that induce apoptosis in TNBC in culture, co-inhibition of either ß1 or ß2 is also observed. These concentrations exceed clinically relevant concentrations, potentially explaining why proteasome inhibitors have no in vivo activity in solid tumors, and suggesting that stronger co-inhibition of either ß1 or ß2 would improve in vivo activity. It is not known whether the ß2 or the ß1 site is a better target. We developed specific inhibitors of the ß1 and ß2 sites and CRISPR- generated mutations of these sites. We found that LU-102 and mutations in the ß2 sites are much stronger sensitizers of TNBC cells to Cfz and Btz than the ß1 inhibitor NC-021 or ß1 mutations; ß2 mutations sensitize orthotopic xenografts of TNBC cells to sub-toxic doses of Cfz. LU-102 and ß2 mutations block recovery of proteasome activity in Cfz-treated cells. In contrast to myeloma cells, proteasome activity recovers in TNBC cells treated with clinically relevant concentrations of Cfz. We found that LU-102 and ß2 mutations inhibit recovery of proteasome activity by causing aggregation of Nrf1 (NFE2L1/TCF11), a transcription factor that up- regulates proteasome genes expression in cells treated with ß5 inhibitors. NC-021 and ß1 mutations do not block recovery of proteasome activity. Therefore, we hypothesize that ß2 inhibitors will sensitize TNBCs to FDA-approved ß5 inhibitors, by the mechanism which involves tumor-specific inhibition of Nrf1-mediated recovery of proteasome activity. We further hypothesize that targeting enzymes involved in Nrf1 activation will also sensitize TNBC to proteasome inhibitors. The specific aims of this proposal are: (i) to determine whether ß2 inhibitors sensitize TNBC to Cfz, Btz, and Ixz in vivo; (ii) to determine whether ß2 inhibitors block recovery of proteasome activity in a tumor-specific fashion in vivo and determine whether alternative approaches to target this recovery sensitize tumors to FDA-approved inhibitors. Accomplishing these aims will demonstrate that the ß2 sites and Nrf1 are effective drug targets in TNBC, establish the mechanism of action of ß2 inhibitors, pave the road to clinical trials of ß2 inhibitors in TNBC, and launch future work in solid tumors of different organs." |