Revealing the hidden phosphoproteome of drug resistant triple negative breast cancer

Triple negative breast cancer (TNBC) represents the most aggressive form of breast cancer, associated with poor prognosis and an overall survival lower than 5 years. Currently, targeted therapies for TNBC are lacking, necessitating the reliance on chemotherapy as the primary treatment strategy. Alarmingly, over 50% of patients display resistance to standard treatments, leading to the development of drug-resistant and metastatic tumors. Phosphorylation- dependent signaling is emerging as a key player in TNBC drug resistance, but current phosphoproteomic techniques fail in comprehensive analysis of this phenomenon. To address this need, we are developing an innovative phosphoproteomic approach that combines protein engineering and advanced mass spectrometry techniques for unbiased and in-depth phosphoproteomic analysis. SH2 and FHA domains naturally interact, with weak affinity, with cellular phosphotyrosine (pTyr) and phosphothreonine (pThr)-containing proteins, respectively. We recently engineered SH2 domains for ultra-sensitive and selective binding of pTyr-peptides and demonstrated unprecedented pTyr-proteome coverage. We hypothesize that the development of novel, highly sensitive pThr probes will unlock underexplored regions of the phosphoproteome, enabling a deeper understanding of drug resistance in TNBC. We propose two specific aims to (1) develop FHA domain superbinders for ultra-deep analysis of pThr-mediated signaling pathways, and (2) combine FHA domain superbinders with our engineered SH2 domains and conventional immobilized metal-affinity chromatography (TMAC, primarily recognizing pSer) for in-depth analysis of pThr, pTyr and pSer landscapes in drug- resistant TNBC. The novel FHA domain super-binders developed in this work will provide a useful tool for the scientific community and enable to advance our understanding of pThr-mediated signaling. Combination of FHA superbinders with engineered SH2 domain and TMAC chromatography will enable unprecedented analysis of the human phosphoproteome and will allow us to investigate in detail the mechanisms underlying drug resistance in TNBC, potentially identifying novel biomarkers and therapeutic targets.