Phosphoproteomics reveals a crucial role of FLT3 in BCR-ABL1 independent resistant chronic myeloid leukemia

Chronic Myeloid Leukemia is a myeloproliferative neoplasm characterized by the malignant transformation of hematopoietic stem cell precursors within the myeloid lineage. The Philadelphia chromosome, caused by the t(9;22)(q34;q11) translocation, fuses the BCR and ABL1 genes, encoding for the BCR::ABL1 oncoprotein that drives the pathogenesis of CML. (Sampaio et al., 2021). BCR::ABL1 is a constitutively active tyrosine kinase that triggers downstream signaling pathways, such as MAPK, JAK/STAT, and PI3K/AKT, promoting uncontrolled proliferation, growth factor-independent survival, and resistance to apoptosis (Meenakshi Sundaram et al., 2019). The commercialization of imatinib, a selective tyrosine kinase inhibitor (TKI) targeting BCR::ABL1, revolutionized CML therapy. However, 20-30% of CML cases display resistance to TKI treatment, that leads to disease progression and relapse (Jabbour and Kantarjian, 2016). Resistance mechanisms in CML are categorized as either BCR::ABL1-dependent, when mutations or overexpression of the oncogene occur, or BCR::ABL1-independent, when alternative signaling pathways sustain survival and proliferation (Alves et al., 2021). In this study, we employed state-of-the-art mass spectrometry-based (phospho)proteomics, coupled with SignalingProfiler 2.0 algorithm (Venafra et al., 2024), to comprehensively characterize i) signaling network rewiring following imatinib treatment in CML sensitive cell lines, ii) key signaling pathways in resistant cells able to survive independently of BCR::ABL1 activity. To identify therapeutic vulnerabilities in resistant cells, we developed the Druggability Score algorithm, a computational tool that ranks proteins based on their ability to effectively kill resistant cells when treated with specific inhibitors. Our integrative analysis identified several high-priority druggable targets, highlighting the potential of unbiased, system-level approaches to elucidate resistance mechanisms. These findings hold promise for the development of more effective therapeutic strategies, with the final aim of improving clinical outcomes for non-responder CML patients.