Investigation of cellular resistance mechanisms in Chronic Myeloid Leukemia using proteomics strategiesPhD thesis -Ribeirão Preto Medical School, University of São Paulo. 2023.Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL1 oncoprotein, with constitutive activation of its tyrosine kinase activity, and the neoplastic transformation of hematopoietic stem cells. The development of BCR-ABL1-specific tyrosine kinase inhibitors (TKIs), such as imatinib mesylate, represented an important advancement in the treatment of CML. However, the current therapeutic challenges are: i-offering patients a long-term cure, without ongoing maintenance of treatment, and, iireducing the impact of resistance to treatment observed in up to 20% of patients. The T315I mutation in BCR-ABL1 produces resistance to all available TKIs. From these demands, the logical rationale for therapeutic complementation based on molecular targets supporting the transforming action of BCR-ABL1 emerges. The identification of the association of the IRS1 adapter protein with BCR-ABL1 and the demonstration of the antineoplastic effects of the pharmacological inhibitor NT157 of IGF1R-IRS1/2 in BCR-ABL1 cell models, regardless of the mutational status for T315I, opened new perspectives for reducing the impact of resistance in patients. Thus, we sought to identify new molecular mechanisms of NT157 and imatinib in BCR-ABL1 cells, mutated or not for T315I, through proteomic analysis. For this, we used in vitro models of murine cell lines Ba/F3 BCR-ABL1 (WT e T315I) that were submitted to treatment with NT157 and/or imatinib and also primary cells from patients with mutation T315I. In order to elucidate the early molecular processes triggered by NT157, we used conditions that do not interfere with cell viability, but that alter survival mechanisms. Detailed proteomic analysis in Ba/F3 cells demonstrated proteins that are NT157 and imatinib-dependent, highlighting regulated processes such as the ubiquitin proteasome system, cellular metabolism and regulation of gene transcription. In primary cells, our results indicated that treatment with NT157 (6.4 µM) decreased viability and increased apoptosis in PBMCs carrying the T315I mutation. Also, through a quantitative proteomic analysis, proteins in the patient's cells stand out, such as the CD70 cytokine, which had its levels increased with NT157 treatment. Changes were also identified in other proteins known to be involved in CML, such as CD44, CD14 and CD33. Our findings suggest that inhibition of IGF1R/IRS signaling has important specificity and opens up the prospect of possible combination therapies with TKI for resistant patients.