Reduced levels of ITGB1 cause activation of p38MAPK-ERK-LYN axis of BCR::ABL1 signaling despite inactivation of the oncoprotein by imatinib - novel resistance mechanism in blast crisis of chronic myeloid leukemia unraveled.

内容摘要

Oncogenic tyrosine kinase activity of BCR::ABL1 is causally associated with chronic phase (CP) of chronic myeloid leukemia (CML) and its inhibition by tyrosine kinase inhibitors (TKIs) induces remission in majority of patients. However, in the terminal blast crisis (BC) phase, 80% patients are resistant to TKIs. Forty percent among them are resistant despite inhibition of BCR::ABL1 activity by TKIs and in earlier study, we observed similar antithesis in K562 cells resistant to TKI-imatinib. We demonstrated active BCR::ABL1 signaling downstream of inactivated BCR::ABL1 and its causal association with resistance. This study explores the involvement of ITGB1, integrin with significant role in genesis of CML and differentially expressed in resistant K562 cells, in mediation of atypical activation of BCR::ABL1 pathway and thus resistance. Imatinib-sensitive and resistant K562 cells as well as sensitive cells with ITGB1 knockdown (KD) to mimic resistant cells, were analyzed for proteome and phosphoproteome by mass spectrometry and kinome was profiled by quantification of kinase activities in an array-based assay. The commonality between differentially expressed proteins (DEPs) identified in resistant cells and ITGB1-KD cells together with ITGB1 interactors, identified from ITGB1-immunoprecipitate by mass spectrometry, provided a comprehensive sketch of ITGB1-mediated signaling in resistant cells. The proteins identified as key players in ITGB1-mediated activation of BCR::ABL1 signaling were validated in K562 and KU812 cells, CD34+ cells from patient samples and their functional significance was assessed by inhibition studies in K562 cells. It was observed that the reduced levels of ITGB1 in resistant cells activated BCR::ABL1 downstream signaling kinases- p38MAPK and ERK. Though not influenced by ITGB1, LYN was active in resistant cells. Inhibition studies revealed that p38MAPK, ERK and LYN regulate the activity of each other, thereby explaining ITGB1-independent activation of LYN. The three kinases in turn activate members of CDK, MAPK, AKT and PKC families while ERK alone modulates EPH activity. Thus, reduction in level of ITGB1 in resistant cells leads to activation of ERK and p38MAPK belonging to BCR::ABL1 pathway, despite inactivation of BCR::ABL1, which in turn leads to activation of LYN. Together, these kinases activate members of vital pro-survival pathways, thereby imparting imatinib resistance in CML-BC cells.