Multiomic Profiling of Tyrosine Kinase Inhibitor-Resistant K562 Cells Suggests Metabolic Reprogramming To Promote Cell Survival.

Resistance to chemotherapy can occur through a wide variety of mechanisms. Resistance to tyrosine kinase inhibitors (TKIs) often arises from kinase mutations-however, "off-target" resistance occurs but is poorly understood. Previously, we established cell line resistance models for three TKIs used in chronic myeloid leukemia treatment, and found that resistance was not attributed entirely to failure of kinase inhibition. Here, we performed global, integrated proteomic and transcriptomic profiling of these cell lines to describe mechanisms of resistance at the protein and gene expression level. We used whole transcriptome sequencing and SWATH-based data-independent acquisition mass spectrometry (DIA-MS), which does not require isotopic labels and provides quantitative measurements of proteins in a comprehensive, unbiased fashion. The proteomic and transcriptional data were correlated to generate an integrated understanding of the gene expression and protein alterations associated with TKI resistance. We defined mechanisms of resistance and two novel markers, CA1 and alpha-synuclein, that were common to all TKIs tested. Resistance to all of the TKIs was associated with oxidative stress responses, hypoxia signatures, and apparent metabolic reprogramming of the cells. Metabolite profiling and glucose-dependence experiments showed that resistant cells had routed their metabolism through glycolysis (particularly through the pentose phosphate pathway) and exhibited disruptions in mitochondrial metabolism. These experiments are the first to report a global, integrated proteomic, transcriptomic, and metabolic analysis of TKI resistance. These data suggest that although the mechanisms are complex, targeting metabolic pathways along with TKI treatment may overcome pan-TKI resistance.

A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance.

Kinase enzymes are an important class of drug targets, particularly in cancer. Cell-based kinase assays are needed to understand how potential kinase inhibitors act on their targets in a physiologically relevant context. Current cell-based kinase assays rely on antibody-based detection of endogenous substrates, inaccurate disease models, or indirect measurements of drug action. Here we expand on previous work from our lab to introduce a 96-well plate compatible approach for measuring cell-based kinase activity in disease-relevant human chronic myeloid leukemia cell lines using an exogenously added, multi-functional peptide substrate. Our cellular models natively express the BcrAbl oncogene and are either sensitive or have acquired resistance to well-characterized BcrAbl tyrosine kinase inhibitors. This approach measures IC50 values comparable to established methods of assessing drug potency, and its robustness indicates that it can be employed in drug discovery applications. This medium-throughput assay could bridge the gap between single target focused, high-throughput in vitro assays and lower-throughput cell-based follow-up experiments.