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

Abstract

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.

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

“…We performed transcriptomic and proteomic analyses of the TKI-sensitive human CML cell line K562 and three TKIresistant derivatives developed in our laboratory: K562-IR (imatinib resistant), K562-NR (nilotinib resistant), and K562-DR (dasatinib resistant). 17 These cell lines were generated by continuous, increasing dosage exposure to kinase inhibitors over >90 days until a resistant population was generated (Figure 1). Quantitative, whole transcriptome RNaseq and data-independent acquisition (DIA) SWATH-MS data sets were generated.…”

Assays for tyrosine phosphorylation in human cells

“…For example, the chronic myeloid leukemia (CML) cell line K562 is characterized by the constitutively active fusion protein BCR-ABL (Grosveld et al, 1986). As we have previously demonstrated, it is possible to measure BCR-ABL activity in K562 cells using a peptide substrate without any additional stimulation (Ouellette et al, 2016;T. Y. Yang et al, 2013).Additionally, in many cellular settings, inhibition of phosphatases will also be critical and can assist in verifying phosphorylation of the substrate.…”

“…As an alternative, substrate peptides can be conjugated with cell penetrating peptides and delivered to cells without the need for expression of a genetically engineered sensor construct. Phosphorylation of the substrate peptide can then be measured using many types of read-outs, including single cell electrophoresis (Soughayer et al, 2004;Turner et al, 2016), mass spectrometry (Placzek, Plebanek, Lipchik, Kidd, & Parker, 2010;Yang, Eissler, Hall, & Parker, 2013), fluorescence lifetime imaging (Damayanti, Parker, & Irudayaraj, 2013), or generic antiphosphotyrosine antibodies (Lipchik, Killins, Geahlen, & Parker, 2012;Ouellette, Noel, & Parker, 2016), depending on the particular substrate design and application. A unique advantage of these kinds of synthetic substrates is the ability to incorporate non-natural amino acids and other chemical moieties that would not be compatible with genetic encoding into sensor proteins, such as D amino acids to improve peptide stability (Proctor, Wang, Lawrence, & Allbritton, 2012a) or constrained phosphotyrosine analogs that are resistant to dephosphorylation (Turner et al, 2016).…”

“…Accordingly, there is no one-size-fits-all solution, and researchers will need to put time and effort into identifying the best substrate for their kinase target and cell-based application. Alternatively, it may be simplest to use a substrate already characterized by others if one is available, such as those we have previously reported for Abl and Syk family kinases (Damayanti et al, 2013;Lipchik et al, 2012;Ouellette et al, 2016;Placzek et al, 2010). Overall, while identifying a suitable substrate is not trivial and requires an empirical process, ultimately it pays off in ease-of-use and can provide clear, interpretable results in cell-based assays.…”

“…Tip: When designing these types of experiments reagents to be tested (such as inhibitors, siRNA, or DMSO controls) can be pre-incubated with the cells in a culture flask in bulk, or set up in a 6-to 12-well tissue culture plate or deep-well 96-well filter plate (Ouellette et al, 2016) at 2X concentration in media, depending on the number and nature of conditions to be tested, volume of media, and number of cells being used in the assay. Imatinib stock concentration is designed to result in 0.05% v/v DMSO in each assay, thus an equivalent volume of DMSO is used as a vehicle control in non-inhibitor wells.…”