Phosphoflow-Based Evaluation of Mek Inhibitors as Small-Molecule Therapeutics for B-Cell Precursor Acute Lymphoblastic Leukemia

Abstract

Upstream mutations that lead to constitutive activation of Erk in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are relatively common. In the era of personalized medicine, flow cytometry could be used as a rapid method for selection of optimal therapies, which may include drugs that target the Erk pathway. Here, we evaluated the utility of phospho-flow, compared to Western blotting, to monitor Erk pathway activation and its inhibition by targeted Mek kinase inhibitors in human BCP ALL. Because the Erk pathway is not only activated endogenously, by mutations, but also by normal extracellular stimulation through stromal contact and serum growth factors, we compared Erk activation ex vivo in ALL cells in the presence and absence of stroma and serum. Phospho-flow was able to readily detect changes in the pool of pErk1/2 that had been generated by normal microenvironmental stimuli in patient-derived BCP-ALL cells passaged in NSG mice, in viably frozen primary patient samples, and in fresh patient samples. Treatment with the Mek1/2 inhibitor selumetinib resulted in a rapid, complete and persistent reduction of microenvironment-generated pErk1/2. Imaging flow cytometry confirmed reduction of nuclear pErk1/2 upon selumetinib treatment. An ALL relapsing with an activating KRasG12V mutation contained higher endogenous as well as serum/stromal-stimulated levels of pErk1/2 than the matched diagnosis sample which lacked the mutation, but selumetinib treatment reduced pErk1/2 to the same level in both samples. Selumetinib and trametinib as Mek inhibitors were mainly cytostatic, but combined treatment with the PI3K∂ inhibitor CAL101 increased cytotoxicity. Thus phospho-flow cytometry could be used as a platform for rapid, individualized in vitro drug sensitivity assessment for leukemia patients at the time of diagnosis.

Cellular Signaling Analysis shows antiviral, ribavirin-mediated ribosomal signaling modulation

“…Analysis of alternations in cellular signaling induced by any one drug, combination of drugs, or the disease state can rapidly elucidate the intricate changes in cellular signaling which is crucial for determining how to therapeutically apply drugs for a proper balance between therapeutic outcomes and potential side effects. Phosphoflow technology is a robust tool that can monitor many cellular signaling pathways that can be modulated during drug treatment (George et al, 2015;Hildebrand and Kubatzky, 2017;Coppin et al, 2017). Using this technology one can rapidly interrogates the state of key signaling nodes in diverse cellular pathways.…”

“…Treatment of different disease states through combinations of multiple drugs is used to achieve better therapeutic results, reduce the impact of drug resistance, and sustain high efficacy with low toxicity often with lower doses of the individual drug components. The use of drug combinations is well documented in both cancer and infectious disease therapy (Al-Lazikani et al, 2012;Arts and Hazuda, 2012;George et al, 2015). Therapeutic drug combinations work by simultaneously controlling several cellular pathways in the complex network system.…”

MEK Inhibition Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy

“…However, unlike in other malignancies, structural abnormalities leading to MEK/ERK activation in B-ALL are uncommon. Activity of MAPK/ERK pathway in B-ALL is also triggered by normal extracellular stimulation through stromal contact and serum growth factors [ 66 ]. Treatment with selumetinib resulted in a rapid, complete and persistent reduction of microenvironment-generated pERK1/2.…”

Recent Approaches Encompassing the Phenotypic Cell Heterogeneity for Anticancer Drug Efficacy Evaluation

“…Cells in suspension and can be readily analyzed in vitro not only for the antigens, but also for cellular signaling. For example, phospho-flow cytometry was applied in the search for B-cell precursor acute lymphoblastic leukemia individualized therapy and enabled to screen for small molecule kinase inhibitors that induced cancer cell death [230]. Functional analysis of single-cell mass accumulation rate became available by means of microchannel resonance [231].…”