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

Aswathi A George,Jonathan Kirzner,Yong-Mi Kim,Hisham Abdel-Azim,Helicia Paz,Nora Heisterkamp,Fei Fei,Linda Bendall

doi:10.1371/journal.pone.0137917

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.

Highlights

Overall survival rates for childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) using traditional chemotherapy have increased to more than 80%
Western blotting with Cell Signaling Technology (CST) pErk1/2 antibodies indicated that the ALL cells cultured with stroma contained higher levels of pErk1/2 than cells which did not receive stromal stimulation (Fig 1)
We first determined the sensitivity of flow cytometry to detect changes in pErk and pMek

Summary

Introduction

Overall survival rates for childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) using traditional chemotherapy have increased to more than 80%. Case et al [1] [2] reported that activation of the Ras pathway, which includes Raf, Mek and Erk, could be detected in 35% of diagnostic and 25% of relapsed samples. As reviewed in [3], because of “oncogene addiction”, cancers with constitutive activation of a specific signal transduction pathway are thought to be more sensitive to inhibitors of such pathway than cancers that lack constitutive activation. Irving et al [5] recently applied this principle to test the non-ATP competitive Mek1/2 inhibitor selumetinib (AZD6244, ARRY-142886) as monotreatment for childhood ALL in preclinical studies and concluded that clinical evaluation of selumetinib is warranted

Methods

Results

Conclusion