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

• Published in: PloS one Vol. 10; no. 9; p. e0137917
• Main Authors: George, Aswathi A, Paz, Helicia, Fei, Fei, Kirzner, Jonathan, Kim, Yong-Mi, Heisterkamp, Nora, Abdel-Azim, Hisham
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 11-09-2015; Public Library of Science (PLoS)
• Subjects: 1-Phosphatidylinositol 3-kinase; Activation; Acute lymphoblastic leukemia; Acute lymphocytic leukemia; Animals; Antineoplastic agents; Antineoplastic Agents - pharmacology; Antineoplastic Agents - therapeutic use; Benzimidazoles - pharmacology; Bone marrow; Cancer therapies; Cell Line, Tumor; Cell Proliferation - drug effects; Cell Survival - drug effects; Cellular signal transduction; Change detection; Chemotherapy; Combined treatment; Comparative analysis; Complications and side effects; Cytometry; Cytotoxicity; Diagnosis; Dosage and administration; Drug therapy; Drugs; Enzyme Activation; Extracellular signal-regulated kinase; Flow Cytometry; Genetic aspects; Growth factors; Hematology; Humans; Inhibitors; Kinases; Leukemia; Lymphatic leukemia; Lymphocytes B; MAP Kinase Signaling System - drug effects; Medical prognosis; MEK inhibitors; MEK kinase; Metabolic pathways; Mice; Mitogen-Activated Protein Kinase 1 - antagonists & inhibitors; Mitogen-Activated Protein Kinase 3 - antagonists & inhibitors; Multiple myeloma; Mutation; Oncology; Patients; Pediatrics; Phosphorylation; Physiological aspects; Precision medicine; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma - drug therapy; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma - genetics; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma - metabolism; Precursors; Protein Kinase Inhibitors - pharmacology; Protein Kinase Inhibitors - therapeutic use; Proto-Oncogene Proteins p21(ras) - genetics; Reduction; Sensitivity analysis; Signal transduction; Stroma; Stromal Cells - drug effects; Stromal Cells - metabolism; Toxicity; Western blotting; United States
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0137917

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.