title: 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.
authors.affiliation: Division of Hematology/Oncology and Bone Marrow Transplantation, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, CA, United States of America.
authors.forename: Aswathi A
authors.initials: AA
authors.lastname: George
authors.affiliation: Division of Hematology/Oncology and Bone Marrow Transplantation, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, CA, United States of America; Section of Molecular Carcinogenesis, Department of Pediatrics, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, CA, United States of America.
authors.forename: Helicia
authors.initials: H
authors.lastname: Paz
authors.affiliation: Division of Hematology/Oncology and Bone Marrow Transplantation, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, CA, United States of America; Section of Molecular Carcinogenesis, Department of Pediatrics, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, CA, United States of America.
authors.forename: Fei
authors.initials: F
authors.lastname: Fei
authors.affiliation: Division of Hematology/Oncology and Bone Marrow Transplantation, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, CA, United States of America.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Kirzner
authors.affiliation: Division of Hematology/Oncology and Bone Marrow Transplantation, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, CA, United States of America.
authors.forename: Yong-Mi
authors.initials: YM
authors.lastname: Kim
authors.affiliation: Division of Hematology/Oncology and Bone Marrow Transplantation, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, CA, United States of America; Section of Molecular Carcinogenesis, Department of Pediatrics, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, CA, United States of America; Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center and Department of Pathology, University of Southern California, Los Angeles, CA, United States of America.
authors.forename: Nora
authors.initials: N
authors.lastname: Heisterkamp
authors.affiliation: Division of Hematology/Oncology and Bone Marrow Transplantation, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, CA, United States of America.
authors.forename: Hisham
authors.initials: H
authors.lastname: Abdel-Azim
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001562:Benzimidazoles / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors*; D009154:Mutation; D010766:Phosphorylation; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism
chemical_list: C517975:AZD 6244; D000970:Antineoplastic Agents; D001562:Benzimidazoles; C117307:KRAS protein, human; D047428:Protein Kinase Inhibitors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D016283:Proto-Oncogene Proteins p21(ras)