Therapeutically Targetable ALK Mutations in Leukemia

Abstract

Genome sequencing is revealing a vast mutational landscape in leukemia, offering new opportunities for treatment with targeted therapy. Here, we identify two patients with acute myelogenous leukemia and B-cell acute lymphoblastic leukemia whose tumors harbor point mutations in the ALK kinase. The mutations reside in the extracellular domain of ALK and are potently transforming in cytokine-independent cellular assays and primary mouse bone marrow colony formation studies. Strikingly, both mutations conferred sensitivity to ALK kinase inhibitors, including the FDA-approved drug crizotinib. On the basis of our results, we propose that tumors harboring ALK mutations may be therapeutically tractable for personalized treatment of certain aggressive leukemias with ALK inhibitors.

Figure 1. Identification of ALK mutations in leukemia patient samples

(A) Schematic of the location of the ALK mutations identified by deep sequencing. Including the location of the following domains: MAM1, LDL-A (LDL), MAM2, glycine Rich (Gly), transmembrane (TM), and Tyrosine Kinase. (B,C) Sanger sequencing confirms that tumor cells from a pediatric B-ALL leukemia sample harbored the ALK A348D mutation in the MAM1 domain, and tumor cells from a patient with Adult AML exhibited the ALK F856S mutation in the glycine-rich domain.

Figure 2. ALK point mutations found in leukemia samples are oncogenic, and sensitive to Crizotinib

(A) The ALK A348D and F856S mutations transforms the murine Ba/F3 pro-B cell line to cytokine independent growth. Ba/F3 cells expressing WT ALK, ALK A348D, F856S, Ba/F3 cells harboring an empty vector (Mig Empty), or parental Ba/F3s were grown in the absence of the cytokine IL3. Total viable cells are plotted over time. (B) ALK mutations are expressed at the same level as WT ALK. Immunoblot analysis of Ba/F3 cells expressing ALK mutations along with their respective WT ALK controls from each Ba/F3 IL3 withdrawal experiment. GAPDH serves as a loading control. (C) ALK point mutations induce colony formation in mouse bone marrow. Mouse bone marrow cells were infected with retrovirus expressing WT ALK, A348D or F856S and then plated in colony formation medium in the absence of cytokines. The average number of colonies formed from three replicates are shown. Error bars represent the standard error of the mean. (D) ALK point mutant expressing Ba/F3 cells are sensitive to Crizotinib. IL3-independent Ba/F3 cells expressing the ALK A348D or F856S mutations were treated with Crizotinib in triplicate. Cell viability was determined using a tetrazolamine based viability assay. Viability is represented as a percentage of the untreated control. The mean of three replicates are shown, along with the standard error.

Figure 3. The ALK A348D and F856S mutations confer sensitivity to ALK inhibitors

IL3-independent Ba/F3 cells expressing the ALK A348D or F856S mutations were treated with NVPTAE684 (A), AP2631 (B), LDK378 (C) or GSK1838705A (D). Parental BaF3 cells in IL3 containing medium were used as a control. All drugs were used at the following concentrations: 500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.95, 0.98, 0.49 and 0 nM. Cell viability was determined using a tetrazolamine based viability assay. Viability is represented as a percentage of the untreated control. The mean of three replicates is plotted, along with the standard error.