title: ALK mutants in the kinase domain exhibit altered kinase activity and differential sensitivity to small molecule ALK inhibitors.
abstract: Abnormal expression of constitutively active anaplastic lymphoma kinase (ALK) chimeric proteins in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is well established. Recent studies with small molecule kinase inhibitors have provided solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK (+) ALCL cells. In this study, several missense mutants of ALK in the phosphate anchor and gatekeeper regions were generated and their kinase activity was measured. NPM-ALK L182M, L182V, and L256M mutants displayed kinase activity in cells comparable to or higher than that of NPM-ALK wild type (WT) and rendered BaF3 cells into IL-3-independent growth, while NPM-ALK L182R, L256R, L256V, L256P, and L256Q displayed much weaker or little kinase activity in cells. Similar kinase activities were obtained with corresponding GST-ALK mutants with in vitro kinase assays. With regard to inhibitor response, NPM-ALK L182M and L182V exhibited sensitivity to a fused pyrrolocarbazole (FP)-derived ALK inhibitor comparable to that of NPM-ALK WT but were dramatically less sensitive to a diaminopyrimidine (DAP)-derived ALK inhibitor. On the other hand, NPM-ALK L256M exhibited >30-fold lower sensitivity to both FP-derived and DAP-derived ALK inhibitors. The growth inhibition and cytotoxicity of BaF3/NPM-ALK mutant cells induced by ALK inhibitors were consistent with inhibition of cellular NPM-ALK autophosphorylation. In a mouse survival model, treatment with the orally bioavailable DAP-ALK inhibitor substantially extended the survival of the mice inoculated with BaF3/NPM-ALK WT cells but not those inoculated with BaF3/NPM-ALK L256M cells. Binding of ALK inhibitors to ALK WT and mutants was analyzed using ALK homology models. In summary, several potential active ALK mutants were identified, and our data indicate that some of these mutants are resistant to select small molecule ALK inhibitors. Further characterization of these mutants may help to identify and develop potent ALK inhibitors active against both WT and resistant mutants of ALK.
authors.affiliation: Worldwide Discovery Research, Cephalon Inc., West Chester, Pennsylvania 19380, USA.
authors.forename: Lihui
authors.initials: L
authors.lastname: Lu
authors.forename: Arup K
authors.initials: AK
authors.lastname: Ghose
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Quail
authors.forename: Mark S
authors.initials: MS
authors.lastname: Albom
authors.forename: John T
authors.initials: JT
authors.lastname: Durkin
authors.forename: Beverly P
authors.initials: BP
authors.lastname: Holskin
authors.forename: Thelma S
authors.initials: TS
authors.lastname: Angeles
authors.forename: Sheryl L
authors.initials: SL
authors.lastname: Meyer
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Ruggeri
authors.forename: Mangeng
authors.initials: M
authors.lastname: Cheng
mesh_terms: D000595:Amino Acid Sequence; D000077548:Anaplastic Lymphoma Kinase; D000818:Animals; D002460:Cell Line; D006801:Humans; D017728:Lymphoma, Large-Cell, Anaplastic / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D020125:Mutation, Missense; D009368:Neoplasm Transplantation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015996:Survival Rate
chemical_list: D011993:Recombinant Fusion Proteins; C000626173:ALK protein, human; C000626174:Alk protein, mouse; D000077548:Anaplastic Lymphoma Kinase; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases