Differential protein stability and ALK inhibitor sensitivity of EML4-ALK fusion variants.
OBJECTIVE
ALK rearrangement-positive lung cancers can be effectively treated with ALK inhibitors. However, the magnitude and duration of response is heterogeneous. In addition, acquired resistance limits the efficacy of ALK inhibitors, with most upfront resistance mechanisms being unknown.


METHODS
By making use of the Ba/F3 cell line model, we analyzed the cytotoxic efficacy of ALK kinase inhibitors as a function of different EML4-ALK fusion variants v1, v2, v3a, and v3b as well as of three artificially designed EML4-ALK deletion constructs and the ALK fusion genes KIF5b-ALK and NPM1-ALK. In addition, the intracellular localization, the sensitivity to HSP90 inhibition and the protein stability of ALK fusion proteins were studied.


RESULTS
Different ALK fusion genes and EML4-ALK variants exhibited differential sensitivity to the structurally diverse ALK kinase inhibitors crizotinib and TAE684. In addition, differential sensitivity correlated with differences in protein stability in EML4-ALK-expressing cells. Furthermore, the sensitivity to HSP90 inhibition also varied depending on the ALK fusion partner but differed from ALK inhibitor sensitivity patterns. Finally, combining inhibitors of ALK and HSP90 resulted in synergistic cytotoxicity.


CONCLUSIONS
Our results might explain some of the heterogeneous responses of ALK-positive tumors to ALK kinase inhibition observed in the clinic. Thus, targeted therapy of ALK-positive lung cancer should take into account the precise ALK genotype. Furthermore, combining ALK and HSP90 inhibitors might enhance tumor shrinkage in EML4-ALK-driven tumors.
Department of Translational Genomics, University of Cologne, c/o MPI for Neurological Research, Gleueler Str. 50, 50931 Cologne, Germany.
Johannes M
JM
Heuckmann
Hyatt
H
Balke-Want
Florian
F
Malchers
Martin
M
Peifer
Martin L
ML
Sos
Mirjam
M
Koker
Lydia
L
Meder
Christine M
CM
Lovly
Lukas C
LC
Heukamp
William
W
Pao
Ralf
R
Küppers
Roman K
RK
Thomas
D000230:Adenocarcinoma; D000077192:Adenocarcinoma of Lung; D000077548:Anaplastic Lymphoma Kinase; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000077547:Crizotinib; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016547:Kinesins / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D051379:Mice; D041681:NIH 3T3 Cells; D000090243:Nucleophosmin; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage; D055550:Protein Stability / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011720:Pyrazoles / Q000008:administration & dosage; D011725:Pyridines / Q000008:administration & dosage; D011743:Pyrimidines / Q000008:administration & dosage; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D015398:Signal Transduction / Q000187:drug effects
C522504:EML4-ALK fusion protein, human; D018841:HSP90 Heat-Shock Proteins; C488101:KIF5B protein, human; C000716967:NPM1 protein, human; C516714:NVP-TAE684; C000715570:Npm1 protein, mouse; D015514:Oncogene Proteins, Fusion; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011725:Pyridines; D011743:Pyrimidines; D000090243:Nucleophosmin; D000077547:Crizotinib; C107023:p80(NPM-ALK) protein; C000626173:ALK protein, human; C000626174:Alk protein, mouse; D000077548:Anaplastic Lymphoma Kinase; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D016547:Kinesins