TY - JOUR
T1 - Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays
T2 - Findings and recommendations
AU - Watanabe-Smith, Kevin
AU - Godil, Jamila
AU - Agarwal, Anupriya
AU - Tognon, Cristina
AU - Druker, Brian
PY - 2017
Y1 - 2017
N2 - The identification and functional validation of potentially oncogenic mutations in leukemia is an essential step toward a future of personalized targeted therapy. To assess the oncogenic capacity of individual mutations, reliable and scalable in vitro experimental approaches are required. Since 1988, researchers have used the IL-3 dependent Ba/F3 transformation assay to validate the oncogenic potential of mutations to drive factor-independent growth. Here we report a previously unrecognized phenomenon whereby Ba/F3 cells, engineered to express weakly transforming mutations, present with additional acquired mutations in the expressed transgene following factor withdrawal. Using four mutations with known transformative capacity in three cytokine receptors (CSF2RB, CSF3R and IL7R), we demonstrate that the mutated receptors are highly susceptible to acquiring additional mutations. These acquired mutations of unknown functional significance are selected by factor withdrawal but appear to exist prior to the removal of growth factor. This anomaly has the potential to confound efforts to both validate and characterize oncogenic mutations in leukemia, particularly when it is not standard practice to sequence validate cDNAs from transformed Ba/F3 lines. We present specific recommendations to detect and mitigate this phenomenon in future research using Ba/F3 transformation assays, along with methods to make the Ba/F3 assay more quantitative.
AB - The identification and functional validation of potentially oncogenic mutations in leukemia is an essential step toward a future of personalized targeted therapy. To assess the oncogenic capacity of individual mutations, reliable and scalable in vitro experimental approaches are required. Since 1988, researchers have used the IL-3 dependent Ba/F3 transformation assay to validate the oncogenic potential of mutations to drive factor-independent growth. Here we report a previously unrecognized phenomenon whereby Ba/F3 cells, engineered to express weakly transforming mutations, present with additional acquired mutations in the expressed transgene following factor withdrawal. Using four mutations with known transformative capacity in three cytokine receptors (CSF2RB, CSF3R and IL7R), we demonstrate that the mutated receptors are highly susceptible to acquiring additional mutations. These acquired mutations of unknown functional significance are selected by factor withdrawal but appear to exist prior to the removal of growth factor. This anomaly has the potential to confound efforts to both validate and characterize oncogenic mutations in leukemia, particularly when it is not standard practice to sequence validate cDNAs from transformed Ba/F3 lines. We present specific recommendations to detect and mitigate this phenomenon in future research using Ba/F3 transformation assays, along with methods to make the Ba/F3 assay more quantitative.
KW - Ba/F3 transformation assay
KW - Functional validation
KW - Leukemia
KW - Oncogenes
KW - Reproducibility in research
UR - http://www.scopus.com/inward/record.url?scp=85013486070&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85013486070&partnerID=8YFLogxK
U2 - 10.18632/oncotarget.15392
DO - 10.18632/oncotarget.15392
M3 - Article
C2 - 28208123
AN - SCOPUS:85013486070
SN - 1949-2553
VL - 8
SP - 12596
EP - 12606
JO - Oncotarget
JF - Oncotarget
IS - 8
ER -