Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays: findings and recommendations

Abstract

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.

Systematic Functional Annotation of Somatic Mutations in Cancer

“…Thus, the number of potential false-positive annotations obtained from our platform could be as low as 4 out of 301 (1.3%) activating mutations. Moreover, a recent study (Watanabe-Smith et al, 2017) suggested that Ba/F3 cells transfected with weak activating mutations can acquire extra mutations on the transgene during prolonged culturing under IL-3-replete conditions. Importantly, each of our constructs was from an individual clone and was sequenced prior to use, which limited the potential for pre-existing mutations in the construct.…”

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

“…In this case, choice of other Cas9 constructs with different selection cassettes may be necessary. A caveat of using cytokine-dependent cell lines, such as Ba/F3 cells is that they are susceptible to spontaneous cytokine independent growth, sometimes as a result of the acquisition of mutations in the ectopically expressed gene of interest following cytokine withdrawal 21 . Accordingly, the use of appropriate controls is required in order to be confident that the observed cellular transformation is due to on-target CRISPR gene editing.…”

Gain-of-function mutations in granulocyte colony–stimulating factor receptor (CSF3R) reveal distinct mechanisms of CSF3R activation

“…The CSF3R T618I mutation was previously found to induce constitutive receptor activation and transform Ba/F3 cells with fast kinetics (around 3-4 days) (17,19,23), whereas ectopic expression of CSF3R WT could sometimes lead to Ba/F3 transformation upon extended culture (Ͼ9 days) (22). We sequenced these transformed CSF3R WT Ba/F3 cells with primers covering most of the transgene.…”

“…In the past, we have observed that Ba/F3 cells harboring CSF3R WT can sometimes spontaneously transform at later time points in a Ba/F3 IL-3 withdrawal assay due to the acquisition of bona fide oncogenic mutations in CSF3R, such as the T618I mutation (22). We therefore took advantage of this Ba/F3 spontaneous transformation model and performed sequencing of the outgrown clones to identify novel CSF3R activating mutations that would further inform us about the biology of this receptor.…”