Unwanted acquired mutations in Ba/F3 transformation assays

Cancer genome sequencing has unraveled the huge variety of DNA alterations acquired during cell transformation. Only a small part of these alterations are thought to promote cancer development, while others represent mere random passenger mutations. It is essential to decipher which mutations are driving the oncogenic process to translate cancer sequencing data into clinically meaningful information, which can be used for precision medicine. Prediction software, such as Polyphen or MutationTaster, almost instantly give a useful estimate of the probability that a newly-identified variant affects the corresponding gene function. Nevertheless, experimental work is still required to definitively establish the importance of each mutation [1]. In this respect, a number of cell-based assays are available to test the functional impact of newly discovered mutations. In hematology, one of the most popular transformation assays is based on Ba/F3 cells and was first described in 1988 by George Daley and David Baltimore to demonstrate the oncogenic potential of BCR-ABL [2]. Ba/F3 cells have been subsequently used in more than one thousand publications referenced in PubMed. The Ba/F3 cell line was isolated from mouse normal bone marrow cells cultured in the presence of interleukin-3 (IL-3) [3]. These cells undergo massive apoptosis as soon as IL-3 is removed from the culture medium. However, Ba/F3 cells that are engineered to express an oncogene, such as BCR-ABL or a mutant receptor, can proliferate indefinitely in the absence of IL-3 (Figure 1). These cells are considered as transformed, because, unlike parental cells, they induce leukemia when reinjected in syngeneic Balb/c or immunodeficient mice [4, 5]. One key advantage of the Ba/F3 model is the absence of spontaneous transformation of untransfected cells. After Ba/F3 transfection, only a fraction of the cells expressing the oncogene adapt to IL-3-independent proliferation. The selection of these cells in culture takes a few days to a few weeks, depending on the potency of the tested oncogene. The molecular events underlying this selection process have remained unclear for many years. In this issue of Oncotarget, Kevin Watanabe-Smith et al. [6] show that spontaneous additional mutations arise in some oncogenic transgenes during the adaptation to IL-3 independence. Strikingly, they also observe that a wild-type receptor transgene, CSF3R, also acquires mutations and transforms Ba/F3 cells. This does not occur with empty control vectors, in agreement with previous publications. This report is of critical importance to the community of researchers who use Ba/F3 cells to study mutants and might have to revise the conclusions of some of their experiments. The publication of Daley and Baltimore is not at stake here: Watanabe-Smith and colleagues did not detect any additional mutation in this potent oncogene after transduction in Ba/F3 cells. Instead, unexpected acquired mutations occur in weakly transforming constructs, which require longer periods of time to transform cells. One problem is that many publications do not provide information regarding the potency of the tested transforming transgene and the efficiency of the Ba/F3 selection step. Instead, a large part of the scientific literature is based on experiments performed on cells that are fully transformed and have been growing in the absence of IL-3 for quite some time. The authors also studied the mechanism whereby transgenes acquired extra mutations. They first ruled out the possibility that widespread genomic instability occurs in transduced Ba/F3 cells. They also showed that the mutations were already present in a small proportion of News