Leukemias are generally clonal in origin, but develop significant heterogeneity as they expand and acquire genetic and epigenetic alterations. Detecting this heterogeneity and tracking the dynamics and evolution of clones within a population are important for our understanding of these cancers and the phenomena of drug resistance and relapse. Attempts to track the contributions of individual cells to the clonality of large populations, however, have been constrained by limitations in sensitivity and complexity. We have created an efficient and high throughput method to overcome these limitations by harnessing the power of viral marking and next generation sequencing technology, allowing us to track the clonal contributions of many thousands of cells with minimal perturbations to the population as a whole. K562s are a common patient-derived CML line featuring rapidly proliferating, highly aneuploid cells. We stably and heritably marked individual cells via the lentiviral-mediated insertion of one of ∼14,000 random, non-coding, 20 basepair DNA “barcodes.” Using Illumina next-generation sequencing we determined the relative frequency of every barcode, thus directly measuring the contribution of each clone to the overall population over 90 population doublings. We anticipated that the the K562 cell line, while very highly aneuploid, would have stable clonal dynamics because it has been grown in culture for years, thus allowing potentially dominant clones to reach equilibrium. We found, however, that K562 cells continue to undergo dramatic changes in clonal representation. As expected, in analysis of our clonally marked population, there was a broad distribution of clones. After culturing under ideal conditions for 90 population doublings, we found that a single clone now represented 9% of the population and that less than 100 clones now represented more than 50% of the population, a massive clonal skewing after just a brief period of time. Cell lines are often assumed to be a homogenous group of identical clones, a seemingly reasonable assumption considering the years of passaging/subculturing. However, our results show that ongoing genomic and/or epigenomic instability leads to proliferative instability, resulting in unexpected clonal dynamics and rapid clonal evolution. These results have profound implications for the experimental use of cell lines, as well as broad applications for clonal analysis of leukemias, other malignancies, and stem cells.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.