Genetic screening for single-cell variability modulators driving therapy resistance

Non-genetic fluctuations in the molecular state of a cell can lead to different cell fate choices in response to environmental perturbations. In the context of cancer, such fluctuations can lead to the formation of rare cells within the population that are able to survive treatment with targeted therapies. However, the biological processes governing the formation of these cells and their subsequent impact on therapy resistance remains largely unknown. We performed genetic screens using CRISPR/Cas9 to identify over 80 factors that modulate these rare cell fluctuations. These screens revealed several novel targets affecting cellular variability and, in turn, therapy resistance. Transcriptomic analysis revealed that many of these novel targets appeared to act via distinct mechanisms from those previously identified; for instance, knockout of either DOT1L or LATS2 appeared to increase resistance by increasing the degree of cellular differentiation, as opposed to knockout of MITF or SOX10, which achieve the same effect by decreasing cellular differentiation. We show that the timing of inhibition of these new variability-altering factors can affect the degree of resistance to targeted therapies. Our results suggest that cellular plasticity may be subject to regulatory processes that play critical roles in cell fate determination.