Transcriptome analysis of rigosertib resistant MDA-MB-453 cells, obtained from a CRISPR screen with WNK1 depletion.

In a genome-wide CRISPR-Cas9 resistance drug screen, we identified the master osmostress regulator WNK1 kinase as a modulator of the response to the mitotic drug rigosertib. Osmotic stress and WNK1 inactivation lead to an altered response not only to rigosertib but also to other microtubule-related drugs, minimizing the prototypical mitotic arrest produced by these drugs. This effect is due to an alteration in microtubule stability and polymerization dynamics, likely maintained by fluctuations in intracellular molecular crowding upon WNK1 inactivation. This promotes resistance to microtubule depolymerizing drugs, and increased sensitivity to microtubule stabilizing drugs. In summary, our data proposes WNK1 osmoregulation activity as a biomarker for microtubule-associated chemotherapy response. Overall design: MDA-MB-453 breast cancer cells, carrying the Yusa sgRNA CRISPR library, were subjected to a RIGOSERTIB drug resistance screen. Cas9 is activated in a TET-ON inducible manner with doxycycline (DOX) for 3 days, an then cells are incubated with 100nM rigosertib during 3 days, and 4 days of drug holiday, repeating this cycle for 4 to 5 weeks. Once resistant colony cells were observed, they were allowed to grow in fresh media for 10 days more. Total RNA was extracted from three resistant colonies (Res) and two parental controls (no DOX and +DOX) to evaluate de transcriptome of the resistant cells.