Figure 2

CDK combinatorial disruption reveals conserved and context-dependent interaction networks. (a) Mean fitness for cells receiving each CDK knockout, pooled across three TNBC cell lines. AAVS1, sgRNA targeting adeno-associated virus integration site 1, a safe-harbor control locus; NTC, non-targeting control. Error bars correspond to standard deviations across measurements from three cell lines: Hs578T, MDA-MB-231, and MDA-MB-468. (b) Fitness trajectories for CDK4/6 dual knockout vs. single knockouts (pairing CDK4 or CDK6 with AAVS) in each TNBC cell background. Error bars correspond to standard deviation of fitness measurements across replicates and 32 guide pairs targeting the same gene pair. (c) Heatmap of significant genetic interactions for each cell line and a pan-cell line analysis. (d) Complete CDK synthetic lethality networks discovered across all experiments. Single gene knockout fitness is defined as the log₂ growth relative to non-targeting control. (e) Schematic of validation of genetic interactions. sgRNAs paired with two different fluorophores are transduced at high MOI and grown in competition. Cells are colored according to the sgRNA a cell received: blue for sgRNA1-BFP, red for sgRNA2-mCherry, yellow for both sgRNA1-BFP and sgRNA2-mCherry, and gray for no viral integration. (f) CDK4/6 single and dual knockout populations 4 days and 11 days after infection. (g–i) Validation of synthetic lethal interactions for (g) CDK4-CDK6, (h) CDK2-CDK6, (i) CDK12-PRMT5 in MDA-MB-231 cells by fold enrichment (positive values) or depletion (negative values) of single and dual knockouts on day 11 vs. day 4 post infection. Error bars represent standard deviation across two replicates. Dual knockouts showed marked reduction in growth relative to single knockouts.