Figure 1. SOD1 is a synthetic lethal vulnerability of PPM1D-mutant leukemia cells.

(A) Schematic of whole-genome CRISPR dropout screen. WT Cas9-expressing OCI-AML2 and two isogenic PPM1D-mutant lines were transduced with the Human Improved Whole Genome Knockout CRISPR library V1 containing 90,709 guide RNAs (gRNAs) targeting 18,010 human genes at low multiplicity of infection (MOI~0.3). Each condition was performed in technical triplicates. Three days post-transduction, cells underwent puromycin selection for three days. Cells were harvested at day 10 as the initial timepoint and then harvested every three days afterwards. sgRNA sequencing was performed on cells collected on day 28. (B) Top biological processes based on gene ontology analysis of the top 37 genes essential for PPM1D-mutant cell survival. Enrichment and depletion of guides and genes were analyzed using MAGeCK-VISPR by comparing read counts from each PPM1D-mutant cell line replicate with counts from the initial starting population at day ten. (C) Volcano plot of synthetic lethal hits ranked by fitness score with a negative score indicating genes for which their knockout leads to decreased growth or survival. SOD1 (highlighted) was the top hit from the screen. (D) Left: Schematic of competitive proliferation assays used for validation of CRISPR targets. Right: WT and PPM1D-mutant Cas9-OCI-AML2 and Cas9-OCI-AML3 cells were transduced with lentiviruses containing a single SOD1-gRNA with a blue fluorescent protein (BFP) reporter. Cells were assayed by flow cytometry every 3–4 days and normalized to the BFP percentage at day 3 post-transduction. Two unique gRNAs against SOD1 were used per cell line and each condition was performed in technical duplicates; multiple unpaired t-tests, **p<0.01, ***p<0.001. E) Left: Cas9-expressing WT and PPM1D-mutant cells were transduced with control or sgSOD1-containing lentiviruses and underwent puromycin (3 ug/mL) selection for three days prior to transplantation. Sublethally-irradiated (250 cGy) NSG mice were intravenously transplanted with 3 x 10⁶ cells. Right: Kaplan-Meier survival curve of mice transplanted with WT or PPM1D-mutant (grey) leukemia cells with or without SOD1-deletion. The median survival of mice transplanted with WT, WT/SOD1^–/–, PPM1D^mut, and PPM1D^mut/SOD1^–/– leukemia cells was 32, 43, 32, and 55 days, respectively; Mantel-Cox test, **p<0.01, ***p<0.001.