Pairwise CRISPR screen reveals combinations of synthetic lethal tyrosine kinase ablations.

(A) Schematic diagram of combinatorial screens performed in TNBC cell line MDA-MB-231. (B) Scatter plot of expected growth phenotype Z score and observed growth phenotype Z score of each gene combination. Green dots indicate gene combinations where the identical gene is targeted by the two sgRNA. Red dots indicate candidate synthetic lethal gene pairs listed in table S1. (C) Scatter plot of growth phenotype Z score and normalized GI score of each gene combination. (D) Scatter plot of gene level GI score and RIGER p value calculated with GI scores of each sgRNA pairs that target the given gene pair.

FYN is critical mediator of TKI resistance.

(A) Schematic diagram of in vitro validation of synthetic lethal gene pairs using sgRNAs. (B) Summary of synergistic killing by sgRNAs targeting indicated gene pairs (n=3). (C) Network analysis of the 30 candidate synthetic lethal gene pairs. The size of each node is proportional to the number of connections the gene has. (D-E) FYN and SRC mRNA expressions in (D) microarray data of primary breast cancers of GSE25066 cohort, and (E) in cancer cell line encyclopedia, for indicated subtypes. (F) Summary of MTT assay with MDA-MB-231 cells treated with the TKI combinations at indicated concentrations (n=2). Synergistic killing is calculated using SynergyFinder with Bliss independence model. (G) Dose response curve of the indicated TKI in the presence and absence of PP2 (n=3). (H) MTT assay with MDA-MB-231 Cas9 cells expressing indicated sgRNAs treated with indicated TKIs (n=3). (I) Cell death and cell proliferation in MDA MB 231 cells treated with NVP-ADW742, gefitinib and PP2 either as single agent or as combination (n=3). (J) western blot analysis of MDA-MB-231 cells treated with indicated drugs. (K) western blot analysis of MDA-MB-231 Cas9 cells expressing indicated sgRNA and treated with indicated drugs. (L) MTT assay of MDA-MB-231 cells treated with indicated drugs (n=3). All data are plotted as mean±s.d. One sample t-test for B, and unpaired two-sided Student’s t-test in D,E,H and L. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., p>0.05. All replicates are biological replicates.

Activation of KDM4 upregulates FYN, conferring drug resistance.

(A) Western blot analysis of MDA-MB-231 cells treated with indicated drugs. (B) RT-qPCR analysis of FYN expression levels in MDA-MB-231 cells treated with indicated drugs (n=3). (C) RT-qPCR analysis of indicated jumonji family histone demethylase expression levels (n=3). (D) Changes in FYN mRNA levels upon NVP-ADW742 treatment in MDA-MB-231 Cas9 cells expressing indicated sgRNAs (n=4). (E) KDM4A mRNA levels in primary tumor tissues of indicated subtypes in GSE25066 cohort. (F) Positive correlation of FYN and KDM4A mRNA levels in CCLE database. (G) western blot analysis of MDA-MB-231 Cas9 cells expressing indicated sgRNA and treated with indicated drugs. (H) MTT assay of MDA-MB-231 Cas9 cells expressing indicated sgRNAs and treated with indicated drugs (n=3). (I) SynergyFinder analysis of MDA-MB-231 cells treated with indicated drug combinations (n=2). (J-K) western blot analysis (J) and RT-qPCR analysis (K) of MDA-MB-231 cells treated with indicated drugs (n=3). (L) mRNA expression levels of SRC family kinases in breast cancer stem cells treated with QC6352 in RNA sequencing data described in Metzger et. al.(29) (M) H3K9me3 and KDM4A enrichment at genomic locus encoding FYN promoter in ChIP sequencing data described in the same study as (L). (N) H3K9me3 Chromatin immunoprecipitation-qPCR analysis of MDA-MB-231 cells treated with indicated drug at specified genomic loci (n=3). All data are plotted as mean±s.d. Unpaired two-sided Student’s t-test in B,C,D,E,H and K. Paired two-sided Student’s t-test in N. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., p>0.05. All replicates are biological replicates.

Combination therapy targeting FYN+IGF1R and KDM4+EGFR synergistically eliminates tumor in vivo.

(A-B) Tumor volume for MDA-MB-231 xenografts treated with indicated drugs. Additive effects were calculated by Bliss independence model (n=5). (C) Distant relapse free survival of GSE25066 patient cohort classified by FYN (left) and KDM4A (right) mRNA expression. (D) Schematics diagram of the mechanism of KDM4-FYN conferring TKI resistance. All data are plotted as mean±s.d. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., p>0.05. All replicates are biological replicates.

FYN and KDM4 are associated with drug tolerance.

(A) MDA-MB-231 cells treated with indicated drugs for short (acute: 2 days) and long (DTP: 10 days) time periods. (B) Summary of mRNA expressions of indicated genes in EGFR mutant lung cancer cells (parental) and their derivative Osimertinib tolerant persisters (DTP). (C-D) western blots (C) and RT-qPCR (D) analyses of indicated parental and EGFR inhibitor resistant lung cancer cells. (E) FYN mRNA expression levels of parental and DTP populations in various cancers treated with indicated drugs (n=3). (F) FYN mRNA expression levels of residual disease after indicated treatments (n=3). (G-H) MTT assay with PC9 parental (par.) and gefitinib resistant (GR) cells treated with indicated drug combinations. (I) western blot analysis with PC9 cells treated with QC6352. All data are plotted as mean±s.d. Paired two-sided Student’s t-test in B, E (HER2+ BRCA set and HGSOC carboplatin set), and F (BRCA set and ESCA set), and unpaired two-sided Student’s t-test in E (COAD and PAAD sets) and F (COAD set). *, p<0.05; **, p<0.01; ***/, p<0.001; n.s., p>0.05. All replicates are biological replicates.