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 sgRNAs. 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 highlighted in figure 1D. The size of each node is manually drawn to be 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 5μM PP2 treated for 72 hours (n=3). (H) MTT assay with MDA-MB-231 Cas9 cells expressing indicated sgRNAs treated with indicated TKIs for 72 hours (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 for 72 hours (n=3). (J) western blot analysis of MDA-MB-231 cells treated with indicated drugs for 48 hours (K) western blot analysis of MDA-MB-231 Cas9 cells expressing indicated sgRNA and treated with indicated drugs for 48 hours. (L) MTT assay of MDA-MB-231 cells treated with indicated drugs for 72 hours (SB203580: 10μ M, NVP-ADW742: 4μM, gefitinib: 10μM, imatinib: 10μM) (n=3). PP2, Saracatinib, NVP-ADW742, gefitinib and imatinib were treated at 5μM, 5μM, 4μM, 12μM, 12μM unless otherwise indicated. 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. Numbers below blots indicate quantification of average±s.d. expression level normalized to GAPDH in three independent experiments. (B) RT-qPCR analysis of FYN expression levels in MDA-MB-231 cells treated with indicated drugs for 48 hours (n=3). (C) RT-qPCR analysis of indicated jumonji family histone demethylase expression levels after 48 hours treatment of NVP-ADW742 (n=3). (D) Changes in FYN mRNA levels upon 48 hours of 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 for 48 hours. (H) MTT assay of MDA-MB-231 Cas9 cells expressing indicated sgRNAs and treated with indicated drugs for 72 hours (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 for 48 hours (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.(38) (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 for 48 hours at specified genomic loci (n=3). QC6352, PP2, NVP-ADW742, gefitinib and imatinib were treated at 10μM, 5μM, 4μM, 12μM, 12μM, respectively, unless otherwise indicated. 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. A: 1μM NVP-ADW742, G: 5μM gefitinib, I: 5μM imatinib, D: 100nM doxorubicin, P: 5nM paclitaxel. Numbers below blots indicate quantification of average±s.d. expression level normalized to GAPDH in three independent experiments. (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-F) MTT assay with PC9 parental (par.) and gefitinib resistant (GR) cells treated with indicated drug combinations (gefi: 2μM gefitinib, PP2: 5μM PP2, Sara: 5μM saracatinib, QC6352: 10μM QC6352) for 72 hours. (G) western blot analysis with PC9 cells treated with 10μM QC6352 for 48 hours. (H) FYN mRNA expression levels of parental and DTP populations in various cancers treated with indicated drugs.. (I) FYN mRNA expression levels of residual disease after indicated treatments. All data are plotted as mean±s.d. Paired two-sided Student’s t-test in B, H (HER2+ BRCA set and HGSOC carboplatin set), and I (BRCA set and ESCA set), and unpaired two-sided Student’s t-test in H (COAD and PAAD sets) and I (COAD set). The expression data in B,H,I are obtained from NCBI gene expression omnibus. The accession numbers of the expression data analyzed are listed in Supplementary table S2. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., p>0.05. All replicates are biological replicates.