Fig. 2: Genetic screens reveal SAC-dependent mechanisms of cyclin A/B RxL inhibitor killing.
From: Targeting G1–S-checkpoint-compromised cancers with cyclin A/B RxL inhibitors
a, Schematic of the genome-wide CRISPR–Cas9 knockout resistance screen in NCI-H1048 cells transduced with the Brunello sgRNA library. After selection (day 10), cells were treated with CIRc-004 (200 nM), CIRc-001 (200 nM), PF-07104091 (CDK2i, 500 nM) or inactive enantiomer (I.E.) CIRc-005 (200 nM) and collected at day 26 (LTP). b, The top enriched and depleted hits from Apron analysis of CIRc-004 LTP versus CIRc-005 LTP. n = 2 biological replicates. c, The top enriched hits (q < 0.25) across treatments. Right, STRING network (https://string-db.org; CC BY 4.0)66 of shared hits highlights SAC proteins. d–g, Dose–response curves of NCI-H1048 cells expressing two non-targeting sgRNAs (sgCtrl) or two sgRNAs targeting CCNB1 (d), CDK2 (e), KNTC1 (f) or MAD1L1 (g), treated with CIRc-004 for 6 days. h, Flow cytometry quantification of cleaved PARP in cells from d–g treated with CIRc-004 (20 nM). Data are the fold change relative to vehicle. i, Immunoblot of the indicated SCLC and NSCLC cell lines treated for 24 h with increasing doses of CIRc-004. In d–h, data are mean ± s.d. In d–i, n = 3 biological replicates. j, RNA-seq analysis of NCI-H1048 cells treated for 24 h with CIRc-004 (200 nM), CIRc-005 (200 nM) or PF-07104091 (500 nM); the top 150 differentially expressed genes are shown (adjusted P (Padj) < 0.05). k, Immunoblot analysis of p-KNL1 in NCI-H1048 cells treated with CIRc-004 (20 nM), BAY-1217389 (MPS1i, 3 nM) or both for 24 h. l,m, CIRc-004 dose–response for proliferation (l) and cleaved PARP FACS assays (m) in the presence or absence of 3 nM BAY-1217389 for 3 days in NCI-H1048 cells. Data are mean ± s.d. n = 3 biological replicates. n, Immunoblot analysis of NCI-H1048 cells expressing sgCtrl or CCNB1 sgRNAs treated with CIRc-004 (20 nM) or DMSO for 24 h (n = 3 biological replicates). o, Cyclin B1 immunoprecipitations from NCI-H1048 cells treated for 2 h with CIRc-004 (004), CIRc-028 (028), CIRc-005 (005), CIRc-018 (018) or CIRc-019 (019) (300 nM); MYT1 binding normalized to cyclin B1 is shown. n = 3 biological replicates for all except for CIRc-028, for which n = 2. p, Dose–response analysis of NCI-H1048 cells treated with CIRc-018 with or without RP-6306 (100 nM) for 5 days. Data are mean ± s.d. from two technical replicates; n = 3 biological replicates. In d–g, l and m, the arrows indicate the DMSO-treated normalization controls. Statistical significance in h was calculated using unpaired two-tailed Student’s t-tests.
