Extended Data Fig. 7. ALK is critical for nuclear localization of CDK9 and transcriptional activates HR repair genes.
(a) Expression of the indicated proteins in 293 T cells expressing Y19F and WT CDK9 was examined by WB after IP with FLAG antibody. Data are representative of two repeats with similar results. (b) Representative images of CDK9 with DAPI staining (left panel) and quantification of cells with nuclear CDK9 positive signal (right panel) in PARPi resistant (OVCA433 and SKOV3) or PARPi sensitive (A2780 and DOV13) ovarian cancer cells. Bar, 20 µm. Data are represented as mean ± SEM of N = 3 independent experiments. One-way ANOVA analysis, A2780 vs SKOV3: ***P = 0.0003, A2780 vs OVCA433: ***P = 0.0005, DOV13 vs SKOV3: ***P = 0.0001, DOV13 vs OVCA433: ***P = 0.0002. (c) Western blot analysis of indicated proteins (left) and real-time PCR analysis of indicated genes (right) in PARPi-resistant SKOV3 cells treated with 0.5 μM PARPi (talazoparib) or 0.5 μM ALK inhibitor (lorlatinib), either alone or in combination, for 24 hours. Data are represented as mean ± SD of N = 3 independent experiments. Con vs ALKi: ***P = 0.0001 in RAD51, **P = 0.0079 in RAD50, ***P < 0.0001 in RBBP8, ***P = 0.0005 in BRCA2, **P = 0.0013 in BRCA1; PARPi vs PARPi+ALKi: ***P < 0.0001 in RAD51, ***P < 0.0001 in RAD50, *P = 0.0241in RBBP8, **P = 0.0097in BRCA2, *P = 0.0122 in BRCA1,two-way ANOVA analysis. (d) Transcriptome analysis of treatment with ALK inhibitor and Y19F CDK9 mutant. Heatmap of genes that were up- or down-regulated in both WT CDK9-ALKi (ALKi treatment) and Y19F-mutant CDK9 (Y19F) cells relative to WT CDK9 (WT) cells. (e) Metascape results showing gene sets related to DNA repair in ALKi treated WT CDK9 cells and Y19F-mutant CDK9 cells relative to WT CDK9 cells. Pre-ranked gene lists for pathway enrichment analysis were generated using the results (P-value) from DEseq2. (f) Metascape report of other Reactome gene sets that were ranked before DNA repair related gene sets and enriched in genes down-regulated in both WT CDK9-ALKi and Y19F-mutant CDK9 cells. Metascape utilizes the well-adopted hypergeometric test and Benjamini-Hochberg p-value correction algorithm for statistical analysis. WT, wild-type; Ai, ALK inhibitor treatment; Y19F, Tyr19Phe mutation.