Extended Data Fig. 5. ALK tyrosine phosphorylates CDK9 at Y19 to promote resistance to PARP inhibitor.
(a) Schematic of the strategy using the indicated criteria (1 and 2) to identify proteins with DNA repair function that potentially regulated by ALK. (b) Cells were infected with control shRNA (pLKO.1) or shRNAs targeting the CDK9 (shCDK9 #1/ shCDK9#2). Relative expression levels of total CDK9 in stable clones were determined by WB. Data are representative of two repeats with similar results. (c) Half maximal inhibitory concentration (IC50) of PARPi (talazoparib). CDK9-knockdown, PARPi-resistant, and PARPi-sensitive cells were treated with talazoparib for 6 days and subjected to MTT assay to determine cell viability. Error bars represent mean ± SEM of N = 3 independent experiments. (d) Cell growth of CDK9-knockdown PARPi-resistant and PARPi-sensitive cells on indicated days. Data represent three independent experiments. (e) Representative images of clonogenic assay in TNBC cells with acquired resistance to PARPi in the presence of the indicated inhibitor for 12 days. The mean percentage survival derived from N = 3 independent experiments was used to calculate the combination index (CI) value. Synergistic inhibition of cell proliferation is defined as CI < 1. (f) Detection of p-ALK and CDK9 binding (red dots) in PARPi-sensitive TNBC parental cells and TNBC cells with acquired resistance to PARPi (#6 and #15), determined by Duo-link assay. Bar, 20 µm. Bar diagram, the percentage of cells showing positive interaction calculated. Error bar represent mean ± SD of N = 3 independent experiments. (g) WB of tyrosine phosphorylation (p-Tyr) signal in in vitro kinase assay results in which purified ALK was incubated with GST-CDK9 protein. Data are representative of two repeats with similar results. (h) Coomassie blue staining of purified ALK and GST-CDK9 protein. Data are representative of two repeats with similar results. (i) Stable re-constitution of PCDH (vector control), wild type (WT), Y19E, or Y19F CDK9 in SKOV3 cells depleted of endogenous CDK9. Expression of Flag-tagged CDK9 was determined by Western blot analysis. Data are representative of two repeats with similar results. Statistical analysis was carried out using the one-way ANOVA analysis (c, d) and two-tailed unpaired t test (f). (c) n.s., not significant. (d) pLKO.1 vs shCDK9#1: ***P = 0.0003 in Parental, ***P < 0.0001 in R#6 and R#15; pLKO.1 vs shCDK9#2: ***P < 0.0001 in Parental, R#6 and R#15.