Fig. 4: ALK promotes PARP inhibitor resistance and HR via phosphorylation of CDK9 at Tyr19. | Nature Cancer

Fig. 4: ALK promotes PARP inhibitor resistance and HR via phosphorylation of CDK9 at Tyr19.

From: Targeting the ALK–CDK9-Tyr19 kinase cascade sensitizes ovarian and breast tumors to PARP inhibition via destabilization of the P-TEFb complex

Fig. 4

a, Relative cell viability of CDK9-knockdown SKOV3 cells re-expressing WT, Tyr19Glu or Tyr19Phe CDK9. Cells were treated with the indicated concentration of talazoparib for 6 d. Error bars represent mean ± s.d. of n = 3 independent experiments, two-tailed, unpaired Student’s t-test: Tyr19Phe versus WT at 312 nM: *P = 0.012; Tyr19Phe versus Tyr19Glu at 312 nM: *P = 0.013; Tyr19Phe versus WT at 625 nM: **P = 0.002; Tyr19Phe versus Tyr19Glu at 625 nM: **P = 0.008; Tyr19Phe versus WT at 1,250 nM: *P = 0.0117; Tyr19Phe versus Tyr19Glu at 1,250 nM: *P = 0.0111. b, Chou–Talalay analysis of CDK9-knockdown SKOV3 cells re-expressing WT, Tyr19Glu or Tyr19Phe CDK9. Cells were treated with varying concentrations of PARP inhibitor (talazoparib) and ALK inhibitor (lorlatinib) for 6 d. The mean percentage of growth inhibition derived from n = 3 independent MTT experiments was used to calculate the CI value. Strong synergism showed as CI < 0.5 at an optimal effect level (Fa > 0.75, region highlighted in orange). The CI values at Fa > 0.75: WT < 0.5 < Tyr9Glu < Tyr19Phe. c, Representative images of DAPI, RAD51 and EdU staining in PARP inhibitor-resistant SKOV3 cells depleted of endogenous CDK9 and re-constituted with pCDH (vector control), WT, Tyr19Glu or Tyr19Phe CDK9. These cells were cultured with 0.25 μM PARP inhibitor (talazoparib) or 0.5 μM ALK inhibitor (lorlatinib), either alone or in combination, for 48 h. Insets: ×3.3 magnification. Scale bar, 20 µm. Data represent n = 3 independent experiments with similar results. d,e, Quantification of EdU-positive cells with RAD51 foci (d) and γH2AX (e) foci in PARP inhibitor-resistant SKOV3 cells depleted of endogenous CDK9 and re-constituted with WT, Tyr19Glu or Tyr19Phe CDK9. These cells were cultured with 0.25 μM PARP inhibitor (talazoparib) or 0.5 μM ALK inhibitor (lorlatinib), either alone or in combination, for 48 h. Error bars represent mean ± s.d. of n = 3 independent experiments. Two-way ANOVA analysis: control versus ALK inhibitor in WT: **P = 0.0027, PARP inhibitor versus PARP inhibitor + ALK inhibitor in WT: **P = 0.0028 (d); control versus PARP inhibitor in PCDH: ***P < 0.001, control versus PARP inhibitor in Tyr19Phe: ***P < 0.001 (e). NS, not significant.

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