CDK5 Inhibitor Downregulates Mcl-1 and Sensitizes Pancreatic Cancer Cell Lines to Navitoclax

Abstract

Developing small molecules that indirectly regulate Mcl-1 function has attracted a lot of attention in recent years. Here, we report the discovery of an aminopyrazole, 2-([1,1'-biphenyl]-4-yl)-N-(5-cyclobutyl-1H-pyrazol-3-yl)acetamide (analog 24), which selectively inhibited cyclin-dependent kinase (CDK) 5 over CDK2 in cancer cell lines. We also show that analog 24 reduced Mcl-1 levels in a concentration-dependent manner in cancer cell lines. Using a panel of doxycycline inducible cell lines, we show that CDK5 inhibitor 24 selectively modulates Mcl-1 function while the CDK4/6 inhibitor 6-acetyl-8-cyclopentyl-5-methyl-2-(5-(piperazin-1-yl)pyridin-2-ylamino)pyrido[2,3-day]pyrimidin-7(8H)-one does not. Previous studies using RNA interference and CRISPR showed that concurrent elimination of Bcl-xL and Mcl-1 resulted in induction of apoptosis. In pancreatic cancer cell lines, we show that either CDK5 knockdown or expression of a dominant negative CDK5 results in synergistic induction of apoptosis. Moreover, concurrent pharmacological perturbation of Mcl-1 and Bcl-xL in pancreatic cancer cell lines using a CDK5 inhibitor analog 24 that reduced Mcl-1 levels and 4-(4-{[2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-[(4-{[(2R)-4-(4-morpholinyl)-1-(phenylsulfanyl)-2-butanyl]amino}-3-[(trifluoromethyl)sulfonyl]phenyl)sulfonyl] benzamide (navitoclax), a Bcl-2/Bcl-xL/Bcl-w inhibitor, resulted in synergistic inhibition of cell growth and induction of apoptosis. In conclusion, we demonstrate targeting CDK5 will sensitize pancreatic cancers to Bcl-2 protein inhibitors. SIGNIFICANCE STATEMENT: Mcl-1 is stabilized by CDK5-mediated phosphorylation in pancreatic ductal adenocarcinoma, resulting in the deregulation of the apoptotic pathway. Thus, genetic or pharmacological targeting of CDK5 sensitizes pancreatic cancers to Bcl-2 inhibitors, such as navitoclax.

Graphical abstract

Fig. 1.

(A) Chemical structure of aminopyrazole analog 24 and its cell-free kinase activity. (B) Concentration-dependent studies with analog 24 in MIA PaCa-2 and HeLa cells treated for 6 hours to assess its activity against CDK2 and CDK5 using western blot analyses. (C) Concentration-response study to determine the cellular CDK5 IC₅₀ value for analog 24 in MIA PaCa-2 cells. (D) Concentration-dependent studies with analog 24 in MIA PaCa-2 and HeLa cells treated for 6 hours to assess its activity against Bcl-xL and Mcl-1 using western blot analyses. Presented blots are representative of at least two independent experiments.

Fig. 2.

Chemical genetic screening strategy to determine selective inactivation of Mcl-1 by analog 24 using doxycycline-inducible cell lines.

Fig. 3.

Chemical genetic screens with caspase 3/7 assay readout. Studies in HeLa-Dox cell lines: (A) concentration-response results with ABT-263 (n = 3, ±S.D.); (B) time course with analog 24 (n = 3, ±S.D.). (C) Concentration-response results with analog 24 (n = 3, ±S.D.). (D) Western blot analyses of concentration-response studies in HeLa-Dox cell lines with analog 24 and palbociclib. Blots are representative of at least two independent experiments. (E) Concentration-response studies in HeLa-GFP cells treated for 6 hours with analog 24 and ABT-263 individually and in combination (n = 3, ±S.D.). (F) Concentration-response studies in HeLa-GFP cells treated for 6 hours with palbociclib and ABT-263 individually and as a combination (n = 3, ±S.D.).

Fig. 4.

Combination studies in pancreatic cancer cell lines with caspase 3/7 assay readout. (A) Concentration-response studies with analog 24 and ABT-263 individually and as a combination in the MIA PaCa-2 cell lines (n = 3, ±S.D.). (B) Analog 24 and ABT-737 individually and as a combination in the MIA PaCa-2 cell lines (n = 3, ±S.D.). (C) Analog 24 and ABT-263 individually and as a combination in the S2-013 cell lines (n = 3, ±S.D.). (D) Analog 24 and ABT-737 individually and as a combination in the S2-013 cell lines (n = 3, ±S.D.).

Fig. 5.

Synergism studies in pancreatic cancer cell lines. (A) Western blot analyses of cleaved PARP and Mcl-1 levels in S2-013 cells treated with ABT compounds, analog 24, or the combination for 12 hours. (B) Combination studies of 4-(2,6-dichlorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide (AT7519), analog 24, and palbociclib with ABT-263 and WEHI-539 in MIA PaCa-2 cells treated for 6 hours. (C) CDK5 knockdown (pooled siRNA) and synergism studies with ABT-263 in S2-013 and MIA PaCa-2 cells. (D) Effect of ABT-263 in S2013 CDK5DN doxycycline-inducible cells. For all of the aforementioned experiments, the presented blots are representative of at least two independent experiments. (E) Dose-effect curve for analog 24 and ABT-263 individually and in combination in MIA PaCa-2 cells. (F) Tables summarizing the median-effective dose (D_m) for analog 24 and ABT-263 individually and in combination in MIA PaCa-2 cells, along with effective dose (ED) and CI values derived from growth inhibition studies in MIA PaCa-2 cells (n = 2); a CI value of 0.7–1.0 is considered mildly synergistic, a CI value of 0.7–0.3 is considered moderately synergistic, and a CI value <0.3 is considered strongly synergistic.

Fig. 6.

Rationale for using analog 24 in pancreatic cancer therapy. (A–D) Analyses of TCGA and GTEx data, mRNA Mcl-1, Bcl-w, Bcl-xL, and Bcl-2 levels in normal pancreas tissue and pancreatic cancer. (E) Basal levels of Mcl-1 in human pancreatic nestin expressing (HPNE) and a panel of pancreatic cancer cell lines. Blots are representative of at least two independent experiments. (F) Our model for combining analog 24 and navitoclax, a Food and Drug Administration–approved Bcl-xL/Bcl-2/Bcl-w inhibitor, in pancreatic cancer therapy.