Combined depletion of cdk2, cdk1 and cdk9 activities induces apoptosis in cancer cells

Proc Amer Assoc Cancer Res, Volume 47, 2006 4898 Cyclin-dependent kinases (cdks) are universally overactive in tumor cells and represent potential anti-neoplastic drug targets. However, it remains unclear whether selective vs. pan-cdk inhibition is superior for inducing anti-tumor effects. Recent data have suggested the depletion of cdk2 activity alone does not lead to antiproliferative effects in cancer cells. On the other hand, expression of cdk2 dominant-negative mutants, cdk2 inhibitory peptides and targeted degradation of cyclin A all lead to effects on S/G2 progression and apoptosis. The latter approaches, however, target both cdk2 and cdk1 suggesting potential benefit for combined depletion of these cdks. To investigate these issues further, we have used siRNA technology to inducibly deplete cdk2, cdk1 and both together from NCI-H1299 non-small cell lung cancer and U2OS osteosarcoma cells. Depletion of cdk2 activity alone leads to a subtle antiproliferative defect in NCI-1299 cells, with slowing of G1 progression following release from a nocodazole-induced mitotic block. Cell cycle effects after cdk2 depletion are not evident in U2OS cells, in which rapid compensation by D-cyclin overexpression occurs. Depletion of cdk1 activity alone leads only to a weak G2/M arrest in NCI-H1299 cells, while depletion of both cdk1 and cdk2 together induces a potent G2/M arrest, eventually followed by a small amount of cell death. In contrast, cdk1 depletion results in G2/M arrest and endoreduplication in U2OS cells, while combined cdk2 and cdk1 depletion slows progression through S phase as well, and is accompanied by a dramatic induction of apoptosis. Combined cdk2 and cdk1 depletion in U2OS cells is preceded by reduced expression of the anti-apoptotic protein XIAP, encoded by an mRNA of short half-life that is depleted after inhibition of cdk-mediated phosphorylation of the C-terminal domain of RNA polymerase II. The CTD is phosphorylated by cdks7 and 9, but may be phosphorylated by other cdks as well in some cellular contexts. XIAP depletion is critical to cell death, as combined depletion of cdk2, cdk1 and cdk9 or cdk2, cdk1 and XIAP substantially increases the degree of apoptosis induced in NCI-H1299 cells compared to the combined depletion of cdk2 and cdk1 alone. Similarly, depletion of cdk1 and cdk2 sensitizes NCI-H1299 cells to flavopiridol, used at concentrations only expected to potently inhibit cdk9. Our data indicate that combined depletion of cdk2, cdk1 and cdk9 can induce apoptosis in tumor cells and suggest that these cdks represent a rational subset of the cdk family for drug targeting.