Induction of Sustained Early G1 Arrest by Selective Inhibition of CDK4 and CDK6 Primes Myeloma Cells for Synergistic Killing by Proteasome Inhibitors Carfilzomib and PR-047

Targeting the cell cycle in combination with cytotoxic killing is a rational approach to cancer therapy. Dysregulation of the cyclin-dependent kinases CDK4 and CDK6 precedes uncontrolled proliferation of myeloma cells in vivo, in particular during relapse and drug resistance. This finding reinforces the critical importance of targeting CDK4/6 in myeloma, but success with broad-spectrum CDK inhibitors has been modest. Using the only known selective inhibitor of CDK4/6, PD 0332991, we have developed a novel approach to prime chemoresistant myeloma cells for synergistic killing by diverse cytotoxic agents. We show that selective inhibition of CDK4/6 by PD 0332991 leads to sustained cell cycle arrest in early G1 in the absence of apoptosis. However, it markedly augments cytotoxic killing by PR-171 (carfilzomib), a selective inhibitor of the chymotrypsin-like activity of the proteasome, or PR-047, an orally bioavailable analog of carfilzomib. Synergistic killing of myeloma cells arrested in early G1 by carfilzomib (or PR-047) is caspase-dependent, and requires only a brief (one hour) exposure to the proteasome inhibitor at concentrations as low as 60 nM. This effect is mediated by synergistic and rapid induction of mitochondrial membrane depolarization and activation of downstream caspase-9 within 6 hours of removal of carfilzomib or PR-047. As PD 0332991 acts as an ATP-competitive inhibitor of the CDK4/6 kinase domain, inhibition of CDK4/6 and the cell cycle by PD 0332991 is reversible. Importantly, targeting CDK4/6 with PD 0332991 in combination with either carfilzomib or PR-047 leads to complete eradication of myeloma cells ex vivo, in contrast to the combination of PD 0332991 with other proteasome inhibitors. Selective inhibition of CDK4/6 in combination with carfilzomib (or PR-047), therefore, not only halts cell proliferation but also potently induces synergistic killing that is likely to eliminate cell cycle reentry and generation of resistant cells. PD 0332991 is a small molecule with bio-availability and proven tumor suppressing activity in both human myeloma xenograft and immunocompetent mouse myeloma models. It is well tolerated in humans as shown by the ongoing Phase I/II clinical trials in myeloma and previous phase I trials in mantle cell lymphoma and solid tumors. Evidence from Phase I trials of carfilzomib indicates that it is also well tolerated, in fact, the peripheral neuropathy that is commonly observed with proteasome inhibitor bortezomib appears to be less severe and possibly less frequent. Mechanism-based targeting of CDK4/6 in combination with selective proteasome inhibitors, like carfizomib and PR-047, thus represents a new and promising therapeutic strategy for multiple myeloma and potentially other hematopoietic malignancies.