Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2

Abstract

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138⁺ cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138⁺ primary samples, but spared normal CD138⁻ and CD34⁺ cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G₀/G₁ arrest and increased apoptosis in all cell-cycle phases, including G₀/G₁. To determine whether this regimen is active against quiescent G₀/G₁ MM cells, cells were cultured in low-serum medium to enrich the G₀/G₁ population. G₀/G₁–enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G₀/G₁–enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G₀) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst⁺), low pyronin Y (PY)–staining (2N Hst⁺/PY⁻) G₀ population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.