Abstract 5367: Simultaneous inhibition of Chk1 and MEK1/2 targets non-proliferating multiple myeloma cells
Authors: Pei Xin-Yan , Dai Yun , Youssefian Leena E. , Chen Shuang , Bodie Wesley , Takabatake Yukie , Almenara George , Kramer Lora , Dent Paul , Grant Steven 
Published: June 2012

Abstract
One potential barrier to successful multiple myeloma (MM) treatment stems from the observation that it generally represents a low growth fraction disease with cells resting in a quiescent, non-proliferative state (i.e., &lt; 5% of cells actively cycling) before progressing to the terminal phase. We have previously described synergistic interactions between Chk1 and MEK1/2 inhibitors in cycling malignant cells. The present goal was to investigate the activity of specific and clinically relevant Chk1 (e.g., AZD7762) and MEK1/2 (e.g., selumetinib, AZD6244) inhibitor regimens in low or non-proliferating MM cells. Methods: MM cell lines cultured in standard (10%) or low (0.05-0.2%) serum and primary non-cycling CD138+ MM cells were treated (24-48 h) with Chk1(50-600 nM) and MEK1/2 (0.5-5uM) inhibitors alone or in combination. Studies included apoptosis (annexin V/PI), clonogenicity, comet assays, Western blot, RNAi and transfection, cell cycle and caspase-3 cleavage/DNA content quantitative analysis, immunofluorescence (IFC) staining, and multi-parameter (Hst/PY/7AAD) FCM analysis. Results: AZD7762/AZD6244 (or CEP3891/PD184352) co-exposure arrested cells in G0/G1 and synergistically induced apoptosis in IL-6-dependent or -independent cells and CD138+ primary samples, but spared normal CD138- and CD34+ cells. MM cells cultured in low serum (0.05-0.2%) underwent G0/G1 arrest/enrichment without reduced viability. G0/G1-enriched cells exhibited diminished sensitivity to conventional agents (e.g., taxol, VP-16) but significantly increased susceptibility to Chk1 inhibitors alone or in combination with MEK1/2 inhibitors, or Chk1 siRNA knockdown. Exposure of G0/G1-enriched cells to Chk1/MEK1/2 inhibitor regimens displayed increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knockdown markedly attenuated lethality. Flow cytometric analysis of asynchronized MM cells by cleaved caspase-3/DNA content quantification indicated sharply increased Chk1/MEK1/2 inhibitor-mediated apoptosis in G0/G1 cells. IFC and FCM analysis of growth-arrested or primary MM cells demonstrated co-localization of activated caspase-3 and statin, a quiescent (G0) nuclear envelope protein. Finally, multi-parameter flow cytometric analysis indicated that Chk1/MEK1/2 inhibitor regimens increased cell death (7AAD+) in Hst+, low pyronin-staining (2N Hst+/PY-) G0 MM cells. Conclusions: G0/G1 cells are highly sensitive to simultaneous Chk1 and MEK1/2 inhibition, potentially providing a mechanistic basis for the pronounced susceptibility of low- or non-proliferating primary MM cells to this strategy. Notably, tumor stem or initiating cells are cytokinetically quiescent, which may cause resistance to cytotoxic agents. The present findings raise the possibility that such cells might be susceptible to Chk1/MEK1/2 inhibitor strategies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5367. doi:10.1158/1538-7445.AM2011-5367