Abstract
Multiple myeloma (MM) is proposed to be driven by a CD138− CD20+ population of “MM stem cells” (MMSCs) which give rise to the more “differentiated” CD138+ malignant plasma cells (PCs), which have historically been the main target of anti-MM therapeutics. We hypothesized that inhibition of “differentiation” of these MMSCs to CD138+ derivative MM PCs could allow for expansion of the MMSC compartment and facilitate its cellular and molecular characterization. To test this hypothesis, we performed in vitro studies of MM cells (cell lines and primary MM cells) with reversine (2-(4-Morpholinoanilino)-6-cyclohexylaminopurine), a small molecule known to suppress differentiation (e.g. of myoblasts to myocytes) and/or cause de-differentiation of lineage-committed cells. We observed no reversine-induced expansion in CD138− populations deemed to represent MMSCs and no de-differentiation of CD138+ CD20− MM PCs to more “immature” CD138− CD20+ phenotype. Instead, sub-uM doses of reversine (as low as 0.1 uM, and significantly lower than the >5 uM required for its de-differentiation effects in other tissue type models) triggered major increases in MM cell death, including cells resistant to conventional (e.g. Dex, alkylating agents, anthracyclines, thalidomide, bortezomib, immunomodulatory thalidomide derivatives) and/or investigational agents (e.g. Apo2L/TRAIL) anti-MM agents. PBMCs, stromal cells, and immortalized hepatocytes were less sensitive to reversine than MM cells. Reversine was also active against a broad panel of neoplastic cells, e.g. various leukemia and lymphoma cell lines, suggesting a potentially broader spectrum of anti-neoplastic properties. Reversine was active against MM cells which are independent from or unresponsive to bone marrow stromal cell (BMSC) and was also able to overcome the effect of cytokine- (e.g.IL-6, IGF-1) or stroma-derived protection to BMSCs-responsive MM cells. The anti-MM effect of reversine involved G2 cell cycle arrest and commitment to cell death by drug exposure as short as 8hrs. Cleavage of PARP and caspase 3 were observed, and protein levels for diverse signaling effectors in the B-Raf and Akt cascades were decreased. Reversine enhanced responses of primary MM cells to other anti-MM agents, e.g. Dex, and had additive effects with cytotoxic chemotherapeutics or bortezomib. Reversine affected the molecular profiles of MM cells in a manner distinct from other anti-MM agents studied to date. Unlike bortezomib, reversine did not decrease NF-κB transcriptional activity, or affect the levels of several Bcl-2 family members, inhibitors of apoptosis, or heat shock proteins. These data suggest that reversine kills MM cells in an NF-κB independent manner, and also supports a broader conclusion that reversine has distinct profiles of molecular effects and anti-MM activity. Differential transcriptional profiling of MM cells with >1 vs. <1 log differential sensitivity to the normal tissues tested identified candidate markers correlating with increased reversine sensitivity, including molecules with known roles in malignant transformation, e.g. PKCiota, polo-like kinase-1 and c-myb. These results, coupled with ongoing in vivo studies, suggest that reversine may represent a distinct class of agents with therapeutic potential for specific subset(s) of MM and perhaps other hematologic neoplasias.
THE MODIFICATION OF REPRODUCTION IN INSECTS TREATED WITH ALKYLATING AGENTS. II. DIFFERENTIAL SENSITIVITY OF OOCYTE MEIOTIC STAGES TO THE INDUCTION OF DOMINANT LETHALS