Cancer Stem Cell Targeting in Multiple Myeloma by GRN163L, a Novel and Potent Telomerase Inhibitor.

Telomerase activity (TA) is required within normal cells capable of long-term replication, including stem cells and is upregulated in many cancers. In the absence of TA or presence of TA inhibitors, the progressive shortening of telomeres ultimately results in cellular senescence and/or apoptosis. These observations support that TA inhibitors represent a novel class of anti-tumor agents. Much evidence suggests that human cancers display a hierarchical cellular organization that mirrors normal tissues. Cancer stem cells (CSC) are derived from the malignant transformation of normal stem cells and progenitors and retain the capacity to self-renew. Moreover, CSC give rise to differentiated tumor cells that form the bulk of the tumor mass, but have little or no capacity for long-term proliferation. We recently demonstrated that the malignant CD138⁺ plasma cells in multiple myeloma (MM) have limited replicative potential; instead they arise from the differentiation of clonogenic CSC that resemble normal memory B cells (CD138^negCD19⁺CD27⁺). In addition, several groups have demonstrated that telomerase inhibitors are active against human MM cell lines in vitro and in vivo. We examined TA in CD138⁺ plasma cells and CD138^neg precursors, and studied the effects of telomerase inhibition against both cell populations. We isolated CD138⁺ and CD138^neg cells by FACS from three human MM cell lines (RPMI 8225, NCI-H929, and U266) and measured TA using a PCR-based assay of activity. For each cell line, TA was detectable within both the CD138^neg and CD138⁺ cell populations. GRN163L is a lipid conjugated 13 nucleotide thio-phosphoramidate oligonucleotide that acts as a potent and specific active site inhibitor of telomerase. We found that treatment with GRN163L (0.1–5μM) markedly reduced TA within 48 hours. To examine the effects of telomerase inhibition on clonogenic growth, we continuously cultured CD138⁺ and CD138^neg RPMI 8226 cells with GRN163L (1μM). Cells were collected weekly, washed to remove GRN163L, and then plated in methylcellulose to assess colony formation. We found that GRN163L was active against both CD138⁺ and CD138^neg cells and eliminated the colony forming potential of both by 5 weeks. Similarly, we found that GRN163L inhibited the in vitro clonogenic growth of CD138^neg MM CSC isolated from the bone marrow aspirates of patients with MM. These data demonstrate that TA is detectable within both immature MM CSC and mature MM plasma cells, and that CSC from both cell lines and primary clinical samples are targeted by the telomerase inhibitor GRN163L. Therefore, this agent may offer a novel therapeutic approach to myeloma as well as other diseases in which CSC have been identified.