Telomerase inhibition targets clonogenic multiple myeloma cells through telomere length-dependent and independent mechanisms

Abstract

Background: Plasma cells constitute the majority of tumor cells in multiple myeloma (MM) but lack the potential for sustained clonogenic growth. In contrast, clonotypic B cells can engraft and recapitulate disease in immunodeficient mice suggesting they serve as the MM cancer stem cell (CSC). These tumor initiating B cells also share functional features with normal stem cells such as drug resistance and self-renewal potential. Therefore, the cellular processes that regulate normal stem cells may serve as therapeutic targets in MM. Telomerase activity is required for the maintenance of normal adult stem cells, and we examined the activity of the telomerase inhibitor imetelstat against MM CSC. Moreover, we carried out both long and short-term inhibition studies to examine telomere length-dependent and independent activities.

Methodology/principal findings: Human MM CSC were isolated from cell lines and primary clinical specimens and treated with imetelstat, a specific inhibitor of the reverse transcriptase activity of telomerase. Two weeks of exposure to imetelstat resulted in a significant reduction in telomere length and the inhibition of clonogenic MM growth both in vitro and in vivo. In addition to these relatively long-term effects, 72 hours of imetelstat treatment inhibited clonogenic growth that was associated with MM CSC differentiation based on expression of the plasma cell antigen CD138 and the stem cell marker aldehyde dehydrogenase. Short-term treatment of MM CSC also decreased the expression of genes typically expressed by stem cells (OCT3/4, SOX2, NANOG, and BMI1) as revealed by quantitative real-time PCR.

Conclusions: Telomerase activity regulates the clonogenic growth of MM CSC. Moreover, reductions in MM growth following both long and short-term telomerase inhibition suggest that it impacts CSC through telomere length-dependent and independent mechanisms.

Figure 1. Telomerase is active and can be inhibited in MM CD138⁺ and CD138^neg cells.

(A) Relative telomerase activity in CD138⁺ plasma cells and CD138^neg CSC isolated from MM cell lines. (B) Relative telomerase activity in CD138⁺ plasma cells and CD19⁺CD27⁺ CSC isolated from clinical MM bone marrow samples. (C) Relative telomerase activity in bulk MM cell lines following 72 hours of treatment with vehicle, mismatch control oligonucleotide, or imetelstat (1 uM). (D) Relative telomerase activity in CD138⁺ and CD138^neg NCI-H929 cells after 72 hours of treatment with mismatch control oligonucleotide or imetelstat (1 uM).

Figure 2. Prolonged telomerase inhibition decreases MM CSC telomere length and clonogenic potential.

(A) Percentage of telomeres less than 1.4 kb as determined by STELA after 1, 2, or 3 weeks with mismatch control oligonucleotide or imetelstat (*p-value <0.05 and NS; not significant). (B) Relative clonogenic growth of CD138^neg NCI-H929 and RPMI8226 cells after 3 and 5 weeks of treatment with mismatch control oligonucleotide or imetelstat (1 uM). (C) Relative clonogenic recovery of CD138^neg cells isolated from primary clinical specimens following 3 weeks of treatment with vehicle, mismatch control oligonucleotide or imetelstat (1 uM). (D) Survival of NOD/SCID mice injected with NCI-H929 cells then treated with imetelstat (solid line) or mismatch control oligonucleotide (dashed line) for two weeks in vivo (p<0.01, n = 8,). (E) Survival of NOD/SCID mice after injection with NCI-H929 cells following in vitro treatment with imetelstat (solid line) or mismatch control oligonucleotide (dashed line) for two weeks (P<0.001, n = 20 per group).

Figure 3. Short-term TA inhibition reduces MM CSC activity and promotes differentiation.

(A) Relative clonogenic recovery of CD138^neg NCI-H929, RPMI8226, and U266 cells after treatment with vehicle, mismatch control oligonucleotide or imetelstat for 72 hrs (* p-value<0.01). (B) Relative clonogenic recovery of CD138^neg cells isolated from primary MM specimens following 72 hrs of treatment with vehicle, mismatch control oligonucleotide or imetelstat (1 uM). (C) Percentage of CD138^neg NCI-H929 cells following treatment with imetelstat (1 uM) for 72 hrs (* p-value<0.01). (D) Percentage of ALDH⁺ NCI-H929 cells following treatment with imetelstat (1 uM) for 72 hrs (* p-value<0.01). (E) Expression of OCT3/4, SOX2, NANOG, HES, GAS1 and BMI1 by CD138^neg NCI-H929 cells after 72 hrs of treatment with imetelstat or mismatched control oligonucleotide (1 uM). Data represent fold change in expression relative to mismatch control treated values.