Imetelstat inhibits TA in MM plasma cells and CSC

MM plasma cells express the characteristic surface antigen CD138, but the self-renewing cells responsible for tumor initiation in immunodeficient mice phenotypically resemble memory B cells and are CD138^neg [2]. We initially examined relative TA within CSC and bulk tumor cells and isolated CD138^neg precursors and mature CD138⁺ plasma cells from pre-sorted MM cell lines. In the RPMI8226 cell line, CD138^neg CSC displayed greater TA than CD138⁺ plasma cells, while this pattern was reversed in NCI-H929 cells (Figure 1A). In the U266 cell line, TA was similar between the two cell compartments. We also studied primary clinical specimen from three distinct patients and found that the levels of TA were similar in CD138^negCD19⁺CD27⁺ precursors and CD138⁺ plasma cells (Figure 1B). Taken together, these results demonstrate that telomerase is active in both MM CSC and plasma cells, but relative levels within each cell compartment may vary within individual cell lines and patient specimens.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

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).

https://doi.org/10.1371/journal.pone.0012487.g001

Several approaches have been developed to target telomerase including antisense RNA directed against TERT, small molecules that stabilize G-quadruplexes, and oligonucleotides acting as direct inhibitors of the reverse transcriptase activity [27]–[31]. Imetelstat is a synthetic lipid-conjugated 13-base oligonucleotide N3′ P5′-thio-phosphoramidate complementary to the template region of telomerase RNA (TERC) that acts as a competitive enzyme inhibitor by binding and blocking the active site of the enzyme [10], [11], [14]. This highly specific inhibitor of TA has been clinically studied in patients with MM, chronic lymphocytic leukemia, non-small cell lung cancer, and breast cancer [26], [32]. We studied the ability of imetelstat to inhibit TA in MM and treated RPMI8226, NCI-H929, and U266 cells for 48 hours. Using the TRAP assay, we detected at least a 60% reduction in TA in cells treated with imetelstat compared to a mismatched control oligonucleotide (Figure 1C). Similar degrees of telomerase inhibition have been reported in MM and glioblastoma cells [11], [33]. We also examined telomerase inhibition in CD138^neg precursors and CD138⁺ plasma cells isolated from the NCI-H929 cell line and found that imetelstat inhibited TA by 58% and 74% in each of these cell populations, respectively (Figure 1D).

Imetelstat reduces telomere length and limits the clonogenic growth of CD138^neg MM CSC

Several reports have demonstrated that telomerase inhibition in CD138⁺ MM plasma cells can reduce telomere length to a critical threshold resulting in senescence and/or apoptosis[10]–[14], [34]. We examined the impact of imetelstat on MM CSC telomere length and continuously treated pre-sorted CD138^neg cells isolated from the NCI-H929 cell line. By two weeks, cells treated with imetelstat displayed a significantly increased proportion (p<0.01, Figure 2A) of shortened telomeres (<1.4 kb) using the single telomere length assay (STELA)[35]. In order to examine the effects of telomerase inhibition and progressive telomere shortening on the clonogenic growth of MM CSC, CD138^neg cells were isolated from the RPMI8226 and NCI-H929 cell lines, continuously treated with imetelstat, and plated weekly in methylcellulose. Colony formation was progressively inhibited with a 5-fold reduction at three weeks and greater than 100-fold reduction after five weeks of treatment (Figure 2B). We also examined primary clinical MM specimens and treated pre-sorted CD138^neg bone marrow cells with imetelstat. Compared to the control oligonucleotide, MM clonogenic growth was significantly inhibited by approximately 5-fold after 3 weeks of imetelstat treatment compared to the mismatch control group (P<0.01, Figure 2C).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

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).

https://doi.org/10.1371/journal.pone.0012487.g002

We also examined the effects of imetelstat on a MM growth in vivo. Previous studies demonstrated that imetelstat reduced the growth of subcutaneous tumors in SCID mice [11], [14]. In order to examine the effects of imetelstat on systemic MM growth, NOD/SCID mice were injected with NCI-H929 cells then treated with imetelstat two weeks later. Treatment with imetelstat given three times a week for a total of two weeks significantly extended survival compared to the control mismatch oligonucleotide (p<0.01, Figure 2D). Since this study could not distinguish between the inhibition of bulk tumor growth or the self-renewal capacity of CSC, we next examined the effect of imetelstat on MM engraftment, a surrogate measure of self-renewal potential. We treated NCI-H929 cells with imetelstat or mismatch control for two weeks in vitro and subsequently injected them intravenously into NOD/SCID mice. Pre-treatment of MM cells with imetelstat significantly reduced the proportion and survival of mice engrafting with MM compared to control oligonucleotide treated cells (p<0.001, Figure 2E). Therefore, telomerase inhibition by imetelstat significantly reduces MM clonogenic potential both in vitro and in vivo in parallel with its effects on telomere length.

Short-term telomerase inhibition promotes MM differentiation and depletes CSC activity

During the long-term treatment studies, we found that the clonogenic growth of pre-sorted CD138^neg RPMI8226, NCI-H929, and U266 MM CSC was significantly inhibited by 11–30% following just 72 hours of imetelstat exposure (p<0.01, Figure 3A), although telomere length was not significantly affected in NCI-H929 cells after 1 week of treatment (p>0.05, Figure 2A). Moreover, this effect was not mediated by the induction of cell death as we found no difference in the proportion of apoptotic cells following 72 hours of exposure to imetelstat (data not shown). Similarly, we found that short-term treatment of pre-sorted CD138^neg bone marrow cells from primary clinical specimens with imetelstat significantly inhibited MM colony formation by 19-29% compared to mismatch control treated samples (p<0.05, Figure 3B). These data suggest that short-term imetelstat treatment in MM impacts the functional capacity of MM CSC independent of its effects on telomere length.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

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.

https://doi.org/10.1371/journal.pone.0012487.g003

Several recent reports have suggested that telomerase may regulate the differentiation and self-renewal of normal stem cells independent of its effects on telomere length [33], [36]. Therefore, we examined the impact of short-term imetelstat treatment on the differentiation of MM CSC. Treating presorted CD138^neg with imetelstat, control oligo or vehicle, resulted in increased expression of CD138 after just 72 hours without a significant difference between the groups (Data not shown). To specifically evaluate the effects of imetelstat on this process we evaluated the proportion of CD138^neg cells in unsorted bulk cultures after 72 hours. We found that the proportion of CD138^neg cells was decreased by approximately 40% after short-term imetelstat treatment of unsorted H929 cells (p<0.01, Figure 3C). MM CSC also express relatively higher levels of ALDH activity compared to CD138⁺ plasma cells [2], and short-term imetelstat treatment significantly decreased the ALDH⁺ population from 1.3% to less than 0.4% (p<0.01, Figure 3D). These data suggest that short term TA inhibition depletes the population of MM CSC by inducing their terminal differentiation.

We hypothesized that telomerase may globally influence pathways that regulate CSC cell-fate decisions and self-renewal. Within embryonic stem cells, specific transcriptional regulators, such as OCT3/4, NANOG, and SOX2 are critical for regulating self-renewal and pluripotency [37]–[39]. Moreover, developmental signaling pathways, such as Notch and Hedgehog, as well as the polycomb gene BMI1 are thought to regulate both normal and cancer stem cells [40]–[43]. We examined the expression of OCT3/4, NANOG, SOX2 and BMI1 as well as the Hedgehog and Notch target genes, GAS1 and HES1 in NCI-H929 cells and found that all were detectable in the CD138^neg CSC, but not the bulk CD138⁺ plasma cells (data not shown). Following treatment of CD138^neg NCI-H929 CSC with imetelstat for 72 hours, the expression of five out of six of these stem cell genes was inhibited compared to mismatch control treated cells (Figure 3E). These results suggest that short-term telomerase inhibition may directly impact stem cell programs that regulate CSC self-renewal.

Patient samples, cell lines and cell culture

Bone marrow samples were obtained from patients with active MM granting signed consent for sample acquisition using a protocol and consent forms approved by the Johns Hopkins Medical Institutes Review Board. All patients were newly diagnosed and included those with Stage II (n = 2) and Stage 3 (n = 4) disease according to International Staging System (ISS) criteria [64]. Mononuclear cells were isolated by density centrifugation (Ficoll-Paque, Pharmacia, Piscataway, NJ) and depleted of CD34⁺ hematopoietic progenitors using CD34 magnetic microbeads (Miltenyi Biotech, Auburn, CA). Plasma cells were further isolated by positive magnetic selection using CD138 selection beads (Miltenyi Biotech). CD138^negCD19⁺CD27⁺ MM CSC were isolated by FACS (see below). Isolated cells were also assessed for clonotypic immunoglobulin light chain expression by flow cytometry and demonstrated >90% isotype restriction matching the malignant plasma cells.

The human MM cells lines NCI-H929, RPMI8226 and U266 were obtained from the American Type Tissue Collection (Manassas, VA). All cells were cultured in complete media consisting of RPMI 1640, 2 mM L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin, and 10% fetal bovine serum (FBS). For drug treatment studies cells were treated with 1 µM imetelstat, GRN163L (5′-palm-TAGGGTTAGACAA-3′) or 1 µM mismatch control oligonucleotide, GRN140833 (5′-palm-TAGGTGTAAGCAA-3′) (Geron Corporation, Menlo Park, CA) prepared in phosphate buffered saline.

For the analysis of in vitro clonogenic growth treated cells were washed free of drug and plated at a density of 1,000 cells/mL for cell lines or 200,000 cells/mL for CD138^negCD34^neg bone marrow mononuclear cells in 1.2% methylcellulose, 30% FBS, 1% bovine serum albumin (BSA), 10⁻⁴ M 2-mercaptoethanol, and 2 mM L-glutamine. For clinical specimens, the methylcellulose cultures also contained 10% lymphocyte conditioned media as a source of growth factors [2]. Samples were plated in triplicate onto 35 mm² tissue culture dishes and incubated at 37°C and 5% CO₂. Colonies consisting of more than 40 cells were scored between 14 and 21 days with an inverted microscope. Serial replating was performed by collecting methylcellulose and washing cells in complete media then resuspending a fixed proportion (1/10–1/1,000) of the cells in 1 ml of methylcellulose as described above.

Telomerase activity and telomere length quantification

Telomerase activity was quantified using a real-time PCR based quantitative telomerase detection kit according to manufacturer's instructions (Allied Biotech Inc., Ijamsville, MD). Equal cell numbers (1×10⁵–10⁶ cells) were used for each experiment and relative telomerase activity was calculated based on the value of 2 ∧ difference in C_T value. XpYp single telomere length assay (STELA) was performed using the methods described by Baird et al.[35] Telomere signals were detected using TeloTAGGG Telomere Length Assay kit (Roche, San Francisco, CA). Four separate PCR reactions (1–2 ng genomic DNA per reaction) were set up for each sample and run in separate lanes on agarose gels. Total number of telomere bands from the lanes for each sample were pooled and calculated. Telomere shortening was quantified by determining the percentage of telomere bands less than 1.4 kb to the total number of bands in the sample.

Fluorescence-activated cell sorting and flow cytometry

For antigen expression evaluation and cell sorting, anti-human CD138-fluourescein isothyocyanate (FITC), CD19-FITC, and CD27-allophycocyanin (APC) antibodies were used (BD Pharmingen, San Diego, CA). Aldehyde dehydrogenase (ALDH) activity was detected using the Aldefluor reagent (Stem Cell Technologies, Vancouver, Canada) according to manufacturer's instructions. Control staining reactions consisted of isotypic antibodies or addition of the specific ALDH inhibitor diethylaminobenzaldehyde (DEAB). CD138⁺ and CD138^neg cells were isolated from human MM cell lines by staining with anti-CD138 as well as 2 µg/ml propidium iodide (PI, BD Biosciences, San Jose, CA). Cells were initially gated on a FACSAria cell sorter (BD Biosciences) by forward and orthogonal light scatter, then analysed to collect CD138^negPI^neg and CD138⁺PI^neg cells. For clinical specimens, MM CSC were isolated by staining peripheral blood mononuclear cells with anti-CD19, anti-CD27, and PI. Cells were initially gated to eliminate cellular aggregates and necrotic material by forward and orthogonal light scatter and PI exclusion, then analyzed for CD19 and CD27 expression. Apoptosis analysis was performed using Annexin V (BD Biosciences) and PI staining. Briefly, cells were incubated with Annexin V-FITC in Annexin binding buffer followed by staining with 5 µg/ml PI and analysis by flow cytometry. Annexin^negPI^neg cells were considered viable and compared across treatment groups. Cell surface antigen or ALDH expression was examined by counterstaining cells with 2 µg/ml PI followed by analysis of cells initially gated to eliminate cellular aggregates and necrotic material by forward and orthogonal light scatter and PI exclusion quantification. Cells were analyzed using either a FACSCalibur flow cytometer (BD Biosciences) or FACSAria cell sorter.

Transplantation of MM cells into NOD/SCID mice

All animal studies were approved by the Johns Hopkins Medical Institutes Animal Care Committee. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were bred and maintained in the Johns Hopkins animal core facility. NCI-H929 cells (1×10⁶–10⁷ cells/animal) were injected into the tail vein of 6–10 week old mice. For in vivo treatment experiments, imetelstat or mismatch control oligonucleotide (30 mg/kg) was injected intraperitoneally three times a week for two weeks. Mice were observed daily and sacrificed following the development of symptoms. MM engraftment was confirmed by detecting human κ immunoglobulin light chain in mouse sera using ELISA (Bethyl Labs, Montgomery, TX) and human CD138⁺ plasma cells in the mouse bone marrow by flow cytometry.

Reverse transcriptase PCR

RNA was isolated using RNAqueous (Ambion, Austin, TX), and cDNA made using Superscript II (Invitrogen) with random hexamers according to the manufacture's protocol. For quantitative RT-PCR Taqman real-time primer/probes sets (Applied Biosystems, Foster City, CA) were used to detect cDNA for GAS1 (Hs00266715_s1), NANOG (Hs0238700_g1), SOX2 (Hs01053049_s1), HES (Hs00707120_s1), OCT3/4 (Hs00999652_g1) and BMI1 (Hs00180411_m1) and β-actin (4352935E). PCR was performed using TaqMan Universal Master Mix (4304437, ABI) on an I-Cycler Real-Time PCR machine (BioRad, Hercules, CA). Expression levels were normalized to β-actin and compared with the ΔΔC_T method.

Statistical analysis

Results are presented as the mean ± SEM. Comparisons between groups were performed using a 2-tailed, paired Student t test. Kaplan-Meier analysis was carried out using a log rank test. P values <0.05 were considered significant.