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. 2017 Nov;31(11):2458-2467.
doi: 10.1038/leu.2017.78. Epub 2017 Mar 8.

Imetelstat, a Telomerase Inhibitor, Differentially Affects Normal and Malignant Megakaryopoiesis

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Free PMC article

Imetelstat, a Telomerase Inhibitor, Differentially Affects Normal and Malignant Megakaryopoiesis

G Mosoyan et al. Leukemia. .
Free PMC article

Abstract

Imetelstat (GRN163L) is a specific telomerase inhibitor that has demonstrated clinical activity in patients with myeloproliferative neoplasms (MPN) and in patients with solid tumors. The antitumor effects were associated with the development of thrombocytopenia, one of the common side effects observed in patients treated with imetelstat. The events underlying these adverse effects are not apparent. In this report, we investigated the potential mechanisms that account for imetelstat's beneficial effects in MPN patients and the manner by which imetelstat treatment leads to a reduction in platelet numbers. Using a well-established system of ex vivo megakaryopoiesis, we demonstrated that imetelestat treatment affects normal megakaryocyte (MK) development by exclusively delaying maturation of MK precursor cells. By contrast, additional stages along MPN MK development were affected by imetelstat resulting in reduced numbers of assayable colony-forming unit MK and impaired MK maturation. In addition, treatment with imetelstat inhibited the secretion of fibrogenic growth factors by malignant but not by normal MK. Our results indicate that the delay observed in normal MK maturation may account for imetelstat-induced thrombocytopenia, while the more global effects of imetelstat on several stages along the hierarchy of MPN megakaryopoiesis may be responsible for the favorable clinical outcomes reported in MPN patients.

Conflict of interest statement

CONFLICT OF INTEREST

KE is a former employee of Geron Corporation. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effects of imetelstat on CFU-MK colony formation. (a) CFU-MK colonies formed by untreated CD34+ cells or by CD34+ cells treated with 7.5 and 15 μM of either mismatched control oligonucleotide (MM1) or imetelstat (GRN163L) were enumerated after 14–16 days of incubation in a collagen-based semisolid media in the presence of thrombopoietin, IL-6 and IL-3. Each column represents the average number of small (3–10 MKs), medium (10–50 MKs) and large (450 MKs) colonies ± s.d. enumerated in three independent experiments. (b) CFU-MK formation by PB-MNCs from HCs (n = 8) and from patients with MPN (n = 15) treated with 15 μM of either mismatched control oligonucleotide (MM1) or imetelstat (GRN163L). The vertical axis indicates CFU-MK clonogenic efficiency after normalization to that of untreated cultures. (c) Representative microphotographs of collagen slabs containing CFU-MK colonies (upper panels) and of individual CFU-MK (lower panels) formed by untreated, MM1- and GRN163L-treated PB-MNCs from one representative MPN patient.
Figure 2
Figure 2
Effects of imetelstat on the normal MK phenotype. (a) MK cultures that were untreated and treated with 7.5 and 15 μM of either MM1 or imetelstat (GRN163L) for 14 days were labeled with FITC-conjugated anti-CD41 and APC-conjugated anti-CD34 antibodies and analyzed by flow cytometry. The columns represent fold changes in the frequency (left y axis) and the absolute number (right y axis) of CD34+/CD41+ MK precursors quantified in the indicated culture conditions. (b) Cells from the same cultures as in panel (a) were labeled with FITC-conjugated anti-CD41 and APC-conjugated anti-CD42b antibodies and analyzed by flow cytometry. The columns represent fold changes in the frequency (left y axis) and the absolute number (right y axis) of mature CD41+/CD42b+ MK quantified in the indicated culture conditions. (c) Representative flow cytometric analyses of MK cultures treated continuously with imetelstat for 9 days (left panel) and of MK cultures in which imetelstat was removed after 6 days (right panel). Immature CD34+/CD41+ and more mature CD34/CD41+ MK populations are indicated by blue and red colors, respectively. The results in panels (a and b) represent the mean ± s.d. of the frequency and corresponding absolute numbers detected in three independent experiments utilizing CD34+ cells from different healthy donors. NS, not statistically significant.
Figure 3
Figure 3
Effects of imetelstat on normal MK maturation. (a) Representative flow cytometric analyses of CD41/CD34 expression in MK cultures that were cultured in the absence of drugs for 7 days (upper panel) and then allowed to mature in the absence (untreated) and presence of 15 μM of either MM1 or imetelstat (GRN163L) for 7 additional days (lower panels). Upper-right quadrants contains CD34+/CD41+ MK precursors. (b) Frequency of CD34+/CD41+ MK precursors quantified in MK cultures that were untreated and treated with 7.5 and 15 μM of either MM1 or imetelstat (GRN163L) as indicated in panel (a). The columns represent the mean ± s.d. of three independent experiments initiated with CD34+ cells from three different healthy donors.
Figure 4
Figure 4
Evaluation of hTERT expression and TA in normal MK. (a) Western blot analyses of protein lysates isolated from MK cultures generated on days 4, 7 and 11 in culture during differentiation and maturation into MK of normal CD34+ cells. After blotting, the membranes were incubated with anti-hTERT and p21 antibodies. Actin expression was utilized as control for protein loading. (b) TA measured in MK cultures on days 7 and 11 during MK maturation. (c) TA measured in primary MK cultures untreated or treated with GRN163L for 7 days. The columns represent the results of duplicate experiments utilizing CD34+ cells from three different normal donors. The y axis in panels (b, c) indicates the number of PCR cycles (Ct) required for the amplification of telomere repeats. A low Ct value indicates a cell extract with high TA, whereas a high Ct value indicates a cell extract with low TA.
Figure 5
Figure 5
Effects of imetelstat on normal MK polyploidization. (a) Representative Wright-Giemsa stained cytospin preparations of cells from control cultures (untreated) and from cultures incubated for 14 days in the presence of 15 μM MM1 or GRN163L. Large, multilobulated MK are present in untreated cultures and in cultures treated with MM1 (arrows in panels (a, b), whereas smaller hypolobulated MK are present in GRN163L-treated cultures (arrow in panel (c)). Microphotographs were obtained using an Olympus BX40 microscope with a dry 40 × /0.75 objective. (b) Quantification of MK ploidy detected after 14 days of incubation in control cultures and in cultures generated in the presence of 7.5 and 15 μM of MM1 or GRN163L. The numbers represent the fraction of CD41+ MK with DNA content 44N normalized to that detected in untreated cultures. (c) CD41+ MK immunomagnetically isolated from cultures generated in the absence of drugs for 9 days were treated with either MM1 or GRN163L and allowed to undergo polyploidization for three additional days. Representative histograms illustrating DNA content (x axis) of untreated and MM1-treated MK indicate the presence of distinct ploidy peaks ranging from 4N to 32N DNA content (arrows in panel (a, b)); such ploidy peaks are absent in imetelstat-treated MK (asterisk in panel (c)).
Figure 6
Figure 6
Effects of imetelstat on DNA synthesis during endomitosis. (a) BrdU incorporation ability of MK cultures grown in the absence (untreated) and presence of 15 μM MM1 or GRN163L. The columns indicate the fractions of BrdU positive MK with low (solid) and high (pattern) ploidy levels, that is, o4N and 44N DNA content, respectively. (b) Representative dot plot histograms of BrdU-labeled MK generated in untreated cultures and in cultures exposed to 15 μM of MM1 or GRN163L for 48 h during MK polyploidization. BrdU incorporation, indicating active DNA synthesis, is represented on the y axis while DNA content as evaluated by 7-AAD staining is represented on the x axis. The fraction of BrdU-positive MK with o4N DNA content and with 44N DNA content are indicated in the blue (P2) and red (P3) gates, respectively.
Figure 7
Figure 7
Effects of imetelstat on MPN megakaryopoiesis. (a) Total number viable cells in untreated or drug-treated (MM1 or GRN163L) MK cultures initiated with PB-MNCs from HCs (n = 4) and from MPN patients (n = 12). (b) Quantification of the absolute number of CD34+/CD41+ MK precursors generated by PB-MNCs from HCs (n = 5) or MPN patients (n = 12) after 7 days in cultures untreated and treated with MM1 or GRN163L. (c) Quantification of the absolute number of CD34/CD41+ MK generated after 7 days in cultures treated with control oligonucleotide (MM1) or GRN163L by PB-MNCs from HCs (n = 6) or MPN patients (n = 12). NS, not statistically significant. *P-value ≤ 0.05, **P-value ≤ 0.005, ****P-value ≤ 0.00005.
Figure 8
Figure 8
Quantitative analysis of the GFs present in MK conditioned media. Media conditioned (CM) by MK cultures generated from untreated HCs and MPN PB-MNCs (a) and by MK cultures treated with MM1 or GRN163L for 7 days (b) were analyzed by multiplex immunoassays to measure the secreted levels of BDNF, FGF-2, PDGF-BB and VEGF-A. The results represent the mean ± s.d. measured in CM generated by cultures from HCs (n = 4) and MPN patients (n = 8). NS, not statistically significant.

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