Dear Editor,
Myeloproliferative neoplasms (MPNs) are caused by somatic driver mutations, such as JAK2V617F, which might also affect cellular aging and senescence. Here, we analyzed the heterogeneity of aging in MPN patients and if this can be used to specifically target malignant cells. Our results indicate that cellular aging is accelerated in malignant MPN clones and this can provide a target for treatment with senolytic drugs or telomerase inhibitors.
Aging is reflected by epigenetic modifications, such as changes in DNA methylation (DNAm). We utilized an epigenetic age–predictor based on amplicon deep-sequencing of three age-associated regions (Supplementary Methods) [1]. In healthy donors, there was a high correlation of predictions with chronological age (R2 = 0.86) with a mean age deviation (MAD) of 0.8 years (Supplementary Fig. S1a). In contrast, epigenetic age-predictions of 128 MPN patients showed much higher offsets, particularly in the advanced MPN entity primary myelofibrosis (PMF; Fig. 1a). Overall, epigenetic aging was rather increased, resulting in a positive delta-age (predicted age—chronological age; Fig. 1b).
Telomere length (TL) attrition represents another denominator of cellular aging and its impact has been studied extensively in chronic myeloid leukemia (CML), where TL is shortened in the malignant stem cell clone [2]. Flow-FISH analysis of TL in granulocytes revealed a clear association with chronological age in 134 healthy donors, albeit the correlation was lower than for epigenetic age-prediction (R2 = 0.18; Supplementary Fig. S1b). In the 128 MPN samples, the distribution was overall similar (Fig. 1c), while a significant TL attrition was only observed for the PMF samples (p = 0.0009; Fig. 1d). Interestingly, there was only a moderate correlation between TL attrition and epigenetic delta-age (Supplementary Fig. S1c). Thus, TL and epigenetic age seem to reflect different biological properties of aging.
Next, we analyzed if these measures for cellular aging were associated with specific somatic mutations using a next-generation sequencing panel of 32 genes. Epigenetic age deviation showed a significant increase in patients with JAK2V617F (p < 0.001) and CALR mutations (p = 0.013; Supplementary Fig. S1d), and age-adjusted TL in granulocytes showed significantly accelerated shortening in samples with mutation in CALR (p = 0.025) and ASXL1 (p = 0.004; Supplementary Fig. S1e). Notably, in patients carrying the JAK2V617F mutation, there was a significant association of epigenetic age acceleration (p = 0.0031; Fig. 1e), similar to what was shown in polycythemia vera (PV) in another study [3], as well as telomere attrition (p < 0.0001; Fig. 1f) with mutational burden if all MPN subgroups were combined.
To further investigate cellular aging in the malignant clone of patients with MPN, we analyzed single cell-derived colony forming units (CFUs). Epigenetic age predictions were exemplarily performed for 5 wild type (WT) and 5 JAK2V617F colonies for an individual patient per MPN sub-entity. There was some variation between individual colonies, but overall, the epigenetic age predictions were consistently higher for the mutated clones (Fig. 2a). In analogy, we analyzed TL in individual JAK2V617F and JAK2WT colonies with TEL-PCR in 10 PV patients and 7 healthy donors (Fig. 2b and Supplementary Fig. S2a). Overall, the mean TL of colonies with JAK2V617F mutation was significantly shorter than in WT colonies (p = 0.0075). The degree of telomere shortening in JAK2V617F colonies (ΔTLmut-WT) was found to correlate significantly with the patient’s allele burden (Supplementary Fig. S2b). However, these associations were not observed in 7 PMF patients (Fig. 2c and Supplementary Fig. S2c, d).
To understand if the JAK2V617F mutation directly affects cellular aging parameters, we used previously established syngeneic induced pluripotent stem cell (iPSC) models [4]. Corresponding iPSC lines were generated from three MPN patients with WT JAK2, heterozygous and homozygous JAK2V617F mutations. These clones were then differentiated toward the hematopoietic lineage (Supplementary Fig. S3a). Here, we focused particularly on the cellular aging in the iPSC-lines and iPSC-derived hematopoietic cells. To estimate epigenetic age, we used two multi-tissue epigenetic predictors: the Horvath epigenetic clock [5] (Fig. 2d) and Horvath Skin and Blood clock [6] (Supplementary Fig. S3b). There was a tendency towards increased epigenetic age in the JAK2V617F mutation, but the predictions were overall close to 0 years, as generally observed upon reprogramming [7]. Furthermore, TL analysis did not reveal consistent telomere shortening in the JAK2 mutant clones (Supplementary Fig. S3c). Thus, the impact of JAK2V617F on acceleration of cellular aging appears to be minimal upon short-term differentiation in the iPSC model.
Next, we analyzed vav-cre driven Jak2V617F transgenic mice after development of an MPN-like phenotype (Supplementary Fig. 4a) [8]. When we analyzed epigenetic age in unfractionated bone marrow cells, there was a significant acceleration in mice with Jak2V617F mutation (n = 4) as compared to WT Jak2 (n = 9; Fig. 2e, p = 0.017). TL did not differ between Jak2WT and Jak2V617F (Supplementary Fig. S4b), which might be due to different expression of telomerase and regulation of TL in mice.
Senolytic drugs raised hopes to specifically target prematurely aged cells that show signs of senescence and to thereby rejuvenate tissues [9]. We anticipated that particularly the malignant clones, revealing signs of accelerated aging and displaying a senescence-associated gene signature (Supplementary Fig. S5), might be more susceptible to senolytic drugs than the remaining non-mutated cells. We cultured peripheral blood mononuclear cells (PBMCs) of nine MPN patients with JAK2V617F mutation for three days with eight different senolytic drugs using the concentrations below and above the IC50: piperlongumine, ABT-263, RG-7112, nutlin-3a, dasatinib in combination with quercetin, AMG-232, JQ1, and S63845. Furthermore, we tested the telomerase inhibitor BIBR-1532. After three days, the remaining JAK2V617F mutation burden was analyzed with digital droplet PCR. Overall, the senolytic drugs had only a moderate specific effect on the mutated subsets. Only JQ1, a potent inhibitor of the BET family of bromodomain proteins, and piperlongumine, an amide alkaloid constituent of the long pepper, showed a moderate but significant reduction of the JAK2V617F allele burden (for piperlongumine only at low concentration; Fig. 2f). Simultaneously, we analyzed if the treatment would also affect epigenetic age predictions (n = 7 for each compound). A moderate, non-significant reduction of epigenetic age-predictions was observed for RG7112 and again for piperlongumine (Supplementary Fig. S6a), potentially due to the depletion of clonal cells with premature epigenetic age. Furthermore, we measured TL with TEL-PCR (n = 5 for each compound). A significant increase in TL was again observed with JQ1, piperlongumine and nutlin-3a (Supplementary Fig. S6b). Notably, Kleppe and colleagues demonstrated a reduction in disease burden of MPN by JQ1 treatment either alone or in combination with ruxolitinib [10]. Taken together, at least JQ1 and piperlongumine might have a selective effect for malignant cells with accelerated cellular aging, although the compounds may also have non-senolytic effects, particularly at these relatively high concentrations during short-term in vitro treatment.
The use of telomerase inhibitors (TI) is an option to target malignant cells with shorter telomere length. BIBR-1532 is a potent telomerase inhibitor that has been tested previously in CML [11], but not yet systematically on BCR-ABL-negative MPN cells. Although short-term treatment with BIBR-1532 did not reduce the prominent malignant clone with accelerated aging, we hypothesize that inhibition of telomerase mediates proliferation-dependent critical telomere shortening, which eventually leads to telomere-mediated senescence or apoptosis. Therefore, an effect on subclones with particularly short TL might be observed only after longer treatment. To this end, we cultured PMF-derived PBMC (n = 9) in CFU assays with or without BIBR-1532, picked 30 colonies per condition, and analyzed the percentage of mutated colonies (JAK2V617F and CALR rearrangements). Notably, the fraction of mutated colonies declined particularly in those patients with preexisting shorter mean TL in granulocytes by flow-FISH analysis (R2 = 0.68; p = 0.0064; Fig. 2g), indicating that samples with accelerated telomere attrition are more susceptible to telomerase inhibition.
Subsequently, we performed ß-galactosidase staining within individual colonies (Supplementary Fig. S7a). In the three PMF donors with very short telomeres, the fraction of senescent cells clearly increased upon BIBR-1532 treatment (Fig. 2h). In contrast, this effect was not observed in the three patients that revealed longer telomeres (Fig. 2i). To further substantiate if the potential triggering of senescence occurs specifically in the malignant cells, we further analyzed the genotype in these colonies. BIBR-1532 treatment seems to specifically increase the fractions of senescent cells in CFUs carrying JAK2V617F mutation or CALR rearrangements in patients with shorter telomeres (Supplementary Fig. S7b, c). While BIBR-1532 has so far not been evaluated in clinical trials, telomerase inhibition with imetelstat has already demonstrated clinical activity in primary, post-essential thrombocythemia or post-PV myelofibrosis [12,13,14]. Another study on non-small cell lung cancer cell lines demonstrated that particularly those lines with short TL were susceptible to imetelstat treatment [15]. It will, therefore, be important to determine if particularly patients with short telomeres at diagnosis profit from imetelstat treatment and if this telomerase inhibitor might also induce senescence in the mutant compartment of MPN. It might even be conceivable to combine TI and senolytics to accelerate senescence specifically in the malignant stem cell clones in MPN patients (by the TI component) and to finally eradicate it (with a senolytic drug).
Data availability
The datasets used and/or analyzed in this current study are available from the corresponding author on reasonable request.
References
Han Y, Franzen J, Stiehl T, Gobs M, Kuo CC, Nikolic M, et al. New targeted approaches for epigenetic age predictions. BMC Biol. 2020;18:71.
Brummendorf TH, Holyoake TL, Rufer N, Barnett MJ, Schulzer M, Eaves CJ, et al. Prognostic implications of differences in telomere length between normal and malignant cells from patients with chronic myeloid leukemia measured by flow cytometry. Blood. 2000;95:1883–90.
McPherson S, Greenfield G, Andersen C, Grinfeld J, Hasselbalch HC, Nangalia J, et al. Methylation age as a correlate for allele burden, disease status, and clinical response in myeloproliferative neoplasm patients treated with vorinostat. Exp Hematol. 2019;79:26–34.
Satoh T, Toledo MAS, Boehnke J, Olschok K, Flosdorf N, Gotz K, et al. Human DC3 antigen presenting dendritic cells from induced pluripotent stem cells. Front Cell Dev Biol. 2021;9:667304.
Horvath S. DNA methylation age of human tissues and cell types. Genome Biol. 2013;14:R115.
Horvath S, Oshima J, Martin GM, Lu AT, Quach A, Cohen H, et al. Epigenetic clock for skin and blood cells applied to Hutchinson Gilford Progeria Syndrome and ex vivo studies. Aging (Albany NY). 2018;10:1758–75.
Weidner CI, Lin Q, Koch CM, Eisele L, Beier F, Ziegler P, et al. Aging of blood can be tracked by DNA methylation changes at just three CpG sites. Genome Biol. 2014;15:R24.
Dagher T, Maslah N, Edmond V, Cassinat B, Vainchenker W, Giraudier S, et al. JAK2V617F myeloproliferative neoplasm eradication by a novel interferon/arsenic therapy involves PML. J Exp Med. 2021;218:e2020126.
Zhu Y, Tchkonia T, Pirtskhalava T, Gower AC, Ding H, Giorgadze N, et al. The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs. Aging Cell. 2015;14:644–58.
Kleppe M, Koche R, Zou L, van Galen P, Hill CE, Dong L, et al. Dual targeting of oncogenic activation and inflammatory signaling increases therapeutic efficacy in myeloproliferative neoplasms. Cancer cell. 2018;33:785–7.
Brassat U, Balabanov S, Bali D, Dierlamm J, Braig M, Hartmann U, et al. Functional p53 is required for effective execution of telomerase inhibition in BCR-ABL-positive CML cells. Exp Hematol. 2011;39:66–76 e1. -2
Tefferi A, Lasho TL, Begna KH, Patnaik MM, Zblewski DL, Finke CM, et al. A pilot study of the telomerase inhibitor imetelstat for myelofibrosis. N Engl J Med. 2015;373:908–19.
Baerlocher GM, Oppliger Leibundgut E, Ottmann OG, Spitzer G, Odenike O, McDevitt MA, et al. Telomerase inhibitor imetelstat in patients with essential thrombocythemia. N Engl J Med. 2015;373:920–8.
Mascarenhas J, Komrokji RS, Palandri F, Martino B, Niederwieser D, Reiter A, et al. Randomized, single-blind, multicenter phase II study of two doses of imetelstat in relapsed or refractory myelofibrosis. J Clin Oncol. 2021;39:2881–92.
Frink RE, Peyton M, Schiller JH, Gazdar AF, Shay JW, Minna JD. Telomerase inhibitor imetelstat has preclinical activity across the spectrum of non-small cell lung cancer oncogenotypes in a telomere length dependent manner. Oncotarget. 2016;7:31639–51.
Acknowledgements
We thank Anne Abels for technical assistance and Kim Kricheldorf for organizing the patient samples, Isabelle Plo and the Institut Gustave-Roussy, Villejuif, France, for kindly providing the Jak2V617F transgenic mice, and Caroline Küstermann and Janik Böhnke for support in iPSC culture.
Funding
This work was supported by the Flow Cytometry Facility, a core facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University. We thank Lichterzellen (www.lichterzellen.de) and AA/PNH e.V. for their initiative, enthusiasm and continued support to THB and MV; MAST was funded by CAPES-Alexander von Humboldt postdoctoral fellowship (99999.001703/2014-05) and donation by U. Lehmann. This work was supported by funds from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) within CRU344 “Untangling and Targeting Mechanisms of Myelofibrosis in Myeloproliferative Neoplasms” (WA 1706/12-1 and WA1706/14-1; KO2155/7-1; BR1782/5-1; ZE432/10-1) and KO2155/6-1; by Deutsche Krebshilfe (TRACK-AML); and the ForTra gGmbH für Forschungstransfer der Else Kröner-Fresenius-Stiftung.
Author information
Authors and Affiliations
Contributions
MV, VT, and MKa performed experiments and analyzed the results. MN performed BA-seq analysis. MS supported cell culture and sequencing experiments. MKi analyzed NGS analysis of mutational burden. JB supported gene expression analysis and the murine model system. NF, MAST, and MZ generated and characterized the JAK2V617F iPSC lines. SK interpreted data and supported analysis. THB, FB, and WW conceptually designed the study and supervised the research. MV, VT, and WW wrote the first draft of manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Competing interests
WW and VT are involved in Cygenia GmbH (www.cygenia.com), which can provide services for DNA methylation analysis to other scientists. THB and FB have a scientific collaboration with Repeat Dx (Vancouver, Canada). SK reports research funding, advisory board honoraria, honoraria, and other financial support (e.g., travel support) from Janssen and Geron; and a patent for a BET inhibitor at RWTH Aachen University. All other authors declare no competing financial interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Vieri, M., Tharmapalan, V., Kalmer, M. et al. Cellular aging is accelerated in the malignant clone of myeloproliferative neoplasms. Blood Cancer J. 13, 164 (2023). https://doi.org/10.1038/s41408-023-00936-1
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41408-023-00936-1