Gandotinib | P53 Signal

JAK2 inhibition in JAK2V617F-bearing leukemia cells enriches CD34þ leukemic stem cells that are abolished by the telomerase inhibitor GRN163L

Jenny Dahlstro€m a, *, Chuanyou Xia a, Xiangling Xing a, Xiaotian Yuan b, **,
Magnus Bjo€rkholm a, Dawei Xu a
a Department of Medicine, Division of Hematology, Center for Molecular Medicine (CMM) and Bioclinicum, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden
b School of Medicine, Shandong University, Jinan, PR China

A R T I C L E I N F O

Article history:
Received 24 March 2020
Accepted 13 April 2020 Available online xxx

Keywords: JAK2 mutation KLF4
MPN
Leukemia stem cell Telomerase
TERT

A B S T R A C T

The activating-mutation of JAK2V617F drives the development of myeloproliferative neoplasms (MPNs). Several JAK2 inhibitors such as ruxolitinib and gandotinib (LY2784544) currently in clinical trials and, provide improvements in MPNs including myeloﬁbrosis. However, JAK2 inhibitors are non-curative and murine experiments show that JAK2 inhibitors don’t eradicate MPN stem cells and it is currently unclear how they escape. We thus determined the effect of the speciﬁc JAK2V617F inhibitor LY2784544 on
leukemic stem (CD34þ) cells (LSCs) using the JAK2V617F-bearing erythroleukemia cell line HEL. The
LY2784544 treatment caused a transient proliferation inhibition and apoptosis of HEL cells, but a re- covery occurred within a week. Thereafter, the continuous LY2784544 exposure induced the accumu- lation of CD34þ LSCs, and the CD34þ cells increased from 2% to >90% by week 9, which was accompanied by increased clonogenic potentials. LY2784544 was capable of stimulating CD34 expression even in
CD34— HEL cells, which indicated cellular de-differentiation. A signiﬁcantly enhanced expression of the stem cell factor KLF4 was observed in LY2784544-treated HEL cells. Inhibiting KLF4 expression attenu- ated LY2784544-mediated accumulation of CD34þ LSCs. Moreover, the telomerase inhibitor GRN163L abolished the LY2784544-effect. JAK2 inhibitors thus cause enrichment of LSCs and are unlikely to cure
MPN as a monotherapy. Simultaneously targeting JAK2V617F and KLF4 or telomerase may be a novel strategy for MPN therapy, which should be of signiﬁcance both biologically and clinically.
© 2020 Published by Elsevier Inc.

1. Introduction

Myeloproliferative neoplasms (MPNs) bearing no Philadelphia chromosomes are a group of clonal neoplastic diseases derived from myeloid stem cells in the bone marrow, consisting of polycythemia vera (PV), essential thrombocythemia (ET) and primary myeloﬁbrosis (PMF) [1,2]. The hotspot activating- mutation in exon 14 of the JAK2 gene (JAK2V617F) is a driver for the pathogenesis of MPNs, which occurs in the vast majority of PVs, and approximately half of ETs and PMFs [2,3]. Therefore,
* Corresponding author.
** Corresponding author.
E-mail addresses: [email protected] (J. Dahlstro€m), Xiaotian.Yan@ki. se (X. Yuan).

JAK2 might be a rational therapeutic target for MPNs. Indeed, JAK2 inhibitors have been developed to treat JAK2V617F-positive MPNs. Clinical trials of JAK2 inhibitors in PMFs showed a reduction in spleen size and improvements in other symptoms [2,4e7]. However, this effect does not correlate with reduced JAK2V617F allele burden [8]. Xenotransplantation experiments revealed that JAK2 inhibitors target a population of progenitor cells in the human spleen while sparing another progenitor population and disease stem cells [9].
Most JAK2 inhibitors for treatment of MPNs are generally not speciﬁc to JAK2, and they may inhibit other targets including JAK1, JAK3 and TYK as well [10,11]. Certain side effects reported in clinical studies, such as severe anemia, are believed to be partially caused by the inhibition of other targets than JAK2 [4e7]. One inhibitor, LY2784544, is unique as it shows a dose-dependent selectivity for JAK2V617F [11]. The TEL-JAK3 cell-based assay shows that

https://doi.org/10.1016/j.bbrc.2020.04.058 0006-291X/© 2020 Published by Elsevier Inc.
LY2784544 has a negligible effect on the JAK3 signaling, and it also exhibits a greater degree of selection for JAK2 over JAK1 than other JAK2 inhibitors including ruxolitinib. Mechanistically, LY2784544 displays potent ATP-competitive inhibition of JAK2 tyrosine kinase [11]. Despite its highly selective inhibition of JAK2V617F cells, LY2784544 had minimal effects on JAK2V617F allele burden in clin- ical studies [5,8,12]. In general, the results of early phase 1 and 2 clinical trials with JAK2 inhibitors are not satisfying [4,13]. It is currently incompletely understood why MPN patients respond poorly to JAK2 inhibitors.
Telomerase, a RNA-dependent DNA polymerase responsible for telomere lengthening, is silent in human differentiated cells, while activated in the vast majority of malignancies [14e17]. Telomerase or its catalytic component telomerase reverse tran- scriptase (TERT) is required for the development and progression of human cancer by contributing to multiple cancer hallmarks. Telomerase inhibition has therefore been suggested as a therapy for human malignancies. For instance, GRN163L, a 13-mer deoxyribo-oligonucleotide, acts as a direct antagonist to the telomerase RNA template (TERC) to inhibit telomerase activity and has recently entered clinical trials for several human ma- lignancies including MPNs [18e20]. Initial studies show that GRN163L inhibits the formation of megakaryocyte colonies in patients with ET and PMF and reduces the allele burden of JAK2V617F in megakaryocytes in some patients [21]. A promising efﬁcacy of GRN163L in MPNs has been observed in several clin- ical investigations [22]. However, the drug has also been shown to cause severe myelosuppression in some patients [18e20]. A combined therapeutic strategy of GRN163L with other thera- peutic agents, might enhance the treatment efﬁcacy and mitigate telomerase inhibitors’ side-effects [4].
The aforementioned experimental and clinical studies raise a
number of questions: First, why MPN patients have a limited response to speciﬁc JAK2 inhibitors; second, how GRN163L ach- ieves its therapeutic efﬁcacy in MPNs; and ﬁnally, whether the combined inhibition of both JAK2 and telomerase can have synergistic effects on MPNs. The present study was thus designed to address these issues. Speciﬁcally, we wanted to deﬁne the effect of JAK2 and telomerase inhibitors on leukemic stem cells (LSCs) using HEL leukemic cells carrying the JAK2V617F mutation as a model.

2. Materials and methods

2.1. Cell culture

The erythroleukemia cell line HEL at cell density 0.5 millions/ml was treated with 0.5 mM of the JAK2 inhibitor LY2784544 (Selleck chemicals, Houston, TX) or/and 20 mM of the telomerase inhibitor GRN163L (Johnson-Johnson, New Brunswick, NJ). Further detailed treatment of cells is in supplementary materials.

2.2. Colony formation assay

Colony formation assay was performed with cells treated with LY2784544 and/or GRN163L following 4 and 8 week incubation. The detailed protocol is documented in supplementary materials.

2.3. RNA extraction and real time polymerase chain reaction (RT- PCR)

RNA was isolated using Trizol (Life technologies, Carlsbad, CA) according to the manufacturer’s protocol. RT-PCR and primer se- quences are detailed in supplementary materials.

2.4. Whole transcript expression analysis

Microarray analysis was performed using Affymetrix whole- transcript expression analysis and the WT assay gene ST 1.1 (Affy- metrix, Santa Clara, CA) in association with the Bioinformatics and Expression Analysis Core Facility (BEA), Karolinska Institutet. The expression proﬁle was compared between cells with and without LY2784544 or GRN163L exposure and differentially expressed genes were identiﬁed.

2.5. Western blot

cells were lysed in RIPA buffer and protein concentration measured using DC protein assay (Bio-rad, Hercules, CA). The detailed protocol is documented in supplementary materials.

2.6. Flow cytometry

CD34 and apoptosis assays using ﬂow cytometry are detailed in supplementary materials.

2.7. Lentiviral transfection

Lentiviral particles KLF4 ShRNA with green ﬂuorescent protein (GFP) as a reporter and puromycin as a mammalian selection marker were purchased from Origene (TL316853V). The detailed protocol is documented in supplementary materials.

2.8. Telomerase activity assay

Telomerase activity was measured using a telomeric repeat ampliﬁcation protocol (TRAP)-ELISA KIT (Roche, Basel, Switzerland). The detailed protocol is documented in supplemen- tary materials.

2.9. Telomere length analysis using ﬂow-FISH

Telomere length was measured with ﬂow-FISH as previously described [23]. The detailed protocol is documented in supple- mentary materials.

2.10. Statistical analysis

The data is expressed as means with error bars indicating the standard deviation (SD). For statistical analyses, Student’s t-test was used and a P-value <0.05 was considered signiﬁcant.

3. Results

3.1. LY2784544 or GRN163L treatment of HEL cells induces apoptosis and inhibits proliferation

Treatment with LY2784544, a speciﬁc JAK2 inhibitor, initially reduced both the number and viability of HEL cells (Fig. 1a and b). After an initial dip in viability seen after approximately one week, cells started to recover and at the end of the experiment (9 weeks) the viability was about the same as the control cells (Fig. 1b). A similar pattern was seen when assessing apoptosis after treatment with LY2784544 (Fig. 1c and d). Interestingly, this recovery was not seen in cells that were simultaneously treated with the telomerase inhibitor GRN163L (Figs. S3a and b). Treatment with GRN163L alone severely reduced the cell number and viability with no re- covery with time.

Fig. 1. The JAK2 inhibitor LY2784544 inhibits proliferation and promotes apoptosis of HEL cells. (a) Proliferation of HEL cells after treatment with 0.5 mM LY2784544 expressed as number of cell doublings/24 h. (b) Viability of HEL cells after treatment with 0.5 mM LY2784544 measured using nucleocounter. (c and d) Percentage of late and early apoptotic cells following treatment with 0.5 mM LY2784544, as analyzed using an Annexin V and 7-AAD staining kit. (e) Percentage of CD34þ cells after LY2784544 treatment compared to control, measured with ﬂow cytometry. (f) Histograms of CD34-APC in HEL control and LY2784544 treated cells after 1, 3 and 7 weeks, respectively. Graphed data in ﬁgures aee are shown as mean and standard deviation.

3.2. CD34-positive cells accumulate following JAK2 inhibition in HEL cells

The fraction of CD34-positive (CD34þ) cells drastically increased during LY2784544 treatment. The CD34þ fraction was assessed with ﬂow cytometry every other week for 9 weeks and a gradually
increasing fraction of CD34 cells was seen starting at approxi- mately 10% at 1 week and reaching 85% at 9 weeks of treatment (Fig. 1e and f). To assess the functional signiﬁcance of these increased CD34þ cells, a colony formation assay was performed. Cells treated with LY2784544 indeed gave rise to more colonies, especially colonies containing >50 cells (Fig. 1g). A slight (non-
signiﬁcant) decrease in the number of colonies was seen in cells treated with GRN163L compared to control cells. Simultaneous treatment with GRN163L was able to block the increase in CD34þ cells in the presence of LY2784544 (Fig. S3c) and colony formation potential as well (Fig. 1g). To better understand if LY2784544 causes an induction of CD34þ cells or if the CD34 enrichment is caused by a
decreased sensitivity to the drug, HEL cells were then sorted into
CD34 negative (CD34—) and positive populations before treatment with LY2784544. An increase in CD34þ cell numbers was seen both in the negative and positive populations after treatment for 2 and 4 weeks, respectively (Fig. 2a). The CD34þ cells had a higher viability

compared to the CD34—cells after treatment with LY2784544 (Fig. 2b and c).
3.3. LY2784544-mediated up-regulation of KLF4 expression is responsible for the accumulation of CD34þ HEL cells

To deﬁne the mechanism underlying LY2784544-mediated accumulation of CD34þ cells, we performed Affymetrix whole- transcript expression analysis in HEL cells treated with LY2784544 and/or GRN163L. The mRNA expression results are presented in Supplemental Table 1. A panel of genes was differentially expressed
and we further selected them for veriﬁcation using QRT-PCR and/or Western blot. Altered expressions were seen for a number of genes intimately associated with hematopoiesis and stem cell phenotype; one of which was KLF4 (Fig. 2deg). KLF4 is essential for the gener- ation of induced pluripotent stem cells (iPSCs) and cellular de- differentiation, and we thus sought to determine whether KLF4 played a part in the LY2784544-mediated CD34 enrichment. To this end, we inhibited KLF4 expression in HEL cells using a shRNA spe- ciﬁcally targeting KLF4. Infection with the KLF4 ShRNA lentivirus suppressed KLF4 expression at both mRNA and protein levels in HEL
cells (Fig. 3aec), which subsequently attenuated LY2784544- mediated accumulation of CD34þ LSCs (Fig. 3a).

Fig. 2. LY2784544 increases the CD34þ fraction in CD34¡ HEL cells. (a) Histograms of CD34-APC in control and LY2784544-treated cells after 2 and 4 weeks of incubation. HEL cells were sorted into CD34þ and – populations before starting treatment. (b) Viability in sorted CD34þ and – HEL cells with and without 0.5 mM of LY2784544. (c) Percentage apoptotic cells in CD34-sorted HEL after 72h incubation with 0.5 mM LY2784544. (d) mRNA expression of KLF4 in HEL cells after 48 h incubation with 0.5 mM LY2784544. (e) Protein expression of KLF4 in HEL cells after 48 h incubation with 0.5 mM of LY2784544. (f) mRNA expression of KLF4 after 48 h incubation with 0.5 mM LY2784544 in sorted CD34þ and – HEL populations. (g) mRNA expression of KLF4 in unsorted HEL cells after 48 h incubation with 0.5 mM LY2784544 and/or 20 mM GRN163L. The levels of TERT or KLF4 mRNA were arbitrarily expressed as the ratio of target mRNA/b2-M. All graphed data are shown as mean and standard deviation. Asterisks indicates p-values *<0.05, **≤0.01, ***<0.001.
3.4. JAK2 inhibition in HEL cells causes down-regulation of TERT expression and telomerase activity but paradoxically telomere lengthening

TERT mRNA expression was signiﬁcantly reduced following treatment with LY2784544 for 48 h (P 0.013) (Fig. 4a). Telomerase activity was accordingly reduced following 1 week (P 0.002), 4 weeks (P 0.011) and 9 weeks (P 0.003) of JAK2 inhibition (Fig. 4b). Telomere length following JAK2 inhibition was studied with ﬂow-FISH after 1, 4, 7, and 9 weeks of treatment. Despite down-regulated TERT and telomerase expression, a signiﬁcant in- crease in telomere length was observed at 4 (P 0.03), 7 (P 0.002) and 9 weeks (P 0.003), which indicates a dissociation between
telomerase activity and telomere length in LY2784544-treated HEL cells (Fig. 4c). As JAK2 inhibition leads to CD34þ cell accumulation, the question is whether CD34þ cells carry longer telomeres than CD34—cells, or if the observed telomere lengthening in LY2784554- treated cells is simply due to the increase of the CD34þ fraction. To address this issue, we further separated two cell populations and determined their telomere lengths. Telomeres in the CD34þ HEL cells were longer than those in CD34— HEL cells (P 0.044) (Fig. 4d). We found that telomeres were signiﬁcantly longer in CD34þ cells treated with LY2784544 for 8 weeks compared to CD34

positive cells without treatment (P 0.048) (Fig. 4d). Telomerase activity was signiﬁcantly reduced in both CD34þ and – populations after 8 weeks of incubation (P 0.012 and 0.016, respectively).
Telomerase activity was reduced in both populations already after 4 weeks of treatment with LY2784544, but only signiﬁcantly lower in the CD34þ cells (P 0.026) (Fig. 4e). In concordance with this, TERT
mRNA expression was also signiﬁcantly lower in both CD34þ and -
cells treated with LY2784544 (at 4 weeks: P 0.03 and 0.003, respectively; at 8 weeks: P or <0.001 in both populations) (Fig. 4f). Taken together, both CD34 cell selection and other un- known mechanisms contribute to telomere lengthening in HEL cells treated with LY2784554.

4. Discussion

The activating-mutation of JAK2V617F is a key genetic event to drive the development of MPNs, however, the therapeutic efﬁcacy of JAK2 inhibitors in clinical trial has not lived up to expectations. The accumulated evidence indicates that JAK2 inhibitors fail to eradicate the disease clone [5], with a minimal effect of JAK2V617F allele burden on progenitors and stem cells [24]. To explore the underlying mechanism(s), we designed the present study. We show that JAK2V617F-carrying HEL cells undergo apoptosis and

Fig. 3. Inhibition of KLF4 expression attenuates the LY2784544 mediated increase in CD34þ cells. (a and b) Veriﬁcation of KLF4 suppression after transduction with lentivirus KLF4 ShRNA. (c) HEL cells were infected with scrambled control lenti-viral vector and ShKLF4 vector, respectively, and treated with 0.5 mM of LY2784544 for 2 weeks. Cells were then analyzed for KLF4 mRNA expression. The level of KLF4 mRNA was arbitrarily expressed as the ratio of KLF4 mRNA/b2-M. (d) GFP negative and positive populations oh HEL cells represent shKLF4 non-transduced and transduced cells, respectively. Histogram of CD34-APC in cells treated with 0.5 mM of LY2784544 for 2 weeks. Upper right histogram show
cells transduced with scrambled control ShRNA and lower right histogram show cells transduced with KLF4 ShRNA. All graphed data are shown as mean and standard deviation. Asterisks indicates p-values *<0.05, **<0.01, ***<0.001.
diminished proliferation in the presence of the speciﬁc JAK2 in- hibitor LY2784544 for the ﬁrst week, but recover gradually due to the CD34þ cell accumulation. Mechanistically, LY2784544 treat- ment led to the up-regulation of the stem cell factor KLF4 expres- sion. KLF4 inhibition signiﬁcantly attenuated the enrichment of CD34þ cells mediated by LY2784544. Intriguingly, even in the
CD34— HEL cell fraction, LY2784544 treatment still induced the
generation and gradual accumulation of CD34þ cells. It is thus
evident from the present study that LY2784544 may promote stemness by inducing cellular de-differentiation.
KLF4 is one of the four transcription factors that when combined together can be manipulated to create iPSCs [25]. The role of KLF4 in hematopoiesis is not fully understood, but the ability of KLF4 together with other transcription factors to dedifferentiate mature

somatic cells into iPSCs suggests that it is involved in the mainte- nance of tissue-speciﬁc stem cells. In addition, KLF4 is required for sustained cell proliferation by inhibiting cellular senescence [26]. KLF4 is not crucial for the development and maintenance of HSCs in the fetal liver, but this does not exclude its role in the regulation of the stem cell and progenitor niche in the adult bone marrow [27]. In this study, we do not address the mechanism by which inhibition of JAK2 in HEL cells increases the expression of KLF4; this has to be elucidated by further studies. Transcriptional proﬁling of CD34 cells from PV patients has shown that KLF4 is down- regulated and dependent on the action of JAK2V617F [28], which is consistent with our ﬁnding of KLF4 activation following JAK2 inhibition.
JAK/STAT signaling has previously been suggested as a positive

Fig. 4. Alterations in telomere length and telomerase expression in HEL cells after treatment with the JAK2 inhibitor LY2784544. (a) TERT mRNA expression in HEL cells treated with 0.5 mM of LY2784544 for 48 h. (b) Telomerase activity in HEL cells analyzed with TRAP-ELISA after 1, 4, and 9 week incubation with 0.5 mM of LY2784544, respectively.
(c) Telomere length in HEL cells measured with ﬂow-FISH after 1, 4, 7 and 9 weeks of incubation with 0.5 mM of LY2784544. (d) Telomere length in sorted CD34þ and – populations
after 4 and 9 weeks of incubation with 0.5 mM of LY2784544 as determined using ﬂow-FISH. (e) Telomerase activity analyzed with TRAP-ELISA after 4 and 8 weeks of incubation with LY2784544 in sorted CD34þ and e HEL cell populations. (f) TERT mRNA expression in sorted CD34þ and – cells after 4 and 8 weeks incubation with 0.5 mM of LY2784544. The level of TERT mRNA was arbitrarily expressed as the ratio of TERT/b2-M, and telomerase activity was expressed as absorbance assessed using a telomerase TRAP-ELISA kit. All graphed data are shown as mean and standard deviation. Asterisks indicates p-values *<0.05, **<0.01, ***<0.001.
regulator of TERT expression by direct binding of STAT3 or STAT5 to the TERT promoter [29]. On the other hand, Zhang et al. showed that JAK2 inhibition was involved in senescence of hepatocellular carcinoma (HCC) cells, while the ectopic expression of TERT was capable of attenuating senescence by restoring the JAK2 activity [30]. These ﬁndings indicate a close interplay between JAK2 signaling and telomerase or TERT in oncogenesis. Consistent with the studies above, we observed that LY2784544 treatment of HEL cells signiﬁcantly inhibited TERT expression and telomerase activ- ity. However, paradoxically, telomere length increased in HEL cells during JAK2 inhibition, uncouple with diminished levels of TERT and telomerase activity. Likely, telomeres were elongated in a telomerase-independent manner, or alternative lengthening of telomeres (ALT). ALT occurs frequently in sarcomas and high-grade astrocytomas [16,31]. It has been shown that malignant cells with telomerase activation are able to acquire ALT upon telomerase repression [32]. Because telomere over-erosion and dysfunction is

widely present in MPN patient-derived myeloid cells [23,33], JAK2 inhibitor-mediated telomere lengthening may result in treatment failure [34]. Therefore, further studies are required to determine whether telomere elongation is seen in MPN cells from patients treated with JAK2 inhibitors, and if so, whether it will lead to treatment failure.
The observations above provide a rationale to combine JAK2 inhibitors with telomerase inhibitors for MPN therapy. Indeed, GRN163L was able to abolish the CD34þ HEL cell accumulation resulting from LY2748544 treatment. We further demonstrated that telomere shortening did occur in GRN163L-treated HEL cells.
Likely, shortened telomeres induced by GRN163L block the acqui- sition of stemness mediated by LY2784544. Defective telomere lengthening has been shown to promote HSC differentiation [35].
An increase in CD34þ cells by LY2784544 treatment is also seen in the CD34— HEL population, which supports that the cells might have shifted to a more immature state. However, because the

CD34þ fraction could not be eradicated completely using our sorting method, we were unable to fully exclude that such increase was due to an intrinsic resistance of the minimal CD34þ cells to LY2784544. Our results indeed suggest that those CD34þ cells are more tolerable to LY2784544 treatment than CD34—ones. Never- theless, LY2784544-mediated CD34þ cell accumulation provides a putative explanation for MPN patients’ poor response to JAK2 in-
hibitors. Moreover, the identiﬁcation of KLF4 up-regulation by LY2784544 treatment not only gains mechanistic insights, but also suggests KLF4 as a new potential therapeutic target in MPNs. These ﬁndings should have important clinical signiﬁcances.
In summary, we report that treatment with the JAK2 inhibitor LY2784544 leads to an accumulation of CD34þ cells via up- regulation of the stem cell factor KLF4 expression in a JAK2V617F-
bearing leukemia derived cell line. Inhibiting LY2784544-mediated KLF4 induction signiﬁcantly attenuated an increase in CD34þ cells. Therefore, targeting KLF4 together with JAK2 inhibitors may in- crease therapeutic efﬁcacy for MPN patients. Furthermore, the
accumulation of CD34þ cells resulting from LY2784544 was also blocked by simultaneous treatment with the telomerase inhibitor
GRN163L. Both drugs used in this study have shown potential in the treatment of MPNs and our ﬁndings suggest that a combination of JAK2 and telomerase inhibitors might be effective in the treatment of MPNs.

Data availability

Affymetrix whole-transcript expression analysis results are deposited at the Gene Expression Omnibus (GEO) under accession number GSE141602.

Declaration of competing interest

The authors declare that they have no known competing ﬁnancial interests or personal relationships that could have appeared to inﬂuence the work reported in this paper.

Acknowledgments

We thank Johnson-Johnson (New Brunswick, NJ) for GRN163L. This study was supported by grants from Swedish Cancer Society (19 0018 Pj), Swedish Research Council (2018-02993), Cancer So- ciety in Stockholm (171223), Karolinska Institutet Foundation and Gunnar Grimfors’ endowment fund.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.04.058.

References

[1] J.J. Michiels, Z. Berneman, W. Schroyens, H. De Raeve, Changing concepts of diagnostic criteria of myeloproliferative disorders and the molecular etiology and classiﬁcation of myeloproliferative neoplasms: from Dameshek 1950 to Vainchenker 2005 and beyond, Acta Haematol. 133 (2015) 36e51.
[2] K. Zoi, N.C. Cross, Genomics of myeloproliferative neoplasms, J. Clin. Oncol. 35 (2017) 947e954.
[3] R. Kralovics, F. Passamonti, A.S. Buser, et al., A gain-of-function mutation of JAK2 in myeloproliferative disorders, N. Engl. J. Med. 352 (2005) 1779e1790.
[4] A.M. Vannucchi, C.N. Harrison, Emerging treatments for classical myelopro- liferative neoplasms, Blood 129 (2017) 693e703.
[5] S. Verstovsek, R.A. Mesa, J. Gotlib, et al., A double-blind, placebo-controlled trial of ruxolitinib for myeloﬁbrosis, N. Engl. J. Med. 366 (2012) 799e807.
[6] S. Verstovsek, R.A. Mesa, M.E. Salama, et al., A phase 1 study of the Janus ki- nase 2 (JAK2)(V617F) inhibitor, gandotinib (LY2784544), in patients with

primary myeloﬁbrosis, polycythemia vera, and essential thrombocythemia, Leuk. Res. 61 (2017) 89e95.
[7] S. Verstovsek, A.M. Vannucchi, M. Griesshammer, et al., Ruxolitinib versus best available therapy in patients with polycythemia vera: 80-week follow-up from the RESPONSE trial, Haematologica 101 (2016) 821e829.
[8] A. Pardanani, R.R. Laborde, T.L. Lasho, et al., Safety and efﬁcacy of CYT387, a JAK1 and JAK2 inhibitor, in myeloﬁbrosis, Leukemia 27 (2013) 1322e1327.
[9] X. Wang, F. Ye, J. Tripodi, et al., JAK2 inhibitors do not affect stem cells present in the spleens of patients with myeloﬁbrosis, Blood 124 (2014) 2987e2995.
[10] M.B. Sonbol, B. Firwana, A. Zarzour, et al., Comprehensive review of JAK in- hibitors in myeloproliferative neoplasms, Ther. Adv. Hematol. 4 (2013) 15e35.
[11] L. Ma, J.R. Clayton, R.A. Walgren, et al., Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F, Blood Canc. J. 3 (2013) e109.
[12] J. Berdeja, F. Palandri, M.R. Baer, et al., Phase 2 study of gandotinib (LY2784544) in patients with myeloproliferative neoplasms, Leuk. Res. 71 (2018) 82e88.
[13] F. Schain, E. Vago, C. Song, et al., Survival outcomes in myeloﬁbrosis patients treated with ruxolitinib: a population-based cohort study in Sweden and Norway, Eur. J. Haematol. 103 (2019) 614e619.
[14] X. Yuan, C. Larsson, D. Xu, Mechanisms underlying the activation of TERT transcription and telomerase activity in human cancer: old actors and new players, Oncogene 38 (2019) 6172e6183.
[15] X. Yuan, D. Xu, Telomerase reverse transcriptase (TERT) in action: cross- talking with epigenetics, Int. J. Mol. Sci. 20 (2019).
[16] X. Yuan, M. Dai, D. Xu, Telomere- related markers for cancer, Curr. Top. Med. Chem. 20 (2020) 410e432.
[17] P. Yu, X. Shen, W. Yang, et al., ZEB1 stimulates breast cancer growth by up- regulating hTERT expression, Biochem. Biophys. Res. Commun. 495 (2018) 2505e2511.
[18] A. Tefferi, Telomerase inhibitor imetelstat in essential thrombocythemia and myeloﬁbrosis, N. Engl. J. Med. 373 (2015) 2580e2581.
[19] A. Tefferi, T.L. Lasho, K.H. Begna, et al., Pilot study of the telomerase inhibitor imetelstat for myeloﬁbrosis, N. Engl. J. Med. 373 (2015) 908e919.
[20] G.M. Baerlocher, B. Burington, D.S. Snyder, Telomerase inhibitor imetelstat in essential thrombocythemia and myeloﬁbrosis, N. Engl. J. Med. 373 (2015) 2580.
[21] G. Mosoyan, T. Kraus, F. Ye, et al., Imetelstat, a telomerase inhibitor, differ- entially affects normal and malignant megakaryopoiesis, Leukemia 31 (2017) 2458e2467.
[22] I. Joseph, R. Tressler, E. Bassett, et al., The telomerase inhibitor imetelstat depletes cancer stem cells in breast and pancreatic cancer cell lines, Canc. Res. 70 (2010) 9494e9504.
[23] J. Dahlstrom, X. Zhang, M. Ghaderi, et al., Dysregulation of shelterin factors coupled with telomere shortening in Philadelphia chromosome negative myeloproliferative neoplasms, Haematologica 100 (2015) e402ee405.
[24] A. Angona, A. Alvarez-Larran, B. Bellosillo, et al., Dynamics of JAK2 V617F allele burden of CD34 haematopoietic progenitor cells in patients treated with ruxolitinib, Br. J. Haematol. 172 (2016) 639e642.
[25] K. Takahashi, S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult ﬁbroblast cultures by deﬁned factors, Cell 126 (2006) 663e676.
[26] E. Panatta, A.M. Lena, M. Mancini, et al., Kruppel-like factor 4 regulates ker- atinocyte senescence, Biochem. Biophys. Res. Commun. 499 (2018) 389e395.
[27] C.S. Park, Y. Shen, A. Lewis, et al., Role of the reprogramming factor KLF4 in blood formation, J. Leukoc. Biol. 99 (2016) 673e685.
[28] W. Berkofsky-Fessler, M. Buzzai, M.K. Kim, et al., Transcriptional proﬁling of polycythemia vera identiﬁes gene expression patterns both dependent and independent from the action of JAK2V617F, Clin. Canc. Res. 16 (2010) 4339e4352.
[29] O. Yamada, K. Kawauchi, The role of the JAK-STAT pathway and related signal cascades in telomerase activation during the development of hematologic malignancies, JAK-STAT 2 (2013), e25256.
[30] L. Zhang, Z. Yang, A. Ma, et al., Growth arrest and DNA damage 45G down- regulation contributes to Janus kinase/signal transducer and activator of transcription 3 activation and cellular senescence evasion in hepatocellular carcinoma, Hepatology 59 (2014) 178e189.
[31] J.D. Henson, R.R. Reddel, Assaying and investigating Alternative Lengthening of Telomeres activity in human cells and cancers, FEBS Lett. 584 (2010) 3800e3811.
[32] A. Queisser, S. Heeg, M. Thaler, et al., Inhibition of telomerase induces alter- native lengthening of telomeres during human esophageal carcinogenesis, Canc. Genet. 206 (2013) 374e386.
[33] L. Bernard, C. Belisle, L. Mollica, et al., Telomere length is severely and simi- larly reduced in JAK2V617F-positive and -negative myeloproliferative neo- plasms, Leukemia 23 (2009) 287e291.
[34] X. Zhang, B. Li, J. Yu, et al., MYC-dependent downregulation of telomerase by FLT3 inhibitors is required for their therapeutic efﬁcacy on acute myeloid leukemia, Ann. Hematol. 97 (2018) 63e72.
[35] J.I. Park, A.S. Venteicher, J.Y. Hong, et al., Gandotinib Telomerase modulates Wnt signalling by association with target gene chromatin, Nature 460 (2009) 66e72.