Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2023 Feb 9.
Published in final edited form as: Exp Hematol. 2021 Jun 19;100:52–62. doi: 10.1016/j.exphem.2021.06.003

Cell competition between wild-type and JAK2V617F mutant cells in a murine model of a myeloproliferative neoplasm

Melissa Castiglione 1, Haotian Zhang 2, Kenneth Kaushansky 3, Huichun Zhan 1,4
PMCID: PMC9911310  NIHMSID: NIHMS1867686  PMID: 34153382

Abstract

The myeloproliferative neoplasms (MPNs) are characterized by overproduction of mature blood cells and increased risk of transformation to frank leukemia. The acquired kinase mutation JAK2V617F plays a central role in a majority of patients with these diseases. As MPNs are clonal stem cell disorders (i.e. arise from a single stem cell which eventually expands), the hematopoietic stem/progenitor cell (HSPC) compartment in MPNs is heterogeneous with the presence of both JAK2 wild-type and JAK2V617F mutant cells. Mechanisms responsible for the mutant stem cell expansion in MPNs are not fully understood. Utilizing in vitro co-culture assays and in vivo competitive transplantation assays, we show that the presence of wild-type cells alters both the gene expression profile and cellular function of JAK2V617F mutant HSPCs and inhibits the expansion of co-existing JAK2V617F mutant cells in a normal microenvironment. In contrast, we found that a microenvironment bearing the mutant kinase promotes JAK2V617F mutant HSPC expansion over wild-type cells due in part to altered CXCL12/CXCR4 signaling. Further understanding of the molecular mechanisms controlling the competitive interactions between normal and JAK2V617F mutant cells, and how these mechanisms break down during MPN disease progression hold great potential for advances in treating patients with these diseases.

Keywords: Myeloproliferative neoplasm, Cell competition, Microenvironment

Introduction

Virtually all human cancers derive from a single cell that acquires (or rarely is inherited) genetic alterations that provide a growth advantage over its surrounding normal cells. The emergence of cancer is a multi-step process in which clonal succession occurs as secondary and tertiary mutations are selected, eventually allowing cancer cells to escape the normal cell-cell homeostatic mechanisms that prevent normal tissues from overgrowing or invading adjacent tissues. Hence, cancer is the pathological outcome of cancerous cells outcompeting wild-type cells. However, much evidence indicates that the outgrowth of mutation-bearing cells is not relentless; rather, it is clear that, during the earlier stages of the multistep oncogenic process, the presence of normal surrounding cells can slow or halt the growth of the malignant clone. Cell competition is an evolutionarily conserved mechanism involved in development, tissue homeostasis, and stem cell maintenance. It is akin to natural selection between species, in that “fitter” cells win out over their “less-fit” neighbors1–3. Studies from the past several years revealed that human tissues (e.g. skin, esophageal, blood) show high levels of mosaicism with many precancerous mutations, yet such tissues rarely develop frank tumors4–6. These observations suggest that cell competition can protect against cancer7,8. Only when subclones of mutation-bearing cells develop multiple molecular and cellular mechanisms, both cell intrinsic and cell extrinsic, making them more competitive than their normal counterparts, does a frank cancer emerge.

The myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem/progenitor cell (HSPC) disorders characterized by overproduction of mature blood cells, and an increased risk of transformation to acute leukemia. The acquired signaling kinase mutation JAK2V617F plays a central role in most patients with these disorders. The HSPC compartment in MPN is heterogeneous with the presence of both JAK2 wild-type and JAK2V617F mutant cells in most patients with MPN, at least early in their disease process9. Despite mutant cells bearing an in vitro proliferative advantage because of the constitutive kinase activity of JAK2V617F, in many patients, there is little or no change in the mutant/wild-type cell ratio over long periods of follow up10–12. This is especially true in older individuals found to have clonal hematopoiesis of indeterminate potential (CHIP), where from 2% to over 20% of hematopoietic cells bear JAK2V617F or other mutations associated with MPNs, myelodysplastic syndromes or acute leukemia, yet the clonal cells do not expand for years, or ever13–15. In other patients, the MPN can evolve to acute leukemia and patients experience high relapse rates following allogeneic stem cell transplantation, the only curative treatment for patients with MPNs16–19. These features make the JAK2V617F mutant MPN a model disease to study the early stages of clonal competition and tumor progression. In this work, we investigated the competitive interactions between wild-type and JAK2V617F mutant cells, and the impact of microenvironmental factors in driving mutant clonal dominance in the MPNs.

Methods

Experimental mice

JAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter)20 were provided by Radek Skoda (University Hospital, Basal, Switzerland), and Tie2-Cre mice21 by Mark Ginsberg (University of California, San Diego). FF1 mice were crossed with Tie2-Cre mice to express JAK2V617F specifically in hematopoietic cells and endothelial cells (ECs) (Tie2-cre+FF1+ mice)22,23. All mice used were crossed onto a C57BL/6 background and bred in a pathogen-free mouse facility at Stony Brook University. CD45.1+ congenic mice (SJL) were purchased from Taconic Inc. (Albany, NY). Animal experiments were performed in accordance with the guidelines provided by the Institutional Animal Care and Use Committee.

Marrow cell isolation

For marrow cells, murine femurs and tibias were first harvested and cleaned thoroughly. Marrow cells were flushed into PBS with 2% fetal bovine serum using a 25G needle and syringe. Remaining bones were crushed with a mortar and pestle followed by enzymatic digestion with DNase I (25U/ml) and Collagenase D (1mg/ml) at 37 °C for 20 min under gentle rocking. Tissue suspensions were thoroughly homogenized by gentle and repeated mixing using 10ml pipette to facilitate dissociation of cellular aggregates. Resulting cell suspensions were then filtered through a 40uM cell strainer.

For depletion of mature hematopoietic cells, the Lineage Cell Depletion Kit (Miltenyi Biotec, San Diego, CA) was used. The lineage negative cells were collected and then positively selected for CD117+ (cKit+) cells using CD117 microbead (Miltenyi Biotec) to yield Lin−cKit+ HSPCs.

Complete blood counts and colony assays

Peripheral blood was obtained from the facial vein via submandibular bleeding, collected in an EDTA tube, and analyzed using a Hemavet 950FS (Drew Scientific). Mouse methylcellulose complete media (M3434, Stem Cell Technologies, Vancouver, BC) were used to assay hematopoietic colony formation. After 7-10 days in culture, individual colonies (both erythoid and myeloid) were plucked and genomic DNA was extracted for PCR analysis. Tie2-Cre specific primers (5’-CTGCATTACCGGTCGATGCA-3’ and 5’-ACGTTCACCGGCATCAACGT-3’) were used to differentiate colonies derived from wild-type competitor marrow versus those from Tie2-cre+FF1+ marrow.

Flow cytometry

All samples were analyzed by flow cytometry using FACSAria III or LSR (BD biosciences, San Jose, CA, USA). CD45 (Clone 104) (Biolegend, San Diego, CA, USA), CD45.1 (Clone A20) (BD Biosciences), CD45.2 (Clone 104) (Biolegend), Lineage cocktail (include CD3, B220, Gr1, CD11b, Ter119; Biolegend), cKit (Clone 2B8, Biolegend), Sca1 (Clone D7, Biolegend), EPCR (CD201) (Clone eBio1560, eBioscience, San Diego, CA, USA), CD150 (Clone mShad150, eBioscience), and CD48 (Clone HM48-1, Biolegend) antibodies were used.

Stem cell transplantation assays

Recipient mice were irradiated with two doses of 540 rad 3h apart. Donor cells were injected into recipients by standard intravenous tail vein injection using a 27G insulin syringe. For competitive transplantation, 5x105 CD45.2 donor marrow cells from Tie2-cre+FF1+ mice were injected intravenously together with 5x105 competitor CD45.1 wild-type marrow cells. For non-competitive transplantation, 1 x106 unfractionated donor marrow cells were transplanted into wild-type recipients by intravenous tail vein injection.

BrdU incorporation analysis

Mice were injected intraperitoneally with a single dose of 5-bromo-2’-deoxyuridine (BrdU; 100 mg/kg body weight) and maintained on 1mg BrdU/ml drinking water for two days. Mice were then euthanized and marrow cells isolated. For analysis of HSPC proliferation, Lineageneg (Lin−) cells were first enriched using the Lineage Cell Depletion Kit (Miltenyi Biotec) before staining with fluorescent antibodies specific for cell surface HSPC markers, followed by fixation and permeabilization using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA), DNase digestion (Sigma, St. Louis, MO), and anti-BrdU antibody (Biolegend, San Diego, CA) staining to analyze BrdU incorporation. For analysis of more abundant cell populations, marrow cells were stained with cell surface antibodies, then fixed and stained with anti-BrdU antibody for BrdU incorporation analysis.

Cell cycle analysis

For HSPC cell cycle analysis, marrow cells were first stained with fluorescent antibodies for cell surface markers (CD45.1, CD45.2, CD150, and CD48), washed, and then stained with Hoechst33342 (10ug/ml) (Sigma) at 37°C in dark for 45 min, followed by staining with Pyronin Y (0.5ug/ml) (Sigma) at 37°C in dark for another 45 min. Cells were kept on ice until flow cytometry analysis on a LSR II (BD biosciences)24,25.

Analysis of apoptosis by active caspase-3 staining

Marrow cells were stained with fluorophore-conjugated Lineage cocktail antibodies (Biolegend) and antibodies against cKit, Sca1, CD150, and CD48. Cells were then washed and fixed using the Cytofix/Cytoperm kit (BD Biosciences). Cells were then stained using a rabbit anti-activated caspase-3 antibody. Data were acquired using a FACSAria II flow cytometer.

In vitro co-cultures

Marrow CD45+CD201+CD150+CD48− cells, a highly purified long-term repopulating stem cell population26,27, were isolated from wild-type mice (CD45.1) or JAK2V617F-positive Tie2-cre+FF1+ mice (CD45.2) by flow cytometry. Cells were cultured either directly or in a Transwell unit with 1.0um pore size (Corning, NY) in StemSpan® serum-free expansion medium (SFEM) containing 100 ng/mL recombinant mouse SCF and 100ng/mL recombinant human TPO for a total of 7 days (all from Stem Cell Technologies, Vancouver, BC, Canada). Cell proliferation was assessed by counting bright live cells in a hemocytometer using trypan blue dye to exclude the dead cells.

Transcriptome analysis using RNA sequencing

For RNA sequencing experiments, wild-type (CD45.1) and JAK2V617F mutant (CD45.2) marrow Lin−cKit+ HSPCs were isolated by magnetic bead isolation (with 90-95% purity) (Miltenyi Biotec, San Diego, CA). Total RNA was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany). RNA integrity and quantitation were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). For each sample, 400ng of RNA was used to generate sequencing libraries using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England BioLabs, MA, USA) following manufacturer’s recommendations. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform. Index of the reference genome was built using hisat2 2.1.0 and paired-end clean reads were aligned to the reference genome using HISAT2. HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. Differential expression analysis was performed using the DESeq R package (1.18.0). The resulting P-values were adjusted using Benjamini and Hochberg’s approach for controlling the falsediscovery rate. Genes with an adjusted P-value < 0.05 found by DESeq were assigned as differentially expressed. For samples without biological replicates, TMM was used for between sample normalization and EdgeR software was used to calculate and determine significance. Specifically, biological coefficient of variation (BCV) = 0.2 and Dispersion value = 0.04 were used as recommended by the EdgeR software. Gene Ontology (GO) (http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (http://www.genome.jp/kegg/) enrichment analysis of differentially expressed genes was implemented by the ClusterProfiler R package. GO terms and KEGG pathways with corrected P-value less than 0.05 were considered significantly enriched.

For marrow HSPC RNA sequencing, wild-type (CD45.1) and JAK2V617F mutant (CD45.2) marrow Lin−cKit+ HSPCs were isolated by magnetic bead isolation (Miltenyi Biotec, San Diego, CA). Messenger RNA samples of wild-type Lin-cKit+ HSPCs transplanted alone (one pooled sample from 3 mice), JAK2V617F mutant HSPCs transplanted together with wild-type cells (one pooled sample from 3 mice), and JAK2V617F mutant HSPCs transplanted alone (one pooled sample from 2 mice) were assessed by RNA sequencing.

For marrow EC RNA sequencing, wild-type and JAK2V617F mutant marrow ECs (CD45−CD31+) were isolated from Tie2-cre control mice (n=3) and Tie2-cre+FF1+ mice (n=4) by microbead isolation. Messenger RNA samples of the 3 wild-type marrow EC samples and 4 mutant marrow EC samples were assessed by RNA-sequencing.

Statistical analysis

Statistical analysis was performed using Student’s t tests (2 tailed) using Excel software (Microsoft). A p value of less than 0.05 was considered significant. Data are presented as mean ± standard error of the mean (SEM).

Results

Competition between wild-type and JAK2V617F mutant HSPCs in vitro

To study how wild-type and JAK2V617F mutant cells interact, we isolated murine wild-type and JAK2V617F mutant CD45+CD201+CD150+CD48− cells, a highly purified long-term repopulating stem cell population26,27, from either wild-type mice (CD45.1) or JAK2V617F-positive Tie2-cre+FF1+ mice (CD45.2)23,28,29 and performed a series of in vitro co-culture experiments. We found that JAK2V617F mutant HSPCs displayed a higher proliferation rate than wild-type HSPCs when cultured alone. In contrast, when mixed in direct co-culture, proliferation of wild-type HSPCs was greatly increased, such that there was no relative growth advantage of the mutant HSPCs over the wild-type HSPCs (Figure 1A–B). This was further confirmed by hematopoietic colony formation assays in which co-cultured JAK2 wild-type and JAK2V617F mutant HSPCs generated equal numbers of wild-type and mutant colonies (Figure 1C). Similar results were also obtained when using a transwell co-culture system (Figure 1D–E), suggesting that direct cell-cell contact was not required for the competitive interactions between wild-type and mutant HSPCs observed in these experiments.

Figure 1.

Figure 1.

Open in a new tab

JAK2 wild-type and JAK2V617F mutant cell competition in vitro. (A) Experimental scheme of direct co-culture. (B) Cell proliferation of JAK2 wild-type (WT) and JAK2V617F (VF) HSPCs (CD45+CD201+CD150+CD48−) when cultured alone and when cultured together for a total of 7 days. Cell proliferation was shown as the relative ratio compared to wild-type HSPCs cultured alone which was set as “1”. Data are from two independent experiments with triplicates in each experiment. (C) Co-cultured JAK2 wild-type and JAK2V617F HSPCs were plated on methocult for hematopoietic progenitor cell assay. Single colony genotyping demonstrated co-existence of both wild-type and JAK2V617F mutant colonies after co-culture. Data are from two independent experiments with duplicate wells in each experiment. (D) Experimental scheme of transwell co-culture experiment. (E) Cell proliferation of JAK2WT and JAK2V617F HSPCs when cultured alone and when cultured together in transwells for a total of 7 days. Cell proliferation was shown as the relative ratio compared to wild-type HSPCs cultured alone which was set as “1”. Data are from two independent experiments with triplicates in each experiment. * P<0.05

The presence of wild-type cells inhibits the expansion of co-existing JAK2V617F mutant cells in vivo

To study the competition between wild-type and JAK2V617F mutant HSPCs in vivo, we performed a series of marrow transplantation experiments. When 100% JAK2V617F mutant marrow cells (from the Tie2-cre+FF1+ mice) were transplanted into lethally irradiated wild-type recipients, recipient mice developed a MPN phenotype with leukocytosis and thrombocytosis ~4-8wks after transplantation. In contrast, when a 50-50 mix of mutant and wild-type marrow cells was transplanted into lethally irradiated wild-type recipient mice, despite there being a higher fraction of HSPCs in JAK2V617F mutant marrow than wild-type marrow22, the JAK2V617F donor cells engrafted to a similar level as wild-type donor cells, with the recipient mice displaying roughly 50-50 chimerism of normal and mutant blood cells, and normal blood counts during more than 4-months of follow up (Figure 2A–C)23. Moreover, marrow Lin−cKit+Sca1+CD150+CD48− HSC30 frequencies in recipients of a 50-50 mix of donor cells were significantly lower compared to recipients of 100% mutant cells (Figure 2D). To be certain that the JAK2V617F mutation is expressed during such competitive transplantation, we isolated CD45.2 cells from the recipient mice and confirmed human JAK2 gene expression by RT-PCR (Figure 2E).

Figure 2.

Figure 2.

Open in a new tab

The presence of wild-type cells inhibits the expansion of co-existing JAK2V617F mutant cells in vivo. (A) Scheme of marrow transplantation experiments. (B) Peripheral blood JAK2V617F mutant (CD45.2) donor chimerism at 12wk post transplantation. (C-D) Peripheral blood cell counts (C) and marrow Lin−cKit+Sca1+CD150+CD48− HSC frequencies (D) in recipient mice of wild-type donor alone, JAK2V617F mutant donor alone, or both wild-type and JAK2V617F mutant donor cells ~16wk after transplantation. (n=5-8 mice in each group in B-C and n=4-5 mice in each group in D from two independent experiments). (E) As determined by reverse transcription polymerase chain reaction (RT-PCR), human JAK2 was expressed in both CD45.2 (JAK2V617F mutant) marrow cells from recipient mice of competitive transplantation (i.e. with co-existing wild-type and JAK2V617F mutant cells but no MPN), and CD45.2 marrow cells from recipient mice of direct transplantation (i.e. with mutant donor cells alone and overt MPN). * P<0.05

A mutant microenvironment overcomes the competition between wild-type and JAK2V617F mutant cells, leading to the development of a MPN

Endothelial cells (ECs) are an essential component of the hematopoietic niche and most HSPCs reside close to a marrow sinusoid (the “vascular niche”)31. The JAK2V617F mutation can be detected in microvascular ECs isolated from liver32 and spleen33 (by laser microdissection), and marrow (by flow cytometry)34 of many patients with MPNs. The mutation can also be detected in 60-80% of EC progenitors derived from the hematopoietic lineage and, in some reports based on in vitro culture assays, in endothelial colony-forming cells from MPN patients33–37. As the Tie2-cre promoter directs JAK2V617F expression in all hematopoietic cells (including HSPCs) and vascular ECs20,21, the Tie2-cre+FF1+ murine model allows us to determine the effects of a “malignant niche” on the competition between wild-type and JAK2V617F mutant HSPCs. When a 50-50 mix of wild-type and JAK2V617F mutant marrow cells were transplanted into lethally irradiated control mice (with normal ECs), or Tie2-cre+FF1+ mice (with mutant ECs), only the latter developed a MPN phenotype at 16 wks post transplantation (Figure 3A–C), results consistent with our previous report.23

Figure 3.

Figure 3.

Open in a new tab

Microenvironmental effects on HSPC competition. (A) Scheme of competitive marrow transplantation experiments where both wild-type and JAK2V617F marrow donors were injected together into lethally irradiated Tie2-cre control mice (with wild-type vascular niche) or Tie2-cre+FF1+ mice (with JAK2V617F mutant vascular niche). (B-C) Peripheral blood CD45.2 donor chimerism and blood cell counts at 16wk post transplantation (n=5-8 mice in each group from two independent experiments). (D-E) Peripheral blood CD45.2 donor chimerism and blood cell counts at 7-8wk post transplantation (n=8 in each group from two independent experiments). (F) Marrow CD150+CD48− HSPC frequencies in Tie2-cre recipient mice and Tie2+FF1+ recipient mice at 7-8wk post transplantation (n=4 mice in each group). (G) Scheme of competitive marrow transplantation experiments. (H) Peripheral blood cell counts 40wk after transplantation (n=6 mice in each group from two independent experiments). * P <0.05

To test whether this effect on wild-type and mutant cell competition was due to any endogenous hematopoiesis from the Tie2-cre+FF1+ recipient mice, we examined the recipient mice at 7-8 weeks after the competitive transplantation. Our previous work showed that the Tie2-cre+FF1+ recipient mice did not develop any recovery of endogenous JAK2V617F mutant hematopoiesis until at least ten weeks after the lethal irradiation and transplantation29. We found that, although there was no significant difference in peripheral blood cell counts between the two groups at this early time point, Tie2-cre+FF1+ recipient mice (with mutant vascular niche) displayed a significant expansion of JAK2V617F mutant HSPCs compared to control recipient mice (with wild-type niche) (Figure 3D–F). These findings are consistent with our previous report that the JAK2V617F mutant HSPC displayed a relative growth advantage over the wild-type HSPCs when co-cultured with mutant ECs in vitro, while no difference in cell proliferation was observed between wild-type and mutant HSPCs when co-cultured with wild-type ECs28.

To test for the possibility that mutant HSPCs may require a longer period of time to develop the disease phenotype during competitive repopulation in a wild-type environment, in an additional experiment we transplanted a 50-50 mix of normal (CD45.1) and Tie2-cre+FF1+ (CD45.2) marrow cells, or a 50-50 mix of normal (CD45.1) and Tie2-cre (CD45.2) marrow cells into lethally irradiated wild-type recipients and followed these mice up to 40 wks post transplantation. We did not observe any difference in peripheral blood cell counts between recipients of Tie2-cre donors and recipients of Tie2-cre+FF1+ donors in this competitive transplantation environment (Figure 3G–H). Taken together, these observations indicate that when both wild-type and JAK2V617F mutant marrow cells are transplanted into a mouse with a normal microenvironment, mutant HSPCs by themselves are insufficient to develop a MPN phenotype, but that a diseased microenvironment (e.g. with JAK2V617F-bearing vascular ECs) can overcome the competition between wild-type and JAK2V617F mutant HSPCs and lead to the development of an overt MPN.

Exposure to wild-type cells alters both gene expression profile and cellular function of the JAK2V617F mutant HSPCs

With the establishment of these two cell competition models in either wild-type niche (with co-existing wild-type and JAK2V617F mutant cells but no MPN, as in Fig 2) or mutant niche (with mutant clonal expansion and overt MPN, as in Fig 3), we next explored the cellular and molecular mechanisms of the competitive interactions between wild-type and mutant cells, and the nature of the microenvironmental factors that drive JAK2V617F mutant cell clonal dominance in the development of MPNs.

First, we measured wild-type and JAK2V617F mutant cell proliferation in vivo by BrdU labeling (Figure 4A–B). Using Lin−cKit+Sca1+CD150+CD48− as the highly primitive HSC phenotype markers, we found that JAK2V617F mutant HSCs proliferated more rapidly than wild-type HSCs when transplanted separately into lethally irradiated wild-type recipients; however, when transplanted together in a 50-50 mix, wild-type HSCs actually displayed a significantly higher rate of proliferation than mutant HSCs (Figure 4C–D). These results echoed our findings derived from the in vitro studies (Figure 1). In contrast, there was no difference in cell proliferation between co-existing wild-type and JAK2V617F mutant Lin−cKit+Sca1+ (LSK) cells, and JAK2V617F mutant unfractionated marrow cells were found to proliferate more rapidly than wild-type cells (Figure 4C–D). In addition, while there were equal numbers of wild-type and JAK2V617F mutant HSCs in recipients of 50% mutant and 50% wild-type donor cells, there were significantly more total mutant marrow cells than wild-type marrow cells, consistent with the fact that whole marrow is predominately maturing cells (Figure 4E). These findings suggest that competition between wild-type and JAK2V617F mutant cells varies at differing stages of differentiation, especially prominent at the most primitive cell level, and point to the importance of testing cell cycle kinetics at multiple levels of cellular maturation.

Figure 4.

Figure 4.

Open in a new tab

The presence of wild-type cells alters the behavior of JAK2V617F-mutant cells in vivo. (A) Scheme of marrow transplantation experiments. (B) Representative flow cytometry plots showing gating strategy used to identify LSKs (Lin−cKit+Sca1+), HSCs (Lin−cKit+Sca1+CD150+CD48−), and BrdU-positive cells. (C-D) Cell proliferation of wild-type (grey) and JAK2V617F-mutant (black) HSCs (Lin−cKit+Sca1+CD150+CD48−), LSKs (Lin−cKit+Sca1+), and unfractionated whole marrow cells in direct transplantation (B) and in competitive transplantation (C). (n= 4-5 mice in each group) (E) Wild-type and JAK2V617F-mutant marrow HSCs (left) and unfractionated marrow cells (right) in recipients of both wild-type and mutant donor cells. (n=5 mice) (F) Scheme of marrow transplantation experiments. (G) Peripheral blood donor chimerism at 4 weeks following competitive repopulation assay in which 5x105 JAK2V617F marrow cells with or without exposure to wild-type cell competition were injected together with 5x105 competitor CD45.1 wild-type marrow cells into lethally irradiated CD45.1 wild-type recipients (n=4-5 in each group). (H-I) Principal Component analysis (H) and heatmap of expression (I) of all differentially expressed genes (with an adjusted P-value<0.05) identified by RNA-seq analysis from isolated wild-type Lin−cKit+ HSPCs transplanted alone (#1) (pooled sample from 3 mice), JAK2V617F mutant HSPCs transplanted together with wild-type cells (#2) (pooled sample from 3 mice), and JAK2V617F mutant HSPCs transplanted alone (#3) (pooled sample from 2 mice). (J) Differentially enriched KEGG pathways in mutant HSPCs with cell competition (#2) compared to mutant HSPCs without cell competition (#3). P values are plotted as the negative of their logarithm.

To evaluate the long-term consequences of such competitive interactions between wild-type and JAK2V617F mutant cells on mutant stem cell function, we performed secondary competitive repopulation assays in which we compared the engraftment potential of the JAK2V617F mutant marrow cells that had been transplanted alone into wild-type recipients (i.e. no exposure to wild-type cell competition), to those that had previously been transplanted in a 50-50 mix (i.e. exposed to cell competition) (Figure 4F). At 4-week post-transplant, there was significant CD45.2 (JAK2V617F mutant) donor chimerism in recipients of JAK2V617F mutant marrow cells previously not subjected to cell competition; in contrast, JAK2V617F mutant marrow cells previously subjected to cell competition did not engraft to a significant extent in these secondary transplantation experiments (Figure 4G). These results indicate that competition with wild-type cells suppressed JAK2V617F mutant stem cell function (i.e. the ability to repopulate hematopoiesis upon secondary transplantation).

To further understand the underlying molecular mechanisms of cell competition on JAK2V617F mutant HSPC function, we performed gene expression profiling using bulk RNA sequencing of wild-type and mutant Lin−cKit+ HSPCs isolated from the recipient mice illustrated in Figure 4A. Principal component analysis and unsupervised hierarchical clustering revealed that mutant HSPCs facing cell competition (i.e. transplanted together with wild-type cells) were distinct from mutant HSPCs without competition (i.e. transplanted alone) (Figure 4H–I). 709 genes were differentially expressed (497 down- and 212 up-regulated) in JAK2V617F mutant HSPCs with cell competition compared to JAK2V617F mutant HSPCs without competition. Changes in a number of molecular signatures, including cytokine-cytokine receptor interactions, the PI3K-Akt pathway, extracellular matrix-receptor interaction, focal adhesion, and JAK-STAT signaling pathways, were highly enriched in mutant HSPCs with cell competition compared to mutant HSPCs without cell competition (Figure 4J). Taken together with the in vitro co-culture assays and in vivo competitive transplantation assays, these results indicate that exposure to wild-type cells alters both the gene expression profile and cellular function of the JAK2V617F mutant HSPCs.

A mutant microenvironment alters the competition between wild-type and JAK2V617F mutant cells

Next, we determined how microenvironmental signals influence the competition between wild-type cells and JAK2V617F mutant cells using the two cell competition models we have established (Figure 5A). We first examined cell cycle status of both mutant and wild-type CD150+CD48− HSPCs, a highly enriched stem/progenitor cell population of which~ 20% display long-term repopulating capacity30, using Hoechst33342 and Pyronin Y staining. We found that, consistent with the myeloproliferative phenotype we observed in the Tie2-cre+FF1+ recipients, JAK2V617F mutant cells were more cycling (less quiescent) than wild-type cells in the Tie2-cre+FF1+ recipients (with mutant ECs), while there were no differences in cell cycle status between wild-type and mutant HSPCs co-existing in control recipients (with normal ECs) (Figure 5B).

Figure 5.

Figure 5.

Open in a new tab

The microenvironment affects wild-type and JAK2V617F mutant cell competition. (A) Scheme of competitive marrow transplantation experiments where both wild-type and JAK2V617F mutant marrow donors were injected together into lethally irradiated Tie2-cre control mice (with wild-type vascular niche but no MPN) or Tie2-cre+FF1+ mice (with JAK2V617F mutant vascular niche and overt MPN). (B-C) Representative flow cytometry plots and quantitative analysis of G0 cell cycle status (B) and cell apoptosis (as measured by anti-activated caspase-3 antibody; C) of wild-type and JAK2V617F mutant CD150+CD48− HSPCs in wild-type niche (top panel) and mutant niche (bottom panel) ~12-16 weeks post-competitive transplantation. (n=4-6 mice in each group) (D) Wild-type and JAK2V617F mutant marrow ECs were isolated from Tie2-cre control mice (n=3) and Tie2-cre+FF1+ mice (n=4) by microbead isolation and EC messenger RNA samples (3 wild-type and 4 mutant marrow EC samples) were assessed by RNA-sequencing. Differentially enriched KEGG pathways in JAK2V617F mutant marrow ECs compared to wild-type marrow ECs were shown with P values plotted as the negative of their logarithm. (E) Scheme of competitive transplantation experiments and CXCL12 treatment. (F) Wild-type and mutant HSC proliferation with and without CXCL12 treatment. * P<0.05

We then measured wild-type and JAK2V617F mutant cell apoptosis by assessing activated-caspase-3 staining. While there was no difference in the levels of cell apoptosis between wild-type and JAK2V617F mutant HSPCs in control mice with a wild-type niche, mutant HSPCs displayed significantly less apoptosis compared to wild-type HSPCs in Tie2-cre+FF1+ recipients mice with a mutant niche (Figure 5C). These findings are consistent with our previous report that the mutant JAK2V617F-bearing vascular niche protects HSPCs from e.g. radiation-induced apoptosis29.

Transcriptomic profiling of marrow CD45−CD31+ ECs isolated from Tie2-cre+FF1+ mice and Tie2-cre control mice using bulk RNA sequencing revealed that 623 genes were differentially expressed (311 down- and 312 up-regulated) in JAK2V617F mutant marrow ECs compared to wild-type marrow ECs. Similar to the HSPCs, extracellular matrix-receptor interaction, cell adhesion molecules, and cytokine-cytokine receptor interaction pathways were highly up-regulated in mutant marrow ECs (Figure 5D).

CXCL12 is an essential vascular niche factor for HSPC maintenance and survival31. CXCL12 and its receptor CXCR4 are also involved in cancer cell proliferation and play important roles in the interactions between cancer cells and their microenvironment38,39. Previously, we reported that CXCL12 was upregulated in JAK2V617F mutant marrow ECs compared to wild-type ECs and its receptor CXCR4 was upregulated in mutant HSPCs compared to wild-type HSPCs23. These observations, together with the dysregulated cytokine-cytokine receptor interaction pathway in both JAK2V617F mutant HSPCs with cell competition (Figure 4J) and JAK2V617F mutant marrow ECs (Figure 5D), prompted us to hypothesize that altered CXCL12 cytokine and its receptor CXCR4 signaling can alter wild-type and JAK2V617F mutant cell competition to promote mutant clone expansion. In support of this hypothesis, we found that a single 400ng dose of CXCL12 (intravenous injection)40 altered competing wild-type and mutant HSPC proliferation differently – it increased mutant cell proliferation while suppressed (though not significant) wild-type cell proliferation (Figure 5E–F), mimicking what we have observed of wild-type and mutant cell competition in the JAK2V617F-bearing vascular niche (Figure 5A–B).

Discussion

The co-existence of wild-type and JAK2V617F mutant cells in conditions that range from CHIP to chronic MPNs, which over long periods of time can remain stable or rapidly progress to frank malignancy, coupled with the high risk of relapse following curative allogeneic stem cell transplantation10–12,16–19, all make MPN a unique model disease to study the early stages of clonal competition and tumor progression. Although there have been conflicting reports on the effects of wild-type cells on JAK2V617F mutant cells during competitive transplantation in different murine models of MPN41–43, our studies provide clear evidence that the presence of wild-type cells alters the behavior of co-existing JAK2V617F mutant cells. Most importantly, our data is supported by clinical observations in patients with MPNs10–12, as well as in individuals with JAK2V617F mutant CHIP who do not have hematological abnormalities and usually do not convert to apparent MPNs13–15,44.

In contrast to the traditional view of cell competition in which more-fit cells out-compete their less-fit neighbors (“survival of the fittest”), our work suggests that competition between wild-type cells and JAK2V617F mutant cells may lead to their stable co-existence (“survival of the stable”). When 50% mutant marrow cells and 50% wild-type marrow cells were transplanted together into lethally irradiated wild-type recipient mice, the JAK2V617F mutant donor cells displayed engraftment similar to the wild-type donor cells, and the recipient mice had normal blood cell counts during up to ~10mo of follow up (Figure 2 and 3G–H). Quantitative measurement of cell proliferation in vivo revealed that the co-existing wild-type cells can suppress JAK2V617F mutant stem cell proliferation while leaving more differentiated mutant cell expansion intact, leading to the stable co-existence of both wild-type and mutant cells without developing an overt MPN phenotype. We noted that the JAK2V617F mutant cells stimulated co-existing wild-type cell proliferation in both co-culture experiments in vitro (Figure 1B and E) and competitive transplantation experiments in vivo (Figure 4D). These observations suggest that the mutant cells can alter the behavior of co-existing wild-type cells and cell competition is indeed a twoway interaction. Similarly, a previous study reported that the JAK2V617F mutation can cause aberrant cytokine production in the co-existing nonmalignant cells45.

In humans, cells carrying oncogenic mutations that are phenotypically silent for many years are not uncommon. It has long been recognized that the microenvironment surrounding tumor cells can provide tumor-suppressive signals to prevent mutant cells from expanding46. Several groups have shown that the JAK2V617F mutation can be detected in isolated liver, spleen, and marrow microvascular ECs from patients with MPNs32–34. We found that, in contrast to the stable co-existence of both wild-type and mutant cells in a wild-type niche, a JAK2 mutation-bearing vascular niche can alter the interactions between wild-type and mutant cells to promote mutant clonal expansion and the development of a MPN (Figures 3 and 5). Previously, we showed that CXCL12, an essential vascular niche factor, was up-regulated in JAK2V617F mutant ECs23. In this study, we demonstrated that CXCL12 treatment increased the proliferation of JAK2V617F mutant HSCs while suppressed wild-type HSC proliferation (Figure 5E–F). These results suggest that CXCL12 and its receptor CXCR4 may contribute to tumor microenvironment-induced JAK2V617F mutant clonal expansion in our MPN murine models. Since activated CXCL12 and its receptor CXCR4 signaling has been reported in patients with MPNs47–49, our studies suggest that the CXCL12/CXCR4 signaling is a potential therapeutic target to control mutant clone expansion and prevent the evolution of MPN to acute leukemia. Due to the limitation of the Tie2-cre model, in which the Cre recombinase is expressed in both hematopoietic cells and endothelial cells, we cannot completely exclude the possibility that some residual mutant hematopoiesis in the Tie2-cre+FF1+ recipient mice may eventually relapse and add to the competition between the wild-type and mutant donor cells. However, our conclusion would remain the same, that is, a mutant microenvironment would add to the competitiveness of mutant cells either through promoting mutant cell proliferation or through protecting them from cell death. Together with our previous work23,28,29, findings from this study further demonstrate the critical roles of a diseased microenvironment in MPN pathogenesis, as well as the dynamic interactions among wild-type cells, mutant cells, and their microenvironment. Further understanding of the molecular mechanisms controlling the competitive interactions between normal and neoplastic stem cells, and how these mechanisms break down during cancer progression and relapse hold great potential for advances in treating cancer.

Highlights.

  • The presence of wild-type cells prevents the expansion of JAK2V617F cells in a normal niche

  • Exposure to wild-type cells alters both gene expression and cellular function of JAK2V617F cells

  • A mutant niche promotes JAK2V617F cell expansion over wild-type cells

  • The CXCL12/CXCR4 signaling affects the competition between wild-type and JAK2V167F cells

ACKNOWLEDGEMENTS

This research was supported by the National Heart, Lung, and Blood Institute grant NIH R01 HL134970 (H.Z.), VA Career Development Award BX001559 (HZ), and VA Merit Award BX003947 (H.Z.).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

REFERENCES

  • 1.Moreno E. Is cell competition relevant to cancer? Nat Rev Cancer 2008;8:141–7. [DOI] [PubMed] [Google Scholar]
  • 2.Green DR. Cell competition: pirates on the tangled bank. Cell Stem Cell 2010;6:287–8. [DOI] [PubMed] [Google Scholar]
  • 3.Johnston LA. Socializing with MYC: cell competition in development and as a model for premalignant cancer. Cold Spring Harb Perspect Med 2014;4:a014274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Martincorena I, Fowler JC, Wabik A, et al. Somatic mutant clones colonize the human esophagus with age. Science 2018;362:911–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Martincorena I, Roshan A, Gerstung M, et al. Tumor evolution. High burden and pervasive positive selection of somatic mutations in normal human skin. Science 2015;348:880–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Jaiswal S, Ebert BL. Clonal hematopoiesis in human aging and disease. Science 2019;366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Menendez J, Perez-Garijo A, Calleja M, Morata G. A tumor-suppressing mechanism in Drosophila involving cell competition and the Hippo pathway. Proc Natl Acad Sci U S A 2010;107:14651–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Martins VC, Busch K, Juraeva D, et al. Cell competition is a tumour suppressor mechanism in the thymus. Nature 2014;509:465–70. [DOI] [PubMed] [Google Scholar]
  • 9.James C, Mazurier F, Dupont S, et al. The hematopoietic stem cell compartment of JAK2V617F-positive myeloproliferative disorders is a reflection of disease heterogeneity. Blood 2008;112:2429–38. [DOI] [PubMed] [Google Scholar]
  • 10.Lambert JR, Gale RE, Linch DC. The production of JAK2 wild-type platelets is not downregulated in patients with JAK2 V617F mutant-positive essential thrombocythaemia. Br J Haematol 2009;145:128–30. [DOI] [PubMed] [Google Scholar]
  • 11.Stein BL, Williams DM, Wang NY, et al. Sex differences in the JAK2 V617F allele burden in chronic myeloproliferative disorders. Haematologica 2010;95:1090–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Gale RE, Allen AJ, Nash MJ, Linch DC. Long-term serial analysis of X-chromosome inactivation patterns and JAK2 V617F mutant levels in patients with essential thrombocythemia show that minor mutant-positive clones can remain stable for many years. Blood 2007;109:1241–3. [DOI] [PubMed] [Google Scholar]
  • 13.Xie M, Lu C, Wang J, et al. Age-related mutations associated with clonal hematopoietic expansion and malignancies. Nat Med 2014;20:1472–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Jaiswal S, Fontanillas P, Flannick J, et al. Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med 2014;371:2488–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.McKerrell T, Park N, Moreno T, et al. Leukemia-associated somatic mutations drive distinct patterns of age-related clonal hemopoiesis. Cell Rep 2015;10:1239–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kroger N, Holler E, Kobbe G, et al. Allogeneic stem cell transplantation after reduced-intensity conditioning in patients with myelofibrosis: a prospective, multicenter study of the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Blood 2009;114:5264–70. [DOI] [PubMed] [Google Scholar]
  • 17.Rondelli D, Goldberg JD, Isola L, et al. MPD-RC 101 prospective study of reduced-intensity allogeneic hematopoietic stem cell transplantation in patients with myelofibrosis. Blood 2014;124:1183–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Guardiola P, Anderson JE, Bandini G, et al. Allogeneic stem cell transplantation for agnogenic myeloid metaplasia: a European Group for Blood and Marrow Transplantation, Societe Francaise de Greffe de Moelle, Gruppo Italiano per il Trapianto del Midollo Osseo, and Fred Hutchinson Cancer Research Center Collaborative Study. Blood 1999;93:2831–8. [PubMed] [Google Scholar]
  • 19.Kroger N Current Challenges in Stem Cell Transplantation in Myelofibrosis. Curr Hematol Malig Rep 2015;10:344–50. [DOI] [PubMed] [Google Scholar]
  • 20.Tiedt R, Hao-Shen H, Sobas MA, et al. Ratio of mutant JAK2-V617F to wild-type Jak2 determines the MPD phenotypes in transgenic mice. Blood 2008;111:3931–40. [DOI] [PubMed] [Google Scholar]
  • 21.Constien R, Forde A, Liliensiek B, et al. Characterization of a novel EGFP reporter mouse to monitor Cre recombination as demonstrated by a Tie2 Cre mouse line. Genesis 2001;30:36–44. [DOI] [PubMed] [Google Scholar]
  • 22.Etheridge SL, Roh ME, Cosgrove ME, et al. JAK2V617F-positive endothelial cells contribute to clotting abnormalities in myeloproliferative neoplasms. Proc Natl Acad Sci U S A 2014;111:2295–300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Zhan H, Lin CHS, Segal Y, Kaushansky K. The JAK2V617F-bearing vascular niche promotes clonal expansion in myeloproliferative neoplasms. Leukemia 2018;32:462–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Zhang Y, Lin CHS, Kaushansky K, Zhan H. JAK2V617F Megakaryocytes Promote Hematopoietic Stem/Progenitor Cell Expansion in Mice Through Thrombopoietin/MPL Signaling. Stem Cells 2018;36:1676–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Shapiro HM. Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y. Cytometry 1981;2:143–50. [DOI] [PubMed] [Google Scholar]
  • 26.Kent DG, Copley MR, Benz C, et al. Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential. Blood 2009;113:6342–50. [DOI] [PubMed] [Google Scholar]
  • 27.Kent DG, Li J, Tanna H, et al. Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion. PLoS Biol 2013;11:e1001576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Lin CH, Kaushansky K, Zhan H. JAK2V617F-mutant vascular niche contributes to JAK2V617F clonal expansion in myeloproliferative neoplasms. Blood Cells Mol Dis 2016;62:42–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lin CHS, Zhang Y, Kaushansky K, Zhan H. JAK2V617F-bearing vascular niche enhances malignant hematopoietic regeneration following radiation injury. Haematologica 2018;103:1160–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005;121:1109–21. [DOI] [PubMed] [Google Scholar]
  • 31.Crane GM, Jeffery E, Morrison SJ. Adult haematopoietic stem cell niches. Nat Rev Immunol 2017. [DOI] [PubMed] [Google Scholar]
  • 32.Sozer S, Fiel MI, Schiano T, Xu M, Mascarenhas J, Hoffman R. The presence of JAK2V617F mutation in the liver endothelial cells of patients with Budd-Chiari syndrome. Blood 2009;113:5246–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Rosti V, Villani L, Riboni R, et al. Spleen endothelial cells from patients with myelofibrosis harbor the JAK2V617F mutation. Blood 2013;121:360–8. [DOI] [PubMed] [Google Scholar]
  • 34.Helman R, Pereira WO, Marti LC, et al. Granulocyte whole exome sequencing and endothelial JAK2V617F in patients with JAK2V617F positive Budd-Chiari Syndrome without myeloproliferative neoplasm. Br J Haematol 2018;180:443–5. [DOI] [PubMed] [Google Scholar]
  • 35.Yoder MC, Mead LE, Prater D, et al. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood 2007;109:1801–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Teofili L, Martini M, Iachininoto MG, et al. Endothelial progenitor cells are clonal and exhibit the JAK2(V617F) mutation in a subset of thrombotic patients with Ph-negative myeloproliferative neoplasms. Blood 2011;117:2700–7. [DOI] [PubMed] [Google Scholar]
  • 37.Piaggio G, Rosti V, Corselli M, et al. Endothelial colony-forming cells from patients with chronic myeloproliferative disorders lack the disease-specific molecular clonality marker. Blood 2009;114:3127–30. [DOI] [PubMed] [Google Scholar]
  • 38.Orimo A, Gupta PB, Sgroi DC, et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 2005;121:335–48. [DOI] [PubMed] [Google Scholar]
  • 39.Domanska UM, Kruizinga RC, Nagengast WB, et al. A review on CXCR4/CXCL12 axis in oncology: no place to hide. Eur J Cancer 2013;49:219–30. [DOI] [PubMed] [Google Scholar]
  • 40.Niswander LM, Fegan KH, Kingsley PD, McGrath KE, Palis J. SDF-1 dynamically mediates megakaryocyte niche occupancy and thrombopoiesis at steady state and following radiation injury. Blood 2014;124:277–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Lundberg P, Takizawa H, Kubovcakova L, et al. Myeloproliferative neoplasms can be initiated from a single hematopoietic stem cell expressing JAK2-V617F. J Exp Med 2014;211:2213–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Li J, Spensberger D, Ahn JS, et al. JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F-positive essential thrombocythemia. Blood 2010;116:1528–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Mullally A, Lane SW, Ball B, et al. Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells. Cancer Cell 2010;17:584–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Genovese G, Kahler AK, Handsaker RE, et al. Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N Engl J Med 2014;371:2477–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Kleppe M, Kwak M, Koppikar P, et al. JAK-STAT pathway activation in malignant and nonmalignant cells contributes to MPN pathogenesis and therapeutic response. Cancer Discov 2015;5:316–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Bissell MJ, Hines WC. Why don’t we get more cancer? A proposed role of the microenvironment in restraining cancer progression. Nat Med 2011;17:320–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Rosti V, Massa M, Vannucchi AM, et al. The expression of CXCR4 is down-regulated on the CD34+ cells of patients with myelofibrosis with myeloid metaplasia. Blood Cells Mol Dis 2007;38:280–6. [DOI] [PubMed] [Google Scholar]
  • 48.Wang X, Cho SY, Hu CS, Chen D, Roboz J, Hoffman R. C-X-C motif chemokine 12 influences the development of extramedullary hematopoiesis in the spleens of myelofibrosis patients. Exp Hematol 2015;43:100–9 e1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Abdelouahab H, Zhang Y, Wittner M, et al. CXCL12/CXCR4 pathway is activated by oncogenic JAK2 in a PI3K-dependent manner. Oncotarget 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES