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Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion.

Kent DG, Li J, Tanna H, Fink J, Kirschner K, Pask DC, Silber Y, Hamilton TL, Sneade R, Simons BD, Green AR - PLoS Biol. (2013)

Bottom Line: Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact.This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells.Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F subclinical clonal expansions and the transition to overt MPNs will illuminate the earliest stages of tumor establishment and subclone competition, fundamentally shifting the way we treat and manage cancers.

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Related in: MedlinePlus

JAK2V617F alters the balance of HSC fate choices.(A) A paired daughter cell analysis of WT and JAK2V617F HSCs shows both daughters differentiate more often from JAK2V617F parent HSCs than from WT HSCs as shown by measuring the percentage of KSL cells remaining after 10 d. Each paired daughter set is connected by a line and the pairs are categorized into symmetric SR (both daughters above the WT average %KSL), asymmetric division (one daughter above and one below the average %KSL), and symmetric differentiation (both daughters below the average %KSL). Note the relative increase in symmetric differentiation at the expense of asymmetric divisions. (B) The same paired daughter pairs are displayed here by the absolute number of KSL cells produced. Here it is clear that some of the JAK2V617F pairs produce very few KSL cells (less than 100 per clone in some of the asymmetric divisions and symmetric differentiation divisions compared to WT HSCs, which are all above 100 KSL cells). (C) The pie graph on the left represents the outcome from 78 WT paired daughters (39 pairs), and the pie on the left represents the outcome from 76 mutant paired daughters (38 pairs). (D) Normally, HSCs will execute one of several programs in concert with the other HSCs to provide the requisite numbers of stem cells, progenitors, and differentiated cells for the organism. JAK2V617F disturbs this balance and increases the likelihood of differentiation. As HSCs with the V617F mutation age, they have both an increased chance of fully exhausting as well as an increased chance of progressing to a more severe disease state, likely due to the acquisition of additional genetic or epigenetic perturbations.
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pbio-1001576-g006: JAK2V617F alters the balance of HSC fate choices.(A) A paired daughter cell analysis of WT and JAK2V617F HSCs shows both daughters differentiate more often from JAK2V617F parent HSCs than from WT HSCs as shown by measuring the percentage of KSL cells remaining after 10 d. Each paired daughter set is connected by a line and the pairs are categorized into symmetric SR (both daughters above the WT average %KSL), asymmetric division (one daughter above and one below the average %KSL), and symmetric differentiation (both daughters below the average %KSL). Note the relative increase in symmetric differentiation at the expense of asymmetric divisions. (B) The same paired daughter pairs are displayed here by the absolute number of KSL cells produced. Here it is clear that some of the JAK2V617F pairs produce very few KSL cells (less than 100 per clone in some of the asymmetric divisions and symmetric differentiation divisions compared to WT HSCs, which are all above 100 KSL cells). (C) The pie graph on the left represents the outcome from 78 WT paired daughters (39 pairs), and the pie on the left represents the outcome from 76 mutant paired daughters (38 pairs). (D) Normally, HSCs will execute one of several programs in concert with the other HSCs to provide the requisite numbers of stem cells, progenitors, and differentiated cells for the organism. JAK2V617F disturbs this balance and increases the likelihood of differentiation. As HSCs with the V617F mutation age, they have both an increased chance of fully exhausting as well as an increased chance of progressing to a more severe disease state, likely due to the acquisition of additional genetic or epigenetic perturbations.

Mentions: To challenge the prediction that JAK2V617F alters the balance between proliferation and differentiation at the apex of the stem cell hierarchy, we undertook a paired daughter cell analysis to assess the fate outcome of the first division of HSCs from JAK2V617F mice and their littermate controls. To this end, the progeny of the first cell division of input HSCs were split into individual cultures and, after 10 d, assessed by flow cytometry (see Methods S1 for splitting procedure). We elected to use the average fraction of KSL cells from the WT as a benchmark for self-renewal, since HSCs have been shown to undergo approximate balanced self-renewal under these culture conditions (previous transplantation data [20],[25] and Figure 3). We estimated the outcome of the first division based on whether progeny of the doublets were individually above or below the average (see Methods S1 for further detail). The frequency (Figure 6A) and absolute number (Figure 6B) of KSL cells per daughter cell were measured for each doublet to assess the degree of symmetry between daughters. Applying this procedure, the data indicate that the divisions of WT HSCs lead to all three possible fate outcomes in roughly equal proportion (Figure 6C). In particular, divisions leading to symmetric self-renewal appear to be in balance with those leading to symmetric differentiation, as expected. Moreover, when referred to their average KSL content, the data from the JAK2V617F HSCs also showed approximate balance (unpublished data), consistent with balanced self-renewal remaining intact at the lower progenitor tiers, despite compromised HSC self-renewal. Analysis of the fate of JAK2V617F doublets using the average fraction of KSL cells from the WT as the benchmark demonstrated a significant increase in symmetric differentiation divisions (p = 0.04), mainly at the expense of fewer asymmetric cell divisions (p = 0.01). These data suggest that JAK2V617F directly affects HSC fate choice in vitro, with consequent loss of HSCs.


Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion.

Kent DG, Li J, Tanna H, Fink J, Kirschner K, Pask DC, Silber Y, Hamilton TL, Sneade R, Simons BD, Green AR - PLoS Biol. (2013)

JAK2V617F alters the balance of HSC fate choices.(A) A paired daughter cell analysis of WT and JAK2V617F HSCs shows both daughters differentiate more often from JAK2V617F parent HSCs than from WT HSCs as shown by measuring the percentage of KSL cells remaining after 10 d. Each paired daughter set is connected by a line and the pairs are categorized into symmetric SR (both daughters above the WT average %KSL), asymmetric division (one daughter above and one below the average %KSL), and symmetric differentiation (both daughters below the average %KSL). Note the relative increase in symmetric differentiation at the expense of asymmetric divisions. (B) The same paired daughter pairs are displayed here by the absolute number of KSL cells produced. Here it is clear that some of the JAK2V617F pairs produce very few KSL cells (less than 100 per clone in some of the asymmetric divisions and symmetric differentiation divisions compared to WT HSCs, which are all above 100 KSL cells). (C) The pie graph on the left represents the outcome from 78 WT paired daughters (39 pairs), and the pie on the left represents the outcome from 76 mutant paired daughters (38 pairs). (D) Normally, HSCs will execute one of several programs in concert with the other HSCs to provide the requisite numbers of stem cells, progenitors, and differentiated cells for the organism. JAK2V617F disturbs this balance and increases the likelihood of differentiation. As HSCs with the V617F mutation age, they have both an increased chance of fully exhausting as well as an increased chance of progressing to a more severe disease state, likely due to the acquisition of additional genetic or epigenetic perturbations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672217&req=5

pbio-1001576-g006: JAK2V617F alters the balance of HSC fate choices.(A) A paired daughter cell analysis of WT and JAK2V617F HSCs shows both daughters differentiate more often from JAK2V617F parent HSCs than from WT HSCs as shown by measuring the percentage of KSL cells remaining after 10 d. Each paired daughter set is connected by a line and the pairs are categorized into symmetric SR (both daughters above the WT average %KSL), asymmetric division (one daughter above and one below the average %KSL), and symmetric differentiation (both daughters below the average %KSL). Note the relative increase in symmetric differentiation at the expense of asymmetric divisions. (B) The same paired daughter pairs are displayed here by the absolute number of KSL cells produced. Here it is clear that some of the JAK2V617F pairs produce very few KSL cells (less than 100 per clone in some of the asymmetric divisions and symmetric differentiation divisions compared to WT HSCs, which are all above 100 KSL cells). (C) The pie graph on the left represents the outcome from 78 WT paired daughters (39 pairs), and the pie on the left represents the outcome from 76 mutant paired daughters (38 pairs). (D) Normally, HSCs will execute one of several programs in concert with the other HSCs to provide the requisite numbers of stem cells, progenitors, and differentiated cells for the organism. JAK2V617F disturbs this balance and increases the likelihood of differentiation. As HSCs with the V617F mutation age, they have both an increased chance of fully exhausting as well as an increased chance of progressing to a more severe disease state, likely due to the acquisition of additional genetic or epigenetic perturbations.
Mentions: To challenge the prediction that JAK2V617F alters the balance between proliferation and differentiation at the apex of the stem cell hierarchy, we undertook a paired daughter cell analysis to assess the fate outcome of the first division of HSCs from JAK2V617F mice and their littermate controls. To this end, the progeny of the first cell division of input HSCs were split into individual cultures and, after 10 d, assessed by flow cytometry (see Methods S1 for splitting procedure). We elected to use the average fraction of KSL cells from the WT as a benchmark for self-renewal, since HSCs have been shown to undergo approximate balanced self-renewal under these culture conditions (previous transplantation data [20],[25] and Figure 3). We estimated the outcome of the first division based on whether progeny of the doublets were individually above or below the average (see Methods S1 for further detail). The frequency (Figure 6A) and absolute number (Figure 6B) of KSL cells per daughter cell were measured for each doublet to assess the degree of symmetry between daughters. Applying this procedure, the data indicate that the divisions of WT HSCs lead to all three possible fate outcomes in roughly equal proportion (Figure 6C). In particular, divisions leading to symmetric self-renewal appear to be in balance with those leading to symmetric differentiation, as expected. Moreover, when referred to their average KSL content, the data from the JAK2V617F HSCs also showed approximate balance (unpublished data), consistent with balanced self-renewal remaining intact at the lower progenitor tiers, despite compromised HSC self-renewal. Analysis of the fate of JAK2V617F doublets using the average fraction of KSL cells from the WT as the benchmark demonstrated a significant increase in symmetric differentiation divisions (p = 0.04), mainly at the expense of fewer asymmetric cell divisions (p = 0.01). These data suggest that JAK2V617F directly affects HSC fate choice in vitro, with consequent loss of HSCs.

Bottom Line: Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact.This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells.Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F subclinical clonal expansions and the transition to overt MPNs will illuminate the earliest stages of tumor establishment and subclone competition, fundamentally shifting the way we treat and manage cancers.

Show MeSH
Related in: MedlinePlus