Limits...
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.

Show MeSH

Related in: MedlinePlus

E-SLAM HSCs do not expand in old JAK2V617F knock-in mice and show reduced functional ability as well as a delayed entry into the cell cycle.(A) E-SLAM HSCs were increased in frequency in wild type (∼2-fold, see Figure 1E) but not JAK2V617F marrow in 18–24-mo-old mice (n = 10) compared to 6–10-mo-old mice resulting in a 3-fold overall reduction in E-SLAM HSCs compared to wild-type (p = 0.002) in three independent experiments. (B) Individual HSCs were cultured and cell counts were recorded on day 1 and day 2 to determine whether or not they had undergone a division in three independent experiments. At day 2, significantly fewer (p = 0.039) old JAK2V617F HSCs had divided. The cloning efficiency (C), number of cells per clone (D), and number of KSL cells per clone (F) were not different, but the JAK2V617F cells still produced more differentiated cell types after 10 d of culture (p = 0.039, E). (G) Competitive transplantation of whole bone marrow from old JAK2V617F mice, transformed JAK2V617F mice, and their respective WT littermate controls. Relative chimerism is calculated by measuring donor chimerism as a percentage of donor+competitor chimerism and normalized to the average of the WT contribution (set to 1). The old JAK2V617F BM displays reduced chimerism (p<0.01), whereas transformed JAK2V617F mice that have undergone transformation reacquire their self-renewal capacity.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3672217&req=5

pbio-1001576-g004: E-SLAM HSCs do not expand in old JAK2V617F knock-in mice and show reduced functional ability as well as a delayed entry into the cell cycle.(A) E-SLAM HSCs were increased in frequency in wild type (∼2-fold, see Figure 1E) but not JAK2V617F marrow in 18–24-mo-old mice (n = 10) compared to 6–10-mo-old mice resulting in a 3-fold overall reduction in E-SLAM HSCs compared to wild-type (p = 0.002) in three independent experiments. (B) Individual HSCs were cultured and cell counts were recorded on day 1 and day 2 to determine whether or not they had undergone a division in three independent experiments. At day 2, significantly fewer (p = 0.039) old JAK2V617F HSCs had divided. The cloning efficiency (C), number of cells per clone (D), and number of KSL cells per clone (F) were not different, but the JAK2V617F cells still produced more differentiated cell types after 10 d of culture (p = 0.039, E). (G) Competitive transplantation of whole bone marrow from old JAK2V617F mice, transformed JAK2V617F mice, and their respective WT littermate controls. Relative chimerism is calculated by measuring donor chimerism as a percentage of donor+competitor chimerism and normalized to the average of the WT contribution (set to 1). The old JAK2V617F BM displays reduced chimerism (p<0.01), whereas transformed JAK2V617F mice that have undergone transformation reacquire their self-renewal capacity.

Mentions: Normally HSCs undergo several qualitative and quantitative changes with increasing age including a variably expanded phenotypically defined HSC pool, delayed proliferative responses in vitro, and reduced functional capacity in vivo as measured by transplantation of purified HSCs [26]–[28]. We therefore analyzed BM from JAK2V617F mice and WT littermate controls that were 18–24 mo after pIpC injection (hereafter called old mice). In WT mice, the E-SLAM HSC compartment was ∼2-fold larger in old mice compared to younger mice (compare Figure 4A to Figure 1F). However, the same comparison in JAK2V617F mice shows that the E-SLAM HSC compartment was not expanded in old mice. As a consequence, there was a 3-fold reduction (p = 0.002) in the frequency of E-SLAM HSCs in old JAK2V617F mice compared to their WT littermate controls.


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)

E-SLAM HSCs do not expand in old JAK2V617F knock-in mice and show reduced functional ability as well as a delayed entry into the cell cycle.(A) E-SLAM HSCs were increased in frequency in wild type (∼2-fold, see Figure 1E) but not JAK2V617F marrow in 18–24-mo-old mice (n = 10) compared to 6–10-mo-old mice resulting in a 3-fold overall reduction in E-SLAM HSCs compared to wild-type (p = 0.002) in three independent experiments. (B) Individual HSCs were cultured and cell counts were recorded on day 1 and day 2 to determine whether or not they had undergone a division in three independent experiments. At day 2, significantly fewer (p = 0.039) old JAK2V617F HSCs had divided. The cloning efficiency (C), number of cells per clone (D), and number of KSL cells per clone (F) were not different, but the JAK2V617F cells still produced more differentiated cell types after 10 d of culture (p = 0.039, E). (G) Competitive transplantation of whole bone marrow from old JAK2V617F mice, transformed JAK2V617F mice, and their respective WT littermate controls. Relative chimerism is calculated by measuring donor chimerism as a percentage of donor+competitor chimerism and normalized to the average of the WT contribution (set to 1). The old JAK2V617F BM displays reduced chimerism (p<0.01), whereas transformed JAK2V617F mice that have undergone transformation reacquire their self-renewal capacity.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1001576-g004: E-SLAM HSCs do not expand in old JAK2V617F knock-in mice and show reduced functional ability as well as a delayed entry into the cell cycle.(A) E-SLAM HSCs were increased in frequency in wild type (∼2-fold, see Figure 1E) but not JAK2V617F marrow in 18–24-mo-old mice (n = 10) compared to 6–10-mo-old mice resulting in a 3-fold overall reduction in E-SLAM HSCs compared to wild-type (p = 0.002) in three independent experiments. (B) Individual HSCs were cultured and cell counts were recorded on day 1 and day 2 to determine whether or not they had undergone a division in three independent experiments. At day 2, significantly fewer (p = 0.039) old JAK2V617F HSCs had divided. The cloning efficiency (C), number of cells per clone (D), and number of KSL cells per clone (F) were not different, but the JAK2V617F cells still produced more differentiated cell types after 10 d of culture (p = 0.039, E). (G) Competitive transplantation of whole bone marrow from old JAK2V617F mice, transformed JAK2V617F mice, and their respective WT littermate controls. Relative chimerism is calculated by measuring donor chimerism as a percentage of donor+competitor chimerism and normalized to the average of the WT contribution (set to 1). The old JAK2V617F BM displays reduced chimerism (p<0.01), whereas transformed JAK2V617F mice that have undergone transformation reacquire their self-renewal capacity.
Mentions: Normally HSCs undergo several qualitative and quantitative changes with increasing age including a variably expanded phenotypically defined HSC pool, delayed proliferative responses in vitro, and reduced functional capacity in vivo as measured by transplantation of purified HSCs [26]–[28]. We therefore analyzed BM from JAK2V617F mice and WT littermate controls that were 18–24 mo after pIpC injection (hereafter called old mice). In WT mice, the E-SLAM HSC compartment was ∼2-fold larger in old mice compared to younger mice (compare Figure 4A to Figure 1F). However, the same comparison in JAK2V617F mice shows that the E-SLAM HSC compartment was not expanded in old mice. As a consequence, there was a 3-fold reduction (p = 0.002) in the frequency of E-SLAM HSCs in old JAK2V617F mice compared to their WT littermate controls.

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