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

JAK2V617F induces a loss of self-renewal activity and HSC numbers and leads to a lineage bias when limited HSCs are transplanted.(A) Relative chimerism following transplantation of 106 or 105 JAK2V617F or wild type (WT) whole bone marrow (BM) 6–10 months post-pIpC along with 5×105 whole BM competitor cells into eight recipient mice. Average chimerism was lower in mice receiving JAK2V617F cells (p = 0.03). (B) The relative myeloid (purple) versus lymphoid (green) contribution in each of the recipient animals were determined by calculating a ratio between the contribution to the myeloid compartment [Donor GM/(Donor GM+Competitor GM)] and lymphoid compartment [Donor BT/(Donor BT+Competitor BT)]. (C) Relative chimerism in primary peripheral blood (PB) (white bars) in the eight animals receiving 105 cells compared to levels of PB chimerism in the 16 secondary recipients (two per primary animal, grey bars) in two independent transplantation experiments. An “X” represents a recipient that showed less than 1% chimerism at 24 wk posttransplantation, and a ∧ represents a recipient that only had contribution to the lymphoid lineages. (D) The relative myeloid (purple) versus lymphoid (green) contribution in each of the secondary recipient animals were determined by calculating a ratio between the contribution to the myeloid and lymphoid compartments as in Figure 1B. (E) The FACS isolation strategy for CD45+/EPCR+/CD48−/CD150+ (E-SLAM) cells. The panels are gated on viable white blood cells and show E-SLAM gates for WT (top) and JAK2V617F (bottom). (F) The frequency of E-SLAM HSCs per 105 viable bone marrow (BM) cells in four WT and four JAK2V617F mice 6–10 mo following pIpC injection from four independent experiments. The frequency is reduced in JAK2V617F animals (p = 0.0288).
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1001576-g001: JAK2V617F induces a loss of self-renewal activity and HSC numbers and leads to a lineage bias when limited HSCs are transplanted.(A) Relative chimerism following transplantation of 106 or 105 JAK2V617F or wild type (WT) whole bone marrow (BM) 6–10 months post-pIpC along with 5×105 whole BM competitor cells into eight recipient mice. Average chimerism was lower in mice receiving JAK2V617F cells (p = 0.03). (B) The relative myeloid (purple) versus lymphoid (green) contribution in each of the recipient animals were determined by calculating a ratio between the contribution to the myeloid compartment [Donor GM/(Donor GM+Competitor GM)] and lymphoid compartment [Donor BT/(Donor BT+Competitor BT)]. (C) Relative chimerism in primary peripheral blood (PB) (white bars) in the eight animals receiving 105 cells compared to levels of PB chimerism in the 16 secondary recipients (two per primary animal, grey bars) in two independent transplantation experiments. An “X” represents a recipient that showed less than 1% chimerism at 24 wk posttransplantation, and a ∧ represents a recipient that only had contribution to the lymphoid lineages. (D) The relative myeloid (purple) versus lymphoid (green) contribution in each of the secondary recipient animals were determined by calculating a ratio between the contribution to the myeloid and lymphoid compartments as in Figure 1B. (E) The FACS isolation strategy for CD45+/EPCR+/CD48−/CD150+ (E-SLAM) cells. The panels are gated on viable white blood cells and show E-SLAM gates for WT (top) and JAK2V617F (bottom). (F) The frequency of E-SLAM HSCs per 105 viable bone marrow (BM) cells in four WT and four JAK2V617F mice 6–10 mo following pIpC injection from four independent experiments. The frequency is reduced in JAK2V617F animals (p = 0.0288).

Mentions: Competitive transplantation studies were performed to investigate the HSC defect observed in JAK2V617F mice. In order to study the effect of transplanting limiting numbers of HSCs and investigate potential lineage biases, transplantations were performed using low numbers of donor cells. When transplanted with 105 donor BM cells, mice receiving either WT or JAK2V617F cells displayed reduced PB chimerism compared to those receiving the corresponding 106 cell dose (Figure 1A). Importantly, whereas mice receiving 106 JAK2V617F cells retained a balanced production of myeloid and lymphoid lineages, those receiving 105 JAK2V617F cells exhibited marked lineage skewing (Figure 1B) as determined by the relative production of myeloid and lymphoid elements by donor and competitor cells.


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 induces a loss of self-renewal activity and HSC numbers and leads to a lineage bias when limited HSCs are transplanted.(A) Relative chimerism following transplantation of 106 or 105 JAK2V617F or wild type (WT) whole bone marrow (BM) 6–10 months post-pIpC along with 5×105 whole BM competitor cells into eight recipient mice. Average chimerism was lower in mice receiving JAK2V617F cells (p = 0.03). (B) The relative myeloid (purple) versus lymphoid (green) contribution in each of the recipient animals were determined by calculating a ratio between the contribution to the myeloid compartment [Donor GM/(Donor GM+Competitor GM)] and lymphoid compartment [Donor BT/(Donor BT+Competitor BT)]. (C) Relative chimerism in primary peripheral blood (PB) (white bars) in the eight animals receiving 105 cells compared to levels of PB chimerism in the 16 secondary recipients (two per primary animal, grey bars) in two independent transplantation experiments. An “X” represents a recipient that showed less than 1% chimerism at 24 wk posttransplantation, and a ∧ represents a recipient that only had contribution to the lymphoid lineages. (D) The relative myeloid (purple) versus lymphoid (green) contribution in each of the secondary recipient animals were determined by calculating a ratio between the contribution to the myeloid and lymphoid compartments as in Figure 1B. (E) The FACS isolation strategy for CD45+/EPCR+/CD48−/CD150+ (E-SLAM) cells. The panels are gated on viable white blood cells and show E-SLAM gates for WT (top) and JAK2V617F (bottom). (F) The frequency of E-SLAM HSCs per 105 viable bone marrow (BM) cells in four WT and four JAK2V617F mice 6–10 mo following pIpC injection from four independent experiments. The frequency is reduced in JAK2V617F animals (p = 0.0288).
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1001576-g001: JAK2V617F induces a loss of self-renewal activity and HSC numbers and leads to a lineage bias when limited HSCs are transplanted.(A) Relative chimerism following transplantation of 106 or 105 JAK2V617F or wild type (WT) whole bone marrow (BM) 6–10 months post-pIpC along with 5×105 whole BM competitor cells into eight recipient mice. Average chimerism was lower in mice receiving JAK2V617F cells (p = 0.03). (B) The relative myeloid (purple) versus lymphoid (green) contribution in each of the recipient animals were determined by calculating a ratio between the contribution to the myeloid compartment [Donor GM/(Donor GM+Competitor GM)] and lymphoid compartment [Donor BT/(Donor BT+Competitor BT)]. (C) Relative chimerism in primary peripheral blood (PB) (white bars) in the eight animals receiving 105 cells compared to levels of PB chimerism in the 16 secondary recipients (two per primary animal, grey bars) in two independent transplantation experiments. An “X” represents a recipient that showed less than 1% chimerism at 24 wk posttransplantation, and a ∧ represents a recipient that only had contribution to the lymphoid lineages. (D) The relative myeloid (purple) versus lymphoid (green) contribution in each of the secondary recipient animals were determined by calculating a ratio between the contribution to the myeloid and lymphoid compartments as in Figure 1B. (E) The FACS isolation strategy for CD45+/EPCR+/CD48−/CD150+ (E-SLAM) cells. The panels are gated on viable white blood cells and show E-SLAM gates for WT (top) and JAK2V617F (bottom). (F) The frequency of E-SLAM HSCs per 105 viable bone marrow (BM) cells in four WT and four JAK2V617F mice 6–10 mo following pIpC injection from four independent experiments. The frequency is reduced in JAK2V617F animals (p = 0.0288).
Mentions: Competitive transplantation studies were performed to investigate the HSC defect observed in JAK2V617F mice. In order to study the effect of transplanting limiting numbers of HSCs and investigate potential lineage biases, transplantations were performed using low numbers of donor cells. When transplanted with 105 donor BM cells, mice receiving either WT or JAK2V617F cells displayed reduced PB chimerism compared to those receiving the corresponding 106 cell dose (Figure 1A). Importantly, whereas mice receiving 106 JAK2V617F cells retained a balanced production of myeloid and lymphoid lineages, those receiving 105 JAK2V617F cells exhibited marked lineage skewing (Figure 1B) as determined by the relative production of myeloid and lymphoid elements by donor and competitor cells.

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