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The JAK2 V617F mutation occurs in hematopoietic stem cell in polycythemia vera and predisposes toward erythroid differentiation.

(Jamieson et al. PNAS 2006; 103: 6224-6229)
Summary by Katherine Liu
Advance Stem Cell Graduate Class, Fall 2006

Lay Summary

Polycythemia Vera (PV) is a bone marrow disorder characterized by a significant increase of products from the three blood cell lines. However, there is a primary overproduction of those of the erythroid lineage which can often lead to abnormal clotting and strokes. The cause of this disorder in a large proportion of PV patients lies in a mutation of the Janus Kinase 2 (JAK2) signaling molecule (Figure 1). The JAK2 signaling molecule maintains an intimate relationship with cytokine receptors. It may contain a mutation of a valine to phenylalanine at amino acid 617 leading it to suffer constitutive activation resulting in unlimited proliferation. Jamieson et al., in an attempted to ascertain at which developmental stage this mutation occurred, sequenced and analyzed the hematopoietic stem cells (HSC) and progenitor populations from the peripheral blood of 16 patients with PV and 14 controls.

In this study, Jamieson et al. discovered that the JAK2 mutation was found on the level of hematopoietic stem cells thereby directing the HSCs to an erythroid lineage.  The significance of this finding is that it points to a stem cell disorder. It was also noted by Jamieson et al. that there was an increase in HSCs in the peripheral blood of PV patients. These HSCs also expressed an abnormal phenotype and were showed to form larger erythroid colonies upon visual inspection. Through DNA sequences it was found that these HSCs all contained the JAK2 V617F mutation.  Most startlingly Jamieson et al.  found that the JAK2 inhibitor AG490 was capable of diminishing the erythroid production of the PV HSCs pointing to a possibly therapeutic target.  

Scientific Summary
It has been posed that Polycythemia Vera (PV) could arise either in self renewing hematopoietic stem cells (HSC), in non-self renewing multipotent progenitors or in myeloerythroid progenitors. Jamieson et al. worked to ascertain that the JAK V617F mutation was found in primitive precursors as early as HSCs. With a better understanding of the molecular basis for PV, Jamieson et al. hope to introduce patient specific treatments including an inhibitor for the mutant allele of Janus Kinase 2 (JAK2).

 Like most cancer stem cells, PV progenitors necessitated a disregard for the negative regulation signals, thus allowing the cells to proliferate indefinitely thereby resulting in the morbidity of the disease. These progenitors are theorized to retain properties resembling primitive stem cells. The researched theorized that an increase in the HSC phenotype cells collected from the peripheral blood of PV patients would denote a disorder that stemmed from an early level of hematopoiesis.

Methods and Materials
HSCS, both normal and PV, were collected from the peripheral blood (PB) of patients. They were plated in vitro and maintained in a media of methylcellulose supplemented with recombinant human cytokines. Cells were sorted using FACS (Fluorescence-activated cell sorting).  The HSC markers used in this study were CD34+. CD38-. CD90+, Lin-.

In the quantitative analysis colonies were sorted on day 14 under a Nikon Eclipse TS100 inverted microscope. In the quantitative analysis of the in vitro differentiation, pictures of the colonies were taken in phase-contrast micrographs. Sequence analysis was done on the HSCs to look for the V617F mutation.

FACS was done on HSCs (the most primitive progenitors used in the study) and more differentiated lineages such as CMP (common myeloid progenitors), GMP (granulocyte/macrophage progenitor), and MEP (megakaryote/erythrocyte progenitor) in order to separate the cells for culturing (Figure 2).

Results
Of the 16 PV patients sampled in the study, 14 had peripheral blood HSCs that showed evidence of the JAK2 V617F mutation. Using FACS analysis it was shown that patients with PV had more HSC in their PB than the control patients. The patients with PV that associated with higher white blood cell (WBC) counts had an even higher HSC count suggesting that the disease originates on a primitive progenitor level.

PV HSC Differentiation
In studying the HSCs, one finds that the PV HSC produces a greater number of BFU-E colonies upon differentiation. Upon visual inspection the PV HSC colonies also tend to be abnormally shaped and darker in color (resulting from increased erythroid production). A large proportion of the PV HSCs showed a mutation in JAK V617F and this mutation seemed to carry over in the greatest frequency to the PV CMP cells. It was noted that the JAK V617F mutation did not carry over to all progenitor derived colonies as it was hardly seen in the PV GMP and PV MEP cells.

JAK2 Inhibition
The effect of JAK2 inhibition on PV HSCs was studied by co-culturing with AG490. The investigators reported no statistical significance in the reduction of BFU-E after AG490 in the control cells. In the PV HSC cells, the investigators reported a downward trend in BFU-E after treatment with AG490 but no statistical significance.  The other colony types were not affected.

Upon investigating the persistence of JAK2 mutation after treatment with AG490, it was found that the proportion of affected cells to normal cells varies with no noticeable trend. In nearly all cases the mutation continued to persist despite treatment with AG490. It was also found that the HSC derived colonies in the PV patients with the JAK2 V617F mutation exhibited varying sensitivities to AG490.

Conclusion
The investigators proved that PV is a disease on the level of HSCs from their extensive sequence analysis although they remain uncertain at what level the disease arises, whether it is the original HSCs gone array or if a committed cell with the mutation regains the ability to self renew.

By culturing the cells in vitro the investigators drew the conclusion that the PV HSCs are not only found in greater numbers in the PB blood but also have altered differentiation potential.  Jamieson et al. posed that the IL-3 rich cell population they found pointed to a constitutive activation of the JAK2 pathway. Therefore they worked to inhibit the pathway through AG490. Jamieson et al. concluded that the downward trend in aberrant colonies in response to AG490 is promising but a more specific inhibitor needs to be developed to bind specifically to the mutant JAK2 allele.

Personal Comments:
The work Jamieson et al. done in sequencing the JAK2 V617F mutation region is valuable in ascertaining the role of a wayward copy of JAK2 in PV. However, it does not prove that the mutation occurs at a certain stage of hematopoiesis. As Jamieson et al. states in their paper, it is possible that a downstream progenitor re-acquired the ability to self-renew after significant mutation thus returning to a stem cell state. Although bone marrow studies have been done in the murine model for JAK2 V617F, it is uncertain if this can be applied to humans. The work in JAK2 inhibitors is promising in producing a patient specific drug. However, the fact that AG490 does not specifically target the wayward erythroid colonies is troubling. Whether or not, from this point onward, researchers will be able to develop an inhibitor specific to the mutant JAK2 remains in doubt. This project is a step in a very auspicious direction to patient specific treatments. Importantly, Jamieson et al.’s work also sheds light on PV stem cells the understanding of which could hold the key to a lasting cure.