ilia and displayed a high percentage of Gr 1 granulocytes both in blood and spleen, whereas the percentage of granulocytes AZD8055 in BM was only slightly affected. An increase in B220 B cells was also observed in the spleen, which may be caused by an increase in lymphopoiesis mediated by the ectopic expression of the G CSF RT617N after retroviral transduction in hematopoietic stem cells. Alternatively, this might be related to cytokine release in the spleen by the granulocytic precursors. All CSF3RT617N mice developed splenomegaly. These data demonstrate that the T617N mutation in the G CSF R is at the origin of the neutrophilia observed in patients.
To directly assess the consequences of this mutation on human HSCs, we performed xenotransplantation in irradiated NOG mice of CD34 To investigate the effects of the T617N mutation on G CSF R signaling, CD34 cells isolated from normal donors or patients were first deprived of cytokines, and then stimulated with G CSF CAL-101 for various periods of time and subjected to Western blot analysis using specific antibodies. Interestingly, patient cells exhibited constitutive phosphorylation of JAK2, STAT3, AKT, and ERK. After G CSF stimulation, JAK2, STAT3, STAT5, ERK, and AKT were rapidly and transiently activated in cells from normal donors, whereas phosphorylation was much more potent and sustained in cells from patients. In addition, a specific JAK2 inhibitor abrogated constitutive and G CSF induced activation, suggesting that the mechanism of neutrophilia was JAK2 dependent, reminiscent of JAK2V617F MPD.
We next infected mouse lineage BM cells with a retrovirus containing CSF3Rwt or CSF3RT617N mutation or Figure 2. Computational searches of wild type and T617N G CSF R dimers. Molecular structure of the helix dimer of the T617N mutant having the overall lowest energy in the computational searches. The helices have a left handed crossing angle and an axial separation of 10. 5 ?. The interfacial amino acids are highlighted. Plot of the helix interaction energies in the wild type and T617N TM helix dimers with axial separations of 10. 5 ?. In the wild type G CSF R, the dominant interaction is a direct Thr617 Thr617 hydrogen bond between side chain hydroxyl groups. In contrast, there are several strong stabilizing interactions in the mutant. The Asn amide side chain forms hydrogen bonds to the thiol side chain of Cys620.
Trp624 forms stabilizing van der Waals contacts with the opposing helix, the indole NH is also able to form an interhelical H bond with Cys620. The total interaction energies were very similar for the lowest energy T617N dimers in computational searches at 10 ? and 10. 5 ?. Cross section of the T617N helix dimer showing hydrogen bonding interactions involving Thr617 and Cys620. The axial separation is 10. 5 ?. Cross section of the T617N helix dimer with an axial separation of 11 ? stabilized by interhelical interactions between Asn617 side chains and between Asn617 and Cys618. The overall interaction energies were generally higher at longer interhelical separations for both the wild type and mutant dimers, although the T617N dimers were consistently lower in energy than those of the wild type dimers because of the ability of Asn617 to form interhelical hydrogen bonds. Figure 3. CSF3RT617N mutation y