Primary myelofibrosis (PMF) is a myeloproliferative disorder characterized by abnormal trafficking of CD34+ cells, resulting in their constitutive mobilization. This abnormal trafficking of PMF CD34+ cells has been related to the reduced expression of the chemokine receptor CXCR4 (

Shi et al, Cancer Research 67: 6417, 2007
). We further tested this hypothesis by studying the homing of PMF CD34+ cells to the marrow and the spleens of sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and determining if this defect could be corrected by treatment with chromatin modifying agents. Following the infusion of PMF (n=10) or G-CSF mobilized peripheral blood (mPB) (n=4) CD34+ cells into NOD/SCID mice, reduced numbers of PMF CD34+ cells and assayable human CFU-GM were detected in the marrow of these mice (139±47 PMF CD34+ cells/106 BMCs vs. 1493±946 mPB CD34+ cells/106 BMCs, 2.0±0.8% of input PMF CFU-GM vs. 12.6±6.6% of input mPB CFU-GM, P<0.05). The CFU-GM were shown to be of human origin by plucking individual colonies and analyzing by RT-PCR for the human 17-alpha satellite gene and the murine GAPDH gene. Of the total CD34+ cells homing to the spleen and marrow of the NOD/SCID mice, 25% of PMF CD34+ cells and 2.4% of mPB CD34+ cells homed to the spleen. The abnormal trafficking of PMF CD34+ cells was associated with reduced expression of CXCR4 (15.3% vs.4.8%, P<0.05). PMF CD34+ cells from patients with (n=6) or without (n=4) the JAK2V617F mutation exhibited similar degrees of defective bone marrow(BM) homing, suggesting that the impairement of PMF CD34+ cell trafficking was not related to the JAK2 mutation. We have previously shown that the sequential treatment of PMF CD34+ cell with the DNA methyltransferase inhibitor, decitabine [5-aza-2′-deoxycytidine (5azaD)], followed by the histone deacetylase inhibitor, trichostatin A (TSA), resulted in the upregulation of CXCR4 (
Shi et al, Cancer Research 67: 6417, 2007
). We next determined if this upregulation of CXCR4 was associated with correction of the impaired trafficking of PMF CD34+ cell to the marrow of NOD/SCID mice. The percentage of CD34+ cells expressing CXCR4 was significantly enhanced by exposure of PMF CD34+ cells to 5azaD/TSA (primary CD34+ cells: 2.55±0.87%, cytokines alone: 6.51±1.80%, cytokines plus 5azaD/TSA: 12.47±3.65%). Treatment of PMF CD34+ cells in vitro with cytokines plus 5azaD/TSA for 9 days resulted in a 3-fold increase in the absolute numbers of CD34+CXCR4+ cells when compared to CD34+ cells exposed to cytokines alone and 61-fold increase of CD34+CXCR4+ cells when compared to primary PMF CD34+ cells. Treatment of PMF CD34+ cells with cytokines alone resulted in 3- to 9-fold greater homing to NOD/SCID marrow than that of primary PMF CD34+ cells (P=0.12), while equal number of PMF CD34+ cells treated with chromatin modifying agents had a 7- to 20-fold greater ability to home to mouse marrow than that of primary PMF CD34+ cells (P<0.05) and a 2- to 3-fold greater marrow homing ability than PMF CD34+ cells treated with cytokines alone (P<0.05). Treatment with 5azaD/TSA reduced by 36% the homing of PMF CD34+ cells to the spleen as compared to the treatment with cytokines alone. We conclude that PMF CD34+ cells preferentially home to the spleen rather than the marrow of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin modifying agents. Chromatin modifying agents represent a novel means of correcting the abnormal cellular trafficking characteristic of PMF, a therapeutic strategy which is about to be evaluated in the clinic.

Topics:
cd34 antigens, chromatin, myelofibrosis, idiopathic, chronic, cxcr4 receptors, cytokine, male-pattern baldness, sequential treatment, severe combined immunodeficiency, chemokine receptors, decitabine

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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