trichostatin-a and Primary-Myelofibrosis

Because primary myelofibrosis (PMF) originates at the level of the pluripotent hematopoietic stem cell (HSC), we examined the effects of various therapeutic agents on the in vitro and in vivo behavior of PMF CD34(+) cells. Treatment of PMF CD34(+) cells with chromatin-modifying agents (CMAs) but not hydroxyurea, Janus kinase 2 (JAK2) inhibitors, or low doses of interferon-α led to the generation of greater numbers of CD34(+) chemokine (C-X-C motif) receptor (CXCR)4(+) cells, which were capable of migrating in response to chemokine (C-X-C motif) ligand (CXCL)12 and resulted in a reduction in the proportion of hematopoietic progenitor cells (HPCs) that were JAK2V617F(+). Furthermore, sequential treatment of PMF CD34(+) cells but not normal CD34(+) cells with decitabine (5-aza-2'-deoxycytidine [5azaD]), followed by suberoylanilide hydroxamic acid (SAHA; 5azaD/SAHA), or trichostatin A (5azaD/TSA) resulted in a higher degree of apoptosis. Two to 6 months after the transplantation of CMAs treated JAK2V617F(+) PMF CD34(+) cells into nonobese diabetic/severe combined immunodeficient (SCID)/IL-2Rγ(null) mice, the percentage of JAK2V617F/JAK2(total) in human CD45(+) marrow cells was dramatically reduced. These findings suggest that both PMF HPCs, short-term and long-term SCID repopulating cells (SRCs), are JAK2V617F(+) and that JAK2V617F(+) HPCs and SRCs can be eliminated by sequential treatment with CMAs. Sequential treatment with CMAs, therefore, represents a possible effective means of treating PMF at the level of the malignant SRC.

Topics: Animals; Antigens, CD34; Antimetabolites, Antineoplastic; Antineoplastic Agents; Azacitidine; Bone Marrow; Cells, Cultured; Chromatin; Decitabine; Hematopoietic Stem Cells; Humans; Hydroxamic Acids; Immunoblotting; Interleukin Receptor Common gamma Subunit; Janus Kinase 2; Mice; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Mutation; Primary Myelofibrosis; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vorinostat

The abnormal trafficking of CD34+ cells is a unique characteristic of primary myelofibrosis (PMF). We have further studied the behavior of PMF CD34+ cells by examining their homing to the marrow and the spleens of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Following the infusion of PMF and normal granulocyte colony-stimulating factor-mobilized peripheral blood (mPB) CD34+ cells into NOD/SCID mice, reduced numbers of PMF CD34+ cells and granulocyte-macrophage colony-forming unit (CFU-GM) compared with mPB were detected in the marrow of these mice, whereas similar numbers of PMF and mPB CD34+ cells and CFU-GM homed to their spleens. The abnormal homing of PMF CD34+ cells was associated with reduced expression of CXCR4, but was not related to the presence of JAK2V617F. The sequential treatment of PMF CD34+ cells with the chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), but not treatment with small molecule inhibitors of JAK2, resulted in the generation of increased numbers of CD34+CXCR4+ cells, which was accompanied by enhanced homing of PMF CD34+ cells to the marrow but not the spleens of NOD/SCID mice. Following 5azaD/TSA treatment, JAK2V617F-negative PMF hematopoietic progenitor cells preferentially homed to the marrow but not the spleens of recipient mice. Our data suggest that PMF CD34+ cells are characterized by a reduced ability to home to the marrow but not the spleens of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin-modifying agents.

Topics: Animals; Antigens, CD34; Azacitidine; Bone Marrow; Chromatin; Decitabine; Down-Regulation; Humans; Hydroxamic Acids; Mice; Mice, Inbred NOD; Mice, SCID; Primary Myelofibrosis; Receptors, CXCR4; Spleen

Idiopathic myelofibrosis (IM) is likely the consequence of both the acquisition of genetic mutations and epigenetic changes that silence critical genes that control cell proliferation, differentiation, and apoptosis. We have explored the effects of the sequential treatment with the DNA methyltransferase inhibitor, decitabine [5-aza-2'-deoxycytidine (5azaD)], followed by the histone deacetylase inhibitor, trichostatin A (TSA), on the behavior of IM CD34(+) cells. Unlike normal CD34(+) cells where 5azaD/TSA treatment leads to the expansion of CD34(+) cells and marrow-repopulating cells, treatment of IM CD34(+) cells results in a reduction of the number of total cells, CD34(+) cells, and assayable hematopoietic progenitor cells (HPC). In IM, HPCs are either heterozygous or homozygous for the JAK2V617F mutation or possess wild-type JAK2 in varying proportions. Exposure of IM CD34(+) cells to 5azaD/TSA resulted in a reduction of the proportion of JAK2V617F-positive HPCs in 83% of the patients studied and the reduction in the proportion of homozygous HPCs in 50% of the patients. 5azaD/TSA treatment led to a dramatic reduction in the number of HPCs that contained chromosomal abnormalities in two JAK2V617F-negative IM patients. IM is characterized by constitutive mobilization of HPCs, which has been partly attributed to decreased expression of the chemokine receptor CXCR4. Treatment of IM CD34(+) cells with 5azaD/TSA resulted in the up-regulation of CXCR4 expression by CD34(+) cells and restoration of their migration in response to SDF-1. These data provide a rationale for sequential therapy with chromatin-modifying agents for patients with IM.

Topics: Antigens, CD34; Antimetabolites, Antineoplastic; Azacitidine; Cell Movement; Chromatin; Decitabine; DNA (Cytosine-5-)-Methyltransferases; Hematopoietic Stem Cells; Homozygote; Humans; Hydroxamic Acids; Mutation; Primary Myelofibrosis; Protein Synthesis Inhibitors; Receptors, CXCR4; Stem Cells