Sequential Treatment of CD34⁺ Cells From Patients with Primary Myelofibrosis with Chromatin Modifying Agents Alters the Behavior of JAK2V617F Positive NOD/SCID Marrow Repopulating Cells.

Abstract 1910

Poster Board I-933

Primary myelofibrosis (PMF) is likely the consequence of both the acquisition of genetic mutations and epigenetic changes which silence critical genes influencing cell proliferation, differentiation, survival and trafficking. The abnormal trafficking of CD34⁺ cells in PMF has been attributed to reduced expression of CXCR4 (Shi et al, Cancer Res, 2007; Wang et al, Cancer Res, 2009). We examined the effects of treatment with chromatin modifying agents (CMA), including sequential treatment with a DNA methyltransferase inhibitor, decitabine “5-aza-2'-deoxycytidine (5azaD)”, followed by a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA) (5azaD/SAHA), or trichostatin A (5azaD/TSA) on the in vitro and in vivo behavior of PMF CD34⁺ cells (PCC). Sequential treatment with 5azaD/SAHA resulted in a reduction in the number of total cells, CD34⁺ cells and assayable hematopoietic progenitor cells (HPC) as compared with PCC treated with cytokines alone. By contrast, treatment of PCC with 5azaD/SAHA led to the generation of 2.4-fold greater numbers of CD34⁺CXCR4⁺ cells as compared to PCC exposed to cytokines alone and a 15.8-fold increase of the number of CD34⁺CXCR4⁺ cells when compared to input PCC (P<0.05). The 5azaD/SAHA treated PCC had a greater migration capacity (20.2±6.9%) toward SDF-1 as compared with PCC exposed to cytokines alone (6.2±2.9%) or primary PCC (2.0±0.5%). These findings suggest that the abnormal trafficking of PCC might be corrected by treatment with 5azaD/SAHA, which is similar to what we had previously reported with 5azaD/TSA. Exposure of PCC to 5azaD/SAHA resulted in a reduction in the proportion of JAK2V617F⁺ HPCs (p=0.08, 5azaD/SAHA vs cytokines alone; p<0.05, 5azaD/SAHA vs primary PCC) and a reduction in the proportion of JAK2V617F homozygous HPCs in 3 of the 4 patients studied. We next evaluated the effect of sequential treatment with CMA on PMF SCID repopulating cells (SRC) by transplanting primary PCC, PCC treated with cytokines alone, 5azaD/SAHA or 5azaD/TSA and determined the JAK2V617F allelic burden of hCD45⁺ cells in the marrow of NOD/SCID/IL2Rγ^null mice. Two months following transplantation, 2.9% of the marrow cells were hCD45⁺ cells in mice receiving primary PCC as compared to 0.2% in mice receiving cells treated with cytokines alone or 5azaD/TSA. The engrafted cells in the BM of recipient mice receiving primary PCC were composed of myeloid cells belonging to multiple hematopoietic lineages (CD33⁺ cells, 2.8%; Glycophorin A⁺ cells, 0.5%; CD41a⁺ cells, 0.1% and CD34⁺ cells, 0.1%) but not CD19⁺ or CD3⁺ cells. The JAK2V617F allelic burden of hCD45⁺ cells isolated from the BM of these mice was analyzed using real-time quantitative PCR. The percentage of JAK2V617F/JAK2_total in the gDNA of hCD45⁺ cells was 71.5% in mice transplanted with primary PCC, 34.7% in mice transplanted with cells treated with cytokines alone and 6.8% in mice transplanted with PCC treated with 5azaD/TSA. Six months following transplantation, a similar degree of human cell chimerism was detected in the BM of recipient mice receiving primary PCC, cells treated with cytokines alone or 5azaD/SAHA. However, the percentage of hCD45⁺ cells in peripheral blood (PB) of mice transplanted with either primary PCC (0.25%) or PCC treated with cytokines alone (0.1%) was greater than that observed in mice transplanted with PCC treated with 5azaD/SAHA (0.05%). HCD34⁺ cells were detected in the PB of mice when primary PCC or PCC treated with cytokines alone were transplanted, 2.8% and 1.7%, respectively, but were not detected in mice transplanted with PCC treated with 5azaD/SAHA. These findings suggest that the abnormal trafficking of hCD34⁺ cells which characterizes PMF, can be recapitulated in NOD/SCID mice. Furthermore, the percentage of JAK2V617F/JAK2_total in the gDNA of BM CD45⁺ cells was 82.5% and 97.6% in mice transplanted with primary PCC as compared to 27.1% and 41.5% in mice transplanted with PCC treated with cytokines alone but was dramatically reduced to 3.6% and 7.4% in mice transplanted with PCC treated with 5azaD/SAHA. These findings suggest that SRC in PMF are JAK2V617F⁺ and that JAK2V617F⁺ SRC can be preferentially suppressed by sequential treatment with CMA, leading to engraftment with JAK2V617F^- SRC. Sequential treatment with CMA therefore represents a novel means of treating PMF at the level of a JAK2V617F⁺ SRC, a primitive HPC which shares many properties with the putative PMF stem cell.