JAK2 Inhibitors Do Not Affect Stem Cells Present in the Spleens of Patients with Myelofibrosis

Abstract 1752

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by abnormal trafficking of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC), resulting in their constitutive mobilization and the establishment of extra-medullary hematopoiesis. At present, there is no known therapeutic approach capable of altering the natural history of MF, except for allogeneic stem cell transplantation. Treatment with JAK2 inhibitors has been shown to lead to a rapid and dramatic reduction of splenomegaly although having only a modest effect on the JAK2V617F allele burden and not resulting in the elimination of cytogenetic abnormalities or correction of histopathological abnormalities (Verstovsek S, et al. N Engl J Med. 2010; 363:1117-27). To date, the mechanism underlying the reduction of splenomegaly observed following the treatment with a JAK2 inhibitor remains the subject of speculation. Recently, we observed the presence of MF-stem cells (MF-SC) in the spleens of MF patients and demonstrated that these splenic MF-SCs have a distinct differentiation program that distinguishes them from MF peripheral blood (PB) counterparts (Wang X, et al. Journal of Clinical Investigation, 2012. In Press). We therefore explored the effect of a JAK2 Inhibitor, AZD1480, on splenic MF-SCs in order to provide an explanation for the dramatic effects on MF spleen size.

Treatment of splenic or PB MF CD34⁺ cells with cytokines+AZD1480 (CAZD, 150nM) for 3 days resulted in a significant reduction in the number of total cells, CD34⁺ cells and assayable HPC (CFU-GM, BFU-E and CFU-Mix) as compared with splenic or PB MF CD34⁺ cells treated with cytokines alone (CA), respectively (P all <0.05, n=6). Moreover, the numbers of CD34⁺CD90⁺ cells and CD34⁺CXCR4⁺ cells generated in cultures of splenic MF CD34⁺ cells with CAZD were each half of that achieved in cultures containing CA. The numbers of CD34⁺CD90⁺ cells and CD34⁺CXCR4⁺ cells present in cultures of PB MF CD34⁺ cells with CAZD were each 1/3 of that observed in cultures with CA. However, exposure of splenic or PB MF CD34⁺ cells to CAZD did not result in an alteration of the proportion of JAK2V617F positive HPCs. Furthermore, the treatment of splenic MF CD34⁺cells with CAZD did not affect the number of colonies with a marker chromosomal abnormality.

We have reported that the transplantation of PB MF CD34⁺ cells into NOD/SCID/IL2R γ^null mice resulted in a limited degree of donor cell chimerism and a differentiation program skewed toward myeloid lineages (Wang X, et al. Blood. 2010; 116: 5972–5982), while the transplantation of splenic MF CD34⁺ cells achieved a higher level of chimerism and generated both myeloid and lymphoid cells which contained molecular or cytogenetic abnormalities indicating their malignant origin. Only splenic MF CD34⁺ cells were able to sustain hematopoiesis for prolonged time periods (9 months) and were able to engraft secondary recipients. These data document the existence of MF-SCs that reside in the spleens of MF patients (Wang X, et al. Journal of Clinical Investigation 2012. In Press). We, therefore, examined the effect of AZD1480 on splenic MF-SCs by analyzing the behavior of splenic MF SCID repopulating cells following the transplantation of splenic MF CD34⁺ cells treated with CA or CAZD (n=3) into NOD/SCID/IL2R γ^null mice. Six months after transplantation of splenic MF CD34⁺ cells treated with CA or CAZD, similar numbers of human CD45⁺ cells were detected in both the BM and spleens of recipient mice, respectively. Furthermore, human CD45⁺ cells were harvested and isolated from the BM and spleen of the mice receiving splenic CD34⁺ cells treated with CA or CAZD from a MF patient who had a deletion of the long arms of chromosome 20 [del (20q)] and the presence of del(20q) in human CD45⁺ cells were determined. All the human BM CD45⁺ cells isolated from the mice transplanted with splenic CD34⁺ cells treated with either CA or CAZD had this specific chromosomal abnormality, del (20q). These findings suggest that JAK2 inhibitor treatment does not affect splenic MF-SCs, indicating that the rapid reduction in splenomegaly following such therapy or it enlargement following the cessation of such therapy is either due to the effects of JAK2 inhibitors on more differentiated hematopoietic cells or on the splenic microenvironment.