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Introduction:

We recently reported that mutant JAK2 (V617F) expression in polycythemia vera (PV) progenitors is associated with skewed erythroid differentiation potential (Jamieson et al, PNAS 2006). We investigated the mechanisms responsible for enhanced erythroid differentiation as well as whether a selective JAK2 inhibitor (TG101348) could normalize PV progenitor differentiation both in vitro and in vivo.

Methods:

Q-PCR of erythroid (GATA-1) versus myeloid (PU.1) transcription factor levels was performed on FACS-purified normal and PV progenitors. PCR analysis of an essential regulator of caspase 8 mediated GATA-1 cleavage, FLIP, was also performed on colonies derived from CD34+ cells transduced with JAK2 V617F. The effects of selective JAK2 inhibition were examined on PV patient progenitors or normal peripheral blood (PB) and cord blood (CB) progenitors transduced with lentiviral wild-type JAK2 (JAK WT), mutant JAK2 (JAK MT), backbone (backbone) or no vector in methocult media +/- TG101348. Colonies were scored on day 14. Normal CB progenitors transduced with JAK WT, JAK MT, or backbone and marked by luciferase-GFP. In addition, PV progenitors were transduced with luciferase GFP. These populations were transplanted intrahepatically into neonatal RAG2-/-γc-/- mice. TG101348 (2.2mg) or vehicle was administered to transplanted mice by oral gavage twice daily between 3 and 5 weeks post-transplantation. Bioluminescence of transplanted animals was assessed (Xenogen IVIS 200) before, during and after treatment. Human engraftment analysis of hematopoietic tissues was performed by FACS using human specific antibodies.

Results:

Q-PCR analysis of PV patient progenitors vs. normal cells, revealed increased GATA-1 transcripts in keeping with their erythroid skewed differentiation potential while PV patients who progressed had higher PU.1 transcripts. PCR analysis of JAK2 MT progenitors revealed increased FLIP expression. Transduction of normal progenitors with JAK MT resulted in skewed erythroid colony formation compared to JAK WT, backbone and untransduced controls. Addition of TG101348 inhibited mutant JAK2 kinase-induced erythroid colony formation in a dose dependent manner. Bioluminescent imaging and FACS analysis revealed that transplantation of JAK2-transduced CB progenitors or PV progenitors resulted in enhanced human erythroid engraftment of PV compared with normal progenitors. Bioluminescent imaging and FACS analysis demonstrated selective inhibition of erythroid skewed engraftment with the JAK2 inhibitor while no effect was seen with vehicle alone.

Conclusion:

JAK2 driven erythroid differentiation is associated with increased GATA-1and FLIP expression and coincides with decreased PU.1 expression. Selective JAK2 inhibition with TG101348 normalizes erythroid differentiation potential both in vitro and in a xenogeneic bioluminescent model of human PV.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA