The Jak2-Inhibitor AG409 Affects Survival of BFU-E and CFU-GM of Myeloproliferative Disorder Cells Carrying the JAK2 V617F Mutation.

In the last few years, point mutation of the Janus Kinase 2 (JAK2) gene (V617F) was found in the majority of patient with Polycythemia Vera and two related diseases ET and MMM. This mutation initiates constitutive activation of JAK2 transduction gene, inducing growth factor- independent hemopoiesis and plays a dominant role in origin of myeloproliferative disorder. Inactivation of this mutated gene may restore the normal hemopoiesis. Based on the evidence that the inhibition of the Jak-Stat axis by AG490 induced apoptosis in leukemic compared with normal cell, we attempt to analyzed the AG490 effect on the survival of MPD cells carrying JAK2 mutation in cultures.

Peripheral blood (PB) samples were collected from 49 MPD patients (PV n=31, ET n=14, MMM n=4) at the time of diagnosis or during the follow up. The JAK2-V617F mutation tested by an allele-specific, semi-quantitative PCR was detected in 35 patients. MNPBC’s from four patients homozygous for the JAK2 mutation and from two normal volunteers were cultured in methylcellulose with 50 ng/ml SCF, 10ng/ml GM-CSF, 10ng/ml IL-3 and 3U/ml EPO, and in the same medium without growth factors. On the sixth day, 50 micromole of AG490 were added to the cultures and dishes with no addition of AG490 were kept for control. Colonies were counted on 6^th day and on 14^th day, and we checked the growth patterns on 14^th day.

Spontaneous colonies without growth factors were found only in the four MPD patients. Although, the number of the colonies was not significantly decreased after the addition of the AG490 inhibitor, we observed an obvious change in the normal growth pattern. The appearance of the colonies became darker reflecting an apoptotic process and death.

Such a phenomenon was not seen in normal controls or in MPD cells without addition of the inhibitor. In conclusion our experiment clearly demonstrated preferential inhibition of MPD cells with JAK2 mutation by AG490, probably by induction of apoptosis. Our results suggest a therapeutic potential of inhibitors of JAK2 for the future.