Interferon-Alpha Preferentially Targets JAK2V617F-Positive Rather Than Wild-Type Early Progenitor Cells in Myeloproliferative Disorders.

Abstract 436

Polycythemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF) are myeloproliferative disorders (MPDs) without curative treatment, unless hematopoietic stem cell (HSC) transplantation is performed. However, for several years the use of interferon-alpha (IFNα) has provided an efficient therapeutic alternative for MPD patients. IFNα was shown to induce complete long-term hematologic and molecular remissions in JAK2V617F-positive MPD patients, suggesting a possible curative effect of IFNα.

In order to better understand mechanisms of action of this drug, experiments were perfomred on cell lines, patient cells and mice cells harboring a JAK2V617F mutation. We hypothesized that IFNα may target directly MPD cells through binding to its specific receptor, in addition to the potential immunological effect of this molecule and could induce cell cycle entry of the pathological quiescent HSCs.

Human cell lines harboring JAK2 mutation or BCR-ABL oncogene were treated with increasing doses of IFNα. A significant anti-proliferative effect at low concentrations (100 IU/ml) on the JAK2V617F-positive HEL cell line was observed. On the contrary, at this low dose IFNα did not influence growth of the BCR-ABL-positive K562 and the non-mutated UT-7 cell lines. This result supported a direct effect of IFNα in JAK2V617F cells. Suppressor of cytokine signaling (SOCS) are potent inhibitors of the JAK-STAT pathway by proceeding to a classic negative regulation loop proteins. Following IFNα stimulation of HEL cells, SOCS1 and SOCS3 mRNAs expression were induced (p=0.00036 and p=0.0012, respectively) and efficient knock-down of either SOCS1 or SOCS3 by shRNAs expression in HEL cells was able to counteract the anti-proliferative effect of IFNα (p=0.028 and p=0.031, respectively). We concluded that SOCS1 and SOCS3 are involved, in IFNα proliferative inhibition activity of HEL cells.

To determine whether JAK2V617F confer hypersensitivity to IFNα inhibitory effect, proliferation and genotyping of CD34+ progenitors isolated from the bone marrow of JAK2V617F–positive MPD patients (n=5) and control subjects (n=4) were plated at one cell per well in 96-well plates and counted and genotyped at Day 10-12. A significant decrease of the patients progenitors clonogenicity was observed when exposed to IFNα (10 000 IU/ml, p<0.05). On the contrary, normal progenitors were not sensitive to the anti-proliferative effect of IFNα (p=0.2). Patients colonies were genotyped for JAK2V617F. After IFNα exposure, the amount of homozygous JAK2V617F clones decreased in 3 over 5 patients in favor of the heterozygous JAK2V617F and/or wild-type clone(s) confirming the preferential action of IFNα on JAK2V617F progenitors. This inhibitory effect was more drastic on progenitors carrying the JAK2V617F mutation at the homozygous state.

Lastly, in order to explain the long term molecular responses observed in PV patients treated by IFNα, we investigated the effect of IFNα on the cell cycle in more immature cells. BrdU assay on JAK2V617F Knock-in (KI) mice was performed. Five-months old JAK2V617F KI and wild-type mice received or not 10,000 UI of murine IFNα during 3 days. Sixteen hours before analysis, BrdU was injected i.p. in the animals. Bone marrow Lin-Sca+cKit+cells (LSK) were stained with CD150 and CD48 antibodies before BrdU labelling was analyzed by flow cytometry. Analysis of BrdU postive cells confirmed that i-JAK2V617F induces LSK CD150+CD48- to enter cell cycle ( 8.55+/-3.12% for WT cells versus 19.92+/-3,19% for JAK2V617F KI cells respectively (p = 0.04)) and ii- That IFNα induces CD150+CD48- LSK to enter cell cycle whatever the JAK2 WT (8.55+/-3.12% for non treated animals versus 15.43+/-3.19% for IFNα receiving mice (p=0.02)) or mutated status but this induction was more statistically significant in JAK2V617F mice (19.92+/-1.82% versus 25.23+/-0.57% respectively( p=0.02)).

In conclusion, we gave rise to a double effect of IFNα in MPD cells: A direct preferential anti-proliferative effect of IFNα on JAK2V617F–positive MPD progenitor cells, possibly through the induction of SOCS over-expressions and a direct cell cycling effect on JAK2V617F stem cells suggesting that IFNa containing treatments could be of interest for JAK2V617F patients cure.