Treatment of JAK2V617F driven myeloproliferative neoplasms (MPN) with FDA approved drug Ruxolitinib (JAK inhibitor, JAKi) has shown limited benefits. The failure of JAKi to eradicate JAK2V617F disease has led to the search for other pathways as potential therapeutic targets for this disease. Hedgehog (Hh) pathway inhibitors targeting Smoothened (SMO) have shown promising activity in MPN by reducing bone marrow fibrosis but are inadequate as single agents.In the present study we tested the effects of SMO inhibitor, PF-04449913 in transgenic mouse model of myeloproliferative disease (vavJak2V617F, gifted by Joe Zhao, University of Oklahoma Health Sciences Center). JAK2V617F transgenic mice exhibit a number of phenotypes that recapitulate those observed in MPN including bone marrow fibrosis and splenomegaly.

Treatment with SMO inhibitor, PF-04449913, reduces splenomegaly. JAK2V617F mice showed pronounced splenomegaly compared to WT mice and treatment with SMO inhibitor resulted in a significant decrease in spleen size (Figure 1A). This was also associated with a drop in IL-4 and IL-7 in the peripheral blood (data not shown).

SMO inhibitor reduces JAK2V617F allelic burden. In the next set of experiment we made mixed chimeras by transplanting one million JAK2V617F or WT bone marrow into lethally irradiated normal CD45.1 congenic hosts. Transplanted recipient mice were treated with PF-04449913 for one month and assessed for changes in peripheral blood chimerism. Treatment with PF-04449913 resulted in no change in WT engraftment. However, we found that PF-04449913 reduces the JAK2V617F allele burden in 3/5 mice.

JAKV617F activates Hh signaling. Proteins were isolated from the spleens of 8-10 week JAK2V617F or WT mice. Western blot analyses demonstrate that JAK2V617F splenocytes express high SHH levels. We next measured levels of the GLI activator (GLI1) and the GLI repressor (GLI3R). Activation of Hh signaling is associated with an increase in GLI1 and a decrease in GLI3R. Treatment of the mice with PF-04449913 by daily gavage at 100mg/kg reduced GLI1 and increased GLI3R.

JAK2V617F is associated with increased pERK, AKT, NF-κB activation. Oh and colleagues recently remonstrated the MPN were associated with activation of MAPK and NF-κB signaling pathways (Fisher et al., Leukemia, 2017). To understand how Hh signaling interacted with these pathways, we assessed ERK, AKT, and NF-κB activity by western blotting. JAK2V617F mice showed increased pERK, AKT and NFKB signaling. Treatment with PF-04449913 reduced pERK and AKT levels, confirming that both ERK and AKT are downstream of SMO, while it had little effect on IκBα levels suggesting that NF-κB activation occurs upstream of SMO.

SMO inhibitor blocks bone marrow fibrosis by reducing TGF-β signaling. Numerous studies have implicated TGF-β in the pathogenesis of fibrosis. We analyzed the bone marrow of patients with JAK2V617F MPN and found them to express high TGF-β levels (Figure 1B). These findings were recapitulated in the JAK2V617F transgenic mice with levels of TGF-β in JAK2V617F splenocytes 15-fold greater than WT mice. Treatment with PF-04449913 normalized the levels of TGF-β (Figure 1C). Using a OP-9 co-culture system we demonstrated that JAK2V617F splenocytes robustly induced pSMAD2, which was completely blocked by PF-04449913 (Figure 1D). To assess for changes in bone marrow fibrosis, mice at 27 weeks were started on daily treatment with PF-04449913 for one month. Treatment with PF-04449913 blocks bone marrow fibrosis as demonstrated by decreased reticulin staining and reduced hydroxyproline. (Figure 1E-F).

In conclusion, these data are the first to report that the Smoothened inhibitor, PF-04449913 modulates TGF-β and MAPK signaling in MPN and this results in decreased splenomegaly and marrow fibrosis. These results provide critical insight into the mechanism of action of SMO inhibitors in JAK2V617F associated MPN, and support the rational development of SMO and JAK inhibitor combinations in MPN.

Figure Legend

(A) SMO antagonist, PF-04449913 reduces splenomegaly. (B) Bone marrow of JAK2V617F MPN patient express high TGF-β levels. (C) Splenocytes of JAK2V617F transgenic mice express high TGF-β levels and PF-04449913 treatment normalizes TGF-β levels. (D) JAK2V617F induces Hh dependent TGF-β/pSMAD2 signaling in stroma. (E-F) PF-04449913 blocks fibrosis as measured by reticulin staining and quantification of Hydroxyproline.


O'Connell: Astex Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees. Merchant: Pfizer: Consultancy, Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.

Sign in via your Institution