Abstract
N.M. and T.D. co-first authors, J.L.V. and Hd.T. co-last authors
Introduction:
We have shown in clinical trials and preclinical studies on Myeloproliferative Neoplasms (MPN) that interferon alpha (IFN) is able to specifically target JAK2V617F cells. However, achievement of complete molecular responses is rare and often requires long-term exposures to IFN, increasing the risk of toxicity. The mechanism implicated in the specific targeting of JAK2V617F cells remains elusive and its elucidation is crucial to improve efficiency of IFN therapy. PML is an IFN transcriptional target that drives the assembly of PML-nuclear bodies (NB), activating P53 or RB checkpoints and ultimately leading to cell cycle arrest or senescence. We explored the hypothesis that PML may contribute to IFN efficacy. This would then predict enhancement of IFN activity by arsenic (AS), a potent inducer of NB formation.
Methods:
To analyze IFN and/or AS effect on JAK2V617F-driven MPN, we developed chimeric animals transplanted with a mixture of bone marrow (BM) from wild type (WT) mice and conditional JAK2V617F KI/vav-Cre/Ubi-GFP mice monitoring allele burden by GFP expression. Animals, suffering from a polycythemia vera (PV)-like disease, were treated by IFN, AS or IFN/AS combination for 8 to 14 weeks. Blood was analyzed every 2 weeks. After treatment, animals were either (1) sacrificed to analyze BM, spleen and disease-initiating cells in secondary recipients or (2) kept alive to study disease recurrence. We also developed an UT-7 human megakaryoblastic cell line isogenic model by transducing JAK2WT or JAK2V617F. MPN patient samples were tested in clonogenic assays. Finally, the role of PML was assessed in the JAK2V617F-positive HEL cell line transduced by different shRNA for PML knock-down.
Results:
In vivo experiments demonstrated that IFN vs non-treated (NT) reduced leukocytosis (p<0.001), the platelet number (p<0.001), erythrocytosis (p=0.005) and the JAK2V617F allele burden in granulocytes (p<0.01), platelets (p=0.03) and RBC (p=0.03). More importantly, addition of AS improved the efficacy of IFN for reducing leukocytosis (p<0.03) and erythrocytosis (p=0.02) at 4-6 weeks, but not the platelet number. The effect of the IFN/AS combination was particularly striking in improving the reduction of allele burden compared to IFN alone in granulocytes (p=0.005), platelets (p=0.001) and RBC (p=0.001) (see figure). Improvement of IFN efficacy by addition of AS was also observed on splenomegaly and SLAM progenitor allele burden. The IFN/As combination efficiently decreased relapse rates (48%, p=0.007 vs NT) compared to IFN alone (73%, p=0.2 vs NT), as assessed on blood cells, allele burden and splenomegaly several weeks after treatment discontinuation. Moreover, none of the secondary mice transplanted with BM from IFN/AS-treated mice developed the disease, while 68% of the mice transplanted with BM from IFN alone-treated mice did. These data demonstrate the dramatic effect of the IFN/AS combination on disease initiating cells.
We then look for underlying molecular mechanism. The number and size of PML-NB were increased in JAK2V617F vs. WT UT-7 cells at baseline (4.0 vs 6.6 dots/cell, p= 0.0003). As expected, IFN/AS significantly increased PML-NB in both cell types, but this increase was more important in JAK2V617F cells. Cell cycle arrest induced by IFN/AS was more prominent in UT-7 JAK2V617F cells compared to isogenic control, showing the specificity of the combination for mutant cells. IFN/AS had synergistic effects on JAK2V617F cells in terms of inhibition of proliferation and induction of senescence in contrast to single therapies. Critically, PML knock-down in the JAK2V617F-positive HEL cell line reduced the anti-proliferative effect and pro-senescent activity of the IFN/AS combination showing the key role of PML in these processes.
Finally, comparison of JAK2V617F-positive CD34+ primary cells from PV patients to normal CD34+ cells demonstrated that IFN/AS specifically decreased proliferation and enhanced senescence as well as NB formation in JAK2V617F cells. Moreover, IFN/AS had a drastic effect on the clonogenic potential of BFU-E progenitors derived from 10 PV patients with a specific targeting of JAK2V617F colonies, which were reduced from 67% to 32%.
Conclusion:
Altogether, our results strongly suggest involvement of PML in IFN therapy of MPN and the potential utility of AS to greatly improve its efficacy.
Kiladjian:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees.
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