PS1449 DISSECTING THE THERAPEUTIC MECHANISM OF RUXOLITINIB IN JAK2V617F MEDIATED MPNS REVEALED AN INHIBITION OF BOTH ONCOGENIC AND NON-ONCOGENIC CELLS BY DOWN REGULATION OF INFLAMMATORY CYTOKINE LEVELS

Abstract

Background: Ruxolitinib is a potent JAK2/JAK1 specific inhibitor approved by the FDA and EMA for the treatment of primary myelofibrosis (PMF). The primary clinical benefits of JAK2 inhibitors in PMF patients are reduction in spleen size, alleviation of constitutional symptoms and a significant improvement in the quality of life. However, no significant improvement of bone marrow fibrosis has been demonstrated so far, and the effect on the JAK2V617F allelic burden is modest. In addition, the beneficial clinical effects were also achieved in JAK2V617F negative PMF patients. Aims: It is unclear at the moment whether Ruxolitinib primarily blocks proliferation of the malignant clone or exerts its effects by inhibiting bystander normal hematopoietic cells or bone marrow niche cells. By clinical investigation it is not possible to dissect between these two modes of inhibitor action. However, JAK2V617F variants which are resistant towards clinically used inhibitors can be expressed in mice. Such a mouse model would allow investigation of the mode of action of the JAK2 inhibitors in PMF patients. Methods: So far no inhibitor resistant JAK2 mutations have been reported in patients. Therefore, a comprehensive cell based screening strategy was used to identify Ruxolitinib resistant JAK2V617F variants. Results: Using cell based screening method, we were able to identify seven specific mutations which confer resistance to Ruxolitinib while retaining the kinase activity. For this study, we used JAK2V617F+L902Q and JAK2V617F+L983F variants since these two variants were highly resistant towards the Ruxolitinib. In order to determine the disease phenotype of JAK2V617F+L902Q and JAK2V617F+L983F we transplanted retrovirally infected JAK2V617F and JAK2V617F+L902Q and JAK2V617F+L983F bone marrow into recipient mice. Similar to JAK2V617F recipients, JAK2V617F+L902Q and JAK2V617F+L983F transplanted mice showed an increase in WBC, HCT, RBC, HB and reticulocyte values. Histopathological analysis revealed that the myeloproliferative phenotype resembles PV with a proliferation of all three lineages albeit with only a moderate increase in megakaryopoeisis. Grade II bone marrow fibrosis was observed in JAK2V617F+L902Q and JAK2V617F+L983F after 2–3 months similar to JAK2V617F mice. Ruxolitinib treatment lead to a decrease in spleen size, length and WBC number in JAK2V617F mice however, surprisingly, JAK2V617F+L902Q and JAK2V617F+L983F mice also decreased the spleen size, length and WBC number after Ruxolitinib treatment. FACS analysis of different hematopoietic cell population from peripheral blood, spleen and bone marrow results suggested that Ruxolitinib mainly act on eGFP+ neutrophils and also on eGFP− neutrophils, B and T cells in JAK2V617F, JAK2V617F+L902Q and JAK2V617F+L983F MPN mouse models. Finally, analysis of pro inflammatory cytokines results from Ruxolitinib treated mice results showed significant down regulation of several cytokines including TNF-alpha, MCP-1, IL-6, INF-beta, IL-27 and IL-23 in both JAK2V617F and JAK2V617F+L902Q MPN mouse models suggesting that ruxolitinib mediated clinical benefits might also be due to the down regulation of inflammatory cytokines by inhibition of JAK1/2 signaling in both hematopoietic as well as non-hematopoietic cells. Summary/Conclusion: Taken together, our results clearly suggest that identification of novel inhibitors that specifically act on malignant cells and spare the non-malignant cells which play a major role in normal hematopoiesis will improve the clinical efficacy in PMF patients.