The NF-KB Inhibitor DMAPT in Combination with Ruxolitinib Displays Efficacy in Jak2V617F Knock-in Mouse Model of Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are a group of hematologic malignancies characterized by excessive proliferation of myeloid/erythroid lineage cells. The JAK2V617F mutation has been found in a majority of patients with MPN including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF and PV. Although ruxolitinib treatment provides some benefits, it does not produce disease remission or reverse bone marrow (BM) fibrosis in patients with MPN/MF. This underscores the need to identify new therapeutic targets and develop novel therapies for treatment of MPN. In order to identify new therapeutic targets in MPN, we performed microarray gene expression analysis on sorted hematopoietic stem cells (HSC) from wild type and JAK2V617F mice. We found significant enrichment of the NF-κB signaling pathway in JAK2V617F HSC compared with wild type HSC. We also observed increased phosphorylation/activation of NF-κB (p65 RelA) in the BM of JAK2V617F mice. Hyper activation of NF-κB also has been observed in patients with MPN. Lentiviral shRNA-mediated knockdown of RelA significantly reduced the proliferation of murine Ba/F3-EpoR cells expressing JAK2V617F. RelA knockdown also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 leukemia cells. Furthermore, forced expression of a dominant negative IκBα mutant markedly reduced proliferation in Ba/F3-EpoR-JAK2V617F cells. These data suggest a role for the NF-κB signaling pathway in MPN pathogenesis. In this study, we investigated the efficacy of the NF-κB inhibitor, dimethylaminoparthenolide (DMAPT), in hematopoietic cells expressing JAK2V617F and in JAK2V617F knock-in mouse model of MPN. DMAPT is a water soluble, orally bioavailable NF-κB inhibitor that has shown promise against several solid tumors in pre-clinical models. Treatment of DMAPT (1-2.5 uM) significantly reduced the proliferation of Ba/F3-EpoR-JAK2V617Fcells. DMAPT (2.5-7.5 uM) treatment also significantly inhibited the human JAK2V617F-positive HEL, SET-2 and UKE-1 leukemia cells. Furthermore, DMAPT treatment significantly inhibited the hematopoietic progenitor colony outgrowth in JAK2V617F mice BM and MPN patient peripheral blood CD34+ cells. We previously generated conditional Jak2V617F knock-in mice. Whereas expression of heterozygous Jak2V617F induces a PV-like disease, homozygous Jak2V617F expression promotes rapid progression to myelofibrosis. We have utilized the homozygous Jak2V617F mice to test the in vivo efficacy of DMAPT alone and in combination with ruxolitinib against MPN. We observed that combined treatment of DMAPT and ruxolitinib significantly reduced the WBC and neutrophil counts and spleen size in Jak2V617F mice compared with vehicle or single drug treatment. Combined treatment of DMAPT and ruxolitinib also significantly reduced hematopoietic stem/progenitors and myeloid precursors in the BM and spleens of Jak2V617F mice. Furthermore, combined treatment of DMAPT and ruxolitinib significantly decreased the Jak2V617F mutant-expressing myeloid precursors in the BM and spleens of transplanted mice. Histopathologic analysis revealed significant reduction of fibrosis in the bone marrow of Jak2V617F mice treated with the combination of DMAPT and ruxolitinib. Overall, our results suggest that simultaneous inhibition of JAK2 and NF-κB pathways might be useful for treatment of MPN/MF. Disclosures Crooks:University of Arkansas for Medical Sciences: Patents & Royalties: Inventor on the DMAPT patents.