The PIM Kinase Inhibitor TP-3654 in Combination with Ruxolitinib Exhibits Marked Improvement of Myelofibrosis in Murine Models
Authors: Nath Dipmoy , Dutta Avik , Yang Yue , Whatcott Clifford , Warner Steven L. , Mohi Golam 
Published: October 2019

Abstract
Myelofibrosis (MF) is the most severe form of myeloproliferative neoplasms (MPN). The median survival for MF patients is ~5 years. A somatic JAK2V617F mutation, which results in constitutive activation of the JAK2 tyrosine kinase, has been found in 50-60% patients with MF. Additional mutations in the genes encoding thrombopoietin receptor (MPL) and calreticulin (CALR) have been found in MF but at a lower frequency than JAK2V617F. Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF. Although ruxolitinib treatment can reduce splenomegaly and constitutional symptoms in patients with MF, it failed to induce disease remission or cure fibrosis. Therefore, there is a critical need to identify new therapeutic target(s) and develop novel targeted therapies for MF. We found that PIM1 expression is significantly increased in mouse and human MPN/MF hematopoietic progenitors. Moreover, PIM1 knockdown markedly inhibited proliferation in JAK2V617F-expressing cells but not in wild type JAK2-expressing cells. In this study, we investigated the efficacy of the second-generation pan-PIM kinase inhibitor, TP-3654, in hematopoietic cells expressing JAK2V617F and MPLW515L and murine models of MF.
TP-3654 (0.25-1.0 uM) treatment significantly reduced proliferation of murine Ba/F3 cells expressing JAK2V617F or MPLW515L. TP-3654 (0.5-1.0 uM) treatment also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 cells. However, TP-3654 only modestly affected proliferation of wild type JAK2-expressing Ba/F3 cells at higher concentrations. TP-3654 treatment also resulted in marked apoptosis in Ba/F3-EpoR-JAK2V617F, HEL and UKE-1 cells but not in wild-type JAK2 expressing Ba/F3-EpoR cells. Additionally, TP-3654 and ruxolitinib synergistically induced apoptosis in JAK2V617F- expressing hematopoietic cells. Moreover, TP-3654 treatment can overcome resistance to JAK2 inhibition in ruxolitinib-resistant Ba/F3-EpoR-JAK2V617F cells. Furthermore, TP-3654 significantly inhibited MPN/MF CD34+ hematopoietic progenitor colony growth.
We previously reported the generation of JAK2V617F knock-in mice. Whereas heterozygous JAK2V617F knock-in mice exhibit a polycythemia vera phenotype, mice expressing homozygous JAK2V617F rapidly develop high-grade MF. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of TP-3654 alone or in combination with ruxolitinib against MF. Treatment of TP-3654 alone significantly reduced the increase in white blood cell (WBC) and neutrophil counts as well as spleen size in mice expressing homozygous JAK2V617F compared with vehicle treatment. Combined treatment of TP-3654 and ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow and spleens of TP-3654-treated mice whereas ruxolitinib treatment did not significantly alter fibrosis. Combined TP-3654 and ruxolitinib treatment almost completely eliminated fibrosis in the bone marrow and spleens of homozygous JAK2V617F mice.
Additionally, we have tested the in vivo efficacy of TP-3654 and TP-3654/ruxolitinib combination using bone marrow transplantation mouse model of MPL mutant (MPLW515L). We observed similar decrease in white blood cell and neutrophil counts in MPL mutant mice upon treatment of TP-3654 alone or in combination with ruxolitinib. We also observed significant decrease in splenomegaly in MPL mutant mice treated with TP-3654 alone and in combination with ruxolitinib. Combined treatment of TP-3654 plus ruxolitinib almost completely ablated the fibrosis in the bone marrow and spleens of MPL mutant mice.
To gain insights into the mechanism of inhibition of myelofibrosis by TP-3654 or TP-3654/ ruxolitinib drug combination, we performed RNA-sequencing on purified LSK (Lin-Sca-1+c-kit+) cells from these drug-treated homozygous JAK2V617F mice. Analysis of RNA-sequencing data revealed that the genes related to HSC maintenance and TNF signaling pathways were significantly down-regulated by TP-3654 alone or TP-3654/ruxolitinib combination compared with vehicle. Overall, our results suggest that TP-3654 alone or in combination with ruxolitinib may have therapeutic potential against MF and support the clinical investigation of TP-3654 and ruxolitinib combination in patients with MF.

Disclosures
Whatcott: Tolero Pharmaceuticals, Inc: Employment. Warner:Tolero Pharmaceuticals: Employment.