Spleen Size In Mice Research Articles

The PIM Kinase Inhibitor TP-3654 in Combination with Ruxolitinib Exhibits Marked Improvement of Myelofibrosis in Murine Models

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. DisclosuresWhatcott:Tolero Pharmaceuticals, Inc: Employment. Warner:Tolero Pharmaceuticals: Employment.

Abstract 1874: The PIM kinase inhibitor TP-3654 demonstrates efficacy in a murine model of myelofibrosis

Abstract Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN). The JAK2V617F mutation has been found in ~50% patients with MF. Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF. However, Ruxolitinib treatment has failed to induce disease remission or cure fibrosis. Therefore, there is a need to develop novel targeted therapies for MF. We found that PIM1 expression is significantly increased in mouse and human MPN/MF hematopoietic progenitor cells. 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 in a murine model of MF. We found that TP-3654 (0.5-1.0 μM) treatment significantly reduced proliferation of murine Ba/F3-EpoR cells expressing JAK2V617F or human JAK2V617F-positive HEL and UKE-1 cells. 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. 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 reduce fibrosis. Combined TP-3654 and Ruxolitinib treatment almost completely eliminated fibrosis in the bone marrow and spleens of homozygous JAK2V617F mice. TP-3654 treatment was well tolerated and did not cause any significant toxicity in wild type mice. RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells from these drug-treated mice revealed that the genes related to TNFα and WNT signaling pathways were significantly down-regulated by treatment of TP-3654 alone or TP-3654 and Ruxolitinib combination compared with vehicle. Overall, our results suggest that TP-3654 alone or in combination with Ruxolitinib may have therapeutic potential for treatment of MF. Citation Format: Avik Dutta, Dipmoy Nath, Yue Yang, Golam Mohi. The PIM kinase inhibitor TP-3654 demonstrates efficacy in a murine model of myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1874.

Efficacy of Vorinostat In a Murine Model of Polycythemia Vera

AbstractAbstract 629The JAK2V617F mutation is present in most patients with polycythemia vera (PV) and 50–60% patients with essential thrombocythemia (ET), and primary myelofibrosis (PMF). Inhibitors of histone deacetylases (HDAC), which regulate gene transcription, cell cycle progression and programmed cell death, are emerging as a new class of potential anticancer drugs. A number of HDAC inhibitors are in clinical trials for various neoplasms. Vorinostat (also known as SAHA or Zolinza) was the first FDA approved HDAC inhibitor for clinical use in treating cutaneous T cell lymphoma (CTCL). In this study, we have investigated the efficacy of vorinostat in hematopoietic cells expressing JAK2V617F and in a murine model of PV. We found that proliferation of Ba/F3-EpoR cells expressing JAK2V617F or human erythroleukemia (HEL) cells harboring JAK2V617F mutation was significantly reduced upon treatment with vorinostat at 0.5–1.0 μM. However, proliferation of wild type JAK2 expressing BCR-ABL-transformed K562 cells was only modestly affected by vorinostat. Vorinostat treatment also resulted in marked apoptosis in HEL cells but not in wild-type JAK2 expressing K562 or UT7 cells as determined by annexin V staining and flow cytometry. Moreover, vorinostat treatment (at 1.0 μM) completely inhibited the clonogenic growth of Ba/F3-EpoR-JAK2V617F and HEL cells in a colony assay. Clonogenic growth of K562 cells was only modestly affected by vorinostat at 1.0 μM. Biochemical analyzes revealed significant inhibition of phosphorylation of JAK2, STAT5, Akt, Erk1/2 in JAK2V617F-positive HEL cells but not in wild-type JAK2-expressing K562 cells. Expression of JAK2 and STAT5 was also significantly downregulated by vorinostat treatment in HEL cells but not in K562 cells. In addition, we observed a marked reduction in c-Myc and Pim-1, and cleavage of caspase 3 and PARP in HEL cells, but not in K562 cells, upon treatment with vorinostat. These results suggest that vorinostat selectively targets JAK2V617F mutated cells and inhibits the activation/expression of JAK2 and STAT5. We reported previously the generation of an inducible JAK2V617F knock-in mouse (Akada et al., Blood 2010; 115: 3589–3597). Expression of the JAK2V617F in the knock-in mice reproducibly produced all the features of human PV. We have utilized this JAK2V617F knock-in mouse model to test the in vivo effects of vorinostat. We found that vorinostat treatment (200 mg/kg/day by IP injection) for 2 weeks significantly reduced the increase in red blood cells (RBC), hemoglobin and hematocrit as well as spleen size in mice expressing JAK2V617F compared with vehicle-treatment. Furthermore, vorinostat markedly inhibited the clonogenic growth of primary erythroid progenitors expressing JAK2V617F without exhibiting any significant toxicity towards wild-type JAK2-expressing normal hematopoietic progenitors in mice. Thus, our in vitro and in vivo results suggest that vorinostat may have therapeutic potential for treatment of PV. Disclosures:No relevant conflicts of interest to declare.

Identification of a genetic locus modulating splenomegaly induced by granulocyte colony-stimulating factor in mice.

Clinically detectable splenomegaly and splenic rupture are uncommon but potentially life-threatening consequences of G-CSF administration. Increased spleen size in mice injected with G-CSF is a complex genetic trait amenable to investigation in experimental inter-strain crosses by quantitative trait analysis. A quantitative trait locus (QTL) with highly significant linkage (LOD 7.9) for splenomegaly was identified within a 22 centimorgan (cM) region on chromosome 1. Inheritance of a C57BL/6 haplotype in this region was associated with a greater spleen weight. The relevance of this locus was confirmed by analysing the responses of mice congenic for the distal 12 cM of this region (C57BL/6 and C57BL/6.SJL-Ptprc(a) Pep3(b)). Consistent with the QTL effect, mice lacking C57BL/6 alleles in this region had reduced splenomegaly induced by G-CSF. Intriguingly, peripheral blood neutrophilia and progenitor cell mobilisation responses to G-CSF were also significantly influenced.

The Effect of Cortisone on the Spleen in Mice.

Summary1. Cortisone causes a significant reduction in the spleen size of mice. The average spleen size of the Cortisone-treated mice was 21% of the controls.2. As little as 4 mg of Cortisone during a 2 day period produced this effect. Continued Cortisone treatment did not increase the original depletion, indicating that the effect is rapid and maximal.3. Histologically there was a reduction in the size of the Malphigian bodies and in the number of cellular elements in the pulp in the Cortisone-treated group.