Splenectomy Normalizes Hematocrit in Murine Polycythemia Vera

Splenic enlargement (splenomegaly) develops in numerous disease states, although a specific pathogenic role for the spleen has rarely been described. In polycythemia vera (PV), an activating mutation in Janus kinase 2 (JAK2V617) induces splenomegaly and an increase in hematocrit. Splenectomy is sparingly performed in patients with PV, however, due to surgical complications. Thus, the role of the spleen in the pathogenesis of human PV remains unknown. We specifically tested the role of the spleen in the pathogenesis of PV by performing either sham (SH) or splenectomy (SPL) surgeries in a murine model of JAK2V617F-driven PV. Compared to SH-operated mice, which rapidly develop high hematocrits after JAK2V617F transplantation, SPL mice completely fail to develop this phenotype. Disease burden (JAK2V617) is equivalent in the bone marrow of SH and SPL mice, however, and both groups develop fibrosis and osteosclerosis. If SPL is performed after PV is established, hematocrit rapidly declines to normal even though myelofibrosis and osteosclerosis again develop independently in the bone marrow. In contrast, SPL only blunts hematocrit elevation in secondary, erythropoietin-induced polycythemia. We conclude that the spleen is required for an elevated hematocrit in murine, JAK2V617F-driven PV, and propose that this phenotype of PV may require a specific interaction between mutant cells and the spleen

Efficacious Intermittent Dosing of a Novel JAK2 Inhibitor in Mouse Models of Polycythemia Vera

A high percentage of patients with the myeloproliferative disorder polycythemia vera (PV) harbor a Val617→Phe activating mutation in the Janus kinase 2 (JAK2) gene, and both cell culture and mouse models have established a functional role for this mutation in the development of this disease. We describe the properties of MRLB-11055, a highly potent inhibitor of both the WT and V617F forms of JAK2, that has therapeutic efficacy in erythropoietin (EPO)-driven and JAK2V617F-driven mouse models of PV. In cultured cells, MRLB-11055 blocked proliferation and induced apoptosis in a manner consistent with JAK2 pathway inhibition. MRLB-11055 effectively prevented EPO-induced STAT5 activation in the peripheral blood of acutely dosed mice, and could prevent EPO-induced splenomegaly and erythrocytosis in chronically dosed mice. In a bone marrow reconstituted JAK2V617F-luciferase murine PV model, MRLB-11055 rapidly reduced the burden of JAK2V617F-expressing cells from both the spleen and the bone marrow. Using real-time in vivo imaging, we examined the kinetics of disease regression and resurgence, enabling the development of an intermittent dosing schedule that achieved significant reductions in both erythroid and myeloid populations with minimal impact on lymphoid cells. Our studies provide a rationale for the use of non-continuous treatment to provide optimal therapy for PV patients

<i>FGFR2</i> Is Amplified in the NCI-H716 Colorectal Cancer Cell Line and Is Required for Growth and Survival

<div><p>Aberrant kinase activation resulting from mutation, amplification, or translocation can drive growth and survival in a subset of human cancer. <i>FGFR2</i> is amplified in breast and gastric cancer, and we report here the first characterization of <i>FGFR2</i> gene amplification in colorectal cancer in the NCI-H716 colorectal cancer cell line. <i>FGFR2</i> is highly expressed and activated in NCI-H716 cells, and FGFR selective small molecule inhibitors or FGFR2 shRNA strongly inhibited cell viability <i>in vitro</i>, indicating “addiction” of NCI-H716 cells to FGFR2. NCI-H716 growth in a xenograft model was also inhibited by an FGFR small molecule inhibitor. FGFR2 was required for activation of multiple downstream signaling proteins including AKT, ERK, S6RP and NFKB. Inhibition of downstream kinases such as AKT or ERK alone had modest effects on proliferation, whereas combined inhibition of AKT and ERK signaling resulted in a loss of viability similar to FGFR2 inhibition. We identified elevated <i>FGFR2</i> expression in a small subset of primary colorectal cancer, however <i>FGFR2</i> amplification was not observed. Although <i>FGFR2</i> amplification is not common in primary colon cancer or lymph node and liver metastases, other subsets of colorectal cancer such as ascites, from which the NCI-H716 cell line was derived, have yet to be tested. These results suggest that emerging FGFR inhibitor therapeutics may have efficacy in a subset of colon cancer driven by <i>FGFR2</i> amplification.</p></div

FGFR2 is required for growth of NCI-H716 cells.

<p>A. PD173074 and MK2461 inhibit FGFR2 phosphorylation. Cells were treated for 1 hour with a titration of PD173074 as described in Materials and methods. Lysates were prepared and 50 ug protein was subjected to SDS-PAGE and western blotting with phospho Y653/654 FGFR and MAB6841 total FGFR2 antibody. B. PD173074 and MK2461 inhibit NCI-H716 cell growth. Cell lines were plated at 4000 cells/well and incubated overnight. NCI-H716 cells were treated with a titration of PD173074 as described in Materials and Methods. Cell growth was measured with Vialight reagent, and growth was presented relative to untreated cells. C. PD173074 and MK2461 selectively inhibit growth of NCI-H716 cells. Colon cancer cell lines listed were plated at 4000 cells/well and 24 hours later were treated with a titration of compounds. 4 days later cell growth was measured with vialight and IC50s were calculated from graph pad prism. D. <i>FGFR2</i> shRNA decreases FGFR2 protein in NCI-H716 cells. <i>FGFR2</i> shRNA was prepared and NCI-H716 cells were infected as described in methods. Left, FGFR2 expression was analyzed with phospho Y653/654 and total protein with MAB6841. E. Growth was analyzed 5 days post infection with Vialight reagent.</p

FGFR2 activates multiple signaling pathways in NCI-H716 cells.

<p>A. Lysates used in Fig. 2A were analyzed by western blotting for phosphorylated or total proteins after a 2 hour treatment with indicated compound titration. “p” indicates phosphoprotein, and the phosphorylation sites are listed in Methods. B. Time course of inhibition for multiple signaling proteins. NCI-H716 cells were treated with 100 nM PD173074 for the indicated times and signaling pathways were analyzed by SDS PAGE and western blotting. 100 nM PD173074 was selected because this amount caused full inhibition of pERK at the 2 hour time point. “p” indicates phosphoprotein, and the phosphorylation sites are listed in Methods. Terminology on the right side of figure groups proteins according to the time at which inhibition or protein loss occurs. C. NCI-H716 cells were treated with 100 nM PD0325901 for the indicated time. Lysates were prepared for SDS PAGE and western blotting with the indicated antibodies. D. NCI-H716 cells plated at 4,000 cells/well were treated 24 hours later with 1 uM L-547 (AKTi), 100 nM PD0325901 (MEKi), 5 nM Rapamycin (rapamycin), 100 nM PD173074, or 1.5 uM MK2461, or the indicated combinations. After 5 days compound and media were removed and replaced with fresh compound and media. After an additional 5 days (10 day total assay) relative cell growth was determined using Vialight reagent.</p

MK2461 has efficacy in NCI-H716 xenografts.

<p>A. 5×10exp6 NCI-H716 cells were implanted subcutaneously into Balb/c nude mice and treated with MK2461 at either 300 mg/kg or 800 mg/kg on a QD dosing schedule. Relative tumor growth is indicated on the Y axis. B. Tumor bearing mice were treated with a single dose of MK2461 at either 300 mg/kg or 800 mg/kg. Tumors were isolated at either 4 or 24 hours post treatment, and placed in liquid nitrogen. Tumors were processed using the tissue lyzer (Qiagen) as described in materials and methods and analyzed by SDS-PAGE and western blotting with the indicated phospho and total antibodies.</p

Exposure and target engagement of MRLB-11055 in the peripheral blood of C57BL/6 mice stimulated with darbepoetin.

<p>A. Effect of MRLB-11055 on phosphoSTAT5 levels at various times post-dose. B. Calculation of IC50 value for inhibition of phosphoSTAT5. C. PK of 3 doses of MRLB-11055 in mouse blood, with calculated IC50 superimposed (dashed line).</p

Effect of MRLB-11055 in a Darbepoetin-Induced PV Efficacy Model.

<p>The ability of MRLB-11055 to prevent darbepoetin-induced increases in hematocrit (Hct) and spleen mass (SPL) over 7 days is shown, as is the impact of MRLB-11055 on white blood cells (WBC) and its concentration in blood (PK). Dashed line indicates <i>in vivo</i> IC50 value.</p