Anjili Mathur

An inhibitor of Janus kinase 2 prevents polycythemia in mice.

Polycythemia vera (PV) is a myeloproliferative disorder characterized by increased red cell mass and splenomegaly in the absence of secondary causes [Tefferi A., Spivak J.L., Polycythemia vera: scientific advances and current practice. Semin Hematol 2005;42(4):206-20.]. Recently, several laboratories have discovered that the vast majority of patients with PV carry a single, activating mutation (V617F) in the pseudokinase domain of Janus kinase 2 (Jak2) [Zhao R, Xing S, Li Z, Fu X, Li Q, Krantz SB, et al., Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 2005;280(24):22788-92; James C, Ugo V, Le Couédic JP, Staerk J, Delhommeau F, Lacout C, et al., A unique clonal JAK2 mutation leading to constitutive signalling causes polycythemia vera. Nature 2005;434(7037):1144-8; Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al., A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352(17):1779-90; Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, et al., Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005;7(4):387-97.]. This discovery has spurred interest in developing therapies for PV via inhibition of Jak2. We induced polycythemia in mice by administering high dose recombinant erythropoietin (Epo) and determined that administration recapitulates almost all of the major and minor diagnostic features of human PV. We then tested a selective, small molecule inhibitor of Jak2 (Jak2i) and showed that this treatment prevents polycythemia. This prevention of polycythemia was accompanied by lower hematocrits, reduced spleen sizes and reductions in Stat5 phosphorylation (pStat5). Surprisingly, Epo rapidly (<1h) induces mobilization of activated erythroid precursors into the blood, thus allowing drug-response relationships to guide discovery. We conclude that inhibition of Jak2 prevents polycythemia in mice, and furthermore present this model as an efficient tool for the discovery of drugs that effectively treat human PV.

FGFR2 is amplified in the NCI-H716 colorectal cancer cell line and is required for growth and survival

Aberrant kinase activation resulting from mutation, amplification, or translocation can drive growth and survival in a subset of human cancer. FGFR2 is amplified in breast and gastric cancer, and we report here the first characterization of FGFR2 gene amplification in colorectal cancer in the NCI-H716 colorectal cancer cell line. FGFR2 is highly expressed and activated in NCI-H716 cells, and FGFR selective small molecule inhibitors or FGFR2 shRNA strongly inhibited cell viability in vitro, 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 FGFR2 expression in a small subset of primary colorectal cancer, however FGFR2 amplification was not observed. Although FGFR2 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 FGFR2 amplification.

The discovery of tricyclic pyridone JAK2 inhibitors. Part 1: hit to lead.

This paper describes the discovery and design of a novel class of JAK2 inhibitors. Furthermore, we detail the optimization of a screening hit using ligand binding efficiency and log D. These efforts led to the identification of compound 41, which demonstrates in vivo activity in our study.

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.

Thiophene carboxamide inhibitors of JAK2 as potential treatments for myleoproliferative neoplasms

A series of carboxamide-substituted thiophenes demonstrating inhibition of JAK2 is described. Development of this chemical series began with the bioisosteric replacement of a urea substituent by a pyridyl ring. Issues of chemical and metabolic stability were solved using the results of both in vitro and in vivo studies, ultimately delivering compounds such as 24 and 25 that performed well in an acute PK/PD model measuring p-STAT5 inhibition.

Development of a Cell-Based Assay for Measurement of c-Met Phosphorylation Using AlphaScreenTM Technology and High-Content Imaging Analysis

c-Met is a receptor tyrosine kinase (RTK) with a critical role in many fundamental cellular processes, including cell proliferation and differentiation. Deregulated c-Met signaling has been implicated in both the initiation and progression of human cancers and therefore represents an attractive target for anticancer therapy. Monitoring the phosphorylation status of relevant tyrosine residues provides an important method of assessing c-Met kinase activity. This report describes a novel assay to monitor c-Met phosphorylation in cells using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen™) technology. Using AlphaScreen™, the authors were able to detect both global and site-specific phosphorylation of c-Met in transformed cell lines. Data obtained from the AlphaScreen™ assay were compared to data obtained from a high-content imaging (HCI) method developed in parallel to monitor c-Met phosphorylation at the single cell level. The AlphaScreen™ assay was miniaturized to a 384-well format with acceptable signal-to-background ratio (S/B) and Z′ statistics and was employed to measure c-Met kinase activity in situ after treatment with potent c-Met-specific kinase inhibitors. The authors discuss the utility of quantifying endogenous cellular c-Met phosphorylation in lead optimization and how the modular design of the AlphaScreen™ assay allows its adaptation to measure cellular activity of other kinases. (Journal of Biomolecular Screening 2009:404-411)

The discovery of reverse tricyclic pyridone JAK2 inhibitors. Part 2: Lead optimization

This communication discusses the discovery of novel reverse tricyclic pyridones as inhibitors of Janus kinase 2 (JAK2). By using a kinase cross screening approach coupled with molecular modeling, a unique inhibitor-water interaction was discovered to impart excellent broad kinase selectivity. Improvements in intrinsic potency were achieved by utilizing a rapid library approach, while targeted structural changes to lower lipophilicity led to improved rat pharmacokinetics. This multi-pronged approach led to the identification of 31, which demonstrated encouraging rat pharmacokinetics, in vivo potency, and excellent off-target kinase selectivity.

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.

Abstract 284: FGFR2 is required for growth and survival in a rare subset of FGFR2 amplified colorectal cancer

FGFR2 amplification has been reported in subsets of gastric cancer and breast cancer. We describe FGFR2 amplification in a cell line derived from a colorectal carcinoma arising in a young patient with ulcerative colitis. The cell line has features of endocrine differentiation, representing a rare ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 284.