Laura J. Bloem

A Novel, Selective Inhibitor of Fibroblast Growth Factor Receptors That Shows a Potent Broad Spectrum of Antitumor Activity in Several Tumor Xenograft Models

The fibroblast growth factor receptors (FGFR) are tyrosine kinases that are present in many types of endothelial and tumor cells and play an important role in tumor cell growth, survival, and migration as well as in maintaining tumor angiogenesis. Overexpression of FGFRs or aberrant regulation of their activities has been implicated in many forms of human malignancies. Therefore, targeting FGFRs represents an attractive strategy for development of cancer treatment options by simultaneously inhibiting tumor cell growth, survival, and migration as well as tumor angiogenesis. Here, we describe a potent, selective, small-molecule FGFR inhibitor, (R)-(E)-2-(4-(2-(5-(1-(3,5-Dichloropyridin-4-yl)ethoxy)-1H-indazol-3yl)vinyl)-1H-pyrazol-1-yl)ethanol, designated as LY2874455. This molecule is active against all 4 FGFRs, with a similar potency in biochemical assays. It exhibits a potent activity against FGF/FGFR-mediated signaling in several cancer cell lines and shows an excellent broad spectrum of antitumor activity in several tumor xenograft models representing the major FGF/FGFR relevant tumor histologies including lung, gastric, and bladder cancers and multiple myeloma, and with a well-defined pharmacokinetic/pharmacodynamic relationship. LY2874455 also exhibits a 6- to 9-fold in vitro and in vivo selectivity on inhibition of FGF- over VEGF-mediated target signaling in mice. Furthermore, LY2874455 did not show VEGF receptor 2–mediated toxicities such as hypertension at efficacious doses. Currently, this molecule is being evaluated for its potential use in the clinic. Mol Cancer Ther; 10(11); 2200–10. ©2011 AACR.

Complete Inhibition of Anisomycin and UV Radiation but Not Cytokine Induced JNK and p38 Activation by an Aryl-substituted Dihydropyrrolopyrazole Quinoline and Mixed Lineage Kinase 7 Small Interfering RNA

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nm for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nm or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1β or tumor necrosis factor-α-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.

Protein kinase C βII activation induces angiotensin converting enzyme expression in neonatal rat cardiomyocytes

Objective: Members of the protein kinase C (PKC) family are important mediators of cell signaling underlying multiple aspects of myocardial function. Activation of the βII isoform of PKC is thought to be involved in the development of congestive heart failure. To investigate the biological effect of PKC-βII, we measured gene expression of angiotensin converting enzyme (ACE) and angiotensin II (AngII) receptors AT1A and AT1B in cardiomyocytes overexpressing PKC-βII. Methods: An adenovirus construct expressing PKC-βII was introduced into cultured neonatal rat ventricular myocytes (NRVMs). Western blot and in situ kinase assay was used to measure PKC-βII level and activity in NRVMs. Real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to measure the mRNA levels of several genes following PMA stimulation of either un-infected or ad-PKC-βII infected cells. Results: Our data show that activation of PKC-βII in cardiomyocytes leads to elevated expression of angiotensin-converting enzyme (ACE) gene. Treatment of adeno-PKC-βII infected cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) resulted in an 8-fold increase of ACE mRNA expression, whereas ACE mRNA levels only increased around 2-fold in uninfected or adeno-GFP (green fluorescent protein) infected cardiomyocytes with similar PMA treatment. The induction of ACE mRNA was blocked by the PKC-β-specific antagonist LY379196. No significant change of angiotensin II receptors AT1a and AT1b could be detected in the cardiomyocytes expressing PKC-βII. Conclusion: These data indicate that ACE is a transcription target of PKC-βII activation in cardiomyocytes, and also suggest a mechanism for the involvement of PKC in cardiac hypertrophy and fibrosis through increased activity of angiotensin converting enzyme in the myocardium.

Capture of Drug Targets from Live Cells Using a Multipurpose Immuno-Chemo-Proteomics Tool

Recently we have described the development of an Immuno-chemo-proteomics method for drug target deconvolution and profiling the toxicity of known drugs ( Saxena , C. ; Zhen , E. ; Higgs , R. E. ; Hale , J. E. J. Proteome Res. 2008, 8 , 3490 - 3497 ). The orthogonal nature and advantage of the newly developed method over existing ones were presented. Most commonly, a small molecule was coupled to an epitope and used as an affinity probe to bind targets and later antibody against the epitope was used to isolate the probe-protein complex. However, such studies performed using cell lysates are prone to false positive identification because the protein source is not in its native physiological condition. Here we describe the development and application of a multipurpose soluble probe where a small molecule was coupled to a fluorophore-tagged cell-permeable peptide epitope, which was used to affinity isolate binding proteins from live cells. Fluorophore coupling allowed direct visualization of the compound in the cells, and cell permeability of the probe provided opportunity to capture the targets from the live cell. The GSK3-beta inhibitor Bisindolylmaleimide-III was coupled to a peptide containing the fluorescein-tagged TAT epitope. Following incubation with the live cells, the compound and associated proteins were affinity isolated using antifluorescein antibody beads. Using this approach, we captured the known Bisindolylmaleimide-III target GSK3-beta and previously unidentified targets from live cells. Dose-dependent inhibition of target binding to probe in the presence of uncoupled compound validated the approach. This method was directly compared with the one where cell lysate was used as the protein source providing an advanced strategy to aid in target deconvolution and help to eliminate false positives originating from non-native protein source.

Discovery of LY2457546: a multi-targeted anti-angiogenic kinase inhibitor with a novel spectrum of activity and exquisite potency in the acute myelogenous leukemia-Flt-3-internal tandem duplication mutant human tumor xenograft model

SummaryLY2457546 is a potent and orally bioavailable inhibitor of multiple receptor tyrosine kinases involved in angiogenic and tumorigenic signalling. In biochemical and cellular assays, LY2457546 demonstrates potent activity against targets that include VEGFR2 (KDR), PDGFRβ, FLT-3, Tie-2 and members of the Eph family of receptors. With activities against both Tie2 and Eph receptors, LY2457546 possesses an activity profile that distinguishes it from multikinase inhibitors. When compared head to head with sunitinib, LY2457546 was more potent for inhibition of endothelial tube formation in an in vitro angiogenesis co-culture model with an intermittent treatment design. In vivo, LY2457546 inhibited VEGF-driven autophosphorylation of lung KDR in the mouse and rat in a dose and concentration dependent manner. LY2457546 was well tolerated and exhibited efficacy in a 13762 syngeneic rat mammary tumor model in both once and twice daily continuous dosing schedules and in mouse human tumor xenograft models of lung, colon, and prostate origin. Additionally, LY2457546 caused complete regression of well-established tumors in an acute myelogenous leukemia (AML) FLT3-ITD mutant xenograft tumor model. The observed efficacy that was displayed by LY2457546 in the AML FLT3-ITD mutant tumor model was superior to sunitinib when both were evaluated using equivalent doses normalized to in vivo inhibition of pKDR in mouse lung. LY2457546 was well tolerated in non-clinical toxicology studies conducted in rats and dogs. The majority of the toxicities observed were similar to those observed with other multi-targeted anti-angiogenic kinase inhibitors (MAKs) and included bone marrow hypocellularity, hair and skin depigmentation, cartilage dysplasia and lymphoid organ degeneration and necrosis. Thus, the unique spectrum of target activity, potent in vivo anti-tumor efficacy in a variety of rodent and human solid tumor models, exquisite potency against a clinically relevant model of AML, and non-clinical safety profile justify the advancement of LY2457546 into clinical testing.

Characterization of LY3023414, a Novel PI3K/mTOR Dual Inhibitor Eliciting Transient Target Modulation to Impede Tumor Growth

The PI3K/AKT/mTOR pathway is among the most frequently altered pathways in cancer cell growth and survival. LY3023414 is a complex fused imidazoquinolinone with high solubility across a wide pH range designed to inhibit class I PI3K isoforms and mTOR kinase. Here, we describe the in vitro and in vivo activity of LY3023414. LY3023414 was highly soluble at pH 2–7. In biochemical testing against approximately 266 kinases, LY3023414 potently and selectively inhibited class I PI3K isoforms, mTORC1/2, and DNA-PK at low nanomolar concentrations. In vitro, inhibition of PI3K/AKT/mTOR signaling by LY3023414 caused G1 cell-cycle arrest and resulted in broad antiproliferative activity in cancer cell panel screens. In vivo, LY3023414 demonstrated high bioavailability and dose-dependent dephosphorylation of PI3K/AKT/mTOR pathway downstream substrates such as AKT, S6K, S6RP, and 4E-BP1 for 4 to 6 hours, reflecting the drugs half-life of 2 hours. Of note, equivalent total daily doses of LY3023414 given either once daily or twice daily inhibited tumor growth to similar extents in multiple xenograft models, indicating that intermittent target inhibition is sufficient for antitumor activity. In combination with standard-of-care drugs, LY3023414 demonstrated additive antitumor activity. The novel, orally bioavailable PI3K/mTOR inhibitor LY3023414 is highly soluble and exhibits potent in vivo efficacy via intermittent target inhibition. It is currently being evaluated in phase I and II trials for the treatment of human malignancies. Mol Cancer Ther; 15(10); 2344–56. ©2016 AACR.

Spironolactone increases integrin β3 gene expression in kidney and heart muscle cells

In clinical trials of heart failure, spironolactone, an antagonist of the mineralocorticoid receptor (MR), reduced mortality rates by unknown mechanisms. We hypothesized that spironolactone functions by upregulating expression of certain cardiovascular genes. An RNA differential display technique was used to identify genes whose expression was increased by spironolactone in an Xenopus kidney epithelial cell line (A6), a known target of aldosterone. We found that integrin beta3 gene expression was increased by spironolactone, and reversed by aldosterone or dexamethasone in a dose dependent manner. Competition binding studies and RT-PCR indicate the presence of MR in A6 cells, suggesting that regulation of expression occurred primarily through MR. Spironolactone also increased integrin beta3 expression in rat neonatal cardiomyocytes. In summary, spironolactone increases integrin beta3 gene expression in kidney epithelial cells and cardiomyocytes. The findings suggest new mechanisms for spironolactone actions with possible relevance to treatment of heart disease.

Chronic inhibition of the MEK/ERK pathway fails to delay disease progression in a transgenic animal model of tauopathy

Background: Neurofibrillary tangles (NFT), one of the hallmarks of Alzheimer’s disease (AD), are composed of paired helical filaments (PHF) of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD.Methods:We have used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with early AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes pS202, pT231, pS235 and pS396/404) were measured in cell lysates using AlphaScreen assays. Results: GSK3a, GSK3b and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all 4 epitopes. Pathway analysis of all the ‘hits’ suggests the Ras family of GTPases (MAPK family) to be a key regulator of tau phosphorylation. Pathway analysis on subgroups of kinases reveals several mechanisms involved in regulating tau expression levels by inhibition or activation of translation.Conclusions: The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies.

Abstract 3624: A novel, selective FGFR inhibitor that causes blockade of FGFR autophosphorylation shows a broad-spectrum of anti-tumor activity in preclinical xenograft tumor models

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Anti-angiogenic agents targeting VEGF/VEGFR including Bevacizumab, Sorafenib, and Sunitinib that target blood vessel formation in rapidly growing cancer cells are very effective cancer therapies in the clinic. However, these anti-angiogenic agents also have debilitating side effects, such as hypertension and bleeding, which thereby limit their use in many patients in which the anti-VEGF/VEGFR based therapies are contraindicated. Furthermore, the emergence of resistance to the anti-VEGF/VEGFR based therapies in the clinic due to over-expression of FGF2 or other growth factors could pose a serious challenge to their current use in the clinic. The FGFRs are receptor tyrosine kinases that are present in many types of tumor cells as well as endothelial cells, and play an important role in tumor cell growth, survival, and migration, and also in maintaining tumor angiogenesis. Over-expression of FGFRs or aberrant regulation of their activities has been implicated in many forms of human malignancies. Therefore, targeting FGFRs for cancer treatment represents an attractive strategy that has the potential to treat a variety of cancers by simultaneously inhibiting tumor cell growth, survival, and migration, and also tumor angiogenesis without the side effects associated with the VEGF/VEGFR based therapies. Here, we describe a selective small molecule inhibitor of FGFRs. The FGFR inhibitor is active against all four FGF receptors (IC50 = 2.6 to 7.7 nM) as revealed by biochemical autophosphorylation assays. It also exhibits a potent activity against FGFR or FGFR-mediated Erk phosphorylation in several cancer cell lines (IC50 = 0.6 to 1.5 nM). In addition, in vitro cell based assays have shown that this inhibitor is much more active at inhibiting the proliferation of FGFR-dependent than FGFR-independent cancer cell lines (IC50s = 3-2,600 nM). Furthermore, in vitro selectivity assays have established that this inhibitor exhibits a robust preferential activity in inhibiting FGF- over VEGF-mediated cord formation (IC50s = 0.6 nM for FGF and 3.6 nM for VEGF). Consistent with these in vitro selectivity data, in vivo target inhibition assays have also demonstrated that the FGFR inhibitor has a potent, yet much higher activity against FGF- than VEGF-mediated target phosphorylation in mice (TEC50s and TEC90s of 6 and 29 nM for FGFR, and 35 and 252 nM for VEGFR, respectively). Finally, the FGFR inhibitor shows an excellent broad-spectrum anti-tumor activity in xenograft tumor models representing several major cancer histologies including bladder cancer when dosed at TED50 (1.3 mg/kg) and especially at TED90 (3.2 mg/kg). Thus, the FGFR inhibitor exhibits a very well behaved PK/PD relationship. Currently, the FGFR inhibitor is being further evaluated for its potential utility in the clinic. 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 3624.