Anne Lewin

Local Control of Granule Cell Generation by Cerebellar Purkinje Cells

Cerebellar Purkinje cells were ablated by the specific expression of diphtheria toxin in these cells in transgenic mice. Purkinje cell degeneration during early postnatal development shows a zonally restricted pattern which has been exploited in order to look for local secondary effects. The most obvious early effect is the alignment of gaps in the Purkinje cell layer with dramatically thinned zones in the overlying EGL, the germinal layer from which granule cells are generated. Within these EGL zones in the transgenic mutant, markers that distinguish matrix from mantle cells demonstrate a preferential loss of the proliferative cells. Comparison of BrdU incorporation in the mutant vs wild-type confirms the reduction in proliferation. In the mutant, in situ labeling of DNA fragmentation associated with apoptotic cell death shows abundant labeling of granule cells that have exited the EGL, but not of progenitor cells in the EGL. Thus, although a trophic role for Purkinje cells has been well documented, these observations further suggest a mitogenic role which can be exerted locally.

The Antiangiogenic Activity in Xenograft Models of Brivanib, a Dual Inhibitor of Vascular Endothelial Growth Factor Receptor-2 and Fibroblast Growth Factor Receptor-1 Kinases

Tumor angiogenesis is a complex and tightly regulated network mediated by various proangiogenic factors. The fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) family of growth factors, and associated tyrosine kinase receptors have a major influence in tumor growth and dissemination and may work synergistically to promote angiogenesis. Brivanib alaninate is the orally active prodrug of brivanib, a selective dual inhibitor of FGF and VEGF signaling. Here, we show that brivanib demonstrates antitumor activity in a broad range of xenograft models over multiple dose levels and that brivanib alaninate shows dose-dependent efficacy equivalent to brivanib in L2987 human tumor xenografts. Brivanib alaninate (107 mg/kg) reduced tumor cell proliferation as determined by a 76% reduction in Ki-67 staining and reduced tumor vascular density as determined by a 76% reduction in anti-CD34 endothelial cell staining. Furthermore, Matrigel plug assays in athymic mice showed that brivanib alaninate inhibited angiogenesis driven by VEGF or basic FGF alone, or combined. Dynamic contrast-enhanced magnetic resonance imaging, used to assess the effects of brivanib alaninate on tumor microcirculation, showed a marked decrease in gadopentetate dimeglumine contrast agent uptake at 107 mg/kg dose, with a reduction in area under the plasma concentration-time curve from time 0 to 60 minutes at 24 and 48 hours of 54% and 64%, respectively. These results show that brivanib alaninate is an effective antitumor agent in preclinical models across a range of doses, and that efficacy is accompanied by changes in cellular and vascular activities. Mol Cancer Ther; 9(2); 369–78

Discovery and Validation of Biomarkers that Respond to Treatment with Brivanib Alaninate, a Small-Molecule VEGFR-2/FGFR-1 Antagonist

The process of neovascularization from preexisting blood vessels, referred to as angiogenesis, plays a critical role in both tumor growth and dissemination in multiple cancer types. Currently, there exists a need to identify biomarkers that can both indicate biological activity and predict efficacy at the molecular level for antiangiogenesis drugs which are anticipated to result in tumor stasis rather than regression. To identify such biomarkers, athymic mice bearing L2987 human tumor xenografts were treated with the antiangiogenic agent brivanib alaninate, which is currently under clinical evaluation. This is an orally available and selective tyrosine kinase inhibitor that targets the key angiogenesis receptors vascular endothelial growth factor receptor 2 (VEGFR-2) and fibroblast growth factor receptor 1. In the described studies, tumor samples were collected from these xenografts and RNA was extracted for gene expression profiling on Affymetrix 430A mouse GeneChips. Statistical analysis was done using a defined set of genes identified to be coexpressed with VEGFR-2 from a clinical tumor gene expression profiling database and between tumor samples isolated from brivanib alaninate-treated and untreated mice. Tyrosine kinase receptor 1 (Tie-1), collagen type IV alpha1 (Col4a1), complement component 1, q subcomponent receptor 1 (C1qr1), angiotensin receptor-like 1 (Agtrl1), and vascular endothelial-cadherin (Cdh5) were all identified to be significantly modulated by treatment with brivanib alaninate. These genes, which may be potentially useful as markers of brivanib alaninate activity, were further studied at the protein level in two separate in vivo human colon tumor xenograft models, HCT116 and GEO, using immunohistochemistry-based approaches.

Synergistic Antitumor Activity of Ixabepilone (BMS-247550) Plus Bevacizumab in Multiple In vivo Tumor Models

Purpose: Angiogenesis is a critical step in the establishment, growth, and metastasis of solid tumors, and combination of antiangiogenic agents with chemotherapy is an attractive therapeutic option. We investigated the potential of ixabepilone, the first in a new class of antineoplastic agents known as epothilones, to synergize with antiangiogenic agents to inhibit tumor growth. Experimental Design:In vitro and in vivo cytotoxicity of ixabepilone as single agent and in combination with two targeted antiangiogenic agents, bevacizumab or sunitinib, were examined in preclinical tumor models. Direct effects of the agents against endothelial cells was also examined and compared with the effects of paclitaxel as single agent and in combination with bevacizumab. Results: Ixabepilone showed robust synergistic antitumor activity in combination with bevacizumab and sunitinib in preclinical in vivo models derived from breast, colon, lung, and kidney cancers. The synergistic antitumor effect was greater with ixabepilone compared with paclitaxel. Furthermore, ixabepilone was more effective than paclitaxel at killing endothelial cells expressing P-glycoprotein in vitro and inhibiting endothelial cell proliferation and tumor angiogenesis in vivo. Conclusions: Ixabepilone may enhance the antitumor effects of antiangiogenic therapy by direct cytotoxicity and also indirectly via the killing of tumor-associated endothelial cells. Given that ixabepilone has reduced susceptibility to drug efflux pumps compared with taxanes, these data may explain the increased antiangiogenic and antitumor activity of ixabepilone in combination with antiangiogenic agents. Phase II studies to assess the efficacy and safety of ixabepilone plus bevacizumab in locally recurrent or metastatic breast cancer are planned.

Characterization of Organic Anion Transporter 2 (SLC22A7): A Highly Efficient Transporter for Creatinine and Species-Dependent Renal Tubular Expression

The contribution of organic anion transporter OAT2 (SLC22A7) to the renal tubular secretion of creatinine and its exact localization in the kidney are reportedly controversial. In the present investigation, the transport of creatinine was assessed in human embryonic kidney (HEK) cells that stably expressed human OAT2 (OAT2-HEK) and isolated human renal proximal tubule cells (HRPTCs). The tubular localization of OAT2 in human, monkey, and rat kidney was characterized. The overexpression of OAT2 significantly enhanced the uptake of creatinine in OAT2-HEK cells. Under physiologic conditions (creatinine concentrations of 41.2 and 123.5 µM), the initial rate of OAT2-mediated creatinine transport was approximately 11-, 80-, and 80-fold higher than OCT2, multidrug and toxin extrusion protein (MATE)1, and MATE2K, respectively, resulting in approximately 37-, 1850-, and 80-fold increase of the intrinsic transport clearance when normalized to the transporter protein concentrations. Creatinine intracellular uptake and transcellular transport in HRPTCs were decreased in the presence of 50 µM bromosulfophthalein and 100 µM indomethacin, which inhibited OAT2 more potently than other known creatinine transporters, OCT2 and multidrug and toxin extrusion proteins MATE1 and MATE2K (IC50: 1.3 µM vs. > 100 µM and 2.1 µM vs. > 200 µM for bromosulfophthalein and indomethacin, respectively) Immunohistochemistry analysis showed that OAT2 protein was localized to both basolateral and apical membranes of human and cynomolgus monkey renal proximal tubules, but appeared only on the apical membrane of rat proximal tubules. Collectively, the findings revealed the important role of OAT2 in renal secretion and possible reabsorption of creatinine and suggested a molecular basis for potential species difference in the transporter handling of creatinine.

Antitumor and Antiangiogenic Activities of BMS-690514, an Inhibitor of Human EGF and VEGF Receptor Kinase Families

Purpose: The extensive involvement of the HER kinases in epithelial cancer suggests that kinase inhibitors targeting this receptor family have the potential for broad spectrum antitumor activity. BMS-690514 potently inhibits all three HER kinases, and the VEGF receptor kinases. This report summarizes data from biochemical and cellular pharmacology studies, as well as antitumor activity of BMS-690514. Experimental Design: The potency and selectivity of BMS-690514 was evaluated by using an extensive array of enzymatic and binding assays, as well as cellular assays that measure proliferation and receptor signaling. Antitumor activity was evaluated by using multiple xenograft models that depend on HER kinase signaling. The antiangiogenic properties of BMS-690514 were assessed in a matrigel plug assay, and effect on tumor blood flow was measured by dynamic contrast-enhanced MRI. Results: BMS-690514 is a potent and selective inhibitor of epidermal growth factor receptor (EGFR), HER2, and HER4, as well as the VEGF receptor kinases. It inhibits proliferation of tumor cells with potency that correlates with inhibition of receptor signaling, and induces apoptosis in lung tumor cells that have an activating mutation in EGFR. Antitumor activity was observed with BMS-690514 at multiple doses that are well tolerated in mice. There was evidence of suppression of tumor angiogenesis and endothelial function by BMS-690514, which may contribute to its efficacy. Conclusions: By combining inhibition of two receptor kinase families, BMS-690524 is a novel targeted agent that disrupts signaling in the tumor and its vasculature. Clin Cancer Res; 17(12); 4031–41. ©2011 AACR.

Abstract B192: Preclinical characterization of BMS‐833923 (XL139), a hedgehog (HH) pathway inhibitor in early clinical development

Background: Aberrant HH pathway signaling has been implicated in human malignancies ranging from semi‐malignant tumors of the skin to highly aggressive cancers of the brain, lung, pancreas, breast, prostate, and lymphohematopoietic lineages. Dysregulation of this pathway contributes to uncontrolled proliferation, invasion, metastasis, evasion of apoptosis, and resistance to chemotherapy. Results: BMS‐833923 is a potent inhibitor of SMO, a GPCR‐like 7‐transmembrane receptor that is a critical regulator of the HH pathway. In vitro, BMS‐833923 inhibits the expression of downstream effectors in the HH pathway (GLI1 and PTCH1) in cell lines that express wild‐type SMO and those which express activated mutant forms of SMO (IC 50 values of 6–35 nM). In FACS‐based binding assays, BMS‐833923 inhibits BODIPY cyclopamine binding to SMO in a dose‐dependent manner with an IC50 of 21 nM. Prior work has demonstrated that HH pathway blockade inhibits the in vitro clonal expansion of multiple myeloma (MM) precursor cells in both established cell lines and in clinical samples from patients with MM (Peacock et al 2007, PNAS 104:4048). Consistent with these observations, BMS‐833923 inhibited the in vitro growth of MM clones and proportion of ALDH+ cancer stem cells derived from bone marrow samples from subjects with MM. Furthermore, BMS‐833923 inhibited the clonogenic growth of multiple tumor cell lines derived from patients with hematological malignancies including CML, ALL and AML, suggesting that targeting the HH pathway may have broad therapeutic utility for patients with hematologic malignancies. In vivo pharmacodynamic studies show that BMS‐833923 robustly inhibits HH pathway activity with a long duration of action after a single oral dose in medulloblastoma and pancreatic carcinoma xenograft models. The pharmacodynamic effects of BMS‐833923 observed in these models translate into tumor growth inhibition at well‐tolerated doses. These results are consistent with pharmacokinetic parameters demonstrated in nonclinical studies. Conclusions: BMS‐833923 is an orally bioavailable, potent and selective inhibitor of the HH pathway as measured by in vitro cell‐based assays and in vivo pharmacodynamic and efficacy models, supporting the clinical development of BMS‐833923 in both hematological and non‐hematological malignancies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B192.

Abstract 1476: A therapeutic antibody that inhibits CD73 activity by dual mechanisms

CD73 has a central role in dictating the adenosine concentration within the tumor as it is the final step in converting extracellular ATP to adenosine. Thus, substantial reduction of CD73 enzymatic activity has the potential to reduce immunosuppression of effector immune cells within the tumor. We present data describing an anti-human CD73 antibody that suppresses CD73 by two mechanisms: 1. direct inhibition of enzymatic activity upon binding to CD73 and 2. rapid, near-complete internalization of the enzyme. Durable reduction of cell-surface CD73 was observed in multiple tumor cell lines both in vitro and in vivo. The unique properties of this antibody are a result of the use of a human IgG2-IgG1 hybrid antibody with effector function eliminated by specific mutations of the Fc. The IgG2 sequence of this antibody drives superior internalization of CD73 and enhanced CD73 inhibition. Syngeneic tumor models demonstrate that CD73 contributes to resistance to anti-tumor therapy. Combination therapy with PD-1 blockade and a surrogate anti-mouse-CD73 antibody resulted in a better anti-tumor efficacy than either treatment alone. Finally, we demonstrate a novel technique for assessing CD73 enzymatic activity in situ that has potential for clinical application. These data support antibody-based anti-CD73 therapy in cancer and highlight a novel mechanism for inhibition of CD73 enzymatic activity. Citation Format: Bryan C. Barnhart, Emanuela Sega, Aaron Yamniuk, Sandra Hatcher, Ming Lei, Haben Ghermazien, Anne Lewin, Xi-Tao Wang, Haichun Huang, Pingping Zhang, Alan Korman. A therapeutic antibody that inhibits CD73 activity by dual mechanisms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1476.

Abstract 5016: Antitumor activity of anti-PD-1 in combination with tyrosine kinase inhibitors in a preclinical renal cell carcinoma model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Nivolumab (BMS-936558; MDX-1106; ONO-4538) is a fully human IgG4 programmed death-1 (PD-1) immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), inhibiting the downregulation of antitumor T-cell functions. Nivolumab has shown activity in advanced solid tumors, including renal cell carcinoma (RCC), melanoma, and non-small cell lung cancer (Topalian SL, et al. NEJM 2012;366:2443-54). Sunitinib and sorafenib are anti-angiogenic tyrosine kinase inhibitors (TKIs) used for the treatment of RCC. We investigated the activity of nivolumab in combination with TKIs in a preclinical RCC murine model. Methods: The murine RCC (Renca) tumor cell line was maintained in vitro and implanted subcutaneously into 8-12 week old female Balb/c mice. When mean tumor volume reached approximately 90-100 mm3, mice were randomized into groups of eight. Vehicle control, sunitinib 120 mg/kg, or sorafenib 200 mg/kg were administered orally once daily for 14 days. The nivolumab surrogate antibody, an IgG1 anti-mouse PD-1 monoclonal antibody (clone 4H2), was administered at 10 mg/kg by intraperitoneal injection every four days for four cycles. Immunohistochemistry and flow cytometry analyses were used to assess immune cell infiltration of tumors. Results: Sunitinib monotherapy showed activity in the Renca murine RCC model producing tumor growth inhibition of 84% by the end of treatment; however, tumors grew progressively after cessation of therapy. Conversely, while the anti-PD-1 monoclonal antibody was inactive in this model, addition of anti-PD-1 to sunitinib produced significant antitumor activity, resulting in complete tumor regressions or marked delay in tumor growth. Combination anti-PD-1 plus sunitinib therapy also had no effect on the body weight of the mice. Immunohistochemical analysis demonstrated that sunitinib monotherapy led to an influx of immune cells predominantly into the tumor periphery, whereas greater infiltration of immune cells throughout the tumor was observed with combination anti-PD-1 and sunitinib therapy. In contrast, combination treatment with anti-PD-1 antibody and sorafenib did not enhance antitumor activity in this murine RCC model. Further exploration of the mechanisms contributing to this synergy will be presented. Conclusions: Combination therapy with sunitinib and anti-PD-1 antibody demonstrated an enhanced effect against murine RCC. Safety and response to nivolumab plus sunitinib, pazopanib, or ipilimumab in patients with metastatic RCC are being assessed in an ongoing phase 1 study ([NCT01472081][1]). Citation Format: Gregg Masters, Gennaro Dito, Becky Penhallow, Anne Lewin, Henry Kao, Maria N. Jure-Kunkel. Antitumor activity of anti-PD-1 in combination with tyrosine kinase inhibitors in a preclinical renal cell carcinoma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5016. doi:10.1158/1538-7445.AM2014-5016 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01472081&atom=%2Fcanres%2F74%2F19_Supplement%2F5016.atom

Abstract C18: Assessment of Gli1 expression during skin regeneration in mouse models and normal healthy volunteers

Background: Inhibition of the Hedgehog pathway using Smoothened antagonists is emerging as a promising targeted therapy for cancers with pathway activating mutations or dysregulated expression. In support of the clinical development of BMS‐833923 (XL139), an orally available, potent Smoothened antagonist, a pharmacodynamic assay was developed to measure Gli1 protein expression during the wound healing process. Results: Gli1 protein and mRNA expression were first examined by immunohistochemistry or RT‐PCR in mouse skin at baseline and in regenerating skin at various time points following the initial punch biopsy. The effects of BMS‐833923 treatment on pathway expression profiles were then determined. The results showed that in control mice, Gli1 protein levels were elevated at as early as 3h and sustained for up to 72h. The intensities of Gli1 staining in both hair follicles and epidermis were greatly enhanced. However, in the mice treated with BMS‐833923 at the time of initial biopsy, the magnitude and duration of Gli1 up‐regulation were greatly reduced. While the Gli1 protein level was elevated within 6h, it returned to nearbaseline levels at 24h and remained low thereafter. Gli1 mRNA expression data support these observations. To better define the timing of potential Hedgehog pathway activation in human skin, an experimental medicine study was conducted to examine the expression of Gli1 after skin wounding and during regeneration in eighteen normal healthy volunteers. The baseline skin was sampled at the nape of the neck and the regenerating skin sample completely encompassed the initial wound. The intensities of both nuclear and cytoplasmic staining, as well as the percent cell staining positivity were scored. The data indicated that Gli1 protein was significantly up‐regulated at 6h and 24h following wounding. Conclusion: Gli1 protein level at 24h post‐skin wounding assessed by immunohistochemistry is currently being used as a surrogate tissue pharmacodynamic measurement in a first‐in‐human multiple ascending dose trial in subjects with advanced or metastatic solid tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C18.