Murray O. Robinson

Development of Second-Generation VEGFR Tyrosine Kinase Inhibitors: Current Status

The vascular endothelial growth factor (VEGF) signaling pathway appears to be the dominant pathway involved in tumor angiogenesis, providing a rationale for targeting the VEGF receptors (VEGFR-1, -2, and -3) in the treatment of cancers. In particular, VEGF signaling is thought to be important in renal cell carcinoma (RCC) because of the deregulation of the pathway through nearly uniform loss of the von Hippel Lindau protein. The tyrosine kinase inhibitors (TKIs) sorafenib, sunitinib, and pazopanib are approved by the US Food and Drug Administration for the treatment of advanced RCC; however, these multitargeted agents inhibit a wide range of kinase targets in addition to the VEGFRs, resulting in a range of adverse effects unrelated to efficient VEGF blockade. This article reviews recent advances in the development of the second-generation VEGFR TKIs, including the more selective VEGFR TKIs tivozanib and axitinib, and focuses on the potential benefits of novel inhibitors with improved potency and selectivity.

Chimeric mouse tumor models reveal differences in pathway activation between ERBB family– and KRAS-dependent lung adenocarcinomas

To recapitulate the stochastic nature of human cancer development, we have devised a strategy for generating mouse tumor models that involves stepwise genetic manipulation of embryonic stem (ES) cells and chimera generation. Tumors in the chimeric animals develop from engineered cells in the context of normal tissue. Adenocarcinomas arising in an allelic series of lung cancer models containing HER2 (also known as ERBB2), KRAS or EGFR oncogenes exhibit features of advanced malignancies. Treatment of EGFRL858R and KRASG12V chimeric models with an EGFR inhibitor resulted in near complete tumor regression and no response to the treatment, respectively, accurately reflecting previous clinical observations. Transcriptome and immunohistochemical analyses reveal that PI3K pathway activation is unique to ERBB family tumors whereas KRAS-driven tumors show activation of the JNK/SAP pathway, suggesting points of therapeutic intervention for this difficult-to-treat tumor category.

Dissecting genetic requirements of human breast tumorigenesis in a tissue transgenic model of human breast cancer in mice

Breast cancer development is a complex pathobiological process involving sequential genetic alterations in normal epithelial cells that results in uncontrolled growth in a permissive microenvironment. Accordingly, physiologically relevant models of human breast cancer that recapitulate these events are needed to study cancer biology and evaluate therapeutic agents. Here, we report the generation and utilization of the human breast cancer in mouse (HIM) model, which is composed of genetically engineered primary human breast epithelial organoids and activated human breast stromal cells. By using this approach, we have defined key genetic events required to drive the development of human preneoplastic lesions as well as invasive adenocarcinomas that are histologically similar to those in patients. Tumor development in the HIM model proceeds through defined histological stages of hyperplasia, DCIS to invasive carcinoma. Moreover, HIM tumors display characteristic responses to targeted therapies, such as HER2 inhibitors, further validating the utility of these models in preclinical compound testing. The HIM model is an experimentally tractable human in vivo system that holds great potential for advancing our basic understanding of cancer biology and for the discovery and testing of targeted therapies.

De novo Discovery of a γ-Secretase Inhibitor Response Signature Using a Novel In vivo Breast Tumor Model

Notch pathway signaling plays a fundamental role in normal biological processes and is frequently deregulated in many cancers. Although several hypotheses regarding cancer subpopulations most likely to respond to therapies targeting the Notch pathway have been proposed, clinical utility of these predictive markers has not been shown. To understand the molecular basis of gamma-secretase inhibitor (GSI) sensitivity in breast cancer, we undertook an unbiased, de novo responder identification study using a novel genetically engineered in vivo breast cancer model. We show that tumors arising from this model are heterogeneous on the levels of gene expression, histopathology, growth rate, expression of Notch pathway markers, and response to GSI treatment. In addition, GSI treatment of this model was associated with inhibition of Hes1 and proliferation markers, indicating that GSI treatment inhibits Notch signaling. We then identified a pretreatment gene expression signature comprising 768 genes that is significantly associated with in vivo GSI efficacy across 99 tumor lines. Pathway analysis showed that the GSI responder signature is enriched for Notch pathway components and inflammation/immune-related genes. These data show the power of this novel in vivo model system for the discovery of biomarkers predictive of response to targeted therapies, and provide a basis for the identification of human breast cancers most likely to be sensitive to GSI treatment.

Relationship of hypoxia signature with variant subgroup of clear cell renal cell carcinoma (ccRCC) and its association with clinical activity on tivozanib hydrochloride.

361 Background: TIVO-1, a randomized Phase III trial in first-line targeted therapy for patients (pts) with ccRCC, demonstrated significant improvement in progression-free survival (PFS) in pts receiving tivozanib hydrochloride (T) vs. sorafenib (S) (11.9 vs. 9.1 months [12.7 vs. 9.1 in treatment-naïve pts]). To further characterize molecular ccRCC subtypes and assess relationships between subtypes and vascular endothelial growth factor tyrosine kinase inhibitor activity, we characterized available molecularly annotated datasets from TIVO-1. METHODS Tumor subtypes were established using hierarchical clustering and evaluated in two microarray ccRCC datasets using gene set enrichment analysis with 51 signatures representing a set of molecular phenotypes. A 9-gene signature comprising genes associated with hypoxia-inducible factor (HIF) transcription was quantified by RT-PCR on all available (69/517) formalin-fixed, paraffin-embedded material from patients using a predefined classifier score and cutoff. RESULTS Hierachical clustering generated 3 distinct tumor classes. Molecular clusters defining HIF gene expression (low), endothelial cell content (low), extracellular matrix (low), proliferation (high) epithelial cell phenotype (high), and metabolism (high) were differentially expressed in cluster 3 tumors, which represented approximately 15% of the populations. Based on predefined analysis, the hypoxia signature was significantly associated with better PFS on T using a previously established classifier (Table). The hypoxia signature was not significantly associated with PFS on S. There was no significant correlation to single largest diameter for either agent. CONCLUSIONS A novel molecular subtype of ccRCC is characterized by a distinct molecular profile and can be classified by a low hypoxia signature. This hypoxia gene signature may help identify T responders. This signature is seen in subsets of other solid tumors supporting the broad exploration of this candidate T response biomarker. CLINICAL TRIAL INFORMATION NCT01030783. [Table: see text].

Abstract A5: Tivozanib, a selective VEGFR TKI, potently blocks angiogenesis and growth in tumors that express a high level of VEGF-C and are refractory to VEGF-A blockade.

Background: Scientific understanding of the role of VEGF-A in tumor angiogenesis has led to the development of antiangiogenic therapies, such as bevacizumab, that selectively target VEGF-A. However, clinical trials across multiple cancer types have resulted in limited positive outcomes. VEGF-C is thought to be a potent lymphangiogenic growth factor and plays a role in tumor angiogenesis through VEGFR3; it has also been shown to bind to VEGFR2, which is important in tumor angiogenesis. Nevertheless, a direct role of VEGF-C in driving tumor angiogenesis has not been established. To explore the potential of VEGF-C as a driver of tumor angiogenesis and its implication in developing antiangiogenic therapies, we assessed the activity of tivozanib, a potent and selective TKI for VEGFR1, 2 and 3, and a VEGF-A targeted antibody in animal tumor models that exhibit distinct VEGF-C and VEGF-A expression. Method: A total of 107 independently derived murine breast tumors were expanded in vivo to establish population-based primary tumor models. The tumors were characterized for their angiogenesis phenotypes, including expression of angiogenic factors, microvasculature (demonstrated by quantitative CD31 IHC analysis), and myeloid infiltration, a previously identified mediator of tivozanib resistance. To examine the role of VEGF-C vs. VEGF-A in tumor angiogenesis and in affecting response to anti-VEGF therapies, a panel of representative tumors were tested with the cross species anti-VEGF-A antibody B20–4.1 and tivozanib. Tivozanib was also compared with B20–4.1 in blocking VEGF-C stimulated VEGFR2 or VEGFR3 activation in endothelial cells, and in VEGF-C stimulated in vivo angiogenesis in matrigel assay. Results: Three distinct subtypes of tumors were identified: those that expressed high level of VEGF-A and low VEGF-C, or high VEGF-C and low VEGF-A, or both high VEGF-A and high VEGF-C. These tumors exhibited characteristic VEGF driven microvasculature. When treated with systemic administration of B20–4.1 or tivozanib, the tumors expressing high VEGF-A showed comparable response to both agents, while the tumors expressing high VEGF-C but low VEGF-A only responded to tivozanib. In tumors that express both high VEGF-A and high VEGF-C, B20–4.1 treatment resulted in only modest response, while tivozanib completely blocked tumor progression accompanied by vast central necrosis. Pharmacodynamic analysis in representative tumors revealed tumor death associated with progressive hypoxia in tivozanib-treated tumors, consistent with antiangiogenic mechanism of action. Consistent with these findings, tivozanib, but not B20–4.1, potently blocked VEGF-C stimulated VEGFR2 and VEGFR3 activation in endothelial cells, and effectively blocked VEGF-C induced in vivo angiogenesis in a matrigel assay. Conclusion: These data suggest that VEGF-C can play a direct role in tumor angiogenesis, and that tumor expressing high level of VEGF-C are sensitive to VEGFR TKI tivozanib but are refractory to VEGF-A targeted antibodies. These findings provides further scientific evidence that pan-VEGFR TKIs may have broader activity than agents that selectively target VEGF-A. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A5.

Abstract 1727: Preclinical evaluation of pan-VEGFR inhibitor tivozanib in combination with capecitabine, sirolimus or erlotinib in genetically engineered tumor models support combinations based on complementary mechanisms

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Tivozanib (AV-951) is a highly potent and selective VEGFR TKI that has been shown to be active in a phase 2 clinical trial in RCC, and importantly, to be combinable in phase 1b trials with temsirolimus, paclitaxel and Folfox. However, given the widely mixed clinical experience in combining anti-VEGF agents with targeted therapies or chemotherapies, it is critical to preclinically identify and evaluate optimal combinations using animal models that are relevant to human cancer and tailored to reflect clinical opportunities. To investigate tivozanib combinations with capecitabine or sirolimus, we exploited a population based murine breast tumor model, in which, multiple primary tumors generated genetically were expanded in vivo, characterized and then stratified to represent responders or non-responders to each single agent. To ask mechanistic questions and elucidate potential synergies, in addition to pharmacological assessment, pharmacodynamic analyses were performed using histopathology and molecular markers. Tivozanib combination with capecitabine was evaluated in tumors that exhibited limited response to capecitabine single agent at clinically relevant dose and showed insignificant tumor inhibition by tivozanib alone. Combination of the two agents resulted in tumor regression accompanied by massive necrosis. Further, this substantially increased efficacy could be achieved with reduced doses of either agent, indicating synergistic mechanisms. In tumors where sirolimus alone blocked tumor progression without efficient tumor killing, adding tivozanib resulted in rapid and near complete tumor reduction. Importantly, while drug resistance emerged in tumors received either agents, it is not observed in the combination therapy, with evidence of both antiangiogenic and antitumor effects. In an acquired tivozanib resistance model in which either agent alone induced only partial growth inhibition, the combination resulted in tumor stasis. We investigated tivozanib combination with erlotinib in lung tumors that acquired the EGFR TKI resistance mutation L858R/T790M spontaneously. In this study, tivozanib elicited antitumor activity but failed to block tumor progression. When combined with erlotinib, rapid tumor regression was achieved. Continued treatment resulted in prolonged total tumor suppression, evident by residual lesions upon necropsy. These preclinical studies provided an opportunity to model clinical combinations in a context where the mechanistic contribution of each agent could be characterized. Tivozanib combined with each of the three agents, capecitabine, sirolimus or erlotinib demonstrated mechanistically complementary antitumor effect that resulted in substantially improved activity, providing support for such clinical combinations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1727. doi:10.1158/1538-7445.AM2011-1727

Abstract A255: Activity of VEGFR inhibitor tivozanib as a single agent or in combination with EGFR inhibitor erlotinib in engineered lung adenocarcinoma models

Tivozanib (AV‐951 or KRN951) is an ATP competitive small molecule VEGFR antagonist that exhibits picomolar inhibitory activity against all three VEGF receptors. It has demonstrated robust clinical activity in renal cell carcinoma and is currently being evaluated in multiple clinical studies including a monotherapy phase 1b/2a trial in NSCLC. NSCLC is a highly heterogeneous disease, in which tumor histology and genetics have defined sensitivities and settings to targeted therapeutics. To model and explore potential clinical settings, we created genetically engineered lung adenocarcinoma models driven by EGFRL858R, EGFRL858R/T790M or KRAS G12V, propagated these models in vivo both subcutaneously and orthotopically and evaluated tumor inhibitory activity of tivozanib as a single agent or in combination with EGFR inhibitor erlotinib. To further explore activity of metastatic lesions to the lung, we also evaluated the activity of tivozanib using an engineered breast tumor metastatic model. Treatment of subcutaneous lung KRAS tumors or lung EGFRL858R/T790M tumors with tivozanib alone resulted in complete tumor growth inhibition. Histological analysis revealed substantial central necrosis with proliferating (Ki67+) tumor cells present only at the tumor periphery, typical of an anti‐angiogenesis mechanism. When tivozanib was given to treat lung EGFRL858R+T790M tumors in an orthotopic setting in the lungs after tumors were well established, significant antiangiogenic and antitumor effects, as demonstrated by histology and extended survival period, were observed. However, the necrotic lung lesions showed no sign of resorption or elimination after 6 weeks of treatment. Similar results were also observed in a HER2 driven breast tumor lung metastatic model. This prompted us to further explore the activity of tivozanib in an orthotopic lung tumor minimal disease model. Tivozanib treatment in this setting did not alter tumor lesion numbers but effectively blocked the progression of micro pre‐angiogenic lesions. Finally, we explored the combined activity of tivozanib and erlotinib in a model constructed to reflect the emergence of erlotinib resistance by implanting tumor material comprising both EGFRL858R and EGFRL858R/T790M tumor cells. In this setting, the combination of tivozanib and erlotinib resulted in significantly greater control of tumor progression than erlotinib alone. Our results suggest that tivozanib may have activity as a single agent in lung tumors with KRAS or EGFR L858R/T790M mutations and that combinations with other targeted lung cancer drugs such as erlotinib may provide meaningful clinical benefit. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A255.

Abstract B143: Distinct antitumor mechanisms of VEGFR inhibitor tivozanib (AV‐951) and mTOR inhibitor rapamycin support rational combination for solid tumors to overcome drug resistance

The approval of an increasing number of molecularly targeted therapies in cancer creates an opportunity to contemplate combinations of such agents. This is particularly important because single agents typically exhibit limited activity and very often drug resistance in clinic. A current challenge is to identify appropriate rational combinations that combine distinct mechanisms to elicit maximal anti‐tumor activity and to overcome drug resistance. Tivozanib (formerly AV‐951 or KRN951) is a small molecule VEGFR inhibitor that has been shown to be active in a phase 2 clinical trial in RCC. Tivozanib inhibits VEGFR 1, 2 and 3 activity at picomolar concentrations (IC50 of 0.21, 0.16 and 0.24 nM respectively), Rapamycin targets mTOR, a protein involved in integrating nutrient availability with cellular functions, including proliferation. mTOR is also known to promote hypoxia inducible factor (HIF1) activity, which in turn drives angiogenesis, such that in some settings, rapamycin is thought to elicit anti‐tumor activity through an anti‐angiogenic mechanism. We studied the activity of tivozanib and rapamycin as single agents and in combination in a genetically engineered HER2 driven breast adenocarcinoma model where multiple primary tumors have been expended in vivo for mechanisms of response and drug resistance biomarker exploration. These tumors exhibit significant variation in response to tivozanib. In one group of tumors, tivozanib as a single agent induced complete tumor growth inhibition persisting for at least 6 weeks. Rapamycin at a clinically relevant dose also induced complete tumor growth inhibition. However, histological analysis after short (5 days) or longer (35 days) term treatment revealed distinct mechanisms of action, i.e., anti‐VEGF for tivozanib but direct antiproliferation for rapamycin. Combination of tivozanib and rapamycin resulted in substantially greater overall tumor reduction with evidence of both anti‐angiogenic and anti‐proliferation activities. Importantly, after 6 weeks of treatment, regions of drug resistance start to emerge in either rapamycin or tivozanib monotherapy tumors. However no evidence of drug resistance was observed with the tivozanib/rapamycin combination. To further explore the combination activity in resistant tumors, we established a resistant sub‐line by long term tivozanib treatment. Both tivozanib and rapamycin as single agents resulted in only partial tumor growth inhibition. The combination treatment induced complete growth inhibition with evidence of both angiogenesis inhibition and direct antitumor effect. The distinct mechanisms of action of these two agents, along with the apparent suppression of drug resistance suggest that tivozanib and mTOR inhibitors may represent an attractive rational combination treatment for solid malignancies. At this point, an on‐going phase 1B trial in RCC has shown that tivozanib can be combined with temsirolimus at full doses of both agents. An all oral combination of tivozanib and everolimus, an oral mTOR inhibitor, is also being developed for RCC and other tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B143.

Abstract A22: Response to the triple VEGFR inhibitor tivozanib (AV‐951) in KRAS and EGFR driven lung tumors developed in chimeric mouse models

Lung cancer is one of the most prevalent and deadly malignancies in the world, with more than 213,000 new cases diagnosed each year in the US and a five year survival rate of 15%. Activating mutations in a number of key signaling genes including KRAS, EGFR, and HER2 have been identified. Mutations in EGFR are highly correlated with sensitivity to small molecule EGFR inhibitors, although resistance tends to develop within a short period of time after initiation of treatment. Targeted inhibition of KRAS has not been successful so far, which in turn fueled efforts to identify tractable alternative therapeutic points of intervention. Because all solid tumors are thought to require neo‐vascularization, pharmacologic angiogenesis inhibition may represent an attractive therapeutic strategy for these challenging tumor settings. One attractive anti‐angiogenesis agent is the ATP competitive small molecule VEGFR inhibitor tivozanib (AV‐951). tivozanib exhibits picomolar inhibitory activity against all three VEGF receptors, a multi‐day T1/2 in humans, and demonstrates robust clinical activity demonstrated in renal cell carcinoma, the signal tumor type for VEGF pathway inhibition. In an effort to recapitulate the stochastic nature of human cancer in genetically engineered mouse models and to maximize their use in preclinical settings, we developed a mouse tumor model strategy involving stepwise genetic manipulation of embryonic stem (ES) cells and chimera formation to enable direct tumor induction in tissues containing both normal and engineered cells. An allelic series of lung cancer models containing EGFR, KRAS, or HER2 oncogenes demonstrated that resultant adenocarcinomas arising within normal lung tissue exhibited features of advanced malignancies. In this study, we tested the response of KRAS and EGFR driven lung tumors to the VEGFR inhibitor tivozanib in tumor bearing chimeric mice identified by bioluminescent imaging. We demonstrated that tivozanib treatment conferred significant survival benefit to the tumor bearing mice. Consistent with previous reports that tumors become more dependent on neo‐angiogenesis for survival and proliferation, we observed sensitivity to tivozanib in advanced adenocarcinomas but not hyperplasia or small adenomas. In addition, we found that upon discontinuation of tivozanib treatment, tumors quickly grew back and became less sensitive to further tivozanib treatment. These results indicate that lung cancer patients could potential benefit from anti‐angiogenesis therapy, but sustained inhibition of all VEGF receptors may be important to prevent development of resistance. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A22.