William M. Rideout

Spontaneous Genomic Alterations in a Chimeric Model of Colorectal Cancer Enable Metastasis and Guide Effective Combinatorial Therapy

Colon cancer is the second most common cause of cancer mortality in the Western world with metastasis commonly present at the time of diagnosis. Screening for propagation and metastatic behavior in a novel chimeric-mouse colon cancer model, driven by mutant p53 and β-Catenin, led to the identification of a unique, invasive adenocarcinoma. Comparison of the genome of this tumor, CB42, with genomes from non-propagating tumors by array CGH and sequencing revealed an amplicon on chromosome five containing CDK6 and CDK14, and a KRAS mutation, respectively. Single agent small molecule inhibition of either CDK6 or MEK, a kinase downstream of KRAS, led to tumor growth inhibition in vivo whereas combination therapy not only led to regression of the subcutaneous tumors, but also near complete inhibition of lung metastasis; thus, genomic analysis of this tumor led to effective, individualized treatment.

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

Genetically Engineered Mouse Models of Human Cancer for Drug Discovery and Development

Animal models for cancer research, although not perfect, have traditionally been crucial to the drug discovery and development process. Recent advances in genetically modified mice have created opportunities to model many aspects of cancer biology, which established xenograft models ignore. Selection of the right model will be of increasing importance in the search for efficacious human therapeutics. These improved mouse models also permit a new concept of preclinical trials in which the efficacy of novel drugs can be tested against spontaneous tumors.

Abstract 2737: Spontaneous genomic alterations identified in a chimeric model of colorectal cancer guide effective combinatorial therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Colon cancer is the second most common cause of cancer mortality in the western world. We generated a mouse model of colon cancer in which a conditional mutation of p53 is combined with inducible delta131-beta-Catenin over-expression in the intestinal epithelium. After 4-12 months of beta-Catenin induction, small adenomas and large solitary nodules of invasive adenocarcinomas developed in either the upper or lower intestinal tract, while the remainder of the intestine appeared normal. Molecular analysis revealed that EGFR and MET were expressed in advanced adenocarcinomas but not early stage adenomas. Although propagating mouse colon tumors has been very challenging, we nevertheless successfully established one tumor line through direct in vivo propagation in the kidney capsule. This tumor line, CB42, grew aggressively and metastasized to ancillary lymph nodes as well as the lung in a subcutaneous metastasis assay and grew in the liver when seeded there by intra splenic injection. Treatment with a MET inhibitor Crizotinib, had no effect on either primary tumor growth, or metastasis. By comparing the genome of CB42 with other colon tumors that did not propagate, we discovered an amplicon on chromosome five containing genes that correlated with propagation and metastasis. In addition, whole genome sequencing revealed that CB42 harbored a mutation in KRAS. Results from combination therapies against key genetic alterations will be presented. Citation Format: Yinghui Zhou, William M. Rideout, Angela Bressel, Sireesha Yalavarthi, Tong Zi, Anthony Monti, Steve Bottega, Bin Feng, M. Isabel Chiu, Marcus Bosenberg, Joerg Heyer. Spontaneous genomic alterations identified in a chimeric model of colorectal cancer guide effective combinatorial therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2737. doi:10.1158/1538-7445.AM2013-2737

Abstract 4358: Monoclonal antibodies to Notch receptors may enable targeting of tumor autonomous and tumor micro-environmental processes

AVEO has developed a series of inducible mouse models of cancer which, through the preservation of critical tumor/stromal interactions, facilitate identification of cell-surface and secreted proteins that represent viable targets for therapeutic antibodies and other biologics. Functional genetic screens performed in vivo in these models identified the Notch pathway as a critical regulator of tumor maintenance. This finding is consistent with emerging evidence that activation of Notch signaling via receptor point mutation, receptor amplification, and elevated receptor and ligand expression, plays a key role in various human cancers. Moreover, the Notch pathway controls diverse aspects of tumorigenesis and tumor maintenance, regulating tumor autonomous processes and interactions with the microenvironment, including angiogenesis. To further understand the role of the Notch pathway in tumor maintenance, and to assess the therapeutic potential of targeting the Notch pathway in cancer, we have generated monoclonal antibodies that inhibit various Notch receptors. Characterization of monoclonal antibodies targeting Notch1 or Notch3 through cell-based and biochemical studies demonstrated that these antibodies bound with high affinity and high specificity to the ligand binding domains of the Notch receptors, prevented ligand mediated activation of the target receptor, and specifically repressed Notch-dependent signaling with high potency. Effective inhibition of functional angiogenesis was observed upon anti-Notch1 antibody treatment in both in vitro and in vivo models. Significantly, specific inhibition of Notch1 by this antibody did not result in the dose-limiting gut toxicity observed with pan-Notch inhibitors such as gamma-secretase inhibitors. Humanized versions of the Notch1 monoclonal antibodies have been generated and characterized. Inhibition of tumor growth by the Notch3 monoclonal antibody was effected through tumor cell autonomous mechanisms. To identify tumors that are dependent upon tumor autonomous Notch signaling, gene expression profiles were correlated with Notch pathway dependence in human cancer cell lines. Expression of specific downstream targets was highly correlated with sensitivity of human cancer cell lines to inhibition of ligand-dependent Notch signal. This biomarker of Notch pathway dependence successfully predicted that a subset of Kras mutant pancreatic and colon cancer cell lines would be highly sensitive to Notch pathway inhibition. Moreover, identification of Notch1 and Notch3-specific target genes further enables selection of tumors that will respond to monoclonal antibodies specifically targeting one or other, or both of these receptors. 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 4358. doi:10.1158/1538-7445.AM2011-4358

Abstract 1378: Preclinical efficacy of the triple VEGFR inhibitor tivozanib (AV-951) in chimeric breast and lung tumor models

All solid tumors are thought to require neo-vascularization. Therefore pharmacologic inhibition of angiogenesis may represent an important component for treating many therapeutically challenging tumor types. 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 in renal cell carcinoma, the signal tumor type for VEGF pathway inhibition. To test preclinical efficacy of tivozanib in other cancer types, we chose primary mouse tumor models due to their ability to capture the complex heterotypic interactions between tumor cells and the microenvironments. Our mouse tumor model strategy involves stepwise genetic manipulation of embryonic stem (ES) cells and chimera formation to enable direct tumor induction in tissues containing both normal and engineered cells. A HER2 driven breast model as well as an allelic series of lung cancer models containing EGFR, KRAS, or HER2 oncogenes demonstrated that resultant adenocarcinomas arose within surrounding normal tissue and exhibited features of advanced malignancies. In this study, we tested the response of HER2 driven breast tumors as well as KRAS and EGFR driven lung tumors to the VEGFR inhibitor tivozanib in tumor bearing chimeric mice. We observed that although tivozanib treatment conferred significant survival benefit to the tumor bearing mice in all three models, it is not able to eradicate all tumor cells. Response to tivozanib in the breast model is much more heterogeneous than in the lung models, with some tumors exhibiting significant regression while others showing progression on treatment, either initially or after a period of response. In contrast, lung tumors showed more uniform response to tivozanib treatment initially, but tumors quickly grew back upon discontinuation of tivozanib treatment. These results indicate that both breast and lung cancer patients could potentially benefit from sustained anti-angiogenesis therapy, and combination of anti-angiogenesis with tumor targeting agents may be required for enduring efficacy. 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 1378.

Abstract 5170: Monoclonal antibodies to Notch receptors inhibit tumor maintenance

AVEO has developed a series of inducible mouse models of cancer which, through the preservation of critical tumor/stromal interactions, facilitate identification of cell-surface and secreted proteins that represent viable targets for therapeutic antibodies and other biologics. Functional genetic screens performed in vivo in these models identified the Notch pathway as a critical regulator of tumor maintenance. This finding is consistent with emerging evidence that activation of Notch signaling via receptor point mutation, receptor amplification, and elevated receptor and ligand expression, plays a key role in various human cancers. Moreover, the Notch pathway controls diverse aspects of tumorigenesis and tumor maintenance, regulating tumor autonomous processes and interactions with the microenvironment, including angiogenesis. To further understand the role of the Notch pathway in tumor maintenance, and to assess the therapeutic potential of targeting the Notch pathway in cancer, we have generated monoclonal antibodies that inhibit various Notch receptors. Characterization of a Notch1-specific monoclonal antibody through cell-based and biochemical studies demonstrated that the antibody bound to the Notch1 ligand binding domain with high affinity, prevented ligand mediated activation of the receptor, and specifically repressed Notch1-dependent signaling with high potency. Mice treated with the Notch1 antibody exhibited altered T cell fate specification and loss of hair pigmentation, as expected for loss of Notch1 function. Significantly, specific inhibition of Notch1 by this antibody did not result in the dose-limiting gut toxicity observed with pan-Notch inhibitors such as gamma-secretase inhibitors. However, strong inhibition of functional angiogenesis was observed upon antibody treatment in both in vitro and in vivo models. The apparent lack of toxicity of this antibody in mouse models suggests that inhibition of Notch1 could be effectively combined with other therapies to enhance anti-angiogenic effects, or to overcome resistance to VEGF/VEGFR inhibition. To identify tumors that are dependent upon tumor autonomous Notch signaling the expression of Notch pathway related genes was correlated with Notch pathway dependence in human cancer cell lines. Active Notch signaling alone did not predict dependence upon Notch, but expression of a single Notch target gene, HeyL, was highly correlated with sensitivity of human cancer cell lines to inhibition of ligand-dependent Notch signal. The utility of this biomarker was further confirmed by the identification of a subset of Kras mutant pancreatic and colon cancer cell lines that were subsequently demonstrated to be highly sensitive to Notch pathway inhibition. Hence, HeyL expression may serve as a predictive biomarker of Notch-dependent tumors. These data support the clinical development of AVEO9s humanized Notch antibodies for the treatment of human cancer. 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 5170.