Cecile Chartier

Colorectal Cancer Stem Cells Are Enriched in Xenogeneic Tumors Following Chemotherapy

Background Patients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells, termed cancer stem cells (CSC), appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed, therefore, that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA+CD44+ phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents, then residual tumors might be expected to contain a higher frequency of CoCSC. Methods and Findings Xenogeneic tumors initiated with CoCSC were allowed to reach ∼400 mm3, at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover, the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones, we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent. Conclusions CoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC, major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes, thereby suggesting avenues for improving cancer therapy.

Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors

The Wnt/β-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several coreceptors, has long been implicated in cancer. Here we demonstrate a therapeutic approach to targeting the Wnt pathway with a monoclonal antibody, OMP-18R5. This antibody, initially identified by binding to Frizzled 7, interacts with five Fzd receptors through a conserved epitope within the extracellular domain and blocks canonical Wnt signaling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, this antibody inhibits the growth of a range of tumor types, reduces tumor-initiating cell frequency, and exhibits synergistic activity with standard-of-care chemotherapeutic agents.

Therapeutic targeting of tumor-derived R-spondin attenuates β-catenin signaling and tumorigenesis in multiple cancer types

Deregulation of the β-catenin signaling has long been associated with cancer. Intracellular components of this pathway, including axin, APC, and β-catenin, are frequently mutated in a range of human tumors, but the contribution of specific extracellular ligands that promote cancer development through this signaling axis remains unclear. We conducted a reporter-based screen in a panel of human tumors to identify secreted factors that stimulate β-catenin signaling. Through this screen and further molecular characterization, we found that R-spondin (RSPO) proteins collaborate with Wnt proteins to activate β-catenin. RSPO family members were expressed in several human tumors representing multiple malignancies, including ovarian, pancreatic, colon, breast, and lung cancer. We generated specific monoclonal antibody antagonists of RSPO family members and found that anti-RSPO treatment markedly inhibited tumor growth in human patient-derived tumor xenograft models, either as single agents or in combination with chemotherapy. Furthermore, blocking RSPO signaling reduced the tumorigenicity of cancer cells based on serial transplantation studies. Moreover, gene-expression analyses revealed that anti-RSPO treatment in responsive tumors strongly inhibited β-catenin target genes known to be associated with cancer and normal stem cells. Collectively, our results suggest that the RSPO family is an important stimulator of β-catenin activity in many human tumors and highlight a new effective approach for therapeutically modulating this fundamental signaling axis.

WNT antagonists exhibit unique combinatorial antitumor activity with taxanes by potentiating mitotic cell death

WNT antagonism displays marked synergy with taxane chemotherapy and reverses taxane-induced enrichment of cancer stem cells. The WNT pathway mediates intercellular signaling that regulates cell fate in both normal development and cancer. It is widely appreciated that the WNT pathway is frequently dysregulated in human cancers through a variety of genetic and epigenetic mechanisms. Targets in the WNT pathway are being extensively pursued for the development of new anticancer therapies, and we have advanced two WNT antagonists for clinical development: vantictumab (anti-FZD) and ipafricept (FZD8-Fc). We examined the antitumor efficacy of these WNT antagonists in combination with various chemotherapies in a large set of patient-derived xenograft models. In responsive models, WNT blockade led to profound synergy with taxanes such as paclitaxel, and the combination activity with taxanes was consistently more effective than with other classes of chemotherapy. Taxane monotherapy increased the frequency of cells with active WNT signaling. This selection of WNT-active chemotherapy-resistant tumorigenic cells was prevented by WNT-antagonizing biologics and required sequential dosing of the WNT antagonist followed by the taxane. The WNT antagonists potentiated paclitaxel-mediated mitotic blockade and promoted widespread mitotic cell death. By blocking WNT/β-catenin signaling before mitotic blockade by paclitaxel, we found that this treatment effectively sensitizes cancer stem cells to taxanes. This combination strategy and treatment regimen has been incorporated into ongoing clinical testing for vantictumab and ipafricept.

Abstract 207: Dual targeting of DLL4 and VEGF signaling by a novel bispecific antibody inhibits tumor growth and reduces cancer stem cell frequency

Both Notch/Delta-like ligand 4 (DLL4) and vascular endothelial growth factor (VEGF) pathways play a critical role in angiogenesis and tumor growth. Due to differential regulatory effects of VEGF and DLL4 on the vasculature, inhibition of DLL4 or VEGF inhibits tumor growth by distinct mechanisms: anti-DLL4 treatment induces an abnormal increase of poorly perfused blood vessels, which results in a nonproductive angiogenesis unable to support tumor growth, whereas the anti-VEGF therapy significantly decreases vasculature reducing the blood supply to tumors. We have recently developed a high binding affinity bispecific monoclonal antibody that targets both human DLL4 and human VEGF. In vitro, this antibody exhibited nanomolar affinity to hVEGF and hDLL4, and reduced HUVEC proliferation induced by VEGF. The bispecific antibody demonstrated significant in vivo anti-tumor efficacy in various solid tumors, delayed tumor recurrence following termination of chemotherapy, and decreased the frequency of tumor initiating cells. Analysis of tumor vasculature after treatment with anti-DLL4/VEGF revealed inhibition of vascular gene expression and endothelial cell proliferation, indicating that the anti-VEGF effect on the vasculature is dominant over the anti-DLL4 effect. Notably, at doses where both anti-DLL4 and anti-VEGF alone produced suboptimal anti-tumor effect, dual targeting resulted in additive tumor growth inhibition. These results indicate that our bispecific anti-DLL4/VEGF is broadly efficacious and may be useful for treatment of a variety of solid tumors. Citation Format: Wan-Ching Yen, Fumiko Axelrod, Chris Bond, Jennifer Cain, Cecile Chartier, Marcus Fischer, Shirley Ma, Rene Meisner, Janak Raval, Jalpa Shah, Austin Gurney, John Lewicki, Timothy Hoey. Dual targeting of DLL4 and VEGF signaling by a novel bispecific antibody inhibits tumor growth and reduces cancer stem cell frequency. [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 207. doi:10.1158/1538-7445.AM2014-207

Abstract 2322: The Hippo signaling pathway mediates BMP inhibition of cancer stem cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Multiple developmental signaling pathways cooperatively regulate cancer stem cell (CSC) function. Among them, the BMP pathway has been implicated in brain CSC tumorigenicity. To examine whether BMP is a general regulator of CSCs across diverse organ systems we utilized lentivirus-mediated BMP4 expression in patient-derived xenograft models in NOD/SCID mice. The prevention of tumor growth was observed in breast, colon, ovarian, and melanoma models. Similarly, adenovirus-mediated systemic delivery of BMP4 inhibited tumor growth, dramatically affected CSC-specific cell surface markers and decreased CSC frequency. Microarray analysis of BMP4-treated tumors showed that a set of genes encoding for components of the actin cytoskeleton was significantly enriched amongst regulated genes and identified Hippo pathway genes as potential mediators of the BMP4 anti-tumor response with up-regulation of SAV1 and LATS2 and down-regulation of BIRC5. Hippo signaling regulates organ size, stem cell biology and cancer through a protein kinase complex including MST1/2, SAV1, and LATS1/2, which phosphorylates the downstream nuclear effector YAP/TAZ. Phosphorylated YAP/TAZ is unable to induce the expression of cell proliferation and anti-apoptotic genes through interaction with transcriptional co-activators such as TEAD. The Hippo pathway, known to cross-talk with BMP signaling at multiple levels, recently emerged as a cellular sensor of the physical environment through the actin cytoskeleton. We thus asked whether modulating Hippo signaling affected tumor growth. YAP1 overexpression resulted in tumor growth decrease. A deletion variant of YAP, YAP1-291, possessing a deletion of the transactivation domain has been shown to act as a dominant-negative through binding to TEAD. Intriguingly YAP1-291 was also sufficient to strongly attenuate growth of diverse BMP4-sensitive and BMP4-insensitive tumor models suggesting that transactivation through TEAD is not the only route by which YAP modulates tumor growth. YAP anti-proliferative properties were reported before. In order to reconcile the dual function of YAP in cancer, we propose that the transcriptional co-activators TEAD-mediated oncogenic function may be counter-balanced by its potentiation of SMAD signaling in response to BMP. Citation Format: Cecile Chartier, Shirley Ma, Dragana Antic, Elsa Quintana, Janak Raval, Min Wang, Cristina Dee-Hoskins, Ann Kapoun, Tim Hoey, John Lewicki, Austin Gurney. The Hippo signaling pathway mediates BMP inhibition of cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2322. doi:10.1158/1538-7445.AM2015-2322

Abstract 1764: Inhibition of R-spondin (RSPO) signaling reduces the growth of multiple human tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Deregulation of the canonical Wnt/beta-catenin signaling pathway has long been associated with cancer. Intracellular components of this pathway, including Axin, APC, and beta-catenin are frequently mutated in a range of human tumors. The identity of specific extracellular ligands that contribute to human cancer development through this signaling axis has remained unclear. Molecular characterization of the secreted beta-catenin signaling activities produced by minimally passaged human tumor xenograft models identified RSPO family members produced by multiple tumor types including ovarian, pancreatic, colon, breast and non-small cell lung cancer. In human tumor xenograft models that had RSPO overexpression, in some instances due to genomic translocation, anti-RSPO treatment markedly inhibited tumor growth. In addition, striking combination activity with standard of care chemotherapy agents resulted in regression of established tumors. These results highlight the potential for therapeutic intervention with this newly appreciated signaling axis. Citation Format: Austin Gurney, Fumiko Axelrod, Chris Bond, Jennifer Cain, Cecile Chartier, Marcus Fischer, May Ji, Chris Murriel, Janak Raval, Jalpa Shah, Min Wang, Wan-Ching Yen, Ann Kapoun, John Lewicki, Timothy Hoey. Inhibition of R-spondin (RSPO) signaling reduces the growth of multiple human tumors. [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 1764. doi:10.1158/1538-7445.AM2014-1764

Abstract 4330: In vivo evaluation of anti-tumor activity by an anti-VEGF and anti-DLL4 bispecific antibody in a humanized model of skin graft.

Both Notch/Delta-like ligand 4 (DLL4) and vascular endothelial growth factor (VEGF) pathways play a critical role in angiogenesis and tumor growth. Due to differential regulatory effects of VEGF and DLL4 on the vasculature, blockage of DLL4 or VEGF inhibits tumor growth by distinct mechanisms: anti-DLL4 treatment induces an abnormal increase of poorly perfused blood vessels, which results in a nonproductive angiogenesis unable to support tumor growth, whereas the anti-VEGF therapy significantly decreases vasculature reducing the blood supply to tumors. In our study, we have developed a bispecific monoclonal antibody that targets both human DLL4 (hDLL4) and human VEGF (hVEGF). In vitro, this antibody demonstrated nanomolar affinity to hVEGF and hDLL4, and reduced HUVEC proliferation induced by VEGF (EC50 0.86 ug/ml). To test the activity of this bispecific antibody in vivo, we developed a human skin graft model in NOD/SCID mice, and implanted human tumor specimen-derived colon cancer cells intradermally into these skin transplants. The tumor model was selected based on its sensitivity to both the anti-human DLL4 antibody, OMP-21M18, and to the human VEGF inhibitor bevacizumab. The skin graft model provides a suitable human microenvironment for evaluating anti-tumor efficacy and anti-angiogenesis of the bispecific antibody directed against the human component of DLL4 and VEGF and allows a comparison to OMP-21M18 and also to bevacizumab. Each of these treatments was administered to mice intraperitoneally at a dose of 25 mg/Kg weekly. The bispecific antibody caused a significant inhibition of tumor growth (87% TGI) compared to control antibody (p=0.00001), and this effect was superior to either OMP-21M18 (45% TGI) or bevacizumab (70%TGI). The inhibition of tumor growth by the bispecific antibody was consistently associated with increased blood vessels, up-regulated VEGFA and VEGFR2, and enhanced hypoxia and these effects were more pronounced compared to OMP-21M18. As expected, in this model bevacizumab caused a significant decrease of blood vessels, down-regulated VEGFR2, and increased hypoxia. In separate experiments with mice bearing subcutaneous human colon tumors, the bispecific antibody delayed tumor recurrence following termination of chemotherapy and impacted tumorigenicity by decreasing the frequency of tumor initiating cells. These results suggest that our bispecific anti-DLL4 and anti-VEGF antibody is a potential candidate in the treatment of tumors driven by both VEGF and Notch/DLL4 signaling pathways. Citation Format: Lucia Beviglia, Pete Yeung, Wang-Ching Yen, Belinda Cancilla, Sato Aaron, Chris Bond, Janak Raval, Fumiko Axelrod, Cecile Chartier, Shirley Ma, Austin Gurney, John Lewicki, Ann M. Kapoun, Timothy Hoey. In vivo evaluation of anti-tumor activity by an anti-VEGF and anti-DLL4 bispecific antibody in a humanized model of skin graft. [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 4330. doi:10.1158/1538-7445.AM2013-4330