Several components of the Wnt signaling cascade have been shown to function either as tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis and the need for further investigation of Wnt signaling components as potential targets for cancer therapy. Here, using expression profiling analysis as well as in vitro and in vivo functional studies, we show that the Wnt pathway component BCL9 is a novel oncogene that is aberrantly expressed in human multiple myeloma as well as colon carcinoma. We show that BCL9 enhances β-catenin–mediated transcriptional activity regardless of the mutational status of the Wnt signaling components and increases cell proliferation, migration, invasion, and the metastatic potential of tumor cells by promoting loss of epithelial and gain of mesenchymal-like phenotype. Most importantly, BCL9 knockdown significantly increased the survival of xenograft mouse models of cancer by reducing tumor load, metastasis, and host angiogenesis through down-regulation of c-Myc, cyclin D1, CD44, and vascular endothelial growth factor expression by tumor cells. Together, these findings suggest that deregulation of BCL9 is an important contributing factor to tumor progression. The pleiotropic roles of BCL9 reported in this study underscore its value as a drug target for therapeutic intervention in several malignancies associated with aberrant Wnt signaling.

Multiple myeloma (MM) is a cancer of plasma cells with complex molecular characteristics that evolves from monoclonal gammopathy of undetermined significance, a highly prevalent premalignant condition. MM is the second most frequent hematologic cancer in the United States, and it remains incurable, thereby highlighting the need for new therapeutic approaches, particularly those targeting common molecular pathways involved in disease progression and maintenance, shared across different MM subtypes. Here we report that Wnt/␤-catenin is one such pathway. We document the involvement of ␤-catenin in cell-cycle regulation, proliferation, and invasion contributing to enhanced proliferative and metastatic properties of MM. The pleiotropic effects of ␤-catenin in MM correlate with its transcriptional function, and we demonstrate regulation of a novel target gene, Aurora kinase A, implicating ␤-catenin in G2/M regulation. ␤-catenin and Aurora kinase A are present in most MM but not in normal plasma cells and are expressed in a pattern that parallels progression from monoclonal gammopathy of undetermined significance to MM. Our data provide evidence for a novel functional link between ␤-catenin and Aurora kinase A, underscoring a critical role of these pathways in MM disease progression. (Blood. 2009;114:2699-2708) IntroductionMultiple myeloma (MM) is a neoplasm of plasma cells that infiltrates the bone marrow (BM). Despite recent advances in its treatment, it remains incurable, with a median survival of 6 years. 1 MM accounts for more than 10% of all hematologic malignancies and is the second most frequent hematologic cancer in the United States. It is typically preceded by an age-progressive condition termed monoclonal gammopathy of undetermined significance (MGUS), which is present in 1% to 10% of adults older than 25 years of age and progresses to malignant MM at a rate of 0.5% to 3% annually. 1,2 This disease is characterized by frequent chromosomal aberrations and mutations in several oncogenes and tumor-suppressor genes. 3,4 The Wnt/␤-catenin pathway is significant in cancer development because numerous human malignancies such as colorectal, hepatocellular, and breast cancer harbor activating mutations in critical components of this pathway. 5,6 Normally, the Wnt signaling pathway is active during embryogenesis, hematopoietic stem cell growth, cell differentiation, and tissue development. ␤-catenin, a central effector of the Wnt pathway, is involved in both nuclear and cytoplasmic functions. 7,8 In the absence of Wnt ligands, ␤-catenin is targeted by a complex consisting of adenomatous polyposis coli, axin, glycogen synthase kinase-3␤, and casein kinase 1␣ that phosphorylate and mark it for degradation by the ubiquitinproteasome pathway. 9,10 Upon Wnt stimulation, however, the kinase complex is dissociated, and ␤-catenin is not targeted for destruction. The active form of ␤-catenin translocates to the nucleus and, in association with lymphoid enhancer factor (LEF)/Tcell factor (TCF) proteins, activates transcription...

The antiapoptotic Bcl-2 family member Bfl-1 is up-regulated in many human tumors in which nuclear factor-kappaB (NF-kappaB) is implicated and contributes significantly to tumor cell survival and chemoresistance. We previously found that NF-kappaB induces transcription of bfl-1 and that the Bfl-1 protein is also regulated by ubiquitin-mediated proteasomal degradation. However, the role that dysregulation of Bfl-1 turnover plays in cancer is not known. Here we show that ubiquitination-resistant mutants of Bfl-1 display increased stability and greatly accelerated tumor formation in a mouse model of leukemia/lymphoma. We also show that tyrosine kinase Lck is up-regulated and activated in these tumors and leads to activation of the IkappaB kinase, Akt, and extracellular signal-regulated protein kinase signaling pathways, which are key mediators in cancer. Coexpression of Bfl-1 and constitutively active Lck promoted tumor formation, whereas Lck knockdown in tumor-derived cells suppressed leukemia/lymphomagenesis. These data demonstrate that ubiquitination is a critical tumor suppression mechanism regulating Bfl-1 function and suggest that mutations in bfl-1 or in the signaling pathways that control its ubiquitination may predispose one to cancer. Furthermore, because bfl-1 is up-regulated in many human hematopoietic tumors, this finding suggests that strategies to promote Bfl-1 ubiquitination may improve therapy.

Multiple myeloma is characterized by frequent chromosomal alterations. Deletion of chr 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. Here, we functionally validate the role of the microRNAs-15a/16-1 cluster, centered at the deleted region, as TSGs and delineate their downstream target genes in MM. Using "sponge" lentiviral vectors to competitive stably inhibit mature microRNAs in vitro and in vivo, we have documented enhanced proliferative and invasive capacity of cells with stably inhibition of miR-16. Importantly, miR-16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load and host angiogenesis. Expression profiling analysis of miR-16-deficient cells identified a large number of downstream target genes including FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. We validated designated genes showing binding sites within the conserved 3'-UTR and also within the mRNA coding region as direct miR-16 targets, thus indicating that the miRNAs may have many more targets than anticipated by conventional prediction methods. This loss-of-function system, which mimics the 13q chromosomal deletion, provides a valuable tool to investigate their function in MM pathogenesis and their potential use as therapeutic targets.

<div>Abstract<p>Several components of the Wnt signaling cascade have been shown to function either as tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis and the need for further investigation of Wnt signaling components as potential targets for cancer therapy. Here, using expression profiling analysis as well as <i>in vitro</i> and <i>in vivo</i> functional studies, we show that the Wnt pathway component BCL9 is a novel oncogene that is aberrantly expressed in human multiple myeloma as well as colon carcinoma. We show that BCL9 enhances β-catenin–mediated transcriptional activity regardless of the mutational status of the Wnt signaling components and increases cell proliferation, migration, invasion, and the metastatic potential of tumor cells by promoting loss of epithelial and gain of mesenchymal-like phenotype. Most importantly, BCL9 knockdown significantly increased the survival of xenograft mouse models of cancer by reducing tumor load, metastasis, and host angiogenesis through down-regulation of c-Myc, cyclin D1, CD44, and vascular endothelial growth factor expression by tumor cells. Together, these findings suggest that deregulation of BCL9 is an important contributing factor to tumor progression. The pleiotropic roles of BCL9 reported in this study underscore its value as a drug target for therapeutic intervention in several malignancies associated with aberrant Wnt signaling. [Cancer Res 2009;69(19):7577–86]</p></div>

Supplementary Figures 1-8, Methods from BCL9 Promotes Tumor Progression by Conferring Enhanced Proliferative, Metastatic, and Angiogenic Properties to Cancer Cells

Protein Tyrosine Kinase 7 (PTK7), also known as colon cancer carcinoma kinase 4 (CCK-4), is a highly conserved catalytically inactive tyrosine kinase with inherent signal transduction activity. PTK7 is a member of the Wnt signaling pathway and thought to function in cell proliferation, adhesion, migration and apoptosis. PTK7 has been shown to be highly expressed in certain cancer types including colon, breast, lung, pancreatic, renal and ovarian. Downregulation of PTK7 by shRNA has been shown to reduce tumor growth and metastases development in xenograft mouse models. Furthermore, high PTK7 expression in triple negative breast cancer and NSCLC patients has been linked to poor prognosis. PTK7 therefore appears to be an attractive target for solid cancer therapeutic intervention and has been targeted clinically with an Antibody Drug Conjugate (PF-06647020). However, the protein is also expressed to a lesser degree in some normal adult tissues, particularly in the stromal cells of the ovary, placenta, uterus and lung. We have developed a human/murine cross-reactive anti-PTK7 CAR construct and assessed the ability of allogeneic anti-PTK7 CAR-T cells bearing that construct to target cancer cells and elicit toxicity in mice. These anti-PTK7 CAR-T cells were shown to be efficacious in vitro and in immunocompromised mouse xenograft models of breast, lung, colon, pancreatic and ovarian cancer. However, consistent in all xenograft models studied, a drop in body weight was observed shortly after injection from which all mice rapidly recovered to above baseline. Latent toxicity that was more variable amongst the xenograft experiments was also observed. Understanding and developing appropriate mitigation strategies to address the observed toxicities may be required to develop further a safe and efficacious allogeneic anti-PTK7 CAR-T cell therapy for solid tumor cancers. Citation Format: Katie Levitsky, Zejun Li, Minh Thu Pham, Kelly Maeng, Luke Hanley, Jason Sagert, Jon Terrett, Jui Dutta-Simmons, Melanie Allen. Allogeneic anti-PTK7 CAR-T cells for the treatment of solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3243.

Supplementary Figures 1-8, Methods from BCL9 Promotes Tumor Progression by Conferring Enhanced Proliferative, Metastatic, and Angiogenic Properties to Cancer Cells