Susan Payne

Colon Tumors with the Simultaneous Induction of Driver Mutations in APC, KRAS, and PIK3CA Still Progress through the Adenoma-to-carcinoma Sequence

Human colorectal cancers often possess multiple mutations, including three to six driver mutations per tumor. The timing of when these mutations occur during tumor development and progression continues to be debated. More advanced lesions carry a greater number of driver mutations, indicating that colon tumors might progress from adenomas to carcinomas through the stepwise accumulation of mutations following tumor initiation. However, mutations that have been implicated in tumor progression have been identified in normal-appearing epithelial cells of the colon, leaving the possibility that these mutations might be present before the initiation of tumorigenesis. We utilized mouse models of colon cancer to investigate whether tumorigenesis still occurs through the adenoma-to-carcinoma sequence when multiple mutations are present at the time of tumor initiation. To create a model in which tumors could concomitantly possess mutations in Apc, Kras, and Pik3ca, we developed a novel minimally invasive technique to administer an adenovirus expressing Cre recombinase to a focal region of the colon. Here, we demonstrate that the presence of these additional driver mutations at the time of tumor initiation results in increased tumor multiplicity and an increased rate of progression to invasive adenocarcinomas. These cancers can even metastasize to retroperitoneal lymph nodes or the liver. However, despite having as many as three concomitant driver mutations at the time of initiation, these tumors still proceed through the adenoma-to-carcinoma sequence. Cancer Prev Res; 8(10); 952–61. ©2015 AACR.

PIK3CA mutations can initiate pancreatic tumorigenesis and are targetable with PI3K inhibitors.

Aberrations in the phosphoinositide 3-kinase (PI3K) signaling pathway have a key role in the pathogenesis of numerous cancers by altering cell growth, metabolism, proliferation and apoptosis. Interest in targeting the PI3K signaling cascade continues, as new agents are being clinically evaluated. PIK3CA mutations result in a constitutively active PI3K and are present in a subset of pancreatic cancers. Here we examine mutant PIK3CA-mediated pancreatic tumorigenesis and the response of PIK3CA mutant pancreatic cancers to dual PI3K/mammalian target of rapamycin (mTOR) inhibition. Two murine models were generated expressing a constitutively active PI3K within the pancreas. An increase in acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasms (PanINs) was identified. In one model these lesions were detected as early as 10 days of age. Invasive pancreatic ductal adenocarcinoma developed in these mice as early as 20 days of age. These cancers were highly sensitive to treatment with dual PI3K/mTOR inhibition. In the second model, PanINs and invasive cancer develop with a greater latency owing to a lesser degree of PI3K pathway activation in this murine model. In addition to PI3K pathway activation, increased ERK1/2 signaling is common in human pancreatic cancers. Phosphorylation of ERK1/2 was also investigated in these models. Phosphorylation of ERK1/2 is demonstrated in the pre-neoplastic lesions and invasive cancers. This activation of ERK1/2 is diminished with dual PI3K/mTOR inhibition. In summary, PIK3CA mutations can initiate pancreatic tumorigenesis and these cancers are particularly sensitive to dual PI3K/mTOR inhibition. Future studies of PI3K pathway inhibitors for patients with PIK3CA mutant pancreatic cancers are warranted.

Versican-Derived Matrikines Regulate Batf3–Dendritic Cell Differentiation and Promote T Cell Infiltration in Colorectal Cancer

Colorectal cancer originates within immunologically complex microenvironments. To date, the benefits of immunotherapy have been modest, except in neoantigen-laden mismatch repair–deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. In this article, we report that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) strongly correlated with CD8+ T cell infiltration in colorectal cancer, regardless of mismatch repair status. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8+ T cell infiltration. Tumor-intrinsic WNT pathway activation was associated with CD8+ T cell exclusion and VCAN accumulation. In addition to regulating VCAN levels at the tumor site, VCAN proteolysis results in the generation of bioactive fragments with novel functions (VCAN-derived matrikines). Versikine, a VCAN-derived matrikine, enhanced the generation of CD103+CD11chiMHCIIhi conventional dendritic cells (cDCs) from Flt3L-mobilized primary bone marrow–derived progenitors, suggesting that VCAN proteolysis may promote differentiation of tumor-seeding DC precursors toward IRF8- and BATF3-expressing cDCs. Intratumoral BATF3-dependent DCs are critical determinants for T cell antitumor immunity, effector T cell trafficking to the tumor site, and response to immunotherapies. Our findings provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker and VCAN-derived matrikines as novel immunotherapy agents.

Colon Cancer Tumorigenesis Initiated by the H1047R Mutant PI3K.

The phosphoinositide 3-kinase (PI3K) signaling pathway is critical for multiple important cellular functions, and is one of the most commonly altered pathways in human cancers. We previously developed a mouse model in which colon cancers were initiated by a dominant active PI3K p110-p85 fusion protein. In that model, well-differentiated mucinous adenocarcinomas developed within the colon and initiated through a non-canonical mechanism that is not dependent on WNT signaling. To assess the potential relevance of PI3K mutations in human cancers, we sought to determine if one of the common mutations in the human disease could also initiate similar colon cancers. Mice were generated expressing the Pik3caH1047R mutation, the analog of one of three human hotspot mutations in this gene. Mice expressing a constitutively active PI3K, as a result of this mutation, develop invasive adenocarcinomas strikingly similar to invasive adenocarcinomas found in human colon cancers. These tumors form without a polypoid intermediary and also lack nuclear CTNNB1 (β-catenin), indicating a non-canonical mechanism of tumor initiation mediated by the PI3K pathway. These cancers are sensitive to dual PI3K/mTOR inhibition indicating dependence on the PI3K pathway. The tumor tissue remaining after treatment demonstrated reduction in cellular proliferation and inhibition of PI3K signaling.

Translational investigations of gastrointestinal malignancies using spheroid cultures.

620 Background: Spheroid cultures are now being used to investigate the biology of multiple types of cancer. This technique allows for a cost-effective, efficient and reliable means to culture tissues of interest. Recent investigations have demonstrated that spheroids maintain high mutational concordance with the cancers they are derived. We sought to determine if spheroid techniques could be utilized to perform translational investigations into subtypes of gastrointestinal malignancies. Methods: Transgenic mice carrying conditional mutations in key genes known to have important roles in colorectal cancer (CRC) tumorigenesis and progression were treated with a Cre-expressing adenovirus to initiate tumorigenesis. The mutant genes of interest included Apc, Trp53, KRAS, Pik3ca, and/or BRAF. Biopsies of these cancers were obtained with the murine endoscope and the tissue processed for spheroid culture. In addition, transgenic E6 and E7 mice were treated with DMBA and the resulting anal squamous cell carcinoma...

Abstract 618: Dual PI3K/mTOR inhibition as a treatment strategy for PIK3CA and APC mutant colorectal cancers

BACKGROUND: Colorectal cancer (CRC) is understood as a diverse group of cancers calling for the personalization of therapeutics to individual subtypes of CRC. Mutations in PIK3CA, which result in a constitutively active phosphoinositide-3-kinase protein (PI3K), are present in 20-30% of CRCs. Aberrations in the Adenomatous Polyposis Coli (APC) gene are found in 86% of PIK3CA mutant CRCs. The potential for treatment of tumors with this specific profile is of great interest. METHODS: Mice expressing a constitutively active PI3K and loss of APC were generated and spheroid cultures were derived from the resulting cancers. PIK3CA and APC mutant spheroids were treated with GDC0941 (GDC), a pan PI3K inhibitor, NVP-BYL719 (BYL), a PI3K alpha isomer specific inhibitor, or NVP-BEZ235 (BEZ), a dual mTOR/PI3K inhibitor. Proliferation was measured by changes in spheroid diameter over time. BEZ treatment of mice with PIK3CA and APC mutant CRCs was performed over 14 days. Tumor response was measured with serial murine colonoscopy and dual hybrid 18F-FDG PET/CT imaging. RESULTS: Persistent PIK3CA and APC mutant CRC spheroid growth was observed with BYL and GDC treatment, while BEZ treatment resulted in a significant reduction in spheroid size. Immunoblotting determined that this correlated with significant suppression of phosphorylated AKT, RPS6 and 4EBP1 in BEZ-treated spheroids and incomplete PI3K pathway inhibition following BYL and GDC treatment. BEZ treatment was then investigated in mice with PIK3CA and APC mutant CRCs. BEZ elicited a dramatic treatment response in these cancers on endoscopy, with a 53% decrease in median lumen occlusion compared to a 60% increase in controls over the 14 day treatment period. PET/CT imaging results confirmed these findings demonstrating a decrease in tumor size and avidity post BEZ treatment. BEZ resulted in inhibition of the PI3K pathway in these tumors. Reactivation of PI3K signaling was observed within 24 hours of the withdrawal of BEZ. BEZ was well-tolerated in these mice and associated with a decrease in the endoscopic anemia score. CONCLUSIONS: PIK3CA and APC mutant CRCs are a potentially targetable subtype of cancer. These cancers are resistant to proximal inhibition of the PI3K pathway, but are responsive to dual PI3K/mTOR inhibition. These results warrant further investigation in clinical studies. Citation Format: Susan Payne, Tyler Foley, Cheri Pasch, Alexander Yueh, Demetra Korkos, Dana Van De Hey, Linda Clipson, Dustin Deming. Dual PI3K/mTOR inhibition as a treatment strategy for PIK3CA and APC mutant colorectal cancers. [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 618.

Abstract 385: mTORC1/2 inhibition as a treatment strategy for subtypes of colorectal cancer

BACKGROUND Colorectal cancer (CRC) remains the second leading cause of cancer-related deaths in the United States. Several key mutations in CRC include APC (80%), TP53 (50%), and PIK3CA (20-30%). Mutations in the PIK3CA gene, resulting in a constitutively active PI3K, often occur concomitantly with loss of the APC gene in human CRCs. Our lab has developed a murine model system where a constitutively active PI3K and loss of APC occur simultaneously in the colon (AK3K) as well as concomitant loss of p53 (AK3KTO). Colon tumors from these models are cultured as three-dimensional spheroids and treatment studies. METHODS AK3K and AK3KTO spheroids were treated with a dual PI3K/mTOR inhibitor, NVP-BEZ235, or a mTORC1/2 inhibitor, MLN0128. Images were taken both pre- and post-treatment and changes in spheroid diameter were measured. Parallel treatment studies were performed on a primary human colon cancer tumor cell line, SW48, which carries a mutant FBXW7 and also on a cell line transfectedwith a PIK3CA mutation (SW48PK). RESULTS Treatment with NVP-BEZ235 and MLN0128 resulted in a significant treatment response as measured by marked change in spheroid diameter in the AK3K spheroids at 100, 200 and 400nM (p Citation Format: Stephanie Fricke, Cheri Pasch, Susan Payne, Alexander Yueh, Tyler Foley, Demetra Korkos, Dana Van De Hey, Linda Clipson, Dustin Deming. mTORC1/2 inhibition as a treatment strategy for subtypes of colorectal cancer. [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 385.