Halbrook, Christopher J.

Abstract Intratumoral genomic heterogeneity in glioblastoma (GBM) is a barrier to overcoming radiation (RT) resistance. To discover genotype-independent mediators of RT resistance, we correlated RT resistance with the concentration of approximately 700 metabolites across 23 GBM cell lines. Purine metabolites, especially those containing the base guanine, were most correlated with RT resistance. Similarly, increased abundance of tumor purines was associated with decreased survival in GBM patients treated with RT. This relationship is causal. Purine supplementation protected RT-sensitive GBMs from RT and promoted the repair of RT-induced double strand DNA breaks (DSBs). In vitro and in vivo stable isotope tracing confirmed that GBM cell lines and orthotopic patient-derived xenografts primarily generated purines through the de novo synthetic pathway. RT treatment further increased de novo purine synthesis in GBM through signaling via the DNA damage response. Inhibition of de novo GTP synthesis with mycophenolic acid (MPA) sensitized multiple GBM cell lines and neurospheres to RT by slowing the repair of RT-induced DSBs. MPA-induced radiosensitization was GTP-dependent as it was rescued by nucleoside supplementation. Modulating pyrimidine metabolism affected neither RT resistance nor DSB repair, suggesting these GTP-specific effects are due to active signaling rather than its ability to act as a physical substrate for DNA repair and candidate signaling molecules have been identified. These results were recapitulated in vivo with mycophenolate mofetil (MMF), the orally bioavailable FDA-approved prodrug of MPA. MMF potentiated RT efficacy, reduced tumor guanylates and slowed the repair of RT-induced DSBs across multiple models. Because de novo purine synthesis is activated by many of the oncogenic alterations that drive GBM, its inhibition is a promising genotype-independent strategy to overcome GBM RT resistance. We have now begun a clinical trial to determine whether combining MMF and RT is safe and potentially efficacious in patients with GBM.

Abstract Prior to PanIN formation, acinar cells harboring oncogenic Kras gradually lose their acinar character and take on a ductal phenotype in a process known as acinar-to-ductal metaplasia. Rather than mimicking a normal pancreatic duct, metaplastic ducts take on a proliferative biliary progenitor phenotype, marked by the expression of SOX17 and PDX1 and the presence of numerous tuft cells, recently identified as a PanIN initiating cells. Manipulation of SOX17 and PDX1 in vivo reveal them to be a transdifferentiation promoter and suppressor, respectively, both greatly affecting tuft cell genesis and tumor formation. These opposing roles of developmental transcription factors during tumorigenesis implicate the usurpation of a differentiation program that significantly contributes to cellular heterogeneity within early pre-cancerous lesions of the pancreas. Citation Format: Kenneth K. Takeuchi, Kathleen E. Delgiorno, Christopher J. Halbrook, Howard C. Crawford. Acinar cell transdifferentiation sets the stage for early tumor heterogeneity. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA13.

Abstract Pancreatic adenocarcinoma (PDAC) is the 4th most common cause of cancer related deaths in the United States and has a dismal 5-year survival rate of 6%. This prognosis is attributed the disease’s fast and asymptomatic progression to metastasis, leading to late diagnosis upon which time there are few treatment options available. The A Disintegrin and Metalloproteinase (ADAM) family are sheddase proteins known to regulate cell adhesion and function. This regulation can allow ADAMs to influence the ability of tumor cells to disseminate from the primary tumor and metastasize to distant organs. ADAM10, which is seen to be up-regulated in both chronic pancreatitis and PDAC, cleaves a variety of substrates including several cell adhesion molecules such as L1-CAM, N-Cadherin, and E-Cadherin. Using a conditional knockout for ADAM10 in the Kras+/G12D;Ptf1a+/Cre (KC) mouse model of pancreatic cancer, we have observed an absence of metastasis when ADAM10 is genetically ablated. To investigate the potential role that ADAM10 plays in this, we used a shRNA approach to knock-down ADAM10 expression in various human pancreatic cancer cells lines. Analysis of these cell lines suggests a partial restoration of a less aggressive, differentiated phenotype, consistent with the increased longevity of the ADAM10 knockout KC mice. Citation Format: Louise V. Peverley, Christopher J. Halbrook, Jason C. Hall, Christine M. Ardito, Howard C. Crawford. Investigating the role of ADAM10 in pancreatic tumor differentiation. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A77.

Abstract The EGFR and Notch signaling pathways have been widely implicated in relation to both chronic pancreatitis and pancreatic ductal adenocarcinoma in humans, however the link between the two pathways has remained obscure. Acinar to ductal metaplasia (ADM,) a phenomenon where acinar cells undergo a morphological change and begin expressing markers normally found only in ductal cells while losing markers typical of acinar cells, has been linked to both the Notch and EGFR signaling pathways. ADM is highly proliferative with the potential to develop into pancreatic intraepithelial neoplasia, a precursor lesion to pancreatic cancer. Recently we and others have shown that genetic ablation of EGFR in the pancreas effectively prevents acinar to ductal metaplasia in cerulein model of pancreatitis in mice, and prevents tumorigenesis driven by oncogenic Kras. Additionally, we have found that acinar cells harvested from EGFRf/f;Ptf1aCre/+ mice (EKO) mice are resistant to ADM in vitro upon cerulein treatment, or by the expression of oncogenic Kras. Activation of EGFR by treatment of WT collagen embedded acinar cell explants with the EGFR ligand TGF-α has previously been shown to induce ADM in a manner requiring Notch pathway activation. Furthermore, it has also been demonstrated that expression of Notch2 intracellular domain (N2ICD) is sufficient to induce ADM in WT acinar cells. To directly investigate the relationship of the EGFR, Kras, and Notch signaling pathways in ADM, we examined the effect of Notch signaling in acinar cells harvested from EKO mice. Interestingly, these acinar cells fail to transdifferentiate in vitro upon transduction of N2ICD in 3D culture; however the transdifferentiation event can be rescued by co-expression of oncogenic Kras with N2ICD in acinar cells lacking EGFR. These results indicate that EGFR is important in Notch induced ADM, however there are other pathways that possibly can be utilized by acinar cells expressing oncogenic Kras to drive ADM. Citation Format: Christopher J. Halbrook, Howard C. Crawford. Redefining the role of Notch in acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A27.

Abstract Nearly all pancreatic ductal adenocarcinomas are caused by oncogenic mutations in the KRAS gene. Pharmacological inhibition of mutant KRAS has thus far been unsuccessful in the clinical setting, precipitating a need to understand the pathways downstream of KRAS which may prove more easily targeted with small molecule inhibitors. Here we show that PI3K p110α is absolutely required for pancreatic tumorigenesis while p110β is dispensable for this process. Surprisingly, ablation of p110α does not impair the ability of KRAS to activate AKT, demonstrating that AKT activation is not sufficient for transformation. Instead we find that p110α is required for robust activation of RAC1, a small GTPase required for pancreatic metaplasia. Consistent with this, our data show that p110α is necessary for regulating epithelial expression and activation of RAC-GEFs including Vav1, Tiam1 and Ect2. Ultimately, these results define the mechanistic role of p110α in pancreatic tumorigenesis and suggest selective inhibition of this PI3K isoform as a promising therapeutic approach to treating patients with pancreas cancer. Citation Format: Kenneth K. Takeuchi, Eileen Carpenter, Claire Wu, Christopher J. Halbrook, Richard Z. Lin, Howard C. Crawford. PI3K regulation of RAC1 is required for Kras-induced pancreatic tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A04.

Abstract Almost all reported cases of pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer, harbor an oncogenic Kras mutation. Recently, targeting oncogenic Kras to specific cell types in the adult pancreas has shown the acinar compartment to be more susceptible to neoplastic transformation than ductal cells. Acinar to ductal metaplasia (ADM) is a phenomenon where acinar cells undergo a morphological change and begin expressing markers normally found only in ductal cells while losing markers typical of acinar cells. ADM is highly proliferative with the potential to develop into pancreatic intraepithelial neoplasias, a precursor lesion to pancreatic cancer. Acinar cells harvested from mice expressing mutant Kras undergo spontaneous ADM when embedded in 3D collagen cultures, however, we and others have found the epidermal growth factor receptor (EGFR) is required for ADM induced by mutant Kras expression in vitro. Activation of EGFR by treatment of WT collagen embedded acinar cell explants with the EGFR ligand TGF-α has also been shown to induce ADM in a manner requiring Notch pathway activation. Furthermore, it has been demonstrated that expression of Notch2 intracellular domain (N2ICD) is sufficient to induce ADM in WT acinar cells. To directly investigate the relationship of the EGFR, Kras, and Notch signaling pathways in ADM, we examined the effect of Notch signaling in acinar cells harvested from EGFRf/f; Ptf1a+/Cre (EKO) mice, which harbor a pancreas specific deletion of EGFR. Surprisingly, we found that expression of N2ICD failed to induce ADM in acinar cells from EKO mice. We also observed that co-expression of a constitutively active p110α mutant or mutant Kras with N2ICD was able to rescue ADM in acinar cells from EKO mice, though neither was sufficient to drive ADM in the absence of N2ICD. Additionally, treatment of acinar cells from WT mice with either the p110α inhibitor Pik75 or the expression of a dominant negative mutant of the PI3K downstream target Rac1 reduced Erk1/2 phosphorylation, whereas expression of constitutively active p110α increased the level of Erk1/2 phosphorylation. These results demonstrate the importance of EGFR signaling in ADM, and identify the need to determine the mechanism of crosstalk between the PI3K and MAPK pathways in acinar cells. Citation Format: Christopher J. Halbrook, Howard C. Crawford. Notch signaling couples with Kras or PI3K to drive EGFR independent pancreatic acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A09. doi: 10.1158/1557-3125.RASONC14-A09

Abstract EGFR and EGFR ligand upregulation has been widely seen in both human chronic pancreatitis and pancreatic ductal adenocarcinoma (PDA) for many years; however the role of EGFR in PDA was poorly understood. Recently we and others have shown that genetic ablation of EGFR effectively prevents both spontaneous and cerulein induced tumorigenesis in the pancreas of the LSL-KrasG12D/+;Ptf1aCre/+ (KC) mouse model of pancreatic tumorigenesis. Pockets of EGFR upregulation localized in single acini of KC mice appear to precede pancreatic intraepithelial neoplasia (PanIN) formation in spontaneous tumorigenesis, coupled with a distinct spatially localized immune response. Ubiquitous EGFR upregulation is observed throughout the pancreas upon induction of pancreatitis in wild-type (WT) and KC mice, returning to normal levels in WT mice but continuing to increase along with the widespread tumorigenesis in cerulein treated KC mice. Comparison of cerulein treated KC mice with the EGFRf/f;LSL-KrasG12D/+;Ptf1aCre/+ mice (EKC), which are largely resistant to cerulein induced pancreatitis, offers an opportunity to identify signals in the KC mouse which could be responsible for the upregulation of EGFR and subsequent tumorigenesis. Citation Format: Christopher J. Halbrook, Kenneth K. Takeuchi, Howard C. Crawford. Regulation of EGFR in pancreatic cancer. [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 3030. doi:10.1158/1538-7445.AM2013-3030