Michele Babicky

Genome-wide mutational landscape of mucinous carcinomatosis peritonei of appendiceal origin

BackgroundMucinous neoplasms of the appendix (MNA) are rare tumors which may progress from benign to malignant disease with an aggressive biological behavior. MNA is often diagnosed after metastasis to the peritoneal surfaces resulting in mucinous carcinomatosis peritonei (MCP). Genetic alterations in MNA are poorly characterized due to its low incidence, the hypo-cellularity of MCPs, and a lack of relevant pre-clinical models. As such, application of targeted therapies to this disease is limited to those developed for colorectal cancer and not based on molecular rationale.MethodsWe sequenced the whole exomes of 10 MCPs of appendiceal origin to identify genome-wide somatic mutations and copy number aberrations and validated significant findings in 19 additional cases.ResultsOur study demonstrates that MNA has a different molecular makeup than colorectal cancer. Most tumors have co-existing oncogenic mutations in KRAS (26/29) and GNAS (20/29) and are characterized by downstream PKA activation. High-grade tumors are GNAS wild-type (5/6), suggesting they do not progress from low-grade tumors. MNAs do share some genetic alterations with colorectal cancer including gain of 1q (5/10), Wnt, and TGFβ pathway alterations. In contrast, mutations in TP53 (1/10) and APC (0/10), common in colorectal cancer, are rare in MNA. Concurrent activation of the KRAS and GNAS mediated signaling pathways appears to be shared with pancreatic intraductal papillary mucinous neoplasm.ConclusionsMNA genome-wide mutational analysis reveals genetic alterations distinct from colorectal cancer, in support of its unique pathophysiology and suggests new targeted therapeutic opportunities.

The RON receptor tyrosine kinase in pancreatic cancer pathogenesis and its potential implications for future targeted therapies

Pancreatic cancer remains a devastating disease with a mortality rate that has not changed substantially in decades. Novel therapies are therefore desperately needed. The RON receptor tyrosine kinase has been identified as an important mediator of KRAS oncogene addiction and is overexpressed in the majority of pancreatic cancers. Preclinical studies show that inhibition of RON function decreases pancreatic cancer cell migration, invasion, and survival and can sensitize pancreatic cancer cells to chemotherapy. This article reviews the current state of knowledge regarding RON biology and pancreatic cancer and discusses its potential as a therapeutic target.

Generation of orthotopic patient-derived xenografts from gastrointestinal stromal tumor

BackgroundGastrointestinal stromal tumor (GIST) is the most common sarcoma and its treatment with imatinib has served as the paradigm for developing targeted anti-cancer therapies. Despite this success, imatinib-resistance has emerged as a major problem and therefore, the clinical efficacy of other drugs has been investigated. Unfortunately, most clinical trials have failed to identify efficacious drugs despite promising in vitro data and pathological responses in subcutaneous xenografts. We hypothesized that it was feasible to develop orthotopic patient-derived xenografts (PDXs) from resected GIST that could recapitulate the genetic heterogeneity and biology of the human disease.MethodsFresh tumor tissue from three patients with pathologically confirmed GISTs was obtained immediately following tumor resection. Tumor fragments (4.2-mm3) were surgically xenografted into the liver, gastric wall, renal capsule, and pancreas of immunodeficient mice. Tumor growth was serially assessed with ultrasonography (US) every 3-4 weeks. Tumors were also evaluated with positron emission tomography (PET). Animals were sacrificed when they became moribund or their tumors reached a threshold size of 2500-mm3. Tumors were subsequently passaged, as well as immunohistochemically and histologically analyzed.ResultsHerein, we describe the first model for generating orthotopic GIST PDXs. We have successfully xenografted three unique KIT-mutated tumors into a total of 25 mice with an overall success rate of 84% (21/25). We serially followed tumor growth with US to describe the natural history of PDX growth. Successful PDXs resulted in 12 primary xenografts in NOD-scid gamma or NOD-scid mice while subsequent successful passages resulted in 9 tumors. At a median of 7.9 weeks (range 2.9-33.1 weeks), tumor size averaged 473±695-mm3 (median 199-mm3, range 12.6-2682.5-mm3) by US. Furthermore, tumor size on US within 14 days of death correlated with gross tumor size on necropsy. We also demonstrated that these tumors are FDG-avid on PET imaging, while immunohistochemically and histologically the PDXs resembled the primary tumors.ConclusionsWe report the first orthotopic model of human GIST using patient-derived tumor tissue. This novel, reproducible in vivo model of human GIST may enhance the study of GIST biology, biomarkers, personalized cancer treatments, and provide a preclinical platform to evaluate new therapeutic agents for GIST.

A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells.

The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription domain. The splice variant is detected in 73% of xenografts derived from pancreatic adenocarcinoma patients and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirm translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing cells, activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target.

Characterization of RON protein isoforms in pancreatic cancer: implications for biology and therapeutics

The RON tyrosine kinase receptor is under investigation as a novel target in pancreatic cancer. While RON mutations are uncommon, RON isoforms are produced in cancer cells via a variety of mechanisms. In this study we sought to: 1) characterize RON isoform expression in pancreatic cancer, 2) investigate mechanisms that regulate isoform expression, and 3) determine how various isoforms effect gene expression, oncogenic phenotypes and responses to RON directed therapies. We quantified RON transcripts in human pancreatic cancer and found expression levels 2500 fold that of normal pancreas with RON isoform expression comprising nearly 50% of total transcript. RNA seq studies revealed that the short form (sfRON) and P5P6 isoforms which have ligand independent activity, induce markedly different patterns of gene expression than wild type RON. We found that transcription of RON isoforms is regulated by promoter hypermethylation as the DNA demethylating agent 5-aza-2′-deoxycytidine decreased all RON transcripts in a subset of pancreatic cancer cell lines. The viability of sfRON-expressing HPDE cells was reduced by a RON specific small molecule inhibitor, while a therapeutic monoclonal antibody had no demonstrable effects. In summary, RON isoforms may comprise half of total RON transcript in human pancreatic cancer and their expression is regulated at least in part by promoter hypermethylation. RON isoforms activate distinct patterns of gene expression, have transforming activity and differential responses to RON directed therapies. These findings further our understanding of RON biology in pancreatic cancer and have implications for therapeutic strategies to target RON activity.

Abstract A26: The RON tyrosine kinase accelerates pancreatic duct neoplasia and activates a positive autocrine/paracrine signaling loop that promotes alternative macrophage activation

Introduction: The RON (receptor d9origine nantais) tyrosine kinase is overexpressed in >80% of human pancreatic cancers, but its effect on pancreatic carcinogenesis is largely unknown. Methods: We developed a transgenic mouse that overexpresses RON specifically in the pancreas (Pdx-1-RON). Additionally, we utilized an existing transgenic mouse with a functionally inactive RON tyrosine kinase domain (Pdx-1-RTK-/-). These strains were crossed with the established CK murine model of pancreatic cancer yielding compound strains, RCK and CK/TK-/-. We performed immunofluorescent staining, flow cytometry, and western blot analysis on the tissues from these animals to study the effects of RON overexpression and the loss of RON signaling in the setting of oncogenic Kras. Results: RON overexpression results in increased acinar-ductal metaplasia, accelerates PanIN progression to invasive cancer, and results in an accumulation of alternatively-activated (MRC1+) macrophages in the tumor microenvironment. We found that RON overexpression results in upregulation of its own ligand, macrophage-stimulating protein (MSP), activating a positive autocrine/paracrine signaling loop that effects tumor growth and the differentiation of tumor-associated macrophages. Knockdown of RON expression and/or MSP expression results in decreased tumor growth in vivo. Additionally, loss of RON signaling in CK/TK-/- mice leads to a profound decrease in alternatively-activated (MRC1+) macrophages in the tumor microenvironment. In contrast, these animals demonstrate an accumulation of classically activated (CD11c+) macrophages. RON signaling is also associated with downregulation of the chemokine, monocyte chemoattractant protein-1 (MCP-1), a known driver of CD11c+ macrophage activation. The correlation of RON and MSP and the inverse correlation with MCP-1 expression were validated in a database analysis of human pancreatic tumor specimens. Conclusion: Our studies demonstrate that murine RON overexpression accelerates the development of invasive pancreatic cancer in the setting of oncogenic Kras. Active RON signaling results in upregulation of its own ligand, MSP, initiating a positive autocrine/paracrine feedback loop that promotes tumor growth and alters the differentiation of macrophages in the tumor microenvironment. Citation Format: Michele L. Babicky, Megan M. Harper, Evangeline S. Mose, Dawn J. Jaquish, Randall P. French, Susan E. Waltz. The RON tyrosine kinase accelerates pancreatic duct neoplasia and activates a positive autocrine/paracrine signaling loop that promotes alternative macrophage activation. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A26.

Abstract A72: Protein isoforms of the RON tyrosine kinase receptor transform human pancreatic ductal epithelial cells and induce acinar to ductal metaplasia

Introduction:The RON tyrosine kinase receptor is increasingly overexpressed during progression to Pancreatic Intraepithelial Neoplasia (PanIN) and pancreatic duct adenocarcinoma (PDAC). RON expression in these neoplastic cells is associated with a highly invasive phenotype and resistance to gemcitabine. Mutations in the gene for RON, MST1R , are rare but alternative splicing produces a wide variety of protein isoforms. Exon skipping is the most common and a skipping of exon 11 produces RONΔ165. Alternative transcription start in intron 10 produces short-form RON (sfRON) which promotes metastasis in breast cancer. Eight RON isoforms have previously been reported in different epithelial cancer types (Colon, Breast, Stomach) and we initially sought to characterize their expression in PDAC. Using low passage patient derived xenografts (PDXs), we found that the known RON isoforms Δ165 and sfRON were expressed in the majority of PDX’s. Results: This study revealed a novel RON isoform resulting from partial splicing of exons 5 and 6 (P5P6) to produce a protein that lacks the first extracellular immunoglobulin-plexin-transcription (IPT) domain. Peptides specific to RON P5P6 were detected in pancreatic cancer specimens by mass spectrometry which confirms translation of the protein. Using end point PCR we determined that RON is overexpressed in 90% of PDX and P5P6 expressed in 73%. In PDXs absolute quantification PCR determined RON isoforms P5P6, sfRON, and RONΔ165 make up 15 – 50% of total RON transcripts. This puts total isoform expression at up to 500 times more than the full length protein in normal pancreas. To determine function of these isoforms we overexpressed sfRON, RON P5P6, and RONΔ165 in a Human Pancreatic Ductal Epithelial (HPDE) cell line using a lentiviral vector. We found that all isoforms are constitutively phosphorylated, present in the cytoplasm in a reticular pattern, and RON P5P6 traffics to the plasma membrane. RON P5P6 is spliced into α and β subunits and can bind its ligand Macrophage Stimulating Protein similar to full length RON. Expression of P5P6 in HPDE cells activates downstream AKT and MAPK signaling pathways and increases cell motility, VEGF secretion, and markers of EMT. HPDE cells expressing P5P6 and short form RON were injected orthotopically into NOD SCID Gamma mouse pancreata and are tumorigenic while the parental cell line is not. P5P6 and short form RON tumors induce a denser stroma not characteristically observed in cell line derived xenografts. In HPDE sfRON tumors, host mouse acini adjacent to the tumor undergo acinar to ductal metaplasia. As the tumor invades into the mouse pancreas these newly formed ducts become increasingly dysplastic and begin to express mesenchymal markers. Conclusion: We have identified protein isoforms of the RON tyrosine kinase receptor which are expressed in human pancreatic cancer. A novel isoform RON P5P6 and short form RON transform human pancreatic ductal epithelial cells to become tumorigenic. Expression of short form RON in these tumors causes host mouse pancreas to undergo acinar to ductal metaplasia. Ultimately these findings demonstrate that RON isoform expression by pancreatic cancer cells has significant biologic consequences and promotes interaction with the adjacent normal pancreas. Further work to characterize the soluble factors responsible for RON paracrine signaling is currently underway. Citation Format: Jeffery M. Chakedis, Randall French, Michele Babicky, Dawn Jaquish, Haleigh Howard, Evangeline Mose, Jaclyn Miyamoto, Zakk Walterscheid, Patrick Holman, Andrew M. Lowy. Protein isoforms of the RON tyrosine kinase receptor transform human pancreatic ductal epithelial cells and induce 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 A72.

Correction: Genome-wide mutational landscape of mucinous carcinomatosis peritonei of appendiceal origin

No abstract.

Abstract 5191: Genome-wide mutational analysis reveals core signaling pathways in mucinous neoplasms of the appendix

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mucinous Neoplasm of the Appendix (MNA) is a rare malignancy often diagnosed after metastasis to the peritoneal surfaces resulting in Mucinous Carcinomatosis Peritonei (MCP). Genetic alterations in MNA are poorly characterized due to its low incidence, the hypo-cellularity of MCPs and a lack of relevant pre-clinical models. As such, application of targeted therapies to this disease is limited to those developed for colorectal cancer (CRC) and not based on molecular rationale. We sequenced the whole exomes of 10 MCPs to identify genome-wide somatic mutations and copy number aberrations and validated significant findings in 19 additional cases using deep sequencing and microdroplet PCR. These studies demonstrate that MNA has a different molecular makeup than CRC. Most tumors have co-existing oncogenic mutations in KRAS (26/29) and GNAS (21/29) and are characterized by cAMP-PKA pathway activation. High-grade tumors are GNAS wild-type (5/6), suggesting they do not progress from low-grade tumors. MNAs do share some genetic alterations with CRC including gain of 1q (5/10), Wnt and TGFβ pathway alteration. In contrast, mutations in TP53 (1/10) and APC (0/10), common in CRC, are rare in MNA. Coincident activation of the Ras-Raf-MEK-ERK and cAMP-PKA signaling pathways appears to be shared with pancreatic intraductal papillary mucinous neoplasm. Thus, MNA genome-wide mutational analysis reveals genetic alterations distinct from CRC, in support its unique pathophysiology and suggests MEK and PKA inhibition as new therapeutic opportunities. Citation Format: Hakan Alakus, Michele Babicky, Pradipta Ghosh, Shawn Yost, Kristen Jepsen, Yang Dai, Angelo Arias, Michael Samuels, Evangeline Mose, Michael Peterson, Andrew Lowy, Kelly Frazer, Olivier Harismendy. Genome-wide mutational analysis reveals core signaling pathways in mucinous neoplasms of the appendix. [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 5191. doi:10.1158/1538-7445.AM2014-5191

Abstract B54: RON overexpression drives macrophage differentiation toward an alternatively activated (M2) phenotype in a mouse model of spontaneous pancreatic carcinogenesis.

The RON receptor is frequently over-expressed in pancreatic cancer. We developed a mouse model of spontaneous pancreatic carcinogenesis driven by RON overexpression and oncogenic KRAS (RCK). RCK mice demonstrate accelerated PanIN progression to invasive cancer marked by desmoplasia that is highly characteristic of human disease. RON signaling in macrophages induces differentiation towards the alternatively activated (M2) phenotype, which promotes tumorigenesis. We hypothesized that pancreatic RON overexpression in RCK mice may promote changes in the tumor microenvironment, specifically by driving the differentiation of infiltrating macrophages towards the M2 phenotype. Methods: Pancreatic tissues from RCK and CK controls (3-12 months) were analyzed via immunofluorescent (IMF) staining to quantify myeloid infiltration. Single cell suspensions were generated from pancreatic and splenic tissues. CD11b+F4/80+ myeloid cell populations, as well as CD11c+ M1 and MRC1+ M2 subpopulations, were analyzed via FACS. Results: RCK pancreata demonstrated increased CD11b+F4/80+ myeloid infiltration at 6-12 months. In RCK, pancreatic myeloid cells were skewed toward the M2 phenotype compared with CK controls (MRC1+ 39% vs. 10% p<0.05, respectively). The expression profile of myeloid cells from the spleens was unchanged in CK vs. RCK (4-15% CD11c+, <1% MRC1+). There was no change in the myeloid derived suppressor cell (MDSC) populations (CD11b+Gr1+) in the spleen or pancreas of these animals. Conclusion: Pancreatic RON overexpression shifts infiltrating myeloid differentiation toward the M2 phenotype in this murine model. This data reveals a direct connection between aberrant RON signaling and the tumor permissive inflammatory infiltrate present within the pancreatic cancer microenvironment. Citation Format: Michele L. Babicky, Megan M. Harper, Evangeline Mose, Dawn Jaquish, Randall French, Andrew M. Lowy. RON overexpression drives macrophage differentiation toward an alternatively activated (M2) phenotype in a mouse model of spontaneous pancreatic carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B54.