Oncogenic KrasG12D Expression Research Articles

DNA methylation-associated allelic inactivation regulates Keratin 19 gene expression during pancreatic development and carcinogenesis.

Within the pancreas, Keratin 19 (KRT19) labels the ductal lineage and is a determinant of pancreatic ductal adenocarcinoma (PDAC). To investigate KRT19 expression dynamics, we developed a human pluripotent stem cell (PSC)-based KRT19-mCherry reporter system in different genetic backgrounds to monitor KRT19 expression from its endogenous gene locus. A differentiation protocol to generate mature pancreatic duct-like organoids was applied. While KRT19/mCherry expression became evident at the early endoderm stage, mCherry signal was present in nearly all cells at the pancreatic endoderm (PE) and pancreatic progenitor (PP) stages. Interestingly, despite homogenous KRT19 expression, mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates, with site-specific DNA methylation patterns occurring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA-methyltransferase inhibitor. In human PDAC cohorts, high KRT19 levels correlate with low locus methylation and decreased survival. At the same time, activation of oncogenic KRASG12D signalling in our reporter system reversed monoallelic back to biallelic KRT19 expression in pancreatic duct-like organoids. Allelic reactivation was also detected in single-cell transcriptomes of human PDACs, which further revealed a positive correlation between KRT19 and KRAS expression. Accordingly, KRAS mutant PDACs had higher KRT19 mRNA but lower KRT19 gene locus DNA methylation than wildtype counterparts. KRT19 protein was additionally detected in plasma of PDAC patients, with higher concentrations correlating with shorter progression-free survival in gemcitabine/nabPaclitaxel-treated and opposing trends in FOLFIRINOX-treated patients. Apart from being an important pancreatic ductal lineage marker, KRT19 appears tightly controlled via a switch from biallelic to monoallelic expression during ductal lineage entry and is aberrantly expressed after oncogenic KRASG12D expression, indicating a role in PDAC development and malignancy. Soluble KRT19 might serve as a relevant biomarker to stratify treatment. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract 701: Atherosclerosis Is A Smooth Muscle Cell-driven Tumor-like Disease

Background: Atherosclerosis, the leading cause of cardiovascular disease (CVD), is a chronic inflammatory disease involving pathological activation of multiple cell types, such as immunocytes (e.g., macrophage, T cells), smooth muscle cells (SMCs), endothelial cells, etc. Multiple lines of evidence have suggested that SMC “phenotypic switching” plays a central role in atherosclerosis development and complications. Yet, how SMCs and SMC-derived cells (SDCs) modulate atherosclerosis progression and clinical CVD remains poorly understood. Methods: We applied SMC lineage tracing mice and implemented comprehensive molecular and cellular biology, histological, computational, and pharmacological studies to reveal how tumor cell-like activities of SDCs drive the development of atherosclerosis. Results: Oxidative DNA damage occurs during SMC phenotypic switching and increases with the progression of atherosclerosis. Extensive DNA damage-induced genomic instability accumulates in both mouse and human atherosclerosis, especially in SDCs. Indeed, SDCs in atherosclerosis harbor multiple tumor cell-like features, including escape of replicative senescence, clonogenicity, invasive capacity, and ex vivo formation of three-dimensional (3D) spheroids like cancer stem cells. Multiple cancer-associated signaling pathways (e.g., p53, TNFa, NFkB, VEGF) are activated in SDCs. SMC-specific expression of oncogenic Kras G12D accelerates SMC phenotypic switching and exacerbates atherosclerosis. Moreover, we provide proof of concept in mouse models that niraparib, an anti-cancer drug targeting DNA damage repair, attenuates atherosclerosis progression and regresses lesions in advanced disease. Conclusions: Our studies in human and mouse models provide the first systematic evidence that SDCs in atherosclerosis share extensive commonalities with tumor, which deepens our understanding of the disease and opens prospects for novel targeted strategies to prevent and treat atherosclerotic CVD.

An ERK1/2-driven RNA-binding switch in nucleolin drives ribosome biogenesis and pancreatic tumorigenesis downstream of RAS oncogene.

Oncogenic RAS signaling reprograms gene expression through both transcriptional and post-transcriptional mechanisms. While transcriptional regulation downstream of RAS is relatively well characterized, how RAS post-transcriptionally modulates gene expression to promote malignancy remains largely unclear. Using quantitative RNA interactome capture analysis, we here reveal that oncogenic RAS signaling reshapes the RNA-bound proteomic landscape of pancreatic cancer cells, with a network of nuclear proteins centered around nucleolin displaying enhanced RNA-binding activity. We show that nucleolin is phosphorylated downstream of RAS, which increases its binding to pre-ribosomal RNA (rRNA), boosts rRNA production, and promotes ribosome biogenesis. This nucleolin-dependent enhancement of ribosome biogenesis is crucial for RAS-induced pancreatic cancer cell proliferation and can be targeted therapeutically to inhibit tumor growth. Our results reveal that oncogenic RAS signaling drives ribosome biogenesis by regulating the RNA-binding activity of nucleolin and highlight a crucial role for this mechanism in RAS-mediated tumorigenesis.

Abstract 24: Oncogenic KrasG12D and Cdkn2a/p16 knockout in LGR5 expressing progenitor cells synergize to advance adenoma and adenocarcinoma phenotypes in murine small intestine

Abstract Background: KRAS activating mutations are reported in 42-54% and inactivation of CDKN2A/p16 in 14-30% of human small intestinal adenocarcinomas, suggesting a role in small intestine adenocarcinogenesis. The phenotypic effects of oncogenic KRAS and p16 loss have not been well characterized in small intestine (SI) adenocarcinogenesis. We targeted LGR5-expressing progenitor cells residing in intestinal crypts for conditional activation of Kras and/or Cdkn2a/p16 deletion to generate a murine model to study the single and combined effects of KRAS and p16 alterations driving gastrointestinal tumorigenesis. We hypothesized that oncogenic Kras and loss of Cdkn2a/p16 drives adenoma-adenocarcinoma sequence in murine SI. Methods: We generated mice with conditional knockout of Cdkn2a/p16 (p16del: IL1bwt/tg;Lgr5-Cre+/−;p16fl/fl;Kraswt/wt), conditional expression of oncogenic KrasG12D (IL1bwt/tg;Lgr5-Cre+/−;KrasG12D/wt), or both alterations (p16del+KrasG12D: IL1bwt/tg;Lgr5-Cre+/−;p16fl/fl;KrasG12D/wt), in mice carrying the human interleukin 1 beta (IL1B) and expressing modified Cre under the Lgr5 promoter. Our control murine model expresses wild type p16 and KRAS (p16wt+Kraswt: IL1bwt/tg;Lgr5-Cre+/−;p16wt/wt;Kraswt/wt). These mice were initially generated to study esophageal adenocarcinogenesis and express the IL1b transgene in esophagus but not in SI. All mice were given bile acid in drinking water daily and were treated with tamoxifen to activate Cre in Lgr5-positive gastrointestinal epithelial progenitor cells. Mice were euthanized and gross and histologic alterations in the SI were examined from 4 to 21 months of age. 3D organoids were isolated from the SI of each mouse model. Results: We examined 89 mice from 4 to 12 months of age. Mice with KRAS activation (KrasG12D) alone infrequently developed SI duodenal adenomas (6.25%: 1/16) compared to 50% (9/18) of mice carrying the combined P16del+KrasG12D genotypes (P=0.005). No adenomas were seen in 27 control and 28 p16del mice. Adenomas with high-grade dysplasia, intramucosal adenocarcinoma, and invasive adenocarcinoma were seen only in older p16del+KrasG12D mice (13-21 months). Single or multiple adenomas and adenocarcinomas were observed only in the duodenal segment of SI and not in the colon. 3D organoids isolated from each mouse model reproduce the intestinal histologic phenotype. Conclusions: Loss of Cdkn2a/p16 and KRASG12D expression synergistically advances adenoma-adenocarcinoma tumorigenesis in the SI duodenum, providing a murine model that recapitulates SI adenocarcinogenesis in vivo. 3D SI organoids provide useful in vitro models for mechanistic studies of SI adenocarcinogenesis. These in vivo and in vitro models will enable identification of key molecular pathways that may be targeted with novel therapies for this rare cancer. Citation Format: Tyler Seckar, Jing Sun, Elena V. Komissarova, Jorge L. Sepulveda, Timothy L. Wang, Antonia R. Sepulveda. Oncogenic KrasG12D and Cdkn2a/p16 knockout in LGR5 expressing progenitor cells synergize to advance adenoma and adenocarcinoma phenotypes in murine small intestine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 24.

Identifying Adult Stomach Tissue Stem/Progenitor Cells Using the Iqgap3-2A-CreERT2 Mouse.

Identification of unique gene markers of normal and cancer stem cells is an effective strategy to study cells of origin and understand tumor behavior. Lineage tracing experiments using the Cre recombinase driven by a stem cell-specific promoter in the CreERT2 reporter mouse model enables identification of adult stem cells and delineation of stem cell activities in vivo. In our recent research on the mouse stomach, Iqgap3 was identified as a homeostatic stem cell marker located in the isthmus of the stomach epithelium. Lineage tracing with the Iqgap3-2A-CreERT2;Rosa26-LSL-tdTomato mouse model demonstrated stem cell activity in Iqgap3-expressing cells. Using the Iqgap3-2A-CreERT2 mouse model to target oncogenic KrasG12D expression to Iqgap3-expressing cells, we observed the rapid development of precancerous metaplasia in the stomach and proposed that aberrant Iqgap3-expressing cells may be critical determinants of early carcinogenesis. In this chapter, we detail a lineage tracing protocol to assess stem cell activity in the murine stomach. We also describe the procedure of inducing KrasG12D expression in Iqgap3-expressing homeostatic stem cells to explore their role as cells of origin and to trace the early cellular changes that precede neoplastic transformation.

AP1/Fra1 confers resistance to MAPK cascade inhibition in pancreatic cancer

Targeting KRAS downstream signaling remains an important therapeutic approach in pancreatic cancer. We used primary pancreatic ductal epithelial cells and mouse models allowing the conditional expression of oncogenic KrasG12D, to investigate KRAS signaling integrators. We observed that the AP1 family member FRA1 is tightly linked to the KRAS signal and expressed in pre-malignant lesions and the basal-like subtype of pancreatic cancer. However, genetic-loss-of-function experiments revealed that FRA1 is dispensable for KrasG12D-induced pancreatic cancer development in mice. Using FRA1 gain- and loss-of-function models in an unbiased drug screen, we observed that FRA1 is a modulator of the responsiveness of pancreatic cancer to inhibitors of the RAF–MEK–ERK cascade. Mechanistically, context-dependent FRA1-associated adaptive rewiring of oncogenic ERK signaling was observed and correlated with sensitivity to inhibitors of canonical KRAS signaling. Furthermore, pharmacological-induced degradation of FRA1 synergizes with MEK inhibitors. Our studies establish FRA1 as a part of the molecular machinery controlling sensitivity to MAPK cascade inhibition allowing the development of mechanism-based therapies.

Abstract C075: The loss of ATRX induces a sex-specific progression of pancreatic ductal adenocarcinoma in the presence of oncogenic KRASG12D

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related deaths primarily due to a lack of effective early screening and the tumor microenvironment increasing resistance to chemotherapy. Somatic mutation in oncogenic KRASG12D appears in 97% of PDAC patients, however, the addition of environmental and epigenetic stressors are required to promote PDAC initiation and progression from acinar cells. We are elucidating the mechanism by which a loss of α-thalassemia, mental-retardation, X-linked (ATRX), a SWI/SNF chromatin remodeling protein, potentiates acinar cells to develop neoplastic lesions in females. Young et al. have confirmed that female mice with a loss of Atrx, combined with KRASG12D, have shown increased acinar-to-duct cell metaplasia (ADM), increased fibrosis, inflammation, and pancreatic intraepithelial neoplasias (PanINs) lesions relative to mice expressing KRASG12D and Atrx. I hypothesize that a loss of ATRX function increases sensitivity to KRASG12D in PDAC by affecting DNA damage and repair pathways and altering gene expression in acinar cells in a sex-specific fashion. Methods: Mice with an acinar-specific Atrx deletion and oncogenic KRASG12D expression were generated by mating to mice targeting an inducible creERT to the Ptf11α gene (PtflacreERT). Mice were gavaged with tamoxifen and pancreatic injury was induced in these mice (Ptf11αcreERT KRASLSLG12DAtrxfl/fl) with cerulein 10 days later. Mice are monitored for two and five weeks following cerulein treatment and pancreatic tissue was collected for molecular, biochemical, and histological analysis. In addition, pre-neoplastic pancreatic organoid cultures +/- ATRX expression were generated to assess differences in growth rates and chemotherapy resistance. Results: Preliminary results have shown increased sensitivity to chronic pancreatitis is predicted to be observed specifically in KRASG12D Atrx-/- female pancreatic tissue and derived organoids. Conversely, a protective effect stimulated by a loss of ATRX is observed in KRASG12D Atrx-/- male mice, indicated by less ADM/PanIN lesions and greater acinar cells. Discussion: A healthier prognosis in Atrx-/-, KRAS activated male mice suggests that the interaction between ATRX and various scaffold proteins (PRC2, DAXX, EZH2, etc.) alters chromosomal architecture, possibly altering gene expression via X chromosome inactivation or reactivation. Sex-specific differences in pancreatic tissue phenotype of Atrx-/- mice are predicted to arise from differential effects of ATRX in triggering acinar cell genomic instability in female mice to prevent the resolution of dsDNA breaks via non-homologous end joining to sensitize acinar cells to oncogenic KRAS. Future experiments will focus on establishing pre-neoplastic organoid lines derived from inflamed murine pancreata to elucidate the signalling pathway of ATRX that likely stimulates a differential gene and protein expression involved in a better prognosis in KRASG12D activated, Atrx-/- male mice. Citation Format: Aswin Sureshkumar, Phyo Win, Christopher Pin. The loss of ATRX induces a sex-specific progression of pancreatic ductal adenocarcinoma in the presence of oncogenic KRASG12D [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C075.

Canonical TGF\u03b2 signaling induces collective invasion in colorectal carcinogenesis through a Snail1- and Zeb1-independent partial EMT

Local invasion is the initial step towards metastasis, the main cause of cancer mortality. In human colorectal cancer (CRC), malignant cells predominantly invade as cohesive collectives and may undergo partial epithelial-mesenchymal transition (pEMT) at the invasive front. How this particular mode of stromal infiltration is generated is unknown. Here we investigated the impact of oncogenic transformation and the microenvironment on tumor cell invasion using genetically engineered organoids as CRC models. We found that inactivation of the Apc tumor suppressor combined with expression of oncogenic KrasG12D and dominant-negative Trp53R172H did not cell-autonomously induce invasion in vitro. However, oncogenic transformation primed organoids for activation of a collective invasion program upon exposure to the prototypical microenvironmental factor TGFβ1. Execution of this program co-depended on a permissive extracellular matrix which was further actively remodeled by invading organoids. Although organoids shed some epithelial properties particularly at the invasive edge, TGFβ1-stimulated organoids largely maintained epithelial gene expression while additionally implementing a mesenchymal transcription pattern, resulting in a pEMT phenotype that did not progress to a fully mesenchymal state. Notably, while TGFβ1 induced pEMT and promoted collective invasion, it abrogated self-renewal capacity of TKA organoids which correlated with the downregulation of intestinal stem cell (ISC) marker genes. Mechanistically, induction of the non-progressive pEMT required canonical TGFβ signaling mediated by Smad transcription factors (TFs), whereas the EMT master regulators Snail1 and Zeb1 were dispensable. Gene expression profiling provided further evidence for pEMT of TGFβ1-treated organoids and showed that their transcriptomes resemble those of human poor prognosis CMS4 cancers which likewise exhibit pEMT features. We propose that collective invasion in colorectal carcinogenesis is triggered by microenvironmental stimuli through activation of a novel, transcription-mediated form of non-progressive pEMT independently of classical EMT regulators.

Loss of Jag1 cooperates with oncogenic Kras to induce pancreatic cystic neoplasms.

Notch signaling exerts both oncogenic and tumor-suppressive functions in the pancreas. In this study, deletion of Jag1 in conjunction with oncogenic Kras G12D expression in the mouse pancreas induced rapid development of acinar-to-ductal metaplasia and early stage pancreatic intraepithelial neoplasm; however, culminating in cystic neoplasms rather than ductal adenocarcinoma. Most cystic lesions in these mice were reminiscent of serous cystic neoplasm, and the rest resembled intraductal papillary mucinous neoplasm. Jag1 expression was lost or decreased in cystic lesions but retained in adenocarcinoma in these mice, so was the expression of Sox9. In pancreatic cancer patients, JAG1 expression is higher in cancerous tissue, and high JAG1 is associated with poor overall survival. Expression of SOX9 is correlated with JAG1, and high SOX9 is also associated with poor survival. Mechanistically, Jag1 regulates expression of Lkb1, a tumor suppressor involved in the development of pancreatic cystic neoplasm. Collectively, Jag1 can act as a tumor suppressor in the pancreas by delaying precursor lesions, whereas loss of Jag1 promoted a phenotypic switch from malignant carcinoma to benign cystic lesions.

Desmoplasia and oncogene driven acinar-to-ductal metaplasia are concurrent events during acinar cell-derived pancreatic cancer initiation in young adult mice.

The five-year survival rate of patients diagnosed with advanced pancreatic ductal adenocarcinoma (PDAC) has remained static at <5% despite decades of research. With the exception of erlotinib, clinical trials have failed to demonstrate the benefit of any targeted therapy for PDAC despite promising results in preclinical animal studies. The development of more refined mouse models of PDAC which recapitulate the carcinogenic progression from non-neoplastic, adult exocrine subsets of pancreatic cells to invasive carcinoma in humans are needed to facilitate the accurate translation of therapies to the clinic. To study acinar cell-derived PDAC initiation, we developed a genetically engineered mouse model of PDAC, called KPT, utilizing a tamoxifen-inducible Cre recombinase/estrogen receptor (ESR1) fusion protein knocked into the Ptf1a locus to activate the expression of oncogenic KrasG12D and Trp53R270H alleles in mature pancreatic acinar cells. Oncogene-expressing acinar cells underwent acinar-to-ductal metaplasia, and formed pancreatic intraepithelial neoplasia lesions following the induction of oncogene expression. After a defined latency period, oncogene-expressing acinar cells initiated the formation of highly differentiated and fibrotic tumors, which metastasized to the lungs and liver. Whole-transcriptome analysis of microdissected regions of acinar-to-ductal metaplasia and histological validation experiments demonstrated that regions of acinar-to-ductal metaplasia are characterized by the deposition of the extracellular matrix component hyaluronan. These results indicate that acinar cells expressing KrasG12D and Trp53R270H can initiate PDAC development in young adult mice and implicate hyaluronan deposition in the formation of the earliest characterized PDAC precursor lesions (and the progression of pancreatic cancer). Further studies are necessary to provide a comprehensive characterization of PDAC progression and treatment response in KPT mice and to investigate whether the KPT model could be used as a tool to study translational aspects of acinar cell-derived PDAC tumorigenesis.

A novel pancreatic cancer model originated from transformation of acinar cells in adult tree shrew, a primate-like animal.

ABSTRACTPancreatic cancer is one of the most lethal common cancers. The cell of origin of pancreatic ductal adenocarcinoma (PDAC) has been controversial, and recent evidence suggested acinar cells as the most probable candidate. However, the genetic alterations driving the transformation of pancreatic acinar cells in fully mature animals remain to be deciphered. In this study, lentivirus was used as a tool to introduce genetic engineering in tree shrew pancreatic acinar cells to explore the driver mutation essential for malignant transformation, establishing a novel tree shrew PDAC model, because we found that lentivirus could selectively infect acinar cells in tree shrew pancreas. Combination of oncogenic KRASG12D expression and inactivation of tumor suppressor genes Tp53, Cdkn2a and Cdkn2b could induce pancreatic cancer with full penetrance. Silencing of Cdkn2b is indispensable for Rb1 phosphorylation and tumor induction. Tree shrew PDAC possesses the main histological and molecular features of human PDAC. The gene expression profile of tree shrew PDAC was more similar to human disease than a mouse model. In conclusion, we established a novel pancreatic cancer model in tree shrew and identified driver mutations indispensable for PDAC induction from acinar cells in mature adults, demonstrating the essential roles of Cdkn2b in the induction of PDAC originating from adult acinar cells. Tree shrew could thus provide a better choice than mouse for a PDAC model derived from acinar cells in fully mature animals.

IκB Kinase α Is Required for Development and Progression of KRAS-Mutant Lung Adenocarcinoma.

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB-activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKβ in KRAS-mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRASG12D Using NFκB reporter mice and conditional deletions of IKKα and IKKβ, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of RelB, IκBβ, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic KRAS-mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant KRAS for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage in vitro and in vivo IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against KRAS-mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results demonstrate an actionable requirement for IKKα in KRAS-mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease.Significance: These findings report a novel requirement for IKKα in mutant KRAS lung tumor formation, with potential therapeutic applications. Cancer Res; 78(11); 2939-51. ©2018 AACR.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

CDKN2B deletion is essential for pancreatic cancer development instead of unmeaningful co-deletion due to juxtaposition to CDKN2A

Pancreatic cancer is among the deadliest malignancies; however, the genetic events that lead to pancreatic carcinogenesis in adults remain unclear. In vivo models in which these genetic alterations occur in adult animals may more accurately reflect the features of human cancer. In this study, we demonstrate that inactivation of Cdkn2b (p15ink4b) is necessary for induction of pancreatic cancer by oncogenic KRASG12D expression and inactivation of Tp53 and Cdkn2a in adult mouse pancreatic ductal cells (P60 or older). KRASG12D overexpression in these cells activated transforming growth factor-β signaling and expression of CDKN2B, which, along with CDKN2A, led to cellular senescence and protected cells from KRAS-mediated transformation via inhibition of retinoblastoma phosphorylation. These results show a critical role of CDKN2B inactivation in pancreatic carcinogenesis, and provide a useful adult animal model by genetic engineering via lentiviral delivery.

Abstract A66: Molecular profiling of regulatory T cells in a genetic mouse model of lung adenocarcinoma

Abstract The clinical success of immune checkpoint blockade in the treatment of various cancer types provides proof-of-principle that targeting immunosuppression in the tumor microenvironment is an effective therapeutic strategy. Regulatory T cells (Tregs) are a CD4+ T lymphocyte population that may play a dominant role in impairing anti-tumor immune responses. Characterized by their expression of the transcriptional regulator FOXP3, Tregs can inhibit adaptive immune responses through the production of inhibitory cytokines, direct cell killing, competition with other T cell subsets for antigen or other substrates, and suppression of antigen presentation. To recapitulate the strong immunosuppression observed in endogenous tumors, we have used an autochthonous, inducible mouse model of lung adenocarcinoma previously generated in the Jacks lab that is driven by expression of oncogenic KrasG12D and loss of p53 (KP model). Indeed, tumor development in the KP model is accompanied by an expansion of lung-resident Tregs, and previous work in the lab has shown that this population actively restrains anti-tumor T cell responses. In this study, we have transcriptionally profiled lung Tregs in the KP model to identify pathways to preferentially target Tregs in tumor-bearing lungs. We have shown that lung Tregs have a distinct transcriptional phenotype that shares characteristics with that of previously described “effector” Treg populations in a variety of tissue and inflammatory contexts. Furthermore, the KP lung Treg gene signature is associated with survival when applied to the bulk transcriptomes of various tumor types, and is better correlated with patient outcome in lung adenocarcinoma than a number of immune parameters, including mutation burden and expression of CD8A, FOXP3, and GZMA. Together, our data demonstrate that a gene expression signature drawn from cancer-associated Tregs with a distinct, functional phenotype may be useful as a biomarker for clinical outcomes in multiple tumor types. Citation Format: Amy Li, Arjun Bhutkar, Nikhil S. Joshi, Tyler E. Jacks. Molecular profiling of regulatory T cells in a genetic mouse model of lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A66.

Context-Dependent Effects of Amplified MAPK Signaling during Lung Adenocarcinoma Initiation and Progression

Expression of oncogenic KrasG12D initiates lung adenomas in a mitogen-activated protein kinase (MAPK) signal-dependent manner from only a subset of celltypes in the adult mouse lung. Amplification of MAPK signaling is associated with progression to malignant adenocarcinomas, but whether this is a cause or a consequence of disease progression isnot known. To better understand the effects of MAPK signaling downstream of KrasG12D expression, we capitalized on the ability of Braf inhibition to selectively amplify MAPK pathway signaling in KrasG12D-expressing epithelial cells. MAPK signal amplification indeed promoted the rapid progressionof established adenomas to malignant adenocarcinomas. However, we observed, surprisingly, a greater number of overall tumor-initiating events after MAPK signal amplification, due to induced proliferation of cell types that are normally refractory to KrasG12D-induced transformation. Thus, MAPK signaling in the lung is thresholded not only during malignant progression but also at the moment of tumor initiation.

Abstract 4191: Alterations in endothelin axis during pancreatic acinar to ductal metaplasia

Abstract Background: Acinar-ductal-metaplasia (ADM) is one the earliest recognizable alterations in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). Inflammation-induced ADM, which is typically reversible upon removal of inflammatory insult, becomes irreversible in the presence of oncogenic K-Ras and progresses to Pancreatic Intraepithelial neoplastic lesions (PanINs) and subsequently PDAC. Endothelin (ET) axis comprising of endothelin isoforms (ET-1, ET-2 and ET-3) and two receptors A (ETAR) and B (ETBR), has been demonstrated to contribute to the pathobiology of pancreatitis and its components exhibit aberrant overexpression in pancreatic cancer PDAC. However, the expression patterns of ET axis in oncogene-associated early lesions remain unknown. We hypothesize that alterations in ET axis contribute to oncogene associated irreversible ADM. Thus, we studied expression pattern of ET axis in the pancreatic inflammation in the presence and absence of oncogenic KRas and in preneoplastic lesions. Methods: Expression of ET-1, ETAR and ETBR was analyzed in murine models of preneoplastic lesions [KC model: (Pdx1-Cre, KrasG12D)) by IHC. To determine the changes in ET axis during ADM, wild type (WT) and KC mutant mice were treated with cerulein to induce pancreatitis and tissues collected at day 0, 2, 7 and 21 days post treatment were analyzed for mRNA and Immunofluorescence analysis. Expression pattern of ET axis components was also determined in the pancreas of KC and WT animals following exposure to cigarette smoke. Results: A progressive increase in the expression of ET-1, ETAR and ETBR was observed in the PanIN lesions in KC mice. In mice with WT Kras, cerulein treatment resulted in a notable increase in the expression of ET-1 and ETAR at day 2, while the levels of ETBR increased marginally; however a recovery to basal levels was observed for all three molecules by day 7. In contrast, significant increase (p&amp;lt;0.005) in ET-1, ETAR and ETBR transcripts was observed following cerulein treatment and these levels continued to remain high even at 21 days post-trauma in KC mice. The changes in the expression ETAR and ETBR were corroborated by confocal microscopy. Cerulein treatment resulted in increased expression of ETAR and ETBR in acinar compartment in both WT and KC mice as indicated by their co-localization with amylase. In KC mice, cerulein induced acinar-to-ductal metaplasia and the resulting CK19 positive ductal components continued to express increased levels of both receptors with distinct magnitude and kinetics. Similarly, in the smoking models, ET-axis components exhibited a more robust and sustained overexpression in KC mice as compared to the WT. Conclusions: The expression of oncogenic KrasG12D results in an enhanced and sustained activation of ET-axis following inflammatory insults in the pancreatic tissues, suggesting its possible role in tumor initiation and progression. Citation Format: Suprit Gupta, Satyanarayana Rachagani, Sushil Kumar, Kavita Mallya, Surinder Kumar Batra, Maneesh Jain. Alterations in endothelin axis during pancreatic acinar to ductal metaplasia. [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 4191.

Abstract B43: Requirement for PI3 kinase interaction in K-RasG12D-driven leukemogenesis.

Abstract Background: Somatic RAS mutations occur in approximately 30% of human cancers. Oncogenic Ras proteins activate multiple downstream effectors including Raf, PI3 kinase (PI3K), and Ral-GDS. The tyrosine 64 residue, which is located in the Switch II effector binding domain of Ras, is an important contact for binding PI3K catalytic subunits. To investigate the role of PI3K binding in hematopoeitic transformation in vivo, we created a “second site” Lox-STOP-Lox (LSL- KrasG12D/Y64G) knock in allele. Methods: LSL-KrasG12D and LSL-KRasG12D/Y64G mice on a 129/Sv background were crossed with C57BL/6 Mx1-Cre mice. These mice were injected with polyIpolyC (pIpC) at 21 days of age to excise the LSL casette, which was confirmed by QPCR. Colony growth potential of primary bone marrow cells was assessed by sacrificing six to ten week old Mx1-Cre;LSL-KrasG12D, Mx1-Cre;LSL-KrasG12D/Y64G, and wild type mice, isolating bone marrow, and plating 50,000 cells in methylcellulose media containing 0, 0.01, 0.1, 1, or 10 ng/ml GM-CSF. Activation of Ras effector pathways was assessed in macrophages derived from primary bone marrow cells harvested from 6-10 week old mice and grown in the presence of M-CSF. Results: As previously described, inducing oncogenic KrasG12D expression in the hematopoietic compartment of Mx1-Cre;KrasG12D mice results in death by three months of age from an aggressive myeloproliferative neoplasm (MPN) that accurately models chronic and juvenile myelomonocytic leukemias. MPN development is greatly attenuated in Mx1-Cre;LSL-KrasG12D/Y64G mice, which develop anemia beginning at six months of age, but do not display elevated blood leukocyte counts and have &amp;gt;90% survival at eight months. Bone marrow cells from Mx1-Cre;LSL-KrasG12D mice form myeloid progenitor colonies in methylcellulose without added cytokines and show profound hypersensitivity to GM-CSF. By contrast, G12D/Y64G doubly mutant progenitors require GM-CSF for colony formation, and display modestly elevated cytokine sensitivity compared to wild type progenitor cells. Whereas cultured Mx1-Cre;LSL-KrasG12D and Mx1-Cre;LSL-KrasG12D/Y64G macrophages contain markedly elevated levels of active Ras-GTP, they exhibit different patterns of effector pathway activation. In particular, basal levels of phosphorylated ERK and Akt (pERK and pAkt) are unexpectedly higher in Mx1-Cre;LSL-KRasG12D/Y64G macrophages, implicating the PI3K pathway in feedback responses to oncogenic Ras output. Conclusions: Our initial data support an important role of efficient PI3K activation in KrasG12D-driven leukemia and uncover unexpected complexity in cellular responses to Ras-GTP. We are continuing to monitor blood counts in aging Mx1-Cre;LSL-KrasG12D/Y64G mice and are analyzing effects of this mutation on eythroid differentiaion and on hematopoieitc stem and progentior cell (HSPC) populations. In addition, this novel conditional mutant allele will be a valuable genetic tool for assessing the importance of oncogenic-Ras-mediated PI3K signaling in other cancers with frequent somatic KRAS mutations including lung, pancreatic, and colon. Citation Format: Yasmine N. White, Ari J. Firestone, Kevin M. Shannon. Requirement for PI3 kinase interaction in K-RasG12D-driven leukemogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B43.

Abstract 891: FGF21 prevents high fat diet-induced pancreatic cancer in mice expressing oncogenic Kras

Abstract The genetic landscape of pancreatic cancer shows nearly ubiquitous mutations of Kras. However, expression of mutant Kras alone is not sufficient to drive pancreatic ductal adenocarcinoma (PDAC) in adult mice. Obesity is a metabolic disorder and is a risk factor for PDAC. One of the main factors that contributes to the development of obesity is high-fat consumption. Recent studies have demonstrated that High-Fat-Diet (HFD) can promote oncogenic Kras mediated development of pancreatic intraepithelial neoplasia and PDAC. However, it is not clear why mice with oncogenic KrasG12D expression are predisposed to HFD mediated pancreatic carcinogenesis. Here, we report that Ras activity dramatically reduces the expression of fibroblast growth factor 21 (FGF21). FGF21 is a key mediator of fatty acid oxidation and lipid metabolism. Pharmacological doses of FGF21 lower serum free fatty acids and lead to weight loss in obese mice. FGF21 is highly expressed in pancreatic acinar cells and is a potential autocrine factor. High levels expression of FGFR1 and β-Klotho, key components for specific sites of FGF21 action, were observed on normal control mouse pancreatic acinar cells using both qRT-PCR and immunohistochemistry technics. FGF21 levels were dramatically reduced (∼6-fold) by Cre-mediated knock-in of oncogenic KrasG12D within one week and further decreased by more than 100-fold within 7 months. This effect of KrasG12D may have been mediated by decreased expression of pancreatic transcription factor Mist1, which was also observed. To dissect obesity and oncogenic KrasG12D mediated PDAC, we fed adult KrasG12D expressing mice with HFD or HFD+FGF21. We observed that KrasG12D expressing mice fed with HFD+FGF21 for 9 weeks significantly lower body weight and pancreatic triglyceride, as well as dramatically fewer PanIN lesions and tumor, compared with KrasG12D mice on a HFD without FGF21. FGF21 treatment extended survival of KrasG12D expressing mice on a HFD. Our results suggest that pancreas-derived FGF21 is an important metabolic regulator which is silenced by oncogenic KrasG12D expression and plays an important role in preventing HFD induced PDAC in KrasG12D expressing mice. Citation Format: Weiqin Lu, Yaying Yang, Xiaojie Wang, Yan Liu, Yongde Luo, Robert A. Wolff, James L. Abbruzzese, Craig D. Logsdon. FGF21 prevents high fat diet-induced pancreatic cancer in mice expressing oncogenic Kras. [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 891. doi:10.1158/1538-7445.AM2015-891

Mutationally Activated PIK3CAH1047R Cooperates with BRAFV600E to Promote Lung Cancer Progression

Abstract Adenocarcinoma of the lung, a leading cause of cancer death, frequently displays mutational activation of the KRAS proto-oncogene but, unlike lung cancers expressing mutated EGFR, ROS1, or ALK, there is no pathway-targeted therapy for patients with KRAS-mutated lung cancer. In preclinical models, expression of oncogenic KRASG12D in the lung epithelium of adult mice initiates development of lung adenocarcinoma through activation of downstream signaling pathways. In contrast, mutationally activated BRAFV600E, a KRAS effector, fails to initiate lung carcinogenesis despite highly efficient induction of benign lung tumorigenesis. To test if phosphoinositide 3-kinase (PI3K)-α (PIK3CA), another KRAS effector, might cooperate with oncogenic BRAFV600E to promote lung cancer progression, we used mice carrying a conditional allele of Pik3ca that allows conversion of the wild-type catalytic subunit of PIK3CA to mutationally activated PIK3CAH1047R. Although expression of PIK3CAH1047R in the lung epithelium, either alone or in combination with PTEN silencing, was without phenotype, concomitant expression of BRAFV600E and PIK3CAH1047R led to dramatically decreased tumor latency and increased tumor burden compared with BRAFV600E alone. Most notably, coexpression of BRAFV600E and PIK3CAH1047R elicited lung adenocarcinomas in a manner reminiscent of the effects of KRASG12D. These data emphasize a role for PI3K signaling, not in lung tumor initiation per se, but in both the rate of tumor growth and the propensity of benign lung tumors to progress to a malignant phenotype. Finally, biologic and biochemical analysis of BRAFV600E/PIK3CAH1047R-expressing mouse lung cancer cells revealed mechanistic clues about cooperative regulation of the cell-division cycle and apoptosis by these oncogenes. Cancer Res; 73(21); 6448–61. ©2013 AACR.