Kras Mice Research Articles

Dose-dependent effects of Dnmt3a in an inducible murine model of KrasG12D-driven leukemia

DNMT3A mutations are frequently found in clonal hematopoiesis and a variety of hematologic malignancies, including acute myeloid leukemia. An assortment of mouse models have been engineered to explore the tumorigenic potential and malignant lineage bias due to loss of function of DNMT3A in consort with commonly comutated genes in myeloid malignancies, such as Flt3, Nras, Kras, and c-Kit. We employed several tamoxifen-inducible Cre-ERT2 murine model systems to study the effects of constitutively active KrasG12D-driven myeloid leukemia (Kras) development together with heterozygous (3aHet) or homozygous Dnmt3a deletion (3aKO). Due to the rapid generation of diverse nonhematologic tumors appearing after tamoxifen induction, we employed a transplantation model. With pretransplant tamoxifen induction, most Kras mice died quickly of T-cell malignancies regardless of Dnmt3a status. Using posttransplant induction, we observed a dose-dependent effect of DNMT3A depletion that skewed the leukemic phenotype toward a myeloid lineage. Specifically, 64% of 3aKO/Kras mice had exclusively myeloid disease compared with 36% of 3aHet/Kras and only 13% of Kras mice. Here, 3aKO combined with Kras led to increased disease burden, multiorgan infiltration, and faster disease progression. DOT1L inhibition exerted profound antileukemic effects in malignant 3aKO/Kras cells, but not malignant cells with Kras mutation alone, consistent with the known sensitivity of DNMT3A-mutant leukemia to DOT1L inhibition. RNAseq from malignant myeloid cells revealed that biallelic Dnmt3a deletion was associated with loss of cell-cycle regulation, MYC activation, and TNF⍺ signaling. Overall, we developed a robust model system for mechanistic and preclinical investigations of acute myeloid leukemia with DNMT3A and Ras-pathway lesions.

Unraveling CASTOR1's tumor suppressive role: Genetic ablation of CASTOR1 and KRAS-driven mouse model of non-small cell lung cancer.

8528 Background: Cytosolic Arginine Sensor for mTORC1 Subunit 1 (CASTOR1) has emerged as a potent suppressor of mTORC1 activity, playing a pivotal role in various cancer contexts. Previously implicated as a tumor suppressor in multiple cancers, CASTOR1 is tightly regulated in cancer, including inhibition by viral miRNAs in KSHV-induced cancer and degradation via AKT-phosphorylation and RNF167-mediated ubiquitination. Notably, CASTOR1's role extends beyond normal cellular contexts, as evidenced by its tumor-suppressive function in breast cancer xenograft models and its prognostic value in lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). This study aims to elucidate CASTOR1’s role in a transgenic Kras-driven LUAD mouse model, shedding light on its tumor-suppressive potential. Methods: Utilizing a novel CASTOR1 knockout (KO) mouse model crossed with the Kras (LSL-KrasG12D/+) NSCLC model, we established a new mouse model with simultaneous Kras activation and homozygous CASTOR1 ablation. Tumor incidence and growth were monitored in Kras mice harboring wild-type (WT) and KO CASTOR1. Further investigations examined the proliferative dynamics and activation status of various oncogenic pathways, and assessed mTORC1 inhibitor sensitivities in human LUAD cell lines with altered CASTOR1 expression levels. Results: CASTOR1 deficiency markedly heightened lung tumor incidence and size, concomitant with an elevated proliferative index. Intriguingly, a novel antibody identified robust pCASTOR1 levels in CASTOR1 WT mouse LUAD tumors, positively correlated with tumor stage and inversely related to total CASTOR1 abundance, hinting at an active degradation mechanism promoting tumor progression. Elevated mTORC1 signaling, evidenced by increased levels of p4EBP and pS6, underscored the pathway's activation consequent to CASTOR1 loss, likely driving tumor development. Furthermore, heightened pERK levels in CASTOR1 KO tumors unveiled a novel feedback loop intertwining CASTOR1/mTORC1 and the Kras/ERK pathway. Notably, treatment with mTORC1 inhibitors sensitized organoids of Kras tumors with mTORC1 hyperactivation to Kras G12D inhibitor. Conclusions: Our findings elucidate CASTOR1's tumor-suppressive role in Kras-dependent mouse LUAD tumors, suggesting the potential efficacy of combined Kras and mTORC1 inhibition in LUAD subsets characterized by hyperactivated Kras and diminished CASTOR1 levels or mTORC1 hyperactivity.

Abstract 7297: Inhibition of lung tumorigenesis and potentiation of cancer preventive vaccines by a small molecule CA170 targeting the immune checkpoint protein VISTA

Abstract The V-domain Ig Suppressor of T cell Activation (VISTA) is expressed on cells of the myeloid and lymphoid lineages and is an emerging target for cancer immunotherapy. Blocking VISTA activates both innate and adaptive immunity to eradicate tumors in mice. Furthermore, a tripeptide small molecule antagonist of VISTA, CA170, exhibited potent anticancer efficacy on carcinogen-induced mouse lung tumorigenesis. We have previously demonstrated that KRAS or EGFR vaccines are effective in preventing lung tumorigenesis driven by either KRAS or EGFR in a pure prevention setting but are less effective in mice carrying existing lesions. Here, we determined if combining an MHC class II-restricted multi-antigen vaccine targeting KRAS or EGFR with CA170 will provide enhanced efficacy in preventing lung cancer progression. Using tetracycline-inducible KRAS and EGFR transgenic mice, we found that lung tumor development was significantly suppressed when CA170, delivered by aerosol inhalation to minimize systemic toxicity, was combined with either KRAS or EGFR MHCII-directed peptide vaccines using a post-initiation model. Flow cytometry and single-cell RNA sequencing (scRNA-seq) revealed that CA170 significantly increases tumor infiltrating CD8+ T cells and enhances effector-memory T cell frequencies and functions of both CD4+ and CD8+ T cells. These changes coincide with significant reductions in G-MDSCs (granulocytic myeloid-derived suppressor cells) and Foxp3+ Treg populations within lung tumors from mice treated with CA170. We found that the KRAS and EGFR preventive vaccines primarily induced the expansion of CD4+ effector T cells. VISTA antagonism by CA170 revealed strong efficacy against lung tumorigenesis with broad immunoregulatory functions that influence effector, memory, and regulatory T cells, and drive an adaptive T cell tumor-specific immune response that enhances the efficacy of the KRAS and EGFR preventive vaccines in transgenic mouse models of lung cancer. Citation Format: Qi Zhang, Jing Pan, Donghai Xiong, Yian Wang, Mark S. Miller, Ming You. Inhibition of lung tumorigenesis and potentiation of cancer preventive vaccines by a small molecule CA170 targeting the immune checkpoint protein VISTA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7297.

Deficient Rnf43 potentiates hyperactive Kras-mediated pancreatic preneoplasia initiation and malignant transformation.

BackgroundLargely due to incidental detection, asymptomatic pancreatic cystic lesions (PCLs) have become prevalent in recent years. Among them, intraductal papillary mucinous neoplasm (IPMN) infrequently advances to pancreatic ductal adenocarcinoma (PDAC). Conservative surveillance versus surgical intervention is a difficult clinical decision for both caregivers and PCL patients. Because RNF43 loss‐of‐function mutations and KRAS gain‐of‐function mutations concur in a subset of IPMN and PDAC, their biological significance and therapeutic potential should be elucidated.MethodsPancreatic Rnf43 knockout and Kras activated mice (Rnf43 −/−; KrasG12D ) were generated to evaluate their clinical significance in pancreatic pre‐neoplastic initiation and malignant transformation.ResultsLoss of Rnf43 potentiated the occurrence and severity of IPMN and PDAC in oncogenic Kras mice. The Wnt/β‐catenin signaling pathway was activated in pancreatic KrasG12D and Rnf43 knockout mice and the PORCN inhibitor LGK974 blocked pancreatic IPMN initiation and progression to PDAC accordingly.ConclusionsRnf43 is a tumor suppressor in the prevention of pancreatic malignant transformation. This genetically reconstituted autochthonous pancreatic Rnf43 −/−; KrasG12D preclinical cancer model recapitulates the pathological process from pancreatic cyst to cancer in humans and can be treated with inhibitors of Wnt/β‐catenin signaling. Since the presence of RNF43 and KRAS mutations in IPMNs predicts future development of advanced neoplasia from PCLs, patients with these genetic anomalies warrant surveillance, surgery, and/or targeted therapeutics such as Wnt/β‐catenin inhibitors.

Abstract 109: Vaccination with Polyclonal Antibody Stimulator (PAS) prevents pancreatic carcinogenesis in the KRAS mouse model

Abstract Background: Survival from pancreatic cancer is poor in part because it is not recognized or treated in the precancerous or early cancer stages. Gastrin is expressed in the fetal pancreas but silenced in the adult pancreas. Gastrin is re-expressed in precancerous pancreatic intraepithelial neoplasia (PanINs) where it acts to promote epithelial growth and fibrosis of the extracellular matrix. When gastrin is knocked-out in KRAS mice, PanINs fail to progress to cancer. Polyclonal Antibody Stimulator (PAS) is a target-specific vaccine to gastrin that activates B and T-cells to neutralize the actions of gastrin Hypothesis: It was hypothesized that treatment of transgenic LSL-KrasG12D/+; P48-Cre (KC) mice with PAS will slow PanIN progression and prevent pancreatic cancer. Methods: Nineteen age-matched littermates of KC mice were divided into two groups: control/untreated (N=9) and PAS-treated (N=10). Starting when the mice were 3 months of age, when PanIN lesions are developing, the PAS-mice received an initiation dose of PAS 250µg subcutaneously (sc) at baseline, week 1 and week 3. The PAS-treated mice then received a booster dose of PAS 250µg sc every 4 weeks until the mice reached 8 months of age for a total of 4 boosters. The control mice were untreated. The pancreata were dissected and paraffin embedded for analysis. H&E stained slides were evaluated for PanIN stage, extent of PanINs, inflammation, and fibrosis by a pathologist blinded to the treatment. Sections were stained with Masson's trichrome for fibrosis analysis and arginase for M2 macrophages. Results: When euthanized at 8-months of age, 67% of the control mice had high grade PanIN-3 lesions and 33.3% had invasive carcinoma. In contrast, the histologic stage in the age-matched PAS- treated mice was significantly lower, with more stage PanIN-2 lesions and only 10% with invasive carcinoma. The volume of normal pancreas present in the PAS-treated mice was 60% greater than in the control mice. Quantitation of the extracellular matrix showed 50% less fibrosis in the mice vaccinated with PAS as compared to controls (P=0.0001). Pancreata of PAS-treated mice also had fewer M2-polarized tumor-promoting macrophages than control mice (P<0.001). Conclusion: Pancreatic cancer and the progression of PanIN lesions can be prevented by a vaccine that targets gastrin. PAS treatment alters the microenvironment rendering it less carcinogenic. This vaccine has the potential to decrease pancreatic cancer in high risk populations and to prevent recurrence in surgically treated patients. Citation Format: Jill P. Smith, Hong Cao, Bhaskar Kallakury, Teresa Phillips, Lynda Sutton, Allen Cato. Vaccination with Polyclonal Antibody Stimulator (PAS) prevents pancreatic carcinogenesis in the KRAS mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 109.

Changes and sex- and age-related differences in the expression of drug metabolizing enzymes in a KRAS-mutant mouse model of lung cancer.

BackgroundThis study aimed to systematically profile the alterations and sex- and age-related differences in the drug metabolizing enzymes (DMEs) in a KRAS-mutant mouse model of lung cancer (KRAS mice).MethodologyIn this study, the LC-MS/MS approach and a probe substrate method were used to detect the alterations in 21 isoforms of DMEs, as well as the enzymatic activities of five isoforms, respectively. Western blotting was applied to study the protein expression of four related receptors.ResultsThe proteins contents of CYP2C29 and CYP3A11, were significantly downregulated in the livers of male KRAS mice at 26 weeks (3.7- and 4.4-fold, respectively, p < 0.05). SULT1A1 and SULT1D1 were upregulated by 1.8- to 7.0- fold at 20 (p = 0.015 and 0.017, respectively) and 26 weeks (p = 0.055 and 0.031, respectively). There were positive correlations between protein expression and enzyme activity for CYP2E1, UGT1A9, SULT1A1 and SULT1D1 (r2 ≥ 0.5, p < 0.001). Western blotting analysis revealed the downregulation of AHR, FXR and PPARα protein expression in male KRAS mice at 26 weeks. For sex-related differences, CYP2E1 was male-predominant and UGT1A2 was female-predominant in the kidney. UGT1A1 and UGT1A5 expression was female-predominant, whereas UGT2B1 exhibited male-predominant expression in liver tissue. For the tissue distribution of DMEs, 21 subtypes of DMEs were all expressed in liver tissue. In the intestine, the expression levels of CYP2C29, CYP27A1, UGT1A2, 1A5, 1A6a, 1A9, 2B1, 2B5 and 2B36 were under the limitation of quantification. The subtypes of CYP7A1, 1B1, 2E1 and UGT1A1, 2A3, 2B34 were detected in kidney tissue.ConclusionsThis study, for the first time, unveils the variations and sex- and age-related differences in DMEs in C57 BL/6 (WT) mice and KRAS mice.

Inhibition of miR-21 Regulates Mutant KRAS Effector Pathways and Intercepts Pancreatic Ductal Adenocarcinoma Development.

Almost all pancreatic ductal adenocarcinomas (PDA) develop following KRAS activation, which triggers epithelial transformation and recruitment of desmoplastic stroma through additional transcriptional and epigenetic regulation, but only a few of these regulatory mechanisms have been described. We profiled dysregulated miRNAs starting with the earliest premalignant pancreatic intraepithelial neoplasias (PanIN) in genetically engineered mutated KRAS and P53 (KPC) mice programmed to recapitulate human PDA tumorigenesis. We identified miR-21 and miR-224 as cell-specific and compartment-specific regulators in PanINs and PDA. miR-21 is overexpressed in tumor epithelial cells of premalignant ducts, while miR-224 is overexpressed in cancer-associated fibroblasts in PDA stroma. Inhibition of miR-21 reverted protumorigenic functionalities to baseline levels. Overexpression of miR-224 induced activated phenotypes in normal fibroblasts. In vivo miR-21 inhibition improved survival in established PDA. Importantly, early systemic miR-21 inhibition completely intercepted premalignant progression. Finally, an evaluation of miR-21 expression in the PDA cohort of The Cancer Genome Atlas identified a correlation between tumor epithelial cell content and miR-21 expression in human tumors providing further rationale for conducting human studies. Thus, miR-21 may be useful for early PanIN detection, and for intercepting developing premalignant pancreatic lesions and other KRAS-driven premalignancies.

Abstract 5222: Repression of neuronal genes protects the pancreas from certain injuries and aberrant plasticity

Abstract Among the major cancer types, pancreatic ductal adenocarcinoma has one of the lowest survival rates with little improvement over the past 3 decades. To address this dire clinical need, this study explores the unique and underexploited neuronal property of the pancreas that correlates with greater injury and pancreatic cancer development. The pancreas has a remarkable innate plasticity, and a shift toward neuroendocrine transdifferentiation has been shown to correlate with poor survival and tumor progression in numerous cancers, such as lung and prostate cancers. Recent reports show a subpopulation of cells within pancreatic tumors that express neuroendocrine-related genes correlate with poor outcome and chemoresistance. We hypothesize one regulator of this ductal-neuroendocrine lineage plasticity may be REST, a transcriptional repressor most known for orchestrating neuronal development. Tissue microarray of human pancreatic cancer shows patients negative for REST had a lower overall survival than patients positive for REST. To investigate the role of REST in the pancreas, we developed a novel transgenic mouse model with REST conditionally knocked out of the pancreas. REST KO resulted in an increase of neuroendocrine markers and an increase of zymogen granules, resulting in a lighter, cloudy appearance of the pancreas. When pancreatic injury was introduced by injections of cholecystokinin analog caerulein to induce acute pancreatitis, the REST KO mice show greater serum amylase levels and higher pancreatic edema indicating greater pancreatitis. Furthermore, an increase in neuronal-related genes was observed within REST KO pancreas relative to control. We then investigated the role of REST during cancer-promoting injuries such as chronic pancreatitis. Mice were administered a 10-week series of caerulein injections, and histology was evaluated. As expected, REST KO mice experienced greater inflammation, injury, and pre-neoplastic lesions. Further directions include investigating REST KO during later stages of pancreatic cancer injury by crossing REST KO mice with mutant KRAS mice, as oncogenic KRAS is a leading driver of pancreatic cancer. We expect REST KO to increase neuroendocrine properties that will accelerate KRAS-induced pancreatic cancer development. In conclusion, we hypothesize that loss of REST increases neuroendocrine-like properties of the pancreas that leads to a greater injury, suggesting that REST plays a protective effect against pancreatic damage and pancreatic cancer development. Furthermore, our novel REST KO mice may prove to be a useful model to investigate how neuroendocrine properties may promote pancreatic cancer development and drug resistance. Understanding neuronal influences during pancreatic cancer development could have multiple implications for cancer patients and warrants further investigation. Citation Format: Julie K. Bray, Ola Elgamal, Lais Da Silva, Dhruvit Sutaria, Xiuli Liu, Kristianna Fredenburg, Thomas D. Schmittgen. Repression of neuronal genes protects the pancreas from certain injuries and aberrant plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5222.

Levels of the Autophagy-Related 5 Protein Affect Progression and Metastasis of Pancreatic Tumors in Mice

Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5(Atg5) protein on pancreatic tumor formation and progression. We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/-;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration andinvasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. A5+/-;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/-;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin Land D. The tumors that formed in A5+/-;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/-;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.

Abstract A35: Vesicular secretion of suppressor of cytokine signaling 3 by alveolar macrophages is dysregulated in NSCLC and its provision inhibits tumor cell function

Abstract Rationale: Inadequate expression of suppressor of cytokine signaling 3 (SOCS3) with subsequent activation of its target, the transcription factor STAT3, has been implicated in tumorigenesis and cancer progression in the lung and other organs. Our lab has recently reported the novel capability of alveolar macrophages (AMs) to secrete SOCS3 within microvesicles (MVs). While AM delivery of MV-encapsulated SOCS3 was shown to suppress inflammatory signaling in recipient lung epithelial cells, the potential significance of this process in restraining the development of lung cancer has not been studied. Methods: A KRAS G12D mutant mouse model was utilized to determine dysfunction of AM SOCS3 secretion in lung cancer. Mice were administered adenoviral Cre recombinase via intratracheal instillation, resulting in formation of lung tumors after 16 weeks. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from the lungs of KRAS and wild-type (WT) mice, and analysis of SOCS3 secretion in BAL or AM cell culture medium was done via ELISA after sonication to disrupt vesicles. In vitro experiments utilized human adenocarcinoma cells (A549) or KRAS mutant rat lung epithelial cells (RLE-G12V). Proliferation, apoptosis and transformation were assessed by Cyquant assay, Annexin V staining, and soft agar assay, respectively. For SOCS3 provision studies, natural AM-derived MVs (isolated by ultracentrifugation) or synthetic liposomes containing recombinant SOCS3 were utilized. Results: Levels of secreted SOCS3 were ~50% lower in KRAS mice BAL than in WT BAL fluid. Additionally, although AMs isolated from KRAS mice contained similar amounts of intracellular SOCS3 and released similar numbers of MVs as those from WT mice, their ex vivo capacity for SOCS3 secretion was significantly lower than that of WT AMs. To determine whether provision of exogenous SOCS3 could inhibit tumorigenesis, synthetic SOCS3 liposomes were administered to RLE-G12V cells prior to chemical transformation with N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG). Addition of exogenous SOCS3 had the capacity to significantly inhibit colony formation in soft agar. To investigate effects of exogenous SOCS3 on established tumor cell function, A549 cells were exposed to both natural AM-derived MVs and SOCS3-containing liposomes and effects on proliferation and apoptosis were measured. Both liposomes and natural MVs significantly induced apoptosis and inhibited proliferation. Finally, the reduction in secreted SOCS3 observed in the mouse model was confirmed in BAL samples of a cohort of NSCLC lung cancer patients compared to healthy volunteers. Conclusion: We report a novel dysregulation of immune surveillance in the form of decreased SOCS3 secretion by AMs that is elicited by the tumor microenvironment, and that may promote tumorigenesis via sustained STAT3 activation. Future studies will focus on the mechanism underlying this defect and whether rescuing SOCS3 secretion can inhibit cancer progression in vivo. Citation Format: Jennifer M. Speth, Loka R. Penke, Joseph Bazzill, Daniel J. Schneider, Douglas A. Arenberg, James J. Moon, Venkateshwar G. Keshamouni, Marc Peters-Golden. Vesicular secretion of suppressor of cytokine signaling 3 by alveolar macrophages is dysregulated in NSCLC and its provision inhibits tumor cell function [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A35.

Abstract 4988: PI3Kγ-deficiency protects against pancreatic tumorigenesis at the expense of diet-Induced hyperlipidemia and hepatotoxicity

Abstract Pancreatic ductal adenocarcinoma (PDAC) presents a significant challenge clinically, with poor overall survival and widespread resistance to conventional therapies. Several novel molecular targets are being considered in the management of PDAC patients. To this end, as hyperactive PI3K signaling is implicated in both disease incidence and progression, the inhibition of PI3K p110γ isoform is showing early promise in vivo and has been proposed as a reasonable target for therapy. Classically, p110γ is expressed predominantly in macrophages. However, using both primary pancreatic cancer samples and established patient databases, we found that p110γ is commonly upregulated in the cancer epithelium. Further, p110γ is expressed in human cell lines, and the pharmacologic inhibition of p110γ with AS-604850 both delayed mtKRAS/mtTP53-induced acinar-to-ductal metaplasia ex vivo, and sensitized tumor cells to gemcitabine in vitro. Given the apparent importance of epithelial p110γ to the neoplastic phenotype, we next generated mice with constitutive activation of p110γ targeted to the exocrine pancreas (PI3KγCA). When combined with a KRAS activating mutation, PI3KγCA mice developed highly advanced disease forms with robust fibrosis, inflammation, and AKT activation/proliferation. In accordance with these observations, both homo- and heterozygous global deletion of p110γ protected against KRAS-induced tumorigenesis, reducing downstream AKT signaling and proliferation in vivo. However, despite the reduction in tumor burden, mutant KRAS mice with p110γ-deficiency displayed early signs of hepatotoxicity. Since p110γ has several key roles in lipid metabolism, we next assessed p110γ loss in the setting of a high-fat diet (HFD). These mtKRAS/p110γKO mice developed pronounced hyperlipidemia and severe hepatic cell injury/steatosis. Additionally, the previously observed protective effect in the pancreas was markedly diminished, and neoplastic tissues displayed pronounced AKT pathway activation, indicating potential compensatory signaling through non-p110γ isoforms. Indeed, how p110γ loss and lipid accumulation in liver impact development of mtKRAS-induced neoplasia and cancer is a very interesting future aim. Combined, these observations suggest that systemic p110γ inhibition should be carefully considered, particularly given the potential for reduced efficacy, hyperlipidemia, and hepatic injury in obese patients, unless specific targeting to the pancreas can be achieved. Citation Format: Carolina Torres, Daniel R. Principe, Alex Park, Ronald McKinney, Matthew J. Dorman, Ajay Rana, Emilio Hirsch, Jose Cordoba-Chacon, Paul J. Grippo. PI3Kγ-deficiency protects against pancreatic tumorigenesis at the expense of diet-Induced hyperlipidemia and hepatotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4988.

Abstract 441: NOX2 inhibition reduces oxidative stress and prolongs survival of mice with Kras induced myeloproliferative disease

Abstract 0 0 1 300 1716 University of Gothenburg 14 4 2012 14.0 Normal 0 false false false SV JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:SV;} Mutations leading to Ras gene activation are important contributors to myeloid leukemogenesis. Kras is one of three homologues of the Ras family, and oncogenic Kras is frequently found in monocytic forms of acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and myeloproliferative diseases (MPD). Earlier studies show that excessive amounts of reactive oxygen species (ROS) are produced in Kras-mutated myeloid malignancies. Myeloid cells, including Kras-mutated leukemic cells, express the ROS-generating enzyme NOX2. Relatively little is known about the impact of ROS formation, in particular NOX2-derived ROS formation, on leukemogenesis. Mice where oncogenic Kras was induced in hematopoietic cells of LSL-KrasG12D and Mx1-Cre double transgenic mice with ensuing myeloproliferation were treated with a histamine H2-receptor selective NOX2 inhibitor, Nα -methyl histamine (NMH) to assess whether NOX2 is a feasible therapeutic target in Kras-driven leukemia. Spleens of diseased Kras mice were infiltrated with mature CD11b+Gr1+ myeloid cells that expressed NOX2 and produced ROS. The NOX2-dependent formation of ROS in CD11b+Gr1+ cells was inhibited by NMH. In vivo administration of NMH delayed the development of myeloproliferative disease and significantly prolonged survival of Kras mice (p=0.003). In contrast, treatment with NMH did not alter the survival of mice when Kras expression was induced in hematopoietic cells of NOX2-deficient mice that did not produce ROS (Nox2-/-, LSL-KrasG12D and Mx1-Cre triple transgenic mice). In Kras-mutated Nox+/+ mice, but not in Kras-mutated Nox-/- mice, the in vivo administration of NMH entailed diminished ROS levels and reduced oxidative stress as reflected by reduced DCFDA and anti-8 Hydroxyguanosine staining. We propose that NOX2-derived ROS in Kras-mutated hematopoetic cells mediate genomic instability that may contribute to the progression of leukemia. Strategies to target NOX2 should be further evaluated in Ras-mutated hematopoietic cancer. &amp;lt;!–EndFragment–&amp;gt; Citation Format: Ebru Aydin, Alexander Hallner, Hanna Grauers Wiktorin, Anna Staffas, Kristoffer Hellstrand, Anna Martner. NOX2 inhibition reduces oxidative stress and prolongs survival of mice with Kras induced myeloproliferative disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 441.

PPARγ-activation increases intestinal M1 macrophages and mitigates formation of serrated adenomas in mutant KRAS mice

ABSTRACTTo identify novel hubs for cancer immunotherapy, we generated C57BL/6J mice with concomitant deletion of the drugable transcription factor PPARγ and transgenic overexpression of the mutant KRASG12V oncogene in enterocytes. Animals developed epithelial hyperplasia, transmural inflammation and serrated adenomas in the small intestine with infiltration of CD3+ FOXP3+ T-cells and macrophages into the lamina propria of the non-malignant mucosa. Within serrated polyps, CD3+ CD8+ T-cells and phosphorylated ERK1/2 were reduced and the senescence marker P21 and macrophage counts up-regulated, indicative of an immunosuppressive tissue microenvironment. Treatment of mutant KRASG12V mice with the PPARγ-agonist rosiglitazone augmented M1 macrophage numbers, reduced IL4 expression and diminished polyp load in mice. Rosiglitazone also promoted M1 polarisation of human THP1-derived macrophages and decreased Il4 mRNA in isolated murine lymphocytes. Thus, inhibition of the oncogenic driver mutant RAS by PPARγ in epithelial and immune cell compartments may be a future target for the prevention or treatment of human malignancies associated with intestinal inflammation.

Hdac7 promotes lung tumorigenesis by inhibiting Stat3 activation

BackgroundLung cancer is the leading cause of cancer death worldwide. However, the molecular mechanisms underlying lung cancer development have not been fully understood. The functions of histone deacetylases (HDACs), a class of total eighteen proteins (HDAC1–11 and SIRT1–7 in mammals) that deacetylate histones and non-histone proteins, in cancers are largely unknown.MethodsHdac7+/−/K-Ras mice and HDAC7-depleted human lung cancer cell lines were used as models for studying the function of Hdac7 gene in lung cancer. Kaplan-Meier survival analysis was performed to explore the relationship between HDAC7 expression and prognosis of human lung cancers. Recombinant lentivirus-mediated in vivo gene expression or knockdown, Western blotting, and pull-down assay were applied to investigate the underlying molecular mechanism by which Hdac7 promotes lung tumorigenesis.ResultsThe number and burden of lung tumor were dramatically reduced in Hdac7+/−/K-Ras mice compared to control K-Ras mice. Also, in Hdac7+/−/K-Ras mice, cell proliferation was significantly inhibited and apoptosis in lung tumors was greatly enhanced. Similarly, cell proliferation and anchorage-independent growth of human lung cancer cell lines expressing shHDAC7 were also significantly suppressed and apoptosis was dramatically elevated respectively. Mechanistic study revealed that Hdac7 mutation in mouse lung tumors or HDAC7 depletion in human tumor cell lines resulted in significantly enhanced acetylation and tyrosine-phosphorylation of Stat3 and HDAC7 protein directly interacted with and deacetylateed STAT3. The Hdac7 mutant-mediated inhibitory effects on lung tumorigenesis in mice and cell proliferation/soft agar colony formation of human lung cancer cell lines were respectively reversed by expressing dnStat3. Finally, the high HDAC7 mRNA level was found to be correlated with poor prognosis of human lung cancer patients.ConclusionOur study suggests that Hdac7 promotes lung tumorigenesis by inhibiting Stat3 activation via deacetylating Stat3 and may shed a light on the design of new therapeutic strategies for human lung cancer.

Abstract LB-133: De novo expression of gastrin and CCK-B receptors in cell lines derived from KPC mice

Abstract Background: Animal models to study cancer progression and therapeutics are essential but these animal models are most useful when they resemble human cancers, particularly with respect to inflammation and the immune system in carcinogenesis and therapy. Hence, athymic nude mice bearing human cancer explants are being replaced by genetically modified animal models with intact immune systems. Panc02 cancer cells have been used to create a syngeneic immune competent murine model of pancreatic cancer. However, unlike human pancreatic cancer, Panc02 cells express wild-type KRAS and CCK-A type receptors (rather than CCK-B type receptors). We previously showed that the Pdx1-Cre / LSL-KrasG12D transgenic mice develop de novo CCK-B receptors with PanIN progression. Aim: The goal of this investigation was to examine cell cultures derived from the KrasG12D mice to determine if they are indeed similar to human cancers in regard to in vivo growth properties and expression of gastrin and CCK-B receptors. Methods: Five different pancreatic cell lines were obtained that had been derived from the KPC mice (KPC1, KPC2) or organoids (MT3-2D, MT4-2D, and MT5-2D) of the KPC mice. RNA was extracted from the cell lines and evaluated for CCK-A and CCK-B receptors and the ligand gastrin. Growth studies were characterized in syngeneic C57BL/6 mice. The effects of CCK receptor blockade with proglumide was evaluated on tumor growth in vivo. Histologic sections were stained with H&amp;E, Trichrome, and a PD-L1 Ab.Results: RT-PCR revealed that the KPC and MT-2D cells no longer express the CCK-A receptor found in normal mouse pancreas but all expressed the CCK-B receptor, characteristic of human pancreatic cancers. Gastrin expression was also detected by RT-PCR in all cells. Subcutaneous tumors grew rapidly to 1000mm3 within 21 days after inoculation of 105 cells. Tumor growth in MT3-2D tumors was significantly inhibited and survival of mice prolonged with the CCK-receptor antagonist proglumide compared to PBS controls (p=0.0048). Tumors showed extensive fibrosis characteristic of the human PDAC microenvironment and all tumors had PD-L1 expression. Conclusion: These studies show that cell lines developed from PanIN lesions in KRAS mice exhibit phenotypic and genetic characteristics comparable to human pancreatic cancers and therefore are promising models to study the relationship of CCK receptor-mediated biology to the development and therapy of pancreatic cancer. De novo expression of the CCK-B receptor and gastrin from mouse pancreas suggests a possible relationship between CCK receptor signaling and mutant KRAS expression.Supported by NCI CA50633, CA51008, and CA194745 Citation Format: Jill P. Smith, Shangzi Wang, Louis Weiner, Sandeep Nadella. De novo expression of gastrin and CCK-B receptors in cell lines derived from KPC mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-133. doi:10.1158/1538-7445.AM2017-LB-133

Interleukin-17A Promotes Lung Tumor Progression through Neutrophil Attraction to Tumor Sites and Mediating Resistance to PD-1 Blockade

IntroductionProinflammatory cytokine interleukin-17A (IL-17A) is overexpressed in a subset of patients with lung cancer. We hypothesized that IL-17A promotes a protumorigenic inflammatory phenotype and inhibits antitumor immune responses. MethodsWe generated bitransgenic mice expressing a conditional IL-17A allele along with conditional KrasG12D and performed immune phenotyping of mouse lungs, a survival analysis, and treatment studies with antibodies either blocking programmed cell death 1 (PD-1) or IL-6 or depleting neutrophils. To support the preclinical findings, we analyzed human gene expression data sets and immune profiled patient lung tumors. ResultsTumors in IL-17:KrasG12D mice grew more rapidly, resulting in a significantly shorter survival as compared with that of KrasG12D mice. IL-6, granulocyte colony-stimulating factor (G-CSF), milk fat globule-EGF factor 8 protein, and C-X-C motif chemokine ligand 1 were increased in the lungs of IL17:Kras mice. Time course analysis revealed that levels of tumor-associated neutrophils were significantly increased, and lymphocyte recruitment was significantly reduced in IL17:KrasG12D mice as compared with in KrasG12D mice. In therapeutic studies PD-1 blockade was not effective in treating IL-17:KrasG12D tumors. In contrast, blocking IL-6 or depleting neutrophils with an anti–Ly-6G antibody in the IL17:KrasG12D tumors resulted in a clinical response associated with T-cell activation. In tumors from patients with lung cancer with KRAS mutation we found a correlation between higher levels of IL-17A and colony- stimulating factor 3 and a significant correlation among high neutrophil and lower T-cell numbers. ConclusionsHere we have shown that an increase in a single cytokine, IL-17A, without additional mutations can promote lung cancer growth by promoting inflammation, which contributes to resistance to PD-1 blockade and sensitizes tumors to cytokine and neutrophil depletion.

RelA regulates CXCL1/CXCR2-dependent oncogene-induced senescence in murine Kras-driven pancreatic carcinogenesis.

Tumor suppression that is mediated by oncogene-induced senescence (OIS) is considered to function as a safeguard during development of pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms that regulate OIS in PDAC are poorly understood. Here, we have determined that nuclear RelA reinforces OIS to inhibit carcinogenesis in the Kras mouse model of PDAC. Inactivation of RelA accelerated pancreatic lesion formation in Kras mice by abrogating the senescence-associated secretory phenotype (SASP) gene transcription signature. Using genetic and pharmacological tools, we determined that RelA activation promotes OIS via elevation of the SASP factor CXCL1 (also known as KC), which activates CXCR2, during pancreatic carcinogenesis. In Kras mice, pancreas-specific inactivation of CXCR2 prevented OIS and was correlated with increased tumor proliferation and decreased survival. Moreover, reductions in CXCR2 levels were associated with advanced neoplastic lesions in tissue from human pancreatic specimens. Genetically disabling OIS in Kras mice caused RelA to promote tumor proliferation, suggesting a dual role for RelA signaling in pancreatic carcinogenesis. Taken together, our data suggest a pivotal role for RelA in regulating OIS in preneoplastic lesions and implicate the RelA/CXCL1/CXCR2 axis as an essential mechanism of tumor surveillance in PDAC.

Abstract 2621: Disruption of mucin synthesis by targeting GCNT3 inhibits pancreatic cancer progression

Abstract Pancreatic cancer (PC) is a lethal disease, and its management is an ongoing challenge. PC is the fourth leading cause of deaths due to cancer in the United States. It is a highly aggressive cancer that is usually diagnosed at an advanced stage, and has the worst prognosis of any malignancy, with a five year survival of &amp;lt;7% due to high chemoresistance. This chemoresistance is due in part to altered expressions of mucins, which form a mesh that makes target sites inaccessible to drugs. Clinical and preclinical studies have shown aberrant expression of mucins during PC development. The mucins may prevent drugs from accessing their sites of action. Although several mucins that lead to chemoresistance have been targeted, to date, no mucin synthesizing genes have been identified as targets. Using human pancreatic cancer patient survival data (90 cases of tumor and matched normal adjacent tissue), next-generation sequencing (NGS) of genetically engineered Kras mouse pancreatic tumors (N = 6/group), human PC cells, we identified novel core mucin synthesizing gene target GCNT3 (core 2 beta 1,6 N-acetylglucosaminyltransferase). NGS revealed that GCNT3 upregulation (103-fold; p&amp;lt;0.0001) was correlated with increased mucins Muc4 (50-fold; p&amp;lt;0.04), Muc5ac (87-fold; p&amp;lt;0.01), Muc6 (67-fold; p&amp;lt;0.008), Muc1 (5-fold; p&amp;lt;0.009), Muc16 (5-fold; p&amp;lt;0.0003) and Muc20 (17-fold; p&amp;lt;0.007)]. Aberrant GCNT3 expression was associated with increased mucin production and aggressive tumorigenesis and reduced patient survival. Patients with low expression of GCNT3 had a longer survival time than patients with high expression of GCNT3 (median survival: 17.5 vs. 10.5 months, p = 0.036). Further, using in-silico approaches of small molecular docking simulations, we identified talniflumate as a novel inhibitor that specifically binds to GCNT3. Our blind docking simulations reveal that talniflumate binds to GNTC3 with a docking affinity of -8.3 kcal/mol and deeper in the pocket of GCNT3. The docking predictions suggest three notable hydrogen bonds between talniflumate and GCNT3: Arg192 (3.0 Angstroms), Try288 (3.5 Angstroms), and Ala287 (2.9 Angstroms). Pancreata from 6-week-old Kras mice treated with talniflumate for 1 week showed a significant decrease in GCNT3 and mucin expression in PanIN lesions. mRNA expression of GCNT3 was also observed to be lower in pancreatic tissues from talniflumate-treated mice. CRISPR knock-out of GCNT3 in PC cells reduced proliferation and spheroid formation. Further, talniflumate alone and in combination with low-dose gefitinib reduced GCNT3 leading to disruption of mucins in vivo and in vitro. Hence, mucin disruption might enhance targeted therapy. These findings suggest a prominent role for Kras activation and aberrant mucin synthesis leading to PC pathogenesis, and warrant consideration of GCNT3 and EGFR inhibitors as a combination treatment for PC. (Grant Support: COMAA, Kerley-Cade Endowed Fund). Citation Format: Altaf Mohammed, Naveena B. Janakiram, Venkateshwar Madka, Gaurav Kumar, Scott Edgar, Gopal Pathuri, Taylor Bryant, Hannah Kutsche, Yuting Zhang, Laura Biddick, Hariprasad Gali, Yan Daniel Zhao, Stan Lightfoot, Chinthalapally V. Rao. Disruption of mucin synthesis by targeting GCNT3 inhibits pancreatic cancer progression. [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 2621.

Abstract 1054: SNAI1 regulates the hexosamine biosynthetic pathway to promote tumorigenesis and oncogene-induced senescence escape in lung cancer

Abstract Lung cancer is the deadliest and most common malignancy worldwide. Tumor invasion and metastasis influence the dismal 16% overall 5-year survival. Evidence suggests malignancies commandeer epithelial plasticity programs like the epithelial-mesenchymal transition (EMT) to promote tumorigenesis, tumor progression, and metastasis. Furthermore, EMT has recently been linked to alterations in cancer metabolism, which can engender adaptation to environmental stress while supporting the macromolecular demand of rapid proliferation. Thus it is important to better understand the emerging roles of EMT in regulating cancer metabolism. SNAI1 is a master EMT regulator and is aberrantly overexpressed in many cancers including lung cancer. To investigate the role of SNAI1 in tumorigenesis, we created a novel inducible autochthonous mouse model. Our model reveals SNAI1 overexpressing mutant Kras mice generate identifiable tumors twice as fast (median = 14 weeks) as mice overexpressing mutant Kras alone (median = 30 weeks). To identify possible mechanisms of SNAI1-accelerated tumorigenesis, we used mRNA expression profiling. We identified significantly altered gene signatures related to metabolic reprogramming. Specifically, SNAI1 upregulates the hexosamine biosynthetic pathway (HBP) in mutant Kras-driven mouse lung tumors. SNAI1 elevates protein and mRNA of the rate limiting step (GFPT2) and final step (UAP1) of this pathway. This SNAI1-driven promotion of the HBP was recapitulated in vitro using human non-small cell lung cancer (NSCLC) SNAI1 isogenic cell lines. NSCLC mRNA expression data from The Cancer Genome Atlas (TCGA) also showed SNAI1 expression in patient tumor samples correlate with a significant tendency towards co-expression with HBP genes (Fisher's exact test, p&amp;lt;0.05). Functionally, the final metabolite generated by the HBP, UDP-GlcNAc, is an essential nucleotide sugar found in a plethora of N- and O-linked glycosylations, regulating protein stability and activity. Here we show SNAI1 promotes flux through the HBP to promote increased total levels of O-GlcNAcylated proteins in lung tumors in vivo. In so doing, modulation of the HBP and specific glycosylations may impact cell death and senescence, suggesting a novel targetable EMT-HBP axis. Numerous oncogenes, including c-Myc and SNAI1 itself, are known to be stabilized by O-GlcNAcylation. This link is becoming of increasing importance in cancer biology, as half of the body of literature on O-GlcNAcylation and cancer has been published in the past three years. Together, our data suggest heightened protein O-GlcNAcylation, mediated by SNAI1 and the HBP, serves as a novel means by which EMT contributes to mutant Kras-induced lung tumorigenesis and tumor progression. Citation Format: Kekoa Taparra, Hailun Wang, Katriana Nugent, Reem Malek, Jen Groves, Gokben Yildirir, Brian Simons, Dean Felsher, Natasha Zachara, Phuoc Tran. SNAI1 regulates the hexosamine biosynthetic pathway to promote tumorigenesis and oncogene-induced senescence escape in lung 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 1054.

Phospho-Aspirin (MDC-22) Prevents Pancreatic Carcinogenesis in Mice.

Pancreatic cancer is a deadly disease with a dismal 5-year survival rate of <6%. The currently limited treatment options for pancreatic cancer underscore the need for novel chemopreventive and therapeutic agents. Accumulating evidence indicates that aspirin use is associated with a decreased risk of pancreatic cancer. However, the anticancer properties of aspirin are restricted by its gastrointestinal toxicity and its limited efficacy. Therefore, we developed phospho-aspirin (MDC-22), a novel derivative of aspirin, and evaluated its chemopreventive efficacy in preclinical models of pancreatic cancer. Phospho-aspirin inhibited the growth of human pancreatic cancer cell lines 8- to 12-fold more potently than aspirin; based on the 24-hour IC50 values. In a Panc-1 xenograft model, phospho-aspirin, at a dose of 100 mg/kg/d 5 times per week for 30 days, reduced tumor growth by 78% (P < 0.01 vs. vehicle control). Furthermore, phospho-aspirin prevented pancreatitis-accelerated acinar-to-ductal metaplasia in mice with activated Kras. In p48-Cre;Kras(G12D) mice, cerulein treatment (6 hourly injections two times per week for 3 weeks) led to a significant increase in ductal metaplasia, replacing the majority of the exocrine compartment. Administration of phospho-aspirin 100 mg/kg/day five times per week for 21 days (starting on the first day of cerulein injection) inhibited the acinar-to-ductal metaplasia, reducing it by 87% (P < 0.01, vs. cerulein-treated control). Phospho-aspirin appeared to be safe, with the animals showing no signs of toxicity during treatment. Mechanistically, phospho-aspirin inhibited EGFR activation in pancreatic cancer, an effect consistently observed in pancreatic cancer cells, primary acinar explants and in vivo In conclusion, our findings indicate that phospho-aspirin has strong anticancer efficacy in preclinical models of pancreatic cancer, warranting its further evaluation. Cancer Prev Res; 9(7); 624-34. ©2016 AACR.