Mouse Model Of Adenocarcinoma Research Articles

Identification of a Clinically Relevant Signature for Early Progression in KRAS-Driven Lung Adenocarcinoma.

Inducible genetically defined mouse models of cancer uniquely facilitate the investigation of early events in cancer progression, however, there are valid concerns about the ability of such models to faithfully recapitulate human disease. We developed an inducible mouse model of progressive lung adenocarcinoma (LuAd) that combines sporadic activation of oncogenic KRasG12D with modest overexpression of c-MYC (KM model). Histological examination revealed a highly reproducible spontaneous transition from low-grade adenocarcinoma to locally invasive adenocarcinoma within 6 weeks of oncogene activation. Laser-capture microdissection coupled with RNA-SEQ (ribonucleic acid sequencing) was employed to determine transcriptional changes associated with tumour progression. Upregulated genes were triaged for relevance to human LuAd using datasets from Oncomine and cBioportal. Selected genes were validated by RNAi screening in human lung cancer cell lines and examined for association with lung cancer patient overall survival using KMplot.com. Depletion of progression-associated genes resulted in pronounced viability and/or cell migration defects in human lung cancer cells. Progression-associated genes moreover exhibited strong associations with overall survival, specifically in human lung adenocarcinoma, but not in squamous cell carcinoma. The KM mouse model faithfully recapitulates key molecular events in human adenocarcinoma of the lung and is a useful tool for mechanistic interrogation of KRAS-driven LuAd progression.

Metabolic Coordination of Tumor Suppression

Increasing evidence demonstrates that intracellular metabolic pathways can be critical drivers of tumor progression. Metabolites provide the biomass required for cancer cell proliferation and contribute to antioxidant defenses that promote survival and metastatic dissemination. Moreover, oncogenic mutations in several metabolic enzymes lead to accumulation of pathological levels of metabolites that interfere with histone and DNA demethylation and impose a block to differentiation. Thus, while the ability of metabolites to contribute directly to malignant progression is well established, whether metabolites can contribute to the cell fate changes that occur during tumor suppression remains an open question. To study the role of metabolism during tumor suppression, we took advantage of a mouse model of pancreatic ductal adenocarcinoma driven by mutant Kras and inducible suppression of p53 to enable temporal control of tumor suppression. Using cell lines derived from these tumors, we found that p53 restoration is accompanied by metabolic alterations that can be uncoupled from changes in cell cycle progression. Recapitulating these metabolic alterations with pharmacologic or genetic interventions in malignant cells is sufficient to phenocopy the effects of p53 restoration on differentiation and tumor progression. These results suggest that p53‐driven metabolic rewiring directly contributes to the cell fate changes that occur during tumor suppression.Support or Funding InformationThis work was supported by the Searle Scholars Program, the Damon Runyon Cancer Research Foundation (DFS‐23‐17) and The Starr Foundation (I11‐0039).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Longitudinal Single Cell Profiling of Regulatory T Cells Identifies IL-33 as a Driver of Tumor Immunosuppression

Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we used single cell RNA-Seq to longitudinally profile conventional CD4+ T cells (Tconv) and Tregs in a genetic mouse model of lung adenocarcinoma. Tissue-infiltrating and peripheral CD4+ T cells differed, highlighting divergent pathways of activation during tumorigenesis. Longitudinal shifts in Tregs heterogeneity suggested stabilization of a specialized effector phenotype over time that had enhanced expression of the interleukin 33 receptor ST2. Tregs-specific deletion of ST2 altered lung effector Tregs diversity, increased infiltration of CD8+ T cells into tumors, and decreased tumor burden. Our study shows that ST2 plays a critical role in Tregs-mediated immunosuppression in cancer, highlighting new potential paths for therapeutic intervention.

Optimized Bexarotene Aerosol Formulation Inhibits Major Subtypes of Lung Cancer in Mice.

Bexarotene has shown inhibition of lung and mammary gland tumorigenesis in preclinical models and in clinical trials. The main side effects of orally administered bexarotene are hypertriglyceridemia and hypercholesterolemia. We previously demonstrated that aerosolized bexarotene administered by nasal inhalation has potent chemopreventive activity in a lung adenoma preclinical model without causing hypertriglyceridemia. To facilitate its future clinical translation, we modified the formula of the aerosolized bexarotene with a clinically relevant solvent system. This optimized aerosolized bexarotene formulation was tested against lung squamous cell carcinoma mouse model and lung adenocarcinoma mouse model and showed significant chemopreventive effect. This new formula did not cause visible signs of toxicity and did not increase plasma triglycerides or cholesterol. This aerosolized bexarotene was evenly distributed to the mouse lung parenchyma, and it modulated the microenvironment in vivo by increasing the tumor-infiltrating T cell population. RNA sequencing of the lung cancer cell lines demonstrated that multiple pathways are altered by bexarotene. For the first time, these studies demonstrate a new, clinically relevant aerosolized bexarotene formulation that exhibits preventive efficacy against the major subtypes of lung cancer. This approach could be a major advancement in lung cancer prevention for high risk populations, including former and present smokers.

PET Imaging of DNA Damage Response following 225Ac-radioimmunotherapy in a Pancreatic Ductal Adenocarcinoma Mouse Model

PET Imaging of DNA Damage Response following 225Ac-radioimmunotherapy in a Pancreatic Ductal Adenocarcinoma Mouse Model

Quantitative Imaging of Morphometric and Metabolic Signatures Reveals Heterogeneity in Drug Response of Three-Dimensional Mammary Tumor Spheroids.

In order to monitor the drug responses of three-dimensional mammary tumor spheroids and to elucidate the role of inter- and intra-spheroid heterogeneity in determining drug sensitivity in the spheroids, an integrated image analysis framework was developed for morphometric and metabolic characterization of the three-dimensional tumor spheroids. Three-dimensional spheroid cultures of primary mammary tumor epithelial cells isolated from freshly excised tumors from a transgenic mouse model of adenocarcinoma (MMTV-PyMT) were imaged by using vital dyes and mitochondrial membrane potential markers. Custom-developed java and python program codes facilitated image processing, numerical computation, and graphical analysis of large datasets generated from the experiments. A panel of cancer drugs (rapamycin, BEZ235, MK2206, and flavopiridol) was tested to determine the degree of drug sensitivity as well as heterogeneity in drug response. A new quantitative metric (growth/toxicity) was developed based on morphometric parameters that were found to track the growth and apoptotic cell populations. Further, this study identified two parameters, namely, skew and kurtosis-which report the spatial heterogeneity in mitochondrial metabolism within the spheroids. The results of this study show that three-dimensional tumor spheroids selectively respond to cancer drugs depending on the specific metabolic pathways (AKT inhibition pathway in the present study), and there exists significant heterogeneity in the untreated tumor spheroids. Drug sensitivity of the spheroids was found to be associated with significant alterations in mitochondrial heterogeneity within the spheroids. In conclusion, the quantitative imaging of morphometric and metabolic analysis in large image datasets can serve as an excellent tool box for characterizing tumor heterogeneity in three-dimensional tumor spheroids and potentially, in intact tumors as well.

Deletion of Atm in Tumor but not Endothelial Cells Improves Radiation Response in a Primary Mouse Model of Lung Adenocarcinoma.

Stereotactic body radiotherapy is utilized to treat lung cancer. The mechanism of tumor response to high-dose radiotherapy (HDRT) is controversial, with competing hypotheses of increased direct tumor cell killing versus indirect effects on stroma including endothelial cells. Here we used dual recombinase technology in a primary murine lung cancer model to test whether tumor cells or endothelial cells are critical HDRT targets. Lenti-Cre deleted one or two copies of ataxia-telangiectasia mutated gene (Atm; KPAFL/+ or KPAFL/FL), whereas adeno-FlpO-infected mice expressed Cre in endothelial cells to delete one or both copies of Atm (KPVAFL/+ or KPVAFL/FL) to modify tumor cell or endothelial cell radiosensitivity, respectively. Deletion of Atm in either tumor cells or endothelial cells had no impact on tumor growth in the absence of radiation. Despite increased endothelial cell death in KPVAFL/FL mice following irradiation, tumor growth delay was not significantly increased. In contrast, a prolonged tumor growth delay was apparent in KPAFL/FL mice. Primary tumor cell lines lacking Atm expression also demonstrated enhanced radiosensitivity as determined via a clonogenic survival assay. These findings indicate that tumor cells, rather than endothelial cells, are critical targets of HDRT in primary murine lung cancer. SIGNIFICANCE: These findings establish radiosensitizing tumor cells rather than endothelial cells as the primary mechanism of tumor response to high-dose radiotherapy, supporting efforts to maximize local control by radiosensitizing tumors cells.See related commentary by Hallahan, p. 704.

Immuno-PET Imaging and Pharmacokinetics of an Anti-CEA scFv-based Trimerbody and Its Monomeric Counterpart in Human Gastric Carcinoma-Bearing Mice.

Monoclonal antibodies (mAbs) are currently used as therapeutic agents in different types of cancer. However, mAbs and antibody fragments developed so far show suboptimal properties in terms of circulation time and tumor penetration/retention. Here, we report the radiolabeling, pharmacokinetic evaluation, and determination of tumor targeting capacity of the previously validated anti-CEA MFE23-scFv-based N-terminal trimerbody (MFE23N-trimerbody), and the results are compared to those obtained for the monomeric MFE23-scFv. Dissection and gamma-counting studies performed with the 131I-labeled protein scaffolds in normal mice showed slower blood clearance for the trimerbody, and accumulation in the kidneys, the spleen, and the liver for both species. These, together with a progressive uptake in the small intestine, confirm a combined elimination scheme with hepatobiliary and urinary excretion. Positron emission tomography studies performed in a xenograft mouse model of human gastric adenocarcinoma, generated by subcutaneous administration of CEA-positive human MKN45 cells, showed higher tumor accumulation and tumor-to-muscle (T/M) ratios for 124I-labeled MFE23N-trimerbody than for MFE23-scFv. Specific uptake was not detected with PET imaging in CEA negative xenografts as indicated by low T/M ratios. Our data suggest that engineered intermediate-sized trivalent antibody fragments could be promising candidates for targeted therapy and imaging of CEA-positive tumors.

P53 Activates the Long Noncoding RNA Pvt1b to Inhibit Myc and Suppress Tumorigenesis

The p53 pathway is a key tumor suppressor mechanism that stalls cell cycle progression in response to stress. It has been proposed that p53 suppresses proliferation in part by downregulating the Myc oncogene but the mechanism has remained unclear. Here, we show that p53 directly represses Myc through a long noncoding RNA expressed from the Pvt1 locus. Pvt1b, a p53-dependent RNA isoform, is rapidly induced upon genotoxic and oncogenic stress and accumulates at its site of transcription in foci that exhibit properties of liquid-phase condensates. Pvt1b uses long-range chromatin interactions between Pvt1b and Myc to locally suppress Myc in an RNA-dependent manner. Loss of p53-dependent Pvt1b induction leads to increased cellular proliferation in vitro and enhanced tumor growth in a mouse model of lung adenocarcinoma in vivo. We propose that Pvt1b-mediated Myc repression contributes to the p53 tumor suppressive network to limit cancer growth.

A Novel Approach for Image-Guided 131I Therapy of Pancreatic Ductal Adenocarcinoma Using Mesenchymal Stem Cell-Mediated NIS Gene Delivery.

The sodium iodide symporter (SLC5A5/NIS) as theranostic gene would allow for non-invasive imaging of functional NIS expression and therapeutic radioiodine application. Genetically engineered mesenchymal stem cells (MSC), based on their tumor-homing abilities, show great promise as tumor-selective NIS gene delivery vehicles for non-thyroidal tumors. As a next step towards clinical application, tumor specificity and efficacy of MSCs were investigated in an advanced genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC). Syngeneic murine MSCs were stably transfected with a NIS-expressing plasmid driven by the CMV-promoter (NIS-MSC). In vivo 123I-scintigraphy and 124I-PET revealed significant perchlorate-sensitive NIS-mediated radioiodide accumulation in PDAC after systemic injection of NIS-MSCs. Active MSC recruitment into the tumor stroma was confirmed using NIS immunohistochemistry (IHC). A therapeutic strategy, consisting of three cycles of systemic MSC-mediated NIS delivery, followed by 131I application, resulted in a significant delay and reduction in tumor growth as compared to controls. Furthermore, IHC analysis of α-SMA and Ki67 revealed differences in the amount and behavior of activated fibroblasts in tumors of mice injected with NIS-MSCs as compared with saline-treated mice. Taken together, MSCs as NIS gene delivery vehicles in this advanced endogenous PDAC mouse model demonstrated high stromal targeting of NIS by selective recruitment of NIS-MSCs after systemic application resulting in an impressive 131I therapeutic effect. IMPLICATIONS: These data expand the prospect of MSC-mediated radioiodine imaging-guided therapy of pancreatic cancer using the sodium iodide symporter as a theranostic gene in a clinical setting.

Blocking CXCLs\u2013CXCR2 axis in tumor\u2013stromal interactions contributes to survival in a mouse model of pancreatic ductal adenocarcinoma through reduced cell invasion/migration and a shift of immune-inflammatory microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense stromal reaction (desmoplasia). We have previously reported that mice with conditional KrasG12D mutation and knockout of TGF-β receptor type II (Tgfbr2), PKF mice, develop PDAC with desmoplasia modulated by CXC chemokines that are produced by PDAC cells through tumor–stromal interaction. In this study, we further discovered that PDAC and cancer-associated fibroblast (CAF) accelerated each other’s invasion and migration through the CXC chemokines-receptor (CXCLs–CXCR2) axis. Heterozygous knockout of Cxcr2 in PKF mice (PKF2h mice) prolonged survival and inhibited both tumor angiogenesis and PDAC microinvasion. Infiltration of neutrophils, myeloid-derived suppressor cells (MDSCs), and arginase-1+ M2-like tumor-associated macrophages (TAMs) significantly decreased in the tumors of PKF2h mice, whereas inducible nitric oxide synthase (iNOS)+ M1-like TAMs and apoptotic tumor cells markedly increased, which indicated that blockade of the CXCLs–CXCR2 axis resulted in a shift of immune-inflammatory microenvironment. These results suggest that blocking of the CXCLs–CXCR2 axis in tumor–stromal interactions could be a therapeutic approach against PDAC progression.

The Superior Efficacy of Anti-PD-1/PD-L1 Immunotherapy in KRAS-Mutant Non-Small Cell Lung Cancer that Correlates with an Inflammatory Phenotype and Increased Immunogenicity

Background: Mutations in KRAS are a common oncogene driver detected in approximately 30% of non-squamous non-small cell lung cancer (NSCLC) cases, conferring a poor prognosis without effective targeted therapy. Immune checkpoint inhibitors against PD-1/ PD-L1 yield improved survival rates of KRAS-mutant NSCLC patients while changing the paradigm of cancer therapy. Yet, the underlying association between KRAS mutations and immune responses remains unclear. Methods: We performed an integrated analysis of the data from publicly available repositories and from clinical center cohorts to explore the association between KRAS mutation status and tumor immunity-associated features, including PD-L1 expression, CD8+ tumor-infiltrating lymphocytes (TILs) and tumor mutational burden (TMB). Three-pool analysis was performed to assess the efficacies of different PD-1/PD-L1 inhibitors regarding objective response rates (ORR), durable clinical benefit, and overall survival (OS) in NSCLC patients with or without KRAS mutations. In addition, a KRAS-mutant lung adenocarcinoma mouse model was established to estimate the relative efficacy of anti-PD-L1 monoclonal antibody (mAb) monotherapy or combination treatment with docetaxel versus docetaxel alone. Findings: Pool analysis of 23 public studies suggested that patients with KRAS mutations had increased PD-L1 expression (OR=1.87; 95% CI: 1.34-2.61; P=0.0002). The combined analysis of PD-L1 and CD8+ TIL in the immunostained group showed increased dual-positive (PD-L1+/TIL+) cells in the KRAS-mutant group (P=0.008), suggesting KRAS mutations induce an inflammatory phenotype. Additional data verified a prominently increased KRAS mutant-associated TMB (TCGA: P=0.0155; Broad database: P=0.0001), indicating KRAS mutations promote tumor immunogenicity in NSCLC. Further molecular analyses revealed that KRAS mutations caused various defects in cell cycle checkpoints and DNA damage response pathways. Two-pool analysis confirmed that PD-1/PD-L1 inhibitors resulted in better ORR (OR=1.51; 95% CI: 1.17-1.96; P=0.002) and improved OS compared with docetaxel chemotherapy (HR=0.64; 95% CI: 0.43-0.95; P=0.03) KRAS-mutant NSCLC patients. Finally, anti-PD-L1 mAb significantly reduced tumor growth compared with docetaxel in KRASG12C lung adenocarcinoma mice (P<0.001), while PD-L1 mAb combined with docetaxel did not promote an anti-tumor response. Interpretation: KRAS-mutant NSCLC patients show remarkable clinical benefit from anti-PD-1/PD-L1 immunotherapy. We also uncovered a complex relationship among KRAS mutations, inflammatory phenotypes, and tumor immunogenicity. Importantly, our findings demonstrate that PD-1/PD-L1 inhibitor-based monotherapy may be more effective in KRAS-mutant NSCLC patients. Funding: This work was mainly supported by CIFMS. Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: This study was approved by the Ethics Committee of CICAMS, and all participants provided written informed consent.

Tumor Suppressor Activity of Selenbp1, a Direct Nkx2-1 Target, in Lung Adenocarcinoma.

The Nkx2-1 transcription factor promotes differentiation of lung epithelial lineages and suppresses malignant progression of lung adenocarcinoma. However, targets of Nkx2-1 that limit tumor growth and progression remain incompletely understood. Here, direct Nkx2-1 targets are identified whose expression correlates with Nkx2-1 activity in human lung adenocarcinoma. Selenium-binding protein 1 (Selenbp1), an Nkx2-1 effector that limits phenotypes associated with lung cancer growth and metastasis, was investigated further. Loss- and gain-of-function approaches demonstrate that Nkx2-1 is required and sufficient for Selenbp1 expression in lung adenocarcinoma cells. Interestingly, Selenbp1 knockdown also reduced Nkx2-1 expression and Selenbp1 stabilized Nkx2-1 protein levels in a heterologous system, suggesting that these genes function in a positive feedback loop. Selenbp1 inhibits clonal growth and migration and suppresses growth of metastases in an in vivo transplant model. Genetic inactivation of Selenbp1, using CRISPR/Cas9, also enhanced primary tumor growth in autochthonous lung adenocarcinoma mouse models. Collectively, these data demonstrate that Selenbp1 is a direct target of Nkx2-1, which inhibits lung adenocarcinoma growth in vivo Implications: Selenbp1 is an important suppressor of lung tumor growth that functions in a positive feedback loop with Nkx2-1, and whose loss is associated with worse patient outcome. Mol Cancer Res; 16(11); 1737-49. ©2018 AACR.

PKM2 is not required for pancreatic ductal adenocarcinoma

BackgroundWhile most cancer cells preferentially express the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2), PKM2 is dispensable for tumor development in several mouse cancer models. PKM2 is expressed in human pancreatic cancer, and there have been conflicting reports on the association of PKM2 expression and pancreatic cancer patient survival, but whether PKM2 is required for pancreatic cancer progression is unknown. To investigate the role of PKM2 in pancreatic cancer, we used a conditional allele to delete PKM2 in a mouse model of pancreatic ductal adenocarcinoma (PDAC).ResultsPDAC tumors were initiated in LSL-KrasG12D/+;Trp53flox/flox;Pdx-1-Cre (KP−/−C) mice harboring a conditional Pkm2 allele. Immunohistochemical analysis showed PKM2 expression in wild-type tumors and loss of PKM2 expression in tumors from Pkm2 conditional mice. PKM2 deletion had no effect on overall survival or tumor size. Loss of PKM2 resulted in pyruvate kinase M1 (PKM1) expression, but did not affect the number of proliferating cells. These findings are consistent with results in other cancer models.ConclusionsPKM2 is not required for initiation or growth of PDAC tumors arising in the KP−/−C pancreatic cancer model. These findings suggest that, in this mouse PDAC model, PKM2 expression is not required for pancreatic tumor formation or progression.

A severe combined immunodeficiency disease mouse model of human adenocarcinoma with lepidic-predominant growth

Severe combined immunodeficiency disease (SCID) mice with human lepidic adenocarcinoma were established by the intrabronchial implantation of fresh surgically resected specimens. Human pulmonary adenocarcinoma tissue from 16 different cases was transplanted into SCID mice, and SCID mouse tumors were established from four of these cases (25%). Among the four tumors, the tumor cells of two SCID mice showed replacement lepidic growth of mouse alveolar structures accompanied by multiple intrapulmonary lesions. Human lung carcinoma cell lines showing lepidic growth are rare and the xenograft models using the SCID mouse model developed in the current study will be useful for analyzing the growth and/or progression patterns and clinical behavior of lepidic adenocarcinoma, the major histological subtype of human carcinoma of the lung.

Cisplatin Effects in Lung Adenocarcinoma-bearing Mice, Alone and in Combination with Erlotinib.

Cisplatin causes nephrotoxicity that can lead to the development of acute kidney injury or chronic kidney disease. However, the current mouse model of cisplatin nephrotoxicity is neither physiologically nor clinically relevant. Our goal was to improve upon these deficits by developing a repeated, low-dose regimen of cisplatin and combining it with a transgenic mouse model of lung adenocarcinoma. This overview details how addressing these deficits have improved our understanding of cisplatin-induced kidney injury.

Abstract PR06: Investigating ectopic lymphoid aggregates in a genetically engineered mouse model of lung adenocarcinoma

Abstract Ectopic lymphoid aggregates (LA) located in the vicinity of solid tumors have been identified in human patients with many types of cancers and have been largely associated with improved outcomes. In particular, LA in patients with non-small cell lung cancer have been associated with longer overall survival, regardless of the presence of intratumoral CD8 T cells, a commonly used prognostic marker. Tumor-associated LA are organized similarly to lymph nodes with distinctive T-cell zones, B-cell zones, and high endothelial venules (HEV), suggesting a role of LA in the regulation of an antitumor immune response. Therefore, therapeutics targeting these LA have potential to strengthen the antitumor immune response and consequently improve patient outcome. Nevertheless, there is an absence of preclinical models that mirror this clinically relevant phenomenon, and thus their formation and precise function(s) remains poorly understood. Using a genetically engineered mouse model (GEMM) of lung adenocarcinoma in immunocompetent KP-T mice (KrasLox-STOP-Lox-G12D;p53flox/flox;Rosa26Lox-STOP-Lox-tdTomato), we have observed that tumor-associated LA develop and serve as critical sites for the regulation of antitumor immune responses. In this model, autochthonous tumors develop about 20 weeks after intratracheal infection with Cre recombinase-encoding lentivirus (Cre LV), and progress in a similar manner to human disease. The expression of an antigenic construct containing three well-characterized T-cell antigens fused to luciferase (LucOS) was introduced into developing tumors via LV (LucOS/Cre LV). Interestingly, tumors expressing these antigens are physically associated with LA, whereas tumors that do not express these antigens are not. We are therefore uniquely positioned to investigate the role of tumor antigens, as well as the subsequent antitumor immune response, in the development of tumor-associated ectopic LA. We hypothesize that tumor antigen-specific, “pioneering” T cells trigger the recruitment of other immune cell types and ultimately lead to the formation of tumor-associated LA and the development of an antitumor immune response. Future studies will investigate the role of tumor-specific CD8 T cells in the formation of LA, using various GEMM and LV infections to manipulate and “turn off” the expression of T cell antigens by tumor cells. To advance our understanding of cancer outcomes and select more effective, individualized treatments for patients, we believe that it is crucial to directly interrogate how and when LA develop. Ultimately, our findings will identify cellular and molecular targets essential to the formation and maintenance of LA, thus providing candidates for therapeutic modulation of these clinically relevant tumor-associated immune structures. This abstract is also being presented as Poster B14. Citation Format: Kelli Connolly, Mursal Nader, Nikhil Joshi. Investigating ectopic lymphoid aggregates in a genetically engineered mouse model of lung adenocarcinoma [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 PR06.

Abstract PR01: Modeling Rb loss and pathway reactivation in lung adenocarcinoma

Abstract Mutations in the Rb tumor suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma. Additionally, recent clinical successes with cyclin dependent kinase inhibitors have reinvigorated interest in reactivating the Retinoblastoma (Rb) pathway to treat lung adenocarcinoma and other tumor types. Remarkably though, Rb’s role in suppressing lung adenocarcinoma remains unclear, and whether Rb pathway reactivation would be efficacious in this disease remains unknown. To model Rb pathway reactivation as treatment strategy in lung adenocarcinoma and to shed light on its role in this disease, we established an RbXTR allele that enables Cre-dependent inactivation of Rb in developing tumors, and allows Flp recombinase-inducible reactivation of Rb after tumors are established. In the KrasLox-Stop-Lox-G12D/+;p53flox/flox (KP) mouse model of lung adenocarcinoma, we show that Rb inactivation facilitates the bypass of two molecularly distinct barriers to tumor progression and dramatically accelerates malignant conversion and the development of metastatic disease. Although, in the presence of Rb, malignant conversion requires amplification of the Raf/Mek/Erk (MAPK) signaling pathway beyond that normally activated by the Kras oncogene, we find that this requirement is abrogated when Rb is inactivated. Mechanistically, we identified Cdk2 as an important effector downstream of amplified MAPK signaling and that this activity suppresses Rb’s ability to limit the adenoma-to-carcinoma transition. Importantly, inactivation of Cdk2 reduces cell proliferation in Rb wild-type cells and confers sensitivity to Cdk4/6 inhibition in both human and mouse lung adenocarcinoma cell lines were intrinsically resistant. Acquiring metastatic competency in Rb wild-type tumors is causally linked to epigenetic changes resulting in loss of lung lineage cell fate-determining transcription factors and concomitant derepression of factors normally restricted to embryonic cell types. However, inactivation of Rb uncouples the onset of metastatic competency from the loss of lung lineage factors, facilitates the early derepression of prometastatic factors, and significantly enhances metastatic proclivity. Finally, we demonstrate that reactivation of Rb in metastatic disease settings reprograms these tumors toward a less aggressive cell state and improves overall survival. Our study highlights an unappreciated role for Rb in regulating metastasis-promoting programs, and the potential of Rb restorative therapies to treat lung adenocarcinoma. Further, we suggest that a renewed investment in the development of specific Cdk2 inhibitors may be necessary for Rb pathway reactivation in certain cancer types. This abstract is also being presented as Poster A27. Citation Format: Travis Yates, Caroline Kim-Kiselak, Walter Wang, David M. Feldser. Modeling Rb loss and pathway reactivation in lung adenocarcinoma [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 PR01.

Abstract A01: Stem cell signals in the initiation and progression of non-small cell lung cancer

Abstract Lung cancer is most commonly diagnosed at a late stage when the disease is in its most aggressive state. To define the key regulators of lung cancer, we have focused on how stem cell signals are hijacked to drive progression to an undifferentiated state. Using a novel reporter mouse designed to reflect expression of the stem cell fate determinant Musashi-2 (Msi2), we found that Msi2 is highly expressed in tumors derived from the KrasG12D/+; p53fl/fl (KP) mouse model of lung adenocarcinoma. To test if Msi2-expressing cells preferentially act as the cell of origin in lung cancer, we isolated lung epithelial cells and tested their tumorigenic potential in vitro and in vivo following activation of Kras and loss of p53. Importantly, Msi2+ cells preferentially formed tumorspheres in vitro as well as tumors in vivo, indicating that Msi2+ tumor cells have increased tumor-initiating capacity. To determine whether Msi2 is required for lung cancer growth and progression, we crossed KP mice to Msi2 knockout mice and monitored tumor growth over time. Loss of Msi2 led to a reduction in both the number of tumors that formed and in overall tumor burden. Moreover, the mice exhibited a reduced frequency of high-grade tumors and an increase in overall survival, suggesting that loss of Msi2 impairs tumor development and disease progression. Finally, inhibition of Msi2 in a primary lung adenocarcinoma cell line led to reduced tumor volume in vivo, suggesting that Msi2 continues to be a dependency in established disease. Collectively, our data identify Msi2 as a stem cell signal that is critically required in lung cancer development and progression. Citation Format: Alison G. Barber, Hatim Husain, Mark A. Valasek, Miriam Scadeng, Tannishtha Reya. Stem cell signals in the initiation and progression of non-small cell lung cancer [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 A01.

Abstract B03: Suppression of lung adenocarcinoma growth and metastasis by stromal Hedgehog pathway activation

Abstract Aberrant activation of the Hedgehog (Hh) signaling pathway drives the tumor growth of Gorlin-type cancers, but its role in other epithelial cancers, including non-small cell lung cancer (NSCLC), has been controversial. We used human lung adenocarcinoma (LAD) cell lines and autochthonous lung adenocarcinoma mouse models to elucidate the pathway’s role in lung cancer pathology. SHH and IHH is expressed heterogeneously among &amp;gt;35 tested LAD cell lines, although mostly in mutant Kras cells. SHH secreted from lung cancer cells activated the pathway in co-cultured fibroblasts in a paracrine, rather than autocrine, manner. Treatment with KAAD-cyclopamine, an Hh pathway antagonist, inhibited the growth of high SHH-expressing tumor cells when co-cultured with NIH-3T3 embryonic fibroblasts but not tumor cells alone, further reinforcing the paracrine nature of Hh pathway activation. However, the growth effect due to fibroblast pathway inhibition was diminished when co-cultured with normal lung fibroblasts. Analysis of potential Hh pathway target genes suggest that normal lung fibroblasts secrete ligands that induce a differentiation program in epithelial cells rather than ligands that induce mitogenic programs from NIH-3T3 fibroblasts. In KrasLSL-G12D/+;Trp53fl/fl; Shhfl/fl \(KPS) mice, genetic loss of SHH, surprisingly, had no effect on survival compared to KrasLSL-G12D/+;Trp53fl/fl;Shh+/+ (KP) mice. However, treatment with 5E1, an anti-SHH/IHH blocking antibody, 2 weeks after tumor induction in KP mice resulted in significantly worse survival with increased metastases compared to KP mice treated with control IgG1 antibody. Furthermore, stromal Hh pathway abrogation by 5E1 led to increased tumor burden and decreased tumor vessel density compared to control mice. To account for the survival differences seen between KP mice with genetic SHH loss and those treated with 5E1, we analyzed KP tumors and found unexpectedly high levels of Ihh mRNA, compared to Shh mRNA, that have not been reported previously in normal or neoplastic murine lungs. Preliminary results using an in vivo CRISPR/Cas9 (pSECC) system to genetically ablate Ihh in KrasLSL-G12D/+;Trp53fl/fl;Rosa26LSL-fluc/+ mice showed significant enhancement of tumor growth compared to control mice treated with sgGFP. These results suggest that IHH, rather than SHH, activates a tumor-suppressive program in lung stroma. These results correlate to human survival data of LAD with low lhh mRNA expression (KMPLotter). We are in the process of isolating and examining murine stromal cells from KP mice to identify the secreted stromal factors that limit tumor growth and metastases. Thus, stromal Hh pathway activation, in response to IHH secreted from lung cancer epithelia, suppresses LAD growth and metastases, increases tumor vessel growth, and prolongs survival. Citation Format: Sahba Kasiri, Baozhi Chen, Alexandra Wilson, Annika Reczek, Ummay Marriam, Zhiqun Zeng, Luc Girard, James Kim. Suppression of lung adenocarcinoma growth and metastasis by stromal Hedgehog pathway activation [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 B03.