KP Mice Research Articles

Abstract 7470: Leukemia inhibitory factor promotes tumor progression in KRAS-driven lung cancer

Abstract Leukemia inhibitory factor (LIF) displays complex effects in different types of cancer but its role in lung cancer remains unknown. Since KRAS mutation is an initial validated oncogenic event in near 30% of non-small cell lung cancer (PanCancer Atlas), we were interested in studying how LIF would influence tumor initiation and progression in KRAS-driven lung cancer.To properly study the roles of LIF throughout KRAS-driven tumorigenesis, we generated a novel genetically engineered mouse model (GEMM), where expression of LIF can be conditionally knocked out by Cre recombinase. Conditional deletion of the Lif allele in oncogenic KRAS-induced lung tumors is achieved by crossing Lif flox/flox mice with KrasLSL-G12D/+, Trp53flox/flox (KP) mouse model. In our lung cancer model, induction of oncogenic KRAS and depletion of LIF is attained through intratracheal infection of adeno-Cre virus.When our Lif flox/floxKP (LKP) and KP mice were infected by adeno-Cre virus, they developed lung tumors at reasonable frequencies within 4-8 months. We generated cell lines from tumors and used ELISA and RT-qPCR analysis to validate the efficiency of the knockout. While control KP tumor cell lines (n=3) showed Lif expression and LIF protein secretion, cell lines from LKP tumors (n=3) presented with undetectable Lif expression and no LIF secretion validating the successful deletion of Lif and functionality of our new mouse model. We generated H&E slides from the tumors and with the help of our pathologist we confirmed the subtype of lung adenocarcinoma. The effects of LIF on cell lines proliferation were evaluated in vitro and we found that LKP cell lines grow significantly slower than the KP lines (p<0.05).Mice in both groups were monitored for their overall survival (OS) rate. LKP mice (n=5) tend to have an increased median overall survival at 176 days when compared to control KP mice (n=7) at 152 days (p=0.07). To estimate the tumor initiation and progression, mice in each group were sacrificed at early endpoints (10- and 15-weeks post adeno-cre infection) and ratios of lung weight/mouse weight were measured as an indicator of tumor burden. At 10 weeks, LKP mice (n=5) presented with significantly smaller ratios with an average of 0.014 compared to KP animals (n=4) with an average of 0.019 (p=0.027).We found that LIF tends to shorten overall survival and augments tumor burden in lung cancer. These data suggest that LIF has a pro-tumorigenic role in KRAS-driven lung cancer proposing this cytokine as a potential prognostic biomarker and its depletion as a promising therapeutic target. Citation Format: Imene Boukhalfa Hanafi, Lisa Miller-Phillips, Ons Zakraoui, Kieren D. Marini, Sarah Umetsu, Man-Tzu Wang, Eric A. Collisson. Leukemia inhibitory factor promotes tumor progression in KRAS-driven lung cancer [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 7470.

Abstract 4294: The emerging role of deubiquitinase enzymes in lung cancer plasticity

Abstract Lung cancer is characterized by a high degree of genetic and molecular heterogeneity. Lineage plasticity has emerged as a source of intratumoral heterogeneity and drug resistance, resulting in poor prognosis and treatment failure. The molecular mechanisms driving lung cancer plasticity remain unclear. Ubiquitin-Specific Peptidase 13 (USP13), a deubiquitinating enzyme, is one of the most amplified genes in lung squamous cell carcinoma (LUSC). We developed a USP13 knock-in overexpressing mouse model in KrasLSL-G12D/+; Trp53fl/fl (KP) background (KPU mice). USP13 overexpression resulted in aggressive tumorigenesis and a shortened survival in Kras/Trp53-driven lung cancer. Notably, while KP mice developed lung adenocarcinoma (LUAD), KPU mice developed LUSC and LUAD. LUSC in KPU mice faithfully recapitulated the key pathohistological, molecular features, and cellular pathways of human LUSC. Bulk RNA-sequencing analysis showed that KPU tumors were heterogeneous and enriched in lineage reprogramming pathways including basal cell signaling, stem cell pluripotency, and epithelial-mesenchymal transition (EMT). Using cell-type-restricted adenoviral Cre to target cells expressing surfactant protein C (SPC) or club cell antigen 10 (CC10), we identified bronchiolar secretory club cells as the predominant cell origin of LUSC. USP13 altered the levels of lineage transcription factors such as TTF-1 and SOX2 in club cells during early tumorigenesis. Altered expression of these factors reinforced the fate of CC10+ club cells to squamous carcinoma development rather than adenocarcinoma. In addition, USP13 directly acted on the K48-linked ubiquitination of c-Myc and increased its protein stability, contributing to the elevation of squamous gene expression (SOX2, CK5, P40) in the primary mouse and advanced human lung cancer cells. These results suggest that USP13 promotes lineage plasticity in club cells during the early stage of cancer development and drives reprogramming into LUSC. We also found a potential functional association between USP13 and EMT pathway. Notably, irrespective of cell origin, USP13 overexpression enhanced EMT marker expression including N-cadherin, SNAI1, and ZEB1 in lung cancer cells, promoting cell migration and invasion. USP13 increased tumorigenic and metastatic abilities of lung cancer cells in 3D organoid culture and syngeneic mouse models. Collectively, our research highlights the pivotal significance of USP13 in unleashing lineage plasticity during lung cancer progression, suggesting the potential role of deubiquitinase enzymes in regulating lineage plasticity, which may lead to novel therapeutics for treating lung cancer. Citation Format: Juntae Kwon, Jinmin Zhang, Boram Mok, Samuel Allsup, Cecil Han. The emerging role of deubiquitinase enzymes in lung cancer plasticity [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 4294.

Abstract 3567: Cancer-epigenetic mouse models for medulloblastoma

Abstract Medulloblastoma (MB) is the most common childhood brain tumors (up to 20% of all childhood brain tumors) and occurs mainly in children (<15 years old. MB is located in the cerebellum in the brain and can spread to the spine or to other parts of brain. Because MB growth is very fast, MB is categorized as a type of high-grade malignant primary tumors. MB development is often associated with the dysregulation of signaling pathways, such as the wingless (WNT) and sonic hedgehog (SHH). Methylated histone H3 lysine 4 (H3K4) is a major methylation mark. Trimethyl H3K4 (H3K4me3) occupies as much as 75% of all human gene promoters. We previously reported that the H3K4 methyltransferase KMT2D (also called MLL4, MLL2, and ALR; a transcriptional coactivator, is required for neuronal differentiation of human neuron-lineage NT2/D1 stem cells. Interestingly, our analysis of several databases has shown that Kmt2d is the most frequently mutated epigenetic modifier (8%‒10%) in medulloblastoma (MB). Recently, we showed that brain-specific knockout of Kmt2d alone induces spontaneous MB in mice. Kmt2d loss highly upregulated several oncogenic signaling programs and downregulated tumor suppressive genes. In this study, we sought to determine whether Kmt2d loss cooperates with another oncogenic event in MB genesis. Particularly, PTCH (also known as PTCH1) is one of the most frequently mutated genes in MB, and KMT2D is known to be a shared driver gene mutation in MB. For these reasons, we examined the effect of Kmt2d loss on of Ptch+/−-driven MB genesis. A heterozygous loss of Kmt2d increased the incidence and progression of Ptch+/−-driven MB genesis. We have shown that Kmt2d loss reduces epigenomic signals for super-enhancers/enhancers and broad H3K4me3 in 4-month-old cerebella. In addition, we determine the effect of heterozygous Kmt2d loss on cell proliferation by comparing the Nes-Cre Kmt2dfl/+ Ptch+/- (KP) cerebella to Ptch+/- cerebella at 2- & 4-month-old time points using IHC staining of Ki-67 (a proliferation marker). MB’s from KP mice showed two different mechanisms by activating oncogenic cellular signaling kinases (e.g., phospho [p]-AKT and p-p38) levels as well as MYCN signaling pathway. Our transcriptomic analysis revealed several tumor suppressive and transcriptional corepressor genes to be downregulated such as Ncor2 and Dnmt3a. We found Ncor2 depletion in Ptch-/+ neurospheres shows upregulation of similar oncogenic genes as in KP mice tumors. Kmt2d loss reduced cerebellar granule cell differentiation. Our analysis for oncogenic kinases may provide a basis for a novel MB treatment plan involving the use of cellular kinase inhibitors. As KMT2D is among the most often mutated genes in human MBs, this mouse model may be useful for future preclinical therapeutic experiments for the treatment of MB patients. KMT2D in medulloblastoma genesis using mouse models will provide epigenetic and mechanistic insights into how heterozygous KMT2D loss promotes medulloblastoma. Citation Format: Shilpa S. Dhar, Ali S. Rizvi, Lauren Reed, Calena Brown, Min Gyu Lee. Cancer-epigenetic mouse models for medulloblastoma. [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 3567.

Ophiopogonin A alleviates acute lung inflammation via NF-κB/MAPK signaling pathway in a mouse model of Klebsiella pneumoniae

Klebsiella pneumoniae (Kp) is a gram-negative bacterium present in the flora of the mouth, skin and intestines. Due to the emergence of antibiotic resistance, the outcome of Kp pneumonia is becoming worse. Exploring an effective treatment method is clinically important. Ophiopogon japonicus is a traditional Chinese herbal medicine for treating inflammation and oxidative stress damage, as ophiopogonin A (OP-A) is its main active ingredient. We investigated the impact of OP-A on Kp pneumonia and further elucidated the underlying mechanism. After the establishment of Kp mouse model, the animals were grouped and received intraperitoneal injection of OP-A, levofloxacin (Lvx) or PBS. After 12 hours, mice were dissected to detect the pathological changes of lung tissues, and the number of inflammatory proteins and inflammatory cells in the bronchoalveolar lavage fluid (BALF). Finally, Western blot and RT-qPCR analyses were carried out to detect MARK, JNK, ERK and NF-κB expression in mouse lung tissues upon treatments. Administration of OP-A attenuated the pathological damage of lung tissues of Kp pneumonia mice, as both Lvx and OP-A significantly controlled and inhibited the wet-to-dry ratio of the lung tissues. Increased protein content and inflammatory cells infiltration were visible in Kp pneumonia mice, while the advent of Lvx and OP-A dramatically diminished inflammatory cells infiltration in BALF of lung tissues with fewer contents of IL-1β, IL-6 and TNF-α. The protective effect of OP-A on Kp pneumonia correlated with the NF-κB/MAPK signaling. In conclusion, OP-A treatment may alleviate the pathological damage and reduce inflammation of Kp mice through inhibition of the NF-κB/MAPK signaling pathway. OP-A has a significant anti-inflammatory effect on Kp mice, and hence it is a promising alternative for treating Kp pneumonia.

Abstract PR010: Aging-induced reprogramming of the cell of origin defines lung cancer evolution

Abstract Aging is one of the most important risk factors for cancer development. However, our understanding of how aging impacts cancer evolution remains at an early stage. We investigated the evolution of lung adenocarcinoma (LUAD) in aged vs. young KrasLSL-G12D/+; Trp53flox/flox (KP) mice. Surprisingly, we found that LUAD initiation and progression were significantly impaired with aging. Using functional in vivo and in vitro assays, we demonstrated that the loss of tumor-initiating potential could be attributed to a loss of stemness in the alveolar type 2 (AT2) cells, which are the predominant cell of origin for LUAD. We next explored the biology of the tumors that formed in aged KP mice using single-cell transcriptomics. We discovered a distinct compositional landscape of cancer cell subsets in aged lung tumors, revealing a delay in the molecular progression of the cancers and an aging-specific gene expression signature. Interestingly, key components of this gene signature could be traced back to specific epigenetic alterations in the AT2 cells. Furthermore, the aged mouse LUAD signature was highly predictive of patient age in human LUAD, indicating conservation of the age-associated changes across species. We hypothesized that the aging-associated changes in gene expression during LUAD tumorigenesis may underpin delayed tumor progression in the aged KP mice. To test this, we performed an in vivo genetic screen in the autochthonous KP model and identified genetic dependencies that were specific to LUAD in aged or young mice. Our results indicate that aging-associated epigenetic changes in the cell of origin are imprinted on evolving tumors, which determines tumor initiation potential, alters trajectory of tumor evolution, and leads to age-dependent vulnerabilities. These findings suggest that the biology of tumors is fundamentally altered by aging, raising new possibilities for cancer treatment and prevention in patients of different ages. Citation Format: Xueqian Zhuang, Simon Joost, Qing Wang, Melissa Blum, Selena Ding, Zhuxuan Li, Yingqian Zhan, Richard Koche, Emily Wong, Tuomas Tammela. Aging-induced reprogramming of the cell of origin defines lung cancer evolution [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR010.

Ketamine Inhalation Alters Behavior and Lower Urinary Tract Function in Mice.

We aimed to evaluate behavioral and lower urinary tract changes in mice using a novel ketamine inhalation model mimicking human ketamine abusers and compare the results to those obtained using a ketamine intraperitoneal injection model. C57BL/6N mice were placed in a transparent acrylic observation cage connected to an ultrasonic nebulizer producing ketamine (KI) or saline (SI) fog. The mice were given KI or SI fog twice a week for three months. In another experiment arm, the mice were given intraperitoneal ketamine injections (KP) or saline injections (SP) twice a week for three months. The presence of urine ketamine (>100 ng/mL) was determined using a quick test kit. Locomotor activity was recorded by video using the open field test. Lower urinary tract function was assessed using urine spots, cystometry and histology. KI and KP mice crossed the center more frequently and traveled farther than SI and SP mice. Only KI mice, however, demonstrated popcorn-like jumping, and frequent center crossing. Detrusor overactivity, reduced cystometric bladder capacity, and denuded mucosa were observed in both KI and KP mice. Ketamine inhalation induces behavioral and lower urinary tract changes in mice that are comparable to intraperitoneal ketamine injections.

Abstract 874: MSI2 regulates VEGFR2 signaling and tumor progression in NSCLC

Abstract Lung cancer holds second place in incidence and first place in mortality among all cancers, both in men and women, with metastasis contributing to the vast majority of deaths. Recently, expression of vascular endothelial growth factor receptor-2 (VEGFR2) on both endothelial and tumor cells was demonstrated as one of the key mechanisms contributing to growth of non-small cell lung cancer (NSCLC), the most common subtype of lung cancer. VEGFR2 dependent signaling is critical both for tumor autocrine functions related to NSCLC migration, and in paracrine control of tumor angiogenesis through action on tumor-proximal endothelial cells, suggesting a potential mechanism for growth control. Several drugs targeting VEGFR2 signaling pathway demonstrated their clinical efficacy in NSCLC setting, including VEGFR2 and VEGF-A inhibitors, VEGF-A/PIGF trap. Recently our group demonstrated that RNA-binding protein Musashi-2 (MSI2) is driving progression and metastasis of NSCLC. MSI2 regulates mRNA translation of multiple targets, its expression is upregulated in the metastasis-competent murine and human lung cancer cell lines and is progressively elevated in lung cancer samples. The goal of this study is to define the role of MSI2 in sustaining VEGFR2 signaling during NSCLC progression. Using reverse RNA immunoprecipitation (RIP) qPCR analysis, we demonstrate that MSI2 directly binds VEGFR2 mRNA. Reverse protein phase array (RPPA) of several NSCLC cell line models with expression or knockdown of MSI2 and subsequent direct validation showed that MSI2 strongly and positively regulates expression of VEGFR2. Subsequent mechanistical analysis revealed that MSI2 knockdown leads to decreased VEGFR2 protein and mRNA levels expression in several NSCLC cell lines in vitro, resulting in increased apoptosis via inhibition of VEGFR2 signaling pathway. To better characterize MSI2 biology, we’ve crossed the B6/129S4-Msi2tm1.1Cjl/J (M2) mice with B6/129S/Sv-Krastm3Tyj/J;Trp53tm1Brn/J (KP) mice and generated novel B6/129S/Sv-KP;Msi2-/- (KPM2) mouse model. Induction of tumorigenesis in KP mice using Cre adenovirus leads to development of lung adenocarcinoma. Induction of lung-specific Cre resulted in tumorigenesis in both KP and KPM2 mice. Interestingly, we observed significant decrease in both total lung tumor number and total lung tumor burden in KPM2 compared to KP mice. Next, we established three novel KP and KPM2 derived cell lines from mouse lung tumors and found that KPM2 cell lines demonstrate reduced proliferation capacity and VEGFR2 mRNA level relative to KP cell lines. Additional studies to understand an impact of MSI2 deletion in KP cell lines are ongoing. Taken together, MSI2 is a promising target for NSCLC treatment, as this protein regulates VEGFR2 signaling and apoptosis in cell models and contributes to KP tumorigenesis in vivo. Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Petr Makhov, Alexander Kudinov, Anna Nikonova, John Karanicolas, Erica Golemis, Christopher Lengner, Hossein Borghaei, Jyoti Patel, Yanis Boumber. MSI2 regulates VEGFR2 signaling and tumor progression in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 874.

Abstract 2937: Nedd9 controls autophagy to limit non-small cell lung cancer (NSCLC) growth

Abstract The goal of this study is to define the role of NEDD9 in formation of progression of non-small cell lung cancer (NSCLC). NSCLC has a low survival rate, with metastasis contributing to the vast majority of deaths. NEDD9 expression has been shown to be repressed by LKB1, and upregulated in LKB1-mutated NSCLC; in these tumors, elevated NEDD9 promotes metastasis, serving as a scaffolding intermediate in the integrin and receptor tyrosine kinase signaling cascades, promoting pro-invasive signaling. In this study, we for the first time assessed the consequences for cancer formation after introduction of a constitutive null allele of Nedd9 (KPN mice) in the Krastm4Tyj/J/Trp53tm1Brn/J (KP mice) Adeno-cre inducible murine model of NSCLC, in the context of intact Lkb1. Unexpectedly, KPN mice presented with significantly accelerated NSCLC tumorigenesis and enhanced tumor invasion. Orthotopic re-implantation of KP and KPN tumor into syngeneic mice with wt and null Nedd9 genotypes demonstrated that the effect of Nedd9 loss on promoting tumor growth is cell-autonomous. Proteomic analysis of KPN versus KP tumors revealed that more aggressive phenotypes occurred in spite of depressed activity of the pro-proliferative, pro-invasive Nedd9 partner proteins Src and Fak. Treatment of mice with the Src inhibitor dasatinib significantly reduced, but did not eliminate, tumor growth in KP and KPN mice. As an alternative hypothesis, we considered that Ras-dependent NSCLC tumors require active autophagy, a process dependent on activation of the kinases Lkb1 and Ampk; given Lkb1 represses Nedd9, we investigated whether Nedd9 reciprocally regulates Lkb1. We found that KPN tumors specifically and significantly upregulated active Lkb1 based on a mechanism of post-transcriptional activation, causing increased expression of the Lkb1 substrate Ampk and downstream effectors of autophagy, including elevated appearance of LC3-positive autophagosomes in KPN NSCLC tumor tissue. NEDD9 depletion in human NSCLC cell lines similarly elevated LKB1 activation. In vivo treatment of KPN and KP mice with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors, emphasizing the importance of autophagy for the growth differential. In sum, these data for the first time identify and define a previously unknown role for the pro-metastatic protein NEDD9 in control of autophagy, based on a role as a negative regulator of the Lkb1-Ampk signaling axis in NSCLC. Citation Format: Alexander Y. Deneka, Meghan Kopp, Anna S. Nikonova, Anna Gaponova, Anna Kiseleva, Douglas Flieder, Ilya Serebriiskii, Harvey Hensley, Erica Golemis. Nedd9 controls autophagy to limit non-small cell lung cancer (NSCLC) growth [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 2937.

Abstract 2507: Does a threshold exist for NRF2 hyperactivation to block tumor progression in KRAS mutant, TP53-deficient NSCLC

Abstract Lung cancer is responsible for the most cancer-related deaths worldwide. Within the most prominent histological subtype, non-small cell lung cancer (NSCLC), there is an unmet clinical need: lung adenocarcinomas (ADCs) driven by mutant KRAS. Within this subset of tumors, KRAS mutations co-occur with mutations in tumor suppressor genes including TP53 and the redox regulator KEAP1. KEAP1 is the negative regulator of transcription factor NRF2, which directs the antioxidant response and multiple facets of metabolism. In NSCLC, alterations in the KEAP1-NRF2 circuit result in constitutive NRF2 activation and are often associated with resistance to therapy and poor outcomes in patients. While NRF2 hyperactivation has been associated with tumor progression, our lab's recent findings suggest that this may be context-dependent, and that too much NRF2 activation may be detrimental. To study the role of NRF2 hyperactivation on tumor progression, we have utilized KRAS mutant genetically engineered mouse models of NSCLC harboring TP53 deletion. These studies are based on our lab's finding that the homozygous KEAP1R554Q loss-of-function mutation decreases tumor size in a Kras mutant, Trp53-deficient (KP) lung ADC model (Kang et al. 2019 eLife). In parallel to these studies, we have also developed a conditional murine allele of the NRF2D29H mutation found in human NSCLC to serve as a secondary model of NRF2 hyperactivation in the KP mouse (KPN). Consistent with our homozygous KEAP1 mutant model (KPKK), we found that KPN mice demonstrated constitutive NRF2 activation, as observed by increased immunohistochemical staining of canonical NRF2 target, NQO1. This degree of NRF2 activation in KPN mice was slightly lower than that of KPKK mice, suggesting that the KPN mouse is an intermediate model of NRF2 activation. Supportingly, we also found that KPN mice had decreased tumor burden, although not to the same extent as KPKK mice. Interestingly, our heterozygous KEAP1 mutant model (KPK) demonstrates only modest NRF2 activation but did not exhibit decreased tumor burden. Importantly, analyses of tumor number suggested that KPKK and KPN tumors are impaired in tumor progression, rather than initiation. KPKK and KPN tumors also exhibited lower proliferative indices when compared to KP mice, in correspondence with their reduced tumor burden. Collectively, these results suggest that there may be a threshold for NRF2 activation to block tumor progression in the KP model. Current studies are focused on determining whether this impediment to tumor burden is NRF2-dependent, and what NRF2-dependent mechanisms may impair tumor progression. Importantly, these studies may help identify whether a threshold for NRF2 hyperactivation to promote or block tumor progression exists, and if this can be therapeutically exploited in patients with KRAS mutant, TP53-deficient lung tumors. Citation Format: Janine M. DeBlasi, Aimee Falzone, Gina M. DeNicola. Does a threshold exist for NRF2 hyperactivation to block tumor progression in KRAS mutant, TP53-deficient NSCLC [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 2507.

Nedd9 Restrains Autophagy to Limit Growth of Early Stage Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common cancer worldwide. With overall 5-year survival estimated at <17%, it is critical to identify factors that regulate NSCLC disease prognosis. NSCLC is commonly driven by mutations in KRAS and TP53, with activation of additional kinases such as SRC promoting tumor invasion. In this study, we investigated the role of NEDD9, a SRC activator and scaffolding protein, in NSCLC tumorigenesis. In an inducible model of NSCLC dependent on Kras mutation and Trp53 loss (KP mice), deletion of Nedd9 (KPN mice) led to the emergence of larger tumors characterized by accelerated rates of tumor growth and elevated proliferation. Orthotopic injection of KP and KPN tumors into the lungs of Nedd9-wild-type and -null mice indicated the effect of Nedd9 loss was cell-autonomous. Tumors in KPN mice displayed reduced activation of SRC and AKT, indicating that activation of these pathways did not mediate enhanced growth of KPN tumors. NSCLC tumor growth has been shown to require active autophagy, a process dependent on activation of the kinases LKB1 and AMPK. KPN tumors contained high levels of active LKB1 and AMPK and increased autophagy compared with KP tumors. Treatment with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors. These data for the first time identify NEDD9 as a negative regulator of LKB1/AMPK-dependent autophagy during early NSCLC tumor growth. SIGNIFICANCE: This study demonstrates a novel role for the scaffolding protein NEDD9 in regulating LKB1-AMPK signaling in early stage non-small cell lung cancer, suppressing autophagy and tumor growth.

Investigating the role of KLRG1 in regulatory T-cells and implications for anti-tumor immunity in Non-small Cell Lung Cancer

Abstract Although it is well established that CD4+CD25+Foxp3+ regulatory T cells (Tregs) dampen anti-tumor T cell responses in cancer, their molecular signature and clonal heterogeneity in NSCLC remains poorly understood. In this study, comparative phenotypic assessment of tumor and lymphoid tissue resident Tregs in KRAS/p53 mutant NSCLC mouse model revealed the presence of a distinct tumor infiltrating Treg population which express KLRG1. Compared to their counterparts, tumor associated KLRG1+ Treg showed higher expression of inhibitory receptors and activation markers, suggesting that KLRG1+ Tregs represent an activated subpopulation which may contribute to immunosuppression. Ex-vivo Treg suppression assay revealed that tumor associated KLGR1+ Tregs exhibited higher suppressive activity compared to KLRG1-Tregs. Although T cell development was normal in in-house generated KLRG1 KO mice including thymic and peripheral Treg numbers, KLRG1 KO Tregs showed less suppressive capacity compared to wild type, implying KLRG1 in Tregs functional program. Furthermore, α-KLRG1 antibody treatment in lung tumor bearing KP mice showed prolonged survival, accompanied by reduced proportion of KLRG1+ Tregs in the tumor. Finally, immunogenomic characterization of KLRG1 KO/wt and tumor infiltrating KLRG1+/− Tregs revealed distinct gene expression patterns and associated immune pathways. Collectively, our findings indicate that KLRG1 is not critical for Treg development and maintenance but may play a non-redundant role in Tregs differentiation and function in inflammatory settings. Therefore, targeting KLRG1 to curtail the inhibitory function of the highly suppressive KLRG1+ Treg subset could facilitate antitumor responses in NSCLC.

CCL7 recruits cDC1 to promote antitumor immunity and facilitate checkpoint immunotherapy to non-small cell lung cancer

The efficacy of checkpoint immunotherapy to non-small cell lung cancer (NSCLC) largely depends on the tumor microenvironment (TME). Here, we demonstrate that CCL7 facilitates anti-PD-1 therapy for the KrasLSL−G12D/+Tp53fl/fl (KP) and the KrasLSL−G12D/+Lkb1fl/fl (KL) NSCLC mouse models by recruiting conventional DC 1 (cDC1) into the TME to promote T cell expansion. CCL7 exhibits high expression in NSCLC tumor tissues and is positively correlated with the infiltration of cDC1 in the TME and the overall survival of NSCLC patients. CCL7 deficiency impairs the infiltration of cDC1 in the TME and the subsequent expansion of CD8+ and CD4+ T cells in bronchial draining lymph nodes and TME, thereby promoting tumor development in the KP mouse model. Administration of CCL7 into lungs alone or in combination with anti-PD-1 significantly inhibits tumor development and prolongs the survival of KP and KL mice. These findings suggest that CCL7 potentially serves as a biomarker and adjuvant for checkpoint immunotherapy of NSCLC.

Abstract PR03: Modeling the genetic heterogeneity of KRAS mutant lung adenocarcinomas for therapeutic discovery

Abstract Lung adenocarcinoma (ADC) is the most common histologic subtype of lung cancer. Oncogenic driver mutations in KRAS are found in 36% of lung ADC cases and with a lack of specific KRAS inhibitors, this subset presents a significant clinical challenge. There is therefore an urgency to identify actionable co-occurring mutations as an alternative therapeutic approach. One of the most critical stress-response pathways in mammals is mediated by the transcription factor Nuclear Factor Erythroid-2-Related Factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-Associated Protein 1 (KEAP1). Loss-of-function mutations in KEAP1 have been identified in 19% of ADC, and often co-occur with KRAS mutations, but appear to be largely mutually exclusive with TP53 mutations. To best model these genetic subgroups of KRAS mutant lung ADC, we generated genetically engineered mouse models (GEMMs) whereby oncogenic KrasG12D was expressed either in combination with loss of p53 (KP mice) or Keap1 (KK mice). Critically, loss of Keap1 promoted KrasG12D-induced malignant transformation, which was associated with a unique metabolic program that could be exploited using pharmacologic inhibitors. Furthermore, bronchiolar epithelial cells appeared to be more sensitive than alveolar epithelial cells to transformation following Keap1 loss. Interestingly, this cell-type bias appeared to directly modulate the immune phenotype of lung tumors. Taken together, we have generated powerful in vivo models that enable us to address the relationship between tumor cells and resident immune cells to evaluate novel treatment strategies aimed at improving the long-term outcomes for patients carrying KRAS alterations. This abstract is also being presented as Poster B10. Citation Format: Sarah A. Best, Sheryl Ding, Boris Reljic, Ji-Ying Song, Kate D. Sutherland. Modeling the genetic heterogeneity of KRAS mutant lung adenocarcinomas for therapeutic discovery [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR03.

Protein Kinase Cι and Wnt/β-Catenin Signaling: Alternative Pathways to Kras/Trp53-Driven Lung Adenocarcinoma

We report that mouse LSL-KrasG12D;Trp53fl/fl (KP)-mediated lung adenocarcinoma (LADC) tumorigenesis can proceed through both PKCι-dependent and PKCι-independent pathways. The predominant pathway involves PKCι-dependent transformation of bronchoalveolar stem cells (BASCs). However, KP mice harboring conditional knock out Prkci alleles (KPI mice) develop LADC tumors through PKCι-independent transformation of Axin2+ alveolar type 2 (AT2) stem cells. Transformed growth of KPI, but not KP, tumors is blocked by Wnt pathway inhibition invitro and invivo. Furthermore, a KPI-derived genomic signature predicts sensitivity of human LADC cells to Wnt inhibition, and identifies a distinct subset of primary LADC tumors exhibiting a KPI-like genotype. Thus, LADC can develop through both PKCι-dependent and PKCι-independent pathways, resulting in tumors exhibiting distinct oncogenic signaling and pharmacologic vulnerabilities.

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.

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 LB-284: Engineering a hypermutant mouse model of NSCLC to modulate immune response

Abstract Immune checkpoint inhibitors are an exciting new class of cancer therapy with unprecedented response rates, and have shed light on the importance of understanding the immune context of tumors. Despite achieving durable clinical responses for a subset of patients, immune checkpoint therapies produce variable response rates, even within tumor type, suggesting a need for improved understanding of the tumor-immune cell axis. Recent clinical trials using checkpoint blockade against CTLA-4 and PD-1 have underscored the relevance of tumor mutational landscapes in predicting these response rates. For example, non-small cell lung cancer (NSCLC) patients with high mutational burden and PD-L1 expression respond better to anti-PD-1 treatment than those with high PD-L1 expression alone. While genetically engineered mouse models (GEMMs) of cancer are powerful platforms for testing new therapies, existing models fail to recapitulate the mutational burden and signatures of human cancers, limiting their ability to model relevant immune responses. We have therefore used CRISPR-Cas9 to knockout the DNA mismatch repair (MMR) gene Msh2 in the widely-used Kras and P53 mutant (KP) GEMM of NSCLC. Specifically, lentivirus expressing a short guide RNA (sgRNA) targeting Msh2, and Cre-recombinase, is delivered intra-tracheally into KP mice carrying a Cas9 allele. Whole-exome sequencing reveals elevated mutational burdens in Msh2KO tumors, as compared to non-targeted controls, and mutational signatures characteristic of MMR deficient human tumors. Histological characterization indicates elevated T cell infiltration of Msh2KO tumors at early time points, while tumors at later stages show minimal infiltration and appear like immune “cold” human tumors. Our future efforts will test checkpoint blockade therapies combined with immunogenic chemotherapy to facilitate neo-antigen presentation in late stage tumors. Development and characterization of this “hypermutant” model should prove valuable for future preclinical development of immunotherapies in NSCLC. Citation Format: Olivia C. Smith, Peter M. Westcott, David A. Canner, William M. Rideout, Arjun Bhutkar, Mary C. Beytagh, Tuomas Tammela, Tyler E. Jacks. Engineering a hypermutant mouse model of NSCLC to modulate immune response [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 LB-284.

Lung Endothelial MicroRNA-1 Regulates Tumor Growth and Angiogenesis.

Vascular endothelial growth factor down-regulates microRNA-1 (miR-1) in the lung endothelium, and endothelial cells play a critical role in tumor progression and angiogenesis. To examine the clinical significance of miR-1 in non-small cell lung cancer (NSCLC) and its specific role in tumor endothelium. miR-1 levels were measured by Taqman assay. Endothelial cells were isolated by magnetic sorting. We used vascular endothelial cadherin promoter to create a vascular-specific miR-1 lentiviral vector and an inducible transgenic mouse. KRASG12D mut/Trp53-/- (KP) mice, lung-specific vascular endothelial growth factor transgenic mice, Lewis lung carcinoma xenografts, and primary endothelial cells were used to test the effects of miR-1. In two cohorts of patients with NSCLC, miR-1 levels were lower in tumors than the cancer-free tissue. Tumor miR-1 levels correlated with the overall survival of patients with NSCLC. miR-1 levels were also lower in endothelial cells isolated from NSCLC tumors and tumor-bearing lungs of KP mouse model. We examined the significance of lower miR-1 levels by testing the effects of vascular-specific miR-1 overexpression. Vector-mediated delivery or transgenic overexpression of miR-1 in endothelial cells decreased tumor burden in KP mice, reduced the growth and vascularity of Lewis lung carcinoma xenografts, and decreased tracheal angiogenesis in vascular endothelial growth factor transgenic mice. In endothelial cells, miR-1 level was regulated through phosphoinositide 3-kinase and specifically controlled proliferation, de novo DNA synthesis, and ERK1/2 activation. Myeloproliferative leukemia oncogene was targeted by miR-1 in the lung endothelium and regulated tumor growth and angiogenesis. Endothelial miR-1 is down-regulated in NSCLC tumors and controls tumor progression and angiogenesis.

P3.02-073 Stromal Hedgehog Pathway Activation Suppresses Growth and Metastasis of Lung Adenocarcinoma

Aberrant activation of the Hedgehog (Hh) signaling pathway, a crucial developmental pathway, drives the tumor growth of Gorlin-type cancers. However, recent data suggest that paracrine activation of the pathway is tumor suppressive rather than oncogenic in sporadic epithelial cancers. The role of the pathway in non-small lung cancer is poorly understood. Thus, we explored the role of stromal Hh pathway activation in growth of lung adenocarcinoma. Human lung adenocarcinoma cell lines were used to probe SHH mRNA and protein expression. Co-culture of high SHH expressing cell lines with murine embryonic and lung fibroblasts were used to confirm and probe the role of paracrine SHH expression on the growth of lung cancer cells. The in vivo role of paracrine SHH was tested using autochthonous lung cancer models with conditional KRASG12Dactivation, p53 loss, and SHH loss compared to wild-type SHH. In human lung adenocarcinoma patients, higher expression of SHH mRNA in lung adenocarcinoma correlated with poor overall and progression free survival. A scan of 35 human lung adenocarcinoma cell lines revealed heterogeneous expression of SHH and IHH with high expression found predominantly in mutant K-Ras lines. Co-culture of high SHH expressing tumor epithelial cells and Shh-Light2 reporter cell lines demonstrated that SHH activated the fibroblast reporter in a paracrine manner, rather than an autocrine effect on cancer cells. Treatment with the SMO inhibitor, KAAD-cyclopamine, also inhibited the growth of tumor epithelial cells in co-culture with NIH-3T3 fibroblast cells but the effect was decreased when co-cultured with lung fibroblasts. Genetic loss of SHH in an autochthonous mouse model, LSL-KrasG12D/+;Trp53fl/fl; Shhfl/fl (KPS) did not affect overall survival compared to LSL-KrasG12D/+; Trp53fl/fl(KP) mice However, early inhibition of the Hh pathway by anti-SHH/IHH antibody, 5E1, on KP mice resulted in significantly worse survival rates with increased metastatic burden compared to IgG treatment. Analysis of KP tumors revealed unexpected high levels of IHH mRNA by in situ hybridization and qPCR that may account for the survival differences seen between genetic ablation and pharmaceutical inhibition of the Hh ligands. The Hh signaling pathway acts upon lung stromal cells in a paracrine fashion and induces distinct transcriptional programs in murine embryonic and lung fibroblasts. Inhibition of paracrine Hh pathway activity in vivo worsened mortality rate due to increase in tumor growth and metastases. Furthermore, mutant Kras lung adenocarcinomas express high levels of IHH that dominates the tumor suppressive effects in our mouse models.

Abstract 1834: Constitutive Nedd9 null genotype promotes lung cancer aggressiveness

Abstract Non-small cell lung cancer (NSCLC) has a low survival rate, with metastasis contributing to the vast majority of deaths. The NEDD9 (HEF1/Cas-L) protein has been reported to be elevated in expression and to promote metastasis in a large subset of lung cancers and in other malignancies. NEDD9 functions as a scaffold for multiple critical effectors in integrin/FAK/SRC and receptor tyrosine kinase signaling cascades, as well as for the mitotic kinase Aurora-A, with overexpression thought to sustain signaling by these pro-oncogenic proteins. Previous studies demonstrated depletion of NEDD9 by RNAi reduced the growth and invasion of established lung cell lines and tumors, based on cell culture and xenograft assays. We have now investigated the consequences of a null genotype for Nedd9 from the earliest stages of tumor formation, crossing Nedd9 null mice to a 129S/Sv-Krastm3Tyj/Trp53tm1Brn (KP) model in which Kras mutation is induced specifically in lung tissue by inhalation of adenovirus bearing the Cre gene. Unexpectedly, based on in vivo imaging, the Nedd9 null genotype significantly accelerated tumor growth in KP mice. Pathological examination of tissues indicated Nedd9 null genotype also was associated with higher invasive capacity in vivo, including direct invasion to the heart, as well as elevated proliferation rate, decreased apoptotic activity, and changes in the expression of proteins such as vimentin, associated with mesenchymal status. Although Nedd9 function has been implicated in immune cell activity, we found minimal differences in tumor infiltration of myeloid cells or macrophages based on Nedd9 genotype. Rather, use of Reverse Phase Protein Array (RPPA) analysis to characterize signaling in isolated tumors suggested tumor intrinsic changes in glycolytic and proliferative signaling pathways, which were subsequently validated using direct functional assays. These results for the first time imply that NSCLC tumor progression beyond early stages in the absence of Nedd9 requires reprogramming of intrinsic tumor signaling to compensate for the absence of this protein, emphasizing activities distinct from those associated with elevated Nedd9 in late stage tumors. Citation Format: Alexander Deneka, Meghan Kopp, Anna S. Nikonova, Anna Gaponova, Alexandra Nagele, Harvey Hensley, Erica Golemis. Constitutive Nedd9 null genotype promotes lung cancer aggressiveness [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 1834. doi:10.1158/1538-7445.AM2017-1834