Targeting Lactate Dehydrogenase-A Inhibits Tumorigenesis and Tumor Progression in Mouse Models of Lung Cancer and Impacts Tumor-Initiating Cells

Introduction: We use SIRM to evaluate metabolic reprogramming of lung cancer cells in monoculture, in mouse xenograft/explant models, and in NSCLC patients in situ (1,2). We have now extended the range of models to fresh human tissue slices, which retain the original tissue architecture and heterogeneity with a paired benign versus cancer tissue design under defined cell culture conditions. β-glucan is a polysaccharide that repolarizes tumor-associated macrophages (TAMs) from the M2 to the M1 phenotype in mice (3). Here we report the activation of TAMs in human NSCLC tissue slices. Experimental: Freshly resected paired tissue slices from individual patients (approx. 1 mm or less thick and 5-40 mg wet weight) were incubated ± particulate β-glucan in standard cell culture conditions, with gentle rocking to enable efficient gas, nutrient, and waste product exchange. Tissue slices could be maintained metabolically viable for at least 48 h of incubation. The metabolic activity was determined by measuring the uptake and transformation of 13C and/or 15N-enriched common nutrient tracers such as glucose and glutamine, using high resolution mass spectrometry, GC-MS, and NMR after a period of incubation (2). Findings: Time-course analysis of the slices by NMR, MS, and histology revealed that NSCLC tissue slices, both benign and tumorous, retained their architecture and a broad spectrum of metabolic activities. Glucose and glutamine metabolism was reprogrammed in the tumor relative to the paired benign tissues ex vivo, as the in vivo case. The paired tissues from different patients showed significantly different metabolic responses to β-glucan, as expected Conclusions: This platform offers a human tissue model for preclinical studies on metabolic reprogramming of human cancer and stromal cells in their tissue context, and response to drug treatment (4). As the microenvironment of the target human tissue is maintained, including the resident immune cells, individualized response to immune-active agents can be determined in a clinically relevant setting. Supported by NCI P01CA163223-01 and NIEHS 1R01ES022191-01 1. Lane, A.N., Fan, T.W.-M., Bousamra II, M., et al. (2011) Clinical Applications of Stable Isotope-Resolved Metabolomics (SIRM) in Non-Small Cell Lung Cancer. Omics, 15, 173-182. 2. Sellers, K., Fox, M.P., Bousamra, M., II, et al. (2015) Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation. Journal of Clinical Investigation, 125, 687-698. 3. Liu, M., Luo, F., Ding, C., et al. (2015) Particulate β-Glucan Converts Immunosuppressive Macrophages into M1 Phenotype Through Dectin-1-induced Syk-Card9-Erk Pathway and Raf-1-c-Maf Pathway. J. Immunol., 195, 5055-5065. 4. Xie, H., Hanai, J., Ren, et al. (2014) Targeting lactate dehydrogenase-A (LDH-A) inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor initiating cells. Cell Metabolism 19, 795-809 Citation Format: Teresa W-M Fan, Andrew N. Lane, Jun Yan, Richard M. Higashi, Jeremiah T. Martin, Michael Bousamra. Beta-glucan activates macrophages in human NSCLC demonstrated by Stable Isotope Resolved Metabolomics (SIRM). [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 4271.

Introduction: All preclinical drug testing models have advantages and drawbacks. We have been using SIRM to evaluate metabolic reprogramming of lung cancer cells in monoculture, in mouse xenograft/explant models, and in NSCLC patients in situ (1), and to determine the influence of the tumor microenvironment using these models. We have now extended the range of models to fresh human tissue slices, similar to those originally described by Warburg (2), which retain the original tissue architecture and heterogeneity with a paired benign versus cancer design under controlled cell culture conditions. Experimental: Freshly resected tissue slices from individuals (ca. 1 mm or less thick and 5-40 mg wet weight) were incubated in standard cell culture conditions with gentle rocking for efficient gas, nutrient and waste product exchange. The metabolic activity was determined by measuring the uptake and transformation of 13C and/or 15N-enriched nutrient tracers such as glucose and glutamine, using high-resolution MS, GC-MS, and NMR after a period of incubation. Acute metabolic and histologic response to inhibitors or drugs was readily detected in treated tissue slices. Findings: Analysis at different time points by NMR, MS and histology shows that the NSCLC tissue slices, both benign and tumorous, retained their architecture and remained metabolically viable for at least 48 h of incubation. Glucose and glutamine metabolism was reprogrammed in the tumor relative to the paired benign tissues. The paired tissue also showed very different responses to Se-containing compounds when incubated at the IC50 established for cell lines. After 24 h of incubation, large scale necrosis was evident in the tumor, but not in the benign slices, which was accompanied by large changes in metabolic activities observed by SIRM analysis. Conclusions: This platform offers a human tissue model for preclinical studies on metabolic reprogramming of human cancer cells in their tissue context, and response to drug treatment (3). As the microenvironment of the target human tissue is retained and individualized response to drugs is obtained, this platform promises to transcend current limitations of drug selection for clinical trials or treatments. Supported by NCI P01CA163223-01A1 and NIEHS 1R01ES022191-01 1. Lane, A.N., Fan, T.W.-M., Bousamra II, M., Higashi, R.M., Yan, J. and Miller, D.M. (2011) Clinical Applications of Stable Isotope-Resolved Metabolomics (SIRM) in Non-Small Cell Lung Cancer. Omics, 15, 173-182. 2. Warburg, O. (1923) Versuche an überlebendem Carcinomgewebe (Methoden). Biochem. Zeitschr., 142, 317-333. 3. Xie, H., Hanai, J., Ren, J.-G., Kats, L., Burgess, K., Bhargava, P., Signoretti, S., Billiard, J., Duffy, K.J., Grant, A. et al. (2014) Targeting lactate dehydrogenase-A (LDH-A) inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor initiating cells. Cell Metabolism 19, 795-809. Citation Format: Andrew N. Lane, Teresa W-M Fan, Alexander C. Belshoff, Richard M. Higashi, Jeremiah Martin, Michael Bousamra. Stable isotope resolved metabolomics (SIRM) on fresh human tissues as a preclinical drug testing platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3199. doi:10.1158/1538-7445.AM2015-3199

Lung cancer is a leading cause of cancer death worldwide. This disease is of serious concern in Kentucky, which leads the nation in both lung cancer incidence and mortality. The past decade of research in cancer metabolism reveals the untapped value of exploring human metabolome for the discovery of novel therapeutic and diagnostic biomarkers for human cancers and other diseases. To better understand metabolic reprogramming in individual tumors of lung cancer patients, we have developed stable isotope tracers (e.g., [13C6]-glucose) coupled with NMR and MS-based stable isotope-resolved metabolomics (SIRM) analysis directly in patient and in patient-derived ex vivo and in vivo models. In the in vivo patient study, we mapped differential metabolic network between paired cancerous (CA) and noncancerous (NC) tissues procured from non–small cell lung cancer (NSCLC) patients infused with [13C6]-glucose and snap-frozen immediately after resection (1). In particular, we uncovered elevated anaplerotic pyruvate carboxylase (PC) activity in CA versus paired NC tissues. Proliferating cancer cells require active Krebs cycle for generating anabolic precursors, in addition to energy production. Diversion of the Krebs cycle intermediates to meet anabolic demands cannot be sustained without anaplerosis. Pyruvate carboxylation represents one of the two major anaplerotic pathways that replenish the Krebs cycle intermediates; the other involves glutaminolysis initiated by glutaminase (GLS). We also found that PC but not GLS protein was overexpressed (median 8-10 fold; n=86) in CA tissues relative to paired NC tissues and that PC expression was functionally important to NSCLC cell growth both in vitro and in vivo (1). We also utilize the “Warburg slice” concept to systematically define metabolic distinctions between thinly sliced paired CA versus NC lung tissues freshly resected from individual NSCLC patients and cultured in stable isotope tracers. This ex vivo tissue slice culture system is excellently suited for delineating reprogrammed metabolic pathways in CA tissues without systemic interferences (2-4). These lung tissue slices were metabolically viable for up to 72 hr, while maintaining their 3D architecture and microenvironment (4). We found that the reprogrammed metabolic network in the cultured human tissue slices recapitulated that in vivo (1). These advantages make the ex vivo human tissue slice systems a unique preclinical model for exploring human target tissue metabolism and how it underlies the response to anticancer agents such as chemopreventive Se compounds, enzyme inhibitors (5), and the immune modular β-glucan (4). We found that selenite blocked PC anaplerosis and elicited massive necrosis in CA but not in NC lung tissues. CA lung tissue slices also responded to β-glucan with perturbed metabolic activity and histopathologic changes, which were consistent with polarization towards M1-type macrophages. We were intrigued to find that the metabolism and histopathology of these CA lung tissue slices from different patients responded distinctly to these anticancer agents, which could be translated into individual patients' response to drugs. Furthermore, to delineate systemic and microenvironmental influences on cancer metabolism, we compared and found that the ex vivo tissue slice cultures incorporated to a higher extent 13C from [13C6]-glucose into glycolytic, PPP, and purine nucleotide products than the corresponding mouse PDX in vivo, illustrating the high metabolic viability of the ex vivo tissue slice cultures. These new developments in preclinical models and mechanistic metabolic interrogations promise to provide rigorous prediction for individual patients' response to therapeutics while revealing new and exciting targets for the next generation of personalized therapeutics. Supported by 1R01CA118434-01A2, 1P01CA163223-01A1, 1R01ES022191-01, 3R01ES022191-04S1, 3R01CA118434-02S1, 1U24DK097215-01A1, P30CA177558; KLCRP, and the KY Challenge for Excellence.

BackgroundWorldwide, lung cancer kills more people than breast, colon and prostate cancer combined. Alterations in macrophage number and function during lung tumorigenesis suggest that these immune effector cells stimulate lung cancer growth. Evidence from cancer models in other tissues suggests that cancer cells actively recruit growth factor-producing macrophages through a reciprocal signaling pathway. While the levels of lung macrophages increase during tumor progression in mouse models of lung cancer, and high pulmonary macrophage content correlates with a poor prognosis in human non-small cell lung cancer, the specific role of alveolar macrophages in lung tumorigenesis is not clear.MethodsAfter culturing either an immortalized lung macrophage cell line or primary murine alveolar macrophages from naïve and lung-tumor bearing mice with primary tumor isolates and immortalized cell lines, the effects on epithelial proliferation and cellular kinase activation were determined. Insulin-like growth factor-1 (IGF-1) was quantified by ELISA, and macrophage conditioned media IGF-1 levels manipulated by IL-4 treatment, immuno-depletion and siRNA transfection.ResultsPrimary macrophages from both naïve and lung-tumor bearing mice stimulated epithelial cell proliferation. The lungs of tumor-bearing mice contained 3.5-times more IGF-1 than naïve littermates, and media conditioned by freshly isolated tumor-educated macrophages contained more IGF-1 than media conditioned by naïve macrophages; IL-4 stimulated IGF-1 production by both macrophage subsets. The ability of macrophage conditioned media to stimulate neoplastic proliferation correlated with media IGF-1 levels, and recombinant IGF-1 alone was sufficient to induce epithelial proliferation in all cell lines evaluated. Macrophage-conditioned media and IGF-1 stimulated lung tumor cell growth in an additive manner, while EGF had no effect. Macrophage-derived factors increased p-Erk1/2, p-Akt and cyclin D1 levels in neoplastic cells, and the combined inhibition of both MEK and PI3K ablated macrophage-mediated increases in epithelial growth.ConclusionsMacrophages produce IGF-1 which directly stimulates neoplastic proliferation through Erk and Akt activation. This observation suggests that combining macrophage ablation therapy with IGF-1R, MEK and/or PI3K inhibition could improve therapeutic response in human lung cancer. Exploring macrophage-based intervention could be a fruitful avenue for future research.

Tumor-associated macrophages (TAMs) are well-known as anti-inflammatory immune cells that contribute to various facets of prostate cancer development. An emerging aspect of TAMs is their “iron-rich” phenotype. This accumulation of iron can contribute to cancer cell initiation and tumor progression. In the current study, we aim to explore the key role of TAMs in regulating iron balance within the prostate tumor microenvironment (TME). We induced prostate tumorigenesis in the NP mice using Tamoxifen. The NP mice (Nkx3.1CreERT2/+ ; Ptenflox/flox ; Rosa26-CAG-LSL-EYFP/+ mice) develop prostate intraepithelial neoplasia and localized prostate adenocarcinoma by 12 months of age. We also utilized male TRAMP which spontaneously develops poorly differentiated prostate tumors reminiscent of the neuroendocrine subtype. We then stained prostate tissues using different immune cell markers and Perls''s method which stains “non-heme” iron to identify cells with high abundance of iron. Our results demonstrate that prostate tissues from two distinct transgenic prostate cancer models (adenocarcinoma and neuroendocrine), spanning different developmental stages (12 and 18 months) exhibited high level of non-heme iron in stromal regions compared with non-tumor bearing controls. The areas with high iron abundance exhibited high expression of F4/80 and the iron exporter Ferroportin-1, also known as solute carrier family 40 member 1 (SLC40A1). In summary, we found that the availability of non-heme iron correlates with the abundance of TAMs in prostate tumor microenvironment. Future studies will focus on understanding how TAMs are regulating iron availability in the dynamic tumor environment. Citation Format: Jennifer Rooks, Caleb Taylor, Asmaa El-Kenawi. Iron accumulation correlates with tumor progression in mouse models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6546.

INTRODUCTION: The focus of this work is to develop new mouse models for metastatic lung cancer. Lung cancer is the leading cause of cancer deaths worldwide and greater than 90% of human cancer deaths are due to metastasis. Metastatic disease in most existing mouse models of lung cancer is typically sporadic and often requires aging mice for several months. These factors limit the usefulness of mouse models for basic and pre-clinical research aimed at effective treatment of metastatic disease. METHODS AND RESULTS: We have developed a new mouse model of lung cancer and are in the process of developing two additional models. In the first model, we added mutations in Dicer1, an RNAse III enzyme within the microRNA (miRNA) biosynthesis pathway to a mouse model of Kras-driven pulmonary adenocarcinoma. When we combined conditional expression of an oncogenic allele of Kras (KrasG12D), deletion of both alleles of Trp53 and one allele of Dicer1 in Club cells with expression of truncated Dicer1 in alveolar type II cells, we generated mice with metastatic pulmonary adenocarcinoma. In 30% of these mice, metastatic tumors were observed in the lymph nodes within 11 weeks of tumor induction. In a second mouse model, we switched the cell types expressing the various mutations to determine the effects of cell of origin on tumor progression and metastasis. Our preliminary results suggest that adenocarcinoma develops even faster when KrasG12D is expressed and Trp53 and one allele of Dicer1 are deleted in alveolar type II cells. Finally, based on an analysis of human lung cancer genomics data from TCGA, we generated a mouse model with mutations in Zfhx4 and are crossing these mice with our models that express oncogenic KrasG12D, delete expression of Trp53 and one allele of Dicer1 and truncate the second allele of Dicer1 to determine the effects of adding Zfhx4 mutations on progression and metastasis of pulmonary adenocarcinoma. CONCLUSIONS: Through cell type specific truncation/deletion of Dicer1 we have generated new mouse models that rapidly develop pulmonary adenocarcinomas and metastatic disease. These models have potential for both understanding the basic processes of metastasis and for pre-clinical studies aimed at preventing and/or treating metastatic lung cancer. Citation Format: Julie Wells, Richard S. Maser, Teresa McGee, Wendy Memishian, Rosalinda Doty, Carol J. Bult. Developing new mouse models of metastatic lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1617.

Ras proteins are one of the most prevalent oncogenic drivers of human cancer and lung, pancreatic and colon cancers frequently harbor mutated forms of the KRAS gene. The oncogenic role of Ras is executed via direct interaction with, and activation of, a number of downstream effectors including PI 3-kinases, Raf kinases, RalGDS and Tiam1. Previously our laboratory has demonstrated that disruption of PI 3-kinase activation by oncogenic K-Ras, via mutation of two key residues (T208D and K227A) in the Ras binding domain of p110alpha ( p110alpha-RBD), prevents tumor development in a mouse model of lung cancer. As tumor maintenance is clinically of more significance than tumor initiation to the treatment of pre-existing human cancers, we have subsequently developed an inducible mouse model to investigate whether interaction of Ras and PI 3-kinase is required for the maintenance of established tumors. For this we utilised K-Ras LA2 mice which spontaneously develop K-Ras mutant lung tumors. We have also introduced one allele of Pik3ca mutated in the Ras binding domain, one allele of Pik3ca WT flanked by lox-p sites, and a separate allele of inducible Cre-ER recombinase. Adult mice harboring lung tumors were then treated with tamoxifen to facilitate Cre mediated removal of the floxed allele and exclusive expression of p110alpha-RBD in established tumors. Using MicroCT scanning to monitor individual tumor growth, we have found that interruption of Ras PI 3-kinase signalling induces partial tumor regression, prevents further tumor growth and results in long-term stabilization of lung tumors. Expression of the p110alpha-RBD mutant decreased downstream signalling and reduced overall tumor burden. Orthotopic transplantation of tumor cells into WT mice with intact PI 3-kinase signaling revealed that the tumor cells are intrinsically sensitive to p110alpha-RBD expression. We also compared the effect of p110alpha-RBD expression with complete removal of p110alpha and found that inhibition of tumor growth occurred to a similar extent indicating that p110alpha signalling in these tumors is dependent upon interaction with Ras. Furthermore, combination of p110alpha-RBD expression with inhibition of the MAPK pathway promoted extensive tumor regression and dramatically reduced tumor burden. This effect was also observed with co-treatment with PI 3-kinase inhibitors and Mek inhibitors. This study demonstrates that the interaction of Ras and PI 3-kinase continues to play an important role in the maintenance of established tumors and in tumor progression. Our results indicate that the PI3K pathway is a viable therapeutic target in Ras mutant tumors, particularly in combination with other therapeutics such as Mek inhibitors. Citation Format: Clare Sheridan, Esther Castellano, May Zaw Thin, Miguel Murillo, Francois Lassailly, Gordon Stamp, Julian Downward. Requirement for interaction of PI 3-kinase p110alpha with Ras in lung tumor maintenance. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A54.

Background: c-Met is a proto-oncogene that encodes the protein Met with intrinsic tyrosine kinase activity. Aberrant Met kinase activity triggers a series of unwarranted phosphorylation events and signalling processes that ultimately lead to the development of cancer. Alteration of the Met kinase signalling cascade represents an attractive approach aimed at blocking invasion and metastasis of cancer cells. Herein, we describe the biological activity and pharmacokinetic properties of RP1400, a novel, selective, and potent Met kinase inhibitor with scope to be developed as a clinical candidate for cancers mediated by dysregulated Met kinase activity. Methods: Met Kinase activity of RP1400 was determined using an HTRF® KinEASE assay kit (Cisbio, Bedford, MA) with modifications. Met-dependent antiproliferative effect was determined in a host of Met amplified cell lines representative of various cancers. Inhibition of constitutive Met kinase phosphorylation in MKN-45 and NCI-H441 cells was measured in an ELISA assay. Subsequently, effect of the compound on Akt and Stat-5a phosphorylation, downstream markers in the Met signalling cascade, was determined. In vivo efficacy of RP1400 was evaluated in subcutaneous MKN-45 (gastric cancer), U87MG (glioblastoma), and MHCC97H (hepatocellular carcinoma) xenografts using SCID or nude mice. Pharmacokinetic behavior of the compound in plasma after single dose oral administration or IV injection was determined in Balb/c mice. Results: RP1400 demonstrated remarkable potency against the purified Met kinase enzyme (8.9 nM) with >50-fold selectivity against other kinases in a 451-kinase panel. Inhibition of Met kinase activity was accompanied by a significant reduction in constitutive Met phosphorylation in MKN-45 (28.6 nM) and NCI-H441 (1.8 nM) cells. As a consequence, Akt and Stat-5a phosphorylation were inhibited half-maximally in MKN-45 cells at 16.2 nM and 11.2 nM respectively. RP1400 caused a significant inhibition in proliferation of Met amplified cell lines including MKN-45, EBC-1, SNU-5, and MHCC97H with IC50 values ranging from 3-80 nM. Compounded with a favorable pharmacokinetic profile, in vitro potency of RP1400 translated into excellent in vivo efficacy with >80% reduction in tumor growth noticed in MKN-45, U87MG, and MHCC97H xenografts at 100 mg/kg/BID/PO dose. Conclusions: Our findings demonstrate the potency of RP1400, a novel and selective small-molecule inhibitor of Met kinase with efficacy values comparable or superior to existing Met kinase inhibitors in development. On lines with selective inhibitors, the compound displayed antiproliferative effect only in cell types harboring amplification of the Met kinase gene. RP1400 is currently undergoing extensive toxicological evaluation with clinical trials anticipated in H1 2014. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B287. Citation Format: Srikant Viswanadha, Babu G, Sridhar Veeraraghavan, Swaroop Vakkalanka. Selective targeting of Met-kinase by RP1400 attenuates tumor progression in mouse models of gastric cancer, glioblastoma, and hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B287.

Targeted therapies against activated oncogenes, such as receptor tyrosine kinases, have significantly prolonged non-small cell lung cancer (NSCLC) patient survival, but the development of resistance limits the durability of clinical response. Genetic alterations which constitutively activate Fibroblast Growth Factor Receptors (FGFR) have been observed in patients with NSCLC. Erdafitinib (JNJ-42756493), an orally bioavailable pan-FGFR inhibitor discovered as part of a collaboration between Janssen and Astex Pharmaceuticals, has been shown to inhibit FGFR signaling pathways resulting in cell death and tumor growth inhibition in both in vitro and in vivo models of FGFR pathway aberration. Further, erdafitinib has been shown to have favorable pharmaceutical properties with manageable side effects in humans and several clinical trials are currently underway. One potential strategy to enhance the durability of response to targeted therapies, such as FGFR inhibitors, is to couple them with immunotherapy. In this setting, T cell responses primed and activated by increased antigen release resulting from the tumor cell targeted therapy could be enhanced and maintained by T-cell directed checkpoint blockade. To test this hypothesis, we evaluated erdafitinib in combination with an anti-programmed death-1 (PD-1) blocking antibody in an autochthonous FGFR2K660N/p53 genetically engineered mouse model (GEMM) of lung cancer, in which tumors develop within the context of an intact immune microenvironment. Cohorts of tumor bearing FGFR2K660N/p53 mutant mice treated with erdafitinib with or without anti-PD-1 showed significant tumor regressions compared to control and anti-PD-1 alone groups. Despite lack of differences in acute tumor responses between erdafitinib monotherapy and combination therapy, we observed significant survival benefit in the combination group erdafitinib alone (median survival 19.7 weeks vs 13.4 weeks, p<0.004). In a separate study, similar tumor regressions were noted in the FGFR-driven GEMM at 1 week of erdafitinib with or without anti-PD-1 treatment, while no such response to these treatments was noted in a KRAS-driven lung cancer GEMM. Immune profiling of tumor specimens revealed high baseline expression of programmed death ligand-1 (PD-L1) expression by IHC and flow cytometry. Following combination treatment, subsequent immunohistochemistry (IHC) analyses showed a significant decrease in Ki67 and PD-L1 positive tumor cells, accompanied by an increase in cluster of differentiation 3 (CD3) positive tumor-infiltrating cells in combination group as compared to control. T cell function is not inhibited by erdafitinib, as measured in vitro by mixed lymphocyte reaction and cytomegalovirus recall assays. Additional changes observed in lung tumors across treatment groups in immune cell infiltration, functionality, and T-cell clonality will be discussed. These data suggest that combination treatment of erdafitinib and PD-1 blockade drives improved survival in FGFR2-driven model of lung cancer by simultaneous inhibition of FGFR pathway in tumor cells and enhancement of anti-tumor immunity. Thus, data here provide rationale for the combined clinical testing of erdafitinib and PD-1 blockade in patients with FGFR-altered lung cancers. Citation Format: Sangeetha Palakurthi, Mari Kuraguchi, Sima Zacharek, Jeff Liu, Dennis Bonal, Wei Huang, Kristin Depeaux, Abha Dhaneshwar, Sam Regan, Dyane Bailey, Martha Gowaski, Mei Zheng, Roderick Bronson, Catherine Ferrante, Enrique Zudaire, Sylvie Laquerre, Mark Bittinger, Kirschmeier Paul, Kathryn Packman, Raluca I. Verona, Kwok-Kin Wong, Matthew V. Lorenzi. Improved survival with erdafitinib (JNJ-42756493) and PD-1 blockade mediated by enhancement of anti-tumor immunity in an FGFR2-driven genetically engineered mouse model of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B27.

The goal of this study was to use an inexpensive macroscopic imaging system to monitor tumor progression in mouse models in real-time with minimal intervention. Illumination is provided via a xenon arc lamp and a fiber optic probe which delivers white light or quasi-monochromatic excitation via specific bandpass filters. Fluorescence emission from SCID and nude mice following mammary fat pad injection of red fluorescence protein (RFP)-expressing human breast cancer cell lines was recorded and quantified using a single lens reflex (SLR) digital camera. This simple system enabled the verification of successful tumor take and temporal quantification of tumor progression in mouse models. The macroscopic fluorescence imaging system represents an inexpensive and portable tool to facilitate non-invasive in situ cancer detection with the potential to monitor fluorescent tumor formation and investigation of the efficacy of potential cancer therapeutics.

Metastasis of solid tumors is associated with poor prognosis and bleak survival rates. Tumor infiltrating myeloid cells (TIMs) are known to promote metastasis but the mechanisms underlying their collaboration with tumor cells remain unknown. Here we report an oncogenic role for microRNA in driving M2 reprogramming in TIMs, characterized by the acquisition of pro-tumor and pro-angiogenic properties. The expression of miR-21, miR-29a, miR-142-3p and miR-223 increased in myeloid cells during tumor progression in mouse models of breast cancer and melanoma metastasis. Further, we show that these miRs are regulated by the CSF1-ETS2 pathway in macrophages. A loss of function approach utilizing selective depletion of the microRNA processing enzyme Dicer in mature myeloid cells blocks angiogenesis and metastatic tumor growth. Ectopic expression of miR-21 and miR-29a promotes angiogenesis and tumor cell proliferation through the down-regulation of anti-angiogenic genes such as Col4a2, Spry1 and Timp3 whereas knockdown of the miRs impedes these processes. miR-21 and miR-29a are expressed in Csf1r+ myeloid cells associated with human metastatic breast cancer and levels of these miRs in CD115+ non-classical monocytes correlates with metastatic tumor burden in patients. Taken together, our results suggest that miR-21 and miR-29a are essential for the pro-tumor functions of myeloid cells and the CSF1-ETS2 pathway upstream of the miRs serves as an attractive therapeutic target for the inhibition of M2 remodeling of macrophages during malignancy. In addition, miR-21 and miR-29a in circulating myeloid cells may potentially serve as biomarkers to measure therapeutic efficacy of targeted therapies for CSF1 signaling.

Effective small molecule therapies are a major unmet need in triple-negative breast cancer. Therefore, we examined the mechanism of action of a novel cancer therapeutic target in preclinical mouse models focusing on the α7 nicotinic acetylcholine receptor (CHRNA7). E0771 breast tumor cells were implanted into CHRNA7KO mice to determine the role of CHRNA7, which is expressed in tumor-associated myeloid immune cells. We observed that tumor-bearing CHRNA7KO mice had decreased survival and increased tumor burden linked to a CHRNA7-mediated reduction in immune cell activation. Based on the tumor permissive phenotype of CHRNA7KO mice, we tested the effect of a small molecule agonist of CHRNA7, AR-R17779, in several mouse models of breast cancer. For example, in both the E0771 tumor model and PyMT tumor models, treatment with AR-R17779 increased survival. In the 4T1 breast tumor model, treatment with AR-R17779 also increased survival, with a well-defined reduction in primary tumor burden and lung metastases. The antitumorigenic effects of AR-R17779 were linked to an adaptive immune response based on in vivo studies showing a survival benefit when AR-R17779 was administered as a combination therapy with anti-PD-L1, demonstrating that the effects of AR-R17779 were dependent on CD8 T cells, and in vitro studies showing AR-R17779 treatment of dendritic cells increased T cell activation. Together these findings supported the importance of CHRNA7 as a novel therapeutic target expressed on dendritic cells based on its role in potentiating the adaptive immune response in mouse models of breast cancer.

Accurately modeling human lung tumor initiation and progression in genetically engineered mouse models (GEMMs) remains a challenge. In contrast to human lung cancer development, where patients typically present with a solitary tumor nodule, the majority of GEMMs produce numerous independent primary lung lesions. Each of these lesions then evolves as an independent tumor, a situation not relevant to human lung cancer biology. The Cre-loxP recombination system is widely used to generate GEMMs, including the inducible, tamoxifen-dependent systems that allow cell-specific conditional activation via promoter regulation. We have used this system extensively to generate KRAS-driven Lung adenocarcinomas arising from club cells via the club cell secretory protein (CCSP) Cre-ERT2 system. While powerful, this model has limitations. Specifically, limiting tumor induction to a single location within the lung is impossible. To address this limitation, we have combined the highly efficient photoactivable Cre (PA-Cre) recombinase mouse (JAX: 033544) with a novel CCSP-FlpO mouse to spatially limit tumor formation based on targeted blue-light exposure. Exploiting the CCSP promoter enables us to limit FlpO recombinase expression to club cells, thereby confining the two light-activatable Cre halves to club cells. Following focal trans-cutaneous blue light exposure, the PA -Cre halves dimerize and induce the oncogenic alleles, KRAS p.G12D, and Serine Threonine Kinase 11 loss, respectively. We assert that utilizing this approach will overcome major limitations inherent in current GEMMs used to study lung cancer and provide a paradigm-shifting platform to study lung cancer initiation and progression. Citation Format: Israel Ifeoluwa Odekunle, Sydney Remington, Allison Racela, David J. Seward. An inducible, spatially restricted mouse model of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7227.

We have developed a preclinical model of lung cancer associated with hyper-activated PI3K/mTOR and KRAS. Non-small cell lung cancer (NSCLC) accounts for ∼80% of all lung cancers; of which >50% have aberrant PI3K/AKT/mTOR signaling and ∼30% harbor oncogenic KRAS, conferring chemo/radio-resistance and poor prognosis. KRAS inhibitors are difficult to develop, and rapalogs targeting mTOR are toxic and induce compensatory activation of AKT, causing resistance to apoptosis. Hence, development of effective inhibitors of KRAS and PI3K/mTOR pathways is imperative. Further, clinical studies implicate that tumors with aberrant KRAS and PI3K/mTOR signaling are associated with epithelial-mesenchymal transition (EMT), metastasis, poor differentiation, and chemo/radio-resistance. However, molecular details of EMT mediated by KRAS and PI3K/mTOR pathway is lacking, limiting the development of drugs targeting EMT-mediators. Using a CCSP-promoter driven Cre/LoxP mediated deletion of PTEN in an oncogenic KRAS status (PTENΔΔ/KRasG12D), we have developed a preclinical mouse model of lung cancer with hyper-activation of PI3K/mTOR and KRAS pathways. Microarray based RNA profiling followed by bioinformatic analysis on PTEN-null lung epithelial cells revealed transcriptional activation of RAS/RAF/MAPK/ERK pathway associated candidate genes. On the other hand, Dox-induced PTENΔΔ/KRasG12D lung tumors showed induction of SNAIL1, SLUG, ZEB-2 and SOX2 genes. Taken together, our analysis reveals that PTEN loss predisposes lung tumors to hyper-activation of the KRAS pathway, which is further accentuated by oncogenic mutation in KRAS, initiating EMT and increased stemness. Citation Format: Prerna Malaney, Vrushank Dave. Loss of PTEN cooperates with mutant KRAS initiating EMT and increased stemness in a mouse model of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B09. doi: 10.1158/1557-3125.RASONC14-B09

Seventy percent of patients with Epidermal Growth Factor Receptor (EGFR) mutant lung cancer respond to treatment with the tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. Despite this high response rate, patients almost inevitably develop resistance to these drugs on average within a year of starting drug treatment. Acquired resistance to EGFR TKIs is most commonly due to the emergence of a secondary mutation (T790M) in EGFR (in 50% of cases), amplification of the genes encoding the ERBB2 and MET receptor tyrosine kinases in 12 and 5% of cases, respectively, and phenotypic transformation of the adenocarcinomas to small cell lung cancer (5% of cases). Previously, in an effort to develop strategies to overcome T790M-mediated resistance, we generated tetracycline-inducible transgenic mice that express the EGFRL858R+T790M in the lung epithelium. Upon administration of doxycycline these mice develop lung adenocarcinomas that are resistant to TKIs. However, the combination of the irreversible TKI afatinib and the EGFR antibody cetuximab showed dramatic responses in these transgenic mice. These preclinical studies led to a clinical trial of these agents, which has showing a promising 30% response rate in patients with EGFR mutant tumors resistant to TKIs. However, tumors also acquire resistance to this drug combination and the mechanisms of resistance to afatinib+cetuximab are currently unknown. We set out to identify these mechanisms using xenograft and transgenic mouse models of EGFR mutant lung cancer. Transgenic mice with EGFRL858R+T790M-induced tumors were treated with afatinib+cetuximab using an intermittent dosing strategy that we had previously used to generate erlotinib-resistant tumors in mice with EGFRL858R and EGFRDEL-induced tumors. 75% of mice develop afatinib+cetuximab resistant tumors after three month-long rounds of treatment. The same treatment strategy applied to xenografts harboring subcutaneous tumors induced by EGFRDEL+T790M gave rise to resistant tumors in 20% of cases. Analysis of the afatinib+cetuximab resistant tumors performed to date has not revealed additional mutations in the EGFR transgene, the ERBB2 kinase domain or KRAS. Additional sequencing studies and examination of signaling pathway alterations in the resistant tumors are ongoing. Uncovering mechanisms of resistance to this drug combination will allow the development of strategies to treat tumors that acquire resistance to EGFR-directed therapies. Citation Format: Valentina Pirazzoli, Elisa de Stanchina, Jungfeng Xia, Zhongming Zhao, William Pao, Katerina A. Politi. Modeling acquired resistance to EGFR-directed therapies in mouse models of lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 933. doi:10.1158/1538-7445.AM2013-933