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Purpose: Patients with advanced non–small cell lung cancer (NSCLC) harboring activating EGFR mutations are initially responsive to tyrosine kinase inhibitors (TKI). However, therapeutic resistance eventually emerges, often via secondary EGFR mutations or EGFR-independent mechanisms such as epithelial-to-mesenchymal transition. Treatment options after EGFR-TKI resistance are limited as anti-PD-1/PD-L1 inhibitors typically display minimal benefit. Given that IL6 is associated with worse outcomes in patients with NSCLC, we investigate whether IL6 in part contributes to this immunosuppressed phenotype. Experimental Design: We utilized a syngeneic genetically engineered mouse model (GEMM) of EGFR-mutant NSCLC to investigate the effects of IL6 on the tumor microenvironment and the combined efficacy of IL6 inhibition and anti-PD-1 therapy. Corresponding in vitro studies used EGFR-mutant human cell lines and clinical specimens. Results: We identified that EGFR-mutant tumors which have oncogene-independent acquired resistance to EGFR-TKIs were more mesenchymal and had markedly enhanced IL6 secretion. In EGFR-mutant GEMMs, IL6 depletion enhanced activation of infiltrating natural killer (NK)- and T-cell subpopulations and decreased immunosuppressive regulatory T and Th17 cell populations. Inhibition of IL6 increased NK- and T cell–mediated killing of human osimertinib-resistant EGFR-mutant NSCLC tumor cells in cell culture. IL6 blockade sensitized EGFR-mutant GEMM tumors to PD-1 inhibitors through an increase in tumor-infiltrating IFNγ+ CD8+ T cells. Conclusions: These data indicate that IL6 is upregulated in EGFR-mutant NSCLC tumors with acquired EGFR-TKI resistance and suppressed T- and NK-cell function. IL6 blockade enhanced antitumor immunity and efficacy of anti-PD-1 therapy warranting future clinical combinatorial investigations.

IL-6 modulates expression of NK receptor and their ligands in EGFR mutant tumors.

Supplementary Fig S9 and legend

Supplementary Figures S10-S20 and figure legends

Somatic KRAS mutations are the most common oncogenic variants in lung cancer and are associated with poor prognosis. Using a Kras-induced lung cancer mouse model, CC-LR, we previously showed a role for inflammation in lung tumorigenesis through activation of the NF-κB pathway, along with induction of interleukin 6 (IL6) and an IL17-producing CD4+ T-helper cell response. IL22 is an effector molecule secreted by CD4+ and γδ T cells that we previously found to be expressed in CC-LR mice. IL22 mostly signals through the STAT3 pathway and is thought to act exclusively on nonhematopoietic cells with basal IL22 receptor (IL22R) expression on epithelial cells. Here, we found that higher expression of IL22R1 in patients with KRAS-mutant lung adenocarcinoma was an independent indicator of poor recurrence-free survival. We then showed that genetic ablation of Il22 in CC-LR mice (CC-LR/IL22KO mice) caused a significant reduction in tumor number and size. This was accompanied by significantly lower tumor cell proliferation, angiogenesis, and STAT3 activation. Il22 ablation was also associated with significant reduction in lung-infiltrating inflammatory cells and expression of protumor inflammatory cytokines. Conversely, this was accompanied with increased antitumor Th1 and cytotoxic CD8+ T-cell responses, while suppressing the protumor immunosuppressive T regulatory cell response. In CC-LR/IL22KO mice, we found significantly reduced expression of core stemness genes and the number of prototypical SPC+CCSP+ stem cells. Thus, we conclude that IL22 promotes Kras-mutant lung tumorigenesis by driving a protumor inflammatory microenvironment with proliferative, angiogenic, and stemness contextual cues in epithelial/tumor cells. Cancer Immunol Res; 6(7); 788–97. ©2018 AACR.

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand–receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment.Significance:The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception.This article is highlighted in the In This Issue feature, p. 2355

This file Includes 2 figures and one table as follow: 1: Supplementary figure S1 showing relative IL-22 mRNA expression (S1A), mutant K-ras protein expression (S1B), representative T regulatory cell flow cytometry plot (S1C), and total CD4 and CD8 flow cytometry plots (S1D) 2: Supplementary figure S2 showing pSTAT3 immunohistochemistry (S2A) and T cell related transcription factor mRNA expression (S2B) 3: Supplementary Table S1: List of Q-PCR primers and sequences

Supplementary Methods

Figure S3. Identification of a TRE (TGCGTCA) in the promoter of NRF2. The different human NRF2 promoter constructs were cloned upstream of a luciferase reporter gene (the sites where primer pairs bind are illustrated). These constructs were co-transfected into HEK293 cells alone with an empty vector, KRASDN, KRASG12D or KRASWT expression vector for 48h. Dual luciferase activities were measured. The experiment was repeated three times, each with triplicate samples. Data are expressed as mean {plus minus} SEM (*p < 0.05 Ctrl group vs. KRAS) (left panel).

Table 1. List of Q-RT-PCR primers. Supplementary methods and supplementary figure legends.

Supplementary Figure 2. (A) Heatmap representing IL-6/STAT3 pathway genes expression status. (B-C) Mass spectrometric quantification of IL-6 and STAT3 in lung cancer cell lines. (D) Siltuximab and Tocilizumab decreased STAT3 activation in NSCLC cell lines.

Table S1. Cisplatin toxicity as a function of KRAS, NRF2, KEAP1, and P53. The response to cisplatin-mediated toxicity of the listed NSCLC was measured by functional impairment of mitochondria using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. LD50s are listed. The coding regions of NRF2 and KRAS were sequenced and the status of each gene is listed. The mutation information for KEAP1 and TP53 was obtained from the literature. The references can be found in the supporting references according to the numbers in the table.

Supporting Data. Supporting reference, Supporting Figure Legends and Supporting Materials and Methods

Supplementary Figure 5. Relative expression of Arg1 mRNA normalized by Cd45 expression in lungs of CC-LR mice.

Supplementary Figure 3. Increased levels of IL-6 in bronchoalveolar lavage fluid (BALF) of CC-LR mice during tumor progression.

Supplementary Figure 1. Gating method for FACS analysis and sorting.

Supplementary Figure 6. Increased levels of IL-6 in bronchoalveolar lavage fluid (BALF) of CC-LR mice after NTHi exposure.

Supplementary Figure 4. Lung lesion distribution in lungs of CC-LR mice.

Supplementary Figure 7. Relative expression of Scgb1 (CCSP) mRNA in lungs of CC-LR mice.

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand–receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment. Significance: The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception. This article is highlighted in the In This Issue feature, p. 2355

Combustible cigarette smoking (CCS) is causally related to ~90% of all lung cancers and induces various tumor-initiating effects, some of which (e.g., inflammation) are not reversible even after smoking cessation. More recently, use of non-combustible smoking vectors, such as electronic cigarette vapors (ECV), have rapidly increased, especially among youth. Despite this emerging public health concern, the long-term impact of ECV exposure is poorly understood. Our lab sought to assess the safety of ECV and determine their influence on immune dysfunction and lung cancer progression by comparing the effects of CCS and ECV in an airway epithelial specific K-ras mutant mouse model of lung adenocarcinoma (CC-LR). Three cohorts of 6-week-old CC-LR mice were exposed to either room air, CCS, or ECV 5 days per week for 8 weeks. For CCS exposure, mice were exposed to CCS from 3R4F research cigarettes at a rate of 3 puffs/min for 2 hours each day using the SCIREQ integrated Cigarette Smoking Robot. For ECV exposure, mice were exposed to 72 mg/ml of liquid nicotine in 50% propylene glycol/vegetable glycerin solution at a rate of 3 puffs/min for 2 hours using the SCIREQ Electronic Nicotine Delivery System. While CCS led to a significant increase in tumor burden (25%), ECV showed no significant changes compared to room air-exposed control. Bronchoalveolar lavage fluid had significantly increased total lung immune cell infiltrates in both CCS (30%) and ECV (25%), particularly in macrophage and lymphocyte populations. Immunophenotyping of CCS and ECV exposed lungs displayed pronounced pro-tumor immunosuppressive phenotypes, characterized by significantly decreased CD4+ IFNγ+ and CD8+ GZMB+ cytotoxic T cells along with a significantly elevated CD4+ FOXP3+ regulatory T cells. Furthermore, the cytotoxic cytokine IFNγ was significantly reduced in lungs of CCS- (7-fold) and ECV-exposed (2-fold) mice compared to room air-exposed controls. We also found increased levels of the immunosuppressive cytokine IL-10 in lungs of CCS- and ECV-exposed mice. Previous studies have shown IL-10 to play an important role in microbiome-associated immune modulation, hence we performed a preliminary gut and lung microbiome study to assess microbiome dissimilarity in these cohorts. Taxonomic profiling via 16S rRNA gene sequencing of matched stool and lung samples showed differences in the relative abundance of several lung Proteobacteria spp. while gut Firmicutes, particularly Turicibacter and Ileibacterium, were increased by CCS and ECV. In conclusion, while both CCS and ECV increased immune suppression, ECV did not significantly promote tumorigenesis during this timeframe of intervention. Future studies probing differences in microbiome-modulated CCS and ECV immune dysfunction can help pave way for identification of new targets that foster new interception and early treatment strategies for K-ras mutant lung cancer. Citation Format: Walter V. Velasco Torrez, Maria T. Grimaldo, Michael J. Clowers, Bo Yuan, Segundo del Aguila Soto, Iman Bouchelkia, Javier Eduardo Moreno Barragan, Umesh C. Karandikar, Joseph F. Petrosino, Florencia McAllister, Humam Kadara, Kristi Louise Hoffman, Seyed Javad Moghaddam. Comparative effects of combustible cigarette versus electronic cigarette exposures on K-ras mutant lung cancer [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 649.

Mounting evidence supports synergistic roles for the gut microbiome in cancer progression. Yet, the interplay between the gut microbiome and immune responses in cancer is still poorly understood. We recently showed that gut microbiome changes are closely associated with development of Kras-mutant lung adenocarcinoma (KM-LUAD) in a human-relevant, tobacco-associated mouse model (Gprc5a-/-; G). Knockout of the antimicrobial protein Lcn2 in these mice (Gprc5a-/-/Lcn2-/-; GL) further reduced microbial diversity while enhancing inflammation and tumor development. We thus hypothesized that microbial dysbiosis in the gut, such as that incurred by loss of Lcn2, may exacerbate LUAD development. Here, we investigated the effects of gut microbiome modulation on LUAD pathogenesis using fecal microbiota transfer (FMT) in both syngeneic and tobacco carcinogenesis models. Syngeneic G mice (transplant of G LUAD cells) that received FMT from GL donors (G < GL) exhibited significantly increased tumor growth relative to littermates with FMT from G mice (G < G). These effects were recapitulated in an independent syngeneic model (KrasG12D LKR13 cells in wild type mice). Tobacco carcinogen-exposed G < GL mice also exhibited increased lung tumor development compared with similarly exposed G < G littermates. 16S rDNA-Seq analysis of fecal pellets revealed significant differences in gut beta diversity between syngeneic G < G and G < GL mice. G < GL mice additionally displayed elevated relative abundance of tumor-promoting Alistipes, while Ruminoccocus and Akkermansia, taxa associated with favorable response to immunotherapy, were reduced. We next performed single-cell RNA-sequencing to comprehensively probe the tumor immune microenvironment (TIME) and the immune milieu near the gut of tumors and mesenteric lymph nodes (MLNs), respectively. The TIME in G < GL mice displayed an overall enhanced immunosuppressive phenotype evidenced by prominently increased fractions of T regulatory and Cd4+ Izumo1r+ exhausted T cells and, conversely, reduced levels of activated Isg15+ Cd8a+ T cells. MLNs from G < GL mice showed markedly increased fractions of memory B cells expressing the immunosuppressor Bank1 and reduced levels of follicular B cells and Cd8a+ Clec9a+ class 1 dendritic cells (cDC1). Flow cytometry further showed enhanced immunosuppression in G < GL relative to G < G mice, including increased fractions of myeloid-derived suppressor cells in the TIME of the former group. Our findings show that gut microbiome dysbiosis fosters lung cancer development by promoting immunosuppression, perhaps via a local and systemic gut microbiota-immune system crosstalk. Modulating the gut microbiome may be a promising strategy for interception or early treatment of lung cancer. Citation Format: Zahraa Rahal, Fuduan Peng, Yuejiang Liu, Matthew C. Ross, Ansam Sinjab, Ke Liang, Jiping Feng, Chidera O. Chukwuocha, Manvi Sharma, Elizabeth Tang, Camille Abaya, Joseph Petrosino, Junya Fujimoto, Seyed Javad Moghaddam, Linghua Wang, Kristi L. Hoffman, Humam Kadara. Gut microbiome dysbiosis promotes immune suppression and lung cancer development [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 2883.

Despite improved diagnosis and treatment strategies such as immunotherapy, lung cancer is still the leading cause of cancer-related deaths worldwide in both men and women. Lung adenocarcinoma (LUAD) with driver mutations in the KRAS oncogene is the most prevalent molecular subtype of lung cancer. KRAS mutant LUAD exhibits aggressive biology in part due to enhanced pro-tumor inflammation mediated by activation of the nuclear factor-κB (NF-κB) and, consequently, elevated expression of various cytokines. While the pro-inflammatory cytokine IL-1β is a product of the NF-κB pathway it also acts as a potent activator, further amplifying the production of protumor cytokines via a positive feedback loop. We have shown that IL-1β blockade enhances anti-tumor immune responses while inhibiting immunosuppression in a mouse model of KRAS mutant LUAD driven by lung epithelial cell-specific expression of KRASG12D (CCSPCre/LSL-KRASG12D, CC-LR mouse). Cell lineage-specific mechanisms that underlie these anti-tumor effects following inhibition of IL-1β are still poorly understood. To fill this void, we here explored the effects of targeting the IL-1R receptor in KRAS mutant lung epithelial cells in the CC-LR mouse model. We studied tumor development and host immune response in 14-week-old CC-LR mice with conditional knockout of IL-1R in KRAS mutant lung epithelial cells (LR/IL-1RΔ/Δ) in comparison to age- and sex-matched control CC-LR littermates. LR/IL-1RΔ/Δ mice displayed markedly reduced tumor multiplicity (~50%) when compared to control CC-LR mice concomitant with decreased cell proliferation and angiogenesis evidenced by attenuated immunohistochemical expression of Ki-67 and ERG. Flow cytometry analysis showed an elevated inflammatory response, most evidently seen through the significant increase in infiltrating monocytes. Interestingly, a shift in type-1 conventional dendritic cells, commonly involved in antigen-cross presentation in tumorigenesis, to type-2 conventional dendritic cells was seen in LR/IL-1RΔ/Δ mice, suggesting a different method of cross-presentation resulting from the conditional knockout of IL-1R that could potentially lead to a protective T-cell response. These results were further confirmed via gene expression analysis of respective markers. Our findings provide further support for the role of the IL-1 cytokine family in the development and progression of KRAS mutant LUAD as well as warrant further studies targeted towards understanding the mechanistic effects IL-1β has on the tumor microenvironment. Citation Format: Avantika Krishna, Michael J. Clowers, Bo Yuan, Milind Mutala, Maria Jose Arredondo Sancristobal, Jocelynn Colunga, Ryan De Maleki, Linda Phan, Humam Kadara, Seyed Javad Moghaddam. Tumor cell-specific IL-1/IL-1R signaling promotes KRAS mutant lung tumorigenesis [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 652.

Understanding cellular processes underlying early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies. While most if not all single-cell RNA sequencing (scRNA-seq) studies of lung cancer provided details on immune and stromal states, little insight is drawn to epithelial cells given their paucity (~4%) when performing unbiased scRNA-seq analysis without prior enrichment. Here, we performed in-depth scRNA-seq of enriched (by sorting for EPCAM+) epithelial cell subsets from 16 early-stage LUADs and 47 matching normal lung (NL) tissues. We also studied tissues from the same LUADs and adjacent NL by whole exome sequencing and a subset by high-resolution spatial protein and transcriptome analysis. We also performed scRNA-seq analysis of murine lungs from a human-relevant model of LUAD development following exposure to tobacco carcinogen, including strains with an alveolar type II (AT2) cell-specific lineage reporter. After extensive quality control, we retained 246,102 high quality human epithelial cells which comprised diverse normal alveolar and airway lineages as well as cancer cell populations. Diversity among cancer cells was strongly linked to LUAD oncogenic drivers. KRAS-mutant cancer cells were unique in their transcriptional features, strikingly reduced differentiation, low levels of copy number changes, and increased variability amongst the cells themselves. The local epithelial niche of LUADs, relative to that of NL, was enriched with intermediary cells in lung alveolar differentiation. A subset of these cells displayed elevated KRT8 expression (KRT8+ alveolar cells; KACs), increased plasticity and frequency of KRASG12D mutations, and its gene expression profiles were enriched in lung precancer and LUAD and signified poor survival. Notably, KACs harboring KRAS mutations were only found in the ecosystem of KRAS-mutant LUADs. Murine KACs were evident in lungs of tobacco carcinogen-exposed mice that develop KRAS-mutant LUADs but not in the saline-treated control group. While murine KACs emerged prior to tumor onset, they persisted for months after carcinogen cessation, and like their human counterparts, acquired driver Kras mutations, were poorly differentiated, and harbored KRAS-specific transcriptional programs. Spatial transcriptomics analysis showed that KAC and KRAS signatures were elevated in both murine and human tumors as well as in KACs that were in the local spatial vicinity of the LUADs. Organoids derived from lungs of tumor-bearing reporter mice were markedly enriched with KACs and were conspicuously sensitive to targeted inhibition of KRAS-G12D. This study provides new insights into the landscape of normal epithelial and malignant cells in LUAD, the role of alveolar intermediate subsets in development of the malignancy, particularly that driven by mutant KRAS, and, thus, potential targets for early interception. Citation Format: Guangchun Han, Ansam Sinjab, Warapen Treekitkarnmongkol, Zahraa Rahal, Yuejiang Liu, Alejandra G. Serrano, Jiping Feng, Ke Liang, Khaja Khan, Wei Lu, Sharia Hernandez, Xuanye Cao, Enyu Dai, Yunhe Liu, Guangsheng Pei, Jian Hu, Lorena I. Gomez Bolanos, Edwin R. Parra, Tina Cascone, Boris Sepesi, Seyed Javad Moghaddam, Paul Scheet, Marcelo V. Negrao, John V. Heymach, Mingyao Li, Jichao Chen, Steven M. Dubinett, Junya Fujimoto, Luisa M. Solis, Ignacio I. Wistuba, Christopher S. Stevenson, Avrum Spira, Linghua Wang, Humam Kadara. An atlas of epithelial cell states and plasticity in lung adenocarcinoma [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 113.

Understanding cellular processes underlying early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here, we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal alveolar and airway lineages as well as cancer cell populations. Diversity among cancer cells was strongly linked to LUAD patient-specific oncogenic drivers. KRAS-mutant cancer cells were unique in their transcriptional features, strikingly reduced differentiation, low levels of DNA copy number changes, and increased variability amongst the cells themselves. The local niche of LUADs, relative to that of normal lungs, was enriched with intermediary cells in lung alveolar differentiation with potential to transition to KRAS-mutant cancer cells. A subset of alveolar intermediate cells with elevated KRT8 expression (KRT8+ alveolar cells; KACs) showed increased plasticity, and their gene expression profiles were enriched in lung precancer and LUAD and signified poor survival. Murine KACs were evident in lungs of tobacco carcinogen-exposed mice that develop KRAS-mutant LUADs but not in the saline-treated control group. While murine KACs emerged prior to tumor onset, they persisted for months after carcinogen cessation, acquired driver Kras mutations and, like their human counterparts, were poorly differentiated and harbored KRAS-specific transcriptional programs. This study provides new insights into early LUAD development and identifies potential targets for treatment.

Bioactive molecule library screening may empirically identify effective combination therapies, but molecular mechanisms underlying favorable drug–drug interactions often remain unclear, precluding further rational design. In the absence of an accepted systems theory to interrogate synergistic responses, we introduce Omics-Based Interaction Framework (OBIF) to reveal molecular drivers of synergy through integration of statistical and biological interactions in synergistic biological responses. OBIF performs full factorial analysis of feature expression data from single versus dual exposures to identify molecular clusters that reveal synergy-mediating pathways, functions and regulators. As a practical demonstration, OBIF analyzed transcriptomic and proteomic data of a dyad of immunostimulatory molecules that induces synergistic protection against influenza A and revealed unanticipated NF-κB/AP-1 cooperation that is required for antiviral protection. To demonstrate generalizability, OBIF analyzed data from a diverse array of Omics platforms and experimental conditions, successfully identifying the molecular clusters driving their synergistic responses. Hence, unlike existing synergy quantification and prediction methods, OBIF is a phenotype-driven systems model that supports multiplatform interrogation of synergy mechanisms.

K-ras mutant lung adenocarcinoma (LUAD) is the most common type of lung cancer, displays abysmal prognosis and is tightly linked to tumor-promoting inflammation, which is increasingly recognized as a target for therapeutic intervention. We have recently shown a gender-specific role for epithelial Stat3 signaling in the pathogenesis of K-ras mutant LUAD. The absence of epithelial Stat3 in male K-ras mutant mice (LR/Stat3Δ/Δ mice) promoted tumorigenesis and induced a nuclear factor-kappaB (NF-κB)-driven pro-tumor immune response while reducing tumorigenesis and enhancing anti-tumor immunity in female counterparts. In the present study, we manipulated estrogen and NF-κB signaling to study the mechanisms underlying this intriguing gender-disparity. In LR/Stat3Δ/Δ females, estrogen deprivation by bilateral oophorectomy resulted in higher tumor burden, an induction of NF-κB-driven immunosuppressive response, and reduced anti-tumor cytotoxicity, whereas estrogen replacement reversed these changes. On the other hand, exogenous estrogen in males successfully inhibited tumorigenesis, attenuated NF-κB-driven immunosuppression and boosted anti-tumor immunity. Mechanistically, genetic targeting of epithelial NF-κB activity resulted in reduced tumorigenesis and enhanced the anti-tumor immune response in LR/Stat3Δ/Δ males, but not females. Our data suggest that estrogen exerts a context-specific anti-tumor effect through inhibiting NF-κB-driven tumor-promoting inflammation and provide insights into developing novel personalized therapeutic strategies for K-ras mutant LUAD.

Lung cancer is the second most common cancers in the world and remains as the cancer with the highest incidence of death (Siegel et al. CA Cancer J Clin 71(1):7-33, 2021). K-RAS mutation is one of the most common mutations in non-small-cell lung cancer (NSCLC), encompassing 15-30% of lung adenocarcinomas (Cancer Genome Atlas Research Network. Nature 511:543-550, 2014). In this chapter, we describe various murine models with the goal of studying the role of inflammation in development and promotion of lung cancer. Immunomodulatory strategies are described in detail as well as the protocols that follow the intervention for harvesting various tissue and fluids for immune-profiling.

Kirsten rat sarcoma viral oncogene (K-ras) is one of the most frequent mutations of non-small cell lung cancer (NSCLC), specifically lung adenocarcinoma. Historically K-ras has been a mutation of great resilience and complexity. This shifted the focus of therapies to several of its related signaling pathways and cytokines. However, due to a greater understanding of the intricacies behind this mutation a greater arsenal of therapies has become available to us in the form of direct K-ras therapy. With this cumulative knowledge, we hope to bring an end to this irrepressible mutation in the future.

Lung adenocarcinoma (LUAD), particularly K-ras mutant LUAD, is a leading cause of cancer mortality. Therefore, strategies to prevent K-ras-mutant LUAD in its earliest stages in high-risk individuals (e.g., smokers) are urgently needed to reduce the public burden of this fatal disease. We and others have shown that K-ras driven tumorigenesis in lung is intimately linked to chronic inflammation and ultimately, tumor cells immune-escape. The antibiotic clarithromycin (CAM) was identified as one of the most promising candidates for repurposing with demonstrated immunomodulatory and anticancer properties. CAM is widely used and belongs to the macrolide class of antibiotics which are among the safest broad spectrum antimicrobials available. Abundant preclinical and clinical evidence exists demonstrating the in vitro and in vivo anticancer effects of CAM. It has been shown that macrolide antibiotics exert suppression of inflammation without overt immunosuppressive effects mostly through the inhibition of proinflammatory cytokines in vitro and in vivo. In these studies, we tested the lung cancer prevention efficacy of CAM using a Kras mutant lung cancer model. In the CCSPCre; LSL-Kras- G12 D (CC-LR) model, activation of the KrasG12 D mutation takes place in club cells by means of removal of the lox-stop-lox genomic sequence via expression of Cre recombinase under the control of the CCSP promoter. This model is excellent for reproducing the various premalignant to malignant progression steps in the lung. CC-LR mice of both genders were randomly enrolled to four experimental arms comparing three CAM doses: 10mg/kg/day, 50mg/kg/day, and 100mg/kg/day, to vehicle control (H2O). Treatment was administered by oral gavage, 5 times per week for 10 wks., starting at 4 wks. of age. At 14 wks. of age mice were euthanized, lung surface tumors were counted, bronchial lavage fluid, as well as lung samples, were obtained for histological, immunohistochemical (IHC) and qRT-PCR analyses. Clarithromycin treatment led to significant lung cancer prevention efficacy, as determined by lung surface tumor counts. A clear dose response with CAM was observed with a mean lung surface tumor count of 30.2 tumors per mouse for vehicle (n=12 mice), 23.5 for CAM 10mg/kg (n=8), 18.8 for CAM 50mg/kg (n=8) and 13.5 tumors per mouse for the CAM 100mg/kg treatment group (n=12, p=0.0014). A significant decrease in the incidence and multiplicity of premalignant and malignant lesions was also observed in histological analyses. For profiling of the lung immune microenvironment, we analyzed by qRT-PCR the expression of 18 genes identifying various cytokines, cell surface markers and proteins characteristic of specific activation states on the various cell subtypes in the tumor microenvironment. We found significant reduction in the expression of pro-inflammatory cytokines, IL-6, TNF, and IL-1β that are known to have a pro-tumor function in lung tumorigenesis, shown previously by us and other groups. We also found a reduction in the expression of myeloid cell-specific immunosuppressive markers, Arg1 and Fizz 1, that could be due to the reduction in the Gr1+ myeloid cell population, which was detected by means of IHC analyses on the same samples, or due to reprograming of pro-tumor M2 type macrophages toward an anti-tumor M1 phenotype. We also see a trend toward reduction of IL-17 cytokine, which we have previously shown to have an essential role in the promotion of K-ras mutant lung tumors. This was associated with an increase, although not significant, in the expression of anti-tumor Th1-specific transcription factor. However, we see an increase in the expression of PD-1, which could be due to the potential induction of an exhausted T cell phenotype. Taken together, we see a reprograming of the lung microenvironment from a tumor-promoting immunosuppressive phenotype toward an anti-tumor phenotype. This work was supported by NCI PREVENT TORFP75N91019F00131 (TORFP 2019 E-05). Citation Format: Seyed Javad Moghaddam, Tabish Hussain, Melody Zarghooni, Walter V. Velasco, Linda Phan, Michelle I. Savage, Jennifer T. Fox, Shizuko Sei, Powel H. Brown, C. Marcelo Aldaz. Repurposing of the macrolide antibiotic clarithromycin for the prevention of lung cancer [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A012.

Lung adenocarcinomas (LUAD) represent the most common lung cancer subtype and frequently harbor somatic mutations in the KRAS oncogene (KM-LUADs). While enhanced screening has improved early diagnosis of KM-LUAD, patient prognosis remains moderate to poor. Decoding the earliest events driving KM-LUADs can inform of ideal targets for its interception. Previous work showed that tobacco carcinogen (NNK) exposure leads to a pervasive field of injury comprised of molecular (e.g., KRAS mutations) and inflammatory changes that are shared between LUADs and their adjacent normal-appearing ecosystem. We and others have also shown that early immune and inflammatory alterations are implicated in the progression of normal lung (NL) epithelia and premalignant lesions (PMLs) to KM-LUAD. Yet, we still do not know the identities of specific epithelial subsets or how they promote a field of injury and inspire KM-LUAD pathogenesis. Here, we performed single-cell RNA-sequencing (scRNA-seq) of lungs from a human-relevant mouse model that develops PMLs and somatic KM-LUADs following NNK exposure. Analysis of 203,991 cells including 19,513 epithelial subsets after NNK cessation and at the onset of KM-LUADs revealed a unique population of alveolar cells that closely associated with tumor inception. These cells were highly evident in NNK- but not in control saline-exposed animals. Trajectory analysis showed that tumor clones developed through these transitionary cells, henceforth referred to as alveolar intermediate cells (AICs). Notably, AICs persisted for months after NNK cessation and acquired the same driver Kras mutations found in the resultant LUADs, thus supporting a role for AICs as KM-LUAD progenitors. Intriguingly, AICs harbored elevated expression of key components of p53 signaling (Trp53, Cdkn2a) and pro-inflammatory responses (IL-1β receptor Il1r1, NF-κB), and augmented cell-cell communication with Il1b+ macrophages which were enriched in LUAD-bearing lungs. Indeed, targeting IL-1β attenuated KM-LUAD development and increased anti-tumor immunity. In parallel, murine AIC expression profiles were significantly enriched in transcriptomes of human PMLs and LUADs. We thus probed our in-house and expanding scRNA-seq cohort of enriched (by sorting) epithelial subsets from human LUADs and NL, including 191,491 alveolar cells. AICs were not only evident in human lung tissues, but their fractions were also significantly increased in LUADs relative to NL. In conclusion, we identified a unique alveolar cell state that typified KM-LUAD progenitors, associated with inflammatory cues, and progressed along the pathologic continuum of damaged epithelium to KM-LUADs. Ongoing studies are evaluating whether AICs, in concert with tumor-initiating inflammation, trigger a field of injury that may underlie early phenotypic initiation and development of KM-LUAD. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Dapeng Hao, Enyu Dai, Luisa M. Solis, Seyed Javad Moghaddam, Junya Fujimoto, Jichao Chen, Matthew Edwards, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma [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 1586.

K-ras mutant lung adenocarcinoma (KM-LUAD) is a difficult-to-drug cancer subtype characterized by a chronic inflammatory tumor microenvironment (TME). Resistance to therapies, including immune checkpoint blockade (ICB), necessitates therapies that target this inflammatory TME. A major transcription factor that mediates chronic inflammation in KM-LUAD is signal transducer and activator of transcription 3 (STAT3). Inhibiting STAT3 may attenuate pro-tumor inflammation. Moreover, STAT3 modulates PD-L1 transcription, so STAT3 inhibitors in conjunction with ICB may increase ICB response rates. Here, we tested the anti-tumor ability of TTI-101, a selective STAT3 inhibitor, in a STAT3 addicted lung cancer cell line (MDA-F471) and in a transgenic mouse model of KM-LUAD (CCSPCre/LSL-KrasG12D, CC-LR). For in vivo experiments, CC-LR mice were treated daily with 50 mg/kg TTI-101 by oral gavage from 10 to 14 weeks of age to model a preventative regimen or from 14 to 18 weeks of age to survey the treatment effect on established tumors. TTI-101 was compared to anti-PD-1 ICB, with 200 μg injected intraperitoneally 3 times per week. In MDA-F471 cells, TTI-101 treatment decreased cell viability, with an IC50 of ~ 20 μM. In mice treated from 10 to 14 weeks of age, TTI-101 therapy significantly reduced the tumor burden compared to ICB. TTI-101 also reduced the number of proliferating cells within tumors. Mice in the 14-18-week group displayed similar trends, but these experiments are ongoing, as are combination TTI-101 and ICB treatment regimens. Our studies show that TTI-101 can reduce K-ras driven tumor cell proliferation in vitro and in vivo, suggesting STAT3 inhibition as an alternative preventive and therapeutic modality for KM-LUAD. The ongoing combination treatments and the 14-18-week cohorts will elucidate the timing of treatments as well as reveal if by targeting the inflammatory TME we are able to improve response to ICB. Funded by: R01 grant from NIH/NCI (R01CA225977) Citation Format: Michael J. Clowers, Cody Chou, Bo Yuan, Walter V. Velasco, Melody Zarghooni, Stephen Peng, T Kris Eckols, Humam Kadara, David J. Tweardy, Seyed Javad Moghaddam. Selective inhibition of the STAT3 pathway suppresses K-ras mutant lung tumorigenesis [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 2095.

Simple Summary The coronavirus disease 2019 (COVID-19) pandemic continues to spread rapidly on a global scale. When presenting with severe respiratory complications, COVID-19 results in markedly high death rates, particularly among patients with comorbidities such as cancer. Motivated by the ongoing global health crisis, we leveraged a growing in-house cohort of pulmonary tissues from lung cancer patients to analyze, at high resolution, the expression of host proteins implicated in the entryway of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into lung epithelial cells. Our results identify key pathways in lung pathobiology and inflammation that offer the potential to identify novel markers and therapeutic targets that can be repurposed for clinical management of COVID-19, particularly among lung cancer patients, a population that represents over half a million individuals in the United States alone. Abstract The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-associated mortality among lung cancer patients. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. The single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. We sought to delineate single-cell expression profiles of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung adenocarcinoma (LUAD) patients. We examined the expression levels and cellular distribution of ACE2 and SARS-CoV-2-priming proteases TMPRSS2 and TMPRSS4 in 5 LUADs and 14 matched normal tissues by single-cell RNA-sequencing (scRNA-seq) analysis. scRNA-seq of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2, and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, the expression of TMPRSS4 was highest and most frequently detected (75%) in lung cells with malignant features. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. We describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, thus comprising potential treatment targets for COVID-19 particularly among lung cancer patients.

Lung adenocarcinomas (LUADs) represent the most common lung cancer subtype and frequently harbor somatic mutations in the KRAS oncogene (KM-LUADs). While enhanced screening has improved early diagnosis of KM-LUAD, patient prognosis remains moderate to poor. Decoding the earliest events driving KM-LUADs can inform of ideal targets for its interception. Previous work showed that tobacco carcinogen (NNK) exposure leads to a pervasive field of injury comprised of molecular (e.g., KRAS mutations) and inflammatory changes that are shared between LUADs and their adjacent normal-appearing ecosystem. We and others have also shown that early immune and inflammatory alterations are implicated in the progression of normal lung (NL) epithelia and premalignant lesions (PMLs) to KM-LUAD. Yet, we still do not know the identities of specific epithelial subsets or how they promote a field of injury and inspire KM-LUAD pathogenesis. Here, we performed single-cell RNA-sequencing (scRNA-seq) of lungs from a human-relevant mouse model that develops PMLs and somatic KM-LUADs following NNK exposure. Analysis of 203,991 cells including 19,513 epithelial subsets after NNK cessation and at the onset of KM-LUADs revealed a unique population of alveolar cells that closely associated with tumor inception. These cells were highly evident in NNK- but not in control saline-exposed animals. Trajectory analysis showed that tumor clones developed through these transitionary cells, henceforth referred to as alveolar intermediate cells (AICs). Notably, AICs persisted for months after NNK cessation and acquired the same driver Kras mutations found in the resultant LUADs, thus supporting a role for AICs as KM-LUAD progenitors. Intriguingly, AICs harbored elevated expression of key components of p53 signaling (Trp53, Cdkn2a) and pro-inflammatory responses (IL-1β receptor Il1r1, NF-κB), and augmented cell-cell communication with Il1b+ macrophages which were enriched in LUAD-bearing lungs. Indeed, targeting IL-1β attenuated KM-LUAD development and increased anti-tumor immunity. In parallel, murine AIC expression profiles were significantly enriched in transcriptomes of human PMLs and LUADs. We thus probed our in-house and expanding scRNA-seq cohort of enriched (by sorting) epithelial subsets from human LUADs and NL, including 191,491 alveolar cells. AICs were not only evident in human lung tissues, but their fractions were also significantly increased in LUADs relative to NL. In conclusion, we identified a unique alveolar cell state that typified KM-LUAD progenitors, associated with inflammatory cues, and progressed along the pathologic continuum of damaged epithelium to KM-LUADs. Ongoing studies are evaluating whether AICs, in concert with tumor-initiating inflammation, trigger a field of injury that may underlie early phenotypic initiation and development of KM-LUAD. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Dapeng Hao, Enyu Dai, Luisa M Solis, Seyed Javad Moghaddam, Junya Fujimoto, Jichao Chen, Matthew Edwards, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR006.

Worldwide, lung cancer, particularly K-ras mutant lung adenocarcinoma (KM-LUAD), is the leading cause of cancer mortality because of its high incidence and low cure rate. To treat and prevent KM-LUAD, there is an urgent unmet need for alternative strategies targeting downstream effectors of K-ras and/or its cooperating pathways. Tumor-promoting inflammation, an enabling hallmark of cancer, strongly participates in the development and progression of KM-LUAD. However, our knowledge of the dynamic inflammatory mechanisms, immunomodulatory pathways, and cell-specific molecular signals mediating K-ras-induced lung tumorigenesis is substantially deficient. Nevertheless, within this signaling complexity, an inflammatory pathway is emerging as a druggable target: signal transducer and activator of transcription 3 (STAT3). Here, we review the cell type-specific functions of STAT3 in the pathogenesis and progression of KM-LUAD that could serve as a new target for personalized preventive and therapeutic intervention for this intractable form of lung cancer.

Rationale: Early pathogenesis of lung adenocarcinoma (LUAD) remains largely unknown. We found that, relative to wild-type littermates, the innate immunomodulator Lcn2 (lipocalin-2) was increased in...

Bioactive molecule library screening strategies may empirically identify effective combination therapies. However, without a systems theory to interrogate synergistic responses, the molecular mechanisms underlying favorable drug-drug interactions remain unclear, precluding rational design of combination therapies. Here, we introduce Omics-Based Interaction Framework (OBIF) to reveal molecular drivers of synergy through integration of statistical and biological interactions in supra-additive biological responses. OBIF performs full factorial analysis of feature expression data from single vs. dual factor exposures to identify molecular clusters that reveal synergy-mediating pathways, functions and regulators. As a practical demonstration, OBIF analyzed a therapeutic dyad of immunostimulatory small molecules that induces synergistic protection against influenza A pneumonia. OBIF analysis of transcriptomic and proteomic data identified biologically relevant, unanticipated cooperation between RelA and cJun that we subsequently confirmed to be required for the synergistic antiviral protection. To demonstrate generalizability, OBIF was applied to data from a diverse array of Omics platforms and experimental conditions, successfully identifying the molecular clusters driving their synergistic responses. Hence, OBIF is a phenotype-driven systems model that supports multiplatform exploration of synergy mechanisms.

Lung adenocarcinomas (LUADs) with mutations in the K-ras oncogene display dismal prognosis. Proinflammatory and immunomodulatory events that drive development of K-ras mutant LUAD are poorly understood. Here, we develop a lung epithelial specific K-ras mutant/Stat3 conditional knockout (LR/Stat3Δ/Δ) mouse model. Epithelial Stat3 deletion results in intriguing sex-associated discrepancies; K-ras mutant tumors are decreased in female LR/Stat3Δ/Δ mice whereas tumor burdens are increased in males. RNA-sequencing and tumor microenvironment (TME) analysis demonstrate increased anti-tumor immune responses following Stat3 deletion in females and, conversely, elevated pro-tumor immune pathways in males. While IL-6 blockade in male LR/Stat3Δ/Δ mice reduces lung tumorigenesis, inhibition of estrogen receptor signaling in female mice augments K-ras mutant oncogenesis and reprograms lung TME toward a pro-tumor phenotype. Our data underscore a critical sex-specific role for epithelial Stat3 signaling in K-ras mutant LUAD, thus paving the way for developing personalized (e.g. sex-based) immunotherapeutic strategies for this fatal disease., Proinflammatory and immunomodulatory events that drive development of K-ras mutant lung adenocarcinoma (LUAD) are poorly understood. Here they develop a lung epithelial specific K-ras mutant/Stat3 conditional knockout mouse model and show a sex-specific role for epithelial Stat3 signaling in K-ras-mutant LUAD.

Somatic KRAS mutations are the most common oncogenic variants in lung cancer and are associated with poor prognosis. Using a Kras-induced lung cancer mouse model, CC-LR, we previously showed a role for inflammation in lung tumorigenesis through activation of the NF-κB pathway, along with induction of interleukin 6 (IL6) and an IL17-producing CD4+ T-helper cell response. IL22 is an effector molecule secreted by CD4+ and γδ T cells that we previously found to be expressed in CC-LR mice. IL22 mostly signals through the STAT3 pathway and is thought to act exclusively on nonhematopoietic cells with basal IL22 receptor (IL22R) expression on epithelial cells. Here, we found that higher expression of IL22R1 in patients with KRAS-mutant lung adenocarcinoma was an independent indicator of poor recurrence-free survival. We then showed that genetic ablation of Il22 in CC-LR mice (CC-LR/IL22KO mice) caused a significant reduction in tumor number and size. This was accompanied by significantly lower tumor cell proliferation, angiogenesis, and STAT3 activation. Il22 ablation was also associated with significant reduction in lung-infiltrating inflammatory cells and expression of protumor inflammatory cytokines. Conversely, this was accompanied with increased antitumor Th1 and cytotoxic CD8+ T-cell responses, while suppressing the protumor immunosuppressive T regulatory cell response. In CC-LR/IL22KO mice, we found significantly reduced expression of core stemness genes and the number of prototypical SPC+CCSP+ stem cells. Thus, we conclude that IL22 promotes Kras-mutant lung tumorigenesis by driving a protumor inflammatory microenvironment with proliferative, angiogenic, and stemness contextual cues in epithelial/tumor cells. Cancer Immunol Res; 6(7); 788–97. ©2018 AACR.

Several promising chemopreventive agents have for lung cancer emerged in preclinical models and in retrospective trials. These agents have been shown to modulate pathways altered in carcinogenesis and reduce markers of carcinogenesis in animal and cell culture models. Cancer-prone transgenic mice with oncogenic Kras expressed in the airway epithelium (CcspCre/+ ; KrasLSL-G12D/+ ) were raised on diets compounded with myo-inositol. These animals form lung premalignant lesions in a stereotypical fashion over the ten weeks following weaning. Mice raised on myo-inositol containing diets showed potent reduction in the number, size, and stage of lesions as compared to those raised on control diets. myo-inositol has previously been reported to inhibit phosphoinositide 3-kinase (PI3K) signaling. However, in mice raised on myo-inositol, total PI3K signaling was largely unaffected. Proteomic and cytokine analyses revealed large reduction in IL-6 related pathways, including STAT3 phosphorylation. This effect was not due to direct inhibition of IL-6 production and autocrine signaling within the tumor cell, but rather through alteration in macrophage recruitment and in phenotype switching, with an increase in antitumoral M1 macrophages.