Faculty Bibliography

Background: There is growing appreciation for the role of tumordraining lymph nodes (TDLN) in the dynamic of immuno-editing orchestrated by non-small cell lung cancers (NSCLC). By comparing Tcell subsets and gene expression in TDLN and non-draining lymph nodes (NDLN), we aim to determine whether there is tumor-regional variation in immunophenotype. Method: Patients undergoing endobronchial ultrasound-guided transbronchial needle aspiration for the diagnosis/staging of NSCLC were recruited. Aspirates were obtained from TDLN (N1/N2 nodes with increased fluorodeoxyglucose-F-18 (FDG) avidity and/or enlarged >1cm) and NDLN (non-enlarged/non- FDG-avid N2/N3 nodes) along with peripheral blood. Samples were stained with fluorophore-conjugated antibodies (CD4-FITC, CD8-V450, CD25-PECy7, CD127-APCR700, CD45RO-PECF594) and analyzed by flow cytometry. CD4+CD25- and CD8+ effector T-cells (Teff) were sorted. Gene expression profiling was performed on sorted Teff using the NanostringTM platform to measure differential expression between TDLN and NDLNs. Result: We compared T-cell subpopulations in TDLN and paired NDLN from 16 subjects. There were significantly fewer CD4+ T-cells in TDLN vs NDLN (10.1% vs 28.9%, p=0.0039), with more Tregs (12.1% vs 7.3%, p=0.1563) suggesting a pattern of tumorregional immunosuppression in the TDLN. This was more consistent when tumor histology was adenocarcinoma compared to squamous cell cancer with respect to both depletion of Teff and higher proportion of Tregs (Fig 1). A more immunosuppressive TDLN phenotype was also observed with high tumor PD-L1 expression (>50%), with 36% fewer CD4+ T-cells in TDLN relative to paired NDLN when PD-L1 expression was high relative to just 3.2% fewer CD4+ T-cells with low PD-L1 expression. Gene expression in Teff has preliminarily demonstrated upregulation of genes mediating T-cell exhaustion (CTLA-4, PD-1, TGFb) and downregulation of co-stimulatory/recruitment factors (CD28, ICOS, ICAM2) in TDLN suggesting impaired activation of tumorregional Teff. Conclusion: Our findings suggest that TDLNs in patients with NSCLC display a tolerogenic phenotype, with more marked immunosuppression in the setting of adenocarcinoma and high tumor PD-L1 expression. (Figure Presented)

Understanding how murine models can elucidate the mechanisms underlying antitumor immune responses and advance immune-based drug development is essential to advancing the field of cancer immunotherapy. The Society for Immunotherapy of Cancer (SITC) convened a workshop titled, "Challenges, Insights, and Future Directions for Mouse and Humanized Models in Cancer Immunology and Immunotherapy" as part of the SITC 31st Annual Meeting and Associated Programs on November 10, 2016 in National Harbor, MD. The workshop focused on key issues in optimizing models for cancer immunotherapy research, with discussions on the strengths and weaknesses of current models, approaches to improve the predictive value of mouse models, and advances in cancer modeling that are anticipated in the near future. This full-day program provided an introduction to the most common immunocompetent and humanized models used in cancer immunology and immunotherapy research, and addressed the use of models to evaluate immune-targeting therapies. Here, we summarize the workshop presentations and subsequent panel discussion.

Cells lacking the tumor suppressor gene LKB1/STK11 alter their metabolism to match the demands of accelerated growth, leaving them highly vulnerable to stress. However, targeted therapy for LKB1-deficient cancers has yet to be reported. In both Kras/p53/Lkb1 cell lines and a genetically engineered mouse model of Kras/p53/Lkb1-induced lung cancer, much higher rates of DNA damage occur, resulting in increased dependence on Chk1 checkpoint function. Here we demonstrate that short-term treatment with the Chk1 inhibitor AZD7762 reduces metabolism in pembrolizumab tumors, synergizing with the DNA-damaging drug gemcitabine to reduce tumor size in these models. Our results offer preclinical proof of concept for use of a Chk1 inhibitor to safely enhance the efficacy of gemcitabine, particularly in aggressive KRAS-driven LKB1-deficient lung adenocarcinomas. Cancer Res; 77(18); 5068-76. (c)2017 AACR.

The bromodomain and extraterminal (BET) family of proteins comprises four members-BRD2, BRD3, BRD4 and the testis-specific isoform BRDT-that largely function as transcriptional coactivators and play critical roles in various cellular processes, including the cell cycle, apoptosis, migration and invasion. BET proteins enhance the oncogenic functions of major cancer drivers by elevating the expression of these drivers, such as c-Myc in leukemia, or by promoting the transcriptional activities of oncogenic factors, such as AR and ERG in prostate cancer. Pathologically, BET proteins are frequently overexpressed and are clinically linked to various types of human cancer; they are therefore being pursued as attractive therapeutic targets for selective inhibition in patients with cancer. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed and have shown promising outcomes in early clinical trials. Although resistance to BET inhibitors has been documented in preclinical models, the molecular mechanisms underlying acquired resistance are largely unknown. Here we report that cullin-3^SPOP earmarks BET proteins, including BRD2, BRD3 and BRD4, for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the degradation of BET proteins, leading to their elevated abundance in SPOP-mutant prostate cancer. As a result, prostate cancer cell lines and organoids derived from individuals harboring SPOP mutations are more resistant to BET-inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor-suppressor role of SPOP in prostate cancer in which it acts as a negative regulator of BET protein stability and also provide a molecular mechanism for resistance to BET inhibitors in individuals with prostate cancer bearing SPOP mutations.

Targeted covalent small molecules have shown promise for cancers driven by KRAS G12C. Allosteric compounds that access an inducible pocket formed by movement of a dynamic structural element in KRAS, switch II, have been reported, but these compounds require further optimization to enable their advancement into clinical development. We demonstrate that covalent quinazoline-based switch II pocket (SIIP) compounds effectively suppress GTP loading of KRAS G12C, MAPK phosphorylation, and the growth of cancer cells harboring G12C. Notably we find that adding an amide substituent to the quinazoline scaffold allows additional interactions with KRAS G12C, and remarkably increases the labeling efficiency, potency, and selectivity of KRAS G12C inhibitors. Structural studies using X-ray crystallography reveal a new conformation of SIIP and key interactions made by substituents located at the quinazoline 2-, 4-, and 7-positions. Optimized lead compounds in the quinazoline series selectively inhibit KRAS G12C-dependent signaling and cancer cell growth at sub-micromolar concentrations.

Effective therapies for non-small cell lung cancer (NSCLC) remain challenging despite an increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now clear that therapeutic agents with potential to impact the tumor immune microenvironment potentiate immune-orchestrated therapeutic benefit. Herein we evaluated the immunoregulatory properties of histone deacetylase (HDAC) and bromodomain inhibitors, two classes of drugs that modulate the epigenome, with a focus on key cell subsets that are engaged in an immune response. By evaluating human peripheral blood and NSCLC tumors, we show that the selective HDAC6 inhibitor ricolinostat promotes phenotypic changes that support enhanced T cell activation and improved function of antigen presenting cells. The bromodomain inhibitor JQ1 attenuated CD4+Foxp3+ T regulatory cell suppressive function and synergized with ricolinostat to facilitate immune-mediated tumor growth arrest, leading to prolonged survival of mice with lung adenocarcinomas. Collectively, our findings highlight the immunomodulatory effects of two epigenetic modifiers that, together, promote T cell-mediated anti-tumor immunity and demonstrate their therapeutic potential for treatment of NSCLC.

HYPOTHESIS: Proinflammatory cytokine Interleukin (IL)-17A (IL-17A) is overexpressed in a subset of patients with lung cancer. We hypothesized that IL-17A promotes a pro-tumorigenic inflammatory phenotype, and inhibits anti-tumor immune responses. EXPERIMENTAL DESIGN: We generated bi-transgenic mice expressing a conditional IL-17A allele along with conditional KrasG12D and performed immune phenotyping of mouse lungs, survival analysis, and treatment studies with antibodies either blocking PD-1 or IL6, or depleting neutrophils. To support preclinical findings, we analyzed human gene expression datasets and immune profiled patient lung tumors. RESULTS: Tumors in IL-17:KrasG12D mice grew more rapidly, resulting in a significantly shorter survival as compared to KrasG12D. IL-6, G-CSF, MFG-E8, and CXCL1 were increased in the lungs of IL17:Kras mice. Time course analysis revealed that tumor-associated neutrophils (TANs) were significantly elevated, and lymphocyte recruitment was significantly reduced in IL17:KrasG12D mice as compared to KrasG12D. In therapeutic studies PD-1 blockade was not effective in treating IL-17:KrasG12D tumors. In contrast, blocking IL-6 or depleting neutrophils with an anti-Ly-6G antibody in the IL17:KrasG12D tumors resulted in a clinical response associated with T cell activation. In tumors from lung cancer patients with KRAS mutation we found a correlation among higher levels of IL-17A and the colony stimulating factor (CSF3), and a significant correlation among high neutrophil and lower T cell numbers. CONCLUSIONS: Here we show that an increase in a single cytokine, IL-17A, without additional mutations, can promote lung cancer growth by promoting inflammation, which contributes to resistance to PD-1 blockade and sensitizes tumors to cytokine/neutrophil depletion.

Adenosquamous lung tumours, which are extremely poor prognosis, may result from cellular plasticity. Here, we demonstrate lineage switching of KRAS+ lung adenocarcinomas (ADC) to squamous cell carcinoma (SCC) through deletion of Lkb1 (Stk11) in autochthonous and transplant models. Chromatin analysis reveals loss of H3K27me3 and gain of H3K27ac and H3K4me3 at squamous lineage genes, including Sox2, DeltaNp63 and Ngfr. SCC lesions have higher levels of the H3K27 methyltransferase EZH2 than the ADC lesions, but there is a clear lack of the essential Polycomb Repressive Complex 2 (PRC2) subunit EED in the SCC lesions. The pattern of high EZH2, but low H3K27me3 mark, is also prevalent in human lung SCC and SCC regions within ADSCC tumours. Using FACS-isolated populations, we demonstrate that bronchioalveolar stem cells and club cells are the likely cells-of-origin for SCC transitioned tumours. These findings shed light on the epigenetics and cellular origins of lineage-specific lung tumours.

OBJECTIVES: Immune checkpoint inhibitors have demonstrated clinical benefit in recurrent, metastatic (R/M) squamous cell carcinoma of the head and neck (SSCHN), but lacking are biomarkers that predict response. We sought to define an inflamed tumor immunophenotype in this R/M SCCHN population and correlate immune metrics with clinical parameters and survival. METHODS: Tumor samples were prospectively acquired from 34 patients to perform multiparametric flow cytometry and multidimensional clustering analysis integrated with next-generation sequencing data, clinical parameters and outcomes. RESULTS: We identified an inflamed subgroup of tumors with prominent CD8+ T cell infiltrates and high PD-1/TIM3 co-expression independent of clinical variables, with improved survival compared with a non-inflamed subgroup (median overall survival 84.0 vs. 13.0months, p=0.004). The non-inflamed subgroup demonstrated low CD8+ T cells, low PD-1/TIM3 co-expression, and higher Tregs. Overall non-synonymous mutational burden did not correlate with response to PD-1 blockade in a subset of patients. CONCLUSION: R/M SCCHN patients with an inflamed tumor immunophenotype demonstrate improved survival. Further prospective studies are needed to validate these findings and explore the use of immunophenotype to guide patient selection for immunotherapeutic approaches.

PURPOSE: To evaluate and compare the volumetric tumor burden changes during crizotinib therapy in mice and human cohorts with ALK-rearranged non-small-cell lung cancer (NSCLC). METHODS: Volumetric tumor burden was quantified on serial imaging studies in 8 bitransgenic mice with ALK-rearranged adenocarcinoma treated with crizotinib, and in 33 human subjects with ALK-rearranged NSCLC treated with crizotinib. The volumetric tumor burden changes and the time to maximal response were compared between mice and humans. RESULTS: The median tumor volume decrease (%) at the maximal response was -40.4% (range: -79.5%-+11.7%) in mice, and -72.9% (range: -100%-+72%) in humans (Wilcoxon p=0.03). The median time from the initiation of therapy to maximal response was 6 weeks in mice, and 15.7 weeks in humans. Overall volumetric response rate was 50% in mice and 97% in humans. Spider plots of tumor volume changes during therapy demonstrated durable responses in the human cohort, with a median time on therapy of 13.1 months. CONCLUSION: The present study described an initial attempt to evaluate quantitative tumor burden changes in co-clinical imaging studies of genomically-matched mice and human cohorts with ALK-rearranged NSCLC treated with crizotinib. Differences are noted in the degree of maximal volume response between the two cohorts in this well-established paradigm of targeted therapy, indicating a need for further studies to optimize co-clinical trial design and interpretation.