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Gene-Specific Primers and Flow Antibodies

Supplemental Figures S1-S6

LKB1 inactivating mutations are commonly observed in patients with KRAS-mutant non–small cell lung cancer (NSCLC). Although treatment of NSCLC with immune checkpoint inhibitors (ICI) has resulted in improved overall survival in a subset of patients, studies have revealed that co-occurring KRAS/LKB1 mutations drive primary resistance to ICIs in NSCLC. Effective therapeutic options that overcome ICI resistance in LKB1-mutant NSCLC are limited. Here, we report that loss of LKB1 results in increased secretion of the C–X–C motif (CXC) chemokines with an NH2-terminal Glu-Leu-Arg (ELR) motif in premalignant and cancerous cells, as well as in genetically engineered murine models (GEMM) of NSCLC. Heightened levels of ELR+ CXC chemokines in LKB1-deficient murine models of NSCLC positively correlated with increased abundance of granulocytic myeloid-derived suppressor cells (G-MDSC) locally within the tumor microenvironment and systemically in peripheral blood and spleen. Depletion of G-MDSCs with antibody or functional inhibition via all-trans-retinoic acid (ATRA) led to enhanced antitumor T-cell responses and sensitized LKB1-deficent murine tumors to PD-1 blockade. Combination therapy with anti–PD-1 and ATRA improved local and systemic T-cell proliferation and generated tumor-specific immunity. Our findings implicate ELR+ CXC chemokine-mediated enrichment of G-MDSCs as a potential mediator of immunosuppression in LKB1-deficient NSCLC and provide a rationale for using ATRA in combination with anti–PD-1 therapy in patients with LKB1-deficient NSCLC refractory to ICIs.Significance:These findings show that accumulation of myeloid-derived suppressor cells in LKB1-deficient non–small cell lung cancer can be overcome via treatment with all-trans-retinoic acid, sensitizing tumors to immunotherapy.

Decresased responses to immune checkpoint inhibitors (ICIs) in NSCLC are associated with low tumor mutational burden (TMB), impaired tumor antigen presentation, decreased baseline CD8+ T lymphocytes tumor infiltration and low PD-L1 expression in the tumor microenvironment (TME). One approach to augment the efficacy of ICIs in NSCLC is to enhance anti-tumor T cell responses by utilizing in situ vaccination (ISV) with professional antigen-presenting dendritic cells (DCs). In preclinical studies as well as a phase I clinical trial, we have shown that ISV with gene-modified DCs expressing CCL21 (CCL21-DC) promotes effector T lymphocyte infiltration into the tumor, PD-L1 upregulation, and systemic tumor-specific immune responses. We hypothesized that in situ vaccination with CCL21-DC could promote T cell priming and activation, thereby sensitizing non-responsive NSCLC tumors to ICI. Utilizing multiple syngeneic murine models of NSCLC with distinct driver mutations [KrasG12D/P53+/-/Lkb1-/- (KPL); KrasG12D/P53+/- (KP); and KrasG12D (K)] and varying TMB, we observed that CCL21-DC ISV synergizes with anti-PD-1 therapy resulting in the eradication of a subset of tumors. Evaluation of KPL tumors by flow cytometry and scRNA-seq revealed that combination therapy reprogrammed the myeloid compartment to be less immunosuppressive, and induced T cell infiltration, Th1 polarization, as well as the enrichment of stem-like TCF1+ T cells within the TME. Intratumoral (IT) CCL21-DC enhanced CD103+DC migration to the local lymph node as determined by flow cytometry. Addition of ICI to IT CCL21-DC resulted in enhanced T cell proliferation and INF-γ secretion within the TME and the spleen. Whole exome sequencing (WES) of tumors revealed immunoediting of tumor subclones after IT CCL21-DC and ICI combination therapy. Tumor rechallenge studies demonstrated the establishment of systemic tumor-specific immunity in a subset of mice that were cured of disease.In conclusion, tumor vaccination with CCL21-DC sensitizes immune-resistant murine NSCLC to anti-PD-1 therapy. Combination therapy enhances anti-tumor T cell responses, resulting in immunoediting of tumor subclones and the generation of systemic tumor-specific immunity. In an ongoing clinical trial, we are evaluating the safety and efficacy of ISV with CCL21-DC in combination with anti-PD-1, pembrolizumab, in patients with advanced NSCLC who have progressed on front-line therapy. Citation Format: Ramin Salehi-Rad, Raymond J. Lim, Linh M. Tran, Yushen Du, Camelia Dumitras, Tianhao Zhang, William Crosson, Jensen Abascal, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situ vaccination with CCL21-DC sensitizes non-responsive murine NSCLCs to anti-PD-1 therapy and generates systemic tumor-specific immune memory [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 4206.

A changing mosaic of natural vegetation and human land uses has evolved within and around the Flint River Watershed (FRW) in Alabama and Tennessee over the past several decades. To determine the cause of change and linkage between human activities and environmental change can prove problematic. Subsequently, there is a need to produce projections of future environments based on planning instruments and socio-economic parameters. Scenarios of potential future land use land cover (LULC) change are required in order to better manage potential impacts on many environmental issues. This study creates future scenarios for the year 2030 from baseline land use of 2001, relative to three projected land use scenarios which include differences related to conservation, planning, and development. The future growth scenarios were created using the ArcGIS tool, Prescott Spatial Growth Model (PSGM). The model allows users to build different future growth scenarios based on socio-economic projections such as population, employment and other controlling factors. The simulation results indicate that LULC changes associated with future urbanization can increase by ~23% - 43% within the FRW, which will lead to significant environmental issues if not managed properly. The overall analysis and model results demonstrate the ability of future growth scenarios to explore and evaluate options for a future environment. Spatial modeling and analysis tools, such as PSGM, provide a powerful approach to evaluate potential impacts of LULC change in the future and should be used to manage urbanization in areas with more intense development.

Flint River watershed is located in northern Alabama and southern Tennessee, USA and is home to several species of rare, threatened, or endangered plants and animals in a rapidly urbanizing area. Dominant land uses are forest and agricultural, with row crops and livestock production as major farm enterprises. Soil and Water Assessment Tool (SWAT), a deterministic hydrologic model that can predict hydrologic conditions over various temporal and spatial scales, was used to simulate the hydrologic response of the watershed to land-use/land cover (LULC) change. Analysis between observed and predicted stream flow demonstrated that the initial SWAT model run requires calibration of stream parameters in order to give a more accurate output from the model. The calibration was performed with sequential uncertainty fitting, ver. 2 (SUFI-2) in the SWAT Calibration Uncertainty Program. After calibration, stream sediment yield values were compared by sub-basin between a current (2001) and three future (2030) land use scenarios, in order to identify areas in the watershed that were the most susceptible to increased sediment yield in the future. The future growth scenarios (smart, plan and sprawl) were created using the ArcGIS extension, Prescott Spatial Growth Model. Sub-basins with the greatest sensitivity for larger sediment yields were identified and prioritized for conservation efforts.

In this study, Moderate Resolution Imaging Spectrometer (MODIS) satellite measurements of aerosol optical depth (AOD) from different retrieval algorithms have been correlated with ground measurements of fine particulate matter less than 2.5 μm (PM

Ambient particulate matter has been shown to be associated with declining human health, although the association between fine particulate matter (PMWe utilized satellite-derived measures of PMAmong 30,239 participants in the REGARDS study, 746 incident events were observed: 72 hemorrhagic, 617 ischemic, and 57 of unknown type. Participants exposed to higher levels of PMIn this large cohort of African-Americans and whites, no association was observed between PM

A new drug, caronamide,1when given orally to patients receiving penicillin produces a physiologic and reversible inhibition of the excretion of this antibiotic, which results in elevation of the concentration of penicillin in the plasma. The primary objective of penicillin therapy is the maintenance of therapeutic concentrations of the antibiotic in the body tissues, but there appears to be no general agreement concerning therapeutic concentrations. Nor has it yet been made clear whether the maintenance of a constant level of penicillin plasma concentration is more desirable than the attainment of intermittent and temporary high levels. There does appear to be general agreement, however, that the attainment of high concentrations of penicillin is desirable. It is difficult to attain elevated concentrations because of the rapidity with which penicillin is absorbed from the site of injection and excreted by the kidneys. Efforts have been made to maintain plasma concentrations of the

A review of 1,997 patients to whom carinamide was administered has demonstrated that the drug possesses a low order of toxicity. Exclusive of nausea (12.1 per cent) and vomiting (5.5 per cent) the incidence of toxic manifestations was less than 2 per cent. True drug sensitivity was confirmed in fourteen patients and may be assumed in twenty-three additional cases although the concurrent administration of penicillin raises some questions as to the etiologic role of carinamide in all of the latter cases. Nausea may preclude therapy with the drug but this symptom may in large measure be ascribed to the mechanical factor of swallowing a large and oft-repeated dose of drug rather than to toxicity of the drug itself. From the observations recorded there is no indication that carinamide interfered with the elimination of normal nitrogenous metabolites or permanently impaired renal functions. Carinamide should be withheld in the presence of renal disease not because it is damaging to the kidneys but because its administration is unnecessary. Since carinamide is excreted almost entirely by glomerular filtration and sub-clinical grades of renal impairment cause accumulation of the drug in the circulation, dosage should be individualized and controlled by plasma carinamide estimations. It is possible that carinamide, by exerting its pharmacologic action upon the renal tubules, may inhibit the tubular excretion of some commonly employed medications. Few drugs in common use, even those that are granted to have little or no toxicity, can be given in the large doses, 18 to 32 Gm. per day, over periods of time up to sixty days, that are recorded here for carinamide.

T HE efficacy of penicillin as a therapeutic agent is impaired by its rapid excretion in the urine, which prevents the attainment and maintenance of high concentrations in the plasma. Relatively large doses must be administered in order to obtain detectable concentrations of penicillin in the blood stream, and massive doses of penicillin heretofore have been necessary if higher plasma concentrations are required. The primary objective of penicillin therapy is the maintenance of therapeutic concentrations of the drug in body tissues, and it is assumed that the higher the concentration of penicillin in the plasma, the higher will be the concentration in the tissues. The commonly accepted minimal effective therapeutic level is 0.03 unit of penicillin per cc. of plasma,’ but certain disease conditions, specifically subacute bacterial endocarditis and osteomyelitis may require much higher levels for curative effects. Attempts to obtain high plasma concentrations (high tissue concentrations) of penicillin include: (1) increase in the dosage; (2) more frequent administration at shorter intervals; (3) variation in the route of administration; (4) attempts to slow the rate of absorption from the site of injection and (5) partial inhibition of the renal excretion of penicillin. This report will describe a new approach to the problem of inhibition of excretion of penicillin by the kidneys by utilizing to advantage the properties and characteristics of a new compound, caronamide, which is a white, crystalline powder with the following structural formula