Suppression of Myeloid Cell Arginase Activity leads to Therapeutic Response in a NSCLC Mouse Model by Activating Anti-Tumor Immunity

Abstract

Background: Tumor orchestrated metabolic changes in the microenvironment limit generation of anti-tumor immune responses. Availability of arginine, a semi-essential amino acid, is critical for lymphocyte proliferation and function. Levels of arginine are regulated by the enzymes arginase 1,2 and nitric oxide synthase (NOS). However, the role of arginase activity in lung tumor maintenance has not been investigated in clinically relevant orthotopic tumor models.

Methods: RNA sequencing (RNA-seq) of sorted cell populations from mouse lung adenocarcinomas derived from immunocompetent genetically engineered mouse models (GEMM)s was performed. To complement mouse studies, a patient tissue microarray consisting of 150 lung adenocarcinomas, 103 squamous tumors, and 54 matched normal tissue were stained for arginase, CD3, and CD66b by multiplex immunohistochemistry. Efficacy of a novel arginase inhibitor compound 9 in reversing arginase mediated T cell suppression was determined in splenocyte ex vivo assays. Additionally, the anti-tumor activity of this compound was determined in vitro and in an autochthonous immunocompetent Kras^G12D GEMM of lung adenocarcinoma model.

Results: Analysis of RNA-seq of sorted myeloid cells suggested that arginase expression is elevated in myeloid cells in the tumor as compared to the normal lung tissue. Accordingly, in the patient samples arginase 1 expression was mainly localized in the granulocytic myeloid cells and significantly elevated in both lung adenocarcinoma and squamous tumors as compared to the controls. Our ex vivo analysis demonstrated that myeloid derived suppressor cell (MDSC)s cause T cell suppression by arginine depletion, and suppression of arginase activity by a novel ARG1/2 inhibitor, compound 9, led to restoration of T cell function by increasing arginine. Treatment of Kras^G12D GEMM of lung cancer model with compound 9 led to a significant tumor regression associated with increased T cell numbers and function, while it had no activity across several murine and human non-small cell (NSCLC) lung cancer lines in vitro.

Conclusions: We show that arginase expression is elevated in mouse and patient lung tumors. In a KRAS^G12D GEMM arginase inhibition diminished growth of established tumors. Our data suggest arginase as an immunomodulatory target that should further be investigated in lung tumors with high arginase activity.

Keywords: Aminoacid; Arginase; Arginine; Autochthonous; Immunocompetent; MDSC; Metabolic checkpoint.

Fig. 1

Arginase expression in the lung tumors. a RNA Sequencing analysis of sorted tumor (Epcam+), macrophage (CD11c+ CD11b- CD103-) and neutrophils (CD11b+, LY6G+) from Kras^G12D lung tumors. T denotes cells isolated from tumor carrying mice and c from healthy control littermates. Heatmaps denote Log2 of fragments per kilobase of transcript (FPKM) per each mouse sample. p values are 0.02 and 0.03 for macrophages and neutrophils for Arg1 and 0.01 and 0.07 for macrophages and neutrophils for Arg2. Log2 (FPKM) values were also used for statistical analysis by student’s T test. Heatmaps represent unscaled values. b High power image of ARG1, CD3, CD66b multiplex immunohistochemistry. c Low power representative images from ARG1, CD3, CD66b multiplex immunohistochemistry staining of the TMA for varying levels of Arg1 and CD3 staining. Left: Arg1 high CD3 low, middle: Arg1 and CD3 high, and right: Arg1 low CD3 high tumor sample. d Correlation of CD66b and ARG1 in lung tumor samples. P < 0.0001, Spearman correlation. e Automated quantification of the ARG1 + CD66b + density in adjacent matched-normals, LUAD, and LUSQ p < 0.0001, determined by Mann-Whitney test f Correlation of CD66b+ ARG1+ double staining and CD3 staining in NSCLC, p < 0.0264, Spearman correlation. G. Kaplan Meier survival analysis for patients whose tumors were CD66b+/ARG1+ high or low density based on multiplex IHC. p = 0.09 (Log Rank test). The thresholds set for fluorescence positive signals for CD66b and CD3 are higher than that for ARG1 therefore the quantity of the expression is relative not exact

Fig. 2

Compound 9 reverses arginine depletion mediated interferon gamma production. a Arginine depletion prevented IFNg secretion by splenocytes. Splenocytes were activated with CD3/28 in the presence of media with arginine levels of 0, 2.5, 10, 40, 160 and 1000 uM. After 24 h IFNg was determined by ELISA in the culture supernatants b-d. Compound 9 mediated restoration of T cell function. Splenocytes were isolated from mixed background mice, dissociated and activated with CD3/28 antibodies in the presence of in the presence of arginine 40 uM. Recombinant Arginase (ARG1, 1 μg/ml) and Cpd9 (50 uM) were added to the indicated wells at the same time. b IFNg was determined by ELISA in the culture supernatants after 24 h c Granzyme B expression CD8 T cells were determined by flow cytometry d IL-2 expressing CD8 T cells were determined by flow cytometry. *p < =0.05, **p < =0.01, and ***p < =0.001. Graphs show representative results from experiments performed at least three times

Fig. 3

M2 macrophages and Cd11b + cells express Arginase 1 and suppress T cell function. a Cpd9 50 uM prevented M2 peritoneal macrophages suppression of IFNg secretion by splenocytes. Splenocytes were activated with CD3/28 in the presence of 25,000 M2 peritoneal macrophages supplemented with arginine. IFNg was determined by ELISA in the culture supernatants after 24 h. Splenocytes 50K, PM 25K. b IC50 of Cpd9 in 15K M2 peritoneal macrophages. c Western blot analysis of Arginase 1 expression in CD11b + cells. CD11b + cells were sorted from ID8 tumor ascites fluid, and Arginase 1 expression was characterized by western blot using Arginase 1 and tubulin antibodies. d Arginine 2 mM prevented CD11b + cells suppression of IFNg secretion by splenocytes. Splenocytes were activated with CD3/28 in the presence of 0.075 mM or 2 mM arginine as indicated. Sorted CD11b + cells were added to the indicated wells. After 24 hs IFNg was determined by ELISA in the culture supernatants. Splenocytes 50K; CD11b: 150K e. Cpd9 50 uM prevented CD11b + MDSC cells suppression of IFNg secretion by splenocytes. Splenocytes were activated with CD3/28 in the presence of arginine 75 uM, sorted CD11b + MDSC and Cpd9 50 uM were added to the indicated wells. IFNg was determined by ELISA in the culture supernatants after 24 h. Splenocytes 50K; CD11b: 150K f IC50 of Cpd9 in CD11b MDSC suppression of splenocytes IFNg secretion. Splenocytes were activated with CD3/28 in the presence of arginine 75 uM arginine, sorted CD11b + MDSC and various concentrations of Cpd9. After 24 hs IFNg was determined by ELISA in the culture supernatants and the % of control (no Cpd9) was calculated. Splenocytes 100K, Arginase 0.35 μg/ml, and Arginine 75 uM. *p < =0.05, **p < =0.01, and ***p < =0.001. Graphs show representative results from experiments performed at least three times

Fig. 4

Inhibiting arginase activity has therapeutic efficacy in Kras mutant Gemms by creating an immune favorable microenvironment. a Representative MRI image of Kras mutant mouse lung tumors from mice treated with Compound 9 for one week. b Quantification of MRI images of lung tumors of mice either treated with vehicle or compound 9 for up to four weeks, each dot represents an individual mouse. p values for each of the 1,2 and 4 week time points are indicated on the graph c Measurement of arginine levels in the lung tumor tissue and blood of mice either treated with vehicle or compound 9 by metabolomics. d % CD3, CD4, and CD8 T cells in all CD45 cells, and e CD8 to regulatory T cell (FoxP3+) ratios in the lung tumor microenvironment of mice treated with vehicle or compound 9. determined by multicolor flow cytometry analysis. f Quantification of interferon gamma production by flow cytometry of ex vivo stimulated and enriched lymphocytes from the lung tumors in mice treated with vehicle or compound 9 for one week. All p values in this figure were determined by Student’s t test. Each data point in the graphs is from a different mouse