American Association for Cancer Research
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Supplementary Tables 1 and 2, Supplementary Figures 1 through 25, and Supplementary Methods from Synergistic Immunostimulatory Effects and Therapeutic Benefit of Combined Histone Deacetylase and Bromodomain Inhibition in Non–Small Cell Lung Cancer

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posted on 2023-04-03, 21:20 authored by Dennis O. Adeegbe, Yan Liu, Patrick H. Lizotte, Yusuke Kamihara, Amir R. Aref, Christina Almonte, Ruben Dries, Yuyang Li, Shengwu Liu, Xiaoen Wang, Tiquella Warner-Hatten, Jessica Castrillon, Guo-Cheng Yuan, Neermala Poudel-Neupane, Haikuo Zhang, Jennifer L. Guerriero, Shiwei Han, Mark M. Awad, David A. Barbie, Jerome Ritz, Simon S. Jones, Peter S. Hammerman, James Bradner, Steven N. Quayle, Kwok-Kin Wong

Supplementary Table 1. HDAC inhibitors tested with healthy donor PBMCs and their biochemical potency (nM) across HDACs 1, 2, 3, and 6. Supplementary Table 2. Information for consented Non-small cell lung cancer (NSCLC) patients that underwent surgical resectioning as part of their treatment plan and whose tumor specimen and blood samples obtained after surgery were analyzed. Supplementary Figure 1. Reduced CD4+FOXP3+ Treg cells in healthy donor and NSCLC patient PBMC in the presence of ricolinostat. Supplementary Figure 2. Up-regulation of CD69 on T cells in healthy donor PBMC cultures in the presence of ricolinostat. Supplementary Figure 3. Viability of immune cells within dissociated tumor specimens cultured in the presence of ricolinostat or entinostat. Supplementary Figure 4. Effector function of T cells within 2-D cultures of disaggregated tumor specimens from NSCLC patients. Supplementary Figure 5. Increased expression of MHC class II and CD86 on monocytes in healthy donor PBMC in the presence of ricolinostat. Supplementary Figure 6. Phenotype of T cells infiltrating lung tumors of genetically engineered mice treated with ricolinostat. Supplementary Figure 7. Gene expression profile of Tumor-infiltrating T cells. Supplementary Figure 8. Ricolinostat promotes up-regulation of MHC class II and CD86 on tumor-associated macrophages. Supplementary figure 9. Immunohistochemical and flow cytometric analyses of acetylated α-tubulin in lung tumors of KP mice. Supplementary Figure 10. Kinetics of tumor growth in KP mice treated with ricolinostat. Supplementary Figure 11. Phenotype of tumor-infiltrating T cell subsets in lung tumors of KP mice treated with JQ1. Supplementary Figure 12. Immunohistochemical and flow cytometric analyses of phospho-STAT5 levels in lung tumors of KP mice. Supplementary Figure 13. Suppressive function of Tregs isolated from the spleen of lung tumor-bearing KP mice treated with JQ1. Supplementary Figure 14. Phenotype of Tregs in the spleen or lung tumors of genetically engineered mouse models (GEMM) of NSCLC. Supplementary Figure 15. Phenotype of Tregs within cultures of dissociated tumor specimen from NSCLC patients. Supplementary Figure 17. Histology and immunohistochemical staining of lung tumor sections from treated KP mice. Supplementary Figure 18. Kinetics of tumor growth in TL or wild-type mice. Supplementary Figure 19. Quantification of T cell subsets and TAMs in tumors of KP mice. Supplementary Figure 20. Phenotype of CD8+T cells infiltrating lung tumors of treated KP mice. Supplementary Figure 21. Gene expression profile of tumor-infiltrating macrophages (TAMs) in treated KP mice. Supplementary Figure 22. Gene expression profile of tumor-infiltrating macrophages T cells in treated KP mice. Supplementary Figure 23. Proportion of CD4+Foxp3+ Tregs present within tumor-infiltrating T cells utilized in gene expression studies. Supplementary Figure 24. Gating strategy. Supplementary Figure 25. Correlation plot for the expression of indicated genes in CD45+ leukocytes in KP tumors as evaluated by single cell RNA-Sequencing.

Funding

National Cancer Institute

Thoracic Foundation

Dana-Farber Cancer Institute

History

ARTICLE ABSTRACT

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 antitumor immunity and demonstrate their therapeutic potential for treatment of NSCLC.Significance: Selective inhibition of HDACs and bromodomain proteins modulates tumor-associated immune cells in a manner that favors improved T-cell function and reduced inhibitory cellular mechanisms. These effects facilitated robust antitumor responses in tumor-bearing mice, demonstrating the therapeutic potential of combining these epigenetic modulators for the treatment of NSCLC. Cancer Discov; 7(8); 852–67. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783