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Abstract
Background Therapies for metastatic Osteosarcoma (OS) are limited and survival rates have remained unchanged. Our goal is to use the histone deacetylase (HDAC) inhibitor, entinostat (MS-275), to epigenetically modulate NK cells to improve trafficking, persistence and therapeutic efficacy against OS lung metastases.
Methods Human NK cells were expanded from donor buffy coats for at least two weeks. RNA seq and ChiPseq was performed in untreated and MS-275 treated NK cells. Culture media and cells from untreated and treated NK cells alone or co-cultured with OS cells were collected for cytokine analysis (IsoLight). Findings were confirmed with flow cytometry and CellScape chip cytometry. The OS-17 human OS lung metastases mouse model was used. OS-17 cells were injected IV. One week after, untreated or pre-treated NK cells (0.5uM MS-275) were infused IV twice a week for three weeks. NK cells were labeled (DiR) and tracked in-vivo using the IVIS animal imaging system. Mice were euthanized 24 hours after the final NK cell treatment for histologic analysis of the lungs and some per group were included in 3D imaging with Xerra cryofluorescence tomography (CFT), to further assess NK cell distribution.
Results We identified 547 differentially expressed genes between the untreated and MS-275 treated NK cells. Four immune related genes (L1CAM, IFNg, C3 and CD28) were upregulated in the MS-275 treated NK cells and histone H3 and H4 acetylation was found at the immunomodulatory genes site (figure 1). MS-275 treated NK cells showed significantly increased MIP-1b, IL-6, IFNg (figure 2), TNFa, NKG2D and Ki67 and a decrease in TGFB1 (figure 3). Additionally, our flow cytometry data showed a phenotypic shift of MS-275 pre-treated NK cells to a greater percentage of the CD56 bright NK cell population (figure 3). There was a significant decrease in the number and size of lung nodules in the MS-275 pre-treated NK cell group relative to the untreated control group (199 vs 97, p=0.05), and Xerra CFT imaging demonstrated greater NK cell fluorescent signal in the lungs of mice that received MS-275 treated NK cells (figure 4), suggesting that MS-275 enhanced persistence of NK cells at tumor sites.
Conclusions We concluded that MS-275 has an immunomodulatory effect on NK cells and increases H3 and H4 acetylation, which leads to transcriptional activation of the immune-related genes that contribute to increased NK cell activation, cytokine production and increased cytolytic function. MS-275 also enhances NK cell persistence and therapeutic efficacy against OS lung metastases.
Ethics Approval All research involving animals was reviewed and approved by the UT MD Anderson Cancer Center IACUC (00000896-RN03).
MS-275 up-regulates immunomodulatory genes in human NK cells. 547 differentially expressed genes were identified including four immune-related genes: L1CAM, IFNg, C3 and CD28. MS-275 promotes H3 and H4 acetylation at L1CAM and IFNg in NK cells
MS-275 increases A. IL-6, an inflammatory cytokine, B. IFNg, an effector cytokine, and C. MIP-1b, a chemoattractive cytokine, in culture media of human NK cells
MS-275 treatment of NK cells (red): (A.) increases TNFa and (B.) decreases TGFB1 in single cell cytokine analysis and (C.) shifts NK cells to a CD56 bright phenotype and increases NKG2D, IFNg and Ki67 in flow cytometry analysis
MS-275 treated NK cells localized and persisted at lung metastases in greater numbers, as shown by increased fluorescent DiR signal, in both Xerra CFT imaging (A.); low signal: blue-purple, high signal: red-yellow; and in IVIS live animal imaging (B.)
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