(P 101) EPIGENETIC MODULATION OF NK CELLS IMPROVES TUMOR TRAFFICKING AND ENHANCES THERAPEUTIC EFFICACY AGAINST OSTEOSARCOMA

Objective: Osteosarcoma (OS) remains a difficult disease to treat with 30-35% of patients dying of pulmonary metastases. Immunotherapy is becoming the main stay treatment in cancer. However, immune therapy and checkpoint inhibitors for relapsed OS has shown limited or unsatisfactory results. Natural Killer (NK) cells have demonstrated anti-tumor activity against OS. However, therapeutic efficacy has been limited due to the inability to penetrate, proliferate, and survive within the tumor. Therefore, strategies to improve NK cell efficacy are needed. We showed that the histone deacetylase inhibitor (HDACI) MS-275, immune-sensitized OS cells to NK cell-mediated lysis. MS-275 has also been shown to augment NK cell function. Therefore, the objective of our studies was to determine if epigenetic modulation of allogeneic NK cells with the HDAC inhibitor MS-275 could improve tumor trafficking and cytolytic function, thus enhancing therapeutic efficacy against OS lung metastases.

Methods: Human NK cells were purified and expanded from donor buffy coats using recombinant human IL-2 and genetically engineered K562 feeder cells. RNA was extracted from untreated and MS-275 pre-treated NK cells, submitted for RNA seq and ChiPseq. Additionally, culture media was collected for cytokine analysis. NK cells were also analyzed by flow cytometry to measure differences in CD56, CD16, NK activating receptor NKG2D, Ki67, Granzyme B and IFN𝝲.
NSG mice were injected with human OS17 luciferase-labeled OS cells and divided into three groups (untreated, NK cell treatment, and MS-275 pre-treated NK cell treatment) when lung tumors were confirmed by bioluminescence. NK cells were treated for 24 hours with 0.5uM MS-275 prior to IV injection into mice and administered twice/week for at least two weeks with some NK doses stained with near-infrared DilC18: 1.1”-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanide iodide (DiR) fluorescent dye prior to injection to allow for tracking of NK cells in-vivo using the IVIS imaging system. Mice were either euthanized 24 hours after the final NK cell treatment for histologic analysis of the lungs or, analyzed for survival across treatment groups. Additional mice from each group were used for 3D imaging [Xerra Cryofluorescent tomography (CFT)].

Results: MS-275 upregulates expression of immunomodulatory genes on NK cells. 547 differentially expressed genes were identified 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 identified immunomodulatory genes. Culture media from MS-275 treated NK cells showed significantly increased MIP-1b, IL-6 and IFN-g compared to untreated NK cells. MS-275 treated NK cells have increased Ki67, Granzyme B and IFN𝝲, measured by flow cytometry, indicating increase proliferation and cytotoxic function.
MS-275 pre-treatment enhanced therapeutic efficacy of NK cells against OS. There was a significant decrease in micro-metastases in the MS-275 pre-treated NK cell group as compared to the untreated control group (199 vs 9 p=0.05). Lastly, Xerra imaging demonstrated greater DiR signal in the lungs of the mice that received MS-275 treated NK cells compared to those that received untreated NK cells, suggesting greater persistence of NK cells at tumor sites.

Conclusion: We conclude that MS-275 has an immunomodulatory effect on NK cells (increased L1CAM, IFN𝝲, C3 and CD28) and increases H3 and H4 acetylation suggesting transcriptional activation of the immune-related genes that contribute to NK cell increased cytolytic function. The increase in Ki67, Granzyme B and IFN𝝲 seen with treatment of NK cells with MS-275 suggests that treated cells have increased proliferation and cytotoxic function. Pre-treatment of NK cells with MS-275 enhances NK cell therapeutic efficacy by enhancing localization and persistence within the lungs.